Genetic structure and inter-generic relationship of closed colony of laboratory rodents based on RAPD markers.

PubMed

Kumar, Mahadeo; Kumar, Sharad

2014-11-01

Molecular genetic analysis was performed using random amplified polymorphic DNA (RAPD) on three commonly used laboratory bred rodent genera viz. mouse (Mus musculus), rat (Rattus norvegicus) and guinea pig (Cavia porcellus) as sampled from the breeding colony maintained at the Animal Facility, CSIR-Indian Institute of Toxicology Research, Lucknow. In this study, 60 samples, 20 from each genus, were analyzed for evaluation of genetic structure of rodent stocks based on polymorphic bands using RAPD markers. Thirty five random primers were assessed for RAPD analysis. Out of 35, only 20 primers generated a total of 56.88% polymorphic bands among mice, rats and guinea pigs. The results revealed significantly variant and distinct fingerprint patterns specific to each of the genus. Within-genera analysis, the highest (89.0%) amount of genetic homogeneity was observed in mice samples and the least (79.3%) were observed in guinea pig samples. The amount of genetic homogeneity was observed very high within all genera. The average genetic diversity index observed was low (0.045) for mice and high (0.094) for guinea pigs. The inter-generic distances were maximum (0.8775) between mice and guinea pigs; and the minimum (0.5143) between rats and mice. The study proved that the RAPD markers are useful as genetic markers for assessment of genetic structure as well as inter-generic variability assessments.

Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis.

PubMed

Midorikawa, G E O; Pinheiro, M R R; Vidigal, B S; Arruda, M C; Costa, F F; Pappas, G J; Ribeiro, S G; Freire, F; Miller, R N G

2008-07-01

The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus-specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus, with a detection limit of 10 fg. Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.

Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

PubMed Central

Pooler, M R; Ritchie, D F; Hartung, J S

1996-01-01

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria. PMID:8795198

RAPD-SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Wu, Li-Ping

2012-06-01

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.

Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas.

PubMed

Damasco, O P; Graham, G C; Henry, R J; Adkins, S W; Smiths, M K; Godwin, I D

1996-11-01

A RAPD marker specific to the dwarf off-type (hereafter known as dwarf) from micropropagation of Cavendish banana (Musa spp. AAA) cultivars New Guinea Cavendish and Williams was identified following an analysis of 57 normal (true-to-type) and 59 dwarf plants generated from several different micropropagation events. Sixty-six random decamer primers were used in the initial screen, of which 19 (28.8%) revealed polymorphisms between normal and dwarf plants. Primer OPJ-04 (5'-CCGAACACGG-3') was found to amplify an approx. 1.5 kb band which was consistently present in all normal but absent in all dwarf plants of both cultivars. Reliable detection of dwarf plants was achieved using this marker, providing the only available means ofin vitro detection of dwarfs. The use of this marker could facilitate early detection and elimination of dwarfs from batches of micropropagated bananas, and may be a useful tool in determining what factors in the tissue culture process lead to this off type production.Other micropropagation-induced RAPD polymorphisms were observed but were not associated with the dwarf trait.

Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India.

PubMed

Ashraf, Kamran; Ahmad, Altaf; Chaudhary, Anis; Mujeeb, Mohd; Ahmad, Sayeed; Amir, Mohd; Mallick, N

2014-04-01

The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers screened were informative and produced 275 amplification products, among which 261 products (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23% to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275 RAPD bands were used to generate Jaccard's similarity coefficients and to construct a dendrogram by means of UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for ginger improvement program.

Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India

PubMed Central

Ashraf, Kamran; Ahmad, Altaf; Chaudhary, Anis; Mujeeb, Mohd.; Ahmad, Sayeed; Amir, Mohd.; Mallick, N.

2013-01-01

The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers screened were informative and produced 275 amplification products, among which 261 products (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23% to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for ginger improvement program. PMID:24600309

Analysis of genetic diversity of Persea bombycina "Som" using RAPD-based molecular markers.

PubMed

Bhau, Brijmohan Singh; Medhi, Kalyani; Das, Ambrish P; Saikia, Siddhartha P; Neog, Kartik; Choudhury, S N

2009-08-01

The utility of RAPD markers in assessing genetic diversity and phenetic relationships in Persea bombycina, a major tree species for golden silk (muga) production, was investigated using 48 genotypes from northeast India. Thirteen RAPD primer combinations generated 93 bands. On average, seven RAPD fragments were amplified per reaction. In a UPGMA phenetic dendrogram based on Jaccard's coefficient, the P. bombycina accessions showed a high level of genetic variation, as indicated by genetic similarity. The grouping in the phenogram was highly consistent, as indicated by high values of cophenetic correlation and high bootstrap values at the key nodes. The accessions were scattered on a plot derived from principal correspondence analysis. The study concluded that the high level of genetic diversity in the P. bombycina accessions may be attributed to the species' outcrossing nature. This study may be useful in identifying diverse genetic stocks of P. bombycina, which may then be conserved on a priority basis.

[RAPD analysis of four species of Cuscuta in Shandong Province].

PubMed

Lin, Huibin; Lin, Jianqun; Lin, Jianqiang

2003-01-01

To explore the genome difference of four species of Cuscuta in different hosts. RAPD was used by 50 primers. Four species of genus Cuscuta can be identified by 8 primers. Both Cuscuta chinensis and C. australis from Subg. Grammica had 3 bands whose molecular weights were 1.3 kb, 1.45 kb and 1.53 kb respectively. C. japonica and C. lupuliformis from Subg. Monogyna had a 1.48 kb specific band. Cuscuta of same subgenus had similar RAPD result and close genetic relationship. Same species of Cuscuta in different hosts showed DNA polymorphism. It indicated that hosts can affect genome of Cuscuta to some extent. RAPD can be used to identify the species of Cuscuta or same Cuscuta in different hosts.

Conservation and multiplication of encapsulated micro shoots of Rauvolfia vomitoria--an endangered medicinal tree: ISSR and RAPD based evaluation of genetic fidelity of converted plantlets.

PubMed

Mehrotra, Shakti; Rahman, Liaq Ur; Mishra, Jahnvi; Kukreja, Arun K

2012-12-01

The in vitro grown axillary micro shoots of Rauvolfia vomitoria were encapsulated in alginate beads. Following 6 months of normal storage at 25 +/- 2 degrees C the regrowth of encapsulated micro shoots, reached 95.2% within 40 days of incubation on MS medium containing 1.0 mg/L BAP and 0.1 mg/L NAA. Among the responding encapsulated explants 69.6% showed emergence of multiple shoots. The developing shoots showed rhizogenesis in two weeks following their transfer to rooting medium. Healthy plants were established in a glass house with 95% survival. Of the 50 RAPD primers tested, 10 produced 23 clear and reproducible amplicons, with an average of 2.3 bands per primer. Eleven ISSR primers produced a total of 42 bands, with a size range of 0.1-1.9 kb. The number of scorable bands for each primer varied from 2 to 6, with an average of 3.81. The similarity matrix, calculated individually from the results obtained from ISSR and RAPD analysis, showed similarity coefficients ranging from 1.0 for RAPD and 0.85 to 1.0 for ISSR.

Impact of gamma rays on the Phaffia rhodozyma genome revealed by RAPD-PCR.

PubMed

Najafi, N; Hosseini, Ramin; Ahmadi, Ar

2011-12-01

Phaffia rhodozyma is a red yeast which produces astaxanthin as the major carotenoid pigment. Astaxanthin is thought to reduce the incidence of cancer and degenerative diseases in man. It also enhances the immune response and acts as a free-radical quencher, a precursor of vitamin A, or a pigment involved in the visual attraction of animals as mating partners. The impact of gamma irradiation was studied on the Phaffia rhodozyma genome. Ten mutant strains, designated Gam1-Gam10, were obtained using gamma irradiation. Ten decamer random amplified polymorphic DNA (RAPD) primers were employed to assess genetic changes. Nine primers revealed scorable polymorphisms and a total of 95 band positions were scored; amongst which 38 bands (37.5%) were polymorphic. Primer F with 3 bands and primer J20 with 13 bands produced the lowest and the highest number of bands, respectively. Primer A16 produced the highest number of polymorphic bands (70% polymorphism) and primer F showed the lowest number of polymorphic bands (0% polymorphism). Genetic distances were calculated using Jaccard's coefficient and the UPGMA method. A dendrogram was created using SPSS (version 11.5) and the strains were clustered into four groups. RAPD markers could distinguish between the parental and the mutant strains of P. rhodozyma. RAPD technique showed that some changes had occurred in the genome of the mutated strains. This technique demonstrated the capability to differentiate between the parental and the mutant strains.

Analysis of the genetic diversity of physic nut, Jatropha curcas L. accessions using RAPD markers.

PubMed

Rafii, M Y; Shabanimofrad, M; Puteri Edaroyati, M W; Latif, M A

2012-06-01

A sum of 48 accessions of physic nut, Jatropha curcas L. were analyzed to determine the genetic diversity and association between geographical origin using RAPD-PCR markers. Eight primers generated a total of 92 fragments with an average of 11.5 amplicons per primer. Polymorphism percentages of J. curcas accessions for Selangor, Kelantan, and Terengganu states were 80.4, 50.0, and 58.7%, respectively, with an average of 63.04%. Jaccard's genetic similarity co-efficient indicated the high level of genetic variation among the accessions which ranged between 0.06 and 0.81. According to UPGMA dendrogram, 48 J. curcas accessions were grouped into four major clusters at coefficient level 0.3 and accessions from same and near states or regions were found to be grouped together according to their geographical origin. Coefficient of genetic differentiation (G(st)) value of J. curcas revealed that it is an outcrossing species.

Genotoxic effects of heavy metal cadmium on growth, biochemical, cyto-physiological parameters and detection of DNA polymorphism by RAPD in Capsicum annuum L. – An important spice crop of India

PubMed Central

Aslam, Rumana; Ansari, M.Y.K.; Choudhary, Sana; Bhat, Towseef Mohsin; Jahan, Nusrat

2014-01-01

The present study was designed to investigate the effects of cadmium (Cd) on biochemical, physiological and cytological parameters of Capsicum annuum L. treated with five different concentrations (20, 40, 60, 80 and 100 ppm) of the metal. Shoot–root length, pigment and protein content showed a continuous decrease with increasing Cd concentrations and the maximal decline was observed at the higher concentration. Proline content was found to be increased upto 60 ppm while at higher concentrations it gradually decreased. MDA content and chromosomal aberrations increased as the concentration increased. Additionally Random amplified polymorphic DNA (RAPD) technique was used for the detection of genotoxicity induced by Cd. A total of 184 bands (62 polymorphic and 122 monomorphic) were generated in 5 different concentrations with 10 primers where primer OPA-02 generated the highest percentage of polymorphism (52.63%). Dendrogram showed that control, R1 and R2 showed similar cluster and R4 and R5 grouped with R3 into one cluster, which showed that plants from higher doses showed much difference than the plants selected at mild doses which resemble control at the DNA level. This investigation showed that RAPD marker is a useful tool for evaluation of genetic diversity and relationship among different metal concentrations. PMID:25313282

RAPD markers linked to eastern filbert blight resistance in Corylus avellana

Treesearch

S.A. Mehlenbacher; R.N. Brown; J.W. Davis; H. Chen; N.V. Bassil; D.C. Smith; Thomas L. Kubisiak

2004-01-01

A total of 1,110 decamer primers were screened for RAPD markers linked to a dominant allele in hazelnut (Corylus avellana) that confers resistance to eastern filbert blight caused by Anisogramma anomnala. Twenty RAPD markers linked in coupling, and five markers linked in repulsion, were found. A seedling population was used to...

[Study on sequence characterized amplified region (SCAR) markers of Cornus officinalis].

PubMed

Chen, Suiqing; Lu, Xiaolei; Wang, Lili

2011-05-01

To establish sequence characterized amplified region markers of Cornus officinalis and provide a scientific basis for molecular identification of C. officinalis. The random primer was screened through RAPD to obtain specific RAPD marker bands. The RAPD marker bands were separated, extracted, cloned and sequenced. Both ends of the sequence of RAPD marker bands were determined. A pair of specific primers was designed for conventional PCR reaction, and SCAR marker was acquired. Four pairs of primers were designed based on the sequence of RAPD marker bands. The DNA of the seven varieties of C. officinalis was amplified by using YST38 and YST43 primer. The results showed that seven varieties of C. officinalis were able to produce a single PCR product. It was an effective way to identify C. officinalis. The varieties with cylindrical and long-pear shape fruits amplified by YST38 showed a specific band, which could be used as the evidence of variety identification. Seven varieties of C. oficinalis were amplified by using primer YST39. But the size of band of the variety with spindly shape fruit (35,0400 bp) was about 300 bp, which was shorter than those of the variety with the other shape fruits of C. officinalis (650-700 bp). The variety with the spindly shape fruit could be identified through this difference. The primer YST92 could produce a fragment from 600-700 bp in the varieties with cylindrical and long-pear shape fruits, a fragment from 200-300 bp in the varieties with oval and short-cylindrical shape fruits and had no fragment in the varieties with long cylindrical, elliptic and short-pear shape fruits, which could be used to select the different shapes of C. officinalis. SCAR mark is established and can be used as the basis for breeding and distinguishing the verieties of C. officinalis.

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus.

PubMed

Matsheka, M I; Lastovica, A J; Zappe, H; Elisha, B G

2006-06-01

DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.

Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment.

PubMed

Arif, I A; Bakir, M A; Khan, H A; Al Farhan, A H; Al Homaidan, A A; Bahkali, A H; Al Sadoon, M; Shobrak, M

2010-11-09

The use of highly discriminatory methods for the identification and characterization of genotypes is essential for plant protection and appropriate use. We utilized the RAPD method for the genetic fingerprinting of 11 plant species of desert origin (seven with known medicinal value). Andrachne telephioides, Zilla spinosa, Caylusea hexagyna, Achillea fragrantissima, Lycium shawii, Moricandia sinaica, Rumex vesicarius, Bassia eriophora, Zygophyllum propinquum subsp migahidii, Withania somnifera, and Sonchus oleraceus were collected from various areas of Saudi Arabia. The five primers used were able to amplify the DNA from all the plant species. The amplified products of the RAPD profiles ranged from 307 to 1772 bp. A total of 164 bands were observed for 11 plant species, using five primers. The number of well-defined and major bands for a single plant species for a single primer ranged from 1 to 10. The highest pair-wise similarities (0.32) were observed between A. fragrantissima and L. shawii, when five primers were combined. The lowest similarities (0) were observed between A. telephioides and Z. spinosa; Z. spinosa and B. eriophora; B. eriophora and Z. propinquum. In conclusion, the RAPD method successfully discriminates among all the plant species, therefore providing an easy and rapid tool for identification, conservation and sustainable use of these plants.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

PCR-Based Identification and Characterization of Fusarium sp. Associated with Mango Malformation

PubMed Central

Arif, M.; Pani, D. R.; Zaidi, N. W.; Singh, U. S.

2011-01-01

Mango malformation is the most serious disease of mango causing considerable damage to the mango orchards worldwide. It is a major threat for mango cultivation in north Indian belt. In recent years, Fusarium sp. is finding wide acceptability in scientific community as a causal agent of this disease. However, little information is known about the variability in Fusarium isolates from malformed mango tissues. Therefore, the major objective of present study was the identification and analysis of genetic diversity among Fusarium isolates collected from malformed mango tissues. Two texon selective primers, ITS-Fu-f and ITS-Fu-r, were used for quick identification of Fusarium spp. The fungal genomic DNA was extracted from using CTAB method and was utilized as template for PCR amplification. Total 224 bands were amplified by 18 RAPD primers at an average of 12.44 bands per primer. The size of the obtained amplicons ranged from 0.264 kb (minimum) to 3.624 kb (maximum). Data scored from 25 isolates of Fusarium sp. with 18 RAPD primers were used to generate similarity coefficients. The similarity coefficient ranged from 0.17 to 0.945. Based on DNA fingerprints, all isolates were categorized into two major clusters. This study indicated a wide variability among different isolates of Fusarium. PMID:21350657

Molecular typing of Epidermophyton floccosum isolated from patients with dermatophytosis by RAPD-PCR.

PubMed

Khosravi, Alireza; Behzad, Forough; Sabokbar, Azar; Shokri, Hojjatollah; Haddadi, Siamak; Masoudi-Nejad, Ali

2010-12-01

We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type Epidermophyton floccosum isolates recovered from patients with dermatophytosis originating from different regions of Iran. A total of 13 clinical isolates of E. floccosum obtained from Iranian patients were analyzed by RAPD with 7 arbitrary primers (OPN16, OPD18' OPU15, OPX19, R28, OPA04 and OPAA17). Among the applied primers, OPN16 produced banding patterns from all the isolates. In addition, some of the isolates had very close relation. The phenon line which represented the mean similarities was at the value of 73%. At this level, 4 groups were characterized. Two isolates of a patient had different molecular patterns, suggesting infection transmission from different sources in the case of a single patient. RAPD-PCR provided a rapid and practical tool for identification of E. floccosum isolates, which was independent of morphological characteristics, and enhanced laboratory diagnosis of dermatophytosis.

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

NASA Astrophysics Data System (ADS)

He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

2003-06-01

Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers.

PubMed

Sreedhar, Reddampalli V; Venkatachalam, Lakshmanan; Bhagyalakshmi, Neelwarne

2007-08-01

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

PubMed

Rossetti, Lia; Giraffa, Giorgio

2005-11-01

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

Identification of genetically diverse genotypes for photoperiod insensitivity in soybean using RAPD markers.

PubMed

Singh, R K; Bhatia, V S; Yadav, Sanjeev; Athale, Rashmi; Lakshmi, N; Guruprasad, K N; Chauhan, G S

2008-10-01

Most of the Indian soybean varieties were found to be highly sensitive to photoperiod, which limits their cultivation in only localized area. Identification of genetically diverse source of photoperiod insensitive would help to broaden the genetic base for this trait. Present study was undertaken with RAPD markers for genetic diversity estimation in 44 accessions of soybean differing in response to photoperiod sensitivity. The selected twenty-five RAPD primers produced a total of 199 amplicons, which generated 89.9 % polymorphism. The number of amplification products ranged from 2 to 13 for different primers. The polymorphism information content ranged from 0.0 for monomorphic loci to 0.5 with an average of 0.289. Genetic diversity between pairs of genotypes was 37.7% with a range of 3.9 to 71.6%. UPGMA cluster analysis placed all the accessions of soybean into four major clusters. No discernable geographical patterns were observed in clustering however; the smaller groups corresponded well with pedigree. Mantel's test (r = 0.915) indicates very good fit for clustering pattern. Two genotypes, MACS 330 and 111/2/1939 made a very divergent group from other accessions of soybean and highly photoperiod insensitive that may be potential source for broadening the genetic base of soybean for this trait.

Comparative analysis of DNA polymorphisms and phylogenetic relationships among Syzygium cumini Skeels based on phenotypic characters and RAPD technique.

PubMed

Singh, Jitendra P; Singh, Ak; Bajpai, Anju; Ahmad, Iffat Zareen

2014-01-01

The Indian black berry (Syzygium cumini Skeels) has a great nutraceutical and medicinal properties. As in other fruit crops, the fruit characteristics are important attributes for differentiation were also determined for different accessions of S. cumini. The fruit weight, length, breadth, length: breadth ratio, pulp weight, pulp content, seed weight and pulp: seed ratio significantly varied in different accessions. Molecular characterization was carried out using PCR based RAPD technique. Out of 80 RAPD primers, only 18 primers produced stable polymorphisms that were used to examine the phylogenetic relationship. A sum of 207 loci were generated out of which 201 loci found polymorphic. The average genetic dissimilarity was 97 per cent among jamun accessions. The phylogenetic relationship was also determined by principal coordinates analysis (PCoA) that explained 46.95 per cent cumulative variance. The two-dimensional PCoA analysis showed grouping of the different accessions that were plotted into four sub-plots, representing clustering of accessions. The UPGMA (r = 0.967) and NJ (r = 0.987) dendrogram constructed based on the dissimilarity matrix revealed a good degree of fit with the cophenetic correlation value. The dendrogram grouped the accessions into three main clusters according to their eco-geographical regions which given useful insight into their phylogenetic relationships.

Evaluation of genetic diversity in Chinese kale (Brassica oleracea L. var. alboglabra Bailey) by using rapid amplified polymorphic DNA and sequence-related amplified polymorphism markers.

PubMed

Zhang, J; Zhang, L G

2014-02-14

Chinese kale is an original Chinese vegetable of the Cruciferae family. To select suitable parents for hybrid breeding, we thoroughly analyzed the genetic diversity of Chinese kale. Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate the genetic diversity across 21 Chinese kale accessions from AVRDC and Guangzhou in China. A total of 104 bands were detected by 11 RAPD primers, of which 66 (63.5%) were polymorphic, and 229 polymorphic bands (68.4%) were observed in 335 bands amplified by 17 SRAP primer combinations. The dendrogram showed the grouping of the 21 accessions into 4 main clusters based on RAPD data, and into 6 clusters based on SRAP and combined data (RAPD + SRAP). The clustering of accessions based on SRAP data was consistent with petal colors. The Mantel test indicated a poor fit for the RAPD and SRAP data (r = 0.16). These results have an important implication for Chinese kale germplasm characterization and improvement.

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding.

PubMed

Hernández, P; Dorado, G; Ramírez, M C; Laurie, D A; Snape, J W; Martín, A

2003-01-01

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.

SCAR marker specific to detect Magnaporthe grisea infecting finger millets (Eleusine coracana).

PubMed

Gnanasing Jesumaharaja, L; Manikandan, R; Raguchander, T

2016-09-01

To determine the molecular variability and develop specific Sequence Characterized Amplified Region (SCAR) marker for the detection of Magnaporthe grisea causing blast disease in finger millet. Random amplified polymorphic DNA (RAPD) was performed with 14 isolates of M. grisea using 20 random primers. SCAR marker was developed for accurate and specific detection of M. grisea infecting only finger millets. The genetic similarity coefficient within each group and variation between the groups was observed. Among the primers, OPF-08 generated a RAPD polymorphic profile that showed common fragment of 478 bp in all the isolates. This fragment was cloned and sequenced. SCAR primers, Mg-SCAR-FP and Mg-SCAR-RP, were designed using sequence of the cloned product. The specificity of the SCAR primers was evaluated using purified DNA from M. grisea isolates from finger millets and other pathogens viz., Pyricularia oryzae, Colletotrichum gloeosporioides, Colletotrichum falcatum and Colletotrichum capcisi infecting different crops. The SCAR primers amplified only specific 460 bp fragment from DNA of M. grisea isolates and this fragment was not amplified in other pathogens tested. SCAR primers distinguish blast disease of finger millet from rice as there is no amplification in the rice blast pathogen. PCR-based SCAR marker is a convenient tool for specific and rapid detection of M. grisea in finger millets. Genetic diversity in fungal population helps in developing a suitable SCAR marker to identify the blast pathogen at the early stage of infection. © 2016 The Society for Applied Microbiology.

Varietal Discrimination and Genetic Variability Analysis of Cymbopogon Using RAPD and ISSR Markers Analysis.

PubMed

Bishoyi, Ashok Kumar; Sharma, Anjali; Kavane, Aarti; Geetha, K A

2016-06-01

Cymbopogon is an important genus of family Poaceae, cultivated mainly for its essential oils which possess high medicinal and economical value. Several cultivars of Cymbopogon species are available for commercial cultivation in India and identification of these cultivars was conceded by means of morphological markers and essential oil constitution. Since these parameters are highly influenced by environmental factors, in most of the cases, it is difficult to identify Cymbopogon cultivars. In the present study, Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) markers were employed to discriminate nine leading varieties of Cymbopogon since prior genomic information is lacking or very little in the genus. Ninety RAPD and 70 ISSR primers were used which generated 63 and 69 % polymorphic amplicons, respectively. Similarity in the pattern of UPGMA-derived dendrogram of RAPD and ISSR analysis revealed the reliability of the markers chosen for the study. Varietal/cultivar-specific markers generated from the study could be utilised for varietal/cultivar authentication, thus monitoring the quality of the essential oil production in Cymbopogon. These markers can also be utilised for the IPR protection of the cultivars. Moreover, the study provides molecular marker tool kit in both random and simple sequence repeats for diverse molecular research in the same or related genera.

Mycelial Propagation and Molecular Phylogenetic Relationships of Commercially Cultivated Agrocybe cylindracea based on ITS Sequences and RAPD

PubMed Central

Alam, Nuhu; Kim, Jeong Hwa; Shim, Mi Ja; Lee, U Youn

2010-01-01

This study evaluated the optimal vegetative growth conditions and molecular phylogenetic relationships of eleven strains of Agrocybe cylindracea collected from different ecological regions of Korea, China and Taiwan. The optimal temperature and pH for mycelial growth were observed at 25℃ and 6. Potato dextrose agar and Hennerberg were the favorable media for vegetative growth, whereas glucose tryptone was unfavorable. Dextrin, maltose, and fructose were the most effective carbon sources. The most suitable nitrogen sources were arginine and glycine, whereas methionine, alanine, histidine, and urea were least effective for the mycelial propagation of A. cylindracea. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The sequence of ITS2 was more variable than that of ITS1, while the 5.8S sequences were identical. The reciprocal homologies of the ITS sequences ranged from 98 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) using 20 arbitrary primers. Fifteen primers efficiently amplified the genomic DNA. The average number of polymorphic bands observed per primer was 3.8. The numbers of amplified bands varied based on the primers and strains, with polymorphic fragments ranging from 0.1 to 2.9 kb. The results of RAPD analysis were similar to the ITS region sequences. The results revealed that RAPD and ITS techniques were well suited for detecting the genetic diversity of all A. cylindracea strains tested. PMID:23956633

Study Of Genetic Diversity Between Grasspea Landraces Using Morphological And Molecular Marker

NASA Astrophysics Data System (ADS)

Sedehi, Abbasali Vahabi; Lotfi, Asefeh; Solooki, Mahmood

2008-01-01

Grass pea is a beneficial crop to Iran since it has some major advantageous such as high grain and forage quality, high drought tolerance and medium level of salinity tolerance and a good native germplasm variation which accessible for breeding programs. This study was carried out to evaluate morphological traits of the grass pea landraces using a randomized complete block design with 3 replications at Research Farm of Isfahan University of Technology. To evaluate genetic diversity of 14 grass pea landraces from various locations in Iran were investigated using 32 RAPD & ISJ primers at Biocenter of University of Zabol. Analysis of variance indicated a highly significant differences among 14 grass pea landrace for the morphological traits. Average of polymorphism percentage of RAPD primer was 73.9%. Among used primer, 12 random primers showed polymorphism and a total of 56 different bands were observed in the genotypes. Jafar-abad and Sar-chahan genotypes with similarity coefficient of 66% and Khoram-abad 2 and Khoram-abad 7 genotypes with similarity coefficient of 3% were the most related and the most distinct genotypes, respectively. Fourteen primers out of 17 semi random primers produced 70 polymorphic bands which included 56% of the total 126 produced bands. Genetic relatedness among population was investigated using Jacard coefficient and unweighted pair group mean analysis (UPGMA) algorithm. The result of this research verified possibility of use of RAPD & ISJ markers for estimation of genetic diversity, management of genetic resources and determination of repetitive accessions in grass pea.

Phylogenetic Relationships in Actinidia as Revealed by RAPD Analysis

Treesearch

Hongwen Huang; Zuozhou Li; Jianqiang Li; Thomas L. Kubiisiak; Desmond R. Lavne

2002-01-01

Phylogenetic relationships within the Actinidia were investigated using randomly amplified polymorphic DNA (RAPD) markers. DNAs from 10 taxa, including31 species encompassing all four sections and four series of the traditional subdivisions within the genus, were amplified using 22 preselected 10-mer oligonucieotide primers. A total 204 DNA bands...

Genetic Diversity of Pinus Roxburghii Sarg. Collected from Different Himalayan Regions of India Assessed by Random Amplified Polymorphic DNA Analysis

PubMed Central

Sinha, Dwaipayan; Singh, Jyotsna; Tandon, P. K.; Kakkar, Poonam

2013-01-01

Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species. PMID:24403729

Systemic Screening of Strains of the Lion's Mane Medicinal Mushroom Hericium erinaceus (Higher Basidiomycetes) and Its Protective Effects on Aβ-Triggered Neurotoxicity in PC12 Cells.

PubMed

Liu, Zongying; Wang, Qinglong; Cui, Jian; Wang, Lili; Xiong, Lili; Wang, Wei; Li, Diqiang; Liu, Na; Wu, Yiran; Mao, Canquan

2015-01-01

Hericium erinaceus possesses multiple medicinal values. To date, however, there have been few studies of the systemic screening of H. erinaceus strains, and the neuroprotective effects of H. erinaceus prepared from homogenized, fresh fruiting bodies are not fully understood. In this study, 4 random primers were selected and used in random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) to screen and evaluate the genetic diversity of 19 commercial strains of H. erinaceus from different localities in China. A total of 66 bands were obtained, and the percentage of polymorphic loci reached 80.30%. Five dendrograms were constructed based on RAPD by Jaccard cluster and within-group linkage analysis. Primer S20 as well as all 4 primers had great potential as specific primers for RAPD-PCR molecular identification and differentiation of H. erinaceus strains. Based on the results of submerged culture and fruiting body cultivation, strains HT-N, HT-J1, HT-C, and HT-M were identified as superior among the 19 H. erinaceus strains. Further study showed that the oral preparation of homogenized, fresh fruiting bodies of H. erinaceus could attenuate the Aβ25-35-triggered damage in PC12 cells by significantly increasing cell viability and by decreasing the release of lactate dehydrogenase. In conclusion, RAPD-PCR combined with liquid and solid cultures can be used well in the screening and identification of H. erinaceus strains, and products prepared from homogenized, fresh fruiting bodies of H. erinaceus had neuroprotective effects on PC12 cells.

Natural hybridization between Phlomis lycia D. Don x P. bourgaei Boiss., (Lamiaceae) revealed by RAPD markers.

PubMed

Yüzbaşioğlu, Ertuğrul; Dadandi, Mehmet Yaşar; Ozcan, Sebahattin

2008-05-01

Randomly Amplified Polymorphic DNA markers (RAPD) were used to assess the hybrid identity of individuals sampled as Phlomis x termessi Davis. Out of 95 primers screened, 11 primers produced reproducible amplification patterns used for discrimination of P. x termessi and their parents. Eleven primers produced 81 bands. Forty two percent of the RAPD bands existed in parents. Of the 54 bands found in P. lycia, 19 were found only in this species and 7 of these were monomorphic. Similarly, of 57 RAPD bands observed in P. bourgaei, 18 were found only in P. bourgaei and 6 of these were monomorphic. Among hybrid individuals, 35 of the 73 markers were monomorphic. Fifteen of these existed in individual parents showing that parents were homozygous for these markers. Of the 35 monomorphic bands observed among hybrid individuals, 5 were present in the samples of one of the parents and completely absent from the samples of the other; therefore, additive inheritance is indicated. Of the 5 additive bands, 1 was inherited from P. bourgaei and 4 were inherited from P. lycia. Among 38 polymorhic markers observed in hybrid individuals, 9 were new and hybrid-specific. Pollen fertility was also investigated. Mean pollen fertility for P. lycia and P. bourgaei was 93% and 97% respectively. However, mean pollen fertility for hybrids was 65% (+/-10.5).

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers.

PubMed

Kim, Suk-Man; Sohn, Jae-Keun

2005-08-31

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.

RAPD analysis of genetic variation in the Australian fan flower, Scaevola.

PubMed

Swoboda, I; Bhalla, P L

1997-10-01

The use of randomly amplified polymorphic DNA (RAPD) to study genetic variability in Scaevola (family Goodeniaceae), a native Australian species used in ornamental horticulture, is demonstrated. Plants of the genus Scaevola are commonly known as "fan flowers," due to the fan-like shape of the flowers. Nineteen accessions of Scaevola (12 cultivated and 7 wild) were studied using 20 random decamer arbitrary primers. Eight primers gave a distinct reproducible amplification profile of 90 scorable polymorphic fragments, enabling the differentiation of the Scaevola accessions. RAPD amplification of genomic DNA revealed a high genetic variability among the different species of Scaevola studied. Molecular markers were used to calculate the similarity coefficients, which were then used for determining genetic distances between each of the accessions. Based on genetic distances, a dendrogram was constructed. Though the dendrogram is in general agreement with the taxonomy, it also highlights discrepancies in the classification. The RAPD data showed that Scaevola aemula (series Pogogynae) is closer to Scaevola glandulifera of series Globuliferae than to the rest of members of series Pogogynae. In addition, the RAPD banding pattern of white flower S. aemula, one of the commercial cultivars, was identical to that of Scaevola albida, indicating their genetic similarity. Our study showed that there is a large genetic distance between commercial cultivars of Scaevola (Purple Fanfare, Pink Perfection, and Mauve Cluster), indicating considerable genetic variation among them. The use of RAPDs in intra- and inter-specific breeding of Scaevola is also explored.

Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants.

PubMed

Doh, Eui Jeong; Oh, Seung-Eun

2012-01-01

Some plants in the genus Artemisia have been used for medicinal purposes. Among them, Artemisia iwayomogi, commonly referred to as "Haninjin," is one of the major medicinal materials used in traditional Korean medicine. By contrast, Artemisia capillaris and both Artemisia argyi and Artemisia princeps, referred to as "Injinho" and "Aeyup," respectively, are used to treat diseases different from those for which "Haninjin" is prescribed. Therefore, the development of a reliable method to differentiate each Artemisia herb is necessary. We found that a random amplified polymorphic DNA (RAPD) method can be used to efficiently discriminate a few Artemisia plants from one another. To improve the reliability of RAPD amplification, we designed primer sets based on the nucleotide sequences of RAPD products to amplify a sequence-characterized amplified region (SCAR) marker of A. iwayomogi. In addition, we designed two other primer sets to amplify SCAR markers of "Aeyup" (A. argyi and A. princeps) along with "Injinho" (A. capillaris) and Artemisia japonica, which are also traded in Korean herbal markets. Using these three primer sets, we developed a multiplex PCR method concurrently not only to discriminate A. iwayomogi from other Artemisia plants, but also to identify Artemisia plants using a single PCR process.

Genetic diversity of worldwide Jerusalem artichoke (Helianthus tuberosus) germplasm as revealed by RAPD markers.

PubMed

Wangsomnuk, P P; Khampa, S; Wangsomnuk, P; Jogloy, S; Mornkham, T; Ruttawat, B; Patanothai, A; Fu, Y B

2011-12-12

Jerusalem artichoke (Helianthus tuberosus) is a wild relative of the cultivated sunflower (H. annuus); it is an old tuber crop that has recently received renewed interest. We used RAPD markers to characterize 147 Jerusalem artichoke accessions from nine countries. Thirty RAPD primers were screened; 13 of them detected 357 reproducible RAPD bands, of which 337 were polymorphic. Various diversity analyses revealed several different patterns of RAPD variation. More than 93% of the RAPD variation was found within accessions of a country. Weak genetic differentiation was observed between wild and cultivated accessions. Six groups were detected in this germplasm set. Four ancestral groups were found for the Canadian germplasm. The most genetically distinct accessions were identified. These findings provide useful diversity information for understanding the Jerusalem artichoke gene pool, for conserving Jerusalem artichoke germplasm, and for choosing germplasm for genetic improvement.

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

PubMed Central

Xian, Zhi-Hong; Cong, Wen-Ming; Zhang, Shu-Hui; Wu, Meng-Chao

2005-01-01

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data. RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene. CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcin-ogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. PMID:15996039

Scar markers in a longleaf pine x slash pine F1 family

Treesearch

C. Weng; Thomas L. Kubisiak; M. Stine

1998-01-01

Sequence characterized amplified region (SCAR) markers were derived from random amplified polymorphic DNAs (RAPDs) that segregate in a longleaf pine x slash pine F1 family. Nine RAPD fragments, five from longleaf pine and four from slash pine, were cloned and end sequenced. A total of 13 SCAR primer pairs, with lengths between 17 and 24...

Preparation of Meloidogyne javanica near-isogenic lines virulent and avirulent against the tomato resistance gene Mi and preliminary analyses of the genetic variation between the two lines.

PubMed

Xu, Jian-Hua; Narabu, Takashi; Li, Hong-Mei; Fu, Peng

2002-01-01

Meloidogyne javanica, reproducing by mitotic parthenogenesis, is an economically important pathogen of a wide range of crops. A pair of near-isogenic lines virulent and avirulent toward the tomato resistance gene Mi were prepared for M. javanica by continuously selecting an avirulent population on the resistant tomato cultivar Momotaro over 19 generations. Random amplified polymorphic DNA (RAPD) analysis with 102 primers revealed that RAPD patterns were highly conserved between the virulent and avirulent lines, confirming that the two lines were genomically very similar. Nevertheless, with one of the primers a distinct polymorphic fragment, specific for the avirulent lines, was amplified. Southern hybridization results indicated that the polymorphic fragment and its homologs were deleted from the genome of the virulent line during the process of virulence acquisition. Sequence analysis and homology searches of public data bases, however, revealed no published sequences significantly similar to the sequence of the fragment, precluding a prediction of the potential function of the sequence. The successful preparation of the near-isogenic Mi-virulent and avirulent lines laid a firm foundation for the further identification and isolation of virulence-related genes in M. javanica.

Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

PubMed

Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

2016-03-01

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

NASA Astrophysics Data System (ADS)

Wang, Tiegu; Huang, Qunce; Feng, Weisen

2007-10-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

Estimation of the genetic diversity in tetraploid alfalfa populations based on RAPD markers for breeding purposes.

PubMed

Nagl, Nevena; Taski-Ajdukovic, Ksenija; Barac, Goran; Baburski, Aleksandar; Seccareccia, Ivana; Milic, Dragan; Katic, Slobodan

2011-01-01

Alfalfa is an autotetraploid, allogamous and heterozygous forage legume, whose varieties are synthetic populations. Due to the complex nature of the species, information about genetic diversity of germplasm used in any alfalfa breeding program is most beneficial. The genetic diversity of five alfalfa varieties, involved in progeny tests at Institute of Field and Vegetable Crops, was characterized based on RAPD markers. A total of 60 primers were screened, out of which 17 were selected for the analysis of genetic diversity. A total of 156 polymorphic bands were generated, with 10.6 bands per primer. Number and percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon's information index were used to estimate genetic variation. Variety Zuzana had the highest values for all tested parameters, exhibiting the highest level of variation, whereas variety RSI 20 exhibited the lowest. Analysis of molecular variance (AMOVA) showed that 88.39% of the total genetic variation was attributed to intra-varietal variance. The cluster analysis for individual samples and varieties revealed differences in their population structures: variety Zuzana showed a very high level of genetic variation, Banat and Ghareh were divided in subpopulations, while Pecy and RSI 20 were relatively uniform. Ways of exploiting the investigated germplasm in the breeding programs are suggested in this paper, depending on their population structure and diversity. The RAPD analysis shows potential to be applied in analysis of parental populations in semi-hybrid alfalfa breeding program in both, development of new homogenous germplasm, and identification of promising, complementary germplasm.

Genetic relatedness of Brazilian Colletotrichum truncatum isolates assessed by vegetative compatibility groups and RAPD analysis.

PubMed

Sant'Anna, Juliane R; Miyamoto, Cláudia T; Rosada, Lúcia J; Franco, Claudinéia C S; Kaneshima, Edilson N; Castro-Prado, Marialba A A

2010-01-01

The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.

Utility of RAPD marker for genetic diversity analysis in gamma rays and ethyl methane sulphonate (EMS)-treated Jatropha curcas plants.

PubMed

Dhakshanamoorthy, Dharman; Selvaraj, Radhakrishnan; Chidambaram, Alagappan

2015-02-01

The presence of important chemical and physical properties in Jatropha curcas makes it a valuable raw material for numerous industrial applications, including the production of biofuel. Hence, the researcher's interest is diversified to develop more and better varieties with outstanding agronomic characteristics using conventional breeding. Among these, mutation breeding is one of the best approaches to bring genetic changes in plant species. The aim of this study is to evaluate the diversity and genetic relationship among J. curcas mutants, which were obtained from different doses of gamma rays (control, 5 Kr, 10 Kr, 15 Kr, 20 Kr and 25 Kr) and EMS (1%, 2%, 3% and 4%), using RAPD marker. Among the 21 random primers, 20 produced polymorphic bands. The primers, OPM-14 and OPAW-13, produced a minimum number of bands (3) each across the ten mutants, while the primer OPF-13 produced the maximum number of bands (10), followed by the primers OPU-13, OPAM-06, OPAW-09 and OPD-05, which produced 9 bands each. The number of amplicons varied from 3 to 10, with an average of 7 bands, out of which 4.57 were polymorphic. The percentage of polymorphism ranged from 0.00 to 100 with an average of 57%. In the present study, RAPD markers were found most polymorphic, with an average polymorphism information content (PIC) value of 0.347, effective multiplex ratio (EMR) of 35.14, marker index (MI) of 14.19, resolution power (Rp) of 11.19, effective marker index (EMI) of 8.21 and genotype index (GI) of 0.36, indicating that random primers are useful in studies of genetic characterization in J. curcas mutant plants. In a dendrogram constructed based on Jaccard's similarity coefficients, the mutants were grouped into three main clusters viz., (a) control, 10 Kr, 15 Kr, 20 Kr, 2% EMS, and 3% EMS, (b) 5 Kr and 1% EMS, and (c) 25 Kr and 4% EMS mutants. Based on the attributes of the random primers and polymorphism studied, it is concluded that RAPD analysis offers a useful molecular marker for the identification of the mutants in gamma rays and EMS treated plants. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Phylogenetic Relationship in Different Commercial Strains of Pleurotus nebrodensis Based on ITS Sequence and RAPD.

PubMed

Alam, Nuhu; Shim, Mi Ja; Lee, Min Woong; Shin, Pyeong Gyun; Yoo, Young Bok; Lee, Tae Soo

2009-09-01

The molecular phylogeny in nine different commercial cultivated strains of Pleurotus nebrodensis was studied based on their internal transcribed spacer (ITS) region and RAPD. In the sequence of ITS region of selected strains, it was revealed that the total length ranged from 592 to 614 bp. The size of ITS1 and ITS2 regions varied among the strains from 219 to 228 bp and 211 to 229 bp, respectively. The sequence of ITS2 was more variable than ITS1 and the region of 5.8S sequences were identical. Phylogenetic tree of the ITS region sequences indicated that selected strains were classified into five clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by RAPD with 20 arbitrary primers. Twelve primers were efficient to applying amplification of the genomic DNA. The sizes of the polymorphic fragments obtained were in the range of 200 to 2000 bp. RAPD and ITS analysis techniques were able to detect genetic variation among the tested strains. Experimental results suggested that IUM-1381, IUM-3914, IUM-1495 and AY-581431 strains were genetically very similar. Therefore, all IUM and NCBI gene bank strains of P. nebrodensis were genetically same with some variations.

A Genetic Linkage Map of Longleaf Pine (Pinus palustris Mill.) Based on Random Amplified Polymorphic DNAs

Treesearch

C.D. Nelson; Thomas L. Kubisiak; M. Stine; W.L. Nance

1994-01-01

Eight megagametophyte DNA samples from a single longleaf pine (Pinus palustris Mill.) tree were used to screen 576 oligonucleotide primers for random amplified polymorphic DNA (RAPD) fragments. Primers amplifying repeatable polymorphic fragments were further characterized within a sample of 72 megagametophytes from the same tree. Fragments...

Identification of a locus characteristic of male individuals of buffalo grass [Buchloe dactyloides (Nutt.) Engelm.] by using an RAPD marker.

PubMed

Li, Y X; Wang, X G; Yang, C H; Cong, L L; Wu, F F; Xue, J G; Han, Y H

2013-09-27

Buffalo grass [Buchloe dactyloides (Nutt.) Engelm.] plants can be either male, female, or hermaphrodite (monoecious). As there is no morphological difference in the early vegetative growth of these three classes of plants, it is worthwhile to use molecular biological methods to attempt to identify the sex of a plant at this early growth period. In this study, we identified 23 plants that had a stable sex for over at least 3 years. Of these, 9 were male plants, 10 were female plants, and 4 were hermaphrodites. Screening of 300 RAPD primers identified a primer, namely S211 (5'-ttccccgcga-3'), which is capable of identifying male plants. The specific fragment was cloned, sequenced, and submitted to the GenBank database (accession No. JN982469). When used to identify the sex of 188 plants during their first growing season, the S211 primer correctly identified 85.8% of all male plants. Our results showed that the S211 primer can identify the male, and in doing so, it facilitates buffalo grass breeding work.

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production.

PubMed

Andrade, M J; Rodríguez, M; Casado, E M; Bermúdez, E; Córdoba, J J

2009-09-01

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.

Analysis of genetic diversity of certain species of Piper using RAPD-based molecular markers.

PubMed

Chowdhury, Utpal; Tanti, Bhaben; Rethy, Parakkal; Gajurel, Padma Raj

2014-09-01

The utility of RAPD markers in assessing genetic diversity and phenetic relationships of six different species of Piper from Northeast India was investigated. Polymerase chain reaction (PCR) with four arbitrary 10-mer oligonucleotide primers applied to the six species produced a total of 195 marker bands, of which, 159 were polymorphic. On average, six RAPD fragments were amplified per reaction. In the UPGMA phenetic dendrogram based on Jaccard's coefficient, the different accessions of Piper showed a high level of genetic variation. This study may be useful in identifying diverse genetic stocks of Piper, which may then be conserved on a priority basis.

Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

PubMed

Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

2015-12-28

The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

Photodynamic Treatment versus Antibiotic Treatment on Helicobacter pylori Using RAPD-PCR

NASA Astrophysics Data System (ADS)

El-Batanouny, M. H.; Amin, R. M.; Ibrahium, M. K.; El Gohary, S.; Naga, M. I.; Salama, M. S.

2009-09-01

Helicobacter pylori is one of the most common causes of chronic bacterial infections in humans and is important in the pathogenesis of gastrointestinal disease, such as duodenal ulcer, gastric ulcer, Gastric adenocarcinoma, and lymphoma. Gastric adenocarcinoma remains one of the leading causes of cancer death in the world. The objective of this study was to assess the effect of photodynamic treatment and medication treatment of Helicobacter pylori using RAPD-PCR. The lethal photosensitization effect was determined by mixing suspensions of H.pylori with Toluidine blue O (TBO) and plating out on blood agar before irradiation with Helium neon (He-Ne) 632.8 nm. The susceptibility of Helicobacter pylori isolates to metronidazole and azithromycin were examined by E-test. Nine random primers were used to screen genetic polymorphism in DNA of different H.pylori groups. Six of them produced RAPD products while three failed to generate any product. The resulting data showed that, although the overall genetic differences between control groups and laser treated groups was higher than that between control groups and azithromycin treated groups yet it still law genetic variability. The main cause of cell death of PDT using TBO as a photosensitizer was mainly cell wall and cytoplasmic membrane.

Evaluation of genetic variability in micropropagated propagules of ornamental pineapple [Ananas comosus var. bracteatus (Lindley) Coppens and Leal] using RAPD markers.

PubMed

Santos, M D M; Buso, G C S; Torres, A C

2008-10-21

The objective of the present study was to evaluate the genetic variability in micropropagated plantlets of ornamental pineapple, after the fourth period of subculture. The basal culture medium consisted of MS salts, vitamins, 3% sucrose, liquid formulation, supplemented with 6-benzylaminopurine (BAP) at concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 mg/L. The addition of BAP influenced the occurrence of genetic variation revealed using random amplified polymorphic DNA (RAPD) markers. Of a total of 520 primers tested, 44 were selected and amplified; 402 monomorphic bands (97.2%) and 18 polymorphic bands (2.8%) resulted among regenerated plantlets. The polymorphic fragments were produced by 12 primers (OPA-01, OPA-20, OPB-01, OPB-19, OPC-19, OPF-13, OPL-17, OPM-13, OPP-16, OPT-07, OPV-19, and OPX-03). Among the primers that identified polymorphism, OPA-01, OPA-20, OPB-19, OPC-19, OPL-17, OPP-16, and OPX-3 each showed, one polymorphic band and OPF-13 amplified a maximum of three bands. In this study, the RAPD technique was effective in showing the occurrence of somaclonal variations that occur during the micropropagation process of ornamental pineapple cultivation in BAP-supplemented medium, and it is possible to detect the presence of genetic variation in early stages of plant development.

Diversity, molecular phylogeny and fingerprint profiles of airborne Aspergillus species using random amplified polymorphic DNA.

PubMed

Kermani, Firoozeh; Shams-Ghahfarokhi, Masoomeh; Gholami-Shabani, Mohammadhassan; Razzaghi-Abyaneh, Mehdi

2016-06-01

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.

[Genetic analysis and estimation of genetic diversity in east-European breeds of swift hounds (Canis familiaris L.) based on the data of genomic studies using RAPD markers].

PubMed

Semenova, S K; Illarionova, N A; Vasil'ev, V A; Shubkina, A V; Ryskov, A P

2002-06-01

The method of polymerase chain reaction with a set of arbitrary primers (RAPD-PCR) was used to describe genetic variation and to estimate genetic diversity in East-European swift hounds, Russian Psovyi and Hortyi Borzois. For comparison, swift hounds of two West-European breeds (Whippet and Greyhound) and single dogs of other breed groups (shepherd, terriers, mastiffs, and bird dogs) were examined. For all dog groups, their closest related species, the wolf Canis lupus, was used as an outgroup. Variation of RAPD markers was studied at several hierarchic levels: intra- and interfamily (for individual families of Russian Psovyi and Hortyi Borzois), intra- and interbreed (for ten dog breeds), and interspecific (C. familiaris-C. lupus). In total, 57 dogs and 4 wolfs were studied. Using RAPD-PCR with three primers, 93 DNA fragments with a length of 150-1500 bp were detected in several Borzoi families with known filiation. These fragments were found to be inherited as dominant markers and to be applicable for estimation of genetic differences between parents and their offspring and for comparison of individuals and families with different level of inbreeding. A high level of intra- and interbreed variation was found in Russian Psovyi and Hortyi Borzois. In these dog groups, genetic similarity indices varied in a range of 72.2 to 93.4% (parents-offspring) and 68.0 to 94.5 (sibs). Based on the patterns of RAPD markers obtained using six primers, a dendrogram of genetic similarity between the wolf and different dog breeds was constructed, and indices of intragroup diversity were calculated. All studied breeds were found to fall into two clusters, swift hounds (Borzoi-like dogs) and other dogs. Russian Borzois represent a very heterogeneous group, in which the Russian Psovyi Borzoi is closer to Greyhound than the Russian Hortyi Borzoi. All studied wolfs constituted a separate cluster. Significant differences were found between the wolf and dogs by the number of RAPD markers (92.8 and 86.1, respectively) and by the indices of genetic diversity (54.3 and 64.8%, respectively). The reasons for the high intraspecific variation of dogs (including Russian Borzois) and the prospects of using the studied group of markers for genetic analysis and differentiation in C. familiaris are discussed.

Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers.

PubMed

Calienes, Aymé Fernandez; Fraga, Jorge; Pointier, Jean-Pierre; Yong, Mary; Sanchez, Jorge; Coustau, Christine; Gutiérrez, Alfredo; Théron, André

2004-09-01

Twelve natural populations of Pseudosuccinea columella snails, sampled in the western and central regions of Cuba, were analyzed using the RAPD-PCR technique to screen for resistance to Fasciola hepatica. Ten OPA primers previously shown to produce marker bands for resistance and susceptibility were tested. A new population of P. columella (El Azufre, Pinar del Río) exhibited the amplification patterns of resistant snails, and its resistant status was confirmed after experimental exposure to miracidia. No genetic variability was detected across or within the susceptible isolates. Similarly, the novel resistant isolate displayed an RAPD profile identical to the profile of two other isolates previously identified as resistant to F. hepatica. However, clear differences in RAPD banding patterns and genetic distance were observed between resistant and susceptible isolates.

Species identification and sex determination of the genus Nepenthes (Nepenthaceae).

PubMed

Mokkamul, Piya; Chaveerach, Arunrat; Sudmoon, Runglawan; Tanee, Tawatchai

2007-02-15

Nepenthes species are well known for their ornamentally attractive pitchers. The species diversity was randomly surveyed in some conservation areas of Thailand and three species were found, namely N. gracilis Korth., N. mirabilis Druce. and N. smilesii Hemsl. Young plants as unknown species from Chatuchak market were added in plant sampled set. Thirty two Inter Simple Sequence Repeat (ISSR) primers were screened and 13 successful primers were used to produce DNA banding patterns for constructing a dendrogram. The dendrogram is potentially power tool to identify unknown species from Chatuchak market, differentiate species population, population by geographical areas and sex determination. The geographical area of N. mirabilis was specified to Southern and Northeastern regions and finally, subdivided into exact areas according to province. Male and female plants of N. gracilis at Phu Wua Wildlife Sanctuary and N. mirabilis at Bung Khonglong non-hunting area were determined. Two unknown species from Chatuchak market were analyzed to be N. mirabilis with the genetic similarities (S) 77.2 to 84.7. Be more sex specific in all sample studied, 37 Random Amplified Polymorphic DNA (RAPD) primers were investigated. The result shows that only one RAPD primer show high resolution results at about 750 bp specific male-related marker.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

PubMed

Bashir, K M I; Awan, F S; Khan, I A; Khan, A I; Usman, M

2014-05-30

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

[Experimental and calculated spectra of the amplicons UBC-85 and UBC-126 (RAPD-PCR)].

PubMed

Glazko, G V; Rogozin, I B; Glazko, V I; Zelenaia, L B; Sozinov, A A

1997-01-01

The comparative analysis of experimental amplification spectrum in 13 Ungulata species and counting ones in DNA sequences of different taxa in GenBank (mammalian, other vertebrate, invertebrate, viruses, prokaryote) with the uses of RAPD-PCR primers UBC-85 and UBC-126 was carried out. The particularities of the distribution of amplicons' frequencies in experimental and counting spectrums were revealed, for some of them the similar increased frequencies in mammalian and prokaryotic species were observed.

RAPD-PCR characterization of lactobacilli isolated from artisanal meat plants and traditional fermented sausages of Veneto region (Italy).

PubMed

Andrighetto, C; Zampese, L; Lombardi, A

2001-07-01

The study was carried out to evaluate the use of randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) as a method for the identification of lactobacilli isolated from meat products. RAPD-PCR with primers M13 and D8635 was applied to the identification and intraspecific differentiation of 53 lactobacilli isolates originating from traditional fermented sausages and artisanal meat plants of the Veneto region (Italy). Most of the isolates were assigned to the species Lactobacillus sakei and Lact. curvatus; differentiation of groups of strains within the species was also possible. RAPD-PCR could be applied to the identification of lactobacilli species most commonly found in meat products. The method, which is easy and rapid to perform, could be useful for the study of the lactobacilli populations present in fermented sausages, and could help in the selection of candidate strains to use as starter cultures in meat fermentation.

Determination of genotoxic effects of boron and zinc on Zea mays using protein and random amplification of polymorphic DNA analyses.

PubMed

Erturk, Filiz Aygun; Nardemir, Gokce; Hilal, A Y; Arslan, Esra; Agar, Guleray

2015-11-01

In this research, we aimed to determine genotoxic effects of boron (B) and zinc (Zn) on Zea mays by using total soluble protein content and random amplification of polymorphic DNA (RAPD) analyses. For the RAPD analysis, 16 RAPD primers were found to produce unique polymorphic band profiles on treated maize seedlings. With increased Zn and B concentrations, increased polymorphism rate was observed, while genomic template stability and total soluble protein content decreased. The treatment with Zn was more effective than that of B groups on the levels of total proteins. The obtained results from this study revealed that the total soluble protein levels and RAPD profiles were performed as endpoints of genotoxicity and these analyses can offer useful biomarker assays for the evaluation of genotoxic effects on Zn and B polluted plants. © The Author(s) 2013.

Development of a rapid screening protocol for selection of strains resistant to spray drying and storage in dry powder.

PubMed

Reimann, S; Grattepanche, F; Baggenstos, C; Rezzonico, E; Berger, B; Arigoni, F; Lacroix, C

2010-06-01

An efficient screening method for selection of Bifidobacterium longum strains resistant to spray drying and storage was developed based on randomly amplified polymorphic DNA (RAPD) for identification of the best survivors in mixed strains bacterial preparations. Three different primers were used to generate RAPD profiles of 22 B. longum strains. All strains were distinguished according to their RAPD profiles except for the strain NCC2705 and its H(2)O(2) resistant derivative variant. The 22 strains were grouped in 3 batches of 7, 7 and 8 strains and subjected to spray drying and storage at 30 and 37 °C under anaerobic conditions. Batch survival rates after spray drying reached 17.1±4.4%. Strains showing the highest prevalence and/or resistance to storage at 37 °C were selected from individual batches for subsequent spray drying and storage testing. After 67 days of storage, NCC572 was identified as the dominant strain in powder. The stability of strain NCC572 was confirmed by performing single spray drying and storage tests. Out of 22 B. longum strains, a robust strain was identified by combining RAPD with a simultaneous screening test for survival under spray drying and storage. The method allowed a fast screening of B. longum strains in mixture for resistance to spray drying and storage compared to traditional screening procedures carried out with individual strains, in the same conditions. This approach could be applied to other stress conditions.

Terbinafine susceptibility and genotypic heterogeneity in clinical isolates of Trichophyton mentagrophytes by random amplified polymorphic DNA (RAPD).

PubMed

Alipour, M; Mozafari, N A

2015-03-01

The four RAPD systems tested in the present study have aimed at investigating DNA fingerprinting of Trichophyton mentagrophytes strains and the correlation between genotyping and antifungal susceptibility to terbinafine. Twenty-nine clinical isolates of T. mentagrophytes were recovered from patients suspected of having active dermatophytosis who were referred to the laboratory of medical mycology department in Tehran university. Then, they were subjected to conventional examination by performing direct microscopic examination, culture on primary media, physiological tests. The in vitro antifungal susceptibility of twenty-nine T. mentagrophytes isolates against terbinafine was evaluated by modified agar dilution method to determine the minimum inhibitory concentration (MIC). Twenty-one sensitive and eight resistant to terbinafine, were submitted to RAPD using 4 decamer primers (A, B, C, D) with the purpose of encountering a genetic marker to terbinafine sensibility and resistance. The UPGMA-Jaccard's correlation coefficient was used to build up dendogram that could represent clusters of similarity. According to their correlation coefficient, the samples were classified as much related (100%), moderately related (80%) and unrelated (<70%). All amplifications revealed distinct polymorphic bands and a total of 34 band positions was scored (0/1) for the 4 primers tested. Genetic distances between each of the isolates were calculated and cluster analysis was used to generate a dendrogram showing relationships between them. The combined dendrogram at an average similarity value of 65% grouped all strains into 2 (A, B) groups corresponding to their susceptibility reactions to terbinafine. All susceptible samples were properly grouped, but a few numbers of resistant isolates were also included. Nevertheless, further biochemical and molecular biological studies will be required to fully elucidate the point that resistance might be the result of a mutation in the gene encoding squalene epoxidase in T. mentagrophytes. This study proved efficacy of applying RAPD molecular technique to complement traditional mycological culture and drug susceptibility tests for accurate and appropriate management of recurrent dermatophytosis and highlights the need for newer antifungals that can combat the emergence of terbinafine-resistant T. mentagrophytes strains. Copyright © 2015. Published by Elsevier Masson SAS.

Classification of plant trypanosomatids (Phytomonas spp.): parity between random-primer DNA typing and multilocus enzyme electrophoresis.

PubMed

Muller, E; Gargani, D; Banuls, A L; Tibayrenc, M; Dollet, M

1997-10-01

The genetic polymorphism of 30 isolates of plant trypanosomatids colloquially referred to as plant trypanosomes was assayed by means of RAPD. The principle objectives of this study were to assess the discriminative power of RAPD analysis for studying plant trypanosomes and to determine whether the results obtained were comparable with those from a previous isoenzyme (MLEE) study. The principle groups of plant trypanosomes identified previously by isoenzyme analysis--intraphloemic trypanosomes, intralaticiferous trypanosomes and trypanosomes isolated from fruits--were also clearly separated by the RAPD technique. Moreover, the results showed a fair parity between MLEE and RAPD data (coefficient of correlation = 0.84) and the two techniques have comparable discriminative ability. Most of the separation revealed by the two techniques between the clusters was associated with major biological properties. However, the RAPD technique gave a more coherent separation than MLEE because the intraphloemic isolates, which were biologically similar in terms of their specific localization in the sieve tubes of the plant, were found to be in closer groups by the RAPD. For both techniques, the existence of the main clusters was correlated with the existence of synapomorphic characters, which could be used as powerful tools in taxonomy and epidemiology.

Analysis of genetic diversity and genome relationships of four eggplant species (Solanum melongena L) using RAPD markers

NASA Astrophysics Data System (ADS)

Susilo; Setyaningsih, M.

2018-01-01

Solanum melongena (eggplant) is one of the diversity of the Solanum family which is grown and widely spread in Indonesia and widely used by the community. This research explored the genetic diversity of four local Indonesian eggplant species namely leuca, tekokak, gelatik and kopek by using RAPD (Random Amplified Polymorphic DNA). The samples were obtained from Agricultural Technology Assessment Institute (BPTP) Bogor, Indonesia. The result of data observation was in the form of Solanum melongena plant’s DNA profile analyzed descriptively and quantitatively. 30 DNA bands (28 polymorphic and 2 monomorphic) were successfully scored by using four primers (OPF-01, OPF-02, OPF-03, and OPF-04). The Primers were used able to amplify all of the four eggplant samples. The result of PCR-RAPD visualization produces bands of 300-1500 bp. The result of cluster analysis showed the existence of three clusters (A, B, and C). Cluster A (coefficient of equal to 49%) consisted of a gelatik, cluster B (coefficient of 65% equilibrium) consisted of TPU (Kopek) and TK (Tekokak), and cluster C (55% equilibrium coefficient) consisted of LC (Leunca). These results indicated that the closest proximity is found in samples of TK (Tekokak) and TPU (Kopek).

Comparisons of molecular karyotype and RAPD patterns of anuran trypanosome isolates during long-term in vitro cultivation.

PubMed

Lun, Z R; Desser, S S

1996-01-01

The patterns of random amplified fragments and molecular karyotypes of 12 isolates of anuran trypanosomes continuously cultured in vitro were compared by random amplified polymorphic DNA (RAPD) analysis and pulsed field gradient gel electrophoresis (PFGE). The time interval between preparation of two series of samples was one year. Changes were not observed in the number and size of sharp, amplified fragments of DNA samples from both series examined with the ten primers used. Likewise, changes in the molecular karyotypes were not detected between the two samples of these isolates. These results suggest that the molecular karyotype and the RAPD patterns of the anuran trypanosomes remain stable after being cultured continuously in vitro for one year.

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

PubMed Central

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively. PMID:10473394

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

PubMed

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Genetic variability in isolates of Chromobacterium violaceum from pulmonary secretion, water, and soil.

PubMed

Santini, A C; Magalhães, J T; Cascardo, J C M; Corrêa, R X

2016-04-28

Chromobacterium violaceum is a free-living Gram-negative bacillus usually found in the water and soil in tropical regions, which causes infections in humans. Chromobacteriosis is characterized by rapid dissemination and high mortality. The aim of this study was to detect the genetic variability among C. violaceum type strain ATCC 12472, and seven isolates from the environment and one from a pulmonary secretion from a chromobacteriosis patient from Ilhéus, Bahia. The molecular characterization of all samples was performed by polymerase chain reaction (PCR) sequencing and 16S rDNA analysis. Primers specific for two ATCC 12472 pathogenicity genes, hilA and yscD, as well as random amplified polymorphic DNA (RAPD), were used for PCR amplification and comparative sequencing of the products. For a more specific approach, the PCR products of 16S rDNA were digested with restriction enzymes. Seven of the samples, including type-strain ATCC 12472, were amplified by the hilA primers; these were subsequently sequenced. Gene yscD was amplified only in type-strain ATCC 12472. MspI and AluI digestion revealed 16S rDNA polymorphisms. This data allowed the generation of a dendogram for each analysis. The isolates of C. violaceum have variability in random genomic regions demonstrated by RAPD. Also, these isolates have variability in pathogenicity genes, as demonstrated by sequencing and restriction enzyme digestion.

Morphological characterization and molecular fingerprinting of Nostoc strains by multiplex RAPD.

PubMed

Hillol, Chakdar; Pabbi, Sunil

2012-01-01

Morphological parameters studied for the twenty selected Nostoc strains were mostly found to be consistent with the earlier reports. But the shape of akinetes observed in this study was a little deviation from the existing descriptions and heterocyst frequency was also found to be different in different strains in spite of growing in the same nitrogen free media. Multiplex RAPD produced reproducible and completely polymorphic amplification profiles for all the strains including some strain specific unique bands which are intended to be useful for identification of those strains. At least one to a maximum of two unique bands was produced by different dual primer combinations. For ten strains out of twenty, strain specific bands were found to be generated. Cluster analysis revealed a vast heterogeneity among these Nostoc strains and no specific clustering based on geographical origin was found except a few strains. It was also observed that morphological data may not necessarily correspond to the genetic data in most of the cases. CCC92 (Nostoc muscorum) and CCC48 (Nostoc punctiforme) showed a high degree of similarity which was well supported by high bootstrap value. The level of similarity of the strains ranged from 0.15 to 0.94. Cluster analysis based on multiplex RAPD showed a good fit revealing the discriminatory power of this technique.

[Genetic variability of the Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae) in vegetable crops in São Luís, state of Maranhão, Brazil].

PubMed

Da Silva, Maria C; De Lemos, Raimunda N S; Lima, Luzia H C; Gourlart Filho, Luiz R; Pereira, Silma R F

2009-01-01

The RAPD technique is widely used to investigate the distinct genetic characteristics of the complex Bemisia tabaci (Gennadius), which is currently constituted of approximately 41 biotypes. The objective of this research was to characterize populations of whitefly collected in crops of agricultural producing areas in São Luís, MA, like okra, beans and pepper, using RAPD molecular markers. Females from nine whitefly populations were analyzed and compared with B. tabaci biotype B taken from poinsettia culture of Embrapa Genetic Resources and Biotechnology (Brasília, DF). Twelve out of the 20 primers tested produced specific band patterns suitable to confirm that the evaluated specimens belong to the biotype B of B. tabaci, despite the high percentage of detected polymorphism. The analysis of the 96 RAPD molecular markers generated indicated that the populations on okra, beans and pepper were grouped according to the host cultures, sharing 80, 76 and 45% of genetic similarity, respectively, when compared with the control population of B. tabaci biotype B. A lower selective pressure was observed with the population of whitefly collected on pepper and minor genetic variability in the whitefly populations collected on okra and bean, when compared with the control population.

Genetic characterization of chayote [Sechium edule (Jacq.) Swartz.] landraces of North Eastern Hills of India and conservation measure.

PubMed

Verma, Veerendra Kumar; Pandey, Avinash; Jha, Anjani Kumar; Ngachan, S V

2017-10-01

Chayote or chow-chow is an underutilized cucurbit vegetable crop, widely cultivated by farmers in the backyards and Jhum lands for its tender leaves, fruits and tuberous root. In order to initiate crop improvement program in this crop, the present study was undertaken to assess the genetic variations in the 74 chow-chow landraces collected from the North Eastern Hill region of India. Wide variations for fruit colors, fruit length (6.5-21.5 cm), fruit width (4.2-10.7 cm), fruit weight (60-560 g), vitamin-C (2.6-13.8 mg/100 g), reducing sugar (0.18-2.77%), total sugar (1.09-2.94%) and phenol content (0.17-3.85 mg/100 g FW) were recorded among the landraces. All the landraces were also characterized using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. In RAPD analyses, out of 28 primers a total of 198 reproducible amplicons were formed at an average of 7.01 per primer and an overall polymorphism of 88.38%. Eight fragments were specific to landraces with light green fruits. Four fragments were observed to be specific to RCSC-22 (dark green fruits) and another four specific to a RCSC-30 (pale yellow fruits). Out of 30 ISSR, only 5 primers generated a total of 32 reproducible amplicons with an average of 6.4 per primer and overall polymorphism of 62.5%. The pair wise similarity coefficient values ranged from 0.55 to 0.96. The grouping of landraces in cluster analysis was found to be independent of their respective geographic locations. The cuttings of suckers and shoot top (2 months old) treated with indole-3-butyric acid (200 mg l -1 ) provide an alternative for the conservation of the diverse genetic materials to the researchers.

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening.

PubMed

Andrade, María J; Rodríguez, Mar; Casado, Eva; Córdoba, Juan J

2010-03-01

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening. Copyright 2009 Elsevier Ltd. All rights reserved.

Genetic relationships among some hawthorn (Crataegus spp.) species and genotypes.

PubMed

Yilmaz, Kadir Ugurtan; Yanar, Makbule; Ercisli, Sezai; Sahiner, Hatice; Taskin, Tuncer; Zengin, Yasar

2010-10-01

The genus Crataegus is well distributed in Turkey as a wild plant, with numerous, inherently variable species and genotypes. RAPD markers were used to study 17 hawthorn genotypes belonging to Crataegus monogyna ssp. monogyna Jacq (2 genotypes), C. monogyna ssp. azarella Jacq (1), Crataegus pontica K.Koch (3), Crataegus orientalis var. orientalis Pallas Ex Bieb (3), Crataegus pseudoheterophylla Pojark (1), Crataegus aronia var. dentata Browicz (1), C. aronia var. aronia Browicz (4), and Crateagus x bornmuelleri Zabel (2). The 10 RAPD primers produced 72 polymorphic bands (88% polymorphism). A dendrogram based on Jaccard's index included four major groups and one outgroup according to taxa. The lowest genetic variability was observed within C. aronia var. aronia genotypes. The study demonstrated that RAPD analysis is efficient for genotyping wild-grown hawthorns.

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

PubMed Central

Hayford, Alice E.; Petersen, Anne; Vogensen, Finn K.; Jakobsen, Mogens

1999-01-01

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates. PMID:10388723

Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human feces.

PubMed

Sato, Naoki; Seo, Genichiro; Benno, Yoshimi

2014-01-01

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Spectrum of bacteriocin activity of Lactobacillus plantarum BS and fingerprinting by RAPD-PCR.

PubMed

Elegado, Francisco B; Guerra, Marie Antonette Ruth V; Macayan, Rommel A; Mendoza, Helen A; Lirazan, Marcelina B

2004-08-15

The spectrum of antimicrobial activity of Lactobacillus plantarum BS against representative bacterial species was established through deferred assay and 'spot-on-lawn' assay using actively growing cells and partially purified bacteriocin extract, respectively. Only lactobacilli, pediococci, enterococci, bacilli and Listeria were inhibited from the test microorganisms. Slight bacteriocinogenic activity through 'spot-on-lawn' assay was detected against Staphylococcus aureus and Escherichia coli O157:H7. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was used to compare the fingerprint of L. plantarum BS with other strains of L. plantarum. Using the 16S rRNA-based primer, P32, the bacteriocinogenic isolate exhibited identical RAPD-PCR fingerprints to L. plantarum ATCC 14917. Dendrograms derived from the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were constructed to show the similarity relationships among the investigated strains based on RAPD-PCR analysis. Bands differentiating L. plantarum BS from L. plantarum ATCC 14917 were also identified by varying the annealing temperature.

Investigation of genetic divergence and polymorphism of nuclear DNA in species and populations of domestic and wild sheep

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mel`nikova, M.N.; Grechko, V.V.; Mednikov, B.M.

1995-08-01

Genetic divergence in repetitive sequences of nuclear DNA of wild and domestic sheep was studied by general restriction endonuclease mapping (i.e., the taxonoprint method). The PCR RAPD method with one and two arbitrary primers was also used to analyze the nuclear DNA polymorphism in some other regions. The taxonoprint method, performed using six endonucleases, showed specificity and virtually complete similarity in the patterns of repetitive DNA sequences of two wild forms, argali and moufflon, and five domestic sheep breeds. Central Asian breeds, Kazakh fine-fleeced, karakuk, ghissar, and eadeelbay, and an English breed, Lincoln, were examined. The results confirm the opinionmore » that wild and domestic sheep may be considered one polytypic species. The PCR-RAPD method, both with one and two arbitrary primers, revealed a closer similarity of all the sheep breeds examined when aragali, rather than with moufflon, was used. These results indicate that the domestication area of sheep was much more broader than was earlier presumed. Otherwise, hybridizations of domestic and wild forms could occasionally occur in the area of their coexistence. The amplification patterns of PCR-RAPD products are the most promising population genetic markers. 27 refs., 4 figs., 7 tabs.« less

RAPD markers on genetic diversity in three populations of pisifera type of oil palm (elaeis guineensis)

NASA Astrophysics Data System (ADS)

Basyuni, M.; Prayogi, H.; Putri, L. A. P.; Syahputra, I.; Siregar, E. S.; Risnasari, I.; Wati, R.; Arifiyanto, D.

2018-03-01

Palm oil (E. guineensis) is one of the major commodity and contributing largely to non-petroleum oil of Indonesian foreign exchange. E. guineensis has three fruit types, dura (female), pisifera (male), and tenera —a hybrid between dura and pisifera. Pisifera plays an important function in the production of seed oil palm. The purpose of this research is to analyze genetic diversity of pisifera type of E. guineensis from three populations, Yangambi, Lame and Lame further cross in Bangun Bandar, North Sumatra, Indonesia. Eighteen samples for each population were analyzed using six RAPD markers. Results showed that RAPD markers were low polymorphic with 1.49, 1.39, and 1.00 average number alleles detected for Yagambi, Lame, and Lame further cross, respectively. The level of genetic diversity detected for each population was 0.28, 0.22, and 0.21 for Yagambi, Lame, and Lame further cross, respectively, indicating that the populations had little genetic variation. The highest of polymorphic information content (PIC) was found on the P11 primer of Yangambi (0.49) and P10 primer for lame further cross (0.49). By contrast, the lowest PIC belongs to P21 for Lame population (0.01). This data is likely to contributing oil palm breeding.

Morpho-molecular analysis as a prognostic model for repulsive feedback of the medicinal plant "Andrographis paniculata" to allogamy.

PubMed

Valdiani, Alireza; Talei, Daryush; Javanmard, Arash; Tan, Soon Guan; Kadir, Mihdzar Abdul; Maziah, Mahmood

2014-06-01

Andrographis paniculata Nees. (AP) is a self-pollinated medicinal herb with a wide range of pharmaceutical properties, facing a low diversity in Malaysia. Cross-pollination of AP accessions leads to considerable rates of heterosis in the agro-morphological characteristics and anticancer phytochemicals of this eminent medicinal herb. However, the poor crossability of the plant at the interpopulation or intraspecific levels is an obstacle from the evolutionary and breeding points of view as an average of 4.56% crossability was recorded for AP in this study. Hence, this research aimed to elicit the impact of parental genetic distances (GDs) on the rate of crossability of AP using seven accessions in 21 possible cross combinations. To this end, a set of 55 randomly amplified polymorphic DNA (RAPD) primers and a total of 13 agro-morphological markers were employed to test the hypothesis. Twenty-two out of the 55 RAPD primers amplified a total of 257 bands of which 107 bands were found to be polymorphic. The principal component analysis (PCA) based on the RAPD markers revealed that the studied AP accessions were distributed to three distinct groups. Furthermore, it was noticed that even a minor increase in GD between two parents can cause a decline in their crossability. Unlike, the morphological-based GDs acted neutrally to crossability. This finding suggests that, despite the low genetic diversity among the Malaysian APs, a population prescreening using RAPD markers would be useful to enhance the rate of fruit set through selecting the genetically adjacent parents. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic and metabolic diversity in Stevia rebaudiana using RAPD and HPTLC analysis.

PubMed

Chester, Karishma; Tamboli, Ennus Tajuddin; Parveen, Rabea; Ahmad, Sayeed

2013-06-01

Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals. To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis. The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360 nm after spraying with anisaldehyde sulphuric acid. Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01-0.08) and polymorphism (67.24-92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration. Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.

Eradication of methicillin resistant S. aureus biofilm by the combined use of fosfomycin and β-chloro-L-alanine.

PubMed

Akbari-Ayezloy, Elham; Hosseini-Jazani, Nima; Yousefi, Saber; Habibi, Nazanin

2017-02-01

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). Fosfomycin is a borad-spectrum antibiotic with inhibitory effects on biofilm production and β-Chloro-L-alanine (β-CLA) is an amino acid analog. The aim of this study was to determine effect of the combination of fosfomycin and β-CLA on biofilm production by MRSA isolates. Also, the clonal relatedness of the isolates was evaluated. To determine the ability of biofilm production by 42 MRSA isolates, microtiter plate method was used. Antibacterial activities of fosfomycin and β-CLA were investigated by determining MICs and MBCs. Antibiofilm activities were measured in the presence of sub-MIC concentrations of fosfomycin, β-CLA or a combination of both. RAPD-PCR was used for investigating the clonal relationship between isolates by the two specific primers. 21.4% of isolates were strong and 5% were moderate biofilm producers. The effect of fosfomycin plus β-CLA treatment on biofilm production was significantly different from non-treated, fosfomycin and β-CLA groups (p=0.00, 0.004 and 0.000 respectively). RAPD-PCR analysis revealed that the RAPD1 primer had more discriminatory power. The Sizes of RAPD-PCR bands ranged from 150 bp to 1500 bp and the number of bands varied from 1 to 13. Clonal relatedness of isolates showed that the majority of biofilm producing isolates had identical pattern and only three isolates showed more than 80% similarity. The combination of fosfomycin and β-CLA could be introduced as an excellent mixture for eradication of MRSA biofilms in vitro.

Pathogenicity for onion and genetic diversity of isolates of the pathogenic fungus Colletotrichum gloeosporioides (Phyllachoraceae) from the State of Pernambuco, Brazil.

PubMed

Nova, M X Vila; Borges, L R; de Sousa, A C B; Brasileiro, B T R V; Lima, E A L A; da Costa, A F; de Oliveira, N T

2011-02-22

Onion anthracnose, caused by Colletotrichum gloeosporioides, is one of the main diseases of onions in the State of Pernambuco. We examined the pathogenicity of 15 C. gloeosporioides strains and analyzed their genetic variability using RAPDs and internal transcribed spacers (ITS) of the rDNA region. Ten of the strains were obtained from substrates and hosts other than onion, including chayote (Sechium edule), guava (Psidium guajava), pomegranate (Punica granatum), water from the Capibaribe River, maracock (Passiflora sp), coconut (Cocus nucifera), surinam cherry (Eugenia uniflora), and marine soil; five isolates came from onions collected from four different regions of the State of Pernambuco and one region of the State of Amazonas. Pathogenicity tests were carried out using onion leaves and bulbs. All strains were capable of causing disease in leaves, causing a variable degree of lesions on the leaves; four strains caused the most severe damage. In the onion bulb tests, only three of the above strains caused lesions. Seven primers of arbitrary sequences were used in the RAPD analysis, generating polymorphic bands that allowed the separation of the strains into three distinct groups. The amplification products generated with the primers ITS1 and ITS4 also showed polymorphism when digested with three restriction enzymes, DraI, HaeIII and MspI. Only the latter two demonstrated genetic variations among the strains. These two types of molecular markers were able to differentiate the strain from the State of Amazonas from those of the State of Pernambuco. However, there was no relationship between groups of strains, based on molecular markers, and degree of pathogenicity for onion leaves and bulbs.

RAPD-SCAR Markers for Genetically Improved NEW GIFT Nile Tilapia (Oreochromis niloticus niloticus L.) and Their Application in Strain Identification.

PubMed

Li, Si-Fa; Tang, Shou-Jie; Cai, Wan-Qi

2010-04-01

The NEW GIFT Nile tilapia (Oreochromis niloticus niloticus L.) is a nationally certificated new strain selected over 14 years and 9 generations from the base strain of GIFT Nile tilapia, introduced in 1994. This new variety has been extended in most of areas of China. The management of genetically improved strains, including the genetic markers for identification is needed urgently. RAPD analysis was conducted and their conversion to SCAR markers was developed. From NEW GIFT Nile tilapia, two strain-specific RAPD bands, S(304 )(624 bp ) and S(36 )(568 bp ) were identified. The strain-specific RAPD bands were gel-purified, cloned, and sequenced. Locus-specific primers were then designed to amplify the strain-specific bands. PCR amplification was conducted to test the variations in allele frequencies of two converted SCAR markers among the NEW GIFT Nile tilapia and its base strains, as well as 7 additional farmed strains worldwide. The frequency of SCAR marker I (553 bp) was 85.7% in NEW GIFT Nile tilapia, but 16.7% in the base strain. The frequency of SCAR marker II (558 bp) was 91.4% in NEW GIFT Nile tilapia, but 0% - 70% in the 7 other strains. In order to confirm the utility of these two markers, an examination was conducted for a wild population from Egypt, resulted the frequency of SCAR I and II was 10% and 70%, respectively, much lower than that of New GIFT strain. The increase in allele frequency of these two SCAR markers suggests that these markers might be genetically linked to the quantitative trait loci (QTL) underlining the performance traits by long term selection, and indicate the bright potential of SCAR marker technology for tracking generations during selection progress and for distinguishing among genetically improved strain and other strains.

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems.

PubMed

Lallawmsanga; Passari, Ajit Kumar; Mishra, Vineet Kumar; Leo, Vincent Vineeth; Singh, Bhim Pratap; Valliammai Meyyappan, Geetha; Gupta, Vijai Kumar; Uthandi, Sivakumar; Upadhyay, Ramesh Chandra

2016-01-01

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems

PubMed Central

Lallawmsanga; Passari, Ajit Kumar; Mishra, Vineet Kumar; Leo, Vincent Vineeth; Valliammai Meyyappan, Geetha; Gupta, Vijai Kumar; Uthandi, Sivakumar; Upadhyay, Ramesh Chandra

2016-01-01

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants. PMID:27902725

Application of the multiplex PCR method for discrimination of Artemisia iwayomogi from other Artemisia herbs.

PubMed

Lee, Mi Young; Doh, Eui Jeong; Kim, Eung Soo; Kim, Young Wha; Ko, Byong Seob; Oh, Seung-Eun

2008-04-01

Some plants classified in the genus Artemisia are used for medicinal purposes. In particular, A. iwayomogi, which is referred to as 'Haninjin,' is used as an important medicinal material in traditional Korean medicine. However, A. capillaris, and both A. argyi and A. princeps, referred to as 'Injinho' and 'Aeyup,' respectively, are used for purposes other than those for which 'Haninjin' is utilized. However, it is occasionally difficult to differentiate 'Haninjin' from 'Injinho' and/or 'Aeyup' on the basis of their morphological features, particularly when in the dried and/or sliced form. Therefore, the development of a reliable method by which to discriminate 'Haninjin' from other Artemisia herbs, especially 'Injinho' and 'Aeyup,' is clearly necessary. We recently determined that the RAPD (random amplified polymorphic DNA) technique can be used to discriminate efficiently between some Artemisia herbs. In particular, when applied to RAPD, the non-specific UBC primer 391 (5'-GCG AAC CTC G-3') was demonstrated to amplify PCR products specific to A. iwayomogi. Based on the nucleotide sequences of the PCR product, we designed a 2F1 (5'-ACC TCG GAC CTA AAT ACA-3')/ 2F3 (5'-TTA TGA TTC ATG TTC AAT TC-3') primer set to amplify a SCAR (sequence-characterized amplified region) marker of A. iwayomogi. Employing this primer set, along with two other primer sets amplifying SCAR markers of 'Aeyup' (A. argyi and A. princeps) and both 'Injinho' (A. capillaris) and A. japonica, which are classified into the same subgroup in a phenogram constructed from RAPD analysis, we developed a multiplex PCR method by which A. iwayomogi could be discriminated with certainty from other Artemisia herbs. Via this method, we determined not only whether the tested Artemisia herb was A. iwayomogi, but also which Artemisia herbs were tested concurrently with A. iwayomogi.

Effects of space environment on biological characters of cultured rose seedlings

NASA Astrophysics Data System (ADS)

Min, L.; Huai, X.; Jinying, L.; Yi, P.; Chunhua, Z.

Cultured rose seedlings were carried into space by SHENZHOU-4 spacecraft and then used as the experimental material to investigate effects of the space environmental conditions on morphology cytology physiology and molecular biology of the seedlings After loaded on the space flight the plant s height number of leaves and fresh weight per seedling were all increased significantly compared to the ground controls The content of chlorophyll was basically unchanged In some cells the ultrastructural changes involved twist contraction and deformation of cell wall curvature and loose arrangement of lamellae of some chloroplasts and a significant increase in number of starch grains per chloroplast In addition the number of mitochondria increased but some mitochondrial outer membrane broke and some mitochondrial cristae disappeared The activities of the defense enzymes such as superoxide dismutase peroxidase and catalyse in rose leaves increased and the content of malondialdehyde decreased In the RAPD analysis with 40 10-mer primers 36 primers generated 148 DNA bands from both of the space flight treated seedlings and the ground controls and five primers amplified polymorphic products The rate of DNA variation was 6 34

Monitoring hydrilla using two RAPD procedures and the nonindigenous aquatic species database

USGS Publications Warehouse

Madeira, Paul T.; Jacono, C.C.; Van, Thai K.

2000-01-01

Hydrilla (Hydrilla verticillata (L.f.) Royle), an invasive aquatic weed, continues to spread to new regions in the United States. Two biotypes, one a female dioecious and the other monoecious have been identified. Management of the spread of hydrilla requires understanding the mechanisms of introduction and transport, an ability to map and make available information on distribution, and tools to distinguish the known U.S. biotypes as well as potential new introductions. Review of the literature and discussions with aquatic scientists and resource managers point to the aquarium and water garden plant trades as the primary past mechanism for the regional dispersal of hydrilla while local dispersal is primarily carried out by other mechanisms such as boat traffic, intentional introductions, and waterfowl. The Nonindigenous Aquatic Species (NAS) database is presented as a tool for assembling, geo-referencing, and making available information on the distribution of hydrilla. A map of the current range of dioecious and monoecious hydrilla by drainage is presented. Four hydrilla samples, taken from three discrete, non-contiguous regions (Pennsylvania, Connecticut, and Washington State) were examined using two RAPD assays. The first, generated using primer Operon G17, and capable of distinguishing the dioecious and monoecious U.S. biotypes, indicated all four samples were of the monoecious biotype. Results of the second assay using the Stoffel fragment and 5 primers, produced 111 markers, indicated that these samples do not represent new foreign introductions. The differences in the monoecious and dioecious growth habits and management are discussed.

Detection of two fungal biocontrol agents against root-knot nematodes by RAPD markers.

PubMed

Zhu, Ming Liang; Mo, Ming He; Xia, Zhen Yuan; Li, Yun Hua; Yang, Shu Jun; Li, Tian Fei; Zhang, Ke Qin

2006-05-01

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 mug, respectively.

Genetic diversity in mesoamerican populations of mahogany (Swietenia macrophylla), assessed using RAPDs.

PubMed

Gillies, A C; Navarro, C; Lowe, A J; Newton, A C; Hernández, M; Wilson, J; Cornelius, J P

1999-12-01

Swietenia macrophylla King, a timber species native to tropical America, is threatened by selective logging and deforestation. To quantify genetic diversity within the species and monitor the impact of selective logging, populations were sampled across Mesoamerica, from Mexico to Panama, and analysed for RAPD DNA variation. Ten decamer primers generated 102 polymorphic RAPD bands and pairwise distances were calculated between populations according to Nei, then used to construct a radial neighbour-joining dendrogram and examine intra- and interpopulation variance coefficients, by analysis of molecular variation (AMOVA). Populations from Mexico clustered closely together in the dendrogram and were distinct from the rest of the populations. Those from Belize also clustered closely together. Populations from Panama, Guatemala, Costa Rica, Nicaragua and Honduras, however, did not cluster closely by country but were more widely scattered throughout the dendrogram. This result was also reflected by an autocorrelation analysis of genetic and geographical distance. Genetic diversity estimates indicated that 80% of detected variation was maintained within populations and regression analysis demonstrated that logging significantly decreased population diversity (P = 0.034). This study represents one of the most wide-ranging surveys of molecular variation within a tropical tree species to date. It offers practical information for the future conservation of mahogany and highlights some factors that may have influenced the partitioning of genetic diversity in this species across Mesoamerica.

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus.

PubMed

Ferri, D V; Munhoz, C F; Neves, P M O; Ferracin, L M; Sartori, D; Vieira, M L C; Fungaro, M H P

2012-12-01

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil.

PubMed

Souza, I G B; Valente, S E S; Britto, F B; de Souza, V A B; Lima, P S C

2011-12-14

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.

DNA-based identification of Brassica vegetable species for the juice industry.

PubMed

Etoh, Kazumi; Niijima, Noritaka; Yokoshita, Masahiko; Fukuoka, Shin-Ichi

2003-10-01

Since kale (Brassica oleracea var. acephala), a cruciferous vegetable with a high level of vitamins and functional compounds beneficial to health and wellness, has become widely used in the juice industry, a precise method for quality control of vegetable species is necessary. We describe here a DNA-based identification method to distinguish kale from cabbage (Brassica oleracea var. capitata), a closely related species, which can be inadvertently mixed with kale during the manufacturing process. Using genomic DNA from these vegetables and combinatory sets of nucleotide primers, we screened for random amplified polymorphic DNA (RAPD) fragments and found three cabbage-specific fragments. These RAPD fragments, with lengths of 1.4, 0.5, and 1.5 kb, were purified, subcloned, and sequenced. Based on sequence-tagged sites (STS), we designed sets of primers to detect cabbage-specific identification (CAI) DNA markers. Utilizing the CAI markers, we successfully distinguished more than 10 different local cabbage accessions from 20 kale accessions, and identified kale juices experimentally spiked with different amounts of cabbage.

An improved micropropagation of Arnebia hispidissima (Lehm.) DC. and assessment of genetic fidelity of micropropagated plants using DNA-based molecular markers.

PubMed

Phulwaria, Mahendra; Rai, Manoj K; Shekhawat, N S

2013-07-01

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3-4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog's (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l(-1) 6-benzylaminopurine and 0.1 mg l(-1) IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50 ± 0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2-4 mm) of shoots was treated with 300 mg l(-1) of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85-90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.

DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

PubMed Central

Brikun, I; Suziedelis, K; Berg, D E

1994-01-01

Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics. Images PMID:8132463

Development of AFLP and RAPD markers linked to a locus associated with twisted growth in corkscrew willow (Salix matsudana 'Tortuosa').

PubMed

Lin, Juan; Gunter, Lee E; Harding, Scott A; Kopp, Richard F; McCord, Rachel P; Tsai, Chung-Jui; Tuskan, Gerald A; Smart, Lawrence B

2007-11-01

Salix matsudana Koidz. cultivar 'Tortuosa' (corkscrew willow) is characterized by extensive stem bending and curling of leaves. To investigate the genetic basis of this trait, controlled crosses were made between a corkscrew female (S. matsudana 'Tortuosa') and a straight-stemmed, wild-type male (Salix alba L. Clone 99010). Seventy-seven seedlings from this family (ID 99270) were grown in the field for phenotypic observation. Among the progeny, 39 had straight stems and leaves and 38 had bent stems and curled leaves, suggesting that a dominant allele at a single locus controls this phenotype. As a first step in characterizing the locus, we searched for amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) markers linked to the tortuosa allele using bulked segregant analysis. Samples of DNA from 10 corkscrew individuals were combined to produce a corkscrew pool, and DNA from 10 straight progeny was combined to make a wild-type pool. Sixty-four AFLP primer combinations and 640 RAPD primers were screened to identify marker bands amplified from the corkscrew parent and progeny pool, but not from the wild-type parent or progeny pool. An AFLP marker and a RAPD marker linked to and flanking the tortuosa locus were placed on a preliminary linkage map constructed based on segregation among the 77 progeny. Sectioning and analysis of shoot tips revealed that the corkscrew phenotype is associated with vascular cell collapse, smaller cell size in regions near the cambium and less developed phloem fibers than in wild-type progeny. Identification of a gene associated with this trait could lead to greater understanding of the control of normal stem development in woody plants.

Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis.

PubMed

Sahilah, A M; Laila, R A S; Sallehuddin, H Mohd; Osman, H; Aminah, A; Ahmad Azuhairi, A

2014-02-01

Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92%, 34/37) and penicillin (89%, 33/37) followed by resistance towards ampicillin (68%, 25/37), cefuroxime (38%, 14/37), amikacin (6%, 2/37) and ceftazidime (14%, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80%. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus.

RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity.

PubMed

Das, I K; Fakrudin, B; Arora, D K

2008-01-01

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

PubMed

Salimath, S S; de Oliveira, A C; Godwin, I D; Bennetzen, J L

1995-08-01

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

Identification and characterization of RAPD-SCAR markers linked to glyphosate-susceptible and -resistant biotypes of Eleusine indica (L.) Gaertn.

PubMed

Cha, Thye San; Anne-Marie, Kaben; Chuah, Tse Seng

2014-02-01

Eleusine indica is one of the most common weed species found in agricultural land worldwide. Although herbicide-glyphosate provides good control of the weed, its frequent uses has led to abundant reported cases of resistance. Hence, the development of genetic markers for quick detection of glyphosate-resistance in E. indica population is imperative for the control and management of the weed. In this study, a total of 14 specific random amplified polymorphic DNA (RAPD) markers were identified and two of the markers, namely S4R727 and S26R6976 were further sequence characterized. Sequence alignment revealed that marker S4R727 showing a 12-bp nucleotides deletion in resistant biotypes, while marker S26R6976 contained a 167-bp nucleotides insertion in the resistant biotypes. Based on these sequence differences, three pairs of new sequence characterized amplified region (SCAR) primers were developed. The specificity of these primer pairs were further validated with genomic DNA extracted from ten individual plants of one glyphosate-susceptible and five glyphosate-resistant (R2, R4, R6, R8 and R11) populations. The resulting RAPD-SCAR markers provided the basis for assessing genetic diversity between glyphosate-susceptible and -resistant E. indica biotypes, as well for the identification of genetic locus link to glyphosate-resistance event in the species.

Molecular Identification of Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) Markers.

PubMed

Al-Khalifah, Nasser S; Shanavaskhan, A E

2017-01-01

Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers. Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible. Various molecular markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) markers have been successfully used as efficient tools for analysis of genetic variation in date palm. This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided.

Genetic variation in Indian populations of Scirpophaga incertulas as revealed by RAPD-PCR analysis.

PubMed

Kumar, L S; Sawant, A S; Gupta, V S; Ranjekar, P K

2001-02-01

Scirpophaga incertulas, commonly referred to as yellow stem borer, is a predominant pest of rice causing serious losses in its yield. Genetic variation among populations of Scirpophaga incertulas collected from 28 hotspot locations in India was examined using the randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In all, 32 primers were used and 354 amplification products were observed. No RAPD-PCR bands diagnostic to the pest population from any specific region were identified. Cluster analysis using UPGMA showed that, with the exception of the pest population from Pattambi, all the populations cluster as one group with GD values in the range of 6-22%, suggesting that gene flow between populations is independent of geographic distance and appears to be unrestricted. The relatively high GD value of 48% exhibited by the pest population from Pattambi was the only exception.

RAPD inheritance and diversity in pawpaw (Asimina triloba)

Treesearch

Hongwen Huang; Desmond R. Layne; Thomas L. Kubisiak

2000-01-01

Twelve, 10-base primers amplified a total of 20 intense and easily scorable polymorphic bands in an interspecific cross of PPFl-5 pawpaw (Asimina triloba (L.) Dunal.) x RET (Asimina reticulata Shuttlew.). In this cross, all bands scored were present in, and inherited from, the A. triloba ...

[Nucleotidic variations of two captive groups of tepezcuintle, Agouti paca (Rodentia: Agoutidae), from two sites in Yucatan, Mexico].

PubMed

Montes-Pérez, Rubén C; García, Adán W Echeverría; Castro, Jorge Zavala; Gamboa, Militza G Alfaro

2006-09-01

The objective of this work was to estimate the nucleotidic variation between two groups of tepezcuintles (Agouti paca) from the states of Campeche and Quintana Roo, Mexico and within members of each group. Blood samples were collected from eleven A. paca kept in captivity. DNA from leukocytic cells was used for Ramdom Amplification of DNA Polimorphism (RAPD). The primers three 5'-d(GTAGACCCGT)- 3' and six 5'-d(CCCGTCAGCA)- 3' were selected from de Amersham kit (Ready.To.Go. RAPD Analysis Beads, Amersham Pharmacia Biotech), because they produced an adequate number of bands. The electrophoretic pattern of bands obtained was analyzed using software for phylogenetic analysis based on the UPGMA method, to estimate the units of nucleotidic variation. The phylogenetic tree obtained with primer three reveals a dicotomic grouping between the animals from both states in the Yucatan Peninsula showing a divergent value of 1.983 nucleotides per hundred. Animals from Quintana Roo show a grouping with primer six; an additional grouping was observed with animals from Campeche. Nucleotidic variation between both groups was 2.118 nucleotides per hundred. The nucleotidic variation for the two primers within the groups from both states, showed fluctuating values from 0.46 to 1.68 nucleotides per hundred, which indicates that nucleotidic variation between the two groups of animals is around two nucleotides per hundred and, within the groups, less than 1.7 nucleotides per hundred.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities.

PubMed

Pérez, Gabriel; Verdejo, Valentina; Gondim-Porto, Clarissa; Orlando, Julieta; Carú, Margarita

2014-11-01

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities* #

PubMed Central

Pérez, Gabriel; Verdejo, Valentina; Gondim-Porto, Clarissa; Orlando, Julieta; Carú, Margarita

2014-01-01

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20–23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community. PMID:25367789

Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

PubMed

Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

2008-12-01

A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

Assessment of genetic characteristics of Aconitum germplasms in Xinjiang Province (China) by RAPD and ISSR markers

PubMed Central

Zhao, Feicui; Nie, Jihong; Chen, Muzhi; Wu, Guirong

2015-01-01

Aconitum is a medicinal treasure trove that grows extensively on fertile pastures in Xinjiang Province (China); however, its molecular genetic characteristics are still poorly studied. We studied Aconitum kusnezoffii Reichb., Aconitum soongaricum Stapf., Aconitum carmichaelii Debx. and Aconitum leucostomum Worosch, using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) techniques, to evaluate their genetic relationship and potential medicinal value. Our results showed that A. kusnezoffii Reichb. and A. soongaricum Stapf. have close genetic relationship and cluster together. Polymorphism rates of 97.25% and 98.92% were achieved by using 15 RAPD and 15 ISSR primers, respectively. Based on Nei's gene diversity (H) and Shannon's index (I), the inter-population diversity (Hs) was higher when compared with the intra-population diversity (Hp). Among the three Aconitum populations, the coefficient of gene differentiation (Gst) was 0.4358 when evaluated by RAPD and 0.5005 by ISSR. The genetic differentiation among the three Aconitum populations was highly significant, suggesting low gene flow (Nm). This was confirmed by the estimates of gene flow (Nm = 0.6473 and Nm = 0.4991, based on ISSR and RAPD data, respectively). Comparing the RAPD and ISSR results, the two DNA markers proved similarly effective in the assessment of the genetic characteristics of the studied Aconitum populations and could be used for reliable fingerprinting and mapping in studies on Aconitum diversity in view of Aconitum suitability for development and protection. PMID:26019645

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

PubMed

Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

2015-05-25

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Genotyping and chlorine-resistance of Methylobacterium aquaticum isolated from water samples in Japan.

PubMed

Furuhata, Katsunori; Banzai, Azusa U; Kawakami, Yasushi; Ishizaki, Naoto; Yoshida, Yoshihiro; Goto, Keiichi; Fukuyama, Masafumi

2011-09-01

For microbial ecological analysis, 14 strains of Methylobacterium aquaticum isolated from water samples were subjected to clustering analysis on the basis of ribotyping and RAPD-PCR tests. The ribopatterns after digestion with EcoRI obtained from 14 strains of M. aquaticum were used to divide the strains into two groups (Groups I and II) with a similarity of 55%. From the analysis of RAPD patterns using primer 208, the 14 strains were divided into 3 groups (A-C) based on a homology of 45% or greater, and from that using primer 272, there were 4 groups (A-D) based on a homology of 50% or greater. The chlorine resistance (99.9% CT values) of these isolates was also experimentally confirmed, and we attempted to define the connection between chlorine resistance and the geno-cluster. The average CT value of group I was 0.89 mg•min/l and the average of group II was 0.69 mg•min/l. No remarkable differences in the CT values for the groups were found.

Population structure of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Thailand based on PCR-RAPD markers.

PubMed

Thaenkham, Urusa; Pakdee, Wallop; Nuamtanong, Supaporn; Maipanich, Wanna; Pubampen, Somchit; Sa-Nguankiat, Surapol; Komalamisra, Chalit

2012-05-01

Angiostrongylus cantonensis is the causative agent of angiostrongyliasis, which is widely distributed throughout the world. It can specifically infect many species of intermediate and definitive hosts. This study examined the genetic differentiation and population structure using the RAPD-PCR method of parasites obtained from 8 different geographical areas of Thailand. Based on 8 primers, high levels of genetic diversity and low levels of gene flow among populations were found. Using genetic distance and neighbor-joining dendrogram methods, A. cantonensis in Thailand could be divided into two groups with statistically significant genetic differentiation of the two populations. However, genotypic variations and haplotype relationships need to be further elucidated using other markers.

RAPD and Internal Transcribed Spacer Sequence Analyses Reveal Zea nicaraguensis as a Section Luxuriantes Species Close to Zea luxurians

PubMed Central

Wang, Pei; Lu, Yanli; Zheng, Mingmin; Rong, Tingzhao; Tang, Qilin

2011-01-01

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species. PMID:21525982

Linkage map of the honey bee, Apis mellifera, based on RAPD markers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hunt, G.J.; Page, R.E. Jr.

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkagemore » groups. We estimate the total genome size to be {approximately}3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species. 71 refs., 6 figs., 1 tab.« less

A report on extensive lateral genetic reciprocation between arsenic resistant Bacillus subtilis and Bacillus pumilus strains analyzed using RAPD-PCR.

PubMed

Khowal, Sapna; Siddiqui, Md Zulquarnain; Ali, Shadab; Khan, Mohd Taha; Khan, Mather Ali; Naqvi, Samar Husain; Wajid, Saima

2017-02-01

The study involves isolation of arsenic resistant bacteria from soil samples. The characterization of bacteria isolates was based on 16S rRNA gene sequences. The phylogenetic consanguinity among isolates was studied employing rpoB and gltX gene sequence. RAPD-PCR technique was used to analyze genetic similarity between arsenic resistant isolates. In accordance with the results Bacillus subtilis and Bacillus pumilus strains may exhibit extensive horizontal gene transfer. Arsenic resistant potency in Bacillus sonorensis and high arsenite tolerance in Bacillus pumilus strains was identified. The RAPD-PCR primer OPO-02 amplified a 0.5kb DNA band specific to B. pumilus 3ZZZ strain and 0.75kb DNA band specific to B. subtilis 3PP. These unique DNA bands may have potential use as SCAR (Sequenced Characterized Amplified Region) molecular markers for identification of arsenic resistant B. pumilus and B. subtilis strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular characterization and RAPD analysis of Juniperus species from Iran.

PubMed

Kasaian, J; Behravan, J; Hassany, M; Emami, S A; Shahriari, F; Khayyat, M H

2011-06-07

The genus Juniperus L. (Cupressaceae), an aromatic evergreen plant, consists of up to 68 species around the world. We classified five species of Juniperus found in Iran using molecular markers to provide a means for molecular identification of Iranian species. Plants were collected (three samples of each species) from two different provinces of Iran (Golestan and East Azarbayejan). The DNA was extracted from the leaves using a Qiagen Dneasy Plant Mini Kit. Amplification was performed using 18 ten-mer RAPD primers. Genetic distances were estimated based on 187 RAPD bands to construct a dendrogram by means of unweighted pair group method of arithmetic means. It was found that J. communis and J. oblonga were differentiated from the other species. Genetic distance values ranged from 0.19 (J. communis and J. oblonga) to 0.68 (J. communis and J. excelsa). Juniperus foetidissima was found to be most similar to J. sabina. Juniperus excelsa subspecies excelsa and J. excelsa subspecies polycarpos formed a distinct group.

Comparative analysis of genetic diversity among Indian populations of Scirpophaga incertulas by ISSR-PCR and RAPD-PCR.

PubMed

Kumar, L S; Sawant, A S; Gupta, V S; Ranjekar, P K

2001-10-01

Genetic variation between 28 Indian populations of the rice pest, Scirpophaga incertulas was evaluated using inter-simple sequence repeats (ISSR)-PCR assay. Nine SSR primers gave rise to 79 amplification products of which 67 were polymorphic. A dendrogram constructed from this data indicates that there is no geographical bias to the clustering and that gene flow between populations appears to be relatively unrestricted, substantiating our earlier conclusion based on the RAPD (random amplified polymorphic DNA) data. The dendrograms obtained using each of these marker systems were poorly correlated with each other as determined by Mantel's test for matrix correlation. Estimates of expected heterozygosity and marker index for each of these marker systems suggests that both these marker systems are equally efficient in determining polymorphisms. Matrix correlation analyses suggest that reliable estimates of genetic variation among the S. incertulas pest populations can be obtained by using RAPDs alone or in combination with ISSRs, but ISSRs alone cannot be used for this purpose.

Genetic diversity analysis of Chrysopidae family (Insecta, Neuroptera) via molecular markers.

PubMed

Yari, Kheirollah; Mirmoayedi, Alinaghi; Marami, Marzieh; Kazemi, Elham; Kahrizi, Danial

2014-09-01

In entomology, improvement of molecular methods would be beneficial tools for accurate identification and detecting the genetic diversity of insect species to discover a corroborative evidence for the traditional classification based on morphology. The aim of this study was focused on RAPD-PCR method for distinguishing the genetic diversity between eight species of Chrysopidae family. In current research, many specimens were collected in different locations of Tehran province (Iran), between them 24 specimens were identified. The wing venation, male genitalia and other morphological characters were used for identification and also the sexing of species was recognized with study of external genitalia. Then, the DNA was extracted with CTAB method. The RAPD-PCR method was carried out with twenty random primers. The agarose gel electrophoresis was used for separation of the PCR products. Based on electrophoresis results, 133 bands were amplified and between them, 126 bands were poly-morph and others were mono-morph. Also, among the applied primers, the primers OPA02 with 19 bands and OPA03 with 8 bands were amplified the maximum and minimum of bands, respectively. The results showed that 80.35 and 73.21 % of genetic similarity existed between Chrysopa pallens-Chrysopa dubitans, and between the Chrysoperla kolthoffi and Chrysoperla carnea, respectively. The minimum (45.53 %) of genetic similarity was observed between C. kolthoffi and C. dubitans, and the maximum (0.80 %) was seen between C. pallens and C. dubitans.

Genetic diversity and gene differentiation among ten species of Zingiberaceae from Eastern India.

PubMed

Mohanty, Sujata; Panda, Manoj Kumar; Acharya, Laxmikanta; Nayak, Sanghamitra

2014-08-01

In the present study, genetic fingerprints of ten species of Zingiberaceae from eastern India were developed using PCR-based markers. 19 RAPD (Rapid Amplified polymorphic DNA), 8 ISSR (Inter Simple Sequence Repeats) and 8 SSR (Simple Sequence Repeats) primers were used to elucidate genetic diversity important for utilization, management and conservation. These primers produced 789 loci, out of which 773 loci were polymorphic (including 220 unique loci) and 16 monomorphic loci. Highest number of bands amplified (263) in Curcuma caesia whereas lowest (209) in Zingiber cassumunar. Though all the markers discriminated the species effectively, analysis of combined data of all markers resulted in better distinction of individual species. Highest number of loci was amplified with SSR primers with resolving power in a range of 17.4-39. Dendrogram based on three molecular data using unweighted pair group method with arithmetic mean classified all the species into two clusters. Mantle matrix correspondence test revealed high matrix correlation in all the cases. Correlation values for RAPD, ISSR and SSR were 0.797, 0.84 and 0.8, respectively, with combined data. In both the genera wild and cultivated species were completely separated from each other at genomic level. It also revealed distinct genetic identity between species of Curcuma and Zingiber. High genetic diversity documented in the present study provides a baseline data for optimization of conservation and breeding programme of the studied zingiberacious species.

[Amplicon density-weighted algorithms for analyzing dissimilarity and dynamic alterations of RAPD polymorphisms of Cordyceps sinensis].

PubMed

Yao, Yi-sang; Gao, Ling; Li, Yu-ling; Ma, Shao-li; Wu, Zi-mei; Tan, Ning-zhi; Wu, Jian-yong; Ni, Lu-qun; Zhu, Jia-shi

2014-08-18

To examine the dynamic maturational alterations of random amplified polymorphic DNA (RAPD) molecular marker polymorphism resulted from differential expressions of multiple fungi in the caterpillar body, stroma and ascocarp portion of Cordyceps sinensis (Cs). Used the fuzzy, integral RAPD molecular marker polymorphism method with 20 random primers; used density-weighted cluster algorithms and ZUNIX similarity equations; compared RAPD polymorphisms of the caterpillar body, stroma and ascocarp of Cs during maturation; and compared RAPD polymorphisms of Cs and Hirsutella sinensis (Hs). Density-unweighted algorithms neglected the differences in density of the DNA amplicons. Use of the density-weighted ZUNIX similarity equations and the clustering method integrated components of the amplicon density differences in similarity computations and clustering construction and prevented from the loss of the information of fungal genomes. An overall similarity 0.42 (< the overall dissimilarity 0.58) was observed for all compartments of Cs at different maturation stages. The similarities for the stromata or caterpillar bodies of Cs at 3 maturational stages were 0.57 or 0.50, respectively. During Cs maturation, there were dynamic Low→High→Low alterations of the RAPD polymorphisms between stromata and caterpillar bodies dissected from the same pieces of Cs. The polymorphic similarity was the highest (0.87) between the ascocarp and mature stroma, forming a clustering clade, while the premature stroma and caterpillar body formed another clade. These 2 clades merged into one cluster. Another clade containing the maturing stroma and caterpillar body merged with mature caterpillar body, forming another cluster. The RAPD polymorphic similarities between Hs and Cs samples were 0.55-0.69. Hs were separated from Cs clusters by the out-group control Paecilomyces militaris. The wealthy RAPD polymorphisms change dynamically in the Cs compartments with maturation. The different RAPD polymorphism for Hs from those for Cs supports the hypothesis of integrated micro-ecosystem Cs with multiple fungi, but does not support the "single fungal species" hypothesis for Cs and the anamorph-teleomorph connection between Hs and Cs.

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC.

PubMed

Chetri, Siva K; Sardar, Pratima Rani; Agrawal, Veena

2014-10-01

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog's medium augmented with different cytokinins, viz. N(6)-benzyladenine (BA), N(6)-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.

Identification of molecular markers associated with mite resistance in coconut (Cocos nucifera L.).

PubMed

Shalini, K V; Manjunatha, S; Lebrun, P; Berger, A; Baudouin, L; Pirany, N; Ranganath, R M; Prasad, D Theertha

2007-01-01

Coconut mite (Aceria guerreronis 'Keifer') has become a major threat to Indian coconut (Coçcos nucifera L.) cultivators and the processing industry. Chemical and biological control measures have proved to be costly, ineffective, and ecologically undesirable. Planting mite-resistant coconut cultivars is the most effective method of preventing yield loss and should form a major component of any integrated pest management stratagem. Coconut genotypes, and mite-resistant and -susceptible accessions were collected from different parts of South India. Thirty-two simple sequence repeat (SSR) and 7 RAPD primers were used for molecular analyses. In single-marker analysis, 9 SSR and 4 RAPD markers associated with mite resistance were identified. In stepwise multiple regression analysis of SSRs, a combination of 6 markers showed 100% association with mite infestation. Stepwise multiple regression analysis for RAPD data revealed that a combination of 3 markers accounted for 83.86% of mite resistance in the selected materials. Combined stepwise multiple regression analysis of RAPD and SSR data showed that a combination of 5 markers explained 100% of the association with mite resistance in coconut. Markers associated with mite resistance are important in coconut breeding programs and will facilitate the selection of mite-resistant plants at an early stage as well as mother plants for breeding programs.

Genetic Analysis of Aedes aegypti Using Random Amplified Polymorphic DNA (RAPD) Markers from Dengue Outbreaks in Pakistan.

PubMed

Ashraf, Hafiz Muhammad; Zahoor, Muhammad Kashif; Nasir, Shabab; Majeed, Humara Naz; Zahoor, Sarwat

2016-12-01

Keeping in view the havoc situation of dengue fever in Pakistan, the current study was designed to demonstrate the genetic variations, gene flow and rate of migration from Lahore and Faisalabad. The larvae were collected from both natural and artificial breeding places from each collection site. The adult mosquitoes were collected by means of sweep net and battery-operated aspirator. DNA extraction was performed using TNE buffer method. Ten GeneLink-A series RAPD primers were used for PCR amplification and the data was analyzed through POPGENE. The number of amplification products produced per primer varied from 8-12, ranging from 200 to 2000 bp with an average of 10.0 bands per primer. The percentage of polymorphic loci amplified by each primer varied from 22.5 to 51%. The UPGMA dendrogram demonstrates two distinct groups from Faisalabad and Lahore populations. The genetic diversity ranged from 0.260 in Faisalabad to 0.294 in Lahore with a total heterozygosity of 0.379. The G ST value for nine populations within Lahore was 0.131 (Nm= 3.317), whereas for nine populations in Faisalabad G ST value was 0.117 (Nm= 3.773). The overall genetic variation among eighteen populations showed G ST = 0.341 and Nm= 1.966. The genetic relatedness and Nm value show that Ae . aegypti populations exhibit intra-population gene flow both in Faisalabad and Lahore. Although, both cities show a distinct pattern of genetic structure; however, few areas from both the cities show genetic similarity. The gene flow and the genetic relatedness in few populations of Lahore and Faisalabad cities need further investigation.

Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers.

PubMed

Goyal, Arvind Kumar; Pradhan, Sushen; Basistha, Bharat Chandra; Sen, Arnab

2015-08-01

Dendrocalamus strictus popularly known as 'Male bamboo' is a multipurpose bamboo which is extensively utilized in pharmaceutical, paper, agricultural and other industrial implements. In this study, in vitro regeneration of D. strictus through nodal culture has been attempted. Murashige and Skoog's medium supplemented with 4 mg/l BAP was found to be most effective in shoot regeneration with 3.68 ± 0.37 shoots per explant. The effect of Kn was found to be moderate. These hormones also had considerable effect on the shoot length. The highest shoot length after 6 weeks (3.11 ± 0.41 cm) was noted with 5 mg/l BAP followed by 3.07 ± 0.28 cm with 5 mg/l Kn, while decrease in the shoot length was noted with other treatments. The effect of IBA and NAA individually or in combination at different concentrations on rooting was evaluated. The highest number of root (1.36 ± 0.04) was regenerated on full-strength MS medium supplemented with 3 mg/l NAA, while maximum length of 1.64 ± 0.03 cm of roots was recorded with combination of 1 mg/l IBA and 3 mg/l NAA. Tissue-cultured plants thus obtained were successfully transferred to the soil. The clonal fidelity among the in vitro-regenerated plantlets was assessed by RAPD and ISSR markers. The ten RAPD decamers produced 58 amplicons, while nine ISSR primers generated a total of 66 bands. All the bands generated were monomorphic. These results confirmed the clonal fidelity of the tissue culture-raised D. strictus plantlets and corroborated the fact that nodal culture is perhaps the safest mode for multiplication of true to type plants.

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

PubMed Central

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Chorianopoulos, Nikos

2015-01-01

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry. PMID:26506345

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

PubMed

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

2015-10-22

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Assessment of genetic relationship in Persea spp by traditional molecular markers.

PubMed

Reyes-Alemán, J C; Valadez-Moctezuma, E; Barrientos-Priego, A F

2016-04-04

Currently, the reclassification of the genus Persea is under discussion with molecular techniques for DNA analysis representing an alternative for inter- and intra-specific differentiation. In the present study, the traditional random-amplified polymorphic DNA (RAPD) and the inter simple sequence repeat (ISSR) markers were used to determine the genomic relationship of different species and hybrids representative of the subgenera Eriodaphne and Persea in a population conserved in a germplasm bank. The data were analyzed statistically using multivariate methods. In the RAPD analysis, a total of 190 polymorphic bands were produced, with an average of 23.7 bands per primer, the percentage contribution of each primer was from 7.66 to 19.63; the polymorphic information content (PIC) ranged from 0.23 to 0.45, with an average of 0.35. In the ISSR analysis, a total of 111 polymorphic bands were considered, with an average of 18.5 bands per primer, the percentage contribution of each was from 11.83 to 19.57; the PIC ranged from 0.35 to 0.48, with an average of 0.42. The phenograms obtained in each technique showed the relationship among the accessions through the clusters formed. In general, both the techniques grouped representatives of the Persea americana races (P. americana var. drymifolia, P. americana var. guatemalensis, and P. americana var. americana). However, it was not possible to separate the species of Persea used as reference into independent clades. In addition, they tended to separate the representatives of subgenera Eriodaphne and Persea.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

PubMed

Abe, H; Seki, M; Ohbayashi, F; Tanaka, N; Yamashita, J; Fujii, T; Yokoyama, T; Takahashi, M; Banno, Y; Sahara, K; Yoshido, A; Ihara, J; Yasukochi, Y; Mita, K; Ajimura, M; Suzuki, M G; Oshiki, T; Shimada, T

2005-08-01

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Relative profile analysis of molecular markers for identification and genetic discrimination of loaches (Pisces, Nemacheilidae).

PubMed

Patil, Tejas Suresh; Tamboli, Asif Shabodin; Patil, Swapnil Mahadeo; Bhosale, Amrut Ravindra; Govindwar, Sanjay Prabhu; Muley, Dipak Vishwanathrao

2016-01-01

Genus Nemacheilus, Nemachilichthys and Schistura belong to the family Nemacheilidae of the order Cypriniformes. The present investigation was undertaken to observe genetic diversity, phylogenetic relationship and to develop a molecular-based tool for taxonomic identification. For this purpose, four different types of molecular markers were utilized in which 29 random amplified polymorphic DNA (RAPD), 25 inter-simple sequence repeat (ISSR) markers, and 10 amplified fragment length polymorphism (AFLP) marker sets were screened and mitochondrial COI gene was sequenced. This study added COI barcodes for the identification of Nemacheilus anguilla, Nemachilichthys rueppelli and Schistura denisoni. RAPD showed higher polymorphism (100%) than the ISSR (93.75-100%) and AFLP (93.86-98.96%). The polymorphic information content (PIC), heterozygosity, multiplex ratio, and gene diversity was observed highest for AFLP primers, whereas the major allele frequency was observed higher for RAPD (0.5556) and lowest for AFLP (0.1667). The COI region of all individuals was successfully amplified and sequenced, which gave a 100% species resolution. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Genetic diversity and environmental associations of sacsaoul ( Haloxylon ammodendron)

NASA Astrophysics Data System (ADS)

Zhang, Linjing; Zhao, Guifang; Yue, Ming; Pan, Xiaoling

2003-07-01

Random amplified polymorphic DNA (RAPD) markers were used to assess levels and patterns of genetic diversity in H. ammodendron (Chenopodiaceae). A total of 117 plants from 6 subpopulations on oasis-desert ecotone was analyzed by 16 arbitrarily chosen primers resulting in highly reproducible RAPD bands. The analysis of molecular variance (AMOVA) with distances among individuals showed that most of the variation (74%) occurred among individuals within subpopulations, which is expected for a crossing organism, and 26% of variation among subpopulations. Estimates of Shannon index and Nei"s index from allele frequencies corroborated AMOVA partitioning in H. ammodendron. UPGMA cluster analyses, based on genetic distance, do not revealed grouping of some geographically proximate populations. This is the first report of the partitioning of genetic variability within and between subpopulations of H. ammodendron and provides important baseline data for optimizing sampling strategies and for conserving the genetic resources of this species. The Percentage of polymorphic loci was as high as 96%, presumably being response to oasis-desert ecotone. There were gene flows (Nm=5.38 individuals/generation), based on gene differentiation coefficient (GST was 0.1567) between subpopulations, and strong habitat selection override the gene flow to maintain the subpopulation differentiation. Correlation analyses showed that there was significant relationship between genetic diversity and soil CL ion.

Phylogenetic analysis, genetic diversity and relationships between the recently segregated species of Corynandra and Cleoserrata from the genus Cleome using DNA barcoding and molecular markers.

PubMed

Tamboli, Asif Shabodin; Patil, Swapnil Mahadeo; Gholave, Avinash Ramchandra; Kadam, Suhas Kishor; Kotibhaskar, Shreya Vijaykumar; Yadav, Shrirang Ramchandra; Govindwar, Sanjay Prabhu

2016-01-01

Cleome is the largest genus in the family Cleomaceae and it is known for its various medicinal properties. Recently, some species from the Cleome genus (Cleome viscosa, Cleome chelidonii, Cleome felina and Cleome speciosa) are split into genera Corynandra (Corynandra viscosa, Corynandra chelidonii, Corynandra felina), and Cleoserrata (Cleoserrata speciosa). The objective of this study was to obtain DNA barcodes for these species for their accurate identification and determining phylogenetic relationships. Out of 10 screened barcoding regions, rbcL, matK and ITS1 regions showed higher PCR efficiency and sequencing success. This study added matK, rbcL and ITS1 barcodes for the identification of Corynandra chelidonii, Corynandra felina, Cleome simplicifolia and Cleome aspera species in existing barcode data. Corynandra chelidonii and Corynandra felina species belong to the Corynandra genus, but they are not grouped with the Corynandra viscosa species, however clustered with the Cleome species. Molecular marker analysis showed 100% polymorphism among the studied plant samples. Diversity indices for molecular markers were ranged from He=0.1115-0.1714 and I=0.2268-0.2700, which indicates a significant amount of genetic diversity among studied species. Discrimination of the Cleome and Corynandra species from Cleoserrata speciosa was obtained by two RAPD primers (OPA-4 and RAPD-17) and two ISSR primers (ISSR-1 and ISSR-2). RAPD and ISSR markers are useful for the genetic characterization of these studied species. The present investigation will be helpful to understand the relationships of Cleome lineages with Corynandra and Cleoserrata species. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

In vitro propagation and assessment of the genetic fidelity of Musa acuminata (AAA) cv. Vaibalhla derived from immature male flowers.

PubMed

Hrahsel, Lalremsiami; Basu, Adreeja; Sahoo, Lingaraj; Thangjam, Robert

2014-02-01

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog's (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L(-1) 6-benzylaminopurine (BAP) + 0.5 mg L(-1) α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L(-1) kinetin + 0.5 mg L(-1) NAA. While MS medium supplemented with a combination of 2 mg L(-1) BAP + 0.5 mg L(-1) NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96% survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.

Genetic diversity of improved salt tolerant calli of maize (Zea mays L.) using RAPD

NASA Astrophysics Data System (ADS)

Saputro, Triono Bagus; Dianawati, Siti; Sholihah, Nur Fadlillatus; Ermavitalini, Dini

2017-06-01

Maize is one of important cultivated plants in the world, in terms of production rates, utilization rates and demands. Unfortunately, the increment of demands were not followed by the increase of production rates since the cultivation area were significantly decrease. Coastal area is the marginal land that have a good potential to extend the cultivation area. The main challenge of this area is the high content of salt. The aims of this research were try to induce a new varian of local maize through in vitro culture and observe its genetic variation using RAPD. Bluto variety from Madura island was used as an explant in callus induction. Induction of callus were conducted using MS basal medium supplemented with 3 mg/L of 2,4 D under dark condition. While the selection stage was conducted using MS basal medium supplemented with 3 mg/L of 2,4 D with the addition of various concentration of NaCl (0 mg/L; 2500 mg/L; 5000 mg/L; and 7500 mg/L). The research were arranged in a completely randomized design with three replications. The exposion of NaCl were significantly decrease the mass of maize callus. The highest addition of callus weight was 210 mgs in control treatment, while the lowest is in 7500 mg/L with 3 mgs. The RAPD technique was utilized to characterize the genotype of maize callus. Out of five primers, only three primers can produce polymorphic bands named OPA10, OPB07 and OPC02. Taken together, the surviving callus of Bluto varians can be further developed as potential somaclone that has high tolerance to salt stress.

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

PubMed Central

Arias, Covadonga R.; Pujalte, María Jesús; Garay, Esperanza; Aznar, Rosa

1998-01-01

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates. PMID:9726889

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

PubMed Central

2012-01-01

Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L.

PubMed Central

Gong, L.; Stift, G.; Kofler, R.; Pachner, M.

2008-01-01

Until recently, only a few microsatellites have been available for Cucurbita, thus their development is highly desirable. The Austrian oil-pumpkin variety Gleisdorfer Ölkürbis (C. pepo subsp. pepo) and the C. moschata cultivar Soler (Puerto Rico) were used for SSR development. SSR-enriched partial genomic libraries were established and 2,400 clones were sequenced. Of these 1,058 (44%) contained an SSR at least four repeats long. Primers were designed for 532 SSRs; 500 primer pairs produced fragments of expected size. Of these, 405 (81%) amplified polymorphic fragments in a set of 12 genotypes: three C. moschata, one C. ecuadorensis, and eight C. pepo representing all eight cultivar groups. On an average, C. pepo and C. moschata produced 3.3 alleles per primer pair, showing high inter-species transferability. There were 187 SSR markers detecting polymorphism between the USA oil-pumpkin variety “Lady Godiva” (O5) and the Italian crookneck variety “Bianco Friulano” (CN), which are the parents of our previous F2 mapping population. It has been used to construct the first published C. pepo map, containing mainly RAPD and AFLP markers. Now the updated map comprises 178 SSRs, 244 AFLPs, 230 RAPDs, five SCARs, and two morphological traits (h and B). It contains 20 linkage groups with a map density of 2.9 cM. The observed genome coverage (Co) is 86.8%. Electronic supplementary material The online version of this article (doi:10.1007/s00122-008-0750-2) contains supplementary material, which is available to authorized users. PMID:18379753

RAPD-PCR and real-time PCR HRM based genetic variation evaluations of Urtica dioica parts, ecotypes and evaluations of morphotypes in Turkey.

PubMed

Uzonur, Irem; Akdeniz, Gamze; Katmer, Zeynep; Ersoy, Seyda Karaman

2013-01-01

Urtica dioica is an ethnobotanically and medicinally important Complementary and Alternative Medicine (CAM) plant worldwide and in Turkey; 90 % of herbal CAM applications depend on it in Turkey. It has a wide range of habitats in nearly all continents. It is found in all three phytogeographical regions in Turkey (Euro-Siberian, Irano-Turanian, Mediterranean) with high adaptivity to heterogeneous geographies such as climate, soil types and altitudes. This fact in relation to the assessment of chemical constituents of the plant and combining with further genetic and morphological variation data can assist and enhance the works for the utility and reliability of CAM applications in effect and activity of this plant species. In this work we have made some preliminary experiments with novel approaches to reveal the ecotypes and genetic variation of mighty ecotypes of Urtica dioica from different phytogeographical regions of Turkey (Euro-Siberian and Mediterranean). The ecotypes have heterogeneity in both its parts (leaf, stem, root) as revealed by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using random primers and High-resolution Melt (HRM) analysis using Urtica dioica specific primers and universal chloroplast DNA (cpDNA) primers and morphological traits such as phenolic contents and antioxidant capacities of plants' leaf infusions as used in medicinal applications in Turkey. This work will contribute a lot for the development of molecular markers to detect the genetic variation and heterogeneity of Urtica dioica to further relate with expected phenotypes that are most useful and relevant in CAM applications.

Characterisation of colletotrichum species associated with anthracnose of banana.

PubMed

Zakaria, Latiffah; Sahak, Shamsiah; Zakaria, Maziah; Salleh, Baharuddin

2009-12-01

A total of 13 Colletotrichum isolates were obtained from different banana cultivars (Musa spp.) with symptoms of anthracnose. Colletotrichum isolates from anthracnose of guava (Psidium guajava) and water apple (Syzygium aqueum) were also included in this study. Based on cultural and morphological characteristics, isolates from banana and guava were identified as Colletotrichum musae and from water apple as Colletotrichum gloeosporiodes. Isolates of C. musae from banana and guava had similar banding patterns in a randomly amplified polymorphic DNA (RAPD) analysis with four random primers, and they clustered together in a UPGMA analysis. C. gloeosporiodes from water apple was clustered in a separate cluster. Based on the present study, C. musae was frequently isolated from anthracnose of different banana cultivars and the RAPD banding patterns of C. musae isolates were highly similar but showed intraspecific variations.

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

PubMed

Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Characterisation of Colletotrichum Species Associated with Anthracnose of Banana

PubMed Central

Zakaria, Latiffah; Sahak, Shamsiah; Zakaria, Maziah; Salleh, Baharuddin

2009-01-01

A total of 13 Colletotrichum isolates were obtained from different banana cultivars (Musa spp.) with symptoms of anthracnose. Colletotrichum isolates from anthracnose of guava (Psidium guajava) and water apple (Syzygium aqueum) were also included in this study. Based on cultural and morphological characteristics, isolates from banana and guava were identified as Colletotrichum musae and from water apple as Colletotrichum gloeosporiodes. Isolates of C. musae from banana and guava had similar banding patterns in a randomly amplified polymorphic DNA (RAPD) analysis with four random primers, and they clustered together in a UPGMA analysis. C. gloeosporiodes from water apple was clustered in a separate cluster. Based on the present study, C. musae was frequently isolated from anthracnose of different banana cultivars and the RAPD banding patterns of C. musae isolates were highly similar but showed intraspecific variations. PMID:24575184

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

PubMed

Dhawan, S S; Rai, G K; Darokar, M P; Lal, R K; Misra, H O; Khanuja, S P S

2011-09-15

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.

Effect of Pollination Timing on the Rate of Apomictic Reproduction Revealed by RAPD Markers in Paspalum notatum

PubMed Central

ESPINOZA, F.; PESSINO, S. C.; QUARÍN, C. L.; VALLE, E. M.

2002-01-01

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1–3 d pre‐anthesis; at anthesis; and 2, 4 and 6 d post‐anthesis. Cytoembryological analyses indicated that approx. 17 % of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety‐five oligonucleotides were assayed on the progenitors in order to search for male‐specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3·4 % before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off‐type plants (16·5 %), of which only one was a BIII hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed. PMID:12099347

Introgression of wheat DNA markers from A, B and D genomes in early generation progeny of Aegilops cylindrica Host x Triticum aestivum L. hybrids.

PubMed

Schoenenberger, N; Felber, F; Savova-Bianchi, D; Guadagnuolo, R

2005-11-01

Introgression from allohexaploid wheat (Triticum aestivum L., AABBDD) to allotetraploid jointed goatgrass (Aegilops cylindrica Host, CCDD) can take place in areas where the two species grow in sympatry and hybridize. Wheat and Ae. cylindrica share the D genome, issued from the common diploid ancestor Aegilops tauschii Coss. It has been proposed that the A and B genome of bread wheat are secure places to insert transgenes to avoid their introgression into Ae. cylindrica because during meiosis in pentaploid hybrids, A and B genome chromosomes form univalents and tend to be eliminated whereas recombination takes place only in D genome chromosomes. Wheat random amplified polymorphic DNA (RAPD) fragments, detected in intergeneric hybrids and introgressed to the first backcross generation with Ae. cylindrica as the recurrent parent and having a euploid Ae. cylindrica chromosome number or one supernumerary chromosome, were assigned to wheat chromosomes using Chinese Spring nulli-tetrasomic wheat lines. Introgressed fragments were not limited to the D genome of wheat, but specific fragments of A and B genomes were also present in the BC1. Their presence indicates that DNA from any of the wheat genomes can introgress into Ae. cylindrica. Successfully located RAPD fragments were then converted into highly specific and easy-to-use sequence characterised amplified regions (SCARs) through sequencing and primer design. Subsequently these markers were used to characterise introgression of wheat DNA into a BC1S1 family. Implications for risk assessment of genetically modified wheat are discussed.

Genetic diversity study of Chromobacterium violaceum isolated from Kolli Hills by amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD).

PubMed

Ponnusamy, K; Jose, S; Savarimuthu, I; Michael, G P; Redenbach, M

2011-09-01

Chromobacterium are saprophytes that cause highly fatal opportunistic infections. Identification and strain differentiation were performed to identify the strain variability among the environmental samples. We have evaluated the suitability of individual and combined methods to detect the strain variations of the samples collected in different seasons. Amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA (RAPD) profiles were obtained using four different restriction enzyme digestions (AluI, HaeIII, MspI and RsaI) and five random primers. A matrix of dice similarity coefficients was calculated and used to compare these restriction patterns. ARDRA showed rapid differentiation of strains based on 16S rDNA, but the combined RAPD and ARDRA gave a more reliable differentiation than when either of them was analysed individually. A high level of genetic diversity was observed, which indicates that the Kolli Hills' C. violaceum isolates would fall into at least three new clusters. Results showed a noteworthy bacterial variation and genetic diversity of C. violaceum in the unexplored, virgin forest area. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Conformation of phylogenetic relationship of Penaeidae shrimp based on morphometric and molecular investigations.

PubMed

Rajakumaran, P; Vaseeharan, B; Jayakumar, R; Chidambara, R

2014-01-01

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly amplified polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric analysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable.

Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin.

PubMed

Srůtková, Dagmar; Spanova, Alena; Spano, Miroslav; Dráb, Vladimír; Schwarzer, Martin; Kozaková, Hana; Rittich, Bohuslav

2011-10-01

Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)(5) primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular characterization of Fasciola species isolated from imported sheep in Taif region (Saudi Arabia).

PubMed

Shalaby, I; Gherbawy, Y; Banaja, A

2013-03-01

Accurate identification of Fasciola species, followed by biological and ecological characterization, is important with concern to the planning for field control. Because there are many variations in morphological characteristics, exact distinguishing of Fasciola species is usually difficult by simple traditional microscopic measurements and, therefore, the morphometric characterization may be insufficient for the species identification. Hence, the present work was proposed to collect 100 liver samples from 100 imported sheep from Sudan from slaughterhouses in Taif region. The samples were firstly examined macroscopically and microscopically to ensure the presence or absence of infection. The collected worms were subjected for RAPD-PCR analysis using different primers and ITS1 sequences for accurate identification. Using RAPD-PCR analysis, two primers were selected to amplify the DNA of each Fasciola. The results show that the amplification fragments were between 500 and 1500 bp and, the use of random genetic markers allowed to discriminate among the different collected species. Using Internal transcribed spacer region (ITS) sequencing, the imported sheep in Taif region consisted of 630 bps including complete ITS1, partial 18S and 5.8S and had 5 variable nucleotide positions. This is the first demonstration of the existence of both F. hepatica, F. gigantica and hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the imported sheep in Saudi Arabia by a genetic approach.

[Establishment of multiple regression model for virulence factors of Saccharomyces albicans by random amplified polymorphic DNA bands].

PubMed

Liu, Qi; Wu, Youcong; Yuan, Youhua; Bai, Li; Niu, Kun

2011-12-01

To research the relationship between the virulence factors of Saccharomyces albicans (S. albicans) and the random amplified polymorphic DNA (RAPD) bands of them, and establish the regression model by multiple regression analysis. Extracellular phospholipase, secreted proteinase, ability to generate germ tubes and adhere to oral mucosal cells of 92 strains of S. albicans were measured in vitro; RAPD-polymerase chain reaction (RAPD-PCR) was used to get their bands. Multiple regression for virulence factors of S. albicans and RAPD-PCR bands was established. The extracellular phospholipase activity was associated with 4 RAPD bands: 350, 450, 650 and 1 300 bp (P < 0.05); secreted proteinase activity of S. albicans was associated with 2 bands: 350 and 1 200 bp (P < 0.05); the ability of germ tube produce was associated with 2 bands: 400 and 550 bp (P < 0.05). Some RAPD bands will reflect the virulence factors of S. albicans indirectly. These bands would contain some important messages for regulation of S. albicans virulence factors.

Genotoxicity of monosodium glutamate.

PubMed

Ataseven, Nazmiye; Yüzbaşıoğlu, Deniz; Keskin, Ayten Çelebi; Ünal, Fatma

2016-05-01

Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro. Copyright © 2016. Published by Elsevier Ltd.

Molecular and biochemical studies on the effect of gamma rays on lead toxicity in cowpea (Vigna sinensis) plants.

PubMed

Mohamed, Heba Ibrahim

2011-12-01

The effect of lead acetate in the presence or absence of cowpea seeds irradiated with gamma rays on morphological criteria, protein electrophoresis, isozymes, and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) of leaves was investigated. A highly significant decrease in shoot and root length was observed upon lead acetate exposure (300 and 600 μM). On the other hand, in seeds irradiated with gamma rays (2, 5, and 8 krad), these morphological parameters were increased after lead acetate treatments. Meanwhile, all treatments (lead acetate and gamma rays) caused variations in number, intensity, and/or density of SDS electrophoretic bands of proteins. In addition, electrophoretic studies of esterase, acid phosphatase, peroxidase, polyphenol oxidase, catalase, and superoxide dismutase isozyme activities were increased with increasing the concentrations of lead acetate and gamma ray doses. The variation in DNA profile in response to lead acetate and gamma irradiation treatments was detected by RAPD-PCR technique. The result of RAPD analysis using the five primers indicated the appearance and disappearance of DNA polymorphic bands at all treatments (gamma rays and lead stress). The relatively high concentrations of lead acetate (600 μM) induced more changes in genomic DNA pattern.

Development of SCAR markers for sex determination in the dioecious shrub Aucuba japonica (Cornaceae).

PubMed

Maki, Masayuki

2009-03-01

Two sex-linked fragments were identified by RAPD analyses in the dioecious diploid shrub Aucuba japonica var. ovoidea and were converted into markers of male-specific sequence characterized amplified region (SCAR) markers. PCRs using the primers designed in this study correctly discriminated 24 flowering males and 24 flowering females at higher annealing temperatures (SCAR markers OPA10-424 at 55 degrees C and OPN11-1095 at 65 degrees C), although at relatively low annealing temperatures, the fragments were amplified in both males and females. These SCAR primers were also tested to see whether they were applicable to sex identification in the conspecific tetraploid Aucuba japonica var. japonica. One set pf SCAR primers could be used for sex identification even in this tetraploid variety, although the other failed. The SCAR markers developed in this study will provide a powerful tool in identifying the sex of immature plants of dioecious A. japonica, which is a commercially valuable shrub due to its conspicuous fruits.

Evidence of sibling species in the brown planthopper complex (Nilaparvata lugens) detected from short and long primer random amplified polymorphic DNA fingerprints.

PubMed

Latif, M A; Soon Guan, Tan; Mohd Yusoh, Omar; Siraj, Siti Shapor

2008-08-01

The inheritance of 31 amplicons from short and long primer RAPD was tested for segregating ratios in two families of the brown planthopper, Nilaparvata lugens, and they were found to be inherited in a simple Mendelian fashion. These markers could now be used in population genetics studies of N. lugens. Ten populations of N. lugens were collected from five locations in Malaysia. Each location had two sympatric populations. Cluster and principal coordinate analyses based on genetic distance along with AMOVA revealed that the rice-infesting populations (with high esterase activity) at five localities clustered together as a group, and Leersia-infesting populations (with low esterase activity) at the same localities formed another distinct cluster. Two amplicons from primers OPD03 (0.65 kb) and peh#6 (1.0 kb) could be considered diagnostic bands, which were fixed in the Leersia-infesting populations. These results represent evidence of a sibling species in the N. lugens complex.

Identification of Species Related to Anopheles (Nyssorhynchus) albitarsis by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (Diptera: Culicidae)

DTIC Science & Technology

1995-11-01

Instituto de Biologia do ExCrcito, Rua Francisco Manuel 102, 2091 l-270 Rio de Janeiro, RJ, Brasil Species-specific Random Amplified Polymorphic DNA...da Panela Manaus Ilha Comprida 6 km SW Registro Ponte Melo Peixoto Capanema Ilha de Marajo Santa Helena nr. Guaira Aguia Branca Rio Socuavo...Brazil; 11, Ponte Melo Peixoto, Brazil. Fig. 3: RAPD amplifications of Albitarsis Complex species A with primer B05. Arrow on left indicates fragment

Application of SCAR (sequence characterized amplified region) analysis to authenticate Lycium barbarum (wolfberry) and its adulterants.

PubMed

Sze, Stephen Cho-Wing; Song, Ju-Xian; Wong, Ricky Ngok-Shun; Feng, Yi-Bin; Ng, Tzi-Bun; Tong, Yao; Zhang, Kalin Yan-Bo

2008-09-01

Fructus Lycii (Gouqizi) is well known in Chinese herbal medicine for its restorative function of benefiting the liver and kidney, replenishing vital essence and improving eyesight. However, ten species and varieties of Lycium have benn found to be substitutes or adulterants of Lycium barbarum (wolfberry) in commercial markets in the Hong Kong Special Administrative Region and in China generally. L. barbarum cv. 'Tianjinense' and Lycium chinense var. potaninii are the most common examples. It is difficult to differentiate among the Lycium species by traditional morphological and histological analyses. An easy and reliable approach based on SCAR (sequence characterized amplified region) analysis was developed in the present study to differentiate L. barbarum from other Lycium species. Two characteristic bands of approx. 700 and 650 bp were detected on the RAPD (random amplification of polymorphic DNA) profiles generated from samples of L. barbarum and L. chinense var. potaninii using the primer OPC-7. They were isolated and sequenced. Two primer sets, based on the sequences, could amplify a single specific band in samples of L. barbarum respectively, whereas no bands were detected in samples of L. chinense var. potaninii. The results confirmed that the SCAR technique can be employed for authenticating L. barbarum and its adulterants.

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Soil Viral Communities Vary Temporally and along a Land Use Transect as Revealed by Virus-Like Particle Counting and a Modified Community Fingerprinting Approach (fRAPD)

PubMed Central

Narr, Anja; Nawaz, Ali; Wick, Lukas Y.; Harms, Hauke; Chatzinotas, Antonis

2017-01-01

Environmental surveys on soil viruses are still rare and mostly anecdotal, i. e., they mostly report on viruses at one location or for only a few sampling dates. Detailed time-series analysis with multiple samples can reveal the spatio-temporal dynamics of viral communities and provide important input as to how viruses interact with their potential hosts and the environment. Such surveys, however, require fast, easy-to-apply and reliable methods. In the present study we surveyed monthly across 13 months the abundance of virus-like particles (VLP) and the structure of the viral communities in soils along a land use transect (i.e., forest, pasture, and cropland). We evaluated 32 procedures to extract VLP from soil using different buffers and mechanical methods. The most efficient extraction was achieved with 1× saline magnesium buffer in combination with 20 min vortexing. For community structure analysis we developed an optimized fingerprinting approach (fluorescent RAPD-PCR; fRAPD) by combining RAPD-PCR with fluorescently labeled primers in order to size the obtained fragments on a capillary sequencing machine. With the concomitantly collected data of soil specific factors and weather data, we were able to find correlations of viral abundance and community structure with environmental variables and sampling site. More specifically, we found that soil specific factors such as pH and total nitrogen content played a significant role in shaping both soil viral abundance and community structure. The fRAPD analysis revealed high temporal changes and clustered the viral communities according to sampling sites. In particular we observed that temperature and rainfall shaped soil viral communities in non-forest sites. In summary our findings suggest that sampling site was a key factor for shaping the abundance and community structure of soil viruses, and when site vegetation was reduced, temperature and rainfall were also important factors. PMID:29067022

Comparison of DNA-based techniques for differentiation of production strains of ale and lager brewing yeast.

PubMed

Kopecká, J; Němec, M; Matoulková, D

2016-06-01

Brewing yeasts are classified into two species-Saccharomyces pastorianus and Saccharomyces cerevisiae. Most of the brewing yeast strains are natural interspecies hybrids typically polyploids and their identification is thus often difficult giving heterogenous results according to the method used. We performed genetic characterization of a set of the brewing yeast strains coming from several yeast culture collections by combination of various DNA-based techniques. The aim of this study was to select a method for species-specific identification of yeast and discrimination of yeast strains according to their technological classification. A group of 40 yeast strains were characterized using PCR-RFLP analysis of ITS-5·8S, NTS, HIS4 and COX2 genes, multiplex PCR, RAPD-PCR of genomic DNA, mtDNA-RFLP and electrophoretic karyotyping. Reliable differentiation of yeast to the species level was achieved by PCR-RFLP of HIS4 gene. Numerical analysis of the obtained RAPD-fingerprints and karyotype revealed species-specific clustering corresponding with the technological classification of the strains. Taxonomic position and partial hybrid nature of strains were verified by multiplex PCR. Differentiation among species using the PCR-RFLP of ITS-5·8S and NTS region was shown to be unreliable. Karyotyping and RFLP of mitochondrial DNA evinced small inaccuracies in strain categorization. PCR-RFLP of HIS4 gene and RAPD-PCR of genomic DNA are reliable and suitable methods for fast identification of yeast strains. RAPD-PCR with primer 21 is a fast and reliable method applicable for differentiation of brewing yeasts with only 35% similarity of fingerprint profile between the two main technological groups (ale and lager) of brewing strains. It was proved that PCR-RFLP method of HIS4 gene enables precise discrimination among three technologically important Saccharomyces species. Differentiation of brewing yeast to the strain level can be achieved using the RAPD-PCR technique. © 2016 The Society for Applied Microbiology.

Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize.

PubMed

Couillerot, O; Poirier, M-A; Prigent-Combaret, C; Mavingui, P; Caballero-Mellado, J; Moënne-Loccoz, Y

2010-08-01

To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 x 10(3) (for UAP-154) and 4 x 10(4) CFU g(-1) (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 10(4) to 10(8) CFU g(-1) soil. BOX-based SCAR markers were useful to find primers for strain-specific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535.

Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers.

PubMed

Abbas, Ghulam; Hameed, Amjad; Rizwan, Muhammad; Ahsan, Muhammad; Asghar, Muhammad J; Iqbal, Nayyer

2015-01-01

Molecular confirmation of interspecific recombinants is essential to overcome the issues like self-pollination, environmental influence, and inadequacy of morphological characteristics during interspecific hybridization. The present study was conducted for genetic confirmation of mungbean (female) and mashbean (male) interspecific crosses using molecular markers. Initially, polymorphic random amplified polymorphic DNA (RAPD), universal rice primers (URP), and simple sequence repeats (SSR) markers differentiating parent genotypes were identified. Recombination in hybrids was confirmed using these polymorphic DNA markers. The NM 2006 × Mash 88 was most successful interspecific cross. Most of true recombinants confirmed by molecular markers were from this cross combination. SSR markers were efficient in detecting genetic variability and recombination with reference to specific chromosomes and particular loci. SSR (RIS) and RAPD identified variability dispersed throughout the genome. In conclusion, DNA based marker assisted selection (MAS) efficiently confirmed the interspecific recombinants. The results provided evidence that MAS can enhance the authenticity of selection in mungbean improvement program.

Assignment of sockeye salmon (Oncorhynchus nerka) to spawning sites using DNA markers.

PubMed

Corley-Smith, Graham E; Wennerberg, Liv; Schembri, Joy A; Lim, Chinten J; Cooper, Karen L; Brandhorst, Bruce P

2005-01-01

Randomly amplified polymorphic DNA (RAPD) markers were used to assign individual adult sockeye salmon to their spawning sites using a genotype assignment test. Six primers were selected for use by screening bulked DNA samples for markers missing in fish from one or more of 5 sites in British Columbia or Alaska. Of 73 markers scored, 54 showed variation between or within sites among the sampled fish. Thirty-seven of the variable markers were not detected in any fish from one or more sites; 18 variable markers were detected in all fish from one or more other sites. Thus 25% of markers scored were found in all fish of some sites and in no fish of some other sites. An assignment test placed all 70 fish tested into their correct populations. Principal coordinate analysis of genetic variation produced clusters of fish corresponding to each sampling site. No sex-specific RAPD markers were detected among more than 1300 screened.

Potential use of random amplified polymorphic DNA (RAPD) technique to study the genetic diversity in Indian mustard (Brassica juncea) and its relationship to heterosis.

PubMed

Jain, A; Bhatia, S; Banga, S S; Prakash, S; Lakshmikumaran, M

1994-04-01

RAPD assays were performed, using 34 arbitrary decamer oligonucleotide primers and six combinations of two primers, to detect inherent variations and genetic relationships among 12 Indian and 11 exotic B. juncea genotypes. Of 595 amplification products identified, 500 of them were polymorphic across all genotypes. A low level of genetic variability was detected among the Indian genotypes, while considerable polymorphism was present among the exotic ones. Based on the pair-wise comparisons of amplification products the genetic similarity was calculated using Jaccard's similarity coefficients and a dendrogram was constructed using an unweighted pair group method was arithmetical averages (UPGMA). On the basis of this analysis the genotypes were clustered into two groups, A and B. Group A comprised only exotic genotypes, whereas all the Indian genotypes and four of the exotic genotypes were clustered in group B. Almost similar genotypic rankings could also be established by computing as few as 200 amplification products. In general, a high per cent of heterosis was recorded in crosses involving Indian x exotic genotypes. On the other hand, when crosses were made amongst Indian or exotic genotypes, about 80% of them exhibited negative heterosis. Results from this study indicate that, despite the lack of direct correlation between the genetic distance and the degree of heterosis, genetic diversity forms a very useful guide not only for investigating the relationships among Brassica genotypes but also in the selection of parents for heterotic hybrid combinations.

Genetic characterization of fig tree mutants with molecular markers.

PubMed

Rodrigues, M G F; Martins, A B G; Desidério, J A; Bertoni, B W; Alves, M C

2012-08-06

The fig (Ficus carica L.) is a fruit tree of great world importance and, therefore, the genetic improvement becomes an important field of research for better crops, being necessary to gather information on this species, mainly regarding its genetic variability so that appropriate propagation projects and management are made. The improvement programs of fig trees using conventional procedures in order to obtain new cultivars are rare in many countries, such as Brazil, especially due to the little genetic variability and to the difficulties in obtaining plants from gamete fusion once the wasp Blastophaga psenes, responsible for the natural pollinating, is not found in Brazil. In this way, the mutagenic genetic improvement becomes a solution of it. For this reason, in an experiment conducted earlier, fig plants formed by cuttings treated with gamma ray were selected based on their agronomic characteristics of interest. We determined the genetic variability in these fig tree selections, using RAPD and AFLP molecular markers, comparing them to each other and to the Roxo-de-Valinhos, used as the standard. For the reactions of DNA amplification, 140 RAPD primers and 12 primer combinations for AFLP analysis were used. The selections did not differ genetically between themselves and between them and the Roxo-de-Valinhos cultivar. Techniques that can detect polymorphism between treatments, such as DNA sequencing, must be tested. The phenotypic variation of plants may be due to epigenetic variation, necessitating the use of techniques with methylation-sensitive restriction enzymes.

Freshwater toxic cyanobacteria induced DNA damage in apple (Malus pumila), rape (Brassica napus) and rice (Oryza sativa).

PubMed

Chen, J Z; Ye, J Y; Zhang, H Y; Jiang, X J; Zhang, Y X; Liu, Z L

2011-06-15

Cyanobacteria in freshwater ecosystems can present a harmful effect on growth and development of plants through irrigation with contaminated water. In this study, the effects of microcystins (MCs)-containing cyanobacteria extract (CE) on DNA damage of apple, rape and rice were investigated to explore the phytotoxic mechanism of MCs through DNA fragmentation and RAPD analysis. Determination of DNA fragmentation by fluorescent dye DAPI showed that significant DNA damage was observed in rice seedlings after exposure to CE while DNA fragmentation in rape seedlings and apple cultures did not differ significantly between treatment and control groups. Qualitative characterization of genomic DNA fragmentation by agarose gel electrophoresis supported the quantitative determination using DAPI. The main changes in RAPD profiles of rape seedlings following exposure of lower doses of CE were variation in band intensity for the primers F03 and S01, while higher doses of CE caused loss of normal bands and appearance of new bands except band intensity changes. The data presented here demonstrate that DNA damage in plants occurs following exposure of microcystins, and the polymorphic RAPDs may be used as an investigation tool for environmental toxicology and as a useful biomarker for the detection of genotoxic effects of microcystins on plants. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Molecular characterization of Candida isolates from intensive care unit patients, Krakow, Poland.

PubMed

Małek, Marianna; Paluchowska, Paulina; Bogusz, Bożena; Budak, Alicja

Over the last decades, Candida species have emerged as important pathogens in immunocompromised patients. Nosocomial infections are mainly of endogenous origin. Nevertheless, some cases of exogenous candidiasis have also been reported. The aim of this study was to evaluate the genetic relatedness between Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida kefyr isolates recovered from intensive care unit (ICU) patients. A total of 132 Candida clinical isolates (62 C. albicans, 40 C. glabrata, 13 C. tropicalis, 11 C. krusei, 6 C. kefyr), obtained from specimens of endotracheal aspirate, urine and blood taken from patients of a tertiary hospital in Poland, were included in the study. Species identification was performed by PCR method and genetic relatedness was assessed by randomly amplified polymorphic DNA assay (RAPD) with five primers. The RAPD analysis revealed high genetic diversity among the studied Candida isolates, indicating that most of the strains were from endogenous sources. Only two clonal strains of C. glabrata isolated from different patients were observed, suggesting a possible cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD assay. This genotyping method can be applied to local epidemiological studies of Candida species. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Genetic Relatedness of Salmonella Serovars Isolated from Catfish (Clarias gariepinus) and Tilapia (Tilapia mossambica) Obtained from Wet Markets and Ponds in Penang, Malaysia.

PubMed

Budiati, Titik; Rusul, Gulam; Wan-Abdullah, Wan Nadiah; Chuah, Li-Oon; Ahmad, Rosma; Thong, Kwai Lin

2016-04-01

A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovismorbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.

IDENTIFICATION OF SEX CHROMOSOME MOLECULAR MARKERS USING RAPDS AND FLUORESCENT IN SITU HYBRIDIZATION IN RAINBOW TROUT

EPA Science Inventory

The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphi...

Genetic diversity and relatedness within and between species of the genus Oligoryzomys (Rodentia; Sigmodontinae).

PubMed

Trott, A; Callegari-Jacques, S M; Oliveira, L F B; Langguth, A; Mattevi, M S

2007-02-01

A RAPD analysis on six species of the rodent genus Oligoryzomys trapped in a wide area (ranging from 01 degrees N to 32 degrees S) of Brazilian territory was performed in order to determine the levels of genetic variability within and between its populations and species. One-hundred and ninety-three animals were collected in 13 different sites (corresponding to 17 samples) located at Pampas, Atlantic Rain Forest, Cerrado, and Amazon domains. Oligoryzomys sp., O. nigripes (8 populations), O. flavescens (4 populations), O. moojeni, O. stramineus, and O. fornesi were the taxa analyzed. Of the 20 primers tested, 4 generated a total of 75 polymorphic products simultaneously amplified in 151 specimens. Various diversity estimators analyzed showed considerable differences between species and populations, indicating a great genetic variation occurring in the Oligoryzomys taxa investigated. A cluster analysis was made using Nei's standard genetic distances, however, it did not correlate the genetic heterogeneity of the species and populations with the geographical areas.

A survey on distribution and toxigenicity of Aspergillus flavus from indoor and outdoor hospital environments.

PubMed

Sepahvand, Asghar; Shams-Ghahfarokhi, Masoomeh; Allameh, Abdolamir; Jahanshiri, Zahra; Jamali, Mojdeh; Razzaghi-Abyaneh, Mehdi

2011-11-01

In the present study, genetic diversity and mycotoxin profiles of Aspergillus flavus isolated from air (indoors and outdoors), levels (surfaces), and soils of five hospitals in Southwest Iran were examined. From a total of 146 Aspergillus colonies, 63 isolates were finally identified as A. flavus by a combination of colony morphology, microscopic criteria, and mycotoxin profiles. No Aspergillus parasiticus was isolated from examined samples. Chromatographic analyses of A. flavus isolates cultured on yeast extract-sucrose broth by tip culture method showed that approximately 10% and 45% of the isolates were able to produce aflatoxin B(1) (AFB(1)) and cyclopiazonic acid (CPA), respectively. Around 40% of the isolates produced sclerotia on Czapek-Dox agar. The isolates were classified into four chemotypes based on the ability to produce AF and CPA that majority of them (55.5%) belonged to chemotype IV comprising non-mycotoxigenic isolates. Random amplified polymorphic DNA (RAPD) profiles generated by a combination of four selected primers were used to assess genetic relatedness of 16 selected toxigenic and non-toxigenic isolates. The resulting dendrogram demonstrated the formation of two separate clusters for the A. flavus comprised both mycotoxigenic and non-toxigenic isolates in a random distribution. The obtained results in this study showed that RAPD profiling is a promising and efficient tool to determine intra-specific genetic variation among A. flavus populations from hospital environments. A. flavus isolates, either toxigenic or non-toxigenic, should be considered as potential threats for hospitalized patients due to their obvious role in the etiology of nosocomial aspergillosis.

Further genetic characterization of the two Trypanosoma cruzi Berenice strains (Be-62 and Be-78) isolated from the first human case of Chagas disease (Chagas, 1909).

PubMed

Cruz, R E; Macedo, A M; Barnabé, C; Freitas, J M; Chiari, E; Veloso, V M; Carneiro, C M; Bahia, M T; Tafuri, Washington L; Lana, M

2006-03-01

We describe here an extension of a previous genetic characterization of Trypanosoma cruzi strains (Be-62 and Be-78) isolated from the patient Berenice, the first human case of Chagas disease [Chagas, C., 1909. Nova Tripanomíase humana. Estudos sobre morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., n. sp., agente etiolójico da nova entidade morbida do homem. Mem. Inst. Oswaldo Cruz 1, 159-218]. We wanted to verify the composition of T. cruzi populations originated from these two isolates. In the present work, 22 enzymatic loci (MLEE), nine RAPD primers and 7 microsatellite loci were analyzed. Clones from both strains were also characterized to verify whether these strains are mono or polyclonal. Be-62 and Be-78 strains were different in 3 out of 22 enzymatic systems, in 3 out of 9 RAPD primers tested and in all microsatellite loci investigated. However, our data suggests that both strains are phylogenetically closely related, belonging to genetic group 32 from Tibayrenc and Ayala [Tibayrenc, M., Ayala, F.J., 1988. Isoenzime variability in Trypanosoma cruzi, the agent of Chagas' disease: genetical, taxonomical, and epidemiological significance. Evolution 42, 277-292], equivalent to zymodeme 2 and T. cruzi II major lineage which, in Brazil, comprises parasites from the domestic cycle of the disease. Microsatellite analyses showed differences between the parental strains but suggested that both populations are monoclonal since each strain and their respective clones showed the same amplification products.

Quizalofop-p-ethyl-induced phytotoxicity and genotoxicity in Lemna minor and Lemna gibba.

PubMed

Doganlar, Zeynep B

2012-01-01

In this study, the effects of the herbicide, quizalofop-p-ethyl, on pigment contents (total chlorophyll, chlorophyll a/b, carotenoid), antioxidant enzyme [superoxide dismutase (SOD) and guaiacol peroxidase (POD)] activities, lipid peroxidation product (malondialdehyde: MDA) and DNA profiles were investigated in Lemna gibba and Lemna minor. Laboratory-acclimatized plants were treated with quizalofop-p-ethyl at 31.375, 62.75, 125 and 250 mg L(-1) for 24 and 96 h. It was determined that quizalofop-p-ethyl affected both the physiological status and the DNA profiles of L. gibba and L. minor. The photosynthetic pigments of L. gibba were more sensitive to the herbicide than were those of L. minor at both treatment times. SOD and POD activities were elevated in both plants at 24 h. However at 96 h, SOD activity decreased in L. minor and had irregular changes in L. gibba.. Significant increases in the amounts of MDA were observed in L. gibba, whereas the levels of this compound decreased in L. minor at 24 and 96 h. Polymorphism in DNA profiles was determined using the Random Amplified Polymorphic DNA (RAPD) technique. Four primers were used for scoring (appearance and disappearance of DNA polymorphic bands), and equally weighted maximum parsimony analyses were performed. Fewer differences were observed at 24 h, and more new bands were observed at 96 h in L. gibba. The RAPD profiles of L. minor produced by all of the primers were slightly less affected by the herbicide treatment than were those of L. gibba.

Study of the generated genetic polymorphisms during the photocatalytic elimination of Klebsiella pneumoniae in water.

PubMed

Venieri, Danae; Fraggedaki, Antonia; Binas, Vassilios; Zachopoulos, Apostolos; Kiriakidis, George; Mantzavinos, Dionissios

2015-03-01

Klebsiella pneumoniae is considered to be an emerging pathogen persisting under extreme environmentally stressed conditions. The aim of the present study is the investigation of inactivation rates of this pathogen in water by means of heterogeneous photocatalytic treatment under solar irradiation and the induced genetic variance applying RAPD-PCR as a molecular typing tool. Novel Mn- and Co-doped TiO2 catalysts were assessed in terms of their disinfection efficiency. The reference strain of K. pneumoniae proved to be readily inactivated, since disinfection occurred rapidly (i.e. after only 10 min of treatment) and low levels of bacterial regrowth were recorded in the dark and under natural sunlight. Binary doped titania exhibited the best photocatalytic activity, verifying the synergistic effect induced by composite dopants. Applying RAPD analysis to viable cells after treatment we concluded that increasing the treatment time led to considerable alteration of RAPD profiles and the homology coefficient ranged almost between 35 and 60%. RAPD-PCR proved to be a useful typing molecular tool that under standardized conditions exhibits highly reproducible results. Genetic variation among isolates increased in relation to the period of treatment and prolonged irradiation in each case affected the overall alteration in band patterns. RAPD patterns were highly diverse between treated and untreated isolates when disinfection was performed with the Co-doped titania. The broad spectrum of genetic variance and generated polymorphisms has the potential to increase the already significant virulence of the species.

Evaluation of powdery mildew-resistance of grape germplasm and rapid amplified polymorphic DNA markers associated with the resistant trait in Chinese wild Vitis.

PubMed

Zhang, J; Zhang, Y; Yu, H; Wang, Y

2014-05-09

The resistance of wild Vitis germplasm, including Chinese and American wild Vitis and Vitis vinifera cultivars, to powdery mildew (Uncinula necator Burr.) was evaluated for two consecutive years under natural conditions. Most of the Chinese and North American species displayed a resistant phenotype, whereas all of the European species were highly susceptible. The Alachua and Conquistador accessions of Vitis rotundifolia species, which originated in North America, were immune to the disease, while Baihe-35-1, one of the accessions of Vitis pseudoreticulata, showed the strongest resistance among all Chinese accessions evaluated. Three rapid amplified polymorphic DNA (RAPD) markers, OPW02-1756, OPO11-964, and OPY13-661, were obtained after screening 520 random primers among various germplasm, and these markers were found to be associated with powdery mildew resistance in Baihe-35-1 and in some Chinese species, but not in any European species. Analysis of F₁ and F₂ progenies of a cross between resistant Baihe-35-1 and susceptible Carignane (V. vinifera) revealed that the three RAPD markers were linked to the powdery resistant trait in Baihe-35-1 plants. Potential applications of the identified RAPD markers for gene mapping, marker-assisted selection, and breeding were investigated in 168 F₂ progenies of the same cross. Characterization of the resistant phenotype of the selected F₂ seedlings for breeding a new disease-resistant grape cultivar is in progress.

Yeast microbiota associated with spontaneous sourdough fermentations in the production of traditional wheat sourdough breads of the Abruzzo region (Italy).

PubMed

Valmorri, Sara; Tofalo, Rosanna; Settanni, Luca; Corsetti, Aldo; Suzzi, Giovanna

2010-02-01

The aims of this study were to describe the yeast community of 20 sourdoughs collected from central Italy and to characterize the sourdoughs based on chemical properties. A polyphasic approach consisting of traditional culture-based tests (spore-forming and physiological tests) and molecular techniques (PCR-RFLP, RAPD-PCR, PCR-DGGE) and chemical analysis (total acidity, acids, and sugar contents), was utilized to describe the yeast population and to investigate the chemical composition of the doughs. PCR-RFLP analysis identified 85% of the isolates as Saccharomyces cerevisiae, with the other dominant species being Candida milleri (11%), Candida krusei (2.5%), and Torulaspora delbrueckii (1%). RAPD-PCR analysis, performed with primers M13 and LA1, highlighted intraspecific polymorphism among the S. cerevisiae strains. The diversity of the sourdoughs from the Abruzzo region is reflected in the chemical composition, yeast species, and strain polymorphism. Our approach using a combination of phenotypic and genotypic methods identified the yeast species in the 20 sourdough samples and provided a complete overview of the yeast populations found in sourdoughs from the Abruzzo region.

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for oxyuroid species (Nematoda).

PubMed

Jobet, E; Bougnoux, M E; Morand, S; Rivault, C; Cloarec, A; Hugot, J P

1998-03-01

Random amplified DNA markers (RAPD; Williams et al., 1990) were used to obtained specific RAPD fragments characterising different species of oxyuroids. We tested six species of worms parasitizing vertebrates or invertebrates: Passalurus ambiguus Rudolphi, 1819, parasite of Leporids; Syphacia obvelata (Rudolphi, 1802) Seurat, 1916, a parasite of rodents; Blatticola blattae (Graeffe, 1860) Chitwood, 1932 parasite of the cockroach Blattella germanica; Hammerschmidtiella diesingi (Hammerschmidt, 1838) Chitwood, 1932 and Thelastoma bulhoesi (Magalhaes, 1990) Travassos, 1929, parasites of the cockroach Periplaneta americana, and an undescribed parasite species of a passalid insect from New Caledonia. Among 15 oligonucleotides tested, nine produced several specific bands allowing the interspecific discrimination.

Distinction between wild and cultivated enset (Ensete ventricosum) gene pools in Ethiopia using RAPD markers.

PubMed

Birmeta, Genet; Nybom, Hilde; Bekele, Endashaw

2004-01-01

In southwest Ethiopia, the cultivation area of Ensete ventricosum (enset) overlaps with the natural distribution area of this species. Analyses of genetic diversity were undertaken using RAPD to provide information for conservation strategies as well as evidence of possible gene flow between the different gene pools, which can be of interest for future improvement of cultivated enset. The extent of RAPD variation in wild enset was investigated in 5 populations in the Bonga area (Kefficho administrative region) and 9 cultivated clones. Comparisons were also made with some Musa samples of potential relevance for crop improvement. Nine oligonucleotide primers amplified 72 polymorphic loci. Population differentiation was estimated with the Shannon index (G'(ST)=0.10), Nei's G(ST) (0.12) and AMOVA (Phi(ST)=0.12), and appears to be relatively low when compared with outbreeding, perennial species in general. Cluster analysis (UPGMA) and principal component analysis (PCA) similarly indicated low population differentiation, and also demonstrated that cultivated clones essentially clustered distinctly from wild enset samples, suggesting that the present-day cultivated enset clones have been introduced to domestication from a limited number of wild progenitors. In addition, subsequent gene flow between wild and cultivated enset may have been prohibited by differences between modes of propagation and harvesting time; cultivated enset is propagated vegetatively through sucker production and the plant is generally harvested before maturity or flower set, thereby hindering pollination by wild enset or vice versa. A significant correlation was not found between genetic and geographical distances. The relatively high total RAPD diversity suggests that wild enset populations in the Bonga area harbour genetic variability which could potentially act as a source for useful or rare genes in the improvement of cultivated enset. As expected, E. ventricosum was clearly differentiated from the analysed Musa samples, that clustered in accordance with the present morphology- and molecular marker-based taxonomy of the genus.

Development of Specific Sequence-Characterized Amplified Region Markers for Detecting Histoplasma capsulatum in Clinical and Environmental Samples

PubMed Central

Frías De León, María Guadalupe; Arenas López, Gabina; Taylor, Maria Lucia; Acosta Altamirano, Gustavo

2012-01-01

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220 and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283220 and 1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283230 SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from different sources. PMID:22189121

First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran.

PubMed

Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat-Aldin; Mohebali, Mehdi

2013-09-01

Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents' ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area.

First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran

PubMed Central

Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat–Aldin; Mohebali, Mehdi

2013-01-01

Background Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. Objectives This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Materials and Methods Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents’ ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. Results All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Conclusions Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area. PMID:24616787

[The application of RAPD technology in genetic diversity detection of Jute].

PubMed

Qi, Jian-Min; Zhou, Dong-Xin; Wu, Wei-Ren; Lin, Li-Hui; Fang, Ping-Ping; Wu, Jian-Mei

2003-10-01

The fingerprints of 10 species including 27 accessions in genus Corchorus were investigated with the technique of RAPD. Twenty-five primers were screened from 119 random primers, and a total of 329 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (87.78%), which were polymorphic. The average number of DNA band produced by each primer was 13.16. UPGMA cluster analysis and Nei's similarity coefficients were carried out and a dendrogram was constructed using software Biol D++. The results showed as follows: (1) There were abundant genetic diversities among 15 wild species and 12 cultivated species in Corchorus with genetic similarity coefficients ranging from 0.49-0.98. (2) The accessions could be clustered into three groups at cultivated species, and their close wild species were obviously different from wild species genetically. (3) At the level of D = 0.850, 27 accessions of Jute could be classified into ten groups, including C. sestuans, C. tridens, C. fascicularis, C. psendo-olitorius, C. psendo-capsularis, C. tilacutaris, Tian Jute (untitled), C. capsularis, C. olitorius and C. uriticifolius. Among which C. capsularis presented closer relationship with C. olitorius and further relationship with C. uriticifolius. The results matched well with that of the morphologic classification. (4) According to the molecular cluster tree, C. uritifolius, Chinese Tina Jute (untitled) and C. aestuans were at the basic level, revealing that these three species could be the primary wild species of Jute. (5) The tree also showed that C. tilacularis 21C from Africa could be a ecological subspecies of C. tilacularis, whilst niannian cai, ma cai and zhu cai collected different ecological types of C. aestuans, C. capsularis from Hainan was a close wild species of round fruit Jute cultivated species, and three species of C. olitorius collected from zhangpu, Henan and Mali were close wild species of long fruit Jute cultivated species. (6) within two cultivated species, the genetic similarity coefficients in round fruit cultivated species was higher than that of in long fruit cultivated species.

Agronomic, chemical and genetic profiles of hot peppers (Capsicum annuum ssp.).

PubMed

De Masi, Luigi; Siviero, Pietro; Castaldo, Domenico; Cautela, Domenico; Esposito, Castrese; Laratta, Bruna

2007-08-01

A study on morphology, productive yield, main quality parameters and genetic variability of eight landraces of hot pepper (Capsicum annuum ssp.) from Southern Italy has been performed. Morphological characters of berries and productivity values were evaluated by agronomic analyses. Chemical and genetic investigations were performed by HPLC and random amplified polymorphic DNA (RAPD)-PCR, respectively. In particular, carotenoid and capsaicinoid (pungency) contents were considered as main quality parameters of hot pepper. For the eight selected samples, genetic similarity values were calculated from the generated RAPD fragments and a dendrogram of genetic similarity was constructed. All the eight landraces exhibited characteristic RAPD patterns that allowed their characterization. Agro-morphological and chemical determinations were found to be adequate for selection, but they resulted useful only for plants grown in the same environmental conditions. RAPD application may provide a more reliable way based on DNA identification. The results of our study led to the identification of three noteworthy populations, suitable for processing, which fitted into different clusters of the dendrogram.

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

PubMed Central

Dutta, Suhrid R.; Kar, Prasanta K.; Srivastava, Ashok K.; Sinha, Manoj K.; Shankar, Jai; Ghosh, Ananta K.

2012-01-01

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F2 progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16905 bp showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F2 progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16826 bp). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk. PMID:23271934

Unraveling the efficiency of RAPD and SSR markers in diversity analysis and population structure estimation in common bean.

PubMed

Zargar, Sajad Majeed; Farhat, Sufia; Mahajan, Reetika; Bhakhri, Ayushi; Sharma, Arjun

2016-01-01

Increase in food production viz-a-viz quality of food is important to feed the growing human population to attain food as well as nutritional security. The availability of diverse germplasm of any crop is an important genetic resource to mine the genes that may assist in attaining food as well as nutritional security. Here we used 15 RAPD and 23 SSR markers to elucidate diversity among 51 common bean genotypes mostly landraces collected from the Himalayan region of Jammu and Kashmir, India. We observed that both the markers are highly polymorphic. The discriminatory power of these markers was determined using various parameters like; percent polymorphism, PIC, resolving power and marker index. 15 RAPDs produced 171 polymorphic bands, while 23 SSRs produced 268 polymorphic bands. SSRs showed a higher PIC value (0.300) compared to RAPDs (0.243). Further the resolving power of SSRs was 5.241 compared to 3.86 for RAPDs. However, RAPDs showed a higher marker index (2.69) compared to SSRs (1.279) that may be attributed to their higher multiplex ratio. The dendrograms generated with hierarchical UPGMA cluster analysis grouped genotypes into two main clusters with various degrees of sub clustering within the cluster. Here we observed that both the marker systems showed comparable accuracy in grouping genotypes of common bean according to their area of cultivation. The model based STRUCTURE analysis using 15 RAPD and 23 SSR markers identified a population with 3 sub-populations which corresponds to distance based groupings. High level of genetic diversity was observed within the population. These findings have further implications in common bean breeding as well as conservation programs.

AFLP analysis of a worldwide collection of Didymella bryoniae.

PubMed

Kothera, Ronald T; Keinath, Anthony P; Dean, Ralph A; Farnham, Mark W

2003-03-01

Didymella bryoniae (anamorph Phoma cucurbitacearum) is an ascomycete that causes gummy stem blight, a foliar disease that occurs on cucurbits in greenhouses and fields throughout the world. In a previous study using RAPD analysis, little genetic diversity was found among isolates of D. bryoniae from New York and South Carolina, USA. Here we report the use of amplified fragment length polymorphism (AFLP) analysis to assess the genetic variation within a worldwide collection of D. bryoniae, 102 field and greenhouse isolates from ten states in the USA (California, Delaware, Florida, Georgia, Indiana, Maryland, Michigan, Oklahoma, South Carolina, and Texas) and seven other countries (Australia, Canada, China, Greece, Israel, Sweden, and The Netherlands) were examined. Seven different AFLP primer-pair combinations generated 450 bands, of which 134 were polymorphic (30%). Using cluster analysis, two groups and a total of seven subgroups were delineated. Representative isolates varied in their virulence on muskmelon and watermelon seedlings, but the degree of virulence was not strongly associated with AFLP groupings. However, isolates from the northern USA grouped separately from isolates originating from the southern USA.

Evaluation of genetic diversity in Piper spp using RAPD and SRAP markers.

PubMed

Jiang, Y; Liu, J-P

2011-11-29

Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) analysis were applied to 74 individual plants of Piper spp in Hainan Island. The results showed that the SRAP technique may be more informative and more efficient and effective for studying genetic diversity of Piper spp than the RAPD technique. The overall level of genetic diversity among Piper spp in Hainan was relatively high, with the mean Shannon diversity index being 0.2822 and 0.2909, and the mean Nei's genetic diversity being 0.1880 and 0.1947, calculated with RAPD and SRAP data, respectively. The ranges of the genetic similarity coefficient were 0.486-0.991 and 0.520-1.000 for 74 individual plants of Piper spp (the mean genetic distance was 0.505 and 0.480) and the within-species genetic distance ranged from 0.063 to 0.291 and from 0.096 to 0.234, estimated with RAPD and SRAP data, respectively. These genetic indices indicated that these species are closely related genetically. The dendrogram generated with the RAPD markers was topologically different from the dendrogram based on SRAP markers, but the SRAP technique clearly distinguished all Piper spp from each other. Evaluation of genetic variation levels of six populations showed that the effective number of alleles, Nei's gene diversity and the Shannon information index within Jianfengling and Diaoluoshan populations are higher than those elsewhere; consequently conservation of wild resources of Piper in these two regions should have priority.

Identification and validation of sex-linked SCAR markers in dioecious Hippophae rhamnoides L. (Elaeagnaceae).

PubMed

Korekar, Girish; Sharma, Ram Kumar; Kumar, Rahul; Meenu; Bisht, Naveen C; Srivastava, Ravi B; Ahuja, Paramvir Singh; Stobdan, Tsering

2012-05-01

The actinorhizal plant seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae) is a wind pollinated dioecious crop. To distinguish male genotypes from female genotypes early in the vegetative growth phase, we have developed robust PCR-based marker(s). DNA bulk samples from 20 male and 20 female plants each were screened with 60 RAPD primers. Two primers, OPA-04 and OPT-06 consistently amplified female-specific (FS) polymorphic fragments of 1,164 and 868 bp, respectively, that were absent in the male samples. DNA sequence of the two markers did not exhibit significant similarity to previously characterized sequences. A sequence-characterized amplified region marker HrX1 (JQ284019) and HrX2 (JQ284020) designed for the two fragments, continued to amplify the FS allele in 120 female plants but not in 100 male plants tested in the current study. Thus, HrX1 and HrX2 are FS markers that can determine the sex of seabuckthorn plants in an early stage and expedite cultivations for industrial applications.

Biochemical and Molecular Analysis of Some Commercial Samples of Chilli Peppers from Mexico

PubMed Central

Troconis-Torres, Ivonne Guadalupe; Rojas-López, Marlon; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes; Maldonado-Mendoza, Ignacio Eduardo; Dorantes-Álvarez, Lidia; Tellez-Medina, Darío; Jaramillo-Flores, María Eugenia

2012-01-01

The genus Capsicum provides antioxidant compounds, such as phenolics and carotenoids, into the diet. In Mexico, there is a wide diversity of species and varieties of chilli peppers, a fruit which has local cultural and gastronomic importance. In the present study, the relationship of the carotenoid and phenolic profiles with the RAPD fingerprint of three different commercial cultivars of chilli peppers of seven regions of Mexico was investigated. Through RAPD, the species of chilli were differentiated by means of different primers (OPE-18, MFG-17, MFG-18, C51, and C52). The genetic distance found with OPE 18 was in the order of 2.6. The observed differences were maintained when the chromatographic profile of carotenoids, and the molecular markers were analyzed, which suggest a close relationship between carotenoids and the genetic profile. While the chromatographic profile of phenols and the molecular markers were unable to differentiate between genotypes of chilli peppers. In addition, by using infrared spectroscopy and statistical PCA, differences explained by geographic origin were found. Thus, this method could be an alternative for identification of chilli species with respect to their geographic origin. PMID:22665993

Biochemical and molecular analysis of some commercial samples of chilli peppers from Mexico.

PubMed

Troconis-Torres, Ivonne Guadalupe; Rojas-López, Marlon; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes; Maldonado-Mendoza, Ignacio Eduardo; Dorantes-Álvarez, Lidia; Tellez-Medina, Darío; Jaramillo-Flores, María Eugenia

2012-01-01

The genus Capsicum provides antioxidant compounds, such as phenolics and carotenoids, into the diet. In Mexico, there is a wide diversity of species and varieties of chilli peppers, a fruit which has local cultural and gastronomic importance. In the present study, the relationship of the carotenoid and phenolic profiles with the RAPD fingerprint of three different commercial cultivars of chilli peppers of seven regions of Mexico was investigated. Through RAPD, the species of chilli were differentiated by means of different primers (OPE-18, MFG-17, MFG-18, C51, and C52). The genetic distance found with OPE 18 was in the order of 2.6. The observed differences were maintained when the chromatographic profile of carotenoids, and the molecular markers were analyzed, which suggest a close relationship between carotenoids and the genetic profile. While the chromatographic profile of phenols and the molecular markers were unable to differentiate between genotypes of chilli peppers. In addition, by using infrared spectroscopy and statistical PCA, differences explained by geographic origin were found. Thus, this method could be an alternative for identification of chilli species with respect to their geographic origin.

A preliminary report on the genetic variation in pointed gourd (Trichosanthes dioica Roxb.) as assessed by random amplified polymorphic DNA.

PubMed

Adhikari, S; Biswas, A; Bandyopadhyay, T K; Ghosh, P D

2014-06-01

Pointed gourd (Trichosanthes dioica Roxb.) is an economically important cucurbit and is extensively propagated through vegetative means, viz vine and root cuttings. As the accessions are poorly characterized it is important at the beginning of a breeding programme to discriminate among available genotypes to establish the level of genetic diversity. The genetic diversity of 10 pointed gourd races, referred to as accessions was evaluated. DNA profiling was generated using 10 sequence independent RAPD markers. A total of 58 scorable loci were observed out of which 18 (31.03%) loci were considered polymorphic. Genetic diversity parameters [average and effective number of alleles, Shannon's index, percent polymorphism, Nei's gene diversity, polymorphic information content (PIC)] for RAPD along with UPGMA clustering based on Jaccard's coefficient were estimated. The UPGMA dendogram constructed based on RAPD analysis in 10 pointed gourd accessions were found to be grouped in a single cluster and may represent members of one heterotic group. RAPD analysis showed promise as an effective tool in estimating genetic polymorphism in different accessions of pointed gourd.

In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers.

PubMed

Ilczuk, Agnieszka; Jacygrad, Ewelina

2016-01-01

Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars 'Aurea' and 'Elegantissima' were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either N 6 -benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L -1 BA, 0.1 mg L -1 NAA, and 20-30 g L -1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L -1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for 'Aurea' and 90% for 'Elegantissima'. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197-199 and 184-187 distinct and reproducible band classes, respectively, in 'Aurea' and 'Elegantissima' plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PubMed

Matray, Olivier; Mouhajir, Abdelmounaim; Giraud, Sandrine; Godon, Charlotte; Gargala, Gilles; Labbé, Franck; Rougeron, Amandine; Ballet, Jean-Jacques; Zouhair, Rachid; Bouchara, Jean-Philippe; Favennec, Loïc

2016-05-01

The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF). © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Haider, Nadia

2017-01-01

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.

Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males.

PubMed

Silva, M H; Nascimento, M D S B; Leonardo, F S; Rebêlo, J M M; Pereira, S R F

2011-01-01

Entomological surveys in the state of Maranhão have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranhão, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.

Initial determination of DNA polymorphism of some Primula veris L. populations from Kosovo and Austria.

PubMed

Berisha, Naim; Millaku, Fadil; Gashi, Bekim; Krasniqi, Elez; Novak, Johannes

2015-01-01

Primula veris L. (Primulaceae) is a long lived perennial and well known pharmaceutical plant, widely collected for these reasons in almost all SE Europe and particularly in Kosovo. The aim of the study is to determine molecular polymorphism of cowslip (P. veris L.) populations from Kosovo. DNA extracted from leaves were  investigated in details for presence of polymorphism. RAPD analyses were conducted using 20 different short primers. Genomic DNA amplification profiles were analyzed and processed using data labelling. Comparison between cowslip populations in genetic composition revealed that samples from Bogaj were too distinct on their own. Molecular variation was observed to be more within populations (73 %) as compared to among populations (27 %). On the other hand, genetic distance of populations revealed that the highest genetic distance is between Leqinat and Maja e Madhe. Mean values of expected heterozygosity were highest in Bogaj population, while lowest in Maja e Madhe population. The obtained results indicated that Bogaj population are more polymorphic. From the obtained data it can be concluded that RAPD markers provided a useful technique to study genetic diversity in P. veris L. populations. This technology allows identification and assessment of the genetic similarities and differences among plant populations.

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon.

PubMed

Ma, Y-Z; Tomita, M

2013-01-01

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai- 1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chromatins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDV-resistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.

Development and characterization of a new TILLING population of common bread wheat (Triticum aestivum L.).

PubMed

Chen, Liang; Huang, Linzhou; Min, Donghong; Phillips, Andy; Wang, Shiqiang; Madgwick, Pippa J; Parry, Martin A J; Hu, Yin-Gang

2012-01-01

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.

Two new species of Undifilum, fungal endophytes of Astragalus (locoweeds) in the United States

PubMed Central

Baucom, Deana L.; Romero, Marie; Belfon, Robert; Creamer, Rebecca

2013-01-01

New species of Undifilum, from locoweeds Astragalus lentiginosus Vitman and Astragalus mollissimus Torr., are described using morphological characteristics and molecular phylogenetic analyses as Undifilum fulvum Baucom & Creamer sp. nov. and Undifilum cinereum Baucom & Creamer sp. nov. Fungi were isolated from dried plants of A. lentiginosus var. araneosus, diphysus, lentiginosus, and wahweapensis collected from Arizona, Oregon, and Utah, USA, and A. mollissimus var. biglovii, earleii, and mollissimus collected from New Mexico, Oklahoma, and Texas, USA. Endophytic fungi from Astragalus locoweeds were compared to Undifilum oxytropis isolates obtained from dried plant material of Oxytropis lamberteii from New Mexico and Oxytropis sericea from Arizona, Colorado, New Mexico, Utah, and Wyoming. Extremely slow growth in vitro was observed for all, and conidia, if present, were ellipsoid with transverse septa. However, in vitro color, growth on four different media, and conidium size differed between fungi from Astragalus spp. and U. oxytropis. Neighbor-joining analyses of internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPD) gene sequences revealed that U. fulvum and U. cinereum formed a clade distinct from U. oxytropis. This was supported by neighbor-joining analyses of results generated from random amplified polymorphic DNA (RAPD) fragments using two different primers. PMID:24223679

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea)

PubMed Central

Gimenes, Marcos A; Hoshino, Andrea A; Barbosa, Andrea VG; Palmieri, Dario A; Lopes, Catalina R

2007-01-01

Background The genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species. Results Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci. Conclusion These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species. PMID:17326826

Molecular characterization of Aspergillus fumigatus isolated from raw cow milk in Argentina: Molecular typing of A. fumigatus from raw cow milk.

PubMed

Pena, Gabriela A; Alonso, Verónica; Manini, Marina Velasco; Pellegrino, M; Cavaglieri, Lilia R

2018-06-20

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a gliotoxinogenic species into section Fumigati commonly found in contaminated animal environments. In dairy herds, exposed areas of lactating cows, as mammalian glandule, can be easily contaminated by them. This study was aimed to identify A. fumigatus sensu lato strains (identified based on morphology) isolated from raw cow milk at species level, by morphological and molecular techniques, and to estimate their genetic variability. Forty-five A. fumigatus strains showed similar RAPD profiles (generated with PELF and URP1F primers) to each other and to A. fumigatus sensu stricto reference strains; also, they were almost identical to clinical human and feed-borne A. fumigatus strains included in the assay, since their similarity coefficient ranged from 0.7 to 1.00. Therefore, all strains were characterized as belonging to A. fumigatus sensu stricto species. This result was supported by sequencing the benA gene of selected strains and by maximum parsimony analysis. In addition, RAPD fingerprinting demonstrated intra-specific genetic variability into the A. fumigatus sensu stricto cluster. The results found in this study strengthen the fact that A. fumigatus sensu stricto is the predominant species in the Aspergillus section Fumigati found in animal environments such as dairy herd environments, while other species such as A. novofumigatus, A. fumigatiaffinis, A. udagawae and A. lentulus may be rarely isolated. Since no differences between animal and human strains were observed they can become pathogenic also for farm handlers'. Moreover, the presence of A. fumigatus sensu stricto in raw cow milk is probably a very important risk factor since milk and its by-products are generally indented for human consumption, then gliotoxin could be transferred to them. Copyright © 2018. Published by Elsevier B.V.

Development of SCAR marker for discrimination of Artemisia princeps and A. argyi from other Artemisia herbs.

PubMed

Lee, Mi Young; Doh, Eui Jeong; Park, Chae Haeng; Kim, Young Hwa; Kim, Eung Soo; Ko, Byong Seob; Oh, Seung-Eun

2006-04-01

Some Artemisia herbs are used for medicinal purposes. In particular, A. princeps and A. argyi are classified as 'Aeyup' and are used as important medicinal material in traditional Korean medicine. On the other hand, A. capillaris and A. iwayomogi, which are classified as 'Injinho' and 'Haninjin', respectively, are used for other purposes distinct from those of 'Aeyup'. However, sometimes 'Aeyup' is not clearly discriminated from 'Injinho' and/or 'Haninjin'. Furthermore, Artemisia capillaris and/or A. iwayomogi have been used in place of A. princeps and A. argyi. In this study, we developed an efficient method to discriminate A. argyi and A. princeps from other Artemisia plants. The RAPD (random amplified polymorphic DNA) method efficiently discriminated various Artemisia herbs. In particular, non-specific primer 329 (5'-GCG AAC CTC C-3'), which shows polymorphism among Artemisia herbs, amplified 838 bp products, which are specific to A. princeps and A. argyi only. Based on nucleotide sequence of the primer 329 product, we designed a Fb (5'-CAT CAA CCA TGG CTT ATC CT-3') and R7 (5'-GCG AAC CTC CCC ATT CCA-3') primer-set to amplify a 254 bp sized SCAR (sequence characterized amplified regions) marker, through which A. princeps and A. argyi can be efficiently discriminated from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi.

A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica.

PubMed Central

Di Stilio, V S; Kesseli, R V; Mulcahy, D L

1998-01-01

The segregation pattern of an 810-bp random amplified polymorphic DNA (RAPD) band in the F1 and backcross generations of a Silene dioica (L.) Clairv. family provides evidence that this molecular marker is located in the pseudoautosomal region (PAR) of the X and Y chromosomes. The marker was found through a combination of bulked segregant analysis (BSA) and RAPD techniques. Recombination rates between this pseudoautosomal marker and the differentiating portion of the Y chromosome are 15% in both generations. Alternative explanations involving nondisjunction or autosomal inheritance are presented and discussed. Chromosome counts provide evidence against the nondisjunction hypothesis, and probability calculations argue against the possibility of autosomal inheritance. This constitutes the first report of a pseudoautosomal DNA marker for plant sex chromosomes. PMID:9691057

ISSR, ERIC and RAPD techniques to detect genetic diversity in the aphid pathogen Pandora neoaphidis.

PubMed

Tymon, Anna M; Pell, Judith K

2005-03-01

The entomopathogenic fungus Pandora neoaphidis is an important natural enemy of aphids. ISSR, ERIC (Enterobacterial Repetitive Intergenic Consensus) and RAPD PCR-based DNA fingerprint analyses were undertaken to study intra-specific variation amongst 30 isolates of P. neoaphidis worldwide, together with six closely related species of Entomophthorales. All methods yielded scorable binary characters, and distance matrices were constructed from both individual and combined data sets. Neighbour-joining was used to construct consensus phylogenetic trees which showed that although P. neoaphidis isolates were highly polymorphic they separated into a monophyletic group compared with the other Entomophthorales tested. Three distinct subclades were found, with UK isolates occupying two of these. No specific correlation with aphid host species was established for any of the isolates apart from those in one cluster which contained isolates obtained from nettle aphid, Microlophium carnosum. ERIC, ISSR and RAPD analysis allowed the rapid genetic characterisation and differentiation of isolates with the generation of potential isolate- and cluster specific-diagnostic DNA markers.

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India.

PubMed

Bhowmick, P P; Khushiramani, R; Raghunath, P; Karunasagar, I; Karunasagar, I

2008-02-01

Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR. Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0.95, while ERIC-PCR showed a DI of 0.94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. The use of protein profiling in combination with other established typing methods such as RAPD and ERIC-PCR generates useful information in the case of V. parahaemolyticus associated with seafood. The study demonstrates the usefulness of nucleic acid and protein-based studies in understanding the relationship between various isolates from seafood.

Population structure of the banana weevil, an introduced pest in the Canary Islands, studied by RAPD analysis.

PubMed

Magaña, C; Beroiz, B; Hernández-Crespo, P; Montes de Oca, M; Carnero, A; Ortego, F; Castañera, P

2007-12-01

The banana weevil (BW), Cosmopolites sordidus (Coleoptera: Curculionidae), is one of the most important insect pests of bananas and plantains. The mobility and the origin of BW infestations at the Canary Islands (Tenerife, La Gomera and La Palma) have been analysed using Random Amplified Polymorphic DNA (RAPD) as molecular markers. Populations from Costa Rica, Colombia, Uganda and Madeira were also included for comparison. One hundred and fifteen reproducible bands from eight primers were obtained. The level of polymorphism in the populations from the Canary Islands (40-62%) was in the range of those found in other populations. Nei's genetic distances, pair-wise fixation index (FST) values indicate that the closest populations are Tenerife populations among themselves (Nei's genetic distance=0.054-0.100; FST=0.091-0.157) and Costa Rica and Colombia populations (Nei's genetic distance=0.049; FST=0.113). Our results indicate the existence of BW local biotypes with limited gene flow and affected by genetic drift. These results are compatible with a unique event of colonization at Tenerife; whereas, the outbreaks in La Gomera and La Palma may come from independent introductions. The Madeira population is phylogenetically and geographically closer to the Canary Islands populations, suggesting that it is the most likely source of the insects introduced in the Canary Islands.

[Genetic variability and differentiation of three Russian populations of yellow potato cyst nematode Globodera rostochiensis as revealed by nuclear markers].

PubMed

Khrisanfova, G G; Kharchevnikov, D A; Popov, I O; Zinov'eva, S V; Semenova, S K

2008-05-01

Genetic variability of yellow potato cyst nematode G. rostochiensis from three Russian populations (Karelia, Vladimir oblast, and Moscow oblast) was investigated using two types of nuclear markers. Using RAPD markers identified with the help of six random primers (P-29, OPA-10, OPT-14, OPA-11, OPB-11, and OPH-20), it was possible to distinguish Karelian population from the group consisting of the populations from two adjacent regions (Moscow oblast and Vladimir oblast). Based on the combined matrix, containing 294 RAPD fragments, dendrogram of genetic differences was constructed, and the indices of genetic divergence and partition (P, H, and G(st)), as well as the gene flow indices N(m) between the nematode samples examined, were calculated. The dendrogram structure, genetic diversity indices, and variations of genetic distances between single individuals in each population from Karelia and Central Russia pointed to genetic isolation and higher genetic diversity of the nematodes from Karelia. Based on polymorphism of rDNA first intergenic spacer ITS1, attribution of all populations examined to the species G. rostochiensis was proved. Small variations of the ITS1 sequence in different geographic populations of nematodes from different regions of the species world range did not allow isolation of separate groups within the species. Possible factors (including interregional transportations of seed potato) affecting nematode population structure in Russia are discussed.

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

PubMed Central

Corroler, D.; Mangin, I.; Desmasures, N.; Gueguen, M.

1998-01-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese. PMID:9835555

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

PubMed

Corroler, D; Mangin, I; Desmasures, N; Gueguen, M

1998-12-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Isozyme, ISSR and RAPD profiling of genotypes in marvel grass (Dichanthium annulatum).

PubMed

Saxena, Raghvendra; Chandra, Amaresh

2010-11-01

Genetic analysis of 30 accessions of marvel grass (Dichanthium annulatum Forsk.), a tropical range grass collected from grasslands and open fields of drier regions, was carried out with the objectives of identifying unique materials that could be used in developing the core germplasm for such regions as well as to explore gene (s) for drought tolerance. Five inter-simple sequence repeat (ISSR) primers [(CA)4, (AGAC), (GACA) 4; 27 random amplified polymorphic DNA (RAPD) and four enzyme systems were employed in the present study. In total, ISSR yielded 61 (52 polymorphic), RAPD 269 (253 polymorphic) and enzyme 55 isozymes (44 polymorphic) bands. The average polymorphic information content (PIC) and marker index (MI) across all polymorphic bands of 3 markers systems ranged from 0.419 to 0.480 and 4.34 to 5.25 respectively Dendrogram analysis revealed three main clusters with all three markers. Four enzymes namely esterase (EST), polyphenoloxidase (PPO), peroxidase (PRX) and superoxide dismutase (SOD) revealed 55 alleles from a total of 16 enzyme-coding loci. Of these, 14 loci and 44 alleles were polymorphic. The mean number of alleles per locus was 3.43. Mean heterozygosity observed among the polymorphic loci ranged from 0.406 (SOD) to 0.836 (EST) and accession wise from 0.679 (1G3108) to 0.743 (IGKMD-10). Though there was intermixing of few accessions of one agro-climatic region to another largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 iterations also showed large numbers of nodes (11 to 17) having strong clustering (> 50 bootstrap values) in all three marker systems. The accessions of the arid and drier regions forming one cluster are assigned as distinct core collection of Dichanthium and can be targeted for isolation of gene (s) for drought tolerance. Variations in isozyme allele numbers and high PIC (0.48) and MI (4.98) as observed with ISSR markers indicated their usefulness for germplasm characterization.

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers.

PubMed

Palmieri, Darío A; Bechara, Marcelo D; Curi, Rogério A; Monteiro, Jomar P; Valente, Sérgio E S; Gimenes, Marcos A; Lopes, Catalina R

2010-01-01

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections.

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

PubMed Central

2010-01-01

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections. PMID:21637613

Resistance to antibiotics in clinical isolates of Pseudomonas aeruginosa.

PubMed

Sevillano, E; Valderrey, C; Canduela, M J; Umaran, A; Calvo, F; Gallego, L

2006-01-01

To analyse the global resistance to some antibiotics used to treat nosocomial infections by Pseudomonas aeruginosa, specially to carbapenems, and its relationship with the presence of carbapenemases, OXA, VIM and IMP. The study included 229 P. aeruginosa isolates from a Hospital in Northern Spain (year 2002). Susceptibility to antimicrobial agents was determined by the analysis of the MIC. Genetic typing was carried out by RAPD-PCR fingerprinting with primer ERIC-2. Genetic experiments to detect class-1 integrons were performed by PCR with primers 5'CS and 3'CS. Detection of carbapenemases was done by phenotypic (Hodge test and DDST) and genotypic methods (PCR with primers for imp, vim1, vim2 and oxa40 genes). 23.9% of isolates were resistant to ceftazidime, 35.9% to cefotaxime, 5.3% to amikacin, 54.9% to gentamicin, 14.6% to imipenem and 6.6% to meropenem. Isolates resistant to imipenem (33) were furtherly tested. Genetic typing didn't show clonal relatedness among the most of the isolates. Class-1 integrons were present in most isolates (sizes 600-1700 bp). Phenotypic methods for carbapenemases showed 5 positive isolates. Genotypic methods showed the presence of two isolates with the oxa40 gene. Meropenem, amikacin and imipenem were the most active agents to treat infections caused by Pseudomonas aeruginosa. In our study, the presence of carbapenemase enzymes wasn't high. Phenotypic tests cannot be considered as accurate screening tool to detect carbapenemases. This is the fist report of the oxa40 gene in Pseudomonas aeruginosa isolates.

DETERMINATION OF GENETIC DIVERSITY AND PATERNITY IN THE GRAY-TAILED VOLE (MICROTUS CANICAUDUS) BY RAPD-PCR

EPA Science Inventory

Genetic relatedness of gray-tailed voles (Microtus canicaudus) was determined by random amplified polymorphic DNA (RAPD). This work is the first reported use of the RAPD method for pedigree analysis of M. canicaudus and demonstrates the feasibility of RAPD for assessing paternity...

RAPD analysis of the genetic diversity among accessions of Fabaceous forages (Poincianella spp) from the Caatinga.

PubMed

Mendes, R F M; Araujo Neto, R B; Nascimento, M P S B C; Lima, P S C

2014-08-01

Among members of the Fabaceae family, native to the Brazilian Caatinga, the species Poincianella pyramidalis and P. bracteosa exhibit particular potential as forage for cattle, sheep and goats. With the aim of establishing genetic relationships within Poincianella, random amplified polymorphic DNA analysis was performed on eight accessions of P. pyramidalis and two accessions of P. bracteosa, originating from the semiarid zone of the state of Piauí, northeastern Brazil, and present in the germplasm bank of Embrapa Meio Norte (Teresina, Piauí, Brazil). Amplification reactions using 11 selected arbitrary sequence primers generated 167 fragments with an overall polymorphism of 70.38%. Five monomorphic loci were generated exclusively in P. pyramidalis accessions, while three unique monomorphic loci were associated with P. bracteosa, and these represented potential species-specific markers. The similarity coefficients between Poincianella accessions were low (mean value 0.59) but with a wide variation (range 0.443 to 0.748). The similarity matrix and the dendrogram constructed using the unweighted pair group method allowed the separation of Poincianella accessions into two major clusters represented by the two distinct species, while the accessions of P. pyramidalis could be separated further into three subgroups. The high level of genetic diversity detected in the genus Poincianella could be used in future breeding programs to produce enhanced cultivars, although the variability could be better exploited if more specimens were collected from other locations within the semiarid region of northeastern Brazil.

A 22-mer primer enhances discriminatory power of AP-PCR fingerprinting technique in characterization of leptospires.

PubMed

Roy, Subarna; Biswas, Debabrata; Vijayachari, P; Sugunan, A P; Sehgal, Subhash C

2004-11-01

To evaluate the discriminatory power and usefulness of arbitrarily primed-polymerase chain reaction (AP-PCR) characterization of leptospires with M16 primer. AP-PCR fingerprints of 20 reference strains of Leptospira representing 20 different serovars belonging to seven genospecies (Leptospira interrogans, 11; L. noguchii, 2; L. borgpetersenii, 1; L. santarosai, 2; L. biflexa, 2; L. kirschneri, 1; L. weilii, 1) were generated by employing M16 primer. Fingerprints generated with this primer were compared with those generated with two other commonly used primers PB1, and L10. An attempt was also made to type 20 leptospiral isolates with the M16 primer. Fingerprints with M16 primer could not only differentiate between strains of different genospecies, but also between strains of the same genospecies belonging to different serovars. While two commonly used primers (PB1 and L10) failed to discriminate between some of the different serovars belonging to the same genospecies, this primer was able to generate discriminatory fingerprints for all strains tested. All 20 Leptospira isolates, recovered from patients in Andaman Islands, could also be typed by fingerprints generated with the M16 primer. The discriminatory power of M16 primer adds more specificity to the rapidity of this system of characterization and can be used as an excellent tool in epidemiological studies on Leptospira.

Targeted mapping and linkage analysis of morphological isozyme, and RAPD markers in peach.

PubMed

Chaparro, J X; Werner, D J; O'Malley, D; Sederoff, R R

1994-02-01

Nine different F2 families of peach [Prunus persica (L.) Batsch] were analyzed for linkage relationships between 14 morphological and two isozyme loci. Linkage was detected between weeping (We) and white flower (W), 33 cM; double flower (Dl) and pillar (Br), 10 cM; and flesh color (Y) and malate dehydrogenase (Mdh1), 26 cM. A leaf variant phenotypically distinct from the previously reported wavy-leaf (Wa) mutant in peach was found in progeny of 'Davie II'. The new willow-leaf character (designated Wa2) was closely linked (0.4 cM) to a new dwarf phenotype (designated Dw3). Two families derived from the pollen-fertile cultivar 'White Glory' segregated for pollen sterility, but segregation did not follow a 3∶1 ratio. Evidence is presented suggesting that 'White Glory' possesses a pollen-sterility gene (designated Ps2) that is non-allelic to the previously reported pollen-sterility gene (Ps) in peach. Ps2 was linked to both weeping (We-Ps2, 15.5 cM) and white flower (Ps2-W, 25.3 cM). A genomic map of peach containing 83 RAPD, one isozyme, and four morphological markers was generated using an F2 family obtained by selfing an NC174RL x 'Pillar' F1. A total of 83 RAPD markers were assigned to 15 linkage groups. Various RAPD markers were linked to morphological traits. Bulked segregant analysis was used to identify RAPD markers flanking the red-leaf (Gr) and Mdh1 loci in the NC174RL x 'Pillar' and 'Marsun' x 'White Glory' F2 families, respectively. Three markers flanking Mdh1 and ten markers flanking Gr were identified. The combination of RAPD markers and bulked segregant analysis provides an efficient method of identifying markers flanking traits of interest. Markers linked to traits that can only be scored late in development are potentially useful for marker-aided selection in trees. Alternatives for obtaining additional map order information for repulsion-phase markers in large F2 populations are proposed.

A population 'consensus', partial linkage map of Picea abies Karst. based on RAPD markers

Treesearch

G. Bucci; Thomas L. Kubisiak; W.L. Nance; P. Menozzi

1997-01-01

The authors built a "consensus" partial linkage map based on RAPD markers using 48 sibships of eight megagametophytes each from a natural population of Norway spruce. A RAPD linkage map for a single individual from the same population had previously been constructed. Using 30 random decamers that had yielded 83 RAPD markers in the single-tree map, eight...

Identification and evaluation of two diagnostic markers linked to Fusarium wilt resistance (race 4) in banana (Musa spp.).

PubMed

Wang, Wei; Hu, Yulin; Sun, Dequan; Staehelin, Christian; Xin, Dawei; Xie, Jianghui

2012-01-01

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (FOC4) results in vascular tissue damage and ultimately death of banana (Musa spp.) plants. Somaclonal variants of in vitro micropropagated banana can hamper success in propagation of genotypes resistant to FOC4. Early identification of FOC4 resistance in micropropagated banana plantlets is difficult, however. In this study, we identified sequence-characterized amplified region (SCAR) markers of banana associated with resistance to FOC4. Using pooled DNA from resistant or susceptible genotypes and 500 arbitrary 10-mer oligonucleotide primers, 24 random amplified polymorphic DNA (RAPD) products were identified. Two of these RAPD markers were successfully converted to SCAR markers, called ScaU1001 (GenBank accession number HQ613949) and ScaS0901 (GenBank accession number HQ613950). ScaS0901 and ScaU1001 could be amplified in FOC4-resistant banana genotypes ("Williams 8818-1" and Goldfinger), but not in five tested banana cultivars susceptible to FOC4. The two SCAR markers were then used to identify a somaclonal variant of the genotype "Williams 8818-1", which lost resistance to FOC4. Hence, the identified SCAR markers can be applied for a rapid quality control of FOC4-resistant banana plantlets immediately after the in vitro micropropagation stage. Furthermore, ScaU1001 and ScaS0901 will facilitate marker-assisted selection of new banana cultivars resistant to FOC4.

Phenotypic and Molecular Aspects of Staphylococcus spp. Isolated from Hospitalized Patients and Beef in the Brazilian Amazon.

PubMed

Pieri, Fabio A; Vargas, Taise F; Galvão, Newton N; Nogueira, Paulo A; Orlandi, Patrícia P

2016-03-01

The aim of this study was to characterize and compare Staphylococcus spp. isolated from hospitalized patients and beef marketed in the city of Porto Velho-RO, Brazil. The isolates were subjected to antibiogram tests, adherence capacity tests, detection of the mecA gene, and epidemiological investigation by the random amplified polymorphic DNA (RAPD) technique, using the primers M13 and H12. Among the 123 Staphylococcus spp. isolates, 50 were identified as S. aureus and 73 as coagulase-negative Staphylococcus; among the latter, 7 species were identified. It was observed that the coagulase-negative Staphylococcus isolates showed greater adhesion ability than S. aureus. The profile of antimicrobial susceptibility was different among isolates, all of which were susceptible to vancomycin and linezolid, and had high penicillin resistance rates, varying according to the bacterial class and the source. In this study, all strains were negative for mecA gene detection; however, 36% of S. aureus and 17% of coagulase-negative Staphylococcus were resistant to oxacillin. The genetic relationship of these bacteria, analyzed by RAPD, was able to discriminate the species of coagulase-negative Staphylococcus strains of S. aureus along its origin. It was concluded that the isolates of Staphylococcus spp. derived from beef and human infections differ genetically. Thus, it is suggested that isolates from beef, which were grouped within hospital isolates, were probably carried via contact with beef in hospital professionals or patients.

Genetic diversity in a Poincianella pyramidalis (Tul.) L.P. Queiroz population assessed by RAPD molecular markers.

PubMed

Belarmino, K S; Rêgo, M M; Bruno, R L A; Medeiros, G D A; Andrade, A P; Rêgo, E R

2017-08-31

Poincianella pyramidalis (Tul.) L.P. Queiroz is an endemic Caatinga (Brazilian savannah biome) species that has been exploited for different purposes, although information is necessary about still existing natural populations. The objective of this study was to evaluate the genetic diversity among 20 P. pyramidalis individuals occurring in a population localized in the Caatinga biome of Paraíba State, aiming at seed collection, using RAPD markers. For the DNA extraction, young shoots of the individuals were used, and amplification was carried out using 20 primers. The obtained markers were converted to a binary matrix, from which a genetic dissimilarity matrix was built using the arithmetic complement of Jaccard's coefficient, and the dendrogram was built by the UPGMA analysis. No amplified fragment was monomorphic, resulting in 100% polymorphism of the analyzed population. The mean genetic diversity among the matrices was 63.28%, ranging from 30.9 to 97.7%. Individuals 09 and 17 showed relevant genetic proximity, and thus planting their seedlings at close sites would not be indicated. The population evaluated in this study showed high genetic diversity, originating twelve groups from the UPGMA hierarchical cluster analysis. Based on the results, individuals 09 and 17 can provide plant material for the evaluation of the physiological performance of P. pyramidalis seeds, and the set of individuals of this population has a high genetic diversity that characterizes them as adequate matrices for projects of restoration and conservation of the seed species.

Characterization and discrimination of three Theileria parva stabilates involved in East Coast fever vaccination.

PubMed

Sparagano, O A; Zanaa, O; Ambrose, N

1998-06-29

Three vaccine stabilates of Theileria parva, of which sporozoites are being used against East Coast fever, were characterized by immunological and molecular biology techniques before being used for a national vaccination campaign in Kenya. T. parva Marikebuni stabilates 316 and 3014, and T. parva Lanet were used in this study and were discriminated from other Kenyan field Theileria isolates. IFAT results showed that all the animals were producing antibodies regardless of the stock used. Primers designed on the TPR1 gene sequence were used for PCR and Decamers were used for RAPD. Specific DNA band patterns (1,877 bp; 1,059 bp, and 443 bp) for the three vaccine stocks were observed. These molecular markers could be used to trace vaccinated animals in Kenya and to identify which isolates are responsible for reactions in animals.

Genotypic intraspecies heterogeneity of Enterococcus italicus: data from dairy environments.

PubMed

Borgo, Francesca; Ferrario, Chiara; Ricci, Giovanni; Fortina, Maria Grazia

2013-01-01

The diversity of a collection of 19 Enterococcus italicus strains isolated from different dairy sources was explored using a molecular polyphasic approach, comprising random amplification of polymorphic DNA (RAPD-PCR), repetitive element PCR (REP-PCR), plasmid profiling and ribotyping. The data obtained showed a high-level of biodiversity, not always correlated to the niche of isolation. Particularly, REP-PCR with primer BOXA1R and plasmid profiling allowed the best discrimination at strain level. Exploiting the genome shotgun sequence of the type strain of the species, available in public database, genes related to insertion sequences present on enterococcal Pathogenic Islands (ISEf1, IS905), determinants related to virulence factors (codifying for hemolysin and cell wall surface proteins), exogenously DNA (conjugal transfer protein, replication plasmid protein, pheromone shutdown protein, phage integrase/recombinase) and penicillin binding proteins system were detected. The presence of most of these genes seemed a common genetic trait in the Enterococcus genus, sur gene (cell wall surface protein) was only detected in strains of E. italicus. To our knowledge, this is the first time that specific primers, with the expection of the species-specific probe targeted to 16S rRNA gene, have been designed for this species. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Effect of BSA on random amplified polymorphic DNA (RAPD) in plants].

PubMed

Bian, Cai-Miao; Li, Jun-Min; Jin, Ze-Xin; Ge, Ming-Ju

2002-05-01

Using Metasequoia glyptostroboides and Heptacodium miconioides DNA as templates,the effect of bovine serum albumin (BSA) on RAPD in plants was studied. The results showed that suitable concentrations of BSA used in Metasequoia glyptostroboides and Heptacodium miconioides RAPD were different, which were 0.6 microg/microl and 1 microg/microl, respectively. The inhibition of acetylated BSA on the amplification of plant RAPD could be relieved by BSA. BSA could reduce the dosage of Taq DNA polymerase.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

PubMed

Tulsiani, S M; Craig, S B; Graham, G C; Cobbold, R C; Dohnt, M F; Burns, M-A; Jansen, C C; Leung, L K-P; Field, H E; Smythe, L D

2010-07-01

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Linkage mapping in a watermelon population segregating for fusarium wilt resistance

Treesearch

Leigh K. Hawkins; Fenny Dane; Thomas L. Kubisiak; Billy B. Rhodes; Robert L. Jarret

2001-01-01

Isozyme, randomly amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers were used to generate a linkage map in an F2 and F3 watermelon (Citrullus lanatus (Thumb.) Matsum. & Nakai) population derived from a cross between the fusarium wilt (Fusarium oxysporum f....

Molecular characterization and biological response to respiration inhibitors of Pyricularia isolates from ctenanthe and rice plants.

PubMed

Paplomatas, Epaminondas J; Pappas, Athanasios C; Syranidou, Elene

2005-07-01

The molecular profile and the biological response of isolates of Pyricularia oryzae Cavara obtained from ctenanthe to two strobilurins (azoxystrobin, kresoxim-methyl) and the phenylpyridinamine fungicide fluazinam were characterized, and compared with isolates from rice plants. Five different isozymes (alpha-esterase, lactate, malate, isocitrate and sorbitol dehydrogenases) and five random decamer primers for RAPD-PCR were used to generate molecular markers. Using unweighted pair-group with arithmetic average analysis, ctenanthe isolates were found to form a separate group distinct from that of the rice isolates for both sets of markers. Amplified polymorphic sequences of mitochondrial cytochrome b that were digested with Fnu4HI or StyI revealed no differences among Pyricularia isolates at amino acid positions 143 or 129 which confer resistance to strobilurins in several fungi. In absence of the alternative respiration inhibitor salicylhydroxamic acid (SHAM) the three fungicides showed inferior and variable efficacy, with a trend toward the rice isolate being less sensitive. The addition of SHAM enhanced the effectiveness of all fungicides against isolates regardless of their origin. Appressorium formation was the most vulnerable target of action of the respiration inhibitors and azoxystrobin the most effective. This is the first report of a comparison between the molecular profiles and sensitivities to respiration inhibitors for Pyricularia oryzae isolates from a non-gramineous host and from rice. Copyright 2005 Society of Chemical Industry

PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

PubMed

Pauthenier, Cyrille; Faulon, Jean-Loup

2014-07-01

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of an RAPD-based SCAR marker for smut disease resistance in commercial sugarcane cultivars of Pakistan

USDA-ARS?s Scientific Manuscript database

Development of RAPD-derived Sequence Characterized Amplified Region (SCAR) marker in order to select Sporisorium scitamineum resistant and susceptible commercial cultivars of sugarcane from Pakistan was achieved. Bulked segregant and RAPD-analysis were conducted using 480 random decamers in initial ...

Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid

PubMed Central

Bhattacharyya, Paromik; Kumaria, Suman; Diengdoh, Reemavareen; Tandon, Pramod

2014-01-01

An efficient genetically stable regeneration protocol with increased phytochemical production has been established for Dendrobium nobile, a highly prized orchid for its economic and medicinal importance. Protocorm like bodies (PLBs) were induced from the pseudostem segments using thidiazuron (TDZ; 1.5 mg/l), by-passing the conventional auxin–cytokinin complement approach for plant regeneration. Although, PLB induction was observed at higher concentrations of TDZ, plantlet regeneration from those PLBs was affected adversely. The best rooting (5.41 roots/shoot) was achieved in MS medium with 1.5 mg/l TDZ and 0.25% activated charcoal. Plantlets were successfully transferred to a greenhouse with a survival rate of 84.3%, exhibiting normal development. Genetic stability of the regenerated plants was investigated using randomly amplified polymorphic DNA (RAPD) and start codon targeted (SCoT) polymorphism markers which detected 97% of genetic fidelity among the regenerants. The PIC values of RAPD and SCoT primers were recorded to be 0.92 and 0.76 and their Rp values ranged between 3.66 and 10, and 4 and 12 respectively. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. A comparative phytochemical analysis among the mother and the micropropagated plants showed a higher yield of secondary metabolites. The regeneration protocol developed in this study provides a basis for ex-situ germplasm conservation and also harnesses the various secondary metabolite compounds of medicinal importance present in D. nobile. PMID:25606433

Comparison of RAPD Linkage Maps Constructed For a Single Longleaf Pine From Both Haploid and Diploid Mapping Populations

Treesearch

Thomas L. Kubisiak; C.Dana Nelson; W.L. Name; M. Stine

1996-01-01

Considerable concern has been voiced regarding the reproducibility/transferability of RAPD markers across different genetic backgrounds in genetic mapping experiments. Therefore, separate gametic subsets (mapping populations) were used to construct individual random amplified polymorphic DNA (RAPD) linkage maps for a single longleaf pine (Pinus palustris...

Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone.

PubMed

Gutacker, Michaela; Conza, Nadine; Benagli, Cinzia; Pedroli, Ambra; Bernasconi, Marco Valerio; Permin, Lise; Aznar, Rosa; Piffaretti, Jean-Claude

2003-06-01

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions I and II). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.

Asymmetric Macular Structural Damage Is Associated With Relative Afferent Pupillary Defects in Patients With Glaucoma

PubMed Central

Gracitelli, Carolina P. B.; Tatham, Andrew J.; Zangwill, Linda M.; Weinreb, Robert N.; Abe, Ricardo Y.; Diniz-Filho, Alberto; Paranhos, Augusto; Baig, Saif; Medeiros, Felipe A.

2016-01-01

Purpose We examined the relationship between relative afferent pupillary defects (RAPDs) and macular structural damage measured by macular thickness and macular ganglion cell-inner plexiform layer (mGCIPL) thickness in patients with glaucoma. Methods A cross-sectional study was done of 106 glaucoma patients and 85 healthy individuals from the Diagnostic Innovations in Glaucoma Study. All subjects underwent standard automated perimetry (SAP) and optic nerve and macular imaging using Cirrus Spectral Domain Optical Coherence Tomography (SDOCT). Glaucoma was defined as repeatable abnormal SAP or progressive glaucomatous changes on stereo photographs. Pupil responses were assessed using an automated pupillometer, which records the magnitude of RAPD (RAPD score), with additional RAPD scores recorded for each of a series of colored stimuli (blue, red, green, and yellow). The relationship between RAPD score and intereye differences (right minus left eye) in circumpapillary retinal nerve fiber layer (cpRNFL) thickness, mGCIPL, macular thickness, and SAP mean deviation (MD), was examined using linear regression. Results There was fair correlation between RAPD score and asymmetric macular structural damage measured by intereye difference in mGCIPL thickness (R2 = 0.285, P < 0.001). The relationship between RAPD score and intereye difference in macular thickness was weaker (R2 = 0.167, P < 0.001). Intereye difference in cpRNFL thickness (R2 = 0.350, P < 0.001) and SAP MD (R2 = 0.594, P < 0.001) had stronger association with RAPD scores compared to intereye difference in mGCIPL and macular thickness. Conclusions Objective assessment of pupillary responses using a pupillometer was associated with asymmetric macular structural damage in patients with glaucoma. PMID:27064394

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

PubMed Central

Ritschel, Patricia Silva; Lins, Tulio Cesar de Lima; Tristan, Rodrigo Lourenço; Buso, Gláucia Salles Cortopassi; Buso, José Amauri; Ferreira, Márcio Elias

2004-01-01

Background Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. Results Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. Conclusions Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon. PMID:15149552

Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation.

PubMed

Trtkova, Jitka; Pavlicek, Petr; Ruskova, Lenka; Hamal, Petr; Koukalova, Dagmar; Raclavsky, Vladislav

2009-11-10

Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification. A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting. Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance.

A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markerswac

Treesearch

Shawn A. Mehlenbacher; Rebecca N. Brown; Eduardo R. Nouhra; Tufan Gokirmak; Nahla V. Bassil; Thomas L. Kubisiak

2006-01-01

A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 x OSU 414.062 was used. RAPD markers in testcross configuration,segregating 1:I, were...

Inheritance of restriction fragment length polymorphisms, random amplified polymorphic DNAs and isozymes in coastal Douglas-fir

Treesearch

K.D. Jermstad; A.M. Reem; J.R. Henifin; N.C. Wheeler; D.B Neale

1994-01-01

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas- fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29...

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

PubMed Central

2013-01-01

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples. PMID:23965160

Genotypic and phenotypic diversity in tropical strains of Aspergillus spp. (section Circumdati) isolated from insects.

PubMed

Moraes, Aurea; Holanda, Veronica; Zahner, Viviane

2006-04-01

The morphology, multilocus enzyme electrophoresis (MLEE), and RAPD-PCR profiles of a panel of 63 strains of Aspergilus section Circumdati, all isoloated from Brazilian insects, were examined. When compared to the descriptions reported in the literature, differences were observed in terms of colony diameter for the representatives studies. Numerical taxonomy based on data generated by MLEE identified two distinct subgroups among the A. ochraceus isolates. In addition, phosphoglucose isomerase (GPI-1) was detected only in A. sclerotiorum, while phosphofructokinase (FK-1) and acid phosphatase (ACP-2) were present only in strains of A. sulphureus, suggesting that these alleles (bands) could be used for species-specific detection. Using RAPD-PCR, species-specific molecular markers were identified for both A. petrakii and A. sulphureus. These results are important from the taxonomic viewpoint and may also be used in the design of screening programs for the isoloation of new strains.

Phenotypic and Molecular Characteristics of Streptococcus agalactiae Isolates Recovered from Milk of Dairy Cows in Brazil

PubMed Central

Duarte, Rafael S.; Miranda, Otávio P.; Bellei, Bruna C.; Brito, Maria Aparecida V. P.; Teixeira, Lúcia M.

2004-01-01

Information on the characteristics of Streptococcus agalactiae obtained from bovine sources in Brazil is still very limited. The aim of this study was to assess the phenotypic and genotypic diversity among S. agalactiae isolates from milk of dairy cows presenting clinical or subclinical mastitis in the southeast region of Brazil. Phenotypic characterization was based on physiological and serological tests. Antimicrobial susceptibility tests were carried out by the disk method. Genetic diversity was evaluated by using random amplified polymorphic DNA-PCR (RAPD-PCR) (by using the primer 1254) and pulsed-field gel electrophoresis (PFGE) (by using SmaI as the restriction enzyme) and by PCRs for detection of genes associated with resistance to erythromycin and tetracycline as well as PCRs for detection of genes coding for cell surface-associated proteins. According to the results of physiologic tests, 45 (52.9%) isolates showed beta-hemolysis and 44 (51.7%) were susceptible to bacitracin. Fourteen different biotypes were detected. The two most frequent biotypes comprised strains that were non-beta-hemolytic; fermented galactose, lactose, and salicin; produced protease; and were negative for DNase production. Serotype III was predominant (66 isolates [77.6%]), followed by serotypes II, Ia, Ib, and VI. Resistance to tetracycline and erythromycin was found in 38 (44.7%) and 9 (10.5%) isolates, respectively, with tet(O) (31.7%) and erm(B) (100%) being the most frequently occurring resistance genes. Three genes coding for surface proteins, bca, lmb, and scpB, were detected in 55 (64.7%), 7 (8.2%), and 43 (50.5%) isolates, respectively. In most cases, isolates from animals in the same herd presented closely related genetic profiles (determined by either RAPD-PCR or PFGE), which were distinct from those of isolates from different herds. PMID:15365014

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil.

PubMed

Qin, X; Miranda, V S; Machado, M A; Lemos, E G; Hartung, J S

2001-06-01

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina.

PubMed

Su, G; Suh, S O; Schneider, R W; Russin, J S

2001-02-01

ABSTRACT To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.

The role of the immunological background of mice in the genetic variability of Schistosoma mansoni as detected by random amplification of polymorphic DNA.

PubMed

Cossa-Moiane, I L; Mendes, T; Ferreira, T M; Mauricio, I; Calado, M; Afonso, A; Belo, S

2015-11-01

Schistosomiasis is a parasitic disease caused by flatworms of the genus Schistosoma. Among the Schistosoma species known to infect humans, S. mansoni is the most frequent cause of intestinal schistosomiasis in sub-Saharan Africa and South America: the World Health Organization estimates that about 200,000 deaths per year result from schistosomiasis in sub-Saharan Africa alone. The Schistosoma life cycle requires two different hosts: a snail as intermediate host and a mammal as definitive host. People become infected when they come into contact with water contaminated with free-living larvae (e.g. when swimming, fishing, washing). Although S. mansoni has mechanisms for escaping the host immune system, only a minority of infecting larvae develop into adults, suggesting that strain selection occurs at the host level. To test this hypothesis, we compared the Belo Horizonte (BH) strain of S. mansoni recovered from definitive hosts with different immunological backgrounds using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). Schistosoma mansoni DNA profiles of worms obtained from wild-type (CD1 and C57BL/6J) and mutant (Jα18- / - and TGFβRIIdn) mice were analysed. Four primers produced polymorphic profiles, which can therefore potentially be used as reference biomarkers. All male worms were genetically distinct from females isolated from the same host, with female worms showing more specific fragments than males. Of the four host-derived schistosome populations, female and male adults recovered from TGFβRIIdn mice showed RAPD-PCR profiles that were most similar to each other. Altogether, these data indicate that host immunological backgrounds can influence the genetic diversity of parasite populations.

Molecular performance of commercial MTG variety oil palm based on RAPD markers

NASA Astrophysics Data System (ADS)

Putri, L. A. P.; Setyo, I. E.; Basyuni, M.; Bayu, E. S.; Setiado, H.; Reynaldi, N. F.; Laia, H.; Puteri, S. A. K.; Arifiyanto, D.; Syahputra, I.

2018-02-01

The oil palm, an economically important tree in Indonesia, has been one of the world’s major sources of edible oil and a significant precursor of biodiesel fuel. This research is conducted by taking individual tree sample of commercial MTG variety germplasm oil palm one years old. The purpose of this research is to analyse molecular performance of some oil palm MTG variety based on RAPD markers. In this experiment, the DNA profile diversity was assessed using markers of oil palm’s random RAPD markers (OPD-20, SB-19, OPM-01 and OPO-11). A total of 15 trees commercial MTG oil palm variety were used for analysis. The results of the experiment indicated out of 4 RAPD markers (OPD-20, SB-19, OPM-01 and OPO-11) showed polymorphic of PCR product. These preliminary results demonstrated RAPD marker can be used to evaluate genetic relatedness among trees of commercial MTG variety oil palm and detecting either genetic variants or mislabelled.

Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

PubMed

Jarocki, Piotr; Podleśny, Marcin; Komoń-Janczara, Elwira; Kucharska, Jagoda; Glibowska, Agnieszka; Targoński, Zdzisław

2016-07-22

Members of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties. Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources. This work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also demonstrated a high discriminatory power of SDS-PAGE fingerprinting of whole-cell proteins. On the other hand, the protein profiles obtained were rather complex, and therefore, difficult to analyze. Among the tested procedures, rep-PCR proved to be the most effective and reliable method allowing rapid differentiation of Bifidobacterium strains. Additionally, the use of the BOXA1R primer in the differentiation of 21 Bifidobacterium strains, newly isolated from infant feces, demonstrated slightly better discriminatory power in comparison to PCR reactions with the (GTG)5 oligonucleotide. Thus, BOX-PCR turned out to be the most appropriate and convenient molecular technique in differentiating Bifidobacterium strains at all taxonomic levels.

Epidemiology and RAPD-PCR typing of thermophilic campylobacters from children under five years and chickens in Morogoro Municipality, Tanzania.

PubMed

Chuma, Idrissa S; Nonga, Hezron E; Mdegela, Robinson H; Kazwala, Rudovick R

2016-11-21

Campylobacter species are gram negative and flagellated bacteria under the genus Campylobacter, family Campylobacteriaceae. These pathogens cause zoonotic infections among human and animal populations. This study was undertaken between December 2006 and May 2007 to determine prevalence, risk factors and genetic diversity of thermophilic Campylobacter isolates from children less than 5 years and chickens in Morogoro Municipality, Tanzania. The Skirrow's protocol was used for isolation and identification of Campylobacter from 268 human stool specimens and 419 chicken cloacal swabs. Patient biodata and risk factors associated with human infection were also collected. Genetic diversity of Campylobacter isolates was determined by a RAPD-PCR technique using OPA 11 primer (5'-CAA TCG CCG T-3'). Phylogenetic analysis and band pattern comparison were done by Bionumerics software and visual inspection. Stool samples from 268 children and 419 cloacal swabs from chickens were analyzed. Prevalence of thermophilic Campylobacters in children was 19% with higher isolation frequency (p = 0.046) in males (23.5%) than females (13.8%). Campylobacter jejuni (78.4%) was more isolated (p = 0.000) than C. coli (19.6%) and 2% were unidentified isolates. In chickens, the prevalence was 42.5% with higher isolation rate (p = 0.000) of C. jejuni (87%) than C. coli (13%). Campylobacters were more frequently recovered (p = 0.000) from indigenous/ local chickens (75.0%) followed by cockerels (52.2%), broilers (50.0%) and lowest in layers (22.7%). Keeping chickens without other domestic animals concurrently (p = 0.000), chicken types (p = 0.000) and flock size (p = 0.007) were risk factors for infection in chickens. One hundred and fifty two (152) thermophillic Campylobacter isolates were genotyped by RAPD-PCR of which 114 were C. jejuni (74 from chickens and 40 humans) and 38 C. coli (28 from chickens and 10 humans). Comparison of Campylobacter isolates from children and chickens revealed high diversity with only 6.1% of C. jejuni and 5.3% of C. coli being 100% genetically similar. This study has recorded high prevalence of thermophilic Campylobacter in children less than 5 years and chickens in Morogoro municipality. The observed genetic similarity among few C. jejuni and C. coli isolates from children and chicken suggests existence of cross transmission of these pathogens between children under 5 years and chickens.

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis▿

PubMed Central

Hizi, Amnon

2008-01-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs. PMID:18753200

The reverse transcriptase of the Tf1 retrotransposon has a specific novel activity for generating the RNA self-primer that is functional in cDNA synthesis.

PubMed

Hizi, Amnon

2008-11-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

Experimental infection of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B.

PubMed

Sanei, B; Barnes, H J; Vaillancourt, J P; Leyc, D H

2007-03-01

During an epidemic of mycoplasmosis in chicken and turkey flocks in North Carolina between 1999 and 2001, isolates of Mycoplasma gallisepticum (MG) from affected flocks were characterized by random amplification of polymorphic DNA (RAPD), and eight distinct RAPD types were identified. MG RAPD type B accounted for more than 90% of the isolates and was associated with moderate-to-severe clinical signs and mortality. The virulence of MG RAPD type B for chickens and turkeys was compared with sham-inoculated negative controls and MG S6 (a virulent strain)-inoculated positive controls. Clinical signs occurred in chickens and turkeys inoculated with either MG RAPD type B or MG S6. However, they were not as frequent or severe as those seen in naturally affected flocks, and there was no mortality in the experimental groups. Based on gross and microscopic findings, MG RAPD type B was equal to or more virulent than MG S6. All MG-inoculated birds were culture and PCR positive at 7 and 14 days postinoculation (PI). Among serological tests, the serum plate agglutination test was positive for the majority of chickens and turkeys (58%-100%) infected with either strain of MG at both 7 and 14 days PI. The hemagglutination inhibition test was negative for all birds at 7 days PI and positive for a few chickens (8%-17%) and several turkey sera (40%-60%) at 14 days PI. Only a single serum was positive by enzyme-linked immunosorbent assay (an MG S6-infected turkey) at 14 days PI.

Analysis of isolates within species of anuran trypanosomes using random amplified polymorphic DNA.

PubMed

Lun, Z R; Desser, S S

1996-01-01

A total of 20 decamer primers were used to generate random applied polymorphic DNA (RAPD) markers from 5 isolates of Trypanosoma fallisi, 3 isolates of T. ranarum, 2 isolates of T. rotatorium, and 2 isolates of T. rotatorium-like trypanosomes in addition to 2 species from the American Type Culture Collection, T. chattoni (ATCC 50294) and Trypanosoma sp. (ATCC 50295). A slight polymorphism was observed among the four isolates of T. fallisi obtained form American toads, Bufo americanus, collected in Algonquin Park, Ontario, Canada, and an isolate obtained from the same species of host collected in Marquette, Michigan, United States, and produced similarity coefficients ranging from 80.7% to 96.9%. Pronounced polymorphism was recorded among the three isolates of T. ranarum from bullfrogs, Rana catesbeiana, collected in Ontario, Canada, and in Maryland, United States, and from a Northern leopard frog, R. pipiens, collected in Minnesota (USA). The similarity coefficients ranged from 54.7% to 59.5%, suggesting that alleles of these isolates were conserved over a wide geographic range. The high degree of polymorphism observed in two isolates of T. rotatorium from a bullfrog collected in Ontario and two isolates of a T. rotatorium-like parasite from the green frog R. clamitans, collected in Louisiana (USA) suggests that they are different species. These results reflect the high similarity among isolates from the same geographic location and the pronounced polymorphism apparent among isolates from distant geographic locations.

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia.

PubMed

Amal, M N A; Zamri-Saad, M; Siti-Zahrah, A; Zulkafli, A R; Nur-Nazifah, M

2013-07-01

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques. A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP-PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP-PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods. Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation. Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

NASA Astrophysics Data System (ADS)

Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

2017-04-01

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Rapid molecular identification and characteristics of Lactobacillus strains.

PubMed

Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

2010-09-01

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines.

PubMed

Agnolucci, M; Vigentini, I; Capurso, G; Merico, A; Tirelli, A; Compagno, C; Foschino, R; Nuti, M

2009-04-15

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l(-1)), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.

A sex-averaged genetic linkage map in coastal Douglas-fir (Pseudotsuga menziesii [Mirb] Franco var menziesii) based on RFLP and RAPD markers

Treesearch

K.D. Jermstad; D.L. Bassoni; N.C. Wheeler; D.B. Neale

1998-01-01

We have constructed a sex-averaged genetic linkage map in coastal Douglas-fir ( Pseudotsuga menziesii [Mirb.] Franco var menziesii) using a three-generation outcrossed pedigree and molecular markers. Our research objectives are to learn about genome organization and to identify markers associated with adaptive traits. The map...

RAPD- and ERIC-Based Typing of Clinical and Environmental Pseudomonas aeruginosa Isolates.

PubMed

Auda, Ibtesam Ghadban; Al-Kadmy, Israa M S; Kareem, Sawsan Mohammed; Lafta, Aliaa Khyuon; A'Affus, Mustafa Hussein Obeid; Khit, Ibrahim Abd Aloahd; Al Kheraif, Abdulaziz Abdullah; Divakar, Darshan Devang; Ramakrishnaiah, Ravikumar

2017-03-01

Pseudomonas aeruginosa is a major cause of nosocomial infection in children and adults, resulting in significant morbidity and mortality due to its ability to acquire drug resistance. The ability of P. aeruginosa in the environment to cause infection in individuals has been reported previously; henceforth, surveillance of the emergence and transmission of P. aeruginosa strains among patients is important for infection control in a clinical setup. Various gene-typing methods have been used for epidemiological typing of P. aeruginosa isolates for the purpose of surveillance. In this work, the suitability and comparability of two typing methods, enterobacterial repetitive intergenic consensus (ERIC)-PCR and random amplification of polymorphic DNA (RAPD)-PCR fingerprinting, were studied to characterize P. aeruginosa strains isolated from clinical and environmental sources. Forty-four clinical and environmental bacterial isolates of P. aeruginosa were collected between October 2015 and January 2016. DNA extraction, ERIC-PCR and RAPD-PCR, agarose gel electrophoresis, and phylogenetic analyses were carried using the unweighted pair-group method with mean. RAPD typing revealed less clonality among clinical isolates, whereas the ERIC method showed greater similarity in comparison with RAPD. Environmental isolates, however, showed greater similarity using RAPD compared with ERIC typing. With only a few exceptions, most clinical isolates were distinct from environmental isolates, irrespective of the typing method. In conclusion, both the RAPD and ERIC typing methods proved to be good tools in understanding clonal diversity. The results also suggest that there is no relationship between clinical and environmental isolates. The absence of clonality among the clinical isolates may indicate that most P. aeruginosa infection cases could be endemic and not epidemic and that endemic infections may be due to nonclonal strains of P. aeruginosa.

Genetic diversity of Schistosoma haematobium parasite IS NOT associated with severity of disease in an endemic area in Sudan.

PubMed

Gasmelseed, Nagla; Karamino, Nhashal E; Abdelwahed, Mohammed O; Hamdoun, Anas O; Elmadani, Ahmed E

2014-08-27

Over 650 million people globally are at risk of schistosomiasis infection, while more than 200 million people are infected of which the higher disease rates occur in children. Eighty three students between 6-20 years (mean 12.45 ± 3.2) from Quran School for boys in Radwan village, Gezira state were recruited to investigate for the relationship between the genetic diversity of Schistosoma haematobium strains and the severity of the disease. Schistosoma haematobium infection was detected by filtration of urine. Ultrasonography was done on each study subject, while PCR technique was used for genotyping via random amplified polymorphic DNA (RAPD) with A01, A02, A12, Y20 and A13 primers. A01 primer gave three different genotypes (A01-1, A01-2 and A01-3). About 54.2% (45/83) were S. haematobium egg positive by urine filtration. On assessment of the upper and lower urinary tract by ultrasound technique, 61.4% (51/83) were positiveand73.3% (60/83) samples were PCR positive. No significant difference was found when comparing the three different genotypes with severity of the disease. This study concludes that no association was found between the different genotypes of S.haemtobium and the severity of the disease. Examination of more samples from different areas to identify any possible differences between the parasites genes and disease severity was recommended.

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Treesearch

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

Genotoxicity Evaluation of an Urban River on Freshwater Planarian by RAPD Assay.

PubMed

Zhang, He-Cai; Liu, Tong-Yi; Shi, Chang-Ying; Chen, Guang-Wen; Liu, De-Zeng

2017-04-01

The aim of this study was to evaluate the genotoxic potential of an urban river - the Wei River in Xinxiang, China using randomly amplified polymorphic DNA (RAPD) assay in planarians. The results showed that the total number of polymorphic bands and varied bands in RAPD patterns of treated planarians decreased with the water sample site far away from the sewage outlet of a factory. In addition, the genome template stability of treated groups decreased and the degree of the decline was negatively related to the distance between the sample site and the sewage outlet, suggesting that the Wei River water had genotoxicity effects on planarians and strengthening the management of the Wei River was necessary. Furthermore, this work also indicated that RAPD assay in planarians was a very promising test for environmental monitoring studies.

CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

PubMed

Yan, Meng; Zhou, Shi-Rong; Xue, Hong-Wei

2015-07-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system. © 2014 Institute of Botany, Chinese Academy of Sciences.

UniPrime2: a web service providing easier Universal Primer design.

PubMed

Boutros, Robin; Stokes, Nicola; Bekaert, Michaël; Teeling, Emma C

2009-07-01

The UniPrime2 web server is a publicly available online resource which automatically designs large sets of universal primers when given a gene reference ID or Fasta sequence input by a user. UniPrime2 works by automatically retrieving and aligning homologous sequences from GenBank, identifying regions of conservation within the alignment, and generating suitable primers that can be used to amplify variable genomic regions. In essence, UniPrime2 is a suite of publicly available software packages (Blastn, T-Coffee, GramAlign, Primer3), which reduces the laborious process of primer design, by integrating these programs into a single software pipeline. Hence, UniPrime2 differs from previous primer design web services in that all steps are automated, linked, saved and phylogenetically delimited, only requiring a single user-defined gene reference ID or input sequence. We provide an overview of the web service and wet-laboratory validation of the primers generated. The system is freely accessible at: http://uniprime.batlab.eu. UniPrime2 is licenced under a Creative Commons Attribution Noncommercial-Share Alike 3.0 Licence.

Sequenced RAPD markers to detect hybridization in the barbary partridge (Alectoris barbara, Phasianidae).

PubMed

Barbanera, Filippo; Guerrini, Monica; Bertoncini, Franco; Cappelli, Fabio; Muzzeddu, Marco; Dini, Fernando

2011-01-01

In the Alectoris partridges (Phasianidae), hybridization occurs occasionally as a result of the natural breakdown of isolating mechanisms but more frequently as a result of human activity. No genetic record of hybridization is known for the barbary partridge (A. barbara). This species is distributed mostly in North Africa and, in Europe, on the island of Sardinia (Italy) and on Gibraltar. The risk of hybridization between barbary and red-legged partridge (A. rufa: Iberian Peninsula, France, Italy) is high in Sardinia and in Spain. We developed two random amplified polymorphic DNA (RAPD) markers to detect A. barbara × A. rufa hybrid partridges. We tested them on 125 experimental hybrids, sequenced the relative species-specific bands and found that the bands and their corresponding sequences were reliably transmitted through a number of generations (F1, F2, F3, BC1, BC2). Our markers represent a highly valuable tool for the preservation of the A. barbara genome from the pressing threat of A. rufa pollution. © 2010 Blackwell Publishing Ltd.

[The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei].

PubMed

Antonov, V A; Altukhova, V V; Savchenko, S S; Zamaraev, V S; Iliukhin, V I; Alekseev, V V

2007-01-01

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.

Molecular characterization and identification of markers for toxic and non-toxic varieties of Jatropha curcas L. using RAPD, AFLP and SSR markers.

PubMed

Sudheer Pamidimarri, D V N; Singh, Sweta; Mastan, Shaik G; Patel, Jalpa; Reddy, Muppala P

2009-07-01

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).

Intra-specific genetic diversity in wild olives (Olea europaea ssp cuspidata) in Hormozgan Province, Iran.

PubMed

Noormohammadi, Z; Samadi-Molayousefi, H; Sheidai, M

2012-03-19

Wild olive (O. europaea ssp cuspidata) plants grow in various regions of Iran and are expected to have considerable genetic diversity due to adaptation to the various environmental conditions. We examined the genetic diversity of four populations of wild olive growing in Hormozgan Province located in southern Iran by using 30 RAPDs and 10 ISSR markers. The mean value of polymorphism for RAPD loci was 73.71%, while the value for ISSR loci was 81.74%. The Keshar population had the highest value of intra-population polymorphism for both RAPD and ISSR loci (66.86 and 62.71%, respectively), while the Tudar population had the lowest values (20.35 and 28.81%, respectively). Similarly, the highest and lowest number of effective alleles, Shannon index and Nei's genetic diversity were also found for these two populations. The highest value of H(pop)/H(sp) within population genetic diversity for RAPD and ISSR loci was found for the Keshar population (H(pop) = 0.85 and H(sp) = 0.90). OPA04-750, OPA13-650 and OPA02-350 RAPD bands were specific for Tudar, Bondon and Keshar populations, respectively, while no specific ISSR bands were observed. Analysis of molecular variance as well as the pairwise F(ST) test showed significant differences for RAPD and ISSR markers among the populations. The NJ and UPGMA trees also separated the wild olive populations from each other, indicating their genetic distinctness. UPGMA clustering of the four wild olive populations placed the Tudar population far from the other populations; Keshar and Bokhoon population samples revealed more similarity and were grouped together. We conclude that there is high genetic diversity among O. europaea ssp cuspidata populations located in southern Iran. We also found RAPD and ISSR markers to be useful molecular tools to discriminate and evaluate genetic variations in wild olive trees.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

PubMed Central

Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

2016-01-01

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

Reforming Science Instruction in California: A Primer

ERIC Educational Resources Information Center

EdSource, 2017

2017-01-01

As schools across the state implement the Next Generation Science Standards, this EdSource primer provides an easy-to-read guide for parents and other community members to understand the rationale for the standards and their potential to affect science instruction in California schools. California adopted the Next Generation Science Standards, or…

Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.

PubMed

Zhang, Huimin; He, Hongkui; Yu, Xiujuan; Xu, Zhaohui; Zhang, Zhizhou

2016-11-01

It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

Determination of the genetic diversity among accessions of Senna spectabilis (canafístula) by using RAPD markers.

PubMed

Santos, M F; Araújo Neto, R B; Nascimento, M P S B C; Lima, P S C

2013-12-02

Senna spectabilis (DC.) H.S. Irwin & Barneby (Fabaceae; Caesalpinioideae), commonly known as "canafístula" or "cassia", is widely used in the semi-arid region of northeastern Brazil as a source of forage and timber. The plant presents a high nutritional content in comparison with other forage species that are native to the Brazilian Caatinga; thus, it represents a valuable resource during periods of drought. The aim of this study was to evaluate the genetic variability among eight accessions of S. spectabilis available in the forage germplasm collection of Embrapa Meio-Norte using the random-amplified polymorphic DNA technique. The 15 primers selected for use in the analysis produced 107 bands, including 59 (55.14%) that were polymorphic. A similarity matrix was generated on the basis of Jaccard coefficients, and a dendrogram was constructed using the unweighted pair group method with arithmetic mean clustering technique. The mean value of the similarity coefficients was 0.73, and the cophenetic correlation coefficient was 83.76%. Accessions CAN. 4 and CAN. 5 presented the greatest genetic similarity, while CAN. 6 and CAN. 8 were the most divergent. The S. spectabilis accessions were classified into two main groups with group I including accessions CAN. 1, CAN. 2, CAN. 4, CAN. 5, CAN. 7, CAN. 8, and CAN. 9, and group II comprising the single accession CAN. 6. The results presented herein revealed that, although the germplasm collection is presently limited, there is sufficient genetic variability among the accessions to permit future breeding programs.

Genetic (RAPD) diversity between Oleria onega agarista and Oleria onega ssp. (Ithomiinae, Nymphalidae, Lepidoptera) in north-eastern Peru.

PubMed

Gallusser, S; Guadagnuolo, R; Rahier, M

2004-05-01

Oleria onega agarista Felder and Felder and Oleria onega ssp. nov. are two Ithomiinae subspecies from north-eastern Peru, that differ for some morphological and behavioural traits. Two contact zones are known near the town of Tarapoto: Ahuashiyacu, where both subspecies cohabit but do not seem to hybridise, and Estero (near the village of Shapaja), where they apparently hybridise. Genetic differences between the two subspecies and between populations were investigated with random amplified polymorphic DNA (RAPD) markers. Both Cluster and Principal Coordinates Analyses (CCoA and PCoA) performed using these data, provided a clear but weak discrimination between the two subspecies. Genetic diversity is much higher within the populations than between them. Moreover, the geographically more distant populations are grouped together by the genetic data. Morphological traits on the wing patterns of the hybrids are intermediary between the two butterflies subspecies, while RAPDs data place them closer to O. onega agarista than to O. onega ssp. The individuals of the Ahuashiyacu population are clearly separated into two groups, those of O. onega ssp. and O. onega agarista, by both morphology and RAPDs data. Moreover, none of those individuals show RAPD similarity with the hybrids, suggesting that hybridisation has not occurred in this population.

Human milk is a source of lactic acid bacteria for the infant gut.

PubMed

Martín, Rocío; Langa, Susana; Reviriego, Carlota; Jimínez, Esther; Marín, María L; Xaus, Jordi; Fernández, Leonides; Rodríguez, Juan M

2003-12-01

To investigate whether human breast milk contains potentially probiotic lactic acid bacteria, and therefore, whether it can be considered a synbiotic food. Study design Lactic acid bacteria were isolated from milk, mammary areola, and breast skin of eight healthy mothers and oral swabs and feces of their respective breast-fed infants. Some isolates (178 from each mother and newborn pair) were randomly selected and submitted to randomly amplified polymorphic DNA (RAPD) polymerase chain reaction analysis, and those that displayed identical RAPD patterns were identified by 16S rDNA sequencing. Within each mother and newborn pair, some rod-shaped lactic acid bacteria isolated from mammary areola, breast milk, and infant oral swabs and feces displayed identical RAPD profiles. All of them, independently from the mother and child pair, were identified as Lactobacillus gasseri. Similarly, among coccoid lactic acid bacteria from these different sources, some shared an identical RAPD pattern and were identified as Enterococcus faecium. In contrast, none of the lactic acid bacteria isolated from breast skin shared RAPD profiles with lactic acid bacteria of the other sources. Breast-feeding can be a significant source of lactic acid bacteria to the infant gut. Lactic acid bacteria present in milk may have an endogenous origin and may not be the result of contamination from the surrounding breast skin.

Phylogenetic analysis of different breeds of domestic chickens in selected area of Peninsular Malaysia inferred from partial cytochrome b gene information and RAPD markers.

PubMed

Yap, Fook Choy; Yan, Yap Jin; Loon, Kiung Teh; Zhen, Justina Lee Ning; Kamau, Nelly Warau; Kumaran, Jayaraj Vijaya

2010-10-01

The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.

Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains

USDA-ARS?s Scientific Manuscript database

Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

Novel Primer Sets for Next Generation Sequencing-Based Analyses of Water Quality

PubMed Central

Lee, Elvina; Khurana, Maninder S.; Whiteley, Andrew S.; Monis, Paul T.; Bath, Andrew; Gordon, Cameron; Ryan, Una M.; Paparini, Andrea

2017-01-01

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination. PMID:28118368

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

PubMed Central

Zhou, Shuntai; Jones, Corbin; Mieczkowski, Piotr

2015-01-01

ABSTRACT Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCE Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. PMID:26041299

Metabolic and structural response of hyporheic microbial communities to variations in supply of dissolved organic matter

USGS Publications Warehouse

Findlay, S.E.G.; Sinsabaugh, R. L.; Sobczak, W.V.; Hoostal, M.

2003-01-01

Hyporheic sediment bacterial communities were exposed to dissolved organic matter (DOM) from a variety of sources to assess the interdependence of bacterial metabolism and community composition. Experiments ranged from small-scale core perfusions with defined compounds (glucose, bovine serum albumin) to mesocosms receiving natural leaf leachate or water from different streams. Response variables included bacterial production, oxygen consumption, extracellular enzyme activity, and community similarity as manifest by changes in banding patterns of randomly amplified polymorphic DNA (RAPD). All DOM manipulations generated responses in at least one metabolic variable. Additions of both labile and recalcitrant materials increased either oxygen consumption, production, or both depending on background DOM. Enzyme activities were affected by both types of carbon addition with largest effects from the labile mixture. Cluster analysis of RAPD data showed strong divergence of communities exposed to labile versus recalcitrant DOM. Additions of leaf leachate to mesocosms representing hyporheic flow-paths caused increases in oxygen consumption and some enzyme activities with weaker effects on production. Community structure yeas strongly affected; samples from the leachate-amended mesocosms clustered separately from the control samples. In mesocosms receiving water from streams ranging in DOC (0.5-4.5 mg L-1), there were significant differences in bacterial growth, oxygen consumption, and enzyme activities. RAPD analysis showed strongest clustering of samples by stream type with more subtle effects of position along the flowpaths. Responses in community metabolism were always accompanied by shifts in community composition, suggesting carbon supply affects both functional and structural attributes of hyporheic bacterial communities.

Genetic stability assessment of wWasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis.

PubMed

Matsumoto, Toshikazu; Akihiro, Takashi; Maki, Shinya; Mochida, Kouhei; Kitagawa, Masaru; Tanaka, Daisuke; Yamamoto, Shin-Ichi; Niino, Takao

2013-01-01

This study compared the effect of cryopreserved storage duration of wasabi shoot tips, which derived from the same in vitro mother-plant. We compared the survival of shoot tips and the genetic stability of regenerated plants originating from four experimental groups: shoot tips stored in a -150°C deep-freezer for 10 years; shoot tips stored in liquid nitrogen for 2 h; shoot tips treated with PVS2 vitrification solution; and untreated controls. No significant difference in survival was observed between the four experimental groups. Survival ranged between 93 and 100%. Genetic stability of plants regenerated from cryopreserved shoot tips was assessed over a period of 24 months using morphological, biochemical and molecular markers. While glucose, fructose and glutamic acid concentrations differed slightly between experimental groups after 16 months, these differences disappeared after 24 months. No significant differences were noted for the morphological markers studied (petiole length, shoot number and leaf index). No differences were observed in RAPD profiles obtained with the six primers tested.

Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

PubMed

Caetano-Anollés, G; Gresshoff, P M

1996-06-01

DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae)

PubMed Central

2012-01-01

Background Japanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians. Results Enriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility. Conclusions This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. PMID:23186361

Fine mapping of the rice Bph1 gene, which confers resistance to the brown planthopper (Nilaparvata lugens stal), and development of STS markers for marker-assisted selection.

PubMed

Cha, Young-Soon; Ji, Hyeonso; Yun, Doh-Won; Ahn, Byoung-Ohg; Lee, Myung Chul; Suh, Seok-Cheol; Lee, Chun Seok; Ahn, Eok Keun; Jeon, Yong-Hee; Jin, Il-Doo; Sohn, Jae-Keun; Koh, Hee-Jong; Eun, Moo-Young

2008-08-31

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

RAPD study on some common species of Porphyra in China

NASA Astrophysics Data System (ADS)

Kuang, Mei; Wang, Su-Juan; Li, Yao; Shen, Da-Leng; Zeng, Cheng-Kui

1998-03-01

RAPD analysis of seven samples of five Porphyra species, P haitanensis (three samples of cultured population), P. katadai var. hemiphylla, P. oligospermatangia, P. suborbiculata and P. yezoensis, showed the closest relationship existing among the three cultured populations of P. haitanensis. The genetic distance between P. haitanensis and P. oligospermatangia was the same as that between P. haitanensis and P. suborbiculata, both were 0.9. The genetic distances, among the other species of Porphyra ranged from 0.7 to 0.8. UPGMA analysis showed P. suborbiculata and P. yezoensis belong to another lineage. Results of this study suggests that RAPD analysis is effective at population level.

Comparison of Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Aquatic Bacterial Communities: an Ecological Perspective▿

PubMed Central

Jones, Stuart E.; Shade, Ashley L.; McMahon, Katherine D.; Kent, Angela D.

2007-01-01

Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used. PMID:17122397

Identification of RAPD marker associated with brown rust resistance in sugarcane

USDA-ARS?s Scientific Manuscript database

Susceptibility to brown rust caused by Puccinia melanocephala is a major reason for the withdrawal of sugarcane cultivars from production. An efficient way to control the disease is to breed cultivars with durable resistance. Our aim was to identify random amplified polymorphic DNA (RAPD) markers ...

Polymorphic sequence-characterized codominant loci in the chestnut blight fungus, Cryphonectria parasitica

Treesearch

J. E. Davis; Thomas L. Kubisiak; M. G. Milgroom

2005-01-01

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we described the development of 11 condominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were...

Molecular Characterization of Cultivated Pawpaw (Asimina triloba) Using RAPD Markers

Treesearch

Hongwen Huang; Desmond R. Layne; Thomas L. Kubisiak

2003-01-01

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely...

Evaluation of different primers for PCR-DGGE analysis of cheese-associated enterococci.

PubMed

Lorbeg, Petra Mohar; Majhenic, Andreja Canzek; Rogelj, Irena

2009-08-01

Enterococci represent an important part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis published so far, six primer pairs amplifying different variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Enterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstrates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distinctive DGGE fingerprints, but with multiple bands patterns; therefore, these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used.

A simple approach to the generation of heterologous competitive internal controls for real-time PCR assays on the LightCycler.

PubMed

Stöcher, Markus; Leb, Victoria; Hölzl, Gabriele; Berg, Jörg

2002-12-01

The real-time PCR technology allows convenient detection and quantification of virus derived DNA. This approach is used in many PCR based assays in clinical laboratories. Detection and quantification of virus derived DNA is usually performed against external controls or external standards. Thus, adequacy within a clinical sample is not monitored for. This can be achieved using internal controls that are co-amplified with the specific target within the same reaction vessel. We describe a convenient way to prepare heterologous internal controls as competitors for real-time PCR based assays. The internal controls were devised as competitors in real-time PCR, e.g. LightCycler-PCR. The bacterial neomycin phosphotransferase gene (neo) was used as source for heterologous DNA. Within the neo gene a box was chosen containing sequences for four differently spaced forward primers, one reverse primer, and a pair of neo specific hybridization probes. Pairs of primers were constructed to compose of virus-specific primer sequences and neo box specific primer sequences. Using those composite primers in conventional preparative PCR four types of internal controls were amplified from the neo box and subsequently cloned. A panel of the four differently sized internal controls was generated and tested by LightCycler PCR using their virus-specific primers. All four different PCR products were detected with the single pair of neo specific FRET-hybridization probes. The presented approach to generate competitive internal controls for use in LightCycler PCR assays proved convenient und rapid. The obtained internal controls match most PCR product sizes used in clinical routine molecular assays and will assist to discriminate true from false negative results.

An equivalent method of mixed dielectric constant in passive microwave/millimeter radiometric measurement

NASA Astrophysics Data System (ADS)

Su, Jinlong; Tian, Yan; Hu, Fei; Gui, Liangqi; Cheng, Yayun; Peng, Xiaohui

2017-10-01

Dielectric constant is an important role to describe the properties of matter. This paper proposes This paper proposes the concept of mixed dielectric constant(MDC) in passive microwave radiometric measurement. In addition, a MDC inversion method is come up, Ratio of Angle-Polarization Difference(RAPD) is utilized in this method. The MDC of several materials are investigated using RAPD. Brightness temperatures(TBs) which calculated by MDC and original dielectric constant are compared. Random errors are added to the simulation to test the robustness of the algorithm. Keywords: Passive detection, microwave/millimeter, radiometric measurement, ratio of angle-polarization difference (RAPD), mixed dielectric constant (MDC), brightness temperatures, remote sensing, target recognition.

SP-Designer: a user-friendly program for designing species-specific primer pairs from DNA sequence alignments.

PubMed

Villard, Pierre; Malausa, Thibaut

2013-07-01

SP-Designer is an open-source program providing a user-friendly tool for the design of specific PCR primer pairs from a DNA sequence alignment containing sequences from various taxa. SP-Designer selects PCR primer pairs for the amplification of DNA from a target species on the basis of several criteria: (i) primer specificity, as assessed by interspecific sequence polymorphism in the annealing regions, (ii) the biochemical characteristics of the primers and (iii) the intended PCR conditions. SP-Designer generates tables, detailing the primer pair and PCR characteristics, and a FASTA file locating the primer sequences in the original sequence alignment. SP-Designer is Windows-compatible and freely available from http://www2.sophia.inra.fr/urih/sophia_mart/sp_designer/info_sp_designer.php. © 2013 John Wiley & Sons Ltd.

Genetic variability among the brown rust resistant and susceptible genotypes of sugarcane by RAPD technique

USDA-ARS?s Scientific Manuscript database

Brown leaf rust in sugarcane is caused by Puccinia melanocephala (Syd. & P. Syd.), which is major cause of cultivar withdrawal. We attempted to analyze the RAPD diversity of two discrete phenotypic classes i.e. rust resistant (R) and rust susceptible (S) of six commercially available sugarcane elite...

Trypanosoma rangeli: RAPD-PCR and LSSP-PCR analyses of isolates from southeast Brazil and Colombia and their relation with KPI minicircles.

PubMed

Marquez, D S; Ramírez, L E; Moreno, J; Pedrosa, A L; Lages-Silva, E

2007-09-01

This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

Study of the coumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) isolates.

PubMed

Godoy, L; Garrido, D; Martínez, C; Saavedra, J; Combina, M; Ganga, M A

2009-04-01

To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern. CD and VR activities were quantified, as well, the growth rate, intracellular protein profile and molecular analysis (RAPD) were determined in 12 isolates of D. bruxellensis. All the isolates studied showed CD activity, but only some showed VR activity. Those isolates with the greatest growth rate did not present a different protein profile from the others. The FASC showed a relationship between RAPD molecular patterns and VR activity. CD activity is common to all of the D. bruxellensis isolates. This was not the case with VR activity, which was detected at a low percentage in the analysed micro-organisms. A correlation was observed between VR activity and the RAPD patterns. This is the first study that quantifies the CD and VR enzyme activities in D. bruxellensis, demonstrating that these activities are not present in all isolates of this yeast.

A DNA-Based Procedure for In Planta Detection of Fusarium oxysporum f. sp. phaseoli.

PubMed

Alves-Santos, Fernando M; Ramos, Brisa; García-Sánchez, M Asunción; Eslava, Arturo P; Díaz-Mínguez, José María

2002-03-01

ABSTRACT We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of >/=4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.

Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress.

PubMed

Wang, Hetong; He, Lei; Song, Jie; Cui, Weina; Zhang, Yanzhao; Jia, Chunyun; Francis, Dennis; Rogers, Hilary J; Sun, Lizong; Tai, Peidong; Hui, Xiujuan; Yang, Yuesuo; Liu, Wan

2016-05-01

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology. Copyright © 2016 Elsevier Ltd. All rights reserved.

MCMC-ODPR: primer design optimization using Markov Chain Monte Carlo sampling.

PubMed

Kitchen, James L; Moore, Jonathan D; Palmer, Sarah A; Allaby, Robin G

2012-11-05

Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base.

MCMC-ODPR: Primer design optimization using Markov Chain Monte Carlo sampling

PubMed Central

2012-01-01

Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base. PMID:23126469

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

PubMed Central

Takahashi, Shunsuke; Tomita, Junko; Nishioka, Kaori; Hisada, Takayoshi; Nishijima, Miyuki

2014-01-01

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples. PMID:25144201

Characterization of local goat breeds using RAP-DNA markers

NASA Astrophysics Data System (ADS)

Al-Barzinji, Yousif M. S.; Hamad, Aram O.

2017-09-01

The present study was conducted on different colors of local goat breeds. A number of 216 does were sampled from the seven groups. Genomic DNA was extracted from the blood samples. From the twenty used RAPD primers 12 of them were amplified, and presence of bands. The total fragment number of 12 primers over all the goat breed samples was 485 fragments. Out of the 485 fragments, 90 of them were Polymorphic fragments numbers (PFN). From all bands obtained, 20 of them possessed unique bands. The highest unique band was found in locus RAP 6 which has 4 unique bands, three of them in the Maraz Brown and one in the local Koor. Nei's gene diversity and Shanon's information index in this study were averaged 0.38 and 0.60, respectively. The genetic distance among several goat breeds ranged from 9.11 to 43.33%. The highest genetic distance 43.33% recorded between Maraz goat and other goat breeds and between local Koor and other goat (except Maraz goats) breeds (37.79%). However, the lowest genetic distance recorded between local white and Pnok. The distance between (local Black and Pnok) and (local Black and local white) was 22.75%. In conclusions, the high distance among these goat breeds, polymorphism and high numbers of unique bands found in present study indicates that these goat breeds have the required amount of genetic variation to made genetic improvement. This study helps us to clarify the image of the genetic diversity of the local goat breeds and the breeders can used it for mating system when need to make the crossing among these goat breeds.

Selection of RAPD markers for investigation of genetic population structure in fusiform rust fungus infecting loblolly pine

Treesearch

James H. Roberds; Thomas L. Kubisiak; Pauline C. Spaine; S.F. Covert; R.L. Doudrick

1997-01-01

research to determine patterns of genetic differentiation among and within field populations of Cronartium quercuum f. sp. fusiforme using RAPD markers is currently underway in the molecular genetics laboratory at the Southern Institute of Forest Genetics. Fungal tissue was collected as a drop of spermatia or scrapings of a...

RAPD linkage mapping in a longleaf pine × slash pine F1 family

Treesearch

Thomas L. Kubisiak; C. Dana. Nelson; W.L. Nance; M. Stine

1995-01-01

Random amplified polymorphic DNAs (RAPDs) were used to construct linkage maps of the parents of a longleaf pine (Pinus palustris Mill.) slash pine (Pinus elliottii Englm.) F1 family. A total of 247 segregating loci [233 (1:1), 14 (3:1)] and 87 polymorphic (between-parents), but non-segregating, loci were...

A complex structure in the mRNA of Tf1 is recognized and cleaved to generate the primer of reverse transcription.

PubMed

Lin, J H; Levin, H L

1997-01-15

All retroviruses and LTR-containing retrotransposons are thought to require specific tRNA molecules to serve as primers of reverse transcription. An exception is the LTR-containing retrotransposon Tf1, isolated from Schizosaccharomyces pombe. Instead of requiring a tRNA, the reverse transcriptase of Tf1 uses the first 11 bases of the Tf1 transcript as the primer for reverse transcription. The primer is generated by a cleavage that occurs between bases 11 and 12 of the Tf1 mRNA. Sequence analysis of the 5' untranslated region of the Tf1 mRNA resulted in the identification of a region with the potential to form an RNA structure of 89 bases that included the primer binding site and the first 11 bases of the Tf1 mRNA. Systematic mutagenesis of this region revealed 34 single-point mutants in the structure that resulted in reduced transposition activity. The defects in transposition correlated with reduced level of Tf1 reverse transcripts as determined by DNA blot analysis. Evidence that the RNA structure did form in vivo included the result that strains with second site mutations that restored complementarity resulted in increased levels of reverse transcripts and Tf1 transposition. The majority of the mutants defective for reverse transcription were unable to cleave the Tf1 mRNA between bases 11 and 12. These data indicate that formation of an extensive RNA structure was required for the cleavage reaction that generated the primer for Tf1 reverse transcription.

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences

PubMed Central

Gao, Xinxin; Yo, Peggy; Keith, Andrew; Ragan, Timothy J.; Harris, Thomas K.

2003-01-01

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (∼0.4–0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (∼0.4–0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4–0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis. PMID:14602936

Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

PubMed Central

2012-01-01

Background Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Results We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Conclusions We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast. PMID:22708584

MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

PubMed Central

Kim, Hyerin; Kang, NaNa; Chon, Kang-Wook; Kim, Seonho; Lee, NaHye; Koo, JaeHyung; Kim, Min-Soo

2015-01-01

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR. PMID:26109350

Identification and characterization of a RAPD-PCR marker for distinguishing Asian and North American gypsy moths

Treesearch

K.J. Garner; J.M. Slavicek

1996-01-01

The recent introduction of the Asian gypsy moth (Lymantria dispar L.) into North America has necessitated the development of genetic markers to distinguish Asian moths from the established North American population, which originated in Europe. We used RAPD-PCR to identify a DNA length polymorphism that is diagnostic for the two moth strains. The...

Genetic variation at allozyme and RAPD markers in Pinus longaeva (Pinaceae) of the White Mountains, California

Treesearch

Seok-Woo Lee; F. Thomas Ledig; David R. Johnson

2002-01-01

We compared genetic diversity estimated from allozymes and from random amplified polymorphic DNA (RAPDs) in a sample of 210 Great Basin bristlecone pines (Pinus longaeva Bailey) from three groves in the White Mountains, California, USA. The White Mountains are the most westerly extension of bristlecone pine and home to the oldest known living trees....

Biofilm formation, antibiotic susceptibility and RAPD genotypes in Pseudomonas aeruginosa clinical strains isolated from single centre intensive care unit patients.

PubMed

Vaněrková, Martina; Mališová, Barbora; Kotásková, Iva; Holá, Veronika; Růžička, Filip; Freiberger, Tomáš

2017-11-01

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD-PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.

[Reticulate evolution of parthenogenetic species of the Lacertidae rock lizards: inheritance of CLsat tandem repeats and anonymous RAPD markers].

PubMed

Chobanu, D; Rudykh, I A; Riabinina, N L; Grechko, V V; Kramerov, D A; Darevskiĭ, I S

2002-01-01

The genetic relatedness of several bisexual and of four unisexual "Lacerta saxicola complex" lizards was studied, using monomer sequences of the complex-specific CLsat tandem repeats and anonymous RAPD markers. Genomes of parthenospecies were shown to include different satellite monomers. The structure of each such monomer is specific for a certain pair of bisexual species. This fact might be interpreted in favor of co-dominant inheritance of these markers in bisexual species hybridogenesis. This idea is supported by the results obtained with RAPD markers; i.e., unisexual species genomes include only the loci characteristic of certain bisexual species. At the same time, in neither case parthenospecies possess specific, autoapomorphic loci that were not present in this or that bisexual species.

Phenetic, genetic diversity and symbiotic compatibility of rhizobial strains nodulating pigeon pea in Northern India.

PubMed

Arora, Naveen Kumar; Khare, Ekta; Singh, Sachin; Tewari, Sakshi

2018-01-01

Pigeon pea ( Cajanus cajan ) is one of the most important legumes grown in the northern province of Uttar Pradesh, India. However, its productively in Uttar Pradesh is lower than the average yield of adjoining states. During the course of the present study, a survey of pigeon pea growing agricultural fields was carried out and it was found that 80% of plants were inadequately nodulated. The study was aimed to evaluate the pigeon pea symbiotic compatibility and nodulation efficiency of root nodulating bacteria isolated from various legumes, and to explore the phenetic and genetic diversity of rhizobial population nodulating pigeon pea growing in fields of Uttar Pradesh. Amongst all the 96 isolates, 40 isolates showed nodulation in pigeon pea. These 40 isolates were further characterized by phenotypic, biochemical and physiological tests. Intrinsic antibiotic resistance pattern was taken to generate similarity matrix revealing 10 phenons. The study shows that most of the isolates nodulating pigeon pea in this region were rapid growers. The dendrogram generated using the NTSYSpc software grouped RAPD patterns into 19 clusters. The high degree of phenetic and genetic diversity encountered is probably because of a history of mixed cropping of legumes. The assessment of diversity is a very important tool and can be used to improve the nodulation and quality of pigeon pea crop. It is also concluded that difference between phenetic and RAPD clustering pattern is an indication that rhizobial diversity of pigeon pea is not as yet completely understood and settled.

Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

PubMed Central

AL-Huqail, Asma A.; Abdelhaliem, Ekram

2015-01-01

The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (R f), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors. PMID:26180815

Development and cross-species/genera transferability of microsatellite markers discovered using 454 genome sequencing in chokecherry (Prunus virginiana L.).

PubMed

Wang, Hongxia; Walla, James A; Zhong, Shaobin; Huang, Danqiong; Dai, Wenhao

2012-11-01

Chokecherry (Prunus virginiana L.) (2n = 4x = 32) is a unique Prunus species for both genetics and disease-resistance research due to its tetraploid nature and X-disease resistance. However, no genetic and genomic information on chokecherry is available. A partial chokecherry genome was sequenced using Roche 454 sequencing technology. A total of 145,094 reads covering 4.8 Mbp of the chokecherry genome were generated and 15,113 contigs were assembled, of which 11,675 contigs were larger than 100 bp in size. A total of 481 SSR loci were identified from 234 (out of 11,675) contigs and 246 polymerase chain reaction (PCR) primer pairs were designed. Of 246 primers, 212 (86.2 %) effectively produced amplification from the genomic DNA of chokecherry. All 212 amplifiable chokecherry primers were used to amplify genomic DNA from 11 other rosaceous species (sour cherry, sweet cherry, black cherry, peach, apricot, plum, apple, crabapple, pear, juneberry, and raspberry). Thus, chokecherry SSR primers can be transferable across Prunus species and other rosaceous species. An average of 63.2 and 58.7 % of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2 % of amplifiable chokecherry primers amplified DNA from other rosaceous species. Using random genome sequence data generated from next-generation sequencing technology to identify microsatellite loci appears to be rapid and cost-efficient, particularly for species with no sequence information available. Sequence information and confirmed transferability of the identified chokecherry SSRs among species will be valuable for genetic research in Prunus and other rosaceous species. Key message A total of 246 SSR primers were identified from chokecherry genome sequences. Of which, 212 were confirmed amplifiable both in chokecherry and other 11 other rosaceous species.

Identification of Saprolegnia Spp. Pathogenic in Chinook Salmon : Final Report.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whisler, Howard C.

1997-06-01

This project has developed procedures to assess the role of the fungal parasite, Saprolegnia in the biology of salmon, particularly adult Chinook, in the Columbia River Basin. Both morphological and DNA ``fingerprinting`` surveys reveal that Saprolegnia parasitica (=S. diclina, Type I) is the most common pathogen of these fish. In the first phase of this study 92% of 620 isolates, from salmon lesions, conformed to this taxa of Saprolegnia. In the current phase, the authors have developed variants of DNA fingerprinting (RAPD and SWAPP analysis) that permit examination of the sub-structure of the parasite population. These results confirm the predominancemore » of S. parasitica, and suggest that at least three different sub-groups of this fungus occur in the Pacific N.W., USA. The use of single and paired primers with PCR amplification permits identification of pathogenic types, and distinction from other species of the genus considered to be more saprophytic in character. A year`s survey of saprolegniaceous fungi from Lake Washington indicated that the fish-pathogen was not common in the water column. Where and how fish encounter this parasite can be approached with the molecular tags identified in this project.« less

Impact assessment of mercury accumulation and biochemical and molecular response of Mentha arvensis: a potential hyperaccumulator plant.

PubMed

Manikandan, R; Sahi, S V; Venkatachalam, P

2015-01-01

The present study was focused on examining the effect of Hg oxidative stress induced physiochemical and genetic changes in M. arvensis seedlings. The growth rate of Hg treated seedlings was decreased to 56.1% and 41.5% in roots and shoots, respectively, compared to the control. Accumulation of Hg level in both roots and shoots was increased with increasing the concentration of Hg. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were found to be increased with increasing the Hg concentration up to 20 mg/L; however, it was decreased at 25 mg/L Hg concentration. The POX enzyme activity was positively correlated with Hg dose. The changes occurring in the random amplification of ploymorphic DNA (RAPD) profiles generated from Hg treated seedlings included variations in band intensity, disappearance of bands, and appearance of new bands compared with the control seedlings. It was concluded that DNA polymorphisms observed with RAPD profile could be used as molecular marker for the evaluation of heavy metal induced genotoxic effects in plant species. The present results strongly suggested that Mentha arvensis could be used as a potential phytoremediator plant in mercury polluted environment.

Impact Assessment of Mercury Accumulation and Biochemical and Molecular Response of Mentha arvensis: A Potential Hyperaccumulator Plant

PubMed Central

Manikandan, R.; Sahi, S. V.; Venkatachalam, P.

2015-01-01

The present study was focused on examining the effect of Hg oxidative stress induced physiochemical and genetic changes in M. arvensis seedlings. The growth rate of Hg treated seedlings was decreased to 56.1% and 41.5% in roots and shoots, respectively, compared to the control. Accumulation of Hg level in both roots and shoots was increased with increasing the concentration of Hg. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were found to be increased with increasing the Hg concentration up to 20 mg/L; however, it was decreased at 25 mg/L Hg concentration. The POX enzyme activity was positively correlated with Hg dose. The changes occurring in the random amplification of ploymorphic DNA (RAPD) profiles generated from Hg treated seedlings included variations in band intensity, disappearance of bands, and appearance of new bands compared with the control seedlings. It was concluded that DNA polymorphisms observed with RAPD profile could be used as molecular marker for the evaluation of heavy metal induced genotoxic effects in plant species. The present results strongly suggested that Mentha arvensis could be used as a potential phytoremediator plant in mercury polluted environment. PMID:25654134

DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

PubMed

Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R

1994-10-01

We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR.

PubMed

Kim, Hyerin; Kang, NaNa; Chon, Kang-Wook; Kim, Seonho; Lee, NaHye; Koo, JaeHyung; Kim, Min-Soo

2015-11-16

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

RExPrimer: an integrated primer designing tool increases PCR effectiveness by avoiding 3' SNP-in-primer and mis-priming from structural variation

PubMed Central

2009-01-01

Background Polymerase chain reaction (PCR) is very useful in many areas of molecular biology research. It is commonly observed that PCR success is critically dependent on design of an effective primer pair. Current tools for primer design do not adequately address the problem of PCR failure due to mis-priming on target-related sequences and structural variations in the genome. Methods We have developed an integrated graphical web-based application for primer design, called RExPrimer, which was written in Python language. The software uses Primer3 as the primer designing core algorithm. Locally stored sequence information and genomic variant information were hosted on MySQLv5.0 and were incorporated into RExPrimer. Results RExPrimer provides many functionalities for improved PCR primer design. Several databases, namely annotated human SNP databases, insertion/deletion (indel) polymorphisms database, pseudogene database, and structural genomic variation databases were integrated into RExPrimer, enabling an effective without-leaving-the-website validation of the resulting primers. By incorporating these databases, the primers reported by RExPrimer avoid mis-priming to related sequences (e.g. pseudogene, segmental duplication) as well as possible PCR failure because of structural polymorphisms (SNP, indel, and copy number variation (CNV)). To prevent mismatching caused by unexpected SNPs in the designed primers, in particular the 3' end (SNP-in-Primer), several SNP databases covering the broad range of population-specific SNP information are utilized to report SNPs present in the primer sequences. Population-specific SNP information also helps customize primer design for a specific population. Furthermore, RExPrimer offers a graphical user-friendly interface through the use of scalable vector graphic image that intuitively presents resulting primers along with the corresponding gene structure. In this study, we demonstrated the program effectiveness in successfully generating primers for strong homologous sequences. Conclusion The improvements for primer design incorporated into RExPrimer were demonstrated to be effective in designing primers for challenging PCR experiments. Integration of SNP and structural variation databases allows for robust primer design for a variety of PCR applications, irrespective of the sequence complexity in the region of interest. This software is freely available at http://www4a.biotec.or.th/rexprimer. PMID:19958502

A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses.

PubMed

Deng, Yi-Mo; Spirason, Natalie; Iannello, Pina; Jelley, Lauren; Lau, Hilda; Barr, Ian G

2015-07-01

Full genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV. To develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans. A set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5'-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions. This minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China. This streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

PubMed

Yang, Jun-Bo; Li, De-Zhu; Li, Hong-Tao

2014-09-01

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms. © 2014 John Wiley & Sons Ltd.

Multiplexing Short Primers for Viral Family PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S N; Hiddessen, A L; Hara, C A

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets for large, diverse, and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ({approx}3700 18-mers or {approx}2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and formore » several diverse species such as foot-and-mouth disease virus, hemagglutinin and neuraminidase segments of influenza A virus, Norwalk virus, and HIV-1.« less

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses

USDA-ARS?s Scientific Manuscript database

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combinat...

Genetic Diversity and Differentiation of Colletotrichum spp. Isolates Associated with Leguminosae Using Multigene Loci, RAPD and ISSR

PubMed Central

Mahmodi, Farshid; Kadir, J. B.; Puteh, A.; Pourdad, S. S.; Nasehi, A.; Soleimani, N.

2014-01-01

Genetic diversity and differentiation of 50 Colletotrichum spp. isolates from legume crops studied through multigene loci, RAPD and ISSR analysis. DNA sequence comparisons by six genes (ITS, ACT, Tub2, CHS-1, GAPDH, and HIS3) verified species identity of C. truncatum, C. dematium and C. gloeosporiodes and identity C. capsici as a synonym of C. truncatum. Based on the matrix distance analysis of multigene sequences, the Colletotrichum species showed diverse degrees of intera and interspecific divergence (0.0 to 1.4%) and (15.5–19.9), respectively. A multilocus molecular phylogenetic analysis clustered Colletotrichum spp. isolates into 3 well-defined clades, representing three distinct species; C. truncatum, C. dematium and C. gloeosporioides. The ISSR and RAPD and cluster analysis exhibited a high degree of variability among different isolates and permitted the grouping of isolates of Colletotrichum spp. into three distinct clusters. Distinct populations of Colletotrichum spp. isolates were genetically in accordance with host specificity and inconsistent with geographical origins. The large population of C. truncatum showed greater amounts of genetic diversity than smaller populations of C. dematium and C. gloeosporioides species. Results of ISSR and RAPD markers were congruent, but the effective maker ratio and the number of private alleles were greater in ISSR markers. PMID:25288981

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains.

PubMed

El-Fiky, Zaki A; Hassan, Gamal M; Emam, Ahmed M

2012-06-01

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories. © 2012 Institute of Food Technologists®

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

PubMed Central

Sulakvelidze, Alexander; Kekelidze, Merab; Gomelauri, Tsaro; Deng, Yingkang; Khetsuriani, Nino; Kobaidze, Ketino; De Zoysa, Aruni; Efstratiou, Androulla; Morris, J. Glenn; Imnadze, Paata

1999-01-01

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates. PMID:10488190

Characterization and zoonotic impact of Shiga toxin producing Escherichia coli in some wild bird species

PubMed Central

Fadel, Hanaa Mohamed; Afifi, Rabab; Al-Qabili, Dheyazan Mohammed

2017-01-01

Aim: Wild birds are considered silent vectors of some zoonotic water and food borne pathogens of public health significance. Owing to the importance of Shiga toxin producing Escherichia coli (STEC) as the most pathogenic among the emerging diarrheagenic E. coli groups that can infect man; the present study was designed to detect the occurrence of STEC among wild birds in Egypt. Materials and Methods: A total of 177 intestinal content swab samples originating from five wild bird species were investigated for the presence of E. coli and STEC by standard culture methods. Suspect STEC isolates were further characterized by serotyping, random amplified polymorphic DNA polymerase chain reaction (RAPD PCR), antimicrobial resistance pattern and PCR detection of stx1, stx2, and eae genes. Results: A total of 30 suspect STEC isolates from 30 positive birds’ samples were detected and identified on STEC CHROMagar (semi-captive pigeons, 15; house crows, 8; cattle egrets, 3; moorhens, 2; and house teals, 2). 25 isolates were grouped into 13 serogroups (O:20, O:25, O:26, O:27, O:63, O:78, O:111, O:114, O:125, O:128, O:142, O:153, and O:158), while five were rough strains. The distribution of STEC virulence genes among wild birds was as follows: 16 birds carried stx1 gene only (nine pigeons [28.1%], six crows [7.1%], and one cattle egret [5.6%]). Stx1 and stx2 genes together were detected in four birds (one cattle egret [5.6%], two moorhens [6.1%], and one house teal, [10%]). Only one pigeon (3.1%) possessed the three alleles. Disk diffusion test results showed that cefixime was the most effective against STEC serotypes with (93.3%) sensitivity, followed by gentamycin (56.7%), and amoxicillin (50%). On the other hand, all the recovered STEC isolates were resistant to cefotaxime, doxycycline, cephalothin, and sulfisoxazole. RAPD fingerprinting using primers OPA-2 and OPA-9 showed that STEC isolates were heterogeneous; they yielded 30 and 27 different clusters, respectively. Conclusions: Wild birds carry STEC and may add to the contamination of the surrounding environment. PMID:29062203

[Molecular epidemiology and antifungal susceptibility of Candida species isolated from urine samples of patients in intensive care unit].

PubMed

Yüksekkaya, Serife; Fındık, Duygu; Arslan, Uğur

2011-01-01

The aims of this study were to analyse the amphotericin B and fluconazole susceptibility and molecular epidemiology of Candida strains (Candida albicans, Candida tropicalis and Candida glabrata) isolated from the urine samples of patients hospitalized in the intensive care unit. Identification of the isolates was done according to microscopic morphology (chlamydospor, blastospor, pseudohyphae and true hyphae) on cornmeal agar, germ tube formation and carbohydrate assimilation patterns (API ID 32C bioMérieux, France). Antifungal susceptibilities of the isolates were determined by in vitro broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI). To investigate the clonal relationship of the isolates, randomly amplified polymorphic DNA (RAPD) analysis was performed by using Cnd3 primer. Of the 56 Candida isolates minimum inhibitory concentration (MIC) ranges, MIC50 and MIC90 values for amphotericin B were 0.125-1 µg/ml, 0.125 and 0.5 µg/ml for C.albicans, 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.tropicalis and 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.glabrata, respectively. Fluconazole MIC ranges, MIC50 and MIC90 values were 0.25-4 µg/ml, 0.25 and 0.5 µg/ml for C.albicans, 0.25-16 µg/ml, 0.5 and 1 µg/ml for C.tropicalis and 0.5-64 µg/ml, 8 and 16 µg/ml for C.glabrata, respectively. For amphotericin B, none of the isolates had high MIC values (MIC > 1 µg/ml). While one of the C.glabrata isolates was resistant to fluconazole (MIC ≥ 64 µg/ml), one C.tropicalis and two C.glabrata isolates were dose-dependent susceptible (MIC: 16-32 µg/ml). The results of RAPD analysis indicated an exogenous spread from two clones for C.albicans, one clone for C.glabrata and one clone for C.tropicalis. This study underlines the importance of molecular epidemiological analysis of clinical samples together with hospital environmental samples in terms of Candida spp. To determine the exogenous origin for the related strains and to prevent nosocomial Candida infections.

Short communication: development and characterization of novel transcriptome-derived microsatellites for genetic analysis of persimmon.

PubMed

Luo, C; Zhang, Q L; Luo, Z R

2014-04-16

Oriental persimmon (Diospyros kaki Thunb.) (2n = 6x = 90) is a major commercial and deciduous fruit tree that is believed to have originated in China. However, rare transcriptomic and genomic information on persimmon is available. Using Roche 454 sequencing technology, the transcriptome from RNA of the flowers of D. kaki was analyzed. A total of 1,250,893 reads were generated and 83,898 unigenes were assembled. A total of 42,711 SSR loci were identified from 23,494 unigenes and 289 polymerase chain reaction primer pairs were designed. Of these 289 primers, 155 (53.6%) showed robust PCR amplification and 98 revealed polymorphism between 15 persimmon genotypes, indicating a polymorphic rate of 63.23% of the productive primers for characterization and genotyping of the genus Diospyros. Transcriptome sequence data generated from next-generation sequencing technology to identify microsatellite loci appears to be rapid and cost-efficient, particularly for species with no genomic sequence information available.

Genetic Variation in the Free-Living Amoeba Naegleria fowleri

PubMed Central

Pélandakis, Michel; De Jonckheere, Johan F.; Pernin, Pierre

1998-01-01

In this study, 30 strains of the pathogenic free-living amoeba Naegleria fowleri were investigated by using the randomly amplified polymorphic DNA (RAPD) method. The present study confirmed our previous finding that RAPD variation is not correlated with geographical origin. In particular, Mexican strains belong to the variant previously detected in Asia, Europe, and the United States. In France, surprisingly, strains from Cattenom gave RAPD patterns identical to those of the Japanese strains. In addition, all of these strains, together with an additional French strain from Chooz, exhibited similarities to South Pacific strains. The results also confirmed the presence of numerous variants in Europe, whereas only two variants were detected in the United States. The two variants found in the United States were different from the South Pacific variants. These findings do not support the previous hypothesis concerning the origin and modes of dispersal of N. fowleri. PMID:9687460

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

PubMed

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

Development and use of tuf gene-based primers for the multiplex PCR detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum in commercial dairy products.

PubMed

Sheu, Sen-Je; Hwang, Wen-zhe; Chen, Hsin-Chih; Chiang, Yu-Cheng; Tsen, Hau-Yang

2009-01-01

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N X 10(3) CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S-23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Short Communication: Analysis of Minor Populations of Human Immunodeficiency Virus by Primer Identification and Insertion-Deletion and Carry Forward Correction Pipelines.

PubMed

Hughes, Paul; Deng, Wenjie; Olson, Scott C; Coombs, Robert W; Chung, Michael H; Frenkel, Lisa M

2016-03-01

Accurate analysis of minor populations of drug-resistant HIV requires analysis of a sufficient number of viral templates. We assessed the effect of experimental conditions on the analysis of HIV pol 454 pyrosequences generated from plasma using (1) the "Insertion-deletion (indel) and Carry Forward Correction" (ICC) pipeline, which clusters sequence reads using a nonsubstitution approach and can correct for indels and carry forward errors, and (2) the "Primer Identification (ID)" method, which facilitates construction of a consensus sequence to correct for sequencing errors and allelic skewing. The Primer ID and ICC methods produced similar estimates of viral diversity, but differed in the number of sequence variants generated. Sequence preparation for ICC was comparably simple, but was limited by an inability to assess the number of templates analyzed and allelic skewing. The more costly Primer ID method corrected for allelic skewing and provided the number of viral templates analyzed, which revealed that amplifiable HIV templates varied across specimens and did not correlate with clinical viral load. This latter observation highlights the value of the Primer ID method, which by determining the number of templates amplified, enables more accurate assessment of minority species in the virus population, which may be relevant to prescribing effective antiretroviral therapy.

A multiplex primer design algorithm for target amplification of continuous genomic regions.

PubMed

Ozturk, Ahmet Rasit; Can, Tolga

2017-06-19

Targeted Next Generation Sequencing (NGS) assays are cost-efficient and reliable alternatives to Sanger sequencing. For sequencing of very large set of genes, the target enrichment approach is suitable. However, for smaller genomic regions, the target amplification method is more efficient than both the target enrichment method and Sanger sequencing. The major difficulty of the target amplification method is the preparation of amplicons, regarding required time, equipment, and labor. Multiplex PCR (MPCR) is a good solution for the mentioned problems. We propose a novel method to design MPCR primers for a continuous genomic region, following the best practices of clinically reliable PCR design processes. On an experimental setup with 48 different combinations of factors, we have shown that multiple parameters might effect finding the first feasible solution. Increasing the length of the initial primer candidate selection sequence gives better results whereas waiting for a longer time to find the first feasible solution does not have a significant impact. We generated MPCR primer designs for the HBB whole gene, MEFV coding regions, and human exons between 2000 bp to 2100 bp-long. Our benchmarking experiments show that the proposed MPCR approach is able produce reliable NGS assay primers for a given sequence in a reasonable amount of time.

Multiplex primer prediction software for divergent targets

PubMed Central

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

2009-01-01

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus. PMID:19759213

Evolution of finger millet: evidence from random amplified polymorphic DNA.

PubMed

Hilu, K W

1995-04-01

Finger millet (Eleusine coracana ssp. coracana) is an annual tetraploid member of a predominantly African genus. The crop is believed to have been domesticated from the tetraploid E. coracana ssp. africana. Cytogenetic and isozyme data point to the allopolyploid nature of the species and molecular information has shown E. indica to be one of the genomic donors. A recent isozyme study questioned the proposed phylogenetic relationship between finger millet and its direct ancestor subspecies africana. An approach using random amplified polymorphic DNA (RAPD) was employed in this study to examine genetic diversity and to evaluate hypotheses concerning the evolution of domesticated and wild annual species of Eleusine. Unlike previous molecular approaches, the RAPD study revealed genetic diversity in the crop. The pattern of genetic variation was loosely correlated to geographic distribution. The allotetraploid nature of the crop was confirmed and molecular markers that can possibly identify the other genomic donor were proposed. Genotypes of subspecies africana did not group closely with those of the crop but showed higher affinities to E. indica, reflecting the pattern of similarity revealed by the isozyme study. The multiple origin of subspecies africana could explain the discrepancy between the isozyme-RAPD evidence and previous information. The RAPD study showed the close genetic affinity of E. tristachya to the E. coracana--E. indica group and understood the distinctness of E. multiflora.

Piezoelectrically Initiated Pyrotechnic Igniter

NASA Technical Reports Server (NTRS)

Quince, Asia; Dutton, Maureen; Hicks, Robert; Burnham, Karen

2013-01-01

This innovation consists of a pyrotechnic initiator and piezoelectric initiation system. The device will be capable of being initiated mechanically; resisting initiation by EMF, RF, and EMI (electromagnetic field, radio frequency, and electromagnetic interference, respectively); and initiating in water environments and space environments. Current devices of this nature are initiated by the mechanical action of a firing pin against a primer. Primers historically are prone to failure. These failures are commonly known as misfires or hang-fires. In many cases, the primer shows the dent where the firing pin struck the primer, but the primer failed to fire. In devices such as "T" handles, which are commonly used to initiate the blowout of canopies, loss of function of the device may result in loss of crew. In devices such as flares or smoke generators, failure can result in failure to spot a downed pilot. The piezoelectrically initiated ignition system consists of a pyrotechnic device that plugs into a mechanical system (activator), which on activation, generates a high-voltage spark. The activator, when released, will strike a stack of electrically linked piezo crystals, generating a high-voltage, low-amperage current that is then conducted to the pyro-initiator. Within the initiator, an electrode releases a spark that passes through a pyrotechnic first-fire mixture, causing it to combust. The combustion of the first-fire initiates a primary pyrotechnic or explosive powder. If used in a "T" handle, the primary would ramp the speed of burn up to the speed of sound, generating a shock wave that would cause a high explosive to go "high order." In a flare or smoke generator, the secondary would produce the heat necessary to ignite the pyrotechnic mixture. The piezo activator subsystem is redundant in that a second stack of crystals would be struck at the same time with the same activation force, doubling the probability of a first strike spark generation. If the first activation fails to ignite, the device is capable of multiple attempts. Another unique aspect is in the design of the pyrotechnic device. There is an electrode that aids the generation of a directed spark and the use of a conductive matrix to support the first-fire material so that the spark will penetrate to the second electrode.

The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase.

PubMed

Herzig, Eytan; Voronin, Nickolay; Hizi, Amnon

2012-06-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

The Removal of RNA Primers from DNA Synthesized by the Reverse Transcriptase of the Retrotransposon Tf1 Is Stimulated by Tf1 Integrase

PubMed Central

Herzig, Eytan; Voronin, Nickolay

2012-01-01

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process. PMID:22491446

Primer design for a prokaryotic differential display RT-PCR.

PubMed Central

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-01-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR. PMID:9108168

Primer design for a prokaryotic differential display RT-PCR.

PubMed

Fislage, R; Berceanu, M; Humboldt, Y; Wendt, M; Oberender, H

1997-05-01

We have developed a primer set for a prokaryotic differential display of mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten 11mer primers generates up to 85 bands from total Escherichia coli RNA, thus covering expressed sequences of a complete bacterial genome. Due to the lack of polyadenylation in prokaryotic RNA the type T11VN anchored oligonucleotides for the reverse transcriptase reaction had to be replaced with respect to the original method described by Liang and Pardee [ Science , 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the new 11mer oligonucleotides were determined by a statistical evaluation of species-specific coding regions extracted from the EMBL database. The 11mer primers used for reverse transcription were selected for localization in the 3'-region of the bacterial RNA. The 10mer primers preferentially bind to the 5'-end of the RNA. None of the primers show homology to rRNA or other abundant small RNA species. Randomly sampled cDNA bands were checked for their bacterial origin either by re-amplification, cloning and sequencing or by re-amplification and direct sequencing with 10mer and 11mer primers after asymmetric PCR.

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India.

PubMed

Prameela, J; Ramakrishnaiah, H; Krishna, V; Deepalakshmi, A P; Naveen Kumar, N; Radhika, R N

2015-07-01

Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated on Murashige and Skoog's (MS) medium supplemented with auxins, Indole-3-acetic acid (IAA), Indole-3- butyric acid (IBA), 1-Naphthaleneacetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and cytokinins, 6-Benzylaminopurine (BAP) and Kinetin (Kn) at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mgl(-1). MS medium with IBA (18.08), NAA (17.83) and IAA (17.58) at 0.5 mgl(-1) concentrations showed efficient regeneration. Regenerated shoots were rooted on half-strength MS medium with and without 0.5 mgl(-1) IBA or NAA. The plantlets were successfully hardened in rooting trays (peat, vermiculite and sand) and transferred to field mileu. The genetic fidelity of in vitro raised plants was assessed by using three different single primer amplification reaction (SPAR) markers namely random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of mini-satellite DNA region (DAMD). The results consistently demonstrated true-to-true type propagation. This is the first report of in vitro propagation and establishment of true-to-true type genetic fidelity in H. incana.

Identification and characterization of wild lactobacilli and pediococci from spontaneously fermented Mountain cheese.

PubMed

Carafa, Ilaria; Nardin, Tiziana; Larcher, Roberto; Viola, Roberto; Tuohy, Kieran; Franciosi, Elena

2015-06-01

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called "Malga" located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants.

PubMed

Szarka, B.; Göntér, I.; Molnár-Láng, M.; Mórocz, S.; Dudits, D.

2002-07-01

Intergeneric somatic hybridization was performed between albino maize ( Zea mays L.) protoplasts and mesophyll protoplasts of wheat ( Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.

Randomly amplified polymorphic-DNA analysis for detecting genotoxic effects of Boron on maize (Zea mays L.).

PubMed

Sakcali, M Serdal; Kekec, Guzin; Uzonur, Irem; Alpsoy, Lokman; Tombuloglu, Huseyin

2015-08-01

This study was carried out to investigate the genotoxic effect of boron (B) on maize using randomly amplified polymorphic DNA (RAPD) method. Experimental design was conducted under 0, 5, 10, 25, 50, 100, 125, and 150 ppm B exposures, and physiological changes have revealed a sharp decrease in root growth rates from 28% to 85%, starting from 25 ppm to 150 ppm, respectively. RAPD-polymerase chain reaction (PCR) analysis shows that DNA alterations are clearly observed from beginning to 100 ppm. B-induced inhibition in root growth had a positive correlation with DNA alterations. Total soluble protein, root and stem lengths, and B content analysis in root and leaves encourage these results as a consequence. These preliminary findings reveal that B causes chromosomal aberration and genotoxic effects on maize. Meanwhile, usage of RAPD-PCR technique is a suitable biomarker to detect genotoxic effect of B on maize and other crops for the future. © The Author(s) 2013.

Genetic analysis of Melipona quadrifasciata LEP. (Hymenoptera: Apidae, Meliponinae) with RAPD markers.

PubMed

Waldschmidt, A M; Marco-Júnior, P; Barros, E G; Campos, L A O

2002-11-01

Melipona quadrifasciata ("mandaçaia") can be subdivided into two subspecies: M. q. anthidioides and M. q. quadrifasciata. In the present study we used RAPD markers to estimate intercolonial genetic variation among 69 colonies of Melipona quadrifasciata. Ten workers per colony were analyzed. The intercolony genetic distances based on RAPD markers ranged from 29.5% (colonies collected in the State of São Paulo vs colonies from the State of Minas Gerais) to 34.2% (São Paulo vs Santa Catarina). These results indicate a high genetic similarity among the colonies analyzed. According to the genetic distances two different groups could be distinguished. The first containing the samples from Santa Catarina region and the second, samples from Paraná, São Paulo, Minas Gerais, and Espírito Santo. Based on the molecular analysis, bees belonging to the different subspecies M. q. quadrifasciata (from Santa Catarina) and M. q. anthidiodes (from the other regions) were distinguished.

[Study on chemical diversity of volatile oils in Houttuynia cordata and their genetic basis].

PubMed

Wu, Lingshang; Si, Jinping; Zhou, Hui; Zhu, Yan; Lan, Yunlong

2009-01-01

To reveal chemical diversify of volatile oils in Houttuynia cordata from major producing areas in China and their genetic basis, lay a foundation for breeding a quality H. cordata variety. The volatile oils in H. cordata from 22 provenances were determined by GC. And the relationship among the peak areas of volatile oils, biological characteristics and RAPD makers were analyzed. There were common and special volatile oils in H. cordata from different provenances. The peak areas of common volatile oils in samples were significantly different. The clustering figure based on the peak areas or the relative peak areas of common volatile oils was almost agreed with the one based on RAPD makers analysis. And the differences in chromatograms could be distinguished according to the biological characteristics. The diversity of volatile oils exists in H. cordata from different provenances which relate with biological characteristics and has genetic basis. H. cordata can be divided into 2 types according to volatile oils, biological characteristics or RAPD marker.

Bi-parentally inherited species-specific markers identify hybridization between rainbow trout and cutthroat trout subspecies

USGS Publications Warehouse

Ostberg, C.O.; Rodriguez, R.J.

2004-01-01

Eight polymerase chain reaction primer sets amplifying bi-parentally inherited species-specific markers were developed that differentiate between rainbow trout (Oncorhynchus mykiss) and various cutthroat trout (O. clarki) subspecies. The primers were tested within known F1 and first generation hybrid backcrosses and were shown to amplify codominantly within hybrids. Heterozygous individuals also amplified a slower migrating band that was a heteroduplex, caused by the annealing of polymerase chain reaction products from both species. These primer sets have numerous advantages for native cutthroat trout conservation including statistical genetic analyses of known crosses and simple hybrid identification.

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

PubMed

Long, Y M; Chao, W S; Ma, G J; Xu, S S; Qi, L L

2017-03-01

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Cytotoxicity and genotoxicity of polyethylenimine and nickel chloride in red sea bream ( Pagrosomus major) fin cell line RSBF

NASA Astrophysics Data System (ADS)

Guo, Hua-Rong; Zhang, Shi-Cui

2002-12-01

A continuous marine fish cell line RSBF (i. c. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC50=1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose-dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl2 posed no acute toxicity but significantly stimulated their growth (107% 214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl2 to RSBF cells; that there was a slight dose-dependent response in the genotoxic effect of PEI but not NiCl2; and that RAPD technique might provide a sensitive, non-specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene, vector in fish gene transfer and human gene therapy.

Phenotypic and Genotypic Characterization of Non-Starter Lactic Acid Bacteria in Mature Cheddar Cheese

PubMed Central

Fitzsimons, N. A.; Cogan, T. M.; Condon, S.; Beresford, T.

1999-01-01

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex. PMID:10427029

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

PubMed Central

Variath, Murali Tottekkad; Joshi, Gopal; Bali, Sapinder; Agarwal, Manu; Kumar, Amar; Jagannath, Arun; Goel, Shailendra

2015-01-01

Background Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing. Principal Findings Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower. Conclusion Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection. PMID:26287743

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

PubMed

Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

2015-07-28

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

A blackberry (Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers

PubMed Central

Lewers, Kim S; Saski, Chris A; Cuthbertson, Brandon J; Henry, David C; Staton, Meg E; Main, Dorrie S; Dhanaraj, Anik L; Rowland, Lisa J; Tomkins, Jeff P

2008-01-01

Background The recent development of novel repeat-fruiting types of blackberry (Rubus L.) cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST) library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR), and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars. Results A cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products. Conclusion This rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry. PMID:18570660

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

PubMed

Hanning, Jennifer E; Groves, Ian J; Pett, Mark R; Coleman, Nicholas

2013-05-21

Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR

PubMed Central

2013-01-01

Background Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects. PMID:23693071

Detection of Ophiocordyceps sinensis and Its Common Adulterates Using Species-Specific Primers

PubMed Central

Liu, Yang; Wang, Xiao-yue; Gao, Zi-tong; Han, Jian-ping; Xiang, Li

2017-01-01

Ophiocordyceps sinensis is a fungus that infects Hepialidae caterpillars, mummifying the larvae and producing characteristic fruiting bodies (stromata) that are processed into one of the most valued traditional Chinese medicines (TCM). The product commands a very high price due to a high demand but a very limited supply. Adulteration with other fungi is a common problem and there is a need to test preparation for the presence of the correct fungus. In the current study, a PCR-based approach for the identification of O. sinensis based on a segment of the internal transcribed spacer (ITS) region was developed. The segments is 146-bp in size and is likely to be amplified even in materials where processing led to DNA fragmentation. Primer development was based on the alignment of sequence data generated from a total of 89 samples of O. sinensis and potential adulterants as well as sequences date from 41 Ophiocordyceps species and 26 Cordyceps species available in GenBank. Tests with primer pair, DCF4/DCR4, demonstrated generation of an amplicon from DNA extracted from O. sinensis stromata, but not from extracts derived from adulterants. Species-specific primer pairs were also developed and tested for detection of the common adulterants, Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, Cordyceps liangshanensis and Ophiocordyceps nutans. The collection of primers developed in the present study will be useful for the authentication of preparation claiming to only contain O. sinensis and for the detection of fungi used as adulterants in these preparations. PMID:28680424

A Next-Generation Sequencing Primer—How Does It Work and What Can It Do?

PubMed Central

Alekseyev, Yuriy O.; Fazeli, Roghayeh; Yang, Shi; Basran, Raveen; Miller, Nancy S.

2018-01-01

Next-generation sequencing refers to a high-throughput technology that determines the nucleic acid sequences and identifies variants in a sample. The technology has been introduced into clinical laboratory testing and produces test results for precision medicine. Since next-generation sequencing is relatively new, graduate students, medical students, pathology residents, and other physicians may benefit from a primer to provide a foundation about basic next-generation sequencing methods and applications, as well as specific examples where it has had diagnostic and prognostic utility. Next-generation sequencing technology grew out of advances in multiple fields to produce a sophisticated laboratory test with tremendous potential. Next-generation sequencing may be used in the clinical setting to look for specific genetic alterations in patients with cancer, diagnose inherited conditions such as cystic fibrosis, and detect and profile microbial organisms. This primer will review DNA sequencing technology, the commercialization of next-generation sequencing, and clinical uses of next-generation sequencing. Specific applications where next-generation sequencing has demonstrated utility in oncology are provided. PMID:29761157

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin H; Giver, Lorraine J.

2000-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Recombination of polynucleotide sequences using random or defined primers

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Affholter, Joseph A.; Zhao, Huimin; Giver, Lorraine J.

2001-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

MIPE: A metagenome-based community structure explorer and SSU primer evaluation tool

PubMed Central

Zhou, Quan

2017-01-01

An understanding of microbial community structure is an important issue in the field of molecular ecology. The traditional molecular method involves amplification of small subunit ribosomal RNA (SSU rRNA) genes by polymerase chain reaction (PCR). However, PCR-based amplicon approaches are affected by primer bias and chimeras. With the development of high-throughput sequencing technology, unbiased SSU rRNA gene sequences can be mined from shotgun sequencing-based metagenomic or metatranscriptomic datasets to obtain a reflection of the microbial community structure in specific types of environment and to evaluate SSU primers. However, the use of short reads obtained through next-generation sequencing for primer evaluation has not been well resolved. The software MIPE (MIcrobiota metagenome Primer Explorer) was developed to adapt numerous short reads from metagenomes and metatranscriptomes. Using metagenomic or metatranscriptomic datasets as input, MIPE extracts and aligns rRNA to reveal detailed information on microbial composition and evaluate SSU rRNA primers. A mock dataset, a real Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) test dataset, two PrimerProspector test datasets and a real metatranscriptomic dataset were used to validate MIPE. The software calls Mothur (v1.33.3) and the SILVA database (v119) for the alignment and classification of rRNA genes from a metagenome or metatranscriptome. MIPE can effectively extract shotgun rRNA reads from a metagenome or metatranscriptome and is capable of classifying these sequences and exhibiting sensitivity to different SSU rRNA PCR primers. Therefore, MIPE can be used to guide primer design for specific environmental samples. PMID:28350876

Strategic conservation of orchard germplasm based on indigenous knowledge and genetic diversity: a case study of sour orange populations in China.

PubMed

Ming, Feng; Liu, Qi-Kun; Shi, Jin-Lei; Wang, Wei; Lu, Bao-Rong

2009-01-01

To effectively conserve sour orange (Citrus aurantium L.) germplasm on two islands at the estuary of the Yangtze River in China, we estimated genetic variation and relationships of the known parental trees and their proposed descendents (young trees) using the fingerprints of random amplified polymorphic DNA (RAPD). Results based on RAPD analyses showed considerable genetic diversity in the parental populations (H(e)=0.202). The overall populations including the parental and young trees showed slightly higher genetic diversity (H(e)=0.298) than the parents, with about 10% variation between populations. An unweighted pair group method with arithmetic mean analysis dendrogram based on cluster analysis of the Jaccard similarity among individuals demonstrated a more complicated relationship of the parental and young trees from the two islands, although the young trees showed a clear association with parental trees. This indicates a significant contribution of parental trees in establishing the sour orange populations on the two islands. According to farmers' knowledge, conservation of only one or two parental trees would be sufficient because they believed that the whole populations were generated from a single mother tree. However, this study suggests that preserving most parental trees and some selected young trees with distant genetic relationships should be an effective conservation strategy for sour orange germplasm on the two islands.

Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

PubMed

Kim, Seong U; Batule, Bhagwan S; Mun, Hyoyoung; Byun, Ju-Young; Shim, Won-Bo; Kim, Min-Gon

2018-02-07

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H 2 O 2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR.

PubMed

Chaumeau, V; Andolina, C; Fustec, B; Tuikue Ndam, N; Brengues, C; Herder, S; Cerqueira, D; Chareonviriyaphap, T; Nosten, F; Corbel, V

2016-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.

Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR

PubMed Central

Chaumeau, V.; Andolina, C.; Fustec, B.; Tuikue Ndam, N.; Brengues, C.; Herder, S.; Cerqueira, D.; Chareonviriyaphap, T.; Nosten, F.; Corbel, V.

2016-01-01

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies. PMID:27441839

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods.

PubMed

Maluping, R P; Ravelo, C; Lavilla-Pitogo, C R; Krovacek, K; Romalde, J L

2005-01-01

The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.

Molecular characterization and determination of antimicrobial resistance of Mycoplasma gallisepticum isolated from chickens.

PubMed

Pakpinyo, Somsak; Sasipreeyajan, Jiroj

2007-11-15

In this study, three consecutive approaches of molecular characterization, determination of minimum inhibitory concentration (MIC) and antimicrobial tested on Mycoplasma gallisepticum (MG) isolated from chicken farms were investigated. These approaches were conducted between 2004 and 2005 to 134 MG samples collected from five different regions of the intensive farming area of Thailand. Twenty MG isolates and four reference strains including S6, F, ts-11, and 6/85 were classified according to Random Amplification of Polymorphic DNA (RAPD) patterns prior to the antimicrobial tests. These isolates exhibited 5 different genotypes (A-E). Consequently, MG isolates representing each genotype were tested on 11 registered antibiotics. The levels of MIC were determined. Three antibiotics, doxycycline (0.20 microg/ml), tiamulin (0.10 microg/ml), and tylosin (0.33 microg/ml), gave the least MICs among all effective drugs. Break point comparisons of each antimicrobial suggested that the MG isolates were most sensitive to lincomycin, oxytetracycline, tiamulin, and tylosin. Some MG isolates had an intermediate effect on josamycin and were resistant to enrofloxacin and erythromycin. Our results also indicated that MG isolated and collected from the region and nearby districts had similar RAPD patterns showing properties of antimicrobial resistance. The RAPD patterns may imply the frequent use of antibiotics and a resistant strain of MG. This is the first report of genetic characterization using RAPD reflected by the levels of MIC against MG. The information is useful to plan for prophylactic and therapeutic impacts on the poultry industry especially in the area of intensive use of antibiotics.

Distribution of Genes Encoding Putative Transmissibility Factors among Epidemic and Nonepidemic Strains of Burkholderia cepacia from Cystic Fibrosis Patients in the United Kingdom

PubMed Central

Clode, Fiona E.; Kaufmann, Mary E.; Malnick, Henry; Pitt, Tyrone L.

2000-01-01

In the last 15 years, Burkholderia cepacia has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients. Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients. These are the cblA gene, encoding giant cable pili; a hybrid of two insertion sequences, IS1356 and IS402; and a 1.4-kb open reading frame known as the B. cepacia epidemic strain marker (BCESM). The latter two are of unknown function. An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern. We searched for the described genetic markers using specific PCR assays with 117 patient isolates of B. cepacia from 40 United Kingdom hospitals. Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272. A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates). All isolates of the epidemic strain were positive for the cblA gene and BCESM, but two lacked the insertion sequence hybrid. None of the 76 sporadic isolates contained cblA or the insertion sequence hybrid, but 11 of them were positive for BCESM. Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4). There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II. We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients. The presence of the cblA gene is the most specific marker for the epidemic strain. We recommend that all isolates of B. cepacia from CF patients should be screened by PCR to influence segregation and infection control strategies. PMID:10790095

Isolation and molecular identification of endophytic diazotrophs from seeds and stems of three cereal crops.

PubMed

Liu, Huawei; Zhang, Lei; Meng, Aihua; Zhang, Junbiao; Xie, Miaomiao; Qin, Yaohong; Faulk, Dylan Chase; Zhang, Baohong; Yang, Shushen; Qiu, Li

2017-01-01

Ten strains of endophytic diazotroph were isolated and identified from the plants collected from three different agricultural crop species, wheat, rice and maize, using the nitrogen-free selective isolation conditions. The nitrogen-fixing ability of endophytic diazotroph was verified by the nifH-PCR assay that showed positive nitrogen fixation ability. These identified strains were classified by 879F-RAPD and 16S rRNA sequence analysis. RAPD analyses revealed that the 10 strains were clustered into seven 879F-RAPD groups, suggesting a clonal origin. 16S rRNA sequencing analyses allowed the assignment of the 10 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Paenibacillus, Enterobacter, Klebsiella and Pantoea. These representative genus are not endophytic diazotrophs in the conventional sense. They may have obtained nitrogen fixation ability through lateral gene transfer, however, the evolutionary forces of lateral gene transfer are not well known. Molecular identification results from 16S rRNA analyses were also confirmed by morphological and biochemical data. The test strains SH6A and MZB showed positive effect on the growth of plants.

RAPD and SCAR markers as potential tools for detection of milk origin in dairy products: Adulterant sheep breeds in Serra da Estrela cheese production.

PubMed

Cunha, Joana T; Ribeiro, Tânia I B; Rocha, João B; Nunes, João; Teixeira, José A; Domingues, Lucília

2016-11-15

Serra da Estrela Protected Designation of Origin (PDO) cheese is the most famous Portuguese cheese and has a high commercial value. However, the adulteration of production with cheaper/lower-quality milks from non-autochthones ovine breeds compromises the quality of the final product and undervalues the original PDO cheese. A Randomly Amplified Polymorphic DNA (RAPD) method was developed for efficient detection of adulterant breeds in milk mixtures used for fraudulent production of this cheese. Furthermore, Sequence Characterized Amplified Region (SCAR) markers were designed envisioning the detection of milk adulteration in processed dairy foods. The RAPD-SCAR technique is here described, for the first time, to be potentially useful for detection of milk origin in dairy products. In this sense, our findings will play an important role on the valorization of Serra da Estrela cheese, as well as on other high-quality dairy products prone to adulteration, contributing to the further development of the dairy industry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of different preservation methods on inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers in botanic samples.

PubMed

Wang, Xiaolong; Li, Lin; Zhao, Jiaxin; Li, Fangliang; Guo, Wei; Chen, Xia

2017-04-01

To evaluate the effects of different preservation methods (stored in a -20°C ice chest, preserved in liquid nitrogen and dried in silica gel) on inter simple sequence repeat (ISSR) or random amplified polymorphic DNA (RAPD) analyses in various botanical specimens (including broad-leaved plants, needle-leaved plants and succulent plants) for different times (three weeks and three years), we used a statistical analysis based on the number of bands, genetic index and cluster analysis. The results demonstrate that methods used to preserve samples can provide sufficient amounts of genomic DNA for ISSR and RAPD analyses; however, the effect of different preservation methods on these analyses vary significantly, and the preservation time has little effect on these analyses. Our results provide a reference for researchers to select the most suitable preservation method depending on their study subject for the analysis of molecular markers based on genomic DNA. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Characterization of Streptococcus constellatus strains recovered from a brain abscess and periodontal pockets in an immunocompromised patient.

PubMed

Marques da Silva, Rafael; Caugant, Dominique A; Josefsen, Roger; Tronstad, Leif; Olsen, Ingar

2004-12-01

There have been a number of reports of brain abscesses suggesting an odontogenic etiology. However, no efforts have been made to compare brain abscess isolates with isolates from the oral cavity using highly discriminative methods. We report a brain abscess caused by Streptococcus constellatus in an immunocompromised patient where oral infection (periodontitis) was suspected to be implicated. The brain abscess and oral isolates were compared by means of one phenotypic and three genetic (restriction fragment length polymorphism [RFLP], ribotyping, and random amplified polymorphic DNA [RAPD]) fingerprinting techniques. The phenotypic method and RFLP showed identical profiles between brain and periodontal isolates, while ribotyping and RAPD showed very close similarity, with only one band difference in one of the three ribotypes and in one of the three polymorphic RAPD. Gene transfer by genetic recombinational events in the periodontal pocket might have been responsible for the emergence of a strain variant of S. constellatus that had the potential to cause an abscess at a distant site (brain). The importance of odontogenic sources as potential foci of infection for brain abscesses is discussed.

[Comparative study of the population structure and population assignment of sockeye salmon Oncorhynchus nerka from West Kamchatka based on RAPD-PCR and microsatellite polymorphism].

PubMed

Zelenina, D A; Khrustaleva, A M; Volkov, A A

2006-05-01

Using two types of molecular markers, a comparative analysis of the population structure of sockeye salmon from West Kamchatka as well as population assignment of each individual fish were carried out. The values of a RAPD-PCR-based population assignment test (94-100%) were somewhat higher than those based on microsatellite data (74-84%). However, these results seem quite satisfactory because of high polymorphism of the microsatellite loci examined. The UPGMA dendrograms of genetic similarity of three largest spawning populations, constructed using each of the methods, were highly reliable, which was demonstrated by high bootstrap indices (100% in the case of RAPD-PCR; 84 and 100%, in the case of microsatellite analysis), though the resultant trees differed from one another. The different topology of the trees, in our view, is explained by the fact that the employed methods explored different parts of the genome; hence, the obtained results, albeit valid, may not correlate. Thus, to enhance reliability of the results, several methods of analysis should be used concurrently.

The relationship between genetic and chemotypic diversity in American ginseng (Panax quinquefolius L.).

PubMed

Schlag, Erin M; McIntosh, Marla S

2013-09-01

Ginseng is one of the world's most important herbals used as an adaptogen and a cure for an impressively large range of ailments. Differences in the medicinal properties of ginseng roots have been attributed to variation in ginsenoside composition. In this study, the association between genetic and chemotypic profiles of wild and cultivated American ginseng (Panax quinquefolius L.) roots grown in Maryland was investigated. Ginseng roots were classified into chemotypes based on their relative composition of Re and Rg1. Genetic profiles of these roots were determined from the analysis of 38 polymorphic RAPD markers and used for a cluster analysis of genetic similarities. The close correspondence between chemotype and genetic cluster provides the first DNA-based evidence for the genetic basis of ginsenoside composition. Results of this research are significant for plant breeding and conservation, phytochemical research, and clinical and pharmacological studies. Also, the correlation between RAPD markers and chemotype indicates the potential to use RAPD markers as a reliable and practical method for identification and certification of ginseng roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers.

PubMed

Lam, Kelly Y C; Chan, Gallant K L; Xin, Gui-Zhong; Xu, Hong; Ku, Chuen-Fai; Chen, Jian-Ping; Yao, Ping; Lin, Huang-Quan; Dong, Tina T X; Tsim, Karl W K

2015-12-15

Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps.

Properties of targeted preamplification in DNA and cDNA quantification.

PubMed

Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

2015-01-01

Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

Genic and Intergenic SSR Database Generation, SNPs Determination and Pathway Annotations, in Date Palm (Phoenix dactylifera L.).

PubMed

Mokhtar, Morad M; Adawy, Sami S; El-Assal, Salah El-Din S; Hussein, Ebtissam H A

2016-01-01

The present investigation was carried out aiming to use the bioinformatics tools in order to identify and characterize, simple sequence repeats within the third Version of the date palm genome and develop a new SSR primers database. In addition single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions were recognized. Moreover, the pathways for the sequences assigned by SSR primers, the biological functions and gene interaction were determined. A total of 172,075 SSR motifs was identified on date palm genome sequence with a frequency of 450.97 SSRs per Mb. Out of these, 130,014 SSRs (75.6%) were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, only 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. A total of 111,403 of SSR primer pairs were designed, that represents 291.9 SSR primers per Mb. Out of the 111,403, only 31,380 SSR primers were in the genic regions, while 80,023 primers were in the intergenic regions. A number of 250,507 SNPs were recognized in 84,172 SSR flanking regions, which represents 75.55% of the total SSR flanking regions. Out of 12,274 genes only 463 genes comprising 896 SSR primers were mapped onto 111 pathways using KEGG data base. The most abundant enzymes were identified in the pathway related to the biosynthesis of antibiotics. We tested 1031 SSR primers using both publicly available date palm genome sequences as templates in the in silico PCR reactions. Concerning in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forwardmore » or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments.« less

Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species.

PubMed

Gonzalez, Lorena B Parra; Straub, Shannon C K; Doyle, Jeff J; Ortega, Paula E Mora; Garrido, Haroldo E Salvo; Butler, Iván J Maureira

2010-08-01

Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. • Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. • These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.

Gender Identification in Date Palm Using Molecular Markers.

PubMed

Awan, Faisal Saeed; Maryam; Jaskani, Muhammad J; Sadia, Bushra

2017-01-01

Breeding of date palm is complicated because of its long life cycle and heterozygous nature. Sexual propagation of date palm does not produce true-to-type plants. Sex of date palms cannot be identified until the first flowering stage. Molecular markers such as random amplified polymorphic DNA (RAPD), sequence-characterized amplified regions (SCAR), and simple sequence repeats (SSR) have successfully been used to identify the sex-linked loci in the plant genome and to isolate the corresponding genes. This chapter highlights the use of three molecular markers including RAPD, SCAR, and SSR to identify the gender of date palm seedlings.

[A comparative analysis of Ungulata species by different molecular genetic markers (proteins, RAPD-PCR)].

PubMed

Glazko, V I; Zelenaia, L B; Iasinetskaia, N A

1997-01-01

The investigation of genetic interrelation between a number of Artiodactyla and Perissodactyla species with the use of different types of molecular-genetic markers (proteins, RAPD-PCR) were carried out. The marker-specific features of interspecific relations and their similarities on the groups of markers of both types were revealed. The distinctions between interspecies genetic relations and ones estimated from the phylogeny on the determined group of different types of markers were observed. It was supposed that these discrepancies may be related with common selection factors and involving this marker group in selection in some species.

Shear Bond Strength of Superficial, Intermediate and Deep Dentin In Vitro with Recent Generation Self-etching Primers and Single Nano Composite Resin.

PubMed

Singh, Kulshrest; Naik, Rajaram; Hegde, Srinidhi; Damda, Aftab

2015-01-01

This in vitro study is intended to compare the shear bond strength of recent self-etching primers to superficial, intermediate, and deep dentin levels. All teeth were sectioned at various levels and grouped randomly into two experimental groups and two control groups having three subgroups. The experimental groups consisted of two different dentin bonding system. The positive control group consisted of All Bond 2 and the negative control group was without the bonding agent. Finally, the specimens were subjected to shear bond strength study under Instron machine. The maximum shear bond strengths were noted at the time of fracture. The results were statistically analyzed. Comparing the shear bond strength values, All Bond 2 (Group III) demonstrated fairly higher bond strength values at different levels of dentin. Generally comparing All Bond 2 with the other two experimental groups revealed highly significant statistical results. In the present investigation with the fourth generation, higher mean shear bond strength values were recorded compared with the self-etching primers. When intermediate dentin shear bond strength was compared with deep dentin shear bond strength statistically significant results were found with Clearfil Liner Bond 2V, All Bond 2 and the negative control. There was a statistically significant difference in shear bond strength values both with self-etching primers and control groups (fourth generation bonding system and without bonding system) at superficial, intermediate, and deep dentin. There was a significant fall in bond strength values as one reaches deeper levels of dentin from superficial to intermediate to deep.

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

PubMed

Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

2015-05-01

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.

PubMed

Chun, Jong-Yoon; Kim, Kyoung-Joong; Hwang, In-Taek; Kim, Yun-Jee; Lee, Dae-Hoon; Lee, In-Kyoung; Kim, Jong-Kee

2007-01-01

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.

Tri-Service Thermal Flash Test Facility. Summary Report.

DTIC Science & Technology

1980-01-15

degradation of materials exposed to the radiant heating generated by a nuclear blast can very enormously. The intense radiation needed to simulate a...Photography - Motion picture photo- graphy of surface degradation would be an asset to data analysis. Although this procedure is relatively straightforward...Polyvinylchloride 68 Rubber 69 Army Systems Camouflage MIL-E-52798A over TTP-636 primer 70 Army Systems Camouflage MIL-E-52835A over TTP-636 primer 71 Army

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PubMed Central

Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

PubMed

Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis.

PubMed

Kokosková, B; Mráz, I; Fousek, J

2010-05-01

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.

Assessment of diversity among populations of Rauvolfia serpentina Benth. Ex. Kurtz. from Southern Western Ghats of India, based on chemical profiling, horticultural traits and RAPD analysis.

PubMed

Nair, Vadakkemuriyil Divya; Raj, Rajan Pillai Dinesh; Panneerselvam, Rajaram; Gopi, Ragupathi

2014-01-01

Genetic, morphological and chemical variations of ten natural populations of Rauvolfia serpentina Benth. Ex. Kurtz. from Southern Western Ghats of India were assessed using RAPD markers reserpine content and morphological traits. An estimate of genetic diversity and differentiation between genotypes of breeding germplasm is of key importance for its improvement. Populations were collected from different geographical regions. Data obtained through three different methods were compared and the correlation among them was estimated. Statistical analysis showed significant differences for all horticultural characteristics among the accessions suggesting that selection for relevant characteristics could be possible. Variation in the content of Reserpine ranges from 0.192 g/100 g (population from Tusharagiri) to 1.312 g/100 g (population from Aryankavu). A high diversity within population and high genetic differentiation among them based on RAPDs were revealed caused both by habitat fragmentation of the low size of most populations and the low level of gene flow among them. The UPGMA dendrogram and PCA analysis based on reserpine content yielded higher separation among populations indicated specific adaptation of populations into clusters each of them including populations closed to their geographical origin. Genetic, chemical and morphological data were correlated based on Mantel test. Given the high differentiation among populations conservation strategies should take into account genetic diversity and chemical variation levels in relation to bioclimatic and geographic location of populations. Our results also indicate that RAPD approach along with horticultural analysis seemed to be best suited for assessing with high accuracy the genetic relationships among distinct R. serpentina accessions. © 2013.

Assessment of genetic stability and instability of tissue culture-propagated plantlets of Aloe vera L. by RAPD and ISSR markers.

PubMed

Rathore, Mangal Singh; Chikara, J; Mastan, Shaik G; Rahman, H; Anand, K G V; Shekhawat, N S

2011-11-01

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis.

PubMed

Perrig, Melina S; Ambroggio, María B; Buzzola, Fernanda R; Marcipar, Iván S; Calvinho, Luis F; Veaute, Carolina M; Barbagelata, María Sol

2015-01-01

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6% concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8% (134/137) and 94.9% (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95% and 100% with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Distribution of Candida albicans in the oral cavity of children aged 3-5 years of Uygur and Han nationality and their genotype in caries-active groups.

PubMed

Wu, N; Lin, J; Wu, L; Zhao, J

2015-01-30

We analyzed the distribution of Candida albicans in the oral cavity of 3-5-year-old children of Uygur and Han nationalities as well as their genotypes in caries-active groups in the Urumqi municipality. CHROMagar Candida was separately cultivated, and we identified 359 Uygur and Han children aged 3-5 years. We randomly selected 20 Han children and 20 Uygur children for this study. We chose a bacterial strain for polymerase chain reaction (PCR) 25S rDNA genotyping and random amplified polymorphic DNA (RAPD) genotyping. The rate of caries-active in Han children was higher than that in Uygur children, with values of 39.6 and 24.3%, respectively. The detection rate of C. albicans was closely correlated to the caries filling index classification (X(2) = 31.037, P = 0.000, r = 0.421; X(2) = 80.454, P = 0.000, r = 0.497). PCR of 25S rDNA from 40 strains of Han and Uygur children revealed 3 genotypes, while RAPD analysis revealed 5 genotypes. The distribution of 25S rDNA genotyping of Han children from PCR differed from that of Uygur children (X(2) = 7.697, P = 0.021), both of which were mainly the A type. RAPD genotyping of both Han and Uygur children showed similar results (X(2) = 1.573, P = 0.814). There were differences in the distributions of C. albicans in children of different nationalities. C. albicans is a key factor causing caries. The PCR 25S rDNA genotyping method is simple and sensitive, while the RAPD genotyping method is reliable and comprehensive.

Beauveria keratitis and biopesticides: case histories and a random amplification of polymorphic DNA comparison.

PubMed

Pariseau, Brett; Nehls, Sarah; Ogawa, Gregory S H; Sutton, Deanna A; Wickes, Brian L; Romanelli, Anna M

2010-02-01

The purposes of this study were to describe 2 contact lens-associated Beauveria keratitis cases and to compare the isolates of 3 contact lens-associated Beauveria keratitis cases with Beauveria-based biopesticides using random amplification of polymorphic DNA (RAPD). A 55-year-old diabetic woman from New Mexico and a 31-year-old healthy woman from southern Wisconsin developed soft contact lens-related corneal ulcers unresponsive to topical moxifloxacin and prednisolone acetate drops. Their corneal cultures grew B. bassiana. These isolates, an isolate from a third soft contact lens-related Beauveria keratitis case, and Beauveria-based biopesticides sold in the United States were analyzed using morphological features, DNA sequencing, and RAPD. A PubMed, Cochrane Library, OVID, UpToDate, and Google search using the term "Beauveria" found only 9 reported Beauveria keratitis infections. Patient 1 responded to topical natamycin, ketoconazole, and 200 mg oral ketoconazole twice daily before developing a secondary bacterial infection requiring penetrating keratoplasty. After subsequent cataract surgery, the best-corrected visual acuity was 20/20. Patient 2 was treated with topical natamycin, topical amphotericin, and 200 mg oral voriconazole twice daily for 1 month with residual scarring and a best-corrected visual acuity of 20/25. RAPD showed that all isolates were unrelated. Although earlier reported Beauveria keratitis cases occurred after corneal injury in patients who did not wear contact lenses, 3 recent patients wore soft contact lenses and denied trauma, mirroring a changing trend in microbial keratitis. RAPD analysis showed that the Beauveria isolates were unrelated to one another and to Beauveria-based biopesticides. In Patient 2, oral voriconazole worked well.

Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer.

PubMed

Hayashi, N; Ito, M; Horiike, S; Taguchi, H

2001-05-01

Random amplified polymorphic DNA (RAPD) PCR analysis of Lactobacillus brevis isolates from breweries revealed that one of the random primers could distinguish beer-spoilage strains of L. brevis from nonspoilage strains. The 1.1-kb DNA fragment amplified from all beer-spoilers included one open reading frame, termed hitA (hop-inducible cation transporter), which encodes an integral membrane protein with 11 putative trans-membrane domains and a binding protein-dependent transport signature of a non-ATP binding membrane transporter common to several prokaryotic and eukaryotic transporters. The hitA polypeptide is homologous to the natural resistance-associated macrophage protein (Nramp) family characterized as divalent-cation transport proteins in many prokaryotic and eukaryotic organisms. Northern blot analysis indicated that the hitA transcripts are expressed in cells cultivated in MRS broth supplemented with hop bitter compounds, which act as mobile-carrier ionophores, dissipating the trans-membrane pH gradient in bacteria sensitive to the hop bitter compounds by exchanging H+ for cellular divalent cations such as Mn2+. This suggests that the hitA gene products may play an important role in making the bacteria resistant to hop bitter compounds in beer by transporting metal ions such as Mn2+ into cells that no longer maintain the proton gradient.

Diversity and genetic distance in populations of Steindachnerina in the upper Paraná river floodplain of Brazil.

PubMed

Oliveira, A V; Prioli, A J; Prioli, S M A P; Pavanelli, C S; Júlio, H F; Panarari, R S

2002-08-01

Whereas four species of the genus Steindachnerina occur in the Paraná river basin, S. insculpta was the only endemic species of the region under analysis, which is the third lower section of the upper Paraná river. Among other factors, this species has been characterised by the absence of spots in the basal region of the dorsal fin. However, various specimens with this characteristic appeared in the region after the construction of the Itaipu Hydroelectric Plant in 1982. An analysis of the genetic variability of Steindachnerina populations with or without spots is provided. Specimens were collected in different sites of the floodplain of the upper Paraná river and samples were compared by random amplified polymorphic DNA (RAPD) technique and morphological analyses. Ninety-eight amplified loci with nine random primers were analysed in 19 specimens of each phenotype. Data for genetic distance showed great divergences between the two phenotypes and indicate two different species. Spotted specimens may be identified as S. brevipinna, found in the region downstream Sete Quedas Falls. The species must have overcome the geographical barrier during the building of the Itaipu hydroelectric dam that submerged the waterfalls and which became an obstacle between the upper and middle Paraná river some 150 km downstream. Since phenotypes do not share dominant alleles, absence of gene flow has been suggested.

Influence of thiourea application on some physiological and molecular criteria of sunflower (Helianthus annuus L.) plants under conditions of heat stress.

PubMed

Akladious, Samia Ageeb

2014-05-01

High temperature is a major factor limiting the growth of plant species during summer. Understanding the mechanisms of plant tolerance to high temperature would help in developing effective management practices and heat-tolerant cultivars through breeding or biotechnology. The present investigation was carried out to study the role of thiourea in enhancing the tolerance of sunflower plants to heat stress. Sunflower plants were subjected to temperature stress by exposing plants to 35 or 45 °C for 12 h. Two levels of thiourea (10 and 20 mM) were applied before sowing (seed treatment). The results indicated that the plants exposed to temperature stress exhibited a significant decline in growth parameters, chlorophylls, relative leaf water content, oil content, leaf nutrient status, and nitrate reductase activity. Treatment with thiourea, especially when applied at 10 mM, improved the above parameters and induced non-enzymatic and enzymatic antioxidants responsible for antioxidation. SDS-PAGE of protein revealed that high-temperature treatments alone or in combination with thiourea were associated with the disappearance of some bands or the appearance of unique ones. The result of RAPD analysis using five primers showed variable qualitative and quantitative changes. These findings confirm the effectiveness of applying thiourea on alleviating heat injuries in sunflower plants.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology(1.).

PubMed

Robarts, Daniel W H; Wolfe, Andrea D

2014-07-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology1

PubMed Central

Robarts, Daniel W. H.; Wolfe, Andrea D.

2014-01-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance. PMID:25202637

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye.

PubMed

Li, Cuiling; Xia, Guangmin; Xiang, Fengning; Zhou, Chuanen; Cheng, Aixia

2004-12-01

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments--cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.

Field Populations of Wild Apis cerana Honey Bees Exhibit Increased Genetic Diversity Under Pesticide Stress Along an Agricultural Intensification Gradient in Eastern India.

PubMed

Chakrabarti, Priyadarshini; Sarkar, Sagartirtha; Basu, Parthiba

2018-05-01

Pesticides have been reported to be one of the major drivers in the global pollinator losses. The large-scale decline in honey bees, an important pollinator group, has resulted in comprehensive studies on honey bee colonies. Lack of information on native wild pollinators has paved the way for this study, which highlights the underlying evolutionary changes occurring in the wild honey bee populations exposed to pesticides along an agricultural intensification landscape. The study reports an increased genetic diversity in native Apis cerana Fabricius (Hymenoptera: Apidae) populations continually exposed to pesticide stress. An increased heterozygosity, evidenced by a higher electrophoretic banding pattern, was observed in the pesticide-exposed populations for two isozymes involved with xenobiotic metabolism-esterase and glucose-6-phosphate dehydrogenase. Differential banding patterns also revealed a higher percentage of polymorphic loci, number of polymorphic bands, Nei's genetic distance, etc. observed in these populations in the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) experiments using three random decamer primers. Higher heterozygosity, being indicative of a more resistant population, implies population survival within the threshold pesticide stress. This study reports such changes for the first time in native wild Indian honey bee populations exposed to pesticides and has far-reaching implications on the population adaptability under pesticide stress.

Molecular monitoring of spoilage yeasts during the production of candied fruit nougats to determine food contamination sources.

PubMed

Martorell, Patricia; Fernández-Espinar, M Teresa; Querol, Amparo

2005-06-15

In the present work, we have analysed the yeast microbiota present in a manufacturing plant of candied fruits and nougats. Four yeasts species (Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Sporobolomyces roseus, and Debaryomyces hansenii) and a filamentous fungi (Nectria mauriiticola) were identified according to restriction analysis of 5.8S-ITS rDNA. These identifications were subsequently confirmed by sequencing the D1/D2 domain of the 26S rRNA gene. Z. rouxii and Z. bailii were isolated at high frequency along the whole manufacturing process. Since food alteration by Z. bailii and Z. rouxii is the cause of important economic losses for the food industry, there is a need for differentiating yeasts at the strain level as an essential part of quality control programs in this industry. For this purpose, we have tested the performance of three molecular techniques (RFLP mtDNA, RAPD-PCR, and microsatellite with (GAC)5 and (GTG)5 primers) to differentiate strains belonging to these two Zygosaccharomyces species. Those techniques with the best discriminatory power were applied to differentiate Zygosaccharomyces species isolates. The results of this analysis indicate that one strain of Z. bailii and two strains of Z. rouxii were involved in the spoilage of candied fruits. Moreover, the Z. bailii strain was also present in the spoiled nougat, hence being responsible of this alteration.

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production.

PubMed

Saxami, Georgia; Papadopoulou, Olga S; Chorianopoulos, Nikos; Kourkoutas, Yiannis; Tassou, Chrysoula C; Galanis, Alex

2016-05-04

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties.

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

PubMed Central

Saxami, Georgia; Papadopoulou, Olga S.; Chorianopoulos, Nikos; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Galanis, Alex

2016-01-01

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties. PMID:27153065

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids.

PubMed

Chupeau, M C; Maisonneuve, B; Bellec, Y; Chupeau, Y

1994-10-28

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.

Robot-assisted duodenum-preserving pancreatic head resection with pancreaticogastrostomy for benign or premalignant pancreatic head lesions: a single-centre experience.

PubMed

Jiang, Yu; Jin, Jia-Bin; Zhan, Qian; Deng, Xia-Xing; Peng, Cheng-Hong; Shen, Bai-Yong

2018-03-02

The purpose of this study was to compare short- and long-term outcomes of modified robot-assisted duodenum-preserving pancreatic head resection (RA-DPPHR) versus robot-assisted pancreaticoduodenectomy (RA-PD). Matched for age, sex, ASA classification, tumour size, history of abdominal surgery and pathological type, 34 patients undergoing RA-DPPHR and 34 patients undergoing RA-PD between January 2010 and December 2016 were retrospectively analyzed. The RA-DPPHR group had shorter surgical time (188.2 vs. 386.3 min, p < 0.001) and less blood loss (168.2 vs. 386.3 ml, p = 0.026) but higher complication rate (47.1% vs. 32.4%, p = 0.105) and pancreatic fistula rate (32.4% vs. 17.6%, p = 0.161). Hospital mortality was 2.9%. Exocrine insufficiency was lower in the RA-DPPHR group (3.0% vs. 24.2%, p = 0.027). Endocrine insufficiency was observed in one RA-DPPHR patient and 5 RA-PD patients (p = 0.197). Modified RA-DPPHR benefits in terms of better conservation of exocrine and endocrine pancreatic functions at the expense of a significant morbidity and non-zero mortality. Copyright © 2018 John Wiley & Sons, Ltd.

Multiple correspondence analysis and random amplified polymorphic DNA molecular typing to assess the sources of Staphylococcus aureus contamination in alheira production lines.

PubMed

Esteves, A; Patarata, L; Aymerich, T; Garriga, M; Martins, C

2007-03-01

Sources and tracing of Staphylococcus aureus in alheira (garlic sausage) production were evaluated by multifactorial correspondence analysis (MCA) of occurrence data and a random amplified polymorphic DNA (RAPD) on S. aureus isolates. Samples from four production lines, four different production batches, and 14 different sampling sites (including raw material, different contact surfaces, and several stages of alheira manufacturing) were analyzed at four sampling times. From the 896 microbial analyses completed, a collection of 170 S. aureus isolates was obtained. Although analysis of the occurrence data alone was not elucidative enough, MCA and RAPD-PCR were able to assess the sources of contamination and to trace the spread of this microorganism along the production lines. MCA results indicated that the presence of S. aureus in alheira was related to its presence in the intermediate manufacturing stages after heat treatment but before stuffing in the casings. It was also possible to associate a cross-contamination path related to handler procedures. RAPD-PCR typing in accordance to MCA results confirmed the cross-contamination path between the raw material and casings and the role of handlers as an important cross-contamination vehicle.

3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

PubMed

Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

2008-09-01

We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

Spatial Genetic Structure and Clonal Diversity in an Alpine Population of Salix herbacea (Salicaceae)

PubMed Central

Reisch, Christoph; Schurm, Sophia; Poschlod, Peter

2007-01-01

Background and Aims Many alpine plant species combine clonal and sexual reproduction to minimize the risks of flowering and seed production in high mountain regions. The spatial genetic structure and diversity of these alpine species is strongly affected by different clonal strategies (phalanx or guerrilla) and the proportion of generative and vegetative reproduction. Methods The clonal structure of the alpine plant species Salix herbacea was investigated in a 3 × 3 m plot of an alpine meadow using microsatellite (simple sequence repeat; SSR) analysis. The data obtained were compared with the results of a random amplified polymorphic DNA (RAPD) analysis. Key Results SSR analysis, based on three loci and 16 alleles, revealed 24 different genotypes and a proportion of distinguishable genotypes of 0·18. Six SSR clones were found consisting of at least five samples, 17 clones consisting of more than two samples and seven single genotypes. Mean clone size comprising at least five samples was 0·96 m2, and spatial autocorrelation analysis showed strong similarity of samples up to 130 cm. RAPD analysis revealed a higher level of clonal diversity but a comparable number of larger clones and a similar spatial structure. Conclusions The spatial genetic structure as well as the occurrence of single genotypes revealed in this study suggests both clonal and sexual propagation and repeated seedling recruitment in established populations of S. herbacea and is thus suggestive of a relaxed phalanx strategy. PMID:17242040

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment.

PubMed

Nardi, Tiziana; Carlot, Milena; De Bortoli, Elena; Corich, Viviana; Giacomini, Alessio

2006-11-01

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.

Novel ITS1 Fungal Primers for Characterization of the Mycobiome

PubMed Central

Usyk, Mykhaylo; Zolnik, Christine P.; Patel, Hitesh; Levi, Michael H.

2017-01-01

ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states. PMID:29242834

Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs

PubMed Central

Housley, Donna JE; Zalewski, Zachary A; Beckett, Stephanie E; Venta, Patrick J

2006-01-01

Background Cross-species primers have been used with moderate success to address a variety of questions concerning genome structure, evolution, and gene function. However, the factors affecting their success have never been adequately addressed, particularly with respect to producing a consistent method to achieve high throughput. Using 1,147 mammalian cross-species primer pairs (1089 not previously reported), we tested several factors to determine their influence on the probability that a given target will amplify in a given species under a single amplification condition. These factors included: number of mismatches between the two species (the index species) used to identify conserved regions to which the primers were designed, GC-content of the gene and amplified region, CpG dinucleotides in the primer region, degree of encoded protein conservation, length of the primers, and the degree of evolutionary distance between the target species and the two index species. Results The amplification success rate for the cross-species primers was significantly influenced by the number of mismatches between the two index species (6–8% decrease per mismatch in a primer pair), the GC-content within the amplified region (for the dog, GC ≥ 50%, 56.9% amplified; GC<50%, 74.2% amplified), the degree of protein conservation (R2 = 0.14) and the relatedness of the target species to the index species. For the dog, 598 products of 930 primer pairs (64.3%) (excluding primers in which dog was an index species) were sequenced and shown to be the expected product, with an additional three percent producing the incorrect sequence. When hamster DNA was used with the single amplification condition in a microtiter plate-based format, 510 of 1087 primer pairs (46.9%) produced amplified products. The primer pairs are spaced at an average distance of 2.3 Mb in the human genome and may be used to produce up to several hundred thousand bp of species-specific sequence. Conclusion The most important factors influencing the proportion of successful amplifications are the number of index species mismatches, GC-richness of the target amplimer, and the relatedness of the target species to the index species, at least under the single PCR condition used. The 1147 cross-species primer pairs can be used in a high throughput manner to generate data for studies on the genetics and genomics of non-sequenced mammalian genomes. PMID:17029642

Development of a molecular approach to describe the composition of Trichoderma communities.

PubMed

Meincke, Remo; Weinert, Nicole; Radl, Viviane; Schloter, Michael; Smalla, Kornelia; Berg, Gabriele

2010-01-01

Trichoderma and its teleomorphic stage Hypocrea play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel Trichoderma-specific primer pair for diversity analysis. Based on a broad range master alignment, specific Trichoderma primers (ITSTrF/ITSTrR) were designed that comprise an approximate 650bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus Trichoderma. This amplicon is suitable for identification with TrichoKey and TrichoBLAST. Moreover, this primer system was successfully applied to study the Trichoderma communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent Trichoderma composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse Trichoderma communities in the rhizosphere of potatoes. High field heterogeneity of Trichoderma communities was revealed by both DGGE. Furthermore, qPCR showed significantly different Trichoderma copy numbers between the sites. Copyright 2009 Elsevier B.V. All rights reserved.

Quantitative analysis of ribosome–mRNA complexes at different translation stages

PubMed Central

Shirokikh, Nikolay E.; Alkalaeva, Elena Z.; Vassilenko, Konstantin S.; Afonina, Zhanna A.; Alekhina, Olga M.; Kisselev, Lev L.; Spirin, Alexander S.

2010-01-01

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture. PMID:19910372

Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers.

PubMed

Chiou, Shu-Jiau; Yen, Jui-Hung; Fang, Cheng-Li; Chen, Hui-Ling; Lin, Tsai-Yun

2007-10-01

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.

Different Strategies for Molecular Differentiation of Mycobacterium bovis Strains Isolated in Sardinia, Italy

PubMed Central

Sechi, Leonardo A.; Leori, Guido; Lollai, Stefano A.; Duprè, Ilaria; Molicotti, Paola; Fadda, Giovanni; Zanetti, Stefania

1999-01-01

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed. PMID:10103282

Hexavalent chromium exposures during full-aircraft corrosion control.

PubMed

Carlton, Gary N

2003-01-01

Aluminum alloys used in the construction of modern aircraft are subject to corrosion. The principal means of controlling this corrosion in the U.S. Air Force are organic coatings. The organic coating system consists of a chromate conversion coat, epoxy resin primer, and polyurethane enamel topcoat. Hexavalent chromium (CrVI) is present in the conversion coat in the form of chromic acid and in the primer in the form of strontium chromate. CrVI inhalation exposures can occur when workers spray conversion coat onto bare metal and apply primer to the treated metal surface. In addition, mechanical abrasion of aircraft surfaces can generate particulates that contain chromates from previously applied primers and conversion coats. This study measured CrVI exposures during these corrosion control procedures. Mean time-weighted average (TWA) exposure to chromic acid during conversion coat treatment was 0.48 microg/m(3), below the current American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV(R)) TWA of 50 microg/m(3) for water-soluble CrVI compounds. Mean TWA exposures to strontium chromate were 5.33 microg/m(3) during mechanical abrasion and 83.8 microg/m(3) during primer application. These levels are in excess of the current ACGIH TLV-TWA of 0.5 microg/m(3) for strontium chromate. In the absence of a change from chromated to nonchromated conversion coats and primers, additional control measures are needed to reduce these exposures.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

PubMed Central

Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

2017-01-01

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

DNA polymerase preference determines PCR priming efficiency.

PubMed

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

PubMed

Karkhane, Ali Asghar; Yakhchali, Bagher; Rastgar Jazii, Ferdous; Bambai, Bijan; Aminzadeh, Saeed; Rahimi, Fatemeh

2015-06-01

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

PubMed

Fatima, Nigar; Ahmad, Naseem; Ahmad, Iqbal; Anis, Mohammad

2015-09-01

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

PubMed Central

Baldi, Paolo; La Porta, Nicola

2017-01-01

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field. PMID:28642764

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

PubMed

Adachi, Noboru; Umetsu, Kazuo; Shojo, Hideki

2014-01-01

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

RAPD/SCAR Approaches for Identification of Adulterant Breeds' Milk in Dairy Products.

PubMed

Cunha, Joana T; Domingues, Lucília

2017-01-01

Food safety and quality are nowadays a major consumers' concern. In the dairy industry the fraudulent addition of cheaper/lower-quality milks from nonlegitimate species/breeds compromises the quality and value of the final product. Despite the already existing approaches for identification of the species origin of milk, there is little information regarding differentiation at an intra-species level. In this protocol we describe a low-cost, sensitive, fast, and reliable analytical technique-Random Amplified Polymorphic DNA/Sequence Characterized Amplified Region (RAPD/SCAR)-capable of an efficient detection of adulterant breeds in milk mixtures used for fraudulent manufacturing of dairy products and suitable for the detection of milk adulteration in processed dairy foods.

[The genetic diversity and homology of Anabaena azollae and its host plant (Azolla) based on rapd analysis].

PubMed

Chen, Jian; Zheng, Wei-wen; Xu, Guo-zhong; Song, Tie-ying; Tang, Long-fei

2002-01-01

Symbiotic Anabeana azollae and its host plant Anabeana-free Azolla were isolated from 16 Azolla accessions representing different Azolla species or geographic origins.DNA polymorphic fragments were obtained by simultaneous RAPD amplification of both symbiont and host. The UPGMA clusters of Anabeana azollae and its host Azolla were established separately based on Dice coefficient caculation and a coordinated relationship was shown between Anabeana azollae and its Azolla host along both individual genetic divergence,but this genetic homology was reduced among different strains within Azolla species while the obvious mutants of Anabeana azollae were detected in some Azolla tested strains collected from different geographic area in the same host species.

Genetic diversity of Iranian honey bee (Apis mellifera meda Skorikow, 1829) populations based on ISSR markers.

PubMed

Rahimi, A; Mirmoayedi, A; Kahrizi, D; Zarei, L; Jamali, S

2016-04-30

Honey bee is one of the most important insects considering its role in agriculture,ecology and economy as a whole. In this study, the genetic diversity of different Iranian honey bee populations was evaluated using inter simple sequence repeat (ISSR) markers. During May to September 2014, 108 young worker honey bees were collected from six different populations in 30 different geoclimatic locations from Golestan, Mazendaran, Guilan, West Azerbaijan, East Azerbaijan, Ardebil provinces of Iran. DNA was extracted from the worker honey bees. The quality and quantity of extracted DNA were measured. A set of ten primers were screened with the laboratory populations of honey bees. The number of fragments produced in the different honey bee populations varied from 3 to 10, varying within 150 to 1500 bp. The used ten ISSR primers generated 40 polymorphic fragments, and the average heterozygosity for each primer was 0.266. Maximum numbers of bands were recorded for primer A1. A dendrogram based on the Unweighted Pair Group Method with Arithmetic mean (UPGMA) method generated two sub-clusters. Honey bee populations of Golestan, Mazendaran, Guilan provinces were located in the first group. The second group included honey bee populations of Ardebil, West Azerbaijan, East Azerbaijan provinces, but this group showed a close relationship with other populations. The results showed obviously the ability of the ISSR marker technique to detect the genetic diversity among the honey bee populations.

Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Leibner-Ciszak, Justyna; Dobrowolska, Anita; Krawczyk, Beata; Kaszuba, Aleksandra; Staczek, Paweł

2010-02-01

In order to identify the source of infections caused by dermatophytes, as well as the pathogen transmission pathway, there is a need to determine methods that allow detailed genetic differentiation of the strains within the dermatophyte genera. In this work, a PCR melting profile (PCR-MP) technique based on the ligation of adaptors and the difference in melting temperatures of DNA restriction fragments was used for the first time for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, scalp, toenails and fingernails were used for this study. PCR-MP and random amplification of polymorphic DNA (RAPD) were used to type 11 isolates of Trichophyton rubrum, 40 isolates of Trichophyton interdigitale and 14 isolates of Microsporum canis. The results distinguished five types (containing one subtype) characteristic for T. rubrum and seven types characteristic for T. interdigitale using the PCR-MP technique. Analysis conducted using RAPD revealed five types for T. rubrum and four types for T. interdigitale isolates. No differentiation was observed for the M. canis isolates with either method. These results demonstrate that PCR-MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strains and yields a discriminatory power that is at least equal to that of RAPD.

Transmission trials, ITS2-PCR and RAPD-PCR show identity of Toxocara canis isolates from red fox and dog.

PubMed

Epe, C; Meuwissen, M; Stoye, M; Schnieder, T

1999-07-01

Toxocara canis isolates from dog and from red fox were compared in transmission trials and with molecular analysis using RAPD-PCR technique and comparison of the ITS2 sequence. After oral infection of bitches with 20,000 embryonated T. canis eggs of vulpine and canine origin, the vertical transmission to pup's was examined. All animals of both groups developed typical clinical symptoms of toxocarosis. The haematological, serological, parasitological and post mortem results showed no differences between both isolates except for the infectivity of T. canis stages in mice where the fox isolate showed a significant higher infectivity than the dog isolate. The RAPD-PCR showed a similarity coefficient of 0.95, similar to the range of intraspecific variation in Toxocara cati and Toxascaris leonina specimens as outgroups. The ITS2 comparison showed a 100% identity between both isolates with no intraspecific variations. Therefore, the study shows that the fox and the dog isolate of T. canis were identical in infectivity, transmission and molecular structure; a host adaptation could not be found and the fox has to be seen as a reservoir for T. canis infections in dogs. Considering the increasing number of foxes in urban areas the importance of helminth control in dogs is stressed.

RAPD of controlled crosses and clones from the field suggests that hybrids are rare in the Salix alba-Salix fragilis complex.

PubMed

Triest, L; De Greef, B; De Bondt, R; Van Slycken, J

2000-05-01

The polyploid Salix alba-Salix fragilis hybrid complex is rather difficult to study when using only morphological characters. Most of the features have a low diagnostic value for unambiguously identifying the hybrids, introgression patterns and population structures, though morphological traits have proved to be useful in making a hybrid index. Morphology and molecular variation from RAPDs were investigated in several case studies on willows from Belgium. A thorough screening of full-sib progenies of interspecific controlled crosses was made to select homologous amplification products. The selected amplified products proved to be useful in a principal coordinate analysis for the estimation of variability of hybrid progenies. On the basis of genetic similarities and ordination analysis, a method for the identification of clones in the field was established using presumed pure species and presumed introgressants. The chosen reference clones were checked against additional European samples of putative pure species to ensure the reliability of the method beyond a regional scale. The RAPDs suggested that both species have kept their gene pools well separated and that hybridization actually does not seem to be a dominating process. The observation that molecular markers do not always follow the morphological traits or allozyme data is discussed.

Molecular serotyping and antimicrobial resistance profiles of Actinobacillus pleuropneumoniae isolated from pigs in South Korea.

PubMed

Kim, Boram; Hur, Jin; Lee, Ji Yeong; Choi, Yoonyoung; Lee, John Hwa

2016-09-01

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP). Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined. Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR. Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains). Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.

Salmonella enteritidis outbreak in Thailand: study by random amplified polymorphic DNA (RAPD) analysis.

PubMed

Kantama, L; Jayanetra, P

1996-03-01

An outbreak of Salmonella enteritidis in Thailand was reported in 1990. The majority of isolates were found in chicken and human throughout the country. The continuation of a high rate of spreading which is presently continuing prompted us to investigate possible clonal involvement in the outbreak. One hundred and twenty five isolates of S. enteritidis which were isolated between 1990-1993 were clonally identified by the technique of Random Amplified Polymorphic DNA (RAPD) analysis. Eight profiles were found indicating the presence of 8 clones, designated no. 1-8. The predominant clone was profile no. 4 which was encountered in 93.6% of tested isolates while the rest of the profile comprised only 0.8-1.6%. The predominant clone was distributed mainly in isolates from chickens and humans which is suggestive that the profile no. 4 is the major clone involved in this outbreak and that chickens were the source of S. enteritidis infection. The information from the Microbiology Laboratory at Ramathibodi Hospital revealed that nearly 40% of S. enteritidis were isolated from blood specimens. This may reflect the invasiveness of S. enteritidis in Thailand. We concluded that the outbreak involved the single clone, RAPD profile no. 4 which may disperse dominantly during the epidemic.

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment.

PubMed

Martín, B; Corominas, L; Garriga, M; Aymerich, T

2009-01-01

Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages. Different points during the production of a traditional fermented sausage type (fuet) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31.4%), Enterococcus faecium (30.7%), Enterococcus sanguinicola (14.9%), Enterococcus devriesei (9.7%), Enterococcus malodoratus (7.2%), Enterococcus gilvus (1.0%), Enterococcus gallinarum (1.3%), Enterococcus casseliflavus (3.4%), Enterococcus hermanniensis (0.2%), and Enterococcus durans (0.2%). A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source. The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin. This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.

Developing biochemical and molecular markers for cyanobacterial inoculants.

PubMed

Prasanna, R; Madhan, K; Singh, R N; Chauhan, A K; Nain, L

2010-09-01

Markers for evaluating the establishment of cyanobacteria based on their sensitivity or resistance to antibiotics, saccharide utilization patterns and PCR generated fingerprints were developed. Four selected strains (isolates from rhizosphere soils of diverse agro-ecosystems) have shown potential as diazotrophs and exhibited plant growth promoting abilities. Different responses were obtained on screening against 40 antibiotics, which aided in developing selectable antibiotic markers for each strain. Biochemical profiles generated using standardized chromogenic identification system (including saccharide utilization tests) revealed that 53 % of the saccharides tested were not utilized by any strain, while some strains exhibited unique ability for utilization of saccharides such as melibiose, cellobiose, maltose and glucosamine. PCR based amplification profiles developed using a number of primers based on repeat sequences revealed the utility of 3 primers in providing unique fingerprints for the strains.

Relationships among superantigen toxin gene profiles, genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis.

PubMed

Wang, Dong; Zhang, Limei; Yong, Changfu; Shen, Mingliang; Ali, Tariq; Shahid, Muhammad; Han, Kun; Zhou, Xuezhang; Han, Bo

2017-06-01

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Primer and short-range releaser pheromone properties of premolt female urine from the shore crab Carcinus maenas.

PubMed

Ekerholm, Mattias; Hallberg, Eric

2005-08-01

The European shore crab Carcinus maenas is considered to rely on a female pheromone when mating. Evidence, however, is scarce on how the urine pheromone in itself affects males. We investigated male primer and releaser responses to female pheromones with methods that minimized effects from females, delivering female urine either as a pump-generated plume or deposited on a polyurethane sponge. We delivered the pheromone at different concentrations in far, near, and close/contact range to get a picture of how distance affects behavioral response. Our results show that substances in premolt female urine (PMU) function as primer and potent short-range releaser pheromones. Based on the olfactometer and sponge tests, we conclude that PMU stimulus in itself is sufficient to elicit increased search and mating-specific behaviors such as posing, posing search, cradle carrying, and stroking. Pheromone concentrations do not seem to be important for attenuating search and posing as long as the level is above a certain threshold concentration. Instead, pheromone levels seem to play a role in male acceptance of females, recruiting more males to respond, and generating better responses with increasing concentration.

PRIMED: PRIMEr Database for Deleting and Tagging All Fission and Budding Yeast Genes Developed Using the Open-Source Genome Retrieval Script (GRS)

PubMed Central

Cummings, Michael T.; Joh, Richard I.; Motamedi, Mo

2015-01-01

The fission (Schizosaccharomyces pombe) and budding (Saccharomyces cerevisiae) yeasts have served as excellent models for many seminal discoveries in eukaryotic biology. In these organisms, genes are deleted or tagged easily by transforming cells with PCR-generated DNA inserts, flanked by short (50-100bp) regions of gene homology. These PCR reactions use especially designed long primers, which, in addition to the priming sites, carry homology for gene targeting. Primer design follows a fixed method but is tedious and time-consuming especially when done for a large number of genes. To automate this process, we developed the Python-based Genome Retrieval Script (GRS), an easily customizable open-source script for genome analysis. Using GRS, we created PRIMED, the complete PRIMEr D atabase for deleting and C-terminal tagging genes in the main S. pombe and five of the most commonly used S. cerevisiae strains. Because of the importance of noncoding RNAs (ncRNAs) in many biological processes, we also included the deletion primer set for these features in each genome. PRIMED are accurate and comprehensive and are provided as downloadable Excel files, removing the need for future primer design, especially for large-scale functional analyses. Furthermore, the open-source GRS can be used broadly to retrieve genome information from custom or other annotated genomes, thus providing a suitable platform for building other genomic tools by the yeast or other research communities. PMID:25643023

Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but Not L. rhamnosus GG in the Human Vagina as Demonstrated by Randomly Amplified Polymorphic DNA

PubMed Central

Gardiner, Gillian E.; Heinemann, Christine; Bruce, Andrew W.; Beuerman, Dee; Reid, Gregor

2002-01-01

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 are well-characterized probiotic strains with efficacy in the prevention and treatment of urogenital infections in women. The aim of the present study was to apply a molecular biology-based methodology for the detection of these strains and L. rhamnosus GG (a commercially available intestinal probiotic) in the human vagina in order to assess probiotic persistence at this site. Ten healthy women inserted vaginally a capsule containing either a combination of strains GR-1 and RC-14 or the GG strain for 3 consecutive nights. Vaginal swabs taken before and at various time points after probiotic insertion were analyzed, and the Lactobacillus flora was assessed by randomly amplified polymorphic DNA (RAPD) analysis. This method generated discrete DNA fingerprints for GR-1, RC-14, and GG and enabled successful detection of these strains in the vagina. Strain GR-1 and/or strain RC-14 was found to persist in the vaginal tract for up to 19 days after vaginal instillation, while L. rhamnosus GG was detectable for up to 5 days postadministration. In conclusion, the fates of probiotic L. rhamnosus and L. fermentum strains were successfully monitored in the human vagina by RAPD analysis. This technique provides molecular biology-based evidence that RC-14 and GR-1, strains selected as urogenital probiotics, persist in the human vagina and may be more suited to vaginal colonization than L. rhamnosus GG. This highlights the importance of proper selection of strains for urogenital probiotic applications. PMID:11777835

Yeasts from autochthonal cheese starters: technological and functional properties.

PubMed

Binetti, A; Carrasco, M; Reinheimer, J; Suárez, V

2013-08-01

The aim of this work was to identify 20 yeasts isolated from autochthonal cheese starters and evaluate their technological and functional properties. The capacities of the yeasts to grow at different temperatures, pH, NaCl and lactic acid concentrations as well as the proteolytic and lipolytic activities were studied. Moreover, survival to simulated gastrointestinal digestion, hydrophobicity, antimicrobial activity against pathogens and auto- and co-aggregation abilities were evaluated. The sequentiation of a fragment from the 26S rDNA gene indicated that Kluyveromyces marxianus was the predominant species, followed by Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces lactis and Galactomyces geotrichum. RAPD with primer M13 allowed a good differentiation among strains from the same species. All strains normally grew at pH 4.7-5.5 and temperatures between 15 and 35°C. Most of them tolerated 10% NaCl and 3% lactic acid. Some strains showed proteolytic (eight isolates) and/or lipolytic (four isolates) capacities. All strains evidenced high gastrointestinal resistance, moderate hydrophobicity, intermediate auto-aggregation and variable co-aggregation abilities. No strains inhibited the growth of the pathogens assayed. Some strains from dairy sources showed interesting functional and technological properties. This study has been the first contribution to the identification and characterization of yeasts isolated from autochthonal cheese starters in Argentina. Many strains could be proposed as potential candidates to be used as probiotics and/or as co-starters in cheese productions. © 2013 The Society for Applied Microbiology.

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLetchie, D.N.; Tuskan, G.A.

Gender, the expression of maleness or femaleness, in dioecious plants has been associated with changes in morphology, physiology, ecological position, and commercial importance of several species, including members of the Salicaceae family. Various mechanisms have been proposed to explain the expression of gender in Salicaceae, including sex chromosomes, simple Mendelian genes, quantitative genes, environment, and genotype-by-environment interactions. Published reports would favor a genetic basis for gender. The objective of this study was to identify molecular markers associated with gender in a segregating family of hybrid poplars. Bulked segregant analysis and chi-squared analysis were used to test for the occurrence ofmore » sex chromosomes, individual loci, and chromosome ratios (i.e., ploidy levels) as the mechanisms for gender determination. Examination of 2488 PCR based RAPD markers from 1219 primers revealed nine polymorphic bands between male and female bulked samples. However, linkage analysis indicated that none of these markers were significantly associated with gender. Chisquared results for difference in male-to-female ratios between diploid and triploid genotypes also revealed no significant differences. These findings suggest gender is not controlled via sex chromosomes, simple Mendelian loci or ratios of autosome to gender-determining loci. It is possible that gender is determined genetically by regions of the genome not sampled by the tested markers or by a complex of loci operating in an additive threshold manner or in an epistatic manner. It is also possible that gender is determined environmentally at an early zygote stage, canalizing gender expression.« less

Genetic comparisons between seed bank and Stipa krylovii plant populations.

PubMed

Han, B; Zhao, M

2011-09-01

The soil seed bank represents the potential plant population since it is the source for population replacement. The genetic structure of a Stipa krylovii (Roshev.) plant population and its soil seed bank was investigated in the Xilinguole Steppe of Inner Mongolia using random amplified polymorphic DNA (RAPD) analyses. The population was sampled at two sites that were in close proximity to each other (0.5 km apart). Thirty plants and 18 seed bank samples were taken from each site to determine the genetic diversity between sites and between sources (plant or seed). The material was analyzed using 13 primers to produce 92 loci. Eighty-six were multi-loci, of which 23 loci (26.74%) of allele frequencies showed significant differences (P < or = 0.05). The genetic similarity between two seed bank sites was 0.9843 while the genetic similarity between two plant sites was 0.9619. Their similarities were all greater than that between the seed bank and plant populations. An analysis of their genetic structure showed that 87.86% of total variation was derived by two-loci. Genetic structures between plant and soil seed bank populations in S. krylovii were different due to the variance of mean gametic disequilibria and mean gene diversity. AMOVA results showed that the majority of variance (88.62%) occurred within sites, 12.75% was from between-groups. Further research is needed to investigate the selective function in maintaining the genetic diversity of Stipa krylovii plant populations.

EPA Lean Government Initiative: How to Replicate Lean Successes

EPA Pesticide Factsheets

This Lean Replication Primer describes how EPA Offices and Regions can identify and adapt successful practices from previous Lean projects to “replicate” their successes and generate further improvements.

Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104:H4 outbreak strains.

PubMed

Pritchard, Leighton; Holden, Nicola J; Bielaszewska, Martina; Karch, Helge; Toth, Ian K

2012-01-01

An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 'positive' E. coli O104:H4 outbreak and 32 'negative' non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.

[The use of RAPD and ITE molecular markers to study genetical structure of the Crimean population of Triticum boeoticum Boiss].

PubMed

Mallabaeva, D Sh; Ignatov, A N; Sheĭko, I A; Isikov, V P; Geliuta, V P; Boĭko, N G; Seriapin, A A; Dorokhov, D B

2007-01-01

Wild wheat Triticum boeoticum Boiss. is the rare species are included in the Red Book of Ukraine. This species are reducing the magnitude of population and the area of distribution under anthropogenic activity. We studied genetic structure of two populations of T. boeoticum, located on Sapun Mountain and in Baidar Valley in Crimea. According RAPD and ITE molecular analysis we have estimated that the population of T. boeoticum on Sapun Mountain is genetically more impoverished than a population from the Baidar Valley. For preservation of maximal natural genetic polymorphism of the rare species it is recommended to direct efforts to preservations of a population of T. boeoticum from the Baidar Valley.

SEM/EDS analysis and characterization of gunshot residues from Brazilian lead-free ammunition.

PubMed

Martiny, Andrea; Campos, Andrea P C; Sader, Marcia S; Pinto, André L

2008-05-02

The exposition to heavy metal-rich airborne due to fire practicing has forced to the development of heavy metal-free environmental ammunition primers all over the world. Here we characterize the GSR elements present in the Brazilian lead-free ammunition produced by Companhia Brasileira de Cartuchos (CBC) and commercialized by MagTech in the U.S. and Europe under the name CleanRange centerfire cartridges. Both first and second generations of CleanRange in calibers 9 mm Luger, .40 S&W, .380 AUTO and .38 SPL were analyzed and compared to regular Brazilian CBC ammunition by scanning electron microscopy/energy dispersive spectroscopy. Differences in composition and morphology of GSR particles from the two generations of CleanRange were observed. The first generation ammunition (found in Europe) presented spherical particles, being strontium the only unique element detected. The second generation (found in the U.S.) produced irregular particles composed mostly by potassium, aluminum, silicon and calcium. We can conclude that identification of GSR derived from CBC second generation lead-free ammunition in suspects' hands may be impossible without the addition of a distinct metallic taggant in the primer composition by the manufacturer.

Do we need primer for orthodontic bonding? A randomized controlled trial.

PubMed

Nandhra, Sarabjit Singh; Littlewood, Simon J; Houghton, Nadine; Luther, Friedy; Prabhu, Jagadish; Munyombwe, Theresa; Wood, Simon R

2015-04-01

To evaluate the clinical performance of APC™II Victory Series™ (3M Unitek) brackets in direct orthodontic bonding with and without the use of primer. A single-operator, two-centre prospective, non-inferiority randomized controlled clinical trial. The Orthodontic departments at the Leeds Dental Institute and St Luke's Hospital, Bradford, UK. Ethical approval was granted by Leeds (East) Research Ethics Committee on 18th of December 2009 (Reference 09/H1306/102). The protocol was not published prior to trial commencement. Ninety-two patients requiring orthodontic treatment with fixed appliances were randomly allocated to the control (bonded with primer) or test groups (bonded without primer). Patients were randomly allocated to either the control or experimental group. This was performed by preparing opaque numbered sealed envelopes in advance using a random numbers table generated by a computer by an independent third party . Once the envelopes were opened, blinding of the operator and the patient was no longer possible due to the nature of the intervention. Patients were approached for inclusion in the trial if they qualified for NHS orthodontic treatment requiring fixed appliances and had no previous orthodontic treatment. Number of bracket failures, time to bond-up appliances, and the adhesive remnant index (ARI) when bracket failure occurred, over a 12-month period Failure rate with primer was 11.1 per cent and without primer was 15.8 per cent. Bonding without primer was shown statistically to be non-inferior to bonding with primer odds ratio 0.95-2.25 (P = 0.08). Mean difference in bond-up time per bracket was 0.068 minutes (4 seconds), which was not statistically significant (P = 0.402). There was a statistically significant difference in the Adhesive Remnant Index - ARI 0 with primer 49.4 per cent, no primer 76.5 per cent, (P < 0.0001). As the study was only performed by one operator, the results can therefore only be truly be applied to their practice. Also this study was powered to ascertain if there was no difference between the 2 groups up to 5%, however orthodontists may consider a change in the bracket failure rate of 2% to be clinically significant. When bonding with APC™II Victory Series™ brackets without primer was shown statistically to be non-inferior to bonding with primer (P =0.08). There was no significant difference in bond-up times. Bond failure was more likely to happen at the composite-enamel interface when bonded without a primer. No conflict of interest for all authors. No funding sources were used. Study was not registered on external databases. © The Author 2014. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

PubMed

Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske

2007-02-14

The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis. We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication.

PubMed

Sasaki, Yohei; Fushimi, Hirotoshi; Cao, Hui; Cai, Shao-Qing; Komatsu, Katsuko

2002-12-01

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.

A Phylogenomic Approach Based on PCR Target Enrichment and High Throughput Sequencing: Resolving the Diversity within the South American Species of Bartsia L. (Orobanchaceae)

PubMed Central

Tank, David C.

2016-01-01

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples. PMID:26828929

Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei City, Taiwan.

PubMed Central

Harn, H J; Shen, K L; Ho, L I; Yu, K W; Liu, G C; Yueh, K C; Lee, J H

1997-01-01

AIMS: To determine, by strain identification of Mycobacterium tuberculosis, whether transmission has occurred between individuals or whether new strains are present. METHODS: A rapid protocol for random amplified polymorphic DNA (RAPD) analysis was developed. This protocol was applied to 64 strains of M tuberculosis that had been confirmed by culture and microbiological methods. RESULTS: There are five groups of M tuberculosis prevalent in Taipei city, Taiwan. The major types are groups I and III. Groups I and II had been prevalent until the end of last year when, according to our group analysis, they had been eradicated. However, group III was continuously present from the middle of 1995 to the middle of 1996, and group IV was present at the end of both years, which indicated that both groups were transmitted continuously. These clustered strains had demographic characteristics consistent with a finding of transmission tuberculosis. Also, there were 13 of 64 strains with unique RAPD fingerprints that were inferred to be due primarily to the reactivation of infection. In the drug resistance analysis, the major type represented included group III and part of group IV. CONCLUSIONS: Our preliminary data imply, not only that the prevalence of M tuberculosis in Taipei city is due to transmission rather than reactivation, but that drug resistance also may play a role in tuberculosis transmission. Images PMID:9378819

Genetic distances between popcorn populations based on molecular markers and correlations with heterosis estimates made by diallel analysis of hybrids.

PubMed

Munhoz, R E F; Prioli, A J; Amaral, A T; Scapim, C A; Simon, G A

2009-08-11

Diallel analysis was used to obtain information on combining ability, heterosis, estimates of genetic distances by random amplified polymorphic DNA (RAPD) and on their correlations with heterosis, for the popcorn varieties RS 20, UNB2, CMS 43, CMS 42, Zélia, UEM J1, UEM M2, Beija-Flor, and Viçosa, which were crossed to obtain all possible combinations, without reciprocals. The genitors and the 36 F(1) hybrids were evaluated in field trials in Maringá during two growing seasons in a randomized complete block design with three replications. Based on the results, strategies for further studies were developed, including the construction of composites by joining varieties with high general combining ability for grain yield (UNB2 and CMS 42) with those with high general combining ability for popping expansion (Zélia, RS 20 and UEM M2). Based on the RAPD markers, UEM J1 and Zélia were the most genetically distant and RS 20 and UNB2 were the most similar. The low correlation between heterosis and genetic distances may be explained by the random dispersion of the RAPD markers, which were insufficient for the exploitation of the popcorn genome. We concluded that an association between genetic dissimilarity and heterosis based only on genetic distance is not expected without considering the effect of dominant loci.

Candidiasis During Pregnancy May Result From Isogenic Commensal Strains

PubMed Central

Daniels, Wayne; Glover, Douglas D.; Essmann, Michael

2001-01-01

Objective: Our laboratory previously demonstrated that asymptomatic vaginal colonization during pregnancy is a factor predisposing patients to subsequent symptomatic vulvovaginal candidiasis. It is unknown whether symptoms result from strain replacement or a change in host relationship to the original colonizing strain. This study was undertaken to determine whether Candida albicans isolates from asymptomatic women could be responsible for subsequent symptomatic vaginitis. Methods: We retained isolates of C. albicans from women followed longitudinally through pregnancy, and identified six pairs of cultures from women who were colonized without symptoms and who later became symptomatic (average time 14 weeks). We used a random amplification of polymorphic DNA (RAPD) analysis to determine whether isolates from our study patients were genetically similar or dissimilar. Results: Analysis of these pairs of yeast strains by RAPD revealed that five of the six women had symptoms apparently due to the same yeast strain that was found initially as a commensal strain. To increase the power of these observations, we also performed RAPD analysis on six randomly selected yeast strains from other women in this study who had not become symptomatic to determine whether any of these unrelated strains matched strains from those women who became symptomatic. Conclusion: Symptomatic yeast vaginitis is usually due to strains of C. albicans already carried in the lower genital tract, underscoring the need to understand regulation of growth and virulence of the organism in vivo. PMID:11495556

Trans-gastric pancreaticogastrostomy reconstruction after pylorus-preserving robotic Whipple: a proposal for a standardized technique.

PubMed

Giulianotti, Pier C; Gonzalez-Heredia, Raquel; Esposito, Sofia; Masrur, Mario; Gangemi, Antonio; Bianco, Francesco M

2018-04-01

A number of technical improvements regarding the pancreatic anastomosis have decreased the morbidity and mortality after pancreaticoduodenectomy. However, postoperative pancreatic fistula (POPF) remains is the most feared complication, and the ideal technique for pancreatic reconstruction is undetermined. This study is a retrospective review of a prospectively maintained database. Data were collected from all consecutive robot-assisted pancreaticoduodenectomies (RAPD), performed by a single surgeon, at the University of Illinois Hospital & Health Sciences System, between September 2007 and January 2016. A total of 28 consecutive patients (16 male and 12 female) who underwent a RAPD were included in this study. Patients had a mean age and mean BMI of 61.5 years (SD = 12.3) and 27 kg/m 2 (SD = 4.9), respectively. The mean operative time was 468.2 min (SD = 73.7) and the average estimated blood loss was 216.1 ml (SD = 113.1). The mean length of hospitalization was 13.1 days (SD = 5.4). There was no clinically significant POPF registered. Trans-gastric pancreaticogastrostomy (TPG) represents a valid and feasible option as a pancreatic digestive reconstruction during RAPD. Initial results showed decreased incidence of POPF with an increased risk of postoperative bleeding. Our experience suggests that TPG might be safer than pancreaticojejunostomy (PJ); further studies are needed in order to confirm.

Genetic Diversity of Vibrio cholerae O1 in Argentina and Emergence of a New Variant

PubMed Central

Pichel, Mariana; Rivas, Marta; Chinen, Isabel; Martín, Fernando; Ibarra, Cristina; Binsztein, Norma

2003-01-01

The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucumán showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose “Tucumán variant” as the designation for this new cluster of cholera toxin-negative V. cholerae O1 strains. PMID:12517837

Genetic diversity of Cosmos species revealed by RAPD and ISSR markers.

PubMed

Rodríguez-Bernal, A; Piña-Escutia, J L; Vázquez-García, L M; Arzate-Fernández, A M

2013-12-04

The genus Cosmos is native of America and is constituted by 34 species; 28 of them are endemic of Mexico. The cosmos are used as a nematicide, antimalarial, and antioxidative agent. The aim of this study was to estimate the genetic diversity among 7 cosmos species based on random amplified polymorphic DNA (RAPD) and inter-simple sequences repeats (ISSR) markers. With RAPD markers, the obtained polymorphism was 91.7 % and the genetic diversity was 0.33, whereas these values were 65.6%, and 0.22 from ISSR markers, respectively, indicating the presence of high genetic diversity among the Cosmos species that were analyzed. The unweighted pair group method with arithmetic mean dendrograms that were obtained with both markers were notably similar, revealing 2 clusters and indicating a clear genetic differentiation among the Cosmos species that were assessed. The first cluster comprised the species Cosmos sulphureus, Cosmos pacificus, and Cosmos diversifolius, while the second cluster included the species Cosmos purpureus, Cosmos crithmifolius, Cosmos bipinnatus, and Cosmos parviflorus. Besides this, the Cosmos species were clustered according to their collection sites. The Mantel test corroborates the correlation between the genetic distance and the geographic altitude of each Cosmos species. The results suggest that it is necessary to preserve the Cosmos species in their natural habitat in addition to the germoplasm collection for ex situ conservation.

Next Generation Loading System for Detonators and Primers

DTIC Science & Technology

Designed , fabricated and installed next generation tooling to provide additional manufacturing capabilities for new detonators and other small...prototype munitions on automated, semi-automated and manual machines. Lead design effort, procured and installed a primary explosive Drying Oven for a pilot...facility. Designed , fabricated and installed a Primary Explosives Waste Treatment System in a pilot environmental processing facility. Designed

TSUNAMI Primer: A Primer for Sensitivity/Uncertainty Calculations with SCALE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rearden, Bradley T; Mueller, Don; Bowman, Stephen M

2009-01-01

This primer presents examples in the application of the SCALE/TSUNAMI tools to generate k{sub eff} sensitivity data for one- and three-dimensional models using TSUNAMI-1D and -3D and to examine uncertainties in the computed k{sub eff} values due to uncertainties in the cross-section data used in their calculation. The proper use of unit cell data and need for confirming the appropriate selection of input parameters through direct perturbations are described. The uses of sensitivity and uncertainty data to identify and rank potential sources of computational bias in an application system and TSUNAMI tools for assessment of system similarity using sensitivity andmore » uncertainty criteria are demonstrated. Uses of these criteria in trending analyses to assess computational biases, bias uncertainties, and gap analyses are also described. Additionally, an application of the data adjustment tool TSURFER is provided, including identification of specific details of sources of computational bias.« less

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

PubMed Central

Levin, Joshua D.; Fiala, Dean; Samala, Meinrado F.; Kahn, Jason D.; Peterson, Raymond J.

2006-01-01

Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, CT) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or CT. ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence. PMID:17071964

A new fungal large subunit ribosomal RNA primer for high throughput sequencing surveys

DOE PAGES

Mueller, Rebecca C.; Gallegos-Graves, La Verne; Kuske, Cheryl R.

2015-12-09

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300–400 bp region of the D2 hypervariable regionmore » of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R–LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Altogether, these findings show that the LR22R–LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.« less

Random amplified polymorphic DNA PCR in the teaching of molecular epidemiology.

PubMed

Reinoso, Elina B; Bettera, Susana G

2016-07-08

In this article, we describe a basic practical laboratory designed for fifth-year undergraduate students of Microbiology as part of the Epidemiology course. This practice provides the students with the tools for molecular epidemiological analysis of pathogenic microorganisms using a rapid and simple PCR technique. The aim of this work was to assay RAPD-PCR technique in order to infer possible epidemiological relationships. The activity gives students an appreciation of the value of applying a simple molecular biological method as RAPD-PCR to a discipline-specific question. It comprises a three-session laboratory module to genetically assay DNAs from strains isolated from a food outbreak. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):391-396, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

Molecular Analysis of Date Palm Genetic Diversity Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSRs).

PubMed

El Sharabasy, Sherif F; Soliman, Khaled A

2017-01-01

The date palm is an ancient domesticated plant with great diversity and has been cultivated in the Middle East and North Africa for at last 5000 years. Date palm cultivars are classified based on the fruit moisture content, as dry, semidry, and soft dates. There are a number of biochemical and molecular techniques available for characterization of the date palm variation. This chapter focuses on the DNA-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) techniques, in addition to biochemical markers based on isozyme analysis. These techniques coupled with appropriate statistical tools proved useful for determining phylogenetic relationships among date palm cultivars and provide information resources for date palm gene banks.

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

PubMed

Yang, G; Ding, J; Wu, L R; Duan, Y D; Li, A Y; Shan, J Y; Wu, Y X

2015-03-13

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts

PubMed Central

Ealba, Erin L.; Schneider, Richard A.

2013-01-01

Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level. PMID:23785056

Highway trust fund primer

DOT National Transportation Integrated Search

1972-01-01

This manual is intended for use as a tool in predicting the noise which will be generated by freely-flowing vehicle traffic along a highway of known characteristics. The manual presents two separate approaches to the prediction problem. The first app...

Development of single-copy nuclear intron markers for species-level phylogenetics: Case study with Paullinieae (Sapindaceae).

PubMed

Chery, Joyce G; Sass, Chodon; Specht, Chelsea D

2017-09-01

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline. Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a "pseudoreference" closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae. This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.

Merlin: Computer-Aided Oligonucleotide Design for Large Scale Genome Engineering with MAGE.

PubMed

Quintin, Michael; Ma, Natalie J; Ahmed, Samir; Bhatia, Swapnil; Lewis, Aaron; Isaacs, Farren J; Densmore, Douglas

2016-06-17

Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off-target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.

[Genetic variation and differentiation in striped field mouse Apodemus agrarius inferred from RAPD-PCR analysis].

PubMed

Atopkin, D M; Bogdanov, A S; Chelomina, G N

2007-06-01

Genetic variation and differentiation of the trans-Palearctic species Apodemus agrarius (striped field mouse), whose range consists of two large isolates-European-Siberian and Far Eastern-Chinese, were examined using RAPD-PCR analysis. The material from the both parts of the range was examined (41 individual of A. agrarius from 18 localities of Russia, Ukraine, Moldova, and Kazakhstan); the Far-Eastern part was represented by samples from the Amur region, Khabarovsk krai, and Primorye (Russia). Differences in frequencies of polymorphic RAPD loci were found between the European-Siberian and the Far Eastern population groups of striped field mouse. No "fixed" differences between them in RAPD spectra were found, and none of the used statistical methods permitted to distinguish with absolute certainty animals from the two range parts. Thus, genetic isolation of the European-Siberian and the Far Eastern population groups of A. agrarius is not strict. These results support the hypothesis on recent dispersal of striped field mouse from East to West Palearctics (during the Holocene climatic optimum, 7000 to 4500 years ago) and subsequent disjunction of the species range (not earlier than 4000-4500 years ago). The Far Eastern population group is more polymorphic than the European-Siberian one, while genetic heterogeneity is more uniformly distributed within it. This is probably explained by both historical events that happened during the species dispersal in the past, and different environmental conditions for the species in different parts of its range. The Far Eastern population group inhabits the area close to the distribution center of A. agrarius. It is likely that this group preserved genetic variation of the formerly integral ancestral form, while some amount of genetic polymorphism could be lost during the species colonization of the Siberian and European areas. To date, the settlement density and population number in general are higher than within the European-Siberian isolate, which seems to account for closer interpopulation associations, intense genetic exchange, and "smoothing" of polymorphism within the Far Eastern population group of A. agrarius.

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

PubMed Central

2009-01-01

Background The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique. Results A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two Lactobacillus strains (Lactobacillus salivarius NCIMB 30211 and Lactobacillus acidophilus NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The L. salivarius strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the L. acidophilus NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the Lactobacillus capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both Lactobacillus strains during capsule consumption was statistically significant (p < 0.05). Conclusion We have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials. PMID:19968877

Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus L.)

PubMed Central

2013-01-01

Background Genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation (Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x = 15). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research. Results We improved the SSR-based genetic linkage map using SSR markers derived from a genomic library, expression sequence tags, and RNA-seq data. Linkage analysis revealed that 412 SSR loci (including 234 newly developed SSR loci) could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x = 15). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits (bacterial wilt resistance and anthocyanin pigmentation in the flower) and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data. Conclusions The improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs. PMID:24160306

Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus L.).

PubMed

Yagi, Masafumi; Yamamoto, Toshiya; Isobe, Sachiko; Hirakawa, Hideki; Tabata, Satoshi; Tanase, Koji; Yamaguchi, Hiroyasu; Onozaki, Takashi

2013-10-26

Genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation (Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x = 15). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research. We improved the SSR-based genetic linkage map using SSR markers derived from a genomic library, expression sequence tags, and RNA-seq data. Linkage analysis revealed that 412 SSR loci (including 234 newly developed SSR loci) could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x = 15). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits (bacterial wilt resistance and anthocyanin pigmentation in the flower) and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data. The improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs.

Molecular epidemiology of Serratia marcescens in two hospitals in Gdańsk, Poland, over a 5-year period.

PubMed

Naumiuk, Lukasz; Baraniak, Anna; Gniadkowski, Marek; Krawczyk, Beata; Rybak, Bartosz; Sadowy, Ewa; Samet, Alfred; Kur, Józef

2004-07-01

The history of the Serratia marcescens population in two hospitals in Danzig, Poland, over a 5-year period was analyzed in a study that combined MIC evaluation, typing by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis, and analysis of extended-spectrum beta-lactamases (ESBLs). We analyzed 354 isolates collected from 341 patients in two teaching hospitals in Danzig, Poland, from 1996 to 2000. The antimicrobial susceptibility profiles varied greatly, and for resistance to newer beta-lactams, probable AmpC cephalosporinase derepression and ESBL production occurred in about 23 and 19% of the isolates, respectively. RAPD typing, by which 69 types were discerned altogether, revealed a high degree of clonal diversity among the populations. However, the four most prevalent types were highly predominant, grouping approximately 71% of the isolates studied. These clones were observed in the two hospitals and were strong contributors to both outbreaks and the background of endemicity of the S. marcescens infections. Some of the strains that were not so widely spread (12 RAPD types; approximately 14% of the isolates) were responsible for several smaller outbreaks, and the remaining isolates represented unique RAPD types (53 types; approximately 15% of the isolates) and were probably sporadic introductions from other environments. ESBLs were identified in several different clones, and some of these had most likely already been introduced into the hospitals as ESBL producers, whereas the others acquired the ESBL-encoding genes from other enterobacterial strains in these environments. The CTX-M-3 enzyme, which is widely observed in Poland, was the most common ESBL type among the S. marcescens isolates, followed by TEM-47 and SHV-5. The complex epidemiology of ESBLs, especially in 1999 and 2000, must have arisen from the introduction of ESBL producers from other centers, their clonal dissemination, and the constant penetration of the S. marcescens populations with plasmids with ESBL genes. Multiple S. marcescens isolates were obtained from 11 patients, who probably represented both patients with recolonizations and reinfections and patients with recurrences of infections with the evolution of the strain's susceptibility.

Molecular Epidemiology of Serratia marcescens in Two Hospitals in Danzig, Poland, over a 5-Year Period

PubMed Central

Naumiuk, Łukasz; Baraniak, Anna; Gniadkowski, Marek; Krawczyk, Beata; Rybak, Bartosz; Sadowy, Ewa; Samet, Alfred; Kur, Józef

2004-01-01

The history of the Serratia marcescens population in two hospitals in Danzig, Poland, over a 5-year period was analyzed in a study that combined MIC evaluation, typing by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis, and analysis of extended-spectrum β-lactamases (ESBLs). We analyzed 354 isolates collected from 341 patients in two teaching hospitals in Danzig, Poland, from 1996 to 2000. The antimicrobial susceptibility profiles varied greatly, and for resistance to newer β-lactams, probable AmpC cephalosporinase derepression and ESBL production occurred in about 23 and 19% of the isolates, respectively. RAPD typing, by which 69 types were discerned altogether, revealed a high degree of clonal diversity among the populations. However, the four most prevalent types were highly predominant, grouping approximately 71% of the isolates studied. These clones were observed in the two hospitals and were strong contributors to both outbreaks and the background of endemicity of the S. marcescens infections. Some of the strains that were not so widely spread (12 RAPD types; ∼14% of the isolates) were responsible for several smaller outbreaks, and the remaining isolates represented unique RAPD types (53 types; ∼15% of the isolates) and were probably sporadic introductions from other environments. ESBLs were identified in several different clones, and some of these had most likely already been introduced into the hospitals as ESBL producers, whereas the others acquired the ESBL-encoding genes from other enterobacterial strains in these environments. The CTX-M-3 enzyme, which is widely observed in Poland, was the most common ESBL type among the S. marcescens isolates, followed by TEM-47 and SHV-5. The complex epidemiology of ESBLs, especially in 1999 and 2000, must have arisen from the introduction of ESBL producers from other centers, their clonal dissemination, and the constant penetration of the S. marcescens populations with plasmids with ESBL genes. Multiple S. marcescens isolates were obtained from 11 patients, who probably represented both patients with recolonizations and reinfections and patients with recurrences of infections with the evolution of the strain's susceptibility. PMID:15243068

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

PubMed Central

2009-01-01

Background The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms. Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers. Results The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism. Conclusion The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils. PMID:20003362

Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104:H4 Outbreak Strains

PubMed Central

Bielaszewska, Martina; Karch, Helge; Toth, Ian K.

2012-01-01

Background An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Methodology/Principal Findings Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 ‘positive’ E. coli O104:H4 outbreak and 32 ‘negative’ non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Conclusions/Significance Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact. PMID:22496820

Use of molecular markers to compare Fusarium verticillioides pathogenic strains isolated from plants and humans.

PubMed

Chang, S C; Macêdo, D P C; Souza-Motta, C M; Oliveira, N T

2013-08-12

Fusarium verticillioides is a pathogen of agriculturally important crops, especially maize. It is considered one of the most important pathogens responsible for fumonisin contamination of food products, which causes severe, chronic, and acute intoxication in humans and animals. Moreover, it is recognized as a cause of localized infections in immunocompetent patients and disseminated infections among severely immunosuppressed patients. Several molecular tools have been used to analyze the intraspecific variability of fungi. The objective of this study was to use molecular markers to compare pathogenic isolates of F. verticillioides and isolates of the same species obtained from clinical samples of patients with Fusarium mycoses. The molecular markers that we used were inter-simple sequence repeat markers (primers GTG5 and GACA4), intron splice site primer (primer EI1), random amplified polymorphic DNA marker (primer OPW-6), and restriction fragment length polymorphism-internal transcribed spacer (ITS) from rDNA. From the data obtained, clusters were generated based on the UPGMA clustering method. The amplification products obtained using primers ITS4 and ITS5 and loci ITS1-5.8-ITS2 of the rDNA yielded fragments of approximately 600 bp for all the isolates. Digestion of the ITS region fragment using restriction enzymes such as EcoRI, DraI, BshI, AluI, HaeIII, HinfI, MspI, and PstI did not permit differentiation among pathogenic and clinical isolates. The inter-simple sequence repeat, intron splice site primer, and random amplified polymorphic DNA markers presented high genetic homogeneity among clinical isolates in contrast to the high variability found among the phytopathogenic isolates of F. verticillioides.

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

PubMed Central

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-01-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment

DOE PAGES

Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.; ...

2016-07-15

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability and biogeochemistry is critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected size was confirmed for all hgcAB+ strains tested via Sanger sequencing.more » Additionally, we developed clade-specific degenerate quantitative primers (qPCR) that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88% and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, the resulting data will be essential in developing accurate and robust predictive models of Hg-methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB.« less

Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis.

PubMed

Papavasileiou, Antonios; Madesis, Panagiotis B; Karaoglanidis, George S

2016-09-01

Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit.

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.

PubMed

Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann

2016-08-01

A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.