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.

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.

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.

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.

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.

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.

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.

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

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.

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

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

PubMed

Taspinar, Mahmut Sinan; Aydin, Murat; Sigmaz, Burcu; Yildirim, Nalan; Agar, Guleray

2017-10-01

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

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).

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

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.

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.

Molecular strategy for identification in Aspergillus section Flavi.

PubMed

Godet, Marie; Munaut, Françoise

2010-03-01

Aspergillus flavus is one of the most common contaminants that produces aflatoxins in foodstuffs. It is also a human allergen and a pathogen of animals and plants. Aspergillus flavus is included in the Aspergillus section Flavi that comprises 11 closely related species producing different profiles of secondary metabolites. A six-step strategy has been developed that allows identification of nine of the 11 species. First, three real-time PCR reactions allowed us to discriminate four groups within the section: (1) A. flavus/Aspergillus oryzae/Aspergillus minisclerotigenes/Aspergillus parvisclerotigenus; (2) Aspergillus parasiticus/Aspergillus sojae/Aspergillus arachidicola; (3) Aspergillus tamarii/Aspergillus bombycis/Aspergillus pseudotamarii; and (4) Aspergillus nomius. Secondly, random amplification of polymorphic DNA (RAPD) amplifications or SmaI digestion allowed us to differentiate (1) A. flavus, A. oryzae and A. minisclerotigenes; (2) A. parasiticus, A. sojae and A. arachidicola; (3) A. tamarii, A. bombycis and A. pseudotamarii. Among the 11 species, only A. parvisclerotigenus cannot be differentiated from A. flavus. Using the results of real-time PCR, RAPD and SmaI digestion, a decision-making tree was drawn up to identify nine of the 11 species of section Flavi. In contrast to conventional morphological methods, which are often time-consuming, the molecular strategy proposed here is based mainly on real-time PCR, which is rapid and requires minimal handling.

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.

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

Assessment of genetic diversity and relationships among Egyptian mango (Mangifera indica L.) cultivers grown in Suez Canal and Sinai region using RAPD markers.

PubMed

Mansour, Hassan; Mekki, Laila E; Hussein, Mohammed A

2014-01-01

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic diversity and relationships in a number of fruit crops. In this study, 10 (7 commercial mango cultivars and 3 accessions) mango genotypes traditionally grown in Suez Canal and Sinai region of Egypt, were selected to assess genetic diversity and relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, eleven primers were selected which gave 92 clear and bright fragments. A total of 72 polymorphic RAPD bands were detected out of 92 bands, generating 78% polymorphisms. The mean PIC values scores for all loci were of 0.85. This reflects a high level of discriminatory power of a marker and most of these primers produced unique band pattern for each cultivar. A dendrogram based on Nei's Genetic distance co-efficient implied a moderate degree of genetic diversity among the cultivars used for experimentation, with some differences. The hybrid which had derived from cultivar as female parent was placed together. In the cluster, the cultivars and accessions formed separate groups according to bearing habit and type of embryo and the members in each group were very closely linked. Cluster analysis clearly showed two main groups, the first consisting of indigenous to the Delta of Egypt cultivars and the second consisting of indigenous to the Suez Canal and Sinai region. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later. The results indicated the potential of RAPD markers for the identification and management of mango germplasm for breeding purposes.

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.

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.

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.

MushBase: A Mushroom Information Database Application

PubMed Central

Le, Vang Quy; Lee, Hyun-Sook

2007-01-01

A database application, namely MushBase, has been built based on Microsoft Access in order to store and manage different kinds of data about mushroom biological information of species, strains and their physiological characteristics such as geometries and growth condition(s). In addition, it is also designed to store another group of information that is experimental data about mushroom classification by Random Amplification of Polymorphic DNA (RAPD). These two groups of information are stored and managed in the way so that it is convenient to retrieve each group of data and to cross-refer between them as well. PMID:24015087

Comparison of RAPD and ISSR markers for assessment of genetic diversity among endangered rare Dalbergia oliveri (Fabaceae) genotypes in Vietnam.

PubMed

Phong, D T; Hien, V T T; Thanh, T T V; Tang, D V

2011-10-06

Dalbergia oliveri is a leguminous tree of the Fabaceae family. This species is popular and valuable in Vietnam and is currently listed on the Vietnam Red List and on the IUCN Red List as endangered. Two PCR techniques using RAPD and inter-simple sequence repeat (ISSR) markers were used to make a comparative analysis of genetic diversity in this species. Fifty-six polymorphic primers (29 RAPD and 27 ISSR) were used. The RAPD primers produced 63 bands across 35 genotypes, of which 24 were polymorphic. The number of amplified bands varied from one to four, with a size range from 250 to 1400 bp. The percentage polymorphism ranged from 0 to 75. Amplification of genomic DNA of the 35 genotypes, using ISSR analysis, yielded 104 fragments, of which 63 were polymorphic. The number of amplified fragments using ISSR primers ranged from one to nine and varied in size from 250 to 1500 bp. The percentage polymorphism ranged from 0 to 100. ISSR markers were relatively more efficient than RAPDs. The mental test between two Jaccard's similarity matrices gave r ≥0.802, showing good fit correlation between ISSRs and RAPDs. Clustering of isolates remained more or less the same for RAPDs compared to combined RAPD and ISSR data. The similarity coefficient ranged from 0.785 to 1.000, 0.698 to 0.956 and 0.752 to 0.964 with RAPD, ISSR, and the combined RAPD-ISSR dendrogram, respectively.

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.

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.

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.

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...

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

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.

[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.

Genetic study of kelp ``901'' strain

NASA Astrophysics Data System (ADS)

Xia, Peng; Wang, Xiuliang; Li, Xiaojie; Zhao, Yushan; Yao, Lin; Duan, Delin

2005-06-01

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F6, F7, F8, and F9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212 0.4767, and the maximum was between F7 and F8 (0.4767). Identity analysis showed that F6 generation was more close to the female parent (0.6593), and F7 generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230 2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.2239), female parent (0.1072), F6 (0.2164), F7(0.2286), F8(0.2296) and F9(0.3172). The nearest genetic distance was 0.083 5(F8, F9). Total heterozygosity (HT) of F6, F7, F8 and F9 generations was 0.3186, the average heterozygosity (HS) for F6, F7, F8 and F9 generations was 0.2480, and deduced coefficient of population differentiation (Gst) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selecting Laminaria.

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

Phytomonas: analysis of polymorphism and genetic relatedness between isolates from plants and phytophagous insects from different geographic regions by RAPD fingerprints and synapomorphic markers.

PubMed

Serrano, M G; Camargo, E P; Teixeira, M M

1999-01-01

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.

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.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

PubMed

Plumed-Ferrer, C; Barberio, A; Franklin-Guild, R; Werner, B; McDonough, P; Bennett, J; Gioia, G; Rota, N; Welcome, F; Nydam, D V; Moroni, P

2015-09-01

In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for β-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

DNA fingerprinting of Brassica juncea cultivars using microsatellite probes.

PubMed

Bhatia, S; Das, S; Jain, A; Lakshmikumaran, M

1995-09-01

The genetic variability in the Brassica juncea cultivars was detected by employing in-gel hybridization of restricted DNA to simple repetitive sequences such as (GATA)4, (GACA)4 and (CAC)5. The most informative probe/enzyme combination was (GATA)4/EcoRI, yielding highly polymorphic fingerprint patterns for the B. juncea cultivars. This technique was found to be dependable for establishing the variety specific patterns for most of the cultivars studied, a prerequisite for germplasm preservation. The results of the present study were compared with those reported in our earlier study in which random amplification of polymorphic DNA (RAPD) was used for assessing the genetic variability in the B. juncea cultivars.

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...

[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.

Biodiversity of Clostridium botulinum Type E Associated with a Large Outbreak of Botulism in Wildlife from Lake Erie and Lake Ontario ▿

PubMed Central

Hannett, George E.; Stone, Ward B.; Davis, Stephen W.; Wroblewski, Danielle

2011-01-01

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected. PMID:21115703

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.

Typing and Subtyping of 83 Clinical Isolates Purified from Surgically Implanted Silicone Feeding Tubes by Random Amplified Polymorphic DNA Amplification

PubMed Central

Dautle, Melanie P.; Ulrich, Ricky L.; Hughes, Thomas A.

2002-01-01

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 “buttons” and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes. PMID:11825951

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.

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 ...

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.

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.

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

Determination of the genotoxic effects of Convolvulus arvensis extracts on corn (Zea mays L.) seeds.

PubMed

Sunar, Serap; Yildirim, Nalan; Aksakal, Ozkan; Agar, Guleray

2013-06-01

In this research, the methanolic extracts of Convolvulus arvensis were tested for genotoxic and inhibitor activity on the total soluble protein content and the genomic template stability against corn Zea mays L. seed. The methanol extracts of leaf, stem and root of C. arvensis were diluted to 50, 75 and 100 μl concentrations and applied to corn seed. The total soluble protein and genomic template stability results were compared with the control. The results showed that especially 100 μl extracts of diluted leaf, stem and root had a strong inhibitory activity on the genomic template stability. The changes occurred in random amplification of polymorphic DNA (RAPD) profiles of C. arvensis extract treatment included variation in band intensity, loss of bands and appearance of new bands compared with control. Also, the results obtained from this study revealed that the increase in the concentrations of C. arvensis extract increased the total soluble protein content in maize. The results suggested that RAPD analysis and total protein analysis could be applied as a suitable biomarker assay for the detection of genotoxic effects of plant allelochemicals.

Short communication: technological and genotypic comparison between Streptococcus macedonicus and Streptococcus thermophilus strains coming from the same dairy environment.

PubMed

Blaiotta, G; Sorrentino, A; Ottombrino, A; Aponte, M

2011-12-01

The species Streptococcus thermophilus is widely used for the preparation of several dairy products, and its technological contribution is clear. On the other hand, although Streptococcus macedonicus was first described more than 10 yr ago and, despite the scientific interest around this issue, the exact role of Strep. macedonicus in cheese making has yet to be clarified. In this study, 121 strains belonging to both species and isolated from the same dairy environment were genetically characterized by random amplification of polymorphic DNA (RAPD)-PCR and compared for the main biochemical features of technological interest, such as acid production, galactose utilization, citrate metabolism, exopolysaccharide production, and lipolytic, ureolytic, exocellular proteolytic, and decarboxylasic activities. Analysis by RAPD-PCR highlighted a remarkable genotypic heterogeneity among strains in both species, and, at a similarity level of 78%, all the isolates and reference strains of Strep. thermophilus grouped together and were well separated from the strains of Strep. macedonicus, confirming that these 2 species are different microbial entities. Comparison between genetic and phenotypic or biotechnological data did not reveal any relationships. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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...

[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.

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.

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...

Assessment of genetic diversity in lettuce (Lactuca sativa L.) germplasm using RAPD markers.

PubMed

Sharma, Shubhangi; Kumar, Pankaj; Gambhir, Geetika; Kumar, Ramesh; Srivastava, D K

2018-01-01

The importance of germplasm characterization is an important link between the conservation and utilization of plant genetic resources in various breeding programmes. In the present study, genetic variability and relationships among 25 Lactuca sativa L. genotypes were tested using random amplified polymorphic DNA (RAPD) molecular markers. A total of 45 random decamer oligonucleotide primers were examined to generate RAPD profiles, out of these reproducible patterns were obtained with 22 primers. A total of 87 amplicon were obtained, out of which all were polymorphic and 7 were unique bands. The level of polymorphism across genotypes was 100% as revealed by RAPD. Genetic similarity matrix, based on Jaccard's coefficients ranged from 13.7 to 84.10% indicating a wide genetic base. Dendrogram was constructed by unweighted pair group method with arithmetic averages method. RAPD technology could be useful for identification of different accessions as well as assessing the genetic similarity among different genotypes of lettuce. The study reveals the limited genetic base and the needs to diversify using new sources from the germplasm.

Antibiotic-Resistant Gram Negative Bacilli in Meals Delivered at a General Hospital, Italy

PubMed Central

Plano, Maria Rosa Anna; Di Noto, Anna Maria; Firenze, Alberto; Sciortino, Sonia; Mammina, Caterina

2009-01-01

This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July—September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked. PMID:19750189

Antibiotic-resistant gram negative bacilli in meals delivered at a general hospital, Italy.

PubMed

Plano, Maria Rosa Anna; Di Noto, Anna Maria; Firenze, Alberto; Sciortino, Sonia; Mammina, Caterina

2009-01-01

This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July-September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked.

Characterization of Treponema spp. isolates from pigs with ear necrosis and shoulder ulcers.

PubMed

Svartström, Olov; Karlsson, Frida; Fellström, Claes; Pringle, Märit

2013-10-25

Ear necrosis and shoulder ulcers in pigs are animal welfare problems and ethical issues that can cause economic losses for producers. Spirochetes have been observed microscopically in scrapings from pig ulcers since the early 1900s, but have until recently not been cultured and therefore not characterized. In this study, 12 Treponema spp. isolates were acquired from porcine ear necrosis, shoulder ulcers and gingiva. DNA analysis of the 16S rRNA-tRNA(Ile) intergenic spacer region (ISR2) or the 16S rRNA gene revealed relatedness to oral treponemes found in dogs and humans. All isolates except one aligned into two clusters, Treponema pedis and Treponema sp. OMZ 840-like. The 16S rRNA gene of the remaining isolate shared 99% nucleotide identity with Treponema parvum. Genetic fingerprinting of the isolates was performed through random amplification of polymorphic DNA (RAPD). In addition, the isolates were characterized by biochemical tests, including api(®)ZYM, tryptophanase and hippuricase activity, and by testing the antimicrobial susceptibility to tiamulin, valnemulin, tylosin, tylvalosin, lincomycin and doxycycline using broth dilution. All isolates except two showed unique RAPD fingerprints, whereas metabolic activity tests could not differentiate between the isolates. The MICs of all antimicrobial agents tested were low. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

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

[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.

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.

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.

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.

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.

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.

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.

Markers and mapping revisited: finding your gene.

PubMed

Jones, Neil; Ougham, Helen; Thomas, Howard; Pasakinskiene, Izolda

2009-01-01

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of this highly dynamic arena of plant science.

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 ...

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...

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...

CRISPR-like sequences in Helicobacter pylori and application in genotyping.

PubMed

Bangpanwimon, Khotchawan; Sottisuporn, Jaksin; Mittraparp-Arthorn, Pimonsri; Ueaphatthanaphanich, Warattaya; Rattanasupar, Attapon; Pourcel, Christine; Vuddhakul, Varaporn

2017-01-01

Many bacteria and archaea possess a defense system called clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas system) against invaders such as phages or plasmids. This system has not been demonstrated in Helicobacter pylori . The numbers of spacer in CRISPR array differ among bacterial strains and can be used as a genetic marker for bacterial typing. A total of 36 H. pylori isolates were collected from patients in three hospitals located in the central (PBH) and southern (SKH) regions of Thailand. It is of interest that CRISPR-like sequences of this bacterium were detected in vlpC encoded for VacA-like protein C. Virulence genes were investigated and the most pathogenic genotype ( cagA vacA s1m1) was detected in 17 out of 29 (58.6%) isolates from PBH and 5 out of 7 (71.4%) from SKH. vapD gene was identified in each one isolate from PBH and SKH. CRISPR-like sequences and virulence genes of 20 isolates of H. pylori obtained in this study were analyzed and CRISPR-virulence typing was constructed and compared to profiles obtained by the random amplification of polymorphic DNA (RAPD) technique. The discriminatory power (DI) of CRISPR-virulence typing was not different from RAPD typing. CRISPR-virulence typing in H. pylori is easy and reliable for epidemiology and can be used for inter-laboratory interpretation.

Virulence profiles of Shiga Toxin-Producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina.

PubMed

Pizarro, M A; Orozco, J H; Degarbo, S M; Calderón, A E; Nardello, A L; Laciar, A; Rüttler, M E

2013-12-01

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

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.

Lactobacillus bobalius sp. nov., a lactic acid bacterium isolated from Spanish Bobal grape must.

PubMed

Mañes-Lázaro, Rosario; Ferrer, Sergi; Rodas, Ana María; Urdiain, Mercedes; Pardo, Isabel

2008-12-01

A Lactobacillus strain, designated 203(T), previously isolated from Bobal grape must was characterized phylogenetically, genotypically and phenotypically in order to establish whether it represents a novel species. On the basis of the 16S rRNA gene sequence, strain 203(T) was shown to belong to the genus Lactobacillus, falling within the Lactobacillus alimentarius-Lactobacillus farciminis group and being closely related to the type strains of L. alimentarius, Lactobacillus kimchii and Lactobacillus paralimentarius. DNA-DNA hybridization results confirmed the separate status of strain 203(T) at the species level. To establish the similarities and differences between 203(T) and the three aforementioned closest species, the following methods were used: amplified rDNA restriction analysis, analysis of the 16S-23S rDNA intergenic spacer region, random amplification of polymorphic DNA (RAPD) profiling, ribotyping, carbohydrate fermentation and physiological tests. Strain 203(T) could be differentiated genetically using RAPD analysis and ribotyping. Phenotypically, it can be distinguished from its closest relatives by its ability to grow at pH 3.3, by gas production from gluconate and by certain carbohydrate fermentations. On the basis of these data, strain 203(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus bobalius sp. nov. is proposed. The type strain is 203(T) (=CECT 7310(T) =DSM 19674(T)).

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.

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

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.

[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.

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.

Epigenetic changes detected in micropropagated hop plants.

PubMed

Peredo, Elena L; Arroyo-García, Rosa; Revilla, M Angeles

2009-07-01

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques: random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested.

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.

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

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...

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.

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.

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.

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...

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....

Diversity of Geotrichum candidum Strains Isolated from Traditional Cheesemaking Fabrications in France

PubMed Central

Marcellino, N.; Beuvier, E.; Grappin, R.; Guéguen, M.; Benson, D. R.

2001-01-01

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in d-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France. PMID:11571181

Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France.

PubMed

Marcellino, N; Beuvier, E; Grappin, R; Guéguen, M; Benson, D R

2001-10-01

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

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.

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.

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.

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.

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.

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.

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.

Effects of Yangtze River source water on genomic polymorphisms of male mice detected by RAPD.

PubMed

Zhang, Xiaolin; Zhang, Zongyao; Zhang, Xuxiang; Wu, Bing; Zhang, Yan; Yang, Liuyan; Cheng, Shupei

2010-02-01

In order to evaluate the environmental health risk of drinking water from Yangtze River source, randomly amplified polymorphic DNA (RAPD) markers were used to detect the effects of the source water on genomic polymorphisms of hepatic cell of male mice (Mus musculus, ICR). After the mice were fed with source water for 90 days, RAPD-polymerase chain reactions (PCRs) were performed on hepatic genomic DNA using 20 arbitrary primers. Totally, 189 loci were generated, including 151 polymorphic loci. On average, one PCR primer produced 5.3, 4.9 and 4.8 bands for each mouse in the control, the groups fed with source water and BaP solution, respectively. Compared with the control, feeding mice with Yangtze River source water caused 33 new loci to appear and 19 to disappear. Statistical analysis of RAPD printfingers revealed that Yangtze River source water exerted a significant influence on the hepatic genomic polymorphisms of male mice. This study suggests that RAPD is a reliable and sensitive method for the environmental health risk of Yangtze River source water.

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: genetic markers and characterization of cellulolytic potential.

PubMed

Koeck, Daniela E; Zverlov, Vladimir V; Liebl, Wolfgang; Schwarz, Wolfgang H

2014-07-01

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach - the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) - was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters. Copyright © 2014 Elsevier GmbH. All rights reserved.

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.

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.

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.

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.

[RAPD analysis of Aspergilli and its application in brewing industry].

PubMed

Pan, Li; Wang, Bin; Guo, Yong

2007-06-01

Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology, using Aspergillus oryzae AS3.951, Aspergillus flavus GIM3.18 and Aspergillus sojae AS3.495 as controls. First, genome DNA of the sixteen test strains were prepared by improved extraction method, and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band (approximately 20 kb) in agarose gel electrophoresis, which conformed to the fact that these strains all belong to Aspergillus. OD260/OD280 of the prepared DNA ranged from 1.80 to 1.90, illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then, three appropriate primers (Primerl, Primer2, Primer5) for RAPD-PCR were screened from nine random primers, and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8-14 bands in their RADP-PCR patterns, where the number of the main bands was 4-9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns, where the percentage of polymorphic bands reached to 40.9% (74 bands). The similarity coefficient between the strains was calculated based on their RAPD-PCR patterns, ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primer1, Primer2 and Primer5. It basically corresponded to traditional morphological taxonomy, demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides, the aflatoxin-producing strains (GIM3.17, CICC2219, CICC2357, CICC2390, CICC2402, CICC2404) could be easily discriminated by RAPD molecular marker, whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently, RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.

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.

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.

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 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.

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.

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

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.

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

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.

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.

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.

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

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers.

PubMed

Cui, Cuiju; Li, Yan; Liu, Yanling; Li, Xiaojie; Luo, Shiju; Zhang, Zhuangzhi; Wu, Ruina; Liang, Guangjin; Sun, Juan; Peng, Jie; Tian, Pingping

2017-02-01

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r=0.78) was better than that of the RAPD marker system (r=0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding. Copyright © 2016 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

An outbreak of ESBL-producing Klebsiella pneumoniae in an Iranian referral hospital: epidemiology and molecular typing.

PubMed

Mahmoudi, Shima; Pourakbari, Babak; Rahbarimanesh, Aliakbar; Abdolsalehi, Mohammad Reza; Ghadiri, Keyghobad; Mamishi, Setareh

2018-05-07

Klebsiella pneumoniae is a common cause of nosocomial infections; however, there is limited information in Iran regarding nosocomial outbreaks due to extended-spectrum β-lactamase (ESBL) producing K pneumoniae strains, particularly using molecular methods. The present study focused on the molecular mechanism of ESBL resistance and genetic relatedness in K. pneumoniae isolates causing nosocomial infections in an Iranian referral hospital. This study was evaluated the antimicrobial resistance and molecular epidemiology of K. pneumoniae causing nosocomial infections between October 2013 and March 2014. The ESBL detection was carried out for all the isolates by the CLSI method and PCR was carried out for the detection of the blaSHV, blaTEM, and blaCTX-M genes among ESBL-producing K. pneumonia. Molecular typing of the K. pneumoniae was performed using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). A total of 30 isolates of K. pneumoniae were used for epidemiological analysis. High rates of resistance to cefotaxime (n=29, 97%), cefazolin (n=29, 97%), cefepime (n=25, 83%) and gentamicin (n=23, 77%) were observed. A total of 29 strains (97%) produced ESBLs. The frequency of blaSHV, blaCTX-M and blaTEM genes among these isolates were 83% (n=25), 70% (n=21) and 57% (n=17), respectively. Surprisingly 11 isolated (37%) carried blaSHV, blaCTX-M and blaTEM genes simultaneously. Moreover, the concurrent presence of "blaSHV and blaCTX-M" and "blaSHV and blaTEM" was seen in 8 (27%) and 4 (13%) isolates, respectively. RAPD-PCR analyses revealed that K. pneumoniae isolates belonged to 2 RAPD-PCR types among which one cluster counted for 28 isolates. To our knowledge this is the first published report of nosocomial outbreak of ESBL-producing K. pneumoniae in children in Iran. Although the epidemiology of nosocomial infections with ESBL-producing organisms has not yet been explored in depth in Iran, our findings suggest that ESBL-producing organisms are already an established public health threat in our country. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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.

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.

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.

Genetic diversity of Palestine landraces of faba bean (Vicia faba) based on RAPD markers.

PubMed

Basheer-Salimia, R; Shtaya, M; Awad, M; Abdallah, J; Hamdan, Y

2013-09-03

Until now, neither phenotypic nor molecular approaches have been used to characterize the landraces of Palestine faba beans (Vicia faba). We used PCR-based RAPD markers to determine the genetic diversity and relatedness among 26 Palestinian faba bean landraces (traditional farmers' varieties) from 8 localities in the West Bank, Palestine. In tests with 37 primers, 14 generated no polymorphic bands, 12 exhibited weak and unclear products, and 11 primers produced good amplification products with high intensity and pattern stability. Ninety-four DNA fragments (loci) were detected, with an average of 8.54 loci per primer and size ranging from 160 to 1370 bp. A minimum of 4 and a maximum of 14 DNA fragments were obtained using (OPA-05 and OPA-09) and (BC-261) primers, respectively. The maximum percentage of polymorphic markers was 71.4 (BC-298) and the minimum was 50.0 (OPA-05, -09, -16). The 11 primers exhibited relatively high collective resolving power (Rp) values of 26.316, and varied from 0.154 for the OPA-09 primer to 5.236 for the BC-261, with an overall mean of 2.392. The primers BC-261, -322, and -298 were found to be the most useful RAPD primers to assess the genetic diversity of Palestinian faba beans, as they revealed relatively high Rp rates (5.236, 3.618, and 3.150, respectively). Based on the Jaccard coefficient, the genetic distance ranged from 0.358 to 0.069, with a mean of 0.213. We conclude that the RAPD technique is useful for determining genetic diversity and for developing suitable fingerprints for faba bean landraces grown in Palestine.

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

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.

Oral Administration of Lactobacillus plantarum 299v Reduces Cortisol Levels in Human Saliva during Examination Induced Stress: A Randomized, Double-Blind Controlled Trial.

PubMed

Andersson, Hannah; Tullberg, Cecilia; Ahrné, Siv; Hamberg, Kristina; Lazou Ahrén, Irini; Molin, Göran; Sonesson, Mikael; Håkansson, Åsa

2016-01-01

Objective . To clarify the effect of Lactobacillus plantarum 299v on the salivary cortisol and salivary IgA levels in young adults under examination stress. Design . Forty-one students with an upcoming academic exam were included in a randomized double-blind, placebo-controlled study. The probiotic bacteria or the placebo product was administered in capsules once a day during 14 days. Saliva was collected and a perceived stress test was filled out at each sampling occasion. Saliva was collected for cortisol analysis by Electrochemiluminescence Immunoassay (ECLI) and salivary IgA was analysed by Enzyme-Linked Immunosorbent Assay (ELISA). Abundance of lactobacilli was evaluated by cultivation of saliva on selective medium and identification of L. plantarum 299v was done on randomly selected colonies by a random amplification of polymorphic DNA (RAPD) typing. Results . A significant difference in cortisol levels was found between the treatment group and the placebo group ( P < 0.05), together with a significant increase in levels of lactobacilli in the treatment group compared with the placebo group ( P < 0.001). No significant changes were found for salivary IgA. Conclusion . A probiotic bacterium with ability to reduce symptoms of irritable bowel syndrome (IBS) prohibited increased levels of the stress marker cortisol during the examination period. The registration number of the study is NCT02974894, and the study is registered at ClinicalTrials.gov.

Oral Administration of Lactobacillus plantarum 299v Reduces Cortisol Levels in Human Saliva during Examination Induced Stress: A Randomized, Double-Blind Controlled Trial

PubMed Central

Andersson, Hannah; Tullberg, Cecilia; Ahrné, Siv; Hamberg, Kristina; Lazou Ahrén, Irini; Molin, Göran; Sonesson, Mikael

2016-01-01

Objective. To clarify the effect of Lactobacillus plantarum 299v on the salivary cortisol and salivary IgA levels in young adults under examination stress. Design. Forty-one students with an upcoming academic exam were included in a randomized double-blind, placebo-controlled study. The probiotic bacteria or the placebo product was administered in capsules once a day during 14 days. Saliva was collected and a perceived stress test was filled out at each sampling occasion. Saliva was collected for cortisol analysis by Electrochemiluminescence Immunoassay (ECLI) and salivary IgA was analysed by Enzyme-Linked Immunosorbent Assay (ELISA). Abundance of lactobacilli was evaluated by cultivation of saliva on selective medium and identification of L. plantarum 299v was done on randomly selected colonies by a random amplification of polymorphic DNA (RAPD) typing. Results. A significant difference in cortisol levels was found between the treatment group and the placebo group (P < 0.05), together with a significant increase in levels of lactobacilli in the treatment group compared with the placebo group (P < 0.001). No significant changes were found for salivary IgA. Conclusion. A probiotic bacterium with ability to reduce symptoms of irritable bowel syndrome (IBS) prohibited increased levels of the stress marker cortisol during the examination period. The registration number of the study is NCT02974894, and the study is registered at ClinicalTrials.gov. PMID:28101105

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.

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

Diverse Geno- and Phenotypes of Persistent Listeria monocytogenes Isolates from Fermented Meat Sausage Production Facilities in Portugal ▿

PubMed Central

Ferreira, V.; Barbosa, J.; Stasiewicz, M.; Vongkamjan, K.; Moreno Switt, A.; Hogg, T.; Gibbs, P.; Teixeira, P.; Wiedmann, M.

2011-01-01

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics. PMID:21378045

Prevalence of Complement-Mediated Cell Lysis-like Gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis Isolates From Japan (2014-2016).

PubMed

Takahashi, Takashi; Fujita, Tomohiro; Shibayama, Akiyoshi; Tsuyuki, Yuzo; Yoshida, Haruno

2017-07-01

Streptococcus dysgalactiae subsp. equisimilis (SDSE; a β-hemolytic streptococcus of human or animal origin) infections are emerging worldwide. We evaluated the clonal distribution of complement-mediated cell lysis-like gene (sicG) among SDSE isolates from three central prefectures of Japan. Group G/C β-hemolytic streptococci were collected from three institutions from April 2014 to March 2016. Fifty-five strains (52 from humans and three from animals) were identified as SDSE on the basis of 16S rRNA sequencing data.; they were obtained from 25 sterile (blood, joint fluid, and cerebrospinal fluid) and 30 non-sterile (skin-, respiratory tract-, and genitourinary tract-origin) samples. emm genotyping, multilocus sequence typing, sicG amplification/sequencing, and random amplified polymorphic DNA (RAPD) analysis of sicG-positive strains were performed. sicG was detected in 30.9% of the isolates (16 human and one canine) and the genes from the 16 human samples (blood, 10; open pus, 3; sputum, 2; throat swab, 1) and one canine sample (open pus) showed the same sequence pattern. All sicG-harboring isolates belonged to clonal complex (CC) 17, and the most prevalent emm type was stG6792 (82.4%). There was a significant association between sicG presence and the development of skin/soft tissue infections. CC17 isolates with sicG could be divided into three subtypes by RAPD analysis. CC17 SDSE harboring sicG might have spread into three closely-related prefectures in central Japan during 2014-2016. Clonal analysis of isolates from other areas might be needed to monitor potentially virulent strains in humans and animals. © The Korean Society for Laboratory Medicine

Characterization and genetic variability of feed-borne and clinical animal/human Aspergillus fumigatus strains using molecular markers.

PubMed

Pena, Gabriela A; Coelho, Irene; Reynoso, María M; Soleiro, Carla; Cavaglieri, Lilia R

2015-09-01

Aspergillus fumigatus, the major etiological agent of human and animal aspergillosis, is a toxigenic fungus largely regarded as a single species by macroscopic and microscopic features. However, molecular studies have demonstrated that several morphologically identified A. fumigatus strains might be genetically distinct. This work was aimed to apply PCR-restriction length fragment polymorphisms (PCR-RFLP) and random amplification of polymorphic DNA (RAPD) molecular markers to characterize a set of feed-borne and clinical A. fumigatus sensu lato strains isolated from Argentina and Brazil and to determine and compare their genetic variability. All A. fumigatus strains had the same band profile and those typical of A. fumigatus sensu stricto positive controls by PCR-RFLP. Moreover, all Argentinian and Brazilian strains typified by RAPD showed similar band patterns to each other and to A. fumigatus sensu stricto reference strains regardless of their isolation source (animal feeds or human/animal clinical cases) and geographic origin. Genetic similarity coefficients ranged from 0.61 to 1.00, but almost all isolates showed 78% of genetic similarly suggesting that genetic variability was found at intraspecific level. Finally, benA sequencing confirmed its identification as A. fumigatus sensu stricto species. These results suggest that A. fumigatus sensu stricto is a predominant species into Aspergillus section Fumigati found in animal environments as well as in human/animal clinical cases, while other species may be rarely isolated. The strains involved in human and animal aspergillosis could come from the environment where this fungus is frequently found. Rural workers and animals would be constantly exposed. © 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.

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.

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.

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.

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.

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.

Molecular contributions to conservation

USGS Publications Warehouse

Haig, Susan M.

1998-01-01

Recent advances in molecular technology have opened a new chapter in species conservation efforts, as well as population biology. DNA sequencing, MHC (major histocompatibility complex), minisatellite, microsatellite, and RAPD (random amplified polymorphic DNA) procedures allow for identification of parentage, more distant relatives, founders to new populations, unidentified individuals, population structure, effective population size, population-specific markers, etc. PCR (polymerase chain reaction) amplification of mitochondrial DNA, nuclear DNA, ribosomal DNA, chloroplast DNA, and other systems provide for more sophisticated analyses of metapopulation structure, hybridization events, and delineation of species, subspecies, and races, all of which aid in setting species recovery priorities. Each technique can be powerful in its own right but is most credible when used in conjunction with other molecular techniques and, most importantly, with ecological and demographic data collected from the field. Surprisingly few taxa of concern have been assayed with any molecular technique. Thus, rather than showcasing exhaustive details from a few well-known examples, this paper attempts to present a broad range of cases in which molecular techniques have been used to provide insight into conservation efforts.

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

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.

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.

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

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.

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 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...

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.

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

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.

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.

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.

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.

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.

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...

Randomly amplified polymorphic DNA linkage relationships in different Norway spruce populations

Treesearch

M. Troggio; Thomas L. Kubisiak; G. Bucci; P. Menozzi

2001-01-01

We tested the constancy of linkage relationships of randomly amplified polymorphic DNA (RAPD) marker loci used to construct a population-based consensus map in material from an Italian stand of Picea abies (L.) Karst. in 29 individuals from three Norwegian populations. Thirteen marker loci linked in the Italian stand did show a consistent locus...

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

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.

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

DNA fingerprinting of jute germplasm by RAPD.

PubMed

Hossain, Mohammad Belayat; Haque, Samiul; Khan, Haseena

2002-07-31

The genotype characteristic of cultivars was investigated, along with varieties of both of the jute species, Corchorus olitorius and Corchorus capsularis, in the germplasm collection at the Bangladesh Jute Research Institute (BJRI). DNA fingerprinting was generated for 9 different varieties and 12 accessions of jute cultivars by using random amplified polymorphic DNA (RAPD). A total of 29 arbitrary oligonucleotide primers were screened. Seven primers gave polymorphism within the varieties, and 6 primers detected polymorphism within the accessions that were tested. A dendrogram was engendered from these data, and this gave a distinct clustering of the cultivated species of jute. Therefore, we generated RAPD markers, which are species-specific. These primers can distinguish between C. olitorius and C. capsularis. From the dendrogram that we generated between the various members of these two species, we found the existing genetic classification that agrees with our molecular marking data. A different dendrogram showed that jute accessions could be clustered into three groups. These data will be invaluable in the conservation and utilization of the genetic pool in the germplasm collection.

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.

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

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

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

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.

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...

Effects of synbiotic fermented milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis ssp. lactis BB-12 on the fecal microbiota of adults with irritable bowel syndrome: A randomized double-blind, placebo-controlled trial.

PubMed

Bogovič Matijašić, Bojana; Obermajer, Tanja; Lipoglavšek, Luka; Sernel, Tjaša; Locatelli, Igor; Kos, Mitja; Šmid, Alenka; Rogelj, Irena

2016-07-01

We conducted a randomized double-blind, placebo-controlled multicentric study to investigate the influence of a synbiotic fermented milk on the fecal microbiota composition of 30 adults with irritable bowel syndrome (IBS). The synbiotic product contained Lactobacillus acidophilus La-5, Bifidobacterium animalis ssp. lactis BB-12, Streptococcus thermophilus, and dietary fiber (90% inulin, 10% oligofructose), and a heat-treated fermented milk without probiotic bacteria or dietary fiber served as placebo. Stool samples were collected after a run-in period, a 4-wk consumption period, and a 1-wk follow-up period, and were subjected to real-time PCR and 16S rDNA profiling by next-generation sequencing. After 4wk of synbiotic (11 subjects) or placebo (19 subjects) consumption, a greater increase in DNA specific for L. acidophilus La-5 and Bifidobacterium animalis ssp. lactis was detected in the feces of the synbiotic group compared with the placebo group by quantitative real-time PCR. After 1wk of follow-up, the content of L. acidophilus La-5 and B. animalis ssp. lactis decreased to levels close to initial levels. No significant changes with time or differences between the groups were observed for Lactobacillus, Enterobacteriaceae, Bifidobacterium, or all bacteria. The presence of viable BB-12- and La-5-like bacteria in the feces resulting from the intake of synbiotic product was confirmed by random amplification of polymorphic DNA (RAPD)-PCR. At the end of consumption period, the feces of all subjects assigned to the synbiotic group contained viable bacteria with a BB-12-like RAPD profile, and after 1wk of follow-up, BB-12-like bacteria remained in the feces of 87.5% of these subjects. The presence of La-5-like colonies was observed less frequently (37.5 and 25% of subjects, respectively). Next-generation sequencing of 16S rDNA amplicons revealed that only the percentage of sequences assigned to Strep. thermophilus was temporarily increased in both groups, whereas the global profile of the fecal microbiota of patients was not altered by consumption of the synbiotic or placebo. In conclusion, daily consumption of a synbiotic fermented milk had a short-term effect on the amount and proportion of La-5-like strains and B. animalis ssp. lactis in the fecal microbiome of IBS patients. Furthermore, both synbiotic and placebo products caused a temporary increase in fecal Strep. thermophilus. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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.

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.

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.

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.

Composting oily sludges: Characterizing microflora using randomly amplified polymorphic DNA

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

Persson, A.; Quednau, M.; Ahrne, S.

1995-12-31

Laboratory-scale composts in which oily sludge was composted under mesophilic conditions with amendments such as peat, bark, and fresh or decomposed horse manure, were studied with respect to basic parameters such as oil degradation, respirometry, and bacterial numbers. Further, an attempt was made to characterize a part of the bacterial flora using randomly amplified polymorphic DNA (RAPD). The compost based on decomposed horse manure showed the greatest reduction of oil (85%). Comparison with a killed control indicated that microbial degradation actually had occurred. However, a substantial part of the oil was stabilized rather than totally broken down. Volatiles, on themore » contrary, accounted for a rather small percentage (5%) of the observed reduction. RAPD indicated that a selection had taken place and that the dominating microbial flora during the active degradation of oil were not the same as the ones dominating the different basic materials. The stabilized compost, on the other hand, had bacterial flora with similarities to the ones found in peat and bark.« less

Genotyping by randomly amplified polymorphic DNA of bacteriocin producing Lactobacillus acidophilus strains from Nigeria.

PubMed

Alli, John Adeolu; Iwalokun, Bamidele A; Oluwadun, Afolabi; Okonko, Iheanyi Omezuruike

2015-01-01

Yogurt and starter culture producers are still searching strains of Lactobacillus acidophilus to produce healthier yogurt with a longer shelf life and better texture, taste, and quality. This study determined the genotyping of bacteriocin producing Lactobacillus acidophilus strains recovered from Nigerian yogurts. Yogurt samples were collected from four different states of South West regions of Nigeria. Isolates were obtained from MRS Medium and biochemically characterized. This was further confirmed by API50CH. The bacteriocin positivity and activity was determined. Genomic characterization of our Lactobacillus acidophilus strains was done with randomly amplified polymorphic DNA-PCR. All yogurt samples containing Lactobacillus acidophilus strains meet the probiotic requirement of ≥10(6) cfu/mL. The gel picture revealed 6 RAPD clonal types of Lactobacillus acidophilus strains with RAPD type C observed to be more common. Significant differences existed in the mean growth inhibition zone (t = -7.32, P < 0.05 for E. coli ATCC; t = -6.19, P < 0.05 for E. coli clinical isolates; t = -6.16, P < 0.05 for Enterobacter sp; t = -11.92, P < 0.05 for Salmonella typhi, t = -1.10, P > 0.05 Staphylococcus aureus). No correlation between the bacteriocin production, activity, and their RAPD clonal division (X(2) = 7.49, P = 0.1610, df = 5). In conclusion, L. acidophilus isolated in Nigeria samples met the probiotic requirements of ≥10(6) cfu/mL and produce bacteriocins with good spectrum of activity.

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....

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.

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.

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.

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.

Use of DNA markers in forest tree improvement research

Treesearch

D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

1992-01-01

DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

Genetic Relatedness of North American Populations of Tomicus piniperda (Coleoptera: Scolytidae)

Treesearch

M. Carol Alosi Carter; Jacqueline L. Robertson; Robert A. Haack; Robert K. Lawrence; Jane L. Hayes

1996-01-01

We used DNA fingerprinting by random amplified polymorphic (RAPD) DNA and electrophoretic characterization of esteraseisozymesto investigate the genetic relatedness of North American populations of the exotic bark beetle Tombspiniperda (L.). Cluster analyses of genetic distances among populations identified the Illinois population as an outlier population with mean...

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.

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

The Prevalence of Mixed Helicobacter pylori Infections in Symptomatic and Asymptomatic Subjects in Dhaka, Bangladesh.

PubMed

Kibria, Khandoker Mohammad K; Hossain, Md Enayet; Sultana, Jinath; Sarker, Shafiqul A; Bardhan, Pradip Kumar; Rahman, Motiur; Nahar, Shamsun

2015-10-01

Helicobacter pylori is a highly genetically diverse bacterial species, which can persist in the gastric environment for decades. Recent studies have shown that single infections predominate in developed countries, whereas mixed infections are more prevalent in developing countries. Mixed infections of this bacterium may be important for adaptation to the hostile gastric environment and may facilitate dyspeptic symptoms. To calculate the prevalence of mixed infections in symptomatic and asymptomatic subjects, 2010 H. pylori isolates collected from 83 symptomatic and 91 asymptomatic subjects from Dhaka, Bangladesh, were analyzed by (i) random amplified polymorphic DNA fingerprinting (RAPD) and (ii) multiplex PCR amplification for cagA and vacA virulence gene alleles. The overall prevalence of mixed H. pylori infection was 60.15% (77/128), indicating substantial co-colonization in this population. We additionally found that symptomatic subjects (53%) had a significantly higher rate of mixed infection than asymptomatic individuals (36.3%) (p = .016) and that the prevalence of the cagA and vacA and vacA m1/s1 and vacA m2/s1 alleles were higher in subjects with mixed infection. Our findings suggest that an increased diversity of the H. pylori strains in the gastric environment may contribute to the development of disease symptoms. © 2015 John Wiley & Sons Ltd.

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.

Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces.

PubMed

Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J; García, Pilar

2012-12-01

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products.

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.

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.

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).

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.

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 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

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.

[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.

Geographic variation, genetic structure, and conservation unit designation in the Larch Mountain salamander (Plethodon larselli).

Treesearch

R. Steven Wagner; Mark P. Miller; Charles M. Crisafulli; Susan M. Haig

2005-01-01

The Larch Mountain salamander (Plethodon larselli Burns, 1954) is an endemic species in the Pacific northwestern United States facing threats related to habitat destruction. To facilitate development of conservation strategies, we used DNA sequences and RAPDs (random amplified polymorphic DNA) to examine differences among populations of this...

PRELIMINARY STUDIES ON THE POPULATION GENETICS OF THE CENTRAL STONEROLLER (CAMPOSTOMA ANOMALUM) FROM THE GREAT MIAMI RIVER BASIN, OHIO

EPA Science Inventory

Molecular approaches are particularly useful for measuring genetic diversity and were applied to samples of central stonerollers obtained from sites along tributaries to the Great Miami River in Ohio. We used Random Amplified Polymorphic DNA (RAPD) analysis to assess the level of...

PRELIMINARY STUDIES ON THE POPULATION GENETICS OF THE CENTRAL STONEROLLER (COMPOSTOMA ANOMALUM) FROM THE GREAT MIAMI RIVER BASIN, OHIO

EPA Science Inventory

Molecular approaches are particularly useful for measuring genetic diversity and were applied to samples of central stonerollers obtained from sites along tributaries to the Great Miami River in Ohio. We used Random Amplified Polymorphic DNA (RAPD) analysis to assess the level o...

The 'Appar' flax release: Origin, distinguishing characteristics, and use; and a native alternative

Treesearch

Rosemary L. Pendleton; Stanley G. Kitchen; E. Durant McArthur; Joann E. Mudge

2008-01-01

This article summarizes information on the taxonomy of 'Appar', a perennial blue flax cultivar (Linum perenne L. [Linaceae]), and characteristics that distinguish it from native Lewis flax (Linum lewisii Pursh [Linaceae]). 'Appar' apparently originated as a European flax that escaped from garden cultivation. Randomly amplified polymorphic DNA (RAPD...

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.

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.

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.

Antibiotic susceptibility profiling and virulence potential of Campylobacter jejuni isolates from different sources in Pakistan.

PubMed

Siddiqui, Fariha Masood; Akram, Muhammad; Noureen, Nighat; Noreen, Zobia; Bokhari, Habib

2015-03-01

To determine antibiotic resistance patterns and virulence potential of Campylobacter jejuni (C. jejuni) isolates from clinical human diarrheal infections, cattle and healthy broilers. Antibiotic sensitivity patterns of C. jejuni isolates were determined by Kirby Bauer Disc Diffusion assay. These isolates were then subjected to virulence profiling for the detection of mapA (membrane-associated protein), cadF (fibronectin binding protein), wlaN (beta-l,3-galactosyltransferase) and neuAB (sialic acid biosynthesis gene). Further C. jejuni isolates were grouped by random amplification of polymorphic DNA (RAPD) profiling. A total of 436 samples from poultry (n=88), cattle (n=216) and humans (n=132) from different locations were collected. Results revealed percentage of C. jejuni isolates were 35.2% (31/88), 25.0% (54/216) and 11.3% (15/132) among poultry, cattle and clinical human samples respectively. Antibiotic susceptibility results showed that similar resistance patterns to cephalothin was ie. 87.0%, 87.1% and 89%among humans, poultry and cattle respectively, followed by sulfamethoxazole+trimethoprim 40.0%, 38.7% and 31.0% in humans, poultry and cattle and Ampicillin 40%, 32% and 20% in humans, poultry and cattle respectively. Beta-lactamase activity was detected in 40.00% humans, 20.37% cattle and 32.25% in poultry C. jejuni isolates. CadF and mapA were present in all poultry, cattle and human C. jejuni isolates, wlaN was not detected in any isolate and neuAB was found in 9/31 (36%) poultry isolates. RAPD profiling results suggested high diversity of C. jejuni isolates. Detection of multidrug resistant C. jejuni strains from poultry and cattle is alarming as they can be potential hazard to humans. Moreover, predominant association of virulence factors, cadF and mapA (100% each) in C. jejuni isolates from all sources and neuAB (36%) with poultry isolates suggest the potential source of transmission of diverse types of C. jejuni to humans. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Variation in Septoria musiva and Implications for Disease Resistance Screening

Treesearch

K.T. Ward; M.E. Ostry

2005-01-01

A set of isolates of Septoria musiva differed in aggressiveness in hybrid poplar leaf disk and stem assays and culture growth in vitro. Clone x isolate interactions were observed in one of the stem assay experiments, but not in the leaf disk assay experiments. Random amplified polymorphic DNA (RAPD) analyses were performed using 52 isolates of

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.

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.

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.

One-step random mutagenesis by error-prone rolling circle amplification

PubMed Central

Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi

2004-01-01

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique. PMID:15507684

Optoenergy storage and random walks assisted broadband amplification in Er3+-doped (Pb,La)(Zr,Ti)O3 disordered ceramics.

PubMed

Xu, Long; Zhao, Hua; Xu, Caixia; Zhang, Siqi; Zou, Yingyin K; Zhang, Jingwen

2014-02-01

A broadband optical amplification was observed and investigated in Er3+-doped electrostrictive ceramics of lanthanum-modified lead zirconate titanate under a corona atmosphere. The ceramic structure change caused by UV light, electric field, and random walks originated from the diffusive process in intrinsically disordered materials may all contribute to the optical amplification and the associated energy storage. Discussion based on optical energy storage and diffusive equations was given to explain the findings. Those experiments performed made it possible to study random walks and optical amplification in transparent ceramics materials.

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.

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.

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.

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.

Identification of a new retrotransposable element in loblolly pine

Treesearch

M.N. Islam-Faridi; A.M. Morse; K.E. Smith; J.M. Davis; S. Garcia; H.V. Amerson; M.A. Majid; T.L. Kubisiak; C.D. Nelson

2005-01-01

We initiated a project to locate the genomic position of fusiform rust resistance gene 1 (Fr1) in loblolly pine using fluorescent in situ hybridization (FISH). Four random amplified polymorphic DNA (RAPD) markers previously found to be tightly linked to Fr1 were cloned and sequenced, providing a total coverage of about 2 Kb. In order to obtain discernible signal of...

Development of SCAR Markers for the DNA-Based Detection of the Asian Long-Horned Beetle; Anoplophora glabripennis (Motschulsky)

Treesearch

Damodar R. Kethidi; David B. Roden; Tim R. Ladd; Peter J. Krell; Arthur Ratnakaran; Qili Feng

2003-01-01

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence charaterized amplified regions (SCARS) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after...

Incidence of Staphylococcus aureus and Analysis of Associated Bacterial Communities on Food Industry Surfaces

PubMed Central

Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J.

2012-01-01

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products. PMID:23023749

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

PubMed

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting.

PubMed

Yokota, Shin-ichi; Konno, Mutsuko; Fujiwara, Shin-ichi; Toita, Nariaki; Takahashi, Michiko; Yamamoto, Soh; Ogasawara, Noriko; Shiraishi, Tsukasa

2015-10-01

The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families. © 2015 John Wiley & Sons Ltd.

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.

Efficient micropropagation and assessment of genetic fidelity of Boerhaavia diffusa L- High trade medicinal plant.

PubMed

Patil, Kapil S; Bhalsing, Sanjivani R

2015-07-01

Boerhaavia diffusa L is a medicinal herb with immense pharmaceutical significance. The plant is used by many herbalist, Ayurvedic and pharmaceutical industries for production biopharmaceuticals. It is among the 46 medicinal plant species in high trade sourced mainly from wastelands and generally found in temperate regions of the world. However, the commercial bulk of this plant shows genetic variations which are the main constraint to use this plant as medicinal ingredient and to obtain high value products of pharmaceutical interest from this plant. In this study, we have regenerated the plant of Boerhaavia diffusa L through nodal explants and evaluated genetic fidelity of the micropropagated plants of Boerhaavia diffusa L with the help of random amplified polymorphic DNA (RAPD) markers. The results obtained using RAPD showed monomorphic banding pattern revealing genetic stability among the mother plant and in vitro regenerated plants of Boerhaavia diffusa L.

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

PubMed

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

Chemometrical characterization of four italian rice varieties based on genetic and chemical analyses.

PubMed

Brandolini, Vincenzo; Coïsson, Jean Daniel; Tedeschi, Paola; Barile, Daniela; Cereti, Elisabetta; Maietti, Annalisa; Vecchiati, Giorgio; Martelli, Aldo; Arlorio, Marco

2006-12-27

This paper describes a method for achieving qualitative identification of four rice varieties from two different Italian regions. To estimate the presence of genetic diversity among the four rice varieties, we used polymerase chain reaction-randomly amplified polymorphic DNA (PCR-RAPD) markers, and to elucidate whether a relationship exists between the ground and the specific characteristics of the product, we studied proximate composition, fatty acid composition, mineral content, and total antioxidant capacity. Using principal component analysis on genomic and compositional data, we were able to classify rice samples according to their variety and their district of production. This work also examined the discrimination ability of different parameters. It was found that genomic data give the best discrimination based on varieties, indicating that RAPD assays could be useful in discriminating among closely related species, while compositional analyses do not depend on the genetic characters only but are related to the production area.

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.

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop.

PubMed

Choudhary, Sumitra Kumari; Patel, Ashok Kumar; Harish; Shekhawat, Smita; Shekhawat, Narpat S

2017-07-01

Momordica dioica Roxb. ex Willd., is a perennial and dioecious (2n = 28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30 ± 3.83) with an average length 6.52 ± 0.89 cm were differentiated on MS medium containing 0.5 mg L -1 BAP, 0.1 mg L -1 IAA and additives (50 mg L -1 ascorbic acid, 25 mg L -1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250 mg L -1 IBA for 5 min produced 12.3 ± 1.33 with a mean length 5.4 ± 0.73 cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica , a popular summer vegetable crop.

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.

Long-term storage and safe retrieval of DNA from microorganisms for molecular analysis using FTA matrix cards.

PubMed

Rajendram, D; Ayenza, R; Holder, F M; Moran, B; Long, T; Shah, H N

2006-12-01

We assessed the potential use of Whatman FTA paper as a device for archiving and long-term storage of bacterial cell suspensions of over 400 bacterial strains representing 61 genera, the molecular applications of immobilised DNA on FTA paper, and tested its microbial inactivation properties. The FTA paper extracted bacterial DNA is of sufficiently high quality to successfully carryout the molecular detection of several key genes including 16S rRNA, esp (Enterococcus surface protein), Bft (Bacteroides fragilis enterotoxin) and por (porin protein) by PCR and for DNA fingerprinting by random amplified polymorphic DNA-PCR (RAPD-PCR). To test the long-term stability of the FTA immobilised DNA, 100 of the 400 archived bacterial samples were randomly selected following 3 years of storage at ambient temperature and PCR amplification was used to monitor its success. All of the 100 samples were successfully amplified using the 16S rDNA gene as a target and confirmed by DNA sequencing. Furthermore, the DNA was eluted into solution from the FTA cards using a new alkaline elution procedure for evaluation by real-time PCR-based assays. The viability of cells retained on the FTA cards varied among broad groups of bacteria. For the more fragile gram-negative species, no viable cells were retained even at high cell densities of between 10(7) and 10(8) colony forming units (cfu) ml(-1), and for the most robust species such as spore-formers and acid-fast bacteria, complete inactivation was achieved at cell densities ranging between 10(1) and 10(4) cfu ml(-1). The inactivation of bacterial cells on FTA cards suggest that this is a safe medium for the storage and transport of bacterial nucleic acids.

Concurrent infection with sibling Trichinella species in a natural host.

PubMed

Pozio, E; Bandi, C; La Rosa, G; Järvis, T; Miller, I; Kapel, C M

1995-10-01

Random amplified polymorphic DNA (RAPD) analysis of individual Trichinella muscle larvae, collected from several sylvatic and domestic animals in Estonia, revealed concurrent infection of a racoon dog with Trichinella nativa and Trichinella britovi. This finding provides strong support for their taxonomic ranking as sibling species. These 2 species appear uniformly distributed among sylvatic animals through Estonia, while Trichinella spiralis appears restricted to the domestic habitat.

Mapping quantitative trait loci controlling early growth in a (longleaf pine × slash pine) × slash pine BC1 family

Treesearch

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

2002-01-01

Random amplified polymorphic DNA (RAPD) markers were employed to map the genome and quantitative trait loci controlling the early growth of a pine hybrid F1 tree (Pinus palustris Mill. × P. elliottii Engl.) and a recurrent slash pine tree (P. ellottii Engl.) in a (longleaf pine × slash pine...

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.

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.

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

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.

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.

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.

[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.

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.

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.

Monitoring of bacterial pathogens at workplaces in power plant using biochemical and molecular methods.

PubMed

Ławniczek-Wałczyk, Anna; Gołofit-Szymczak, Małgorzata; Cyprowski, Marcin; Stobnicka, Agata; Górny, Rafał L

2017-04-01

The aim of this study was to characterize the ways of spreading of the most common bacterial species isolated from workers as well as from the air and raw materials at the workplaces in power plant utilizing biomass sources. To monitor microbial transmission and identify the source of contamination in the working environment, a combination of molecular and biochemical methods was applied. The study was carried out at workplaces in power plant utilizes biomass as a main fuel source. At each of the studied workplaces, bioaerosol particles were collected on sterile Teflon filters using personal conical inhalable samplers (CIS), and biomass samples (straw pellets and briquettes, corn briquettes, sunflower pellets and wood chips) were directly taken from their storage places. Simultaneously with that, the swab samples from the hands of ten workers and their used respiratory masks (of FFP2 class) were also collected after the work shift to evaluate individual workers' microbial contamination. In all collected samples, total bacterial concentrations were assessed and the most common microbial isolates were identified to the species level using both biochemical (API tests) and molecular polymerase chain reaction (PCR), followed by random amplification of polymorphic DNA (RAPD) typing methods. The mean concentrations of culturable bacteria in the air and in biomass samples at the studied workplaces were high, i.e. 1.2 × 10 6 cfu/m 3 and 3.8 × 10 4 cfu/g, respectively. The number of bacteria in the swab and mask samples also reached a high level of 1.4 × 10 4 cfu/ml and 1.9 × 10 3 cfu/cm 2 , respectively. Among the most frequently isolated microorganisms from all types of samples were Gram-positive bacteria of the genus Bacillus and Staphylococcus xylosus. 37 bacterial strains belonging to the genus Bacillus (B. licheniformis 8, B. pumilus 15 and B. subtilis 4) and Staphylococcus (10) were genotyped by the RAPD-PCR method. Based on RAPD-PCR analyses, the genomic similarity among 19 Bacillus strains isolated from biomass, air, protective mask and hand samples as well as 6 S. xylosus strains isolated from air, mask and hand samples exceeded 80%. This study demonstrated that biomass is the primary source of bacteria at power plant workplaces. These results also revealed that biomass-associated bacteria can be easily transferred to workers' hands and mask during their routine activities. To improve health protection at the workplaces, adequate training courses on hand hygiene and how to use and remove respiratory masks correctly for workers should be introduced as a key element of the prevention strategy. From the occupational point of view, the PCR-based methods seem to be an efficient tool for a fast and precise typing of bacterial strains isolated from different sources in the occupational environment. Such methods may help to implement appropriate prophylactic procedures and minimize transmission of infectious agents at workplaces.

Genetic diversity of pigeon pea (Cajanus cajan (l.) Millsp.) based on molecular characterization using randomly amplified polymorphic DNA (RAPD) markers

NASA Astrophysics Data System (ADS)

Khoiriyah, N.; Yuniastuti, E.; Purnomo, D.

2018-03-01

Pigeon pea (Cajanus cajan (L.) Millsp.) is an annual leguminous crop (perennial) which has advantages over other local leguminous crops as drought resistant, hold collapsed and strong pods. The research on drought resistance plant is very important to adapt to climate change adverse impact to support food security. The potential of pigeon pie has not been supported by accurate data. To explore the potential of pigeon pea, it is necessary to record the important properties by characterization, one of which is molecular. Increasing genetic diversity can be done through mutation which widely used gamma ray for the induction. The purpose of this study was to identify the genetic diversity of pigeon pea of black, white and brown seeds type resulted by gamma-ray irradiation with a wavelength of 100, 200 and 300 grays by using RAPD method. The experiment resulted 14 bands, 12 of them are polymorphic bands and 2 of them are monomorphic with size varied from 300 bp to 1.3 kbp. The dendrogram showed from 30 accessions are divided into two main clusters, B shows clear genetical divergence from other clusters and some others split randomly. The range of similarity coefficient is from 0.43 to 1.00

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China.

PubMed

Wang, Dong; Zhang, Limei; Zhou, Xuezhang; He, Yulong; Yong, Changfu; Shen, Mingliang; Szenci, Otto; Han, Bo

2016-12-01

Staphylococcus aureusis the leading pathogen involved inbovine mastitis, but knowledgeabout antimicrobial resistance, virulence factors, and genotypes of Staphylococcus aureus resulting in bovine mastitis in Ningxia, China, is limited. Therefore, antimicrobial susceptibility, virulence gene, and randomly amplified polymorphic DNA (RAPD) analyses of Staph. aureus were carried out. A total of 327 milk samples from cows with clinical and subclinical mastitis in 4 regions of Ningxia were used for the isolation and identification of pathogens according to phenotypic and molecular characteristics. Antimicrobial susceptibility against 22 antimicrobial agents was determined by disk diffusion. The presence of 8 virulence genes in Staph. aureus isolates was tested by PCR. Genotypes of isolates were investigated based on RAPD. Results showed that 35 isolates obtained from mastitis milk samples were identified as Staph. aureus. The isolates were resistant to sulfamethoxazole (100%), penicillin G (94.3%), ampicillin (94.3%), erythromycin (68.6%), azithromycin (68.6%), clindamycin (25.7%), amoxicillin (11.4%), and tetracycline (5.7%). All of the isolates contained one or more virulence genes with average (standard deviation) of 6.6±1.6. The most prevalent virulence genes were hlb (97.1%), followed by fnbpA, hla, coa (94.3% each), nuc (85.7%), fnbpB (80%), clfA (77.1%), and tsst-1 (40%). Nine different gene patterns were found and 3 of them were the dominant gene combinations (77.1%). Staphylococcus aureus isolates (n=35) were divided into 6 genotypes by RAPD tying, the genotypes III and VI were the most prevalent genotypes. There was greatvariation in genotypes of Staph. aureus isolates, not only among different farms, but also within the same herd in Ningxia province. The study showed a high incidence of Staph. aureus with genomic variation of resistance genes, which is matter of great concern in public and animal health in Ningxia province of China. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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...

Application of AFLP fingerprint analysis for studying the biodiversity of Streptococcus thermophilus.

PubMed

Lazzi, Camilla; Bove, Claudio Giorgio; Sgarbi, Elisa; Gatti, Monica; Monica, Gatti; La Gioia, Federica; Torriani, Sandra; Sandra, Torriani; Neviani, Erasmo

2009-10-01

Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. In this work the genetic diversity of S. thermophilus isolates from different sources was analyzed using Amplified Fragment Length Polymorphism fingerprinting (AFLP). Since this is the first report that indicates the application of AFLP in order to study genotypic polymorphism in S. thermophilus species, an optimization of experimental conditions was carried out to decide the optimal AFLP analysis protocol. Furthermore the fingerprinting resolutions of AFLP and RAPD (Random Amplified Polymorphic DNA) were evaluated and compared. The overall data suggest that genotypic characterization performed by AFLP provide a better view of microbial diversity of S. thermophilus, indicating that RAPD is less discriminating than AFLP. The successful use of AFLP analysis in the characterization of S. thermophilus strains reported in this study suggests the potential uses for this technique to define the whole-genome diversity of each specific strain, as an alternative to the fingerprinting methods used till now.

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.

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.

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.

[Comparison of genetic diversity (RAPD) of ex situ collections and natural populations of Naufraga balearica Constance & Cannon].

PubMed

Fridlender, A; Boisselier-Dubayle, M C

2000-04-01

Naufraga balearica Constance & Cannon (Hydrocotyloideae) cultivated in the Botanical Gardens of Lyon, Brest and Porquerolles stem from two or three shoots collected in Corsica in 1981. The genetic diversity of these plants was evaluated using RAPD markers (random amplified polymorphic DNA). It was compared with the diversity found in individuals collected from five natural sites in Majorca. Only a few patterns were present in the collections derived from the Corsican shoots. The plants kept in the Botanical Gardens appeared to be of clonal origin: most individuals (81%) showed a 'dominant pattern'. In contrast, nearly all individuals sampled in the natural populations of the Balearic Islands exhibited a unique pattern. The five populations appeared genetically distinct; the individuals probably resulted from cross-fertilizations. The cultivated Corsican plants from Lyon, Brest and Porquerolles appeared genetically closely related to the individuals sampled in the population of Cala San Vicente in Majorca. The spontaneity of this paleoendemic in Corsica was discussed.

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.

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology.

PubMed

Pérez-Martín, Fátima; Seseña, Susana; Izquierdo, Pedro Miguel; Martín, Raúl; Palop, María Llanos

2012-04-01

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for β-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, β-D-xylopyranoside and α-L-arabinofuranoside although β-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on β-D-glucosidase activity was assayed, a strain-dependent response was observed. The β-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most β-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.

Development of SCAR (sequence-characterized amplified region) markers as a complementary tool for identification of ginger (Zingiber officinale Roscoe) from crude drugs and multicomponent formulations.

PubMed

Chavan, Preeti; Warude, Dnyaneshwar; Joshi, Kalpana; Patwardhan, Bhushan

2008-05-01

Zingiber officinale Roscoe (common or culinary ginger) is an official drug in Ayurvedic, Indian herbal, Chinese, Japanese, African and British Pharmacopoeias. The objective of the present study was to develop DNA-based markers that can be applied for the identification and differentiation of the commercially important plant Z. officinale Roscoe from the closely related species Zingiber zerumbet (pinecone, bitter or 'shampoo' ginger) and Zingiber cassumunar [cassumunar or plai (Thai) ginger]. The rhizomes of the other two Zingiber species used in the present study are morphologically similar to that of Z. officinale Roscoe and can be used as its adulterants or contaminants. Various methods, including macroscopy, microscopy and chemoprofiling, have been reported for the quality control of crude ginger and its products. These methods are reported to have limitations in distinguishing Z. officinale from closely related species. Hence, newer complementary methods for correct identification of ginger are useful. In the present study, RAPD (random amplification of polymorphic DNA) analysis was used to identify putative species-specific amplicons for Z. officinale. These were further cloned and sequenced to develop SCAR (sequence-characterized amplified region) markers. The developed SCAR markers were tested in several non-Zingiber species commonly used in ginger-containing formulations. One of the markers, P3, was found to be specific for Z. officinale and was successfully applied for detection of Z. officinale from Trikatu, a multicomponent formulation.

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

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.

Spread of resistant gram negatives in a Sri Lankan intensive care unit.

PubMed

Tissera, Kavinda; Liyanapathirana, Veranja; Dissanayake, Nilanthi; Pinto, Vasanthi; Ekanayake, Asela; Tennakoon, Manjula; Adasooriya, Dinuka; Nanayakkara, Dulmini

2017-07-11

Infections with multi drug resistant (MDR) organisms are a major problem in intensive care units (ICUs). Proper infection control procedures are mandatory to combat the spread of resistant organisms within ICUs. Well stablished surveillance programmes will enhance the adherence of the staff to infection control protocols. The study was conducted to assess the feasibility of using basic molecular typing methods and routine hospital data for laboratory surveillance of resistance organisms in resource limited settings. A retrospective study was conducted using consecutive Gram negative isolates obtained from an ICU over a six month period. Antibiotic sensitivity patterns and random amplified polymorphic DNA (RAPD) based typing was performed on the given isolates. Of the seventy isolates included in the study, seven were E.coli. All E.coli were MDRs and Extended Spectrum β lactamse (ESBL) producers carrying bla CTX-M . Fourteen isolates were K.pneumoniae, and all were MDRs and ESBL producers. All K.pneumoniae harboured bla SHV while 13 harboured bla CTX-M . The MDR rate among P.aeruginosa was 13% (n=15) while all acinetobacters (n=30) were MDRs. Predominant clusters were identified within all four types of Gram negatives using RAPD and the ICU stay of patients overlapped temporally. We propose that simple surveillance methods like RAPD based typing and basic hospital data can be used to convince hospital staff to adhere to infection control protocols more effectively, in low and middle income countries.

Genetic variability of Brazilian isolates of Alternaria alternata detected by AFLP and RAPD techniques

PubMed Central

Dini-Andreote, Francisco; Pietrobon, Vivian Cristina; Andreote, Fernando Dini; Romão, Aline Silva; Spósito, Marcel Bellato; Araújo, Welington Luiz

2009-01-01

The Alternaria brown spot (ABS) is a disease caused in tangerine plants and its hybrids by the fungus Alternaria alternata f. sp. citri which has been found in Brazil since 2001. Due to the recent occurrence in Brazilian orchards, the epidemiology and genetic variability of this pathogen is still an issue to be addressed. Here it is presented a survey about the genetic variability of this fungus by the characterization of twenty four pathogenic isolates of A. alternata f. sp. citri from citrus plants and four endophytic isolates from mango (one Alternaria tenuissima and three Alternaria arborescens). The application of two molecular markers Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) had revealed the isolates clustering in distinct groups when fingerprintings were analyzed by Principal Components Analysis (PCA). Despite the better assessment of the genetic variability through the AFLP, significant modifications in clusters components were not observed, and only slight shifts in the positioning of isolates LRS 39/3 and 25M were observed in PCA plots. Furthermore, in both analyses, only the isolates from lemon plants revealed to be clustered, differently from the absence of clustering for other hosts or plant tissues. Summarizing, both RAPD and AFLP analyses were both efficient to detect the genetic variability within the population of the pathogenic fungus Alternaria spp., supplying information on the genetic variability of this species as a basis for further studies aiming the disease control. PMID:24031413

[Research progress of molecular genetic analysis in Schistosoma variation].

PubMed

Zheng, Su-Yue; Li, Fei

2014-02-01

The development of molecular biology techniques makes important contributions to the researches of heritable variation of Schistosoma. In recent years, the molecular genetic analysis in the Schistosoma variation researches mainly includes the restriction fragment length polymorphism (RFLP), random amplified polymorphism technology (RAPD), microsatellite anchored PCR (SSR-PCR), and polymerase reaction single-strand conformation polymorphism (PCR-SSCP). This article reviews the research progress of molecular genetic analysis in Schistosoma variation in recent years.

Genetic variation of jointed goatgrass (Aegilops cylindrica Host.) from Iran using RAPD-PCR and SDS-PAGE of seed proteins.

PubMed

Farkhari, M; Naghavi, M R; Pyghambari, S A; Sabokdast

2007-09-01

Genetic variation of 28 populations of jointed goatgrass (Aegilops cylindrica Host.), collected from different parts of Iran, were evaluated using both RAPD-PCR and SDS-PAGE of seed proteins. The diversity within and between populations for the three-band High Molecular Weight (HMW) subunits of glutenin pattern were extremely low. Out of 15 screened primers of RAPD, 14 primers generated 133 reproducible fragments which among them 92 fragments were polymorphic (69%). Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm and separated the 28 populations into two groups. Confusion can happen between populations with the same origin as well as between populations of very diverse geographical origins. Our results show that compare to seed storage protein, RAPD is suitable for genetic diversity assessment in Ae. cylindrica populations.

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.

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.

Differentiation of Trichophyton rubrum clinical isolates from Japanese and Chinese patients by randomly amplified polymorphic DNA and DNA sequence analysis of the non-transcribed spacer region of the rRNA gene.

PubMed

Yang, Xiumin; Sugita, Takashi; Takashima, Masako; Hiruma, Masataro; Li, Ruoyu; Sudo, Hajime; Ogawa, Hideoki; Ikeda, Shigaku

2009-04-01

Trichophyton rubrum is the most common pathogen causing dermatophytosis worldwide. Recent genetic investigations showed that the microorganism originated in Africa and then spread to Europe and North America via Asia. We investigated the intraspecific diversity of T. rubrum isolated from two closely located Asian countries, Japan and China. A total of 150 clinical isolates of T. rubrum obtained from Japanese and Chinese patients were analyzed by randomly amplified polymorphic DNA (RAPD) and DNA sequence analysis of the non-transcribed spacer (NTS) region in the rRNA gene. RAPD analysis divided the 150 strains into two major clusters, A and B. Of the Japanese isolates, 30% belonged to cluster A and 70% belonged to cluster B, whereas 91% of the Chinese isolates were in cluster A. The NTS region of the rRNA gene was divided into four major groups (I-IV) based on DNA sequencing. The majority of Japanese isolates were type IV (51%), and the majority of Chinese isolates were type III (75%). These results suggest that although Japan and China are neighboring countries, the origins of T. rubrum isolates from these countries may not be identical. These findings provide information useful for tracing the global transmission routes of T. rubrum.

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

[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.

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.

Strain variation and geographic endemism in Streptococcus iniae.

PubMed

Kvitt, H; Colorni, A

2004-10-21

Twenty-six Israeli isolates of Streptococcus iniae from both marine and fresh/brackish water sources were compared with each other and with 9 foreign isolates. All the isolates were tentatively identified according to their biochemical profile. Direct sequencing of approximately 600 bp PCR products of the 16S rDNA confirmed their identification as S. iniae at the molecular level and revealed a new (one-nucleotide) variant among Israeli isolates, in addition to 2 variants that had been previously reported. Strain variation was further examined by subjecting the isolates to randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. The RAPD method allowed separation of the isolates into only 2 groups, one including 5 Israeli fresh/brackish water isolates and one including all the other isolates. The AFLP method grouped the Israeli marine isolates into one homogeneous cluster, although they had been obtained in different years (1995 to 2001) from different species of fish, and from wild (Red Sea) as well as cultured (both Mediterranean and Red Sea) sources. The Israeli fresh/brackish water isolates and foreign isolates separated into distinct entities that clustered at generally high degrees of similarity. The distance between the clusters of the Israeli marine and fresh/brackish water isolates indicates that the S. iniae streptococcosis that has been afflicting the aquaculture industries in the 2 environments in recent years was caused by distinct strains. AFLP showed superior discriminative properties over RAPD in detecting intraspecific variation and proved to be an important tool for the characterization of S. iniae. A correlation between strain variation and geographic endemism was established.

The microbial diversity of water kefir.

PubMed

Gulitz, Anna; Stadie, Jasmin; Wenning, Mareike; Ehrmann, Matthias A; Vogel, Rudi F

2011-12-15

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy. Copyright © 2011. Published by Elsevier B.V.

The Evolution of Vicia ramuliflora (Fabaceae) at Tetraploid and Diploid Levels Revealed with FISH and RAPD

PubMed Central

Han, Ying; Liu, Yuan; Wang, Haoyou; Liu, Xiangjun

2017-01-01

Vicia ramuliflora L. is a widely distributed species in Eurasia with high economic value. For past 200 years, it has evolved a tetraploid cytotype and new subspecies at the diploid level. Based on taxonomy, cytogeography and other lines of evidence, previous studies have provided valuable information about the evolution of V. ramuliflora ploidy level, but due to the limited resolution of traditional methods, important questions remain. In this study, fluorescence in situ hybridization (FISH) and random amplified polymorphic DNA (RAPD) were used to analyze the evolution of V. ramuliflora at the diploid and tetraploid levels. Our aim was to reveal the genomic constitution and parents of the tetraploid V. ramuliflora and the relationships among diploid V. ramuliflora populations. Our study showed that the tetraploid cytotype of V. ramuliflora at Changbai Mountains (M) has identical 18S and 5S rDNA distribution patterns with the diploid Hengdaohezi population (B) and the diploid Dailing population (H). However, UPGMA clustering, Neighbor-Joining clustering and principal coordinates analysis based on RAPD showed that the tetraploid cytotype (M) has more close relationships with Qianshan diploid population T. Based on our results and the fact that interspecific hybridization among Vicia species is very difficult, we think that the tetraploid V. ramuliflora is an autotetraploid and its genomic origin still needs further study. In addition, our study also found that Qianshan diploid population (T) had evolved distinct new traits compared with other diploid populations, which hints that V. ramuliflora evolved further at diploid level. We suggest that diploid population T be re-classified as a new subspecies. PMID:28135314

The Evolution of Vicia ramuliflora (Fabaceae) at Tetraploid and Diploid Levels Revealed with FISH and RAPD.

PubMed

Han, Ying; Liu, Yuan; Wang, Haoyou; Liu, Xiangjun

2017-01-01

Vicia ramuliflora L. is a widely distributed species in Eurasia with high economic value. For past 200 years, it has evolved a tetraploid cytotype and new subspecies at the diploid level. Based on taxonomy, cytogeography and other lines of evidence, previous studies have provided valuable information about the evolution of V. ramuliflora ploidy level, but due to the limited resolution of traditional methods, important questions remain. In this study, fluorescence in situ hybridization (FISH) and random amplified polymorphic DNA (RAPD) were used to analyze the evolution of V. ramuliflora at the diploid and tetraploid levels. Our aim was to reveal the genomic constitution and parents of the tetraploid V. ramuliflora and the relationships among diploid V. ramuliflora populations. Our study showed that the tetraploid cytotype of V. ramuliflora at Changbai Mountains (M) has identical 18S and 5S rDNA distribution patterns with the diploid Hengdaohezi population (B) and the diploid Dailing population (H). However, UPGMA clustering, Neighbor-Joining clustering and principal coordinates analysis based on RAPD showed that the tetraploid cytotype (M) has more close relationships with Qianshan diploid population T. Based on our results and the fact that interspecific hybridization among Vicia species is very difficult, we think that the tetraploid V. ramuliflora is an autotetraploid and its genomic origin still needs further study. In addition, our study also found that Qianshan diploid population (T) had evolved distinct new traits compared with other diploid populations, which hints that V. ramuliflora evolved further at diploid level. We suggest that diploid population T be re-classified as a new subspecies.

Biomaterials in light amplification

NASA Astrophysics Data System (ADS)

Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

2017-03-01

Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living cells and tissues or separated phase systems like phosphatydylcholine liposomes. All of the above-mentioned light amplification possibilities of biomaterials also have potential for several interesting applications in biology, medicine, sensing and imaging, which will be described and discussed in this review.

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.

Randomness Amplification under Minimal Fundamental Assumptions on the Devices

NASA Astrophysics Data System (ADS)

Ramanathan, Ravishankar; Brandão, Fernando G. S. L.; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Wojewódka, Hanna

2016-12-01

Recently, the physically realistic protocol amplifying the randomness of Santha-Vazirani sources producing cryptographically secure random bits was proposed; however, for reasons of practical relevance, the crucial question remained open regarding whether this can be accomplished under the minimal conditions necessary for the task. Namely, is it possible to achieve randomness amplification using only two no-signaling components and in a situation where the violation of a Bell inequality only guarantees that some outcomes of the device for specific inputs exhibit randomness? Here, we solve this question and present a device-independent protocol for randomness amplification of Santha-Vazirani sources using a device consisting of two nonsignaling components. We show that the protocol can amplify any such source that is not fully deterministic into a fully random source while tolerating a constant noise rate and prove the composable security of the protocol against general no-signaling adversaries. Our main innovation is the proof that even the partial randomness certified by the two-party Bell test [a single input-output pair (u* , x* ) for which the conditional probability P (x*|u*) is bounded away from 1 for all no-signaling strategies that optimally violate the Bell inequality] can be used for amplification. We introduce the methodology of a partial tomographic procedure on the empirical statistics obtained in the Bell test that ensures that the outputs constitute a linear min-entropy source of randomness. As a technical novelty that may be of independent interest, we prove that the Santha-Vazirani source satisfies an exponential concentration property given by a recently discovered generalized Chernoff bound.

Can environmental pollution by metals change genetic diversity? Ucides cordatus (Linnaeus, 1763) as a study case in Southeastern Brazilian mangroves.

PubMed

Banci, Karina Rodrigues da Silva; Mori, Gustavo Maruyama; Oliveira, Marcos Antonio de; Paganelli, Fernanda Laroza; Pereira, Mariana Rangel; Pinheiro, Marcelo Antonio Amaro

2017-03-15

Industrial areas on estuarine systems are commonly affected by heavy metals, affecting all local biota. Random Amplified Polymorphic DNA (RAPD) was used to evaluate genetic diversity of Ucides cordatus at mangroves in southeastern Brazil (Juréia, J; São Vicente, SV; and Cubatão, C), with distinct pollution levels by metals. The genetic diversity of this species was compared with concentrations of metals (Cd, Pb, Cu, Cr and Hg) in the environment. A pollution gradient was confirmed (SV>C>J), with low levels detected in water, except for mercury in SV. All metals in the sediment samples were below Threshold Effect Level (TEL), without an apparent biological risk to the biota. Genetic distance was very similar between J and C, with SV occurring as an out-group. RAPD was a powerful tool to investigate the effect of metal pollution on genetic diversity of this mangrove crab, and to evaluate the conservation status of the mangrove ecosystem. Copyright © 2017 Elsevier Ltd. All rights reserved.

Systematics of Juniperus section Juniperus based on leaf essential oils and random amplified polymorphic DNAs (RAPDs).

PubMed

Adams

2000-07-01

The composition of the leaf essential oils of all the species of Juniperus in sect. Juniperus (=sect. Oxycedrus) are reported and compared (J. brevifolia, J. cedrus, J. communis, J. c. var. saxatilis, J. c. var. oblonga, J. formosana, J. oxycedrus, J. o. subsp. badia, J. o. subsp. macrocarpa, J. o. subsp. transtagana, J. rigida, J. r. subsp. conferta, J. sibirica, J. taxifolia and J. t. var. lutchuensis). In addition, DNA fingerprinting by RAPDs was utilized. Based on these data, several taxa remained at the same taxonomic level: J. brevifolia, J. cedrus, J. communis, J. c. var. saxatilis, J. formosana, J. oxycedrus, J. rigida, J. r. var. conferta, and J. taxifolia. However, several taxa exhibited considerable differentiation that warranted their recognition at the specific level: J. oblonga M.-Bieb. (=J. communis var. oblonga), J. badia H. Gay (=J. oxycedrus subsp. badia), J. macrocarpa Sibth. and Sm. (=J. oxycedrus subsp. macrocarpa), J. navicularis Gand. (=J. oxycedrus subsp. transtagana), J. sibirica Brugsd. (=J. communis var. saxatilis in part), and J. lutchuensis Koidz. (= J. taxifolia var. lutchuensis).

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

PubMed

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

Comparison of Mediterranean Pistacia lentiscus genotypes by random amplified polymorphic DNA, chemical, and morphological analyses.

PubMed

Barazani, Oz; Dudai, Nativ; Golan-Goldhirsh, Avi

2003-08-01

Characterization of the genetic variability of Mediterranean Pistacia lentiscus genotypes by RAPD, composition of essential oils, and morphology is presented. High polymorphism in morphological parameters was found among accessions, with no significant differences in relation to geographical origin, or to gender. GC-MS analysis of leaves extracted by t-butyl methyl ether, showed 12 monoterpenes, seven sesquiterpenes, and one linear nonterpenic compound. Cluster analysis divided the accessions into two main groups according to the relative content of the major compounds, with no relation to their geographical origin. In contrast, a dendrogram based on RAPD analysis gave two main clusters according to their geographical origins. Low correlation was found between genetic and essential oil content matrices. High morphological and chemical variability on one hand, and genotypic polymorphism on the other, provide ecological advantages that might explain the distribution of Pistacia lentiscus over a wide range of habitats. The plants under study were grown together in the same climatic and environmental conditions, thus pointing to the plausible genetic basis of the observed phenotypic differences.

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.

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

Development of a Sequence-Characterized Amplified Region Marker-Targeted Quantitative PCR Assay for Strain-Specific Detection of Oenococcus oeni during Wine Malolactic Fermentation▿

PubMed Central

Solieri, Lisa; Giudici, Paolo

2010-01-01

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5′ and 3′ flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 102 CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF. PMID:20935116

Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin.

PubMed

Buommino, Elisabetta; Nocera, Francesca Paola; Parisi, Annamaria; Rizzo, Antonietta; Donnarumma, Giovanna; Mallardo, Karina; Fiorito, Filomena; Baroni, Adone; De Martino, Luisa

2016-07-01

Malassezia pachydermatis is a yeast belonging to the microbiota of the skin and mucous membranes of dog and cat, but it can also act as pathogen, causing dermatitis. The aim of this work was to evaluate the genetic variability of M. pachydermatis strains isolated from symptomatic dogs and cats and determine a correlation between genotype and phenotype. For this purpose eleven strains of M. pachydermatis were molecularly classified by nested-polymerase chain reaction (nested-PCR) based on ITS-1 and ITS-2 regions, specific for fungal rRNA genes. Furthermore, random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates identifying four different genotypes. Strains belonging to genotype 1 produced the highest amount of biofilm and phospholipase activity. The inflammatory response induced by M. pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly different when we compared the results obtained from each strain. In particular, HaCat cells infected with the strains belonging to genotypes 1 and 2 triggered the highest levels of increase in TLR-2, IL-1β, IL-6, IL-8, COX-2 and MMP-9 expression. By contrast, cells infected with the strains of genotype 3 and those of genotype 4 did not significantly induce TLR-2 and cytokines. The results obtained might suggest a possible association between genotype and virulence factors expressed by M. pachydermatis strains. This highlights the need for a more accurate identification of the yeast to improve the therapeutic approach and to monitor the onset of human infections caused by this emergent zoonotic pathogen.

Molecular characterisation of four double-flowered mutants of Silene dioica representing four centuries of variation

PubMed Central

Ingle, Elizabeth K. S.; Gilmartin, Philip M.

2015-01-01

Records of double-flowered Silene dioica date from the late sixteenth century and four named varieties are grown today, as previously, for their horticultural interest. Although double-flowered mutants have been characterized in several plants, their study in dioecious species is of particular interest due to influences of the homeotic mutation on the different floral whorl configurations in males and females. We have analysed four double-flowered varieties of Silene dioica: Flore Pleno and Rosea Plena date back to the seventeenth and nineteenth centuries, Thelma Kay and Firefly were recognized in the latter part of the twentieth and early twenty-first centuries. We have analysed the floral structure of the four varieties, which have distinct floral architectures. Based on Y chromosome-specific PCR analysis we show that Firefly is male and that the other three varieties are female: Random Amplification of Polymorphic DNA (RAPD) analyses suggested a common origin for the three female varieties. The double-flowered phenotype in all four varieties is caused by mutation of the C-function MADS-box transcription factor gene SDM1. We show that Firefly carries a unique 44bp insertion into SDM1, revealing an independent origin for this variety. Comparative analysis of SDM1 cDNA and genomic sequences in Flore Pleno, Rosea Plena and Thelma Kay shows that all three are caused by the same 7bp insertion within SDM1 and therefore share a common origin. The three alleles also differ by several single nucleotide polymorphisms, which represent somatic mutations accumulated over four centuries of asexual propagation. PMID:25878355

Intrafamilial transmission of Helicobacter pylori: genotyping of faecal samples.

PubMed

Mamishi, Setareh; Eshaghi, Hamid; Mahmoudi, Shima; Bahador, Abbas; Hosseinpour Sadeghi, Reihaneh; Najafi, Mehri; Farahmand, Fatemeh; Khodadad, Ahmad; Pourakbari, Babak

2016-01-01

After more than 20 years of research, there is a little information about the detailed routes of Helicobacter pylori transmission. The aim of this study was to explore intrafamilial transmission of H. pylori in children who had indication for upper gastrointestinal endoscopy and their parents. Children (aged up to 15 years) were studied during September 2012 to October 2013. The parents of those with positive urea breath test results were asked to provide faecal and blood samples after giving informed consent. Non-invasive tests such as immunoassay for serological antibodies against H. pylori and detection of its antigen in faeces were measured. The genetic similarity of the family strains was investigated by the random amplification of polymorphic DNA (RAPD-PCR) genotyping method. According to the genotyping results of 30 families, in 10 (33.3%) children related H. pylori genotypes to their mothers were found, while only 2 children (6.7%) had similar genotypes to their fathers. Interestingly, children with similar H. pylori genotype with their mothers had higher IgA (35.7 ± 10.8) and IgM antibody titres (87.23 ± 19.15) than other children. In addition, in these children, lower titres of IgG antibodies (9.93 ± 3.31) were found rather than children who had no H. pylori in their faeces or had no similarities with their parents (30.28 ± 6.15). In conclusion, mother-to-child transmission is the main route of intrafamilial transmission of H. pylori in Iranian families. Molecular typing of H. pylori can be useful in identifying a high-risk population.

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

PubMed

Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

2001-03-01

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods

PubMed Central

Betancor, Laura; García, Coralith; Astocondor, Lizeth; Hinostroza, Noemí; Bisio, Julieta; Rivera, Javier; Perezgasga, Lucía; Pérez Escanda, Victoria; Yim, Lucía; Jacobs, Jan; García-del Portillo, Francisco; Chabalgoity, José A.; Puente, José L.

2017-01-01

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed. PMID:29267322

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.

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

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.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2014-12-01

displacement amplification (MDA). 2 adopted multiple displacement amplification (MDA) with random primers for enriched circular DNA by rolling circle ... amplification (RCA) (Fig. 1) and then amplified DNA fragments were subject to deep sequencing. Sequence NO of Reads seq 1 184 seq 2 133 seq 3 2407 seq...prostate cancer cells through multiple displacement amplification .  Clone #7 is the top candidate which has been cloned in an expression vector and it

Cloaks for suppression or enhancement of scattering of diffuse photon density waves

NASA Astrophysics Data System (ADS)

Renthlei, Lalruatfela; Ramakrishna, S. Anantha; Wanare, Harshawardhan

2018-07-01

Enhancement of wave-like characteristics of heavily damped diffuse photon density waves in a random medium by amplification can induce strongly localised resonances. These resonances can be used to either suppress or enhance scattering from an inhomogeneity in the random medium by cloaking the inhomogeneous region by a shell of random medium with the correct levels of absorption or amplification. A spherical core-shell structure consisting of a shell of a random amplifying medium is shown to enhance or suppress specific resonant modes. A shell with an absorbing random medium is also shown to suppress scattering which can also be used for cloaking the core region.

Experimental demonstration of spatially coherent beam combining using optical parametric amplification.

PubMed

Kurita, Takashi; Sueda, Keiichi; Tsubakimoto, Koji; Miyanaga, Noriaki

2010-07-05

We experimentally demonstrated coherent beam combining using optical parametric amplification with a nonlinear crystal pumped by random-phased multiple-beam array of the second harmonic of a Nd:YAG laser at 10-Hz repetition rate. In the proof-of-principle experiment, the phase jump between two pump beams was precisely controlled by a motorized actuator. For the demonstration of multiple-beam combining a random phase plate was used to create random-phased beamlets as a pump pulse. Far-field patterns of the pump, the signal, and the idler indicated that the spatially coherent signal beams were obtained on both cases. This approach allows scaling of the intensity of optical parametric chirped pulse amplification up to the exa-watt level while maintaining diffraction-limited beam quality.

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.

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.

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.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle.

PubMed

Pringle, Märit; Bergsten, Christer; Fernström, Lise-Lotte; Höök, Helena; Johansson, Karl-Erik

2008-10-20

Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

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 and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

PubMed

Kuzmanović, N; Biondi, E; Bertaccini, A; Obradović, A

2015-09-01

To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis. © 2015 The Society for Applied Microbiology.

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.

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.

Alicyclobacillus Contamination in the Production Line of Kiwi Products in China

PubMed Central

Zhang, Jiangbo; Yue, Tianli; Yuan, Yahong

2013-01-01

Alicyclobacillus are spoilage microbes of many juice products, but contamination of kiwi products by Alicyclobacillus is seldom reported. This study aims to investigate the whole production line of kiwi products in China to assess the potential risk of their contamination. A total of 401 samples from 18 commercial products, 1 processing plant and 16 raw material orchards were tested, and 76 samples were positive, from which 76 strains of microbes were isolated and identified as 4 species of Alicyclobacillus, including Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus herbarius and Alicyclobacillus cycloheptanicus, and another 9 strains as 3 species of Bacillus by sequencing of their 16S rDNA. Through phylogenetic tree construction and RAPD-PCR amplification, it was found that there exist genotypic diversities to some extent among these isolates. Four test strains (each from one species of the 4 Alicyclobacillus species isolated in this study) could spoil pH adjusted kiwi fruit juice and some commercial kiwi fruit products with producing guaiacol (11–34 ppb). PMID:23844069

Alicyclobacillus contamination in the production line of kiwi products in China.

PubMed

Zhang, Jiangbo; Yue, Tianli; Yuan, Yahong

2013-01-01

Alicyclobacillus are spoilage microbes of many juice products, but contamination of kiwi products by Alicyclobacillus is seldom reported. This study aims to investigate the whole production line of kiwi products in China to assess the potential risk of their contamination. A total of 401 samples from 18 commercial products, 1 processing plant and 16 raw material orchards were tested, and 76 samples were positive, from which 76 strains of microbes were isolated and identified as 4 species of Alicyclobacillus, including Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus herbarius and Alicyclobacillus cycloheptanicus, and another 9 strains as 3 species of Bacillus by sequencing of their 16S rDNA. Through phylogenetic tree construction and RAPD-PCR amplification, it was found that there exist genotypic diversities to some extent among these isolates. Four test strains (each from one species of the 4 Alicyclobacillus species isolated in this study) could spoil pH adjusted kiwi fruit juice and some commercial kiwi fruit products with producing guaiacol (11-34 ppb).

Efficiency of RAPD versus SSR markers for determining genetic diversity among popcorn lines.

PubMed

Leal, A A; Mangolin, C A; do Amaral, A T; Gonçalves, L S A; Scapim, C A; Mott, A S; Eloi, I B O; Cordovés, V; da Silva, M F P

2010-01-05

Using only one type of marker to quantify genetic diversity generates results that have been questioned in terms of reliability, when compared to the combined use of different markers. To compare the efficiency of the use of single versus multiple markers, we quantified genetic diversity among 10 S(7) inbred popcorn lines using both RAPD and SSR markers, and we evaluated how well these two types of markers discriminated the popcorn genotypes. These popcorn genotypes: "Yellow Pearl Popcorn" (P1-1 and P1-5), "Zélia" (P1-2 and P1-4), "Curagua" (P1-3), "IAC 112" (P9-1 and P9-2), "Avati Pichinga" (P9-3 and P9-5), and "Pisankalla" (P9-4) have different soil and climate adaptations. Using RAPD marker analysis, each primer yielded bands of variable intensities that were easily detected, as well as non-specific bands, which were discarded from the analysis. The nine primers used yielded 126 bands, of which 104 were classified as polymorphic, giving an average of 11.6 polymorphisms per primer. Using SSR procedures, the number of alleles per locus ranged from two to five, giving a total of 47 alleles for the 14 SSR loci. When comparing the groups formed using SSR and RAPD markers, there were similarities in the combinations of genotypes from the same genealogy. Correlation between genetic distances obtained through RAPD and SSR markers was relatively high (0.5453), indicating that both techniques are efficient for evaluating genetic diversity in the genotypes of popcorn that we evaluated, though RAPDs yielded more polymorphisms.

Use of RAPD technique in evolution studies of four species in the family Canidae.

PubMed

Stepniak, Ewa; Zagalska, Maria M; Switoński, Marek

2002-01-01

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption

NASA Astrophysics Data System (ADS)

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-01

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information.

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption

PubMed Central

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-01

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information. PMID:26823196

Quantum Hash function and its application to privacy amplification in quantum key distribution, pseudo-random number generation and image encryption.

PubMed

Yang, Yu-Guang; Xu, Peng; Yang, Rui; Zhou, Yi-Hua; Shi, Wei-Min

2016-01-29

Quantum information and quantum computation have achieved a huge success during the last years. In this paper, we investigate the capability of quantum Hash function, which can be constructed by subtly modifying quantum walks, a famous quantum computation model. It is found that quantum Hash function can act as a hash function for the privacy amplification process of quantum key distribution systems with higher security. As a byproduct, quantum Hash function can also be used for pseudo-random number generation due to its inherent chaotic dynamics. Further we discuss the application of quantum Hash function to image encryption and propose a novel image encryption algorithm. Numerical simulations and performance comparisons show that quantum Hash function is eligible for privacy amplification in quantum key distribution, pseudo-random number generation and image encryption in terms of various hash tests and randomness tests. It extends the scope of application of quantum computation and quantum information.

A point prevalence survey on hand hygiene, with a special focus on Candida species.

PubMed

Brühwasser, Christina; Hinterberger, Guido; Mutschlechner, Wolfgang; Kaltseis, Josef; Lass-Flörl, Cornelia; Mayr, Astrid

2016-01-01

A 1-day point prevalence study evaluated hand hygiene compliance, yeast colonization, and contamination, focusing on the hands of health care workers (HCWs) and patient-oriented surfaces. Hand hygiene compliance was evaluated by applying the direct observation technique and the World Health Organization's compliance program, "My Five Moments for Hand Hygiene." A total of 128 samples from HCWs working in intensive care (n = 11) and intermediate care (n = 2) units and 65 environmental samples from Innsbruck Medical University Hospital were investigated. Hand hygiene compliance was superior for nurses (83.5%) and moderate for medical doctors (45.2%). In general, fungal growth was unique; only 9 of 128 HCW samples and only 4 of 65 environmental samples yielded positive results. The genetic relatedness of yeasts from the same species was investigated by random amplified polymorphic DNA (RAPD) typing. RAPD profiles exhibited the potential for cross-transmission of yeasts. In general, the fungal colonization and contamination rate was low, but a high level of hand hygiene compliance was lacking. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Influence of heavy metal stress on antioxidant status and DNA damage in Urtica dioica.

PubMed

Gjorgieva, Darinka; Kadifkova Panovska, Tatjana; Ruskovska, Tatjana; Bačeva, Katerina; Stafilov, Trajče

2013-01-01

Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity.

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.

Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms.

PubMed

McKay, G J; Egan, D; Morris, E; Scott, C; Brown, A E

1999-02-01

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

Genetic and Morphological Characterization of Cladobotryum Species Causing Cobweb Disease of Mushrooms

PubMed Central

McKay, Gareth J.; Egan, Damian; Morris, Elizabeth; Scott, Carol; Brown, Averil E.

1999-01-01

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin. PMID:9925589

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.

Major Quantitative Trait Loci Affecting Honey Bee Foraging Behavior

PubMed Central

Hunt, G. J.; Page-Jr., R. E.; Fondrk, M. K.; Dullum, C. J.

1995-01-01

We identified two genomic regions that affect the amount of pollen stored in honey bee colonies and influence whether foragers will collect pollen or nectar. We selected for the amount of pollen stored in combs of honey bee colonies, a colony-level trait, and then used random amplified polymorphic DNA (RAPD) markers and interval mapping procedures with data from backcross colonies to identify two quantitative trait loci (pln1 and pln2, LOD 3.1 and 2.3, respectively). Quantitative trait loci effects were confirmed in a separate cross by demonstrating the cosegregation of marker alleles with the foraging behavior of individual workers. Both pln1 and pln2 had an effect on the amount of pollen carried by foragers returning to the colony, as inferred by the association between linked RAPD marker alleles, D8-.3f and 301-.55, and the individual pollen load weights of returning foragers. The alleles of the two marker loci were nonrandomly distributed with respect to foraging task. The two loci appeared to have different effects on foraging behavior. Individuals with alternative alleles for the marker linked to pln2 (but not pln1) differed with respect to the nectar sugar concentration of their nectar loads. PMID:8601492

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile.

PubMed

Valdés, I; Jaureguiberry, B; Romalde, J L; Toranzo, A E; Magariños, B; Avendaño-Herrera, R

2009-04-01

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

Realistic noise-tolerant randomness amplification using finite number of devices.

PubMed

Brandão, Fernando G S L; Ramanathan, Ravishankar; Grudka, Andrzej; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Szarek, Tomasz; Wojewódka, Hanna

2016-04-21

Randomness is a fundamental concept, with implications from security of modern data systems, to fundamental laws of nature and even the philosophy of science. Randomness is called certified if it describes events that cannot be pre-determined by an external adversary. It is known that weak certified randomness can be amplified to nearly ideal randomness using quantum-mechanical systems. However, so far, it was unclear whether randomness amplification is a realistic task, as the existing proposals either do not tolerate noise or require an unbounded number of different devices. Here we provide an error-tolerant protocol using a finite number of devices for amplifying arbitrary weak randomness into nearly perfect random bits, which are secure against a no-signalling adversary. The correctness of the protocol is assessed by violating a Bell inequality, with the degree of violation determining the noise tolerance threshold. An experimental realization of the protocol is within reach of current technology.

Realistic noise-tolerant randomness amplification using finite number of devices

NASA Astrophysics Data System (ADS)

Brandão, Fernando G. S. L.; Ramanathan, Ravishankar; Grudka, Andrzej; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Szarek, Tomasz; Wojewódka, Hanna

2016-04-01

Randomness is a fundamental concept, with implications from security of modern data systems, to fundamental laws of nature and even the philosophy of science. Randomness is called certified if it describes events that cannot be pre-determined by an external adversary. It is known that weak certified randomness can be amplified to nearly ideal randomness using quantum-mechanical systems. However, so far, it was unclear whether randomness amplification is a realistic task, as the existing proposals either do not tolerate noise or require an unbounded number of different devices. Here we provide an error-tolerant protocol using a finite number of devices for amplifying arbitrary weak randomness into nearly perfect random bits, which are secure against a no-signalling adversary. The correctness of the protocol is assessed by violating a Bell inequality, with the degree of violation determining the noise tolerance threshold. An experimental realization of the protocol is within reach of current technology.

Realistic noise-tolerant randomness amplification using finite number of devices

PubMed Central

Brandão, Fernando G. S. L.; Ramanathan, Ravishankar; Grudka, Andrzej; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Szarek, Tomasz; Wojewódka, Hanna

2016-01-01

Randomness is a fundamental concept, with implications from security of modern data systems, to fundamental laws of nature and even the philosophy of science. Randomness is called certified if it describes events that cannot be pre-determined by an external adversary. It is known that weak certified randomness can be amplified to nearly ideal randomness using quantum-mechanical systems. However, so far, it was unclear whether randomness amplification is a realistic task, as the existing proposals either do not tolerate noise or require an unbounded number of different devices. Here we provide an error-tolerant protocol using a finite number of devices for amplifying arbitrary weak randomness into nearly perfect random bits, which are secure against a no-signalling adversary. The correctness of the protocol is assessed by violating a Bell inequality, with the degree of violation determining the noise tolerance threshold. An experimental realization of the protocol is within reach of current technology. PMID:27098302

Genetic relatedness among vaginal and anal isolates of Candida albicans from women with vulvovaginal candidiasis in north-east Brazil.

PubMed

Araújo Paulo de Medeiros, Mariana; Vieira de Melo, Ana Patrícia; Gonçalves, Sarah Santos; Milan, Eveline Pipolo; Chaves, Guilherme Maranhão

2014-11-01

Vulvovaginal candidiasis (VVC) is one of the most common causes of vaginitis and affects about 75% of women of reproductive age. In order to better understand the epidemiology and pathogenesis of this disease, we evaluated genetic relatedness among 62 clinical isolates of Candida albicans sequentially obtained from the anus and vagina of patients with sporadic and recurrent VVC. Evaluation of patients' demographic and clinical data, direct examination, and colony forming units (c.f.u.) counts of vaginal and anal samples were also performed. The genotypes of strains were determined with ABC genotyping and Randomly Amplified Polymorphic DNA (RAPD). Genotype A was the most prevalent (93.6%), followed by genotype C (6.4%), whereas genotype B was not found. We found the maintenance of the same ABC genotype, regardless of the body site of each patient. Most of the vaginal strains suffered microevolution, whereas most of the anal strains were replaced during the period of study. Vaginal and anal isolates of C. albicans obtained simultaneously from the same patient showed the same ABC genotype and high genetic similarity as determined by RAPD. Genotype A seemed to be dominant in both vaginal and anal isolates of patients with VVC. Our results corroborate the hypothesis that there are 'substrains' of the C. albicans vaginal clone successfully established, which dominate in an apparently random manner over the course of time. It is suggested that the anal reservoir constitutes a possible source for vaginal infection in most of the cases. © 2014 The Authors.

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

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.

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.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2016-02-01

circular DNA by rolling circle amplification (RCA) and then amplified DNA fragments were subject to deep sequencing. Deep sequencing of the...demonstrate the existence of microDNAs in prostate cancer. We adopted multiple displacement amplification (MDA) with random 2 primers for enriched...prostate cancer cells through multiple displacement amplification and next generation sequencing. R e la ti v e c e ll g ro w th ( % ) 0 20

Genetic characterization, micropropagation, and potential use for arsenic phytoremediation of Dittrichia viscosa (L.) Greuter.

PubMed

Guarino, Francesco; Conte, Barbara; Improta, Giovanni; Sciarrillo, Rosaria; Castiglione, Stefano; Cicatelli, Angela; Guarino, Carmine

2018-02-01

In the last decade, many scientists have focused their attention on the search for new plant species that can offer improved capacities to reclaim polluted soils and waters via phytoremediation. In this study, seed batches from three natural populations of Dittrichia viscosa, harvested in rural, urban, and industrial areas of central and southern Italy, were used to: (i) evaluate the genetic and morphological diversity of the populations; (ii) develop an efficient protocol for in-vitro propagation from seedling microcuttings; (iii) achieve optimal acclimatization of micropropagated plants to greenhouse conditions; (iv) test the response to arsenic (As) soil contamination of micropropagated plants. The genetic biodiversity study, based on Random Amplification of Polymorphic DNA (RAPD), as well as the morphometric analysis of 20 seedlings from each population revealed some degree of differentiation among populations. Based on these data, the most biodiverse plants from the three populations (10 lines each) were clonally multiplied by micropropagation using microcuttings of in-vitro grown seedlings. Three culture media were tested and Mureshige and Skoog medium was chosen for both seedling growth and micropropagation. The micropropagated plants responded well to greenhouse conditions and over 95% survived the acclimatization phase. Four clones were tested for their capacity to grow on soil spiked with NaAsO 2 and to absorb and accumulate the metalloid. All clones tolerated up to 1.0mg As. At the end of the trial (five weeks), As was detectable only in leaves of As-treated plants and concentration varied significantly among clones. The amount of As present in plants (leaves) corresponded to ca. 0.10-1.7% of the amount supplied. However, As was no longer detectable in soil suggesting that the metalloid was taken up, translocated and probably phytovolatilized. Copyright © 2017 Elsevier Inc. All rights reserved.

[Psilocybin - public available psychodysleptic].

PubMed

Dydak, Karolina; Śliwińska-Mossoń, Mariola; Milnerowicz, Halina

2015-09-07

Substances of plant origin have been used to induce hallucinations for a long time, in religious ceremonies and rituals as well as in pain relief. Psilocybin and psilocin naturally occur in the fungal genus Psilocybe. Due to the psychedelic effects and relative harmlessness of these substances and the fact that they do not cause physical addiction, psilocybin and psilocin recently have been increasingly replacing synthetic psychodysleptics, such as diethylamide D-lysergic acid. Both compounds as psychoactive substances are illegal, but psilocybin, in addition to psychotropic action, also shows positive effects, which from a medical point of view indicate its therapeutic potential and capacity for use in therapy. However, poisoning by psilocin and its derivatives is still a major clinical and social problem, mainly among young people, which is why quick and reliable identification of these substances is very important. Traditional ways of assigning the sample to a particular taxon, such as morphological and biochemical analysis or palynological and sporological studies, are not very universal and often do not provide clear results. Credibility, high speed and lower cost of DNA analysis make genetic methods more often used to determine the species of fungi. These methods are random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and high resolution melting (HRM). Moreover, analysis of the regions ITS1 and nLSU was suggested as a valid method for application in the molecular taxonomy of fungi for forensic purposes. Modern methods of identifying psilocybin and psilocin in fungi and biological material are: zone capillary electrophoresis, high performance liquid chromatography, gas chromatography and liquid chromatography coupled with mass spectrometry. The mentioned methods are successfully used for the identification of psychoactive substances in fungi as well as in blood and urine samples.

Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran.

PubMed

Hajjaran, Homa; Mohebali, Mehdi; Mamishi, Setareh; Vasigheh, Farzaneh; Oshaghi, Mohammad Ali; Naddaf, Saied Reza; Teimouri, Aref; Edrissian, Gholam Hossein; Zarei, Zabiholah

2013-01-01

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

Molecular Identification and Polymorphism Determination of Cutaneous and Visceral Leishmaniasis Agents Isolated from Human and Animal Hosts in Iran

PubMed Central

Mohebali, Mehdi; Mamishi, Setareh; Vasigheh, Farzaneh; Oshaghi, Mohammad Ali; Naddaf, Saied Reza; Teimouri, Aref; Edrissian, Gholam Hossein; Zarei, Zabiholah

2013-01-01

Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran. PMID:24286085

Micropropagation of Pithecellobium dulce (Roxb.) Benth-a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers.

PubMed

Goyal, Pooja; Kachhwaha, Sumita; Kothari, S L

2012-04-01

An efficient and reproducible protocol has been developed for in vitro propagation of Pithecellobium dulce (Roxb.) Benth (a multipurpose leguminous tree) from field grown nodal segments (axillary bud). Shoot bud induction occurred from nodal explants of 15-years-old tree on Murashige and Skoog (MS) basal medium supplemented with 4.4 μM 6-benzyladenine (BA) and multiplication was achieved on MS medium supplemented with 4.4 μM BA + 0.73 μM phenylacetic acid (PAA) i.e. up to 7 shoot buds in the period of 5-6 weeks. Addition of adenine sulphate (AdS) to this medium further enhanced the number of shoot buds up to 10. Proliferating shoot cultures were established by repeatedly subculturing primary culture on fresh medium (MS + 4.4 μM BA + 0.73 μM PAA) after every 25 days. In vitro rooting was achieved on MS medium supplemented with 2.46 μM Indole-3-butyric acid (IBA) + 41.63 μM activated charcoal (AC). The micropropagated shoots with well developed roots were acclimatized in green house in pots containing sand, soil and manure (1:1:1). Genetic stability of micropropagated clones was evaluated using Random amplified polymorphic DNA (RAPD) and Inter simple sequence repeat (ISSR) markers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic uniformity of micropropagated plants. This is the first report of an efficient protocol for regeneration of P. dulce through organogenesis, which can be used for further genetic transformation and pharmaceutical purposes.

Mercury induced oxidative stress, DNA damage, and activation of antioxidative system and Hsp70 induction in duckweed (Lemna minor).

PubMed

Zhang, Tingting; Lu, Qianqian; Su, Chunlei; Yang, Yaru; Hu, Dan; Xu, Qinsong

2017-09-01

Mercury uptake and its effects on physiology, biochemistry and genomic stability were investigated in Lemna minor after 2 and 6d of exposure to 0-30μM Hg. The accumulation of Hg increased in a concentration- and duration-dependent manner, and was positively correlated with the leaf damage. Oxidative stress after Hg exposure was evidenced in L. minor by a significant decrease in photosynthetic pigments, an increase in malondialdehyde and lipoxygenase activities (total enzyme activity and isoenzymes activity). Fronds of L. minor exposed to Hg showed an induction of peroxidase, catalase, and ascorbate peroxidase activities (total enzyme activity and some isoenzymes activities). Exposure of L. minor to Hg reduced the activity (total enzyme activity and some isoenzymes activities) of glutathione reductase, and superoxide dismutase. Exposure to Hg produced a transient increase in the content of glutathione and ascorbic acid. The content of dehydroascorbate and oxidized glutathione in L. minor were high during the entire exposure period. Exposure of L. minor to Hg also caused the accumulation of proline and soluble sugars. The amplification of new bands and the absence of normal DNA amplicons in treated plants in the random amplified polymorphic DNA (RAPD) profile indicated that genomic template stability (GTS) was affected by Hg treatment. The accumulation of Hsp70 indicated the occurrence of a heat shock response at all Hg concentrations. These results suggest that L. minor plants were able to cope with Hg toxicity through the activation of various mechanisms involving enzymatic and non-enzymatic antioxidants, up-regulation of proline, and induction of Hsp70. Copyright © 2017 Elsevier Inc. All rights reserved.

Spoilage-Related Activity of Carnobacterium maltaromaticum Strains in Air-Stored and Vacuum-Packed Meat ▿ †

PubMed Central

Casaburi, Annalisa; Nasi, Antonella; Ferrocino, Ilario; Di Monaco, Rossella; Mauriello, Gianluigi; Villani, Francesco; Ercolini, Danilo

2011-01-01

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall. PMID:21784913

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.

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.

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

NASA Astrophysics Data System (ADS)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

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...

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...

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 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.

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.

Geographic variation, genetic structure and conservation unit designation in the larch mountain salamander( Plethodon larselli)

USGS Publications Warehouse

Wagner, R.S.; Miller, Mark P.; Crisafulli, Charles; Haig, Susan M.

2005-01-01

The Larch Mountain salamander (Plethodon larselli Burns, 1954) is an endemic species in the Pacific northwestern United States facing threats related to habitat destruction. To facilitate development of conservation strategies, we used DNA sequences and RAPDs (random amplified polymorphic DNA) to examine differences among populations of this species. Phylogenetic analyses of cytochrome b revealed a clade of haplotypes from populations north of the Columbia River derived from a clade containing haplotypes from the river's southwestern region. Haplotypes from southeastern populations formed a separate clade. Nucleotide diversity was reduced in northern populations relative to southern populations. These results were corroborated by analyses of RAPD loci, which revealed similar patterns of clustering and diversity. Network analyses suggested that northern populations were colonized following a range expansion mediated by individuals from populations located southwest of the river. Changes in the Columbia River's location during the Pliocene and Pleistocene likely released distributional constraints on this species, permitting their northern range expansion. Based on the barrier presented by the Columbia River's present location and differences in haplotype diversity and population structure observed between northern and southern populations, we suggest that designation of separate management units encompassing each region may assist with mitigating different threats to this species.

Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae.

PubMed

Alavandi, S V; Manoranjita, V; Vijayan, K K; Kalaimani, N; Santiago, T C

2006-11-01

The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.

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.

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1

PubMed Central

Saha, Soumen; Adhikari, Sinchan; Dey, Tulsi; Ghosh, Parthadeb

2015-01-01

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62 ± 0.01), with average length 4.19 ± 0.01 cm, was initially achieved with medium containing 0.5 mg/l N6-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0 mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies. PMID:26693403

Investigation of flurbiprofen genotoxicity and cytotoxicity in rat bone marrow cells.

PubMed

Timocin, Taygun; Ila, Hasan B

2015-01-01

This study was performed to investigate cytogenetic effects of NSAID flurbiprofen which was used as active ingredient in some analgesic, antipyretic and anti-inflammatory drugs. Genotoxic effect of flurbiprofen was investigated using in vivo chromosome aberration (CA) test and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) test. Also, oxidative stress potential of flurbiprofen was determined by measuring total oxidant and antioxidant level which occurred with flurbiprofen treatment in rat peripheral blood. For these purposes, rats were treated with three concentrations of flurbiprofen (29.25, 58.50 and 117 mg/kg, body weight) in single dose at two different treatment periods (12 and 24 h). According to the results, flurbiprofen did not affect chromosome aberrations in rat bone marrow cells with CA test. In RAPD-PCR test, polymorphic bands were unaffected. Also, test substance did not change total oxidant and antioxidant status (except for 58.50 and 117 mg/kg, 12 h) and therefore it did not lead to significant increase on oxidative stress (again except 58.50 and 117 mg/kg, 12 h). However, flurbiprofen reduced to mitotic indexes and these reductions were dose-dependent for 12 h treatment. In summary, flurbiprofen did not show significant genotoxic effect. But it caused cytotoxicity in rat bone marrow cells.

Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA

USGS Publications Warehouse

Theodorakis, Christopher W.; Bickham, John W.; Lamb, Trip; Medica, Philip A.; Lyne, T. Barrett

2001-01-01

We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotype individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations.

Influence of Heavy Metal Stress on Antioxidant Status and DNA Damage in Urtica dioica

PubMed Central

Kadifkova Panovska, Tatjana; Bačeva, Katerina; Stafilov, Trajče

2013-01-01

Heavy metals have the potential to interact and induce several stress responses in the plants; thus, effects of heavy metal stress on DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated. The samples are sampled from areas with different metal exposition. Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used, and genomic DNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant. It was found that heavy metal contents in stems generally changed synchronously with those in leaves of the plant, and extraneous metals led to imbalance of mineral nutrient elements. DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique, and the results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparison with the control sample. This study showed that DNA stability is highly affected by metal pollution which was identified by RAPD markers. Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U. dioica which may help to understand the mechanisms of metals genotoxicity. PMID:23862140

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger.

PubMed

Xu, Defeng; Pan, Li; Zhao, Haifeng; Zhao, Mouming; Sun, Jiaxin; Liu, Dongmei

2011-09-01

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.

Effects of Cry1Ab Transgenic Maize on Lifecycle and Biomarker Responses of the Earthworm, Eisenia Andrei

PubMed Central

van der Merwe, Frances; Bezuidenhout, Carlos; van den Berg, Johnnie; Maboeta, Mark

2012-01-01

A 28-day study was conducted to determine the effects of the Bacillus thuringiensis Cry1Ab toxin on the earthworm Eisenia andrei. Previously, investigations have been limited to life-cycle level effects of this protein on earthworms, and mostly on E. fetida. In this study several endpoints were compared which included biomass changes, cocoon production, hatching success, a cellular metal-stress biomarker (Neutral Red Retention Time; NRRT) and potential genotoxic effects in terms of Randomly Amplified Polymorphic DNA sequences (RAPDs). NRRT results indicated no differences between treatments (p > 0.36), and NRRT remained the same for both treatments at different times during the experiment (p = 0.18). Likewise, no significant differences were found for cocoon production (p = 0.32) or hatching success (p = 0.29). Conversely, biomass data indicated a significant difference between the control treatment and the Bt treatment from the second week onwards (p < 0.001), with the Bt treatment losing significantly more weight than the isoline treatment. Possible confounding factors were identified that might have affected the differences in weight loss between groups. From the RAPD profiles no conclusive data were obtained that could link observed genetic variation to exposure of E. andrei to Cry1Ab proteins produced by Bt maize. PMID:23235452

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.

Antimicrobial resistance and genetic profiling of Escherichia coli from a commercial beef packing plant.

PubMed

Aslam, Mueen; Service, Cara

2006-07-01

The objective of this study was to investigate the extent of antimicrobial resistance and to genetically characterize resistant Escherichia coli recovered from a commercial beef packing plant. E. coli isolates were recovered by a hydrophobic grid membrane filtration method by direct plating on SD-39 medium. A total of 284 isolates comprising 71, 36, 55, 52, and 70 isolates from animal hides, washed carcasses, conveyers, beef trimmings, and ground beef, respectively, were analyzed. The susceptibility of E. coli isolates to 15 antimicrobial agents was evaluated with an automated broth microdilution system, and the genetic characterization of these isolates was performed by the random amplified polymorphic DNA (RAPD) method. Of the 284 E. coli isolates, 56% were sensitive to all 15 antimicrobial agents. Resistance to tetracycline, ampicillin, and streptomycin was observed in 38, 9, and 6% of the isolates, respectively. Resistance to one or more antimicrobial agents was observed in 51% of the E. coli isolates recovered from the hides but in only 25% of the E. coli from the washed carcasses. Resistance to one or more antimicrobial agents was observed in 49, 50, and 37% of the isolates recovered from conveyers, beef trimmings, and ground beef, respectively. The RAPD pattern data showed that the majority of resistant E. coli isolates were genetically diverse. Only a few RAPD types of resistant strains were shared among various sample sources. The results of this study suggest that antimicrobial-resistant E. coli isolates were prevalent during all stages of commercial beef processing and that considerably higher numbers of resistant E. coli were present on conveyers, beef trimmings, and ground beef than on dressed carcasses. This stresses the need for improving hygienic conditions during all stages of commercial beef processing and meatpacking to avoid the risks of transfer of antimicrobial-resistant bacteria to humans.

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity

PubMed Central

Donà, M.; Balestrazzi, A.; Mondoni, A.; Rossi, G.; Ventura, L.; Buttafava, A.; Macovei, A.; Sabatini, M. E.; Valassi, A.; Carbonera, D.

2013-01-01

Background and Aims The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration. Methods Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin–Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length. Key Results The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. Conclusions Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species. PMID:23532044

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.

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.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

PubMed Central

Pringle, Märit; Bergsten, Christer; Fernström, Lise-Lotte; Höök, Helena; Johansson, Karl-Erik

2008-01-01

Background Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Methods Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Results Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. Conclusion This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates. PMID:18937826

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.

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...

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...

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.

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.

[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.

Protection from feed-forward amplification in an amplified RNAi mechanism

PubMed Central

Pak, Julia; Maniar, Jay Mahesh; Mello, Cecilia Cabral; Fire, Andrew

2012-01-01

SUMMARY The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in C. elegans, we find that feed-forward amplification is limited by a critical biosynthetic and structural distinction at the RNA level between (i) triggers that can produce amplification and (ii) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification. PMID:23141544

Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

PubMed Central

Hang, Jun; Forshey, Brett M.; Kochel, Tadeusz J.; Li, Tao; Solórzano, Víctor Fiestas; Halsey, Eric S.; Kuschner, Robert A.

2012-01-01

ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery. PMID:22468136

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.

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Duplication and amplification of antibiotic resistance genes enable increased resistance in isolates of multidrug-resistant Salmonella Typhimurium

USDA-ARS?s Scientific Manuscript database

During normal bacterial DNA replication, gene duplication and amplification (GDA) events occur randomly at a low frequency in the genome throughout a population. In the absence of selection, GDA events that increase the number of copies of a bacterial gene (or a set of genes) are lost. Antibiotic ...

Isolation and Molecular Identification of Streptomyces spp. with Antibacterial Activity from Northwest of Iran

PubMed Central

Maleki, Hadi; Dehnad, Alireza; Hanifian, Shahram; Khani, Sajjad

2013-01-01

Introduction: Streptomyces are a group of prokaryotes that are usually found in all types of ecosystems including water and soil. This group of bacteria is noteworthy as antibiotic producers; so the isolation and characterization of new species seemed to be crucial in introduction of markedly favorable antibiotics. Therefore, in this study we aim to isolate and characterize novel strains of Streptomyces with high antibiotic production capability. Methods: To achieve this goal, from 140 isolates collected throughout northwest of Iran, 12 selected Streptomyces isolates which exhibited high antibacterial activity against pathogenic bacteria were subjected to PCR reaction for identification via 16S rDNA gene and random amplified polymorphic DNA (RAPD) pattern analysis. Results: Analysis of morphological and biochemical characteristics and the 16S rDNA gene sequence indicated that all 12 selected isolates belonged to the genus Streptomyces. Moreover, screening of the isolates with regard to their antimicrobial activity against indicator bacteria as well as their classification using RAPD analysis revealed that G614C1 and K36C5 isolates have considerable antimicrobial activity and high similarity to Streptomyces coelicolor and Sreptomyces albogriseolus, respectively. Conclusion: Since many isolates in this study showed inhibitory effects against pathogenic bacteria, soil of northwest of Iran could be used as a rich source to be explored for novel Streptomyces strains with high potency of antibiotic production. PMID:24163805

Isolation and Molecular Identification of Streptomyces spp. with Antibacterial Activity from Northwest of Iran.

PubMed

Maleki, Hadi; Dehnad, Alireza; Hanifian, Shahram; Khani, Sajjad

2013-01-01

Streptomyces are a group of prokaryotes that are usually found in all types of ecosystems including water and soil. This group of bacteria is noteworthy as antibiotic producers; so the isolation and characterization of new species seemed to be crucial in introduction of markedly favorable antibiotics. Therefore, in this study we aim to isolate and characterize novel strains of Streptomyces with high antibiotic production capability. To achieve this goal, from 140 isolates collected throughout northwest of Iran, 12 selected Streptomyces isolates which exhibited high antibacterial activity against pathogenic bacteria were subjected to PCR reaction for identification via 16S rDNA gene and random amplified polymorphic DNA (RAPD) pattern analysis. Analysis of morphological and biochemical characteristics and the 16S rDNA gene sequence indicated that all 12 selected isolates belonged to the genus Streptomyces. Moreover, screening of the isolates with regard to their antimicrobial activity against indicator bacteria as well as their classification using RAPD analysis revealed that G614C1 and K36C5 isolates have considerable antimicrobial activity and high similarity to Streptomyces coelicolor and Sreptomyces albogriseolus, respectively. Since many isolates in this study showed inhibitory effects against pathogenic bacteria, soil of northwest of Iran could be used as a rich source to be explored for novel Streptomyces strains with high potency of antibiotic production.

Molecular markers shared by diverse apomictic Pennisetum species.

PubMed

Lubbers, E L; Arthur, L; Hanna, W W; Ozias-Akins, P

1994-11-01

Two molecular markers, a RAPD (randomly amplified polymorphic DNA) and a RFLP/STS (restriction fragment length polymorphism/sequence-tagged site), previously were found associated with apomictic reproductive behavior in a backcross population produced to transfer apomixis from Pennisetum squamulatum to pearl millet. The occurrence of these molecular markers in a range of 29 accessions of Pennisetum comprising 11 apomictic and 8 sexual species was investigated. Both markers were specific for apomictic species in Pennisetum. The RFLP/STS marker, UGT 197, was found to be associated with all taxa that displayed apomictic reproductive behavior except those in section Brevivalvula. Neither UGT197 nor the cloned RAPD fragment OPC-04600 hybridized with any sexually reproducing representatives of the genus. The cloned C04600 was associated with 3 of the 11 apomictic species, P. ciliare, P. massaicum, and P. squamulatum. UGT197 was more consistently associated with apomictic reproductive behavior than OPC04600 or cloned C04600, thus it could be inferred that UGT197 is more closely linked to the gene(s) for apomixis than the cloned C04600. The successful use of these probes to survey other Pennisetum species indicates that apomixis is a trait that can be followed across species by using molecular means. This technique of surveying species within a genus will be useful in determining the relative importance of newly isolated markers and may facilitate the identification of the apomixis gene(s).

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.

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.

Characterization of stress induced by copper and zinc on cucumber (Cucumis sativus L.) seedlings by means of molecular and population parameters.

PubMed

Soydam Aydin, Semra; Gökçe, Esra; Büyük, Ilker; Aras, Sümer

2012-07-04

Contamination of plants with heavy metals could result in damage in DNA, such as mutations and cross-links with proteins. These altered DNA profiles may become visible in changes such as the appearance of a new band, or loss of an existing band, in the random amplified polymorphic DNA (RAPD) assay. In this study, various concentrations of copper and zinc salts were applied to cucumber seedlings during germination. Results displayed abnormalities in germination and also changes in root elongation, dry weight and total soluble protein level. All treatment concentrations (40, 80, 160, 240, 320, and 640mg/L) used in the study caused a decrease/delay in germination of the cucumbers to different extents. Inhibition or activation of root elongation was considered to be the first effect of metal toxicity in the tested plants. Application of the metal salts and the combined solutions on cucumber (Cucumis sativus L.) seedlings revealed similar consequences for total soluble protein level, dry weight and ultimately in inhibitory rates as well. The data obtained from RAPD band-profiles and genomic template stability (GTS) showed results that were consistent with the population parameters. In this regard, we conclude that molecular marker assays can be applied in combination with population parameters to measure genotoxic effects of heavy metals on plants. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Population Genetics of the Endemic Hawaiian Species Chrysodracon hawaiiensis and Chrysodracon auwahiensis (Asparagaceae): Insights from RAPD and ISSR Variation.

PubMed

Lu, Pei-Luen; Yorkson, Mitsuko; Morden, Clifford W

2016-08-16

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon's index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.

Epidemiology and molecular characterization of extended-spectrum beta-lactamase-producing Enterobacter spp., Pantoea agglomerans, and Serratia marcescens isolates from a Bulgarian hospital.

PubMed

Markovska, Rumyana Donkova; Stoeva, Temenuga Jekova; Bojkova, Kalina Dineva; Mitov, Ivan Gergov

2014-04-01

Forty-two extended-spectrum beta-lactamase (ESBL)-producing isolates of Enterobacter aerogenes, Enterobacter cloacae, Pantoea agglomerans, and Serratia marcescens, collected consecutively during the period January-November 2011 from the University Hospital in Varna, Bulgaria, were studied to characterize their ESBLs by isoelectric focusing, group-specific PCR, and sequencing. The epidemiological relationship was evaluated by random amplified polymorphic DNA analysis (RAPD). Transferability of ESBL genes was determined by conjugation experiments. Plasmid analysis was done by replicon typing and PstI fingerprinting. The overall rate of ESBL production was 20%. The most widespread enzyme was CTX-M-3, found in 64%. It was dominant in E. aerogenes (100%) and S. marcescens (83%). SHV-12, CTX-M-3, and CTX-M-15 were found among E. cloacae isolates in 50%, 35%, and 45%, respectively. Three main CTX-M-3-producing epidemic clones of E. aerogenes and S. marcescens have been detected. Among E. cloacae isolates, six different RAPD profiles were discerned. The plasmids harboring blaCTX-M-3 belonged to IncL/M type and demonstrated similar PstI fingerprinting profiles. IncFII plasmids were detected in two CTX-M-15-producing E. cloacae isolates. Our results demonstrate wide intrahospital dissemination of clonal E. aerogenes and S. marcescens isolates, carrying IncL/M conjugative plasmids.

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

PubMed

Krajňáková, Jana; Sutela, Suvi; Aronen, Tuija; Gömöry, Dušan; Vianello, Angelo; Häggman, Hely

2011-08-01

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities. Copyright © 2011 Elsevier Inc. All rights reserved.

[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.

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

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.

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.

Horizontal localization and speech intelligibility with bilateral and unilateral hearing aid amplification.

PubMed

Köbler, S; Rosenhall, U

2002-10-01

Speech intelligibility and horizontal localization of 19 subjects with mild-to-moderate hearing loss were studied in order to evaluate the advantages and disadvantages of bilateral and unilateral hearing aid (HA) fittings. Eight loudspeakers were arranged in a circular array covering the horizontal plane around the subjects. Speech signals of a sentence test were delivered by one, randomly chosen, loudspeaker. At the same time, the other seven loudspeakers emitted noise with the same long-term average spectrum as the speech signals. The subjects were asked to repeat the speech signal and to point out the corresponding loudspeaker. Speech intelligibility was significantly improved by HAs, bilateral amplification being superior to unilateral. Horizontal localization could not be improved by HA amplification. However, bilateral HAs preserved the subjects' horizontal localization, whereas unilateral amplification decreased their horizontal localization abilities. Front-back confusions were common in the horizontal localization test. The results indicate that bilateral HA amplification has advantages compared with unilateral amplification.

Lidar using the backscatter amplification effect

NASA Astrophysics Data System (ADS)

Razenkov, Igor A.; Banakh, Victor A.

2018-04-01

Experimental data proving the possibility of lidar measurement of the refractive turbulence strength based on the effect of backscatter amplification (BSA) are reported. It is shown that the values of the amplification factor correlate with the variance of random jitter of optical image of an incoherent light source depending on the value of the structure constant of the air refractive index turbulent fluctuations averaged over the probing path. This paper presents the results of measurements of the BSA factor in comparison with the simultaneous measurements of the BSA peak, which is very narrow and only occurs on the laser beam axis. It is constructed the range-time images of the derivative of the amplification factor gives a comprehensive picture of the location of turbulent zones and their temporal dynamics.

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.

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.

[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.

Genetic relatedness among developing seeds and intra fruit seed abortion in Dalbergia sissoo (Fabaceae).

PubMed

Mohana, G S; Shaanker, R U; Ganeshaiah, K N; Dayanandan, S

2001-07-01

Dalbergia sissoo, a wind-dispersed tropical tree, exhibits high intrafruit seed abortion. Of the four to five ovules in the flower, generally one and occasionally two or three develop to maturity. It has been proposed that the seed abortion is a consequence of intense sibling competition for maternal resources and that this competition occurs as an inverse function of the genetic relatedness among the developing seeds. Accordingly, developing seeds compete intensely when they are genetically less related but tend to develop together when genetically more related. We tested this hypothesis by comparing the genetic similarity among the pairs of seeds developing within a pod with that among (a) random pairs from the pool of all seeds, (b) random pairs from single-seeded pods, and (c) random pairs from two-seeded pods, using both randomly amplified polymorphic DNA (RAPD) and isozymes in five trees. We found that the pairs of seeds developing within a pod are genetically more similar than any random pairs of seeds in a tree. Thus the formation of two-seeded pods appear to be associated with increased genetic relatedness among the developing seeds. We discuss the results in the context of possible fitness advantages and then discuss the possible mechanisms that promote tolerance among related seeds.

Subchronic Toxicity of Copper Oxide Nanoparticles and Its Attenuation with the Help of a Combination of Bioprotectors

PubMed Central

Privalova, Larisa I.; Katsnelson, Boris A.; Loginova, Nadezhda V.; Gurvich, Vladimir B.; Shur, Vladimir Y.; Valamina, Irene E.; Makeyev, Oleg H.; Sutunkova, Marina P.; Minigalieva, Ilzira A.; Kireyeva, Ekaterina P.; Rusakov, Vadim O.; Tyurnina, Anastasia E.; Kozin, Roman V.; Meshtcheryakova, Ekaterina Y.; Korotkov, Artem V.; Shuman, Eugene A.; Zvereva, Anastasia E.; Kostykova, Svetlana V.

2014-01-01

In the copper metallurgy workplace air is polluted with condensation aerosols, which a significant fraction of is presented by copper oxide particles <100 nm. In the scientific literature, there is a lack of their in vivo toxicity characterization and virtually no attempts of enhancing organism’s resistance to their impact. A stable suspension of copper oxide particles with mean (±SD) diameter 20 ± 10 nm was prepared by laser ablation of pure copper in water. It was being injected intraperitoneally to rats at a dose of 10 mg/kg (0.5 mg per mL of deionized water) three times a week up to 19 injections. In parallel, another group of rats was so injected with the same suspension against the background of oral administration of a “bio-protective complex” (BPC) comprising pectin, a multivitamin-multimineral preparation, some amino acids and fish oil rich in ω-3 PUFA. After the termination of injections, many functional and biochemical indices for the organism’s status, as well as pathological changes of liver, spleen, kidneys, and brain microscopic structure were evaluated for signs of toxicity. In the same organs we have measured accumulation of copper while their cells were used for performing the Random Amplification of Polymorphic DNA (RAPD) test for DNA fragmentation. The same features were assessed in control rats infected intraperitoneally with water with or without administration of the BPC. The copper oxide nanoparticles proved adversely bio-active in all respects considered in this study, their active in vivo solubilization in biological fluids playing presumably an important role in both toxicokinetics and toxicodynamics. The BPC proposed and tested by us attenuated systemic and target organs toxicity, as well as genotoxicity of this substance. Judging by experimental data obtained in this investigation, occupational exposures to nano-scale copper oxide particles can present a significant health risk while the further search for its management with the help of innocuous bioprotectors seems to be justified. PMID:25026171

Subchronic toxicity of copper oxide nanoparticles and its attenuation with the help of a combination of bioprotectors.

PubMed

Privalova, Larisa I; Katsnelson, Boris A; Loginova, Nadezhda V; Gurvich, Vladimir B; Shur, Vladimir Y; Valamina, Irene E; Makeyev, Oleg H; Sutunkova, Marina P; Minigalieva, Ilzira A; Kireyeva, Ekaterina P; Rusakov, Vadim O; Tyurnina, Anastasia E; Kozin, Roman V; Meshtcheryakova, Ekaterina Y; Korotkov, Artem V; Shuman, Eugene A; Zvereva, Anastasia E; Kostykova, Svetlana V

2014-07-14

In the copper metallurgy workplace air is polluted with condensation aerosols, which a significant fraction of is presented by copper oxide particles<100 nm. In the scientific literature, there is a lack of their in vivo toxicity characterization and virtually no attempts of enhancing organism's resistance to their impact. A stable suspension of copper oxide particles with mean (±SD) diameter 20±10 nm was prepared by laser ablation of pure copper in water. It was being injected intraperitoneally to rats at a dose of 10 mg/kg (0.5 mg per mL of deionized water) three times a week up to 19 injections. In parallel, another group of rats was so injected with the same suspension against the background of oral administration of a "bio-protective complex" (BPC) comprising pectin, a multivitamin-multimineral preparation, some amino acids and fish oil rich in ω-3 PUFA. After the termination of injections, many functional and biochemical indices for the organism's status, as well as pathological changes of liver, spleen, kidneys, and brain microscopic structure were evaluated for signs of toxicity. In the same organs we have measured accumulation of copper while their cells were used for performing the Random Amplification of Polymorphic DNA (RAPD) test for DNA fragmentation. The same features were assessed in control rats infected intraperitoneally with water with or without administration of the BPC. The copper oxide nanoparticles proved adversely bio-active in all respects considered in this study, their active in vivo solubilization in biological fluids playing presumably an important role in both toxicokinetics and toxicodynamics. The BPC proposed and tested by us attenuated systemic and target organs toxicity, as well as genotoxicity of this substance. Judging by experimental data obtained in this investigation, occupational exposures to nano-scale copper oxide particles can present a significant health risk while the further search for its management with the help of innocuous bioprotectors seems to be justified.

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.

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

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.

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

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.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

Variability in triactinomyxon production from Tubifex tubifex populations from the same mitochondrial DNA lineage infected with Myxobolus cerebralis, the causative agent of whirling disease in salmonids

USGS Publications Warehouse

Rasmussen, C.; Zickovich, J.; Winton, J.R.; Kerans, B.L.

2008-01-01

Myxobolus cerebralis, the causative agent of whirling disease, infects both salmonid fish and an aquatic oligochaete, Tubifex tubifex. Although M. cerebralis has been detected in river drainages throughout the United States, disease severity among wild fish populations has been highly variable. Tubifex tubifex populations have been genetically characterized using sequences from the 16S mitochondrial DNA (mtDNA) gene, the 18S ribosomal RNA gene, the internal transcribed spacer region 1 (ITS1), and randomly amplified polymorphic DNA (RAPD). Our earlier work indicated that large differences in compatibility between the parasite and populations of T. tubifex may play a substantial role in the distribution of whirling disease and resulting mortality in different watersheds. In the present study, we examined 4 laboratory populations of T. tubifex belonging to 16S mtDNA lineage III and 1 population belonging to 16S mtDNA lineage I for triactinomyxon (TAM) production after infection with M. cerebralis myxospores. All 4 16S mtDNA lineage III populations produced TAMs, but statistically significant differences in TAM production were observed. Most individuals in the 16S mtDNA lineage III-infected populations produced TAMs. The 16S mtDNA lineage I population produced few TAMs. Further genetic characterization of the 16S mtDNA lineage III populations with RAPD markers indicated that populations producing similar levels of TAMs had more genetic similarity. ?? American Society of Parasitologists 2008.

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.

Characterization of Novel Trichoderma asperellum Isolates to Select Effective Biocontrol Agents Against Tomato Fusarium Wilt

PubMed Central

El_Komy, Mahmoud H.; Saleh, Amgad A.; Eranthodi, Anas; Molan, Younes Y.

2015-01-01

The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL) is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD) method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and β-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their anta- gonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents. PMID:25774110

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

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.

An insight into the distribution, genetic diversity, and mycotoxin production of Aspergillus section Flavi in soils of pistachio orchards.

PubMed

Jamali, Mojdeh; Ebrahimi, Mohammad-Ali; Karimipour, Morteza; Shams-Ghahfarokhi, Masoomeh; Dinparast-Djadid, Navid; Kalantari, Sanaz; Pilehvar-Soltanahmadi, Yones; Amani, Akram; Razzaghi-Abyaneh, Mehdi

2012-01-01

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB(1) and AFB(2) were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB(1) and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.

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.

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

Population Genetics of the Endemic Hawaiian Species Chrysodracon hawaiiensis and Chrysodracon auwahiensis (Asparagaceae): Insights from RAPD and ISSR Variation

PubMed Central

Lu, Pei-Luen; Yorkson, Mitsuko; Morden, Clifford W.

2016-01-01

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested. PMID:27537876

Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

PubMed Central

Dean, Frank B.; Nelson, John R.; Giesler, Theresa L.; Lasken, Roger S.

2001-01-01

We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications. PMID:11381035

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.

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.

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.

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

PubMed Central

Maruyama, Toru; Yamagishi, Keisuke; Mori, Tetsushi; Takeyama, Haruko

2015-01-01

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. PMID:26389587

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Narrowband random lasing in a Bismuth-doped active fiber

PubMed Central

Lobach, Ivan A.; Kablukov, Sergey I.; Skvortsov, Mikhail I.; Podivilov, Evgeniy V.; Melkumov, Mikhail A.; Babin, Sergey A.; Dianov, Evgeny M.

2016-01-01

Random fiber lasers operating via the Rayleigh scattering (RS) feedback attract now a great deal of attention as they generate a high-quality unidirectional laser beam with the efficiency and performance comparable and even exceeding those of fiber lasers with conventional cavities. Similar to other random lasers, both amplification and random scattering are distributed here along the laser medium being usually represented by a kilometers-long passive fiber with Raman gain. However, it is hardly possible to utilize normal gain in conventional active fibers as they are usually short and RS is negligible. Here we report on the first demonstration of the RS-based random lasing in an active fiber. This became possible due to the implementation of a new Bi-doped fiber with an increased amplification length and RS coefficient. The realized Bi-fiber random laser generates in a specific spectral region (1.42 μm) exhibiting unique features, in particular, a much narrower linewidth than that in conventional cavity of the same length, in agreement with the developed theory. Lasers of this type have a great potential for applications as Bi-doped fibers with different host compositions enable laser operation in an extremely broad range of wavelengths, 1.15–1.78 μm. PMID:27435232

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.

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

PubMed Central

Robert, Catherine; Pascalis, Hervé; Michelle, Caroline; Jardot, Priscilla; Charrel, Rémi; Raoult, Didier; Desnues, Christelle

2015-01-01

Background Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. PMID:26431175

In vitro effect of amoxicillin and clarithromycin on the 3’ region of cagA gene in Helicobacter pylori isolates

PubMed Central

Bustamante-Rengifo, Javier Andrés; Matta, Andrés Januer; Pazos, Alvaro; Bravo, Luis Eduardo

2013-01-01

AIM: To evaluate the in vitro effect of amoxicillin and clarithromycin on the cag pathogenicity island (cag PAI). METHODS: One hundred and forty-nine clinical isolates of Helicobacter pylori (H. pylori) cultured from gastric biopsies from 206 Colombian patients with dyspeptic symptoms from a high-risk area for gastric cancer were included as study material. Antimicrobial susceptibility was determined by the agar dilution method. Resistant isolates at baseline and in amoxicillin and clarithromycin serial dilutions were subjected to genotyping (cagA, vacA alleles s and m), Glu-Pro-Ile-Tyr-Ala (EPIYA) polymerase chain reaction and random amplified polymorphic DNA (RAPD). Images of the RAPD amplicons were analyzed by Gel-Pro Analyzer 4.5 program. Cluster analyses was done using SPSS 15.0 statistical package, where each of the fingerprint bands were denoted as variables. Dendrograms were designed by following Ward’s clustering method and the estimation of distances between each pair of H. pylori isolates was calculated with the squared Euclidean distance. RESULTS: Resistance rates were 4% for amoxicillin and 2.7% for clarithromycin with 2% double resistances. Genotyping evidenced a high prevalence of the genotype cagA-positive/vacA s1m1. The 3’ region of cagA gene was successfully amplified in 92.3% (12/13) of the baseline resistant isolates and in 60% (36/60) of the resistant isolates growing in antibiotic dilutions. Upon observing the distribution of the number of EPIYA repetitions in each dilution with respect to baseline isolates, it was found that in 61.5% (8/13) of the baseline isolates, a change in the number of EPIYA repetitions lowered antibiotic pressure. The gain and loss of EPIYA motifs resulted in a diversity of H. pylori subclones after bacterial adjustment to changing conditions product of antibiotic pressure. RAPD PCR evidenced the close clonal relationship between baseline isolates and isolates growing in antibiotic dilutions. CONCLUSION: Antibiotic pressure does not induce loss of the cag pathogenicity island, but it can lead - in most cases - to genetic rearrangements within the 3’ region cagA of the founding bacteria that can affect the level of tyrosine phosphorylation impacting on its cellular effects and lead to divergence of cagA-positive subclones. PMID:24106405

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.

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.

[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.

Examination of Salmonella and Escherichia coli translocation from hog manure to forage, soil, and cattle grazed on the hog manure-treated pasture.

PubMed

Holley, Richard; Walkty, Joël; Blank, Gregory; Tenuta, Mario; Ominski, Kimberly; Krause, Denis; Ng, Lai-King

2008-01-01

Use of hog (Sus scrofa) manure as a fertilizer is a practical solution for waste re-utilization, however, it may serve as a vehicle for environmental and domestic animal contamination. Work was conducted to determine whether pathogens, naturally present in hog manure could be detected in cattle (Bos taurus) grazed on the manure-treated pasture, and whether forage contamination occurred. During two 3 mo summer trials manure was applied to yield < or = 124 kg available N per hectare in a single spring or split spring and fall application. Samples of hog manure, forage, soil, and cattle feces were analyzed for naturally occurring Salmonella, Yersinia enterocolitica, and Escherichia coli. To follow movement of Salmonella in the environment isolates were identified to serovar and serotyped. Transfer of E. coli from hog manure to soil and cattle was examined by randomly amplified polymorphic DNA (RAPD) analysis of >600 E. coli isolates. While Y. enterocolitica was absent from all samples, in both years S. enterica Derby and S. enterica Krefeld were found in most hog manure samples, but were only on forage samples in the second year. Salmonella enterica Typhimurium, absent from hog manure was present on some forage in the first year. Cattle feces and soil samples were consistently Salmonella negative. These contaminations could not be traced to manure application. During this study, Salmonella and E. coli found in hog manure had different RAPD genomic profiles from those found in the feces of cattle grazing on manure-treated pasture.

Clinical Trichophyton rubrum Strain Exhibiting Primary Resistance to Terbinafine

PubMed Central

Mukherjee, Pranab K.; Leidich, Steven D.; Isham, Nancy; Leitner, Ingrid; Ryder, Neil S.; Ghannoum, Mahmoud A.

2003-01-01

The in vitro antifungal susceptibilities of six clinical Trichophyton rubrum isolates obtained sequentially from a single onychomycosis patient who failed oral terbinafine therapy (250 mg/day for 24 weeks) were determined by broth microdilution and macrodilution methodologies. Strain relatedness was examined by random amplified polymorphic DNA (RAPD) analyses. Data obtained from both broth micro- and macrodilution assays were in agreement and revealed that the six clinical isolates had greatly reduced susceptibilities to terbinafine. The MICs of terbinafine for these strains were >4 μg/ml, whereas they were <0.0002 μg/ml for the susceptible reference strains. Consistent with these findings, the minimum fungicidal concentrations (MFCs) of terbinafine for all six strains were >128 μg/ml, whereas they were 0.0002 μg/ml for the reference strain. The MIC of terbinafine for the baseline strain (cultured at the initial screening visit and before therapy was started) was already 4,000-fold higher than normal, suggesting that this is a case of primary resistance to terbinafine. The results obtained by the broth macrodilution procedure revealed that the terbinafine MICs and MFCs for sequential isolates apparently increased during the course of therapy. RAPD analyses did not reveal any differences between the isolates. The terbinafine-resistant isolates exhibited normal susceptibilities to clinically available antimycotics including itraconazole, fluconazole, and griseofulvin. However, these isolates were fully cross resistant to several other known squalene epoxidase inhibitors, including naftifine, butenafine, tolnaftate, and tolciclate, suggesting a target-specific mechanism of resistance. This is the first confirmed report of terbinafine resistance in dermatophytes. PMID:12499173

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.

Prolonged Outbreak of Mycobacterium chimaera Infection After Open-Chest Heart Surgery.

PubMed

Sax, Hugo; Bloemberg, Guido; Hasse, Barbara; Sommerstein, Rami; Kohler, Philipp; Achermann, Yvonne; Rössle, Matthias; Falk, Volkmar; Kuster, Stefan P; Böttger, Erik C; Weber, Rainer

2015-07-01

Invasive Mycobacterium chimaera infections were diagnosed in 2012 in 2 heart surgery patients on extracorporeal circulation. We launched an outbreak investigation to identify the source and extent of the potential outbreak and to implement preventive measures. We collected water samples from operating theaters, intensive care units, and wards, including air samples from operating theaters. Mycobacterium chimaera strains were characterized by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Case detection was performed based on archived histopathology samples and M. chimaera isolates since 2006, and the patient population at risk was prospectively surveyed. We identified 6 male patients aged between 49 and 64 years with prosthetic valve endocarditis or vascular graft infection due to M. chimaera, which became clinically manifest with a latency of between 1.5 and 3.6 years after surgery. Mycobacterium chimaera was isolated from cardiac tissue specimens, blood cultures, or other biopsy specimens. We were able also to culture M. chimaera from water circuits of heater-cooler units connected to the cardiopulmonary bypass, and air samples collected when the units were in use. RAPD-PCR demonstrated identical patterns among M. chimaera strains from heater-cooler unit water circuits and air samples, and strains in 2 patient clusters. The epidemiological and microbiological features of this prolonged outbreak provided evidence for the airborne transmission of M. chimaera from contaminated heater-cooler unit water tanks to patients during open-heart surgery. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Resistance Potential of Bread Wheat Genotypes Against Yellow Rust Disease Under Egyptian Climate.

PubMed

Mahmoud, Amer F; Hassan, Mohamed I; Amein, Karam A

2015-12-01

Yellow rust (stripe rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive foliar diseases of wheat in Egypt and worldwide. In order to identify wheat genotypes resistant to yellow rust and develop molecular markers associated with the resistance, fifty F8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible bread wheat landraces were obtained. Artificial infection of Puccinia striiformis was performed under greenhouse conditions during two growing seasons and relative resistance index (RRI) was calculated. Two Egyptian bread wheat cultivars i.e. Giza-168 (resistant) and Sakha-69 (susceptible) were also evaluated. RRI values of two-year trial showed that 10 RILs responded with RRI value >6 <9 with an average of 7.29, which exceeded the Egyptian bread wheat cultivar Giza-168 (5.58). Thirty three RILs were included among the acceptable range having RRI value >2 <6. However, only 7 RILs showed RRI value <2. Five RILs expressed hypersensitive type of resistance (R) against the pathogen and showed the lowest Average Coefficient of Infection (ACI). Bulked segregant analysis (BSA) with eight simple sequence repeat (SSR), eight sequence-related amplified polymorphism (SRAP) and sixteen random amplified polymorphic DNA (RAPD) markers revealed that three SSR, three SRAP and six RAPD markers were found to be associated with the resistance to yellow rust. However, further molecular analyses would be performed to confirm markers associated with the resistance and suitable for marker-assisted selection. Resistant RILs identified in the study could be efficiently used to improve the resistance to yellow rust in wheat.

Disease Burden of Invasive Listeriosis and Molecular Characterization of Clinical Isolates in Taiwan, 2000-2013.

PubMed

Huang, Yu-Tsung; Ko, Wen-Chien; Chan, Yu-Jiun; Lu, Jang-Jih; Tsai, Hsih-Yeh; Liao, Chun-Hsing; Sheng, Wang-Huei; Teng, Lee-Jene; Hsueh, Po-Ren

2015-01-01

The information about disease burden and epidemiology of invasive listeriosis in Asia is scarce. From 2000 to 2013, a total of 338 patients with invasive listeriosis (bacteremia, meningitis, and peritonitis) were treated at four medical centers in Taiwan. The incidence (per 10,000 admissions) of invasive listeriosis increased significantly during the 14-year period among the four centers (0.15 in 2000 and >1.25 during 2010-2012) and at each of the four medical centers. Among these patients, 45.9% were elderly (>65 years old) and 3.3% were less than one year of age. More than one-third (36.7%) of the patients acquired invasive listeriosis in the spring (April to June). Among the 132 preserved Listeria monocytogenes isolates analyzed, the most frequently isolated PCR serogroup-sequence type (ST) was IIb-ST87 (23.5%), followed by IIa-ST378 (19.7%) and IIa-ST155 (12.1%). Isolation of PCR serogroups IIb and IVb increased significantly with year, with a predominance of IIb-ST87 isolates (23.5%) and IIb-ST 228 isolates emerging in 2013. A total of 12 different randomly amplified polymorphic DNA (RAPD) patterns (Patterns I to XII) were identified among the 112 L. monocytogenes isolates belonging to eight main PCR serogroup-STs. Identical RAPD patterns were found among the isolates exhibiting the same PCR serogroup-ST. In conclusion, our study revealed that during 2000-2013, listeriosis at four medical centers in Taiwan was caused by heterogeneous strains and that the upsurge in incidence beginning in 2005 was caused by at least two predominant clones.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Error Augmentation on Brain Activation and Motor Learning of a Complex Locomotor Task

PubMed Central

Marchal-Crespo, Laura; Michels, Lars; Jaeger, Lukas; López-Olóriz, Jorge; Riener, Robert

2017-01-01

Up to date, the functional gains obtained after robot-aided gait rehabilitation training are limited. Error augmenting strategies have a great potential to enhance motor learning of simple motor tasks. However, little is known about the effect of these error modulating strategies on complex tasks, such as relearning to walk after a neurologic accident. Additionally, neuroimaging evaluation of brain regions involved in learning processes could provide valuable information on behavioral outcomes. We investigated the effect of robotic training strategies that augment errors—error amplification and random force disturbance—and training without perturbations on brain activation and motor learning of a complex locomotor task. Thirty-four healthy subjects performed the experiment with a robotic stepper (MARCOS) in a 1.5 T MR scanner. The task consisted in tracking a Lissajous figure presented on a display by coordinating the legs in a gait-like movement pattern. Behavioral results showed that training without perturbations enhanced motor learning in initially less skilled subjects, while error amplification benefited better-skilled subjects. Training with error amplification, however, hampered transfer of learning. Randomly disturbing forces induced learning and promoted transfer in all subjects, probably because the unexpected forces increased subjects' attention. Functional MRI revealed main effects of training strategy and skill level during training. A main effect of training strategy was seen in brain regions typically associated with motor control and learning, such as, the basal ganglia, cerebellum, intraparietal sulcus, and angular gyrus. Especially, random disturbance and no perturbation lead to stronger brain activation in similar brain regions than error amplification. Skill-level related effects were observed in the IPS, in parts of the superior parietal lobe (SPL), i.e., precuneus, and temporal cortex. These neuroimaging findings indicate that gait-like motor learning depends on interplay between subcortical, cerebellar, and fronto-parietal brain regions. An interesting observation was the low activation observed in the brain's reward system after training with error amplification compared to training without perturbations. Our results suggest that to enhance learning of a locomotor task, errors should be augmented based on subjects' skill level. The impacts of these strategies on motor learning, brain activation, and motivation in neurological patients need further investigation. PMID:29021739

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.

Extended-spectrum β-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

PubMed

Charfi, Karama; Grami, Raoudha; Ben Jeddou, Abir; Messaoudi, Aziza; Mani, Yosra; Bouallegue, Olfa; Boujaafar, Noureddine; Aouni, Mahjoub; Mammeri, Hedi; Mansour, Wejdène

2017-09-01

This study was conducted to investigate extended-spectrum-β-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The bla CTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission. Copyright © 2017 Elsevier Ltd. All rights reserved.

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Bernardini, Jame N.; Stam, Christina N.

2010-01-01

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.

[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.

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.

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.

Improved acid tolerance of Lactobacillus pentosus by error-prone whole genome amplification.

PubMed

Ye, Lidan; Zhao, Hua; Li, Zhi; Wu, Jin Chuan

2013-05-01

Acid tolerance of Lactobacillus pentosus ATCC 8041 was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type L. pentosus by electroporation and the transformants were screened for growth on low-pH agar plates. After only one round of mutation, one mutant (MT3) was identified that was able to completely consume 20 g/L of glucose to produce lactic acid at a yield of 95% in 1L MRS medium at pH 3.8 within 36 h, whereas no growth or lactic acid production was observed for the wild-type strain under the same conditions. The acid tolerance of mutant MT3 remained genetically stable for at least 25 subcultures. Therefore, the error-prone whole genome amplification technique is a very powerful tool for improving phenotypes of this lactic acid bacterium and may also be applicable for other microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

An analytically soluble problem in fully nonlinear statistical gravitational lensing

NASA Technical Reports Server (NTRS)

Schneider, P.

1987-01-01

The amplification probability distribution p(I)dI for a point source behind a random star field which acts as the deflector exhibits a I exp-3 behavior for large amplification, as can be shown from the universality of the lens equation near critical lines. In this paper it is shown that the amplitude of the I exp-3 tail can be derived exactly for arbitrary mass distribution of the stars, surface mass density of stars and smoothly distributed matter, and large-scale shear. This is then compared with the corresponding linear result.

Micropropagation of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) and assessment of genetic fidelity of micropropagated plants using RAPD markers.

PubMed

Goswami, Komal; Sharma, R; Singh, P K; Singh, Govind

2013-01-01

A micropropagation protocol was developed for multiplication of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) using nodal explants. The maximum shoot regeneration was observed on low level of BAP (0.1 mg l(-1)) or kinetin (0.5 mg l(-1)). BA was recorded to be better than kinetin in terms of number of days taken to bud break. The maximum number of shoots per explant was observed on 0.1 mg l(-1) BA and 0.5 mg l(-1) kinetin. Shoot proliferation decreased with increasing concentration of BA alone, but in case of a combination of BA and NAA (0.1 mg l(-1) each), it increased with increasing concentration of BA up to 10.0 mg l(-1). None of the treatments including BA or kinetin alone or BA in combination with NAA produced significantly more shoots for commercial exploitation. In the case of a combination of BA + kinetin + IBA, the maximum (5.5 shoots per explants) proliferation was observed on MS medium containing 1.0 mg l(-1) BA + 0.5 mg l(-1) kinetin + 0.5 mg l(-1) IBA or 0.25 mg l(-1) BA + 1.0 mg l(-1) kinetin + 1.0 mg l(-1) IBA. Regenerated shoots showed root induction on MS basal medium or on MS medium containing 1.0 mg l(-1) IBA. It is concluded that a five-fold increase (1.0 mg l(-1) BA + 0.5 mg l(-1) kinetin + 0.5 mg l(-1) IBA) in axillary shoot proliferation, while seven-fold increase (0.25 mg/l mg l(-1) BA + 1.0 mg l(-1) kinetin + 1.0 mg l(-1) IBA) during the second cycle of multiplication could be obtained using the two plant growth regulator combinations. PCR amplification with 14 different random primers confirmed no somaclonal variant up to two cycles of shoot multiplication.

Molecular characterization of Histoplasma capsulatum isolated from an outbreak in treasure hunters Histoplasma capsulatum in treasure hunters

PubMed Central

2010-01-01

Background In Mexico, primary pulmonary histoplasmosis is the most relevant clinical form of the disease. The geographical distribution of specific strains of Histoplasma capsulatum circulating in Mexico has not been fully established. Outbreaks must be reported in order to have current, updated information on this disease, identifying new endemic areas, manner of exposure to the fungi, and molecular characterization of the causative agents. We report a recent outbreak of histoplasmosis in treasure hunters and the molecular characterization of two isolates obtained from these patients. Methods Six patients admitted to the National Institute of Respiratory Diseases (INER) in Mexico City presented severe respiratory symptoms suggestive of histoplasmosis. They acquired the infection in the Veracruz (VZ) endemic zone. Diagnosis was made by X-ray and Computed tomography (CT), liver function, immunological techniques, and culture. Identification of H. capsulatum isolates was confirmed by using Polymerase chain reaction (PCR) was conducted with a probe from the M antigen, and the isolates were characterized by means of Random amplification of polymorphic DNA (RAPD)-PCR employed the 1253 oligonucleotide and a mixture of oligonucleotides 1281 and 1283. These were compared to eight reference strain isolates from neighboring areas. Results X-ray and CT revealed disseminated micronodular images throughout lung parenchyma, as well as bilateral retrocaval, prevascular, subcarinal, and hilar adenopathies, hepatosplenomegaly, and altered liver function tests. Five of the six patients developed disseminated histoplasmosis. Two H. capsulatum strains were isolated. The same band profile was detected in both strains, indicating that both isolates corresponded to the sole H. capsulatum strain. Molecular characterization of the isolates was similar in 100% with the EH-53 Hidalgo human (HG) strain (reference strain integrated into the LAm A clade described for Latin America). Conclusions The two isolates appeared to possess the same polymorphic pattern; they are indistinguishable from each other and from EH-53. It is important to remain updated on recent outbreaks of histoplasmosis, the manner of exposure to the fungi, as well as the molecular characterization of the isolates. The severity of cases indicates that this strain is highly virulent and that it is probably prevalent in Hidalgo and Veracruz states. PMID:20825675

Genetic Diversity and Symbiotic Efficiency of Indigenous Common Bean Rhizobia in Croatia.

PubMed

Pohajda, Ines; Babić, Katarina Huić; Rajnović, Ivana; Kajić, Sanja; Sikora, Sanja

2016-12-01

Nodule bacteria (rhizobia) in symbiotic associations with legumes enable considerable entries of biologically fixed nitrogen into soil. Efforts are therefore made to intensify the natural process of symbiotic nitrogen fixation by legume inoculation. Studies of field populations of rhizobia open up the possibility to preserve and probably exploit some indigenous strains with hidden symbiotic or ecological potentials. The main aim of the present study is to determine genetic diversity of common bean rhizobia isolated from different field sites in central Croatia and to evaluate their symbiotic efficiency and compatibility with host plants. The isolation procedure revealed that most soil samples contained no indigenous common bean rhizobia. The results indicate that the cropping history had a significant impact on the presence of indigenous strains. Although all isolates were found to belong to species Rhizobium leguminosarum , significant genetic diversity at the strain level was determined. Application of both random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC- -PCR) methods resulted in similar grouping of strains. Symbiotic efficiency of indigenous rhizobia as well as their compatibility with two commonly grown bean varieties were tested in field experiments. Application of indigenous rhizobial strains as inoculants resulted in significantly different values of nodulation, seed yield as well as plant nitrogen and seed protein contents. The most abundant nodulation and the highest plant nitrogen and protein contents were determined in plants inoculated with R. leguminosarum strains S 17/2 and S 21/6 . Although, in general, the inoculation had a positive impact on seed yield, differences depending on the applied strain were not determined. The overall results show the high degree of symbiotic efficiency of the specific indigenous strain S 21/6 . These results indicate different symbiotic potential of indigenous strains and confirmed the importance of rhizobial strain selection. These are the first studies of indigenous common bean rhizobia in Croatia that provide the basis for further characterization and selection of highly efficient indigenous strains and their potential use in agricultural practice and future research.

Polyploidy creates higher diversity among Cynodon accessions as assessed by molecular markers.

PubMed

Gulsen, Osman; Sever-Mutlu, Songul; Mutlu, Nedim; Tuna, Metin; Karaguzel, Osman; Shearman, Robert C; Riordan, Terrance P; Heng-Moss, Tiffany M

2009-05-01

Developing a better understanding of associations among ploidy level, geographic distribution, and genetic diversity of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to: (1) determine ploidy analysis of Cynodon accessions collected from Turkey, (2) investigate associations between ploidy level and diversity, (3) determine whether geographic and ploidy distribution are related to nuclear genome variation, and (4) correlate among four nuclear molecular marker systems for Cynodon accessions' genetic analyses. One hundred and eighty-two Cynodon accessions collected in Turkey from an area south of the Taurus Mountains along the Mediterranean cost and ten known genotypes were genotyped using sequence related amplified polymorphism (SRAP), peroxidase gene polymorphism (POGP), inter-simple sequence repeat (ISSR), and random amplified polymorphic DNA (RAPD). The diploids, triploids, tetraploids, pentaploids, and hexaploids revealed by flow cytometry had a linear present band frequency of 0.36, 0.47, 0.49, 0.52, and 0.54, respectively. Regression analysis explained that quadratic relationship between ploidy level and band frequency was the most explanatory (r = 0.62, P < 0.001). The AMOVA results indicated that 91 and 94% of the total variation resided within ploidy level and provinces, respectively. The UPGMA analysis suggested that commercial bermudagrass cultivars only one-third of the available genetic variation. SRAP, POGP, ISSR, and RAPD markers differed in detecting relationships among the bermudagrass genotypes and rare alleles, suggesting more efficiency of combinatory analysis of molecular marker systems. Elucidating Cynodon accessions' genetic structure can aid to enhance breeding programs and broaden genetic base of commercial cultivars.

In vitro characterization of Leishmania (Viannia) braziliensis isolates from patients with different responses to Glucantime(®) treatment from Northwest Paraná, Brazil.

PubMed

Fernandes, Andrea Claudia Bekner Silva; Pedroso, Raíssa Bocchi; de Mello, Tatiane França Perles; Donatti, Lucélia; Venazzi, Eneide Aparecida Sabaini; Demarchi, Izabel Galhardo; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Silveira, Thaís Gomes Verzignassi

2016-08-01

Leishmaniasis is a group of diseases that presents various clinical manifestations. Many studies have shown that the parasite plays an important role in the clinical manifestations and prognosis of this disease. The cutaneous and mucosal forms of American tegumentary leishmaniasis (ATL) are associated with Leishmania (Viannia) braziliensis, which exhibits intraspecific genetic polymorphisms and various clinical manifestations. The present study focused on four different L. braziliensis strains that were isolated from patients with distinct Glucantime(®) treatment responses. The isolates were described based on their molecular, biological, and infective characteristics. Growth patterns in culture medium and different grow phases were analyzed, MID-Logarithimic (Mid-LOG), Logarithimic (LOG) and Stationary (STAT) phases. Complement resistance was evaluated using guinea pig serum. Infection to murine peritoneal macrophages, cytokine and nitric oxide were analyzed. Ultrastructural features were determined by transmission electron microscopy, and molecular characteristics were determined based on random amplified polymorphic DNA (RAPD). All of the L. braziliensis isolates showed typical growth and similar complement sensitivity patterns. Markedly lower infectivity indexes were observed for all strains in the LOG phase, with different cytokine profiles. The ultrastructure analysis revealed distinct differences between the MID-LOG, LOG, and STAT phases. The RAPD results showed a divergence between the isolates of the L. braziliensis. The in vitro characterization of L. braziliensis isolates from humans with different treatment responses using various parameters enabled us to observe differences among the isolates. Molecular and in vivo characterizations are currently under study to improve understanding of the parasite-host interaction that can imply in the clinical manifestation differences. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of autochthonous starters to ferment red and yellow peppers (Capsicum annum L.) to be stored at room temperature.

PubMed

Di Cagno, Raffaella; Surico, Rosalinda F; Minervini, Giovanna; De Angelis, Maria; Rizzello, Carlo G; Gobbetti, Marco

2009-03-31

Strains of Lactobacillus curvatus, Leuconostoc mesenteroides, Lactobacillus plantarum and Weissella confusa were identified from raw red and yellow peppers (RYPs) by partial 16S rRNA gene sequence and subjected to typing by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis. L. plantarum PE21, L. curvatus PE4 and W. confusa PE36 were selected based on the kinetics of growth and acidification, and used as the autochthonous mixed starter for the fermentation of RYPs. A protocol which included blanching at 85 degrees C for 2 min, fermentation at 35 degrees C for 15 h in brine (1%, w/v), and heat treatment at 85 degrees C for 15 min, followed by storage at room temperature for 30 days with and without sunflower seeds oil was set up. Unstarted RYPs subjected to the same treatments were used as the control. Cell numbers of autochthonous starter in the RYPs were ca. 1000 times higher than presumptive lactic acid bacteria in unstarted RYPs. As shown by RAPD-PCR analysis, all three autochthonous strains persisted during processing and storage. Presumptive lactic acid bacteria found in started RYPs progressively decreased during storage, leading to a microbiota mainly consisting of autochthonous starters. Started RYPs showed rapid decrease of pH (<3.7), marked consumption of fermentable carbohydrates, and inhibition of total enterobacteria and yeasts. Unstarted RYPs were subjected to slight acidification (pH ca. 4.87) and considerable contamination by total enterobacteria and yeasts throughout storage. After 30 days of storage, started RYPs had significantly (P<0.05) higher firmness and colour indexes with respect to unstarted RYPs. The microbial and sensory features of started RYPs stored with sunflower seeds oil were almost similar to those of RYPs stored without suspending liquid.

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.

Geographic variation and genetic structure in Spotted Owls

USGS Publications Warehouse

Haig, Susan M.; Wagner, R.S.; Forsman, E.D.; Mullins, Thomas D.

2001-01-01

We examined genetic variation, population structure, and definition of conservation units in Spotted Owls (Strix occidentalis). Spotted Owls are mostly non-migratory, long-lived, socially monogamous birds that have decreased population viability due to their occupation of highly-fragmented late successional forests in western North America. To investigate potential effects of habitat fragmentation on population structure, we used random amplified polymorphic DNA (RAPD) to examine genetic variation hierarchically among local breeding areas, subregional groups, regional groups, and subspecies via sampling of 21 breeding areas (276 individuals) among the three subspecies of Spotted Owls. Data from 11 variable bands suggest a significant relationship between geographic distance among local breeding groups and genetic distance (Mantel r = 0.53, P < 0.02) although multi-dimensional scaling of three significant axes did not identify significant grouping at any hierarchical level. Similarly, neighbor-joining clustering of Manhattan distances indicated geographic structure at all levels and identified Mexican Spotted Owls as a distinct clade. RAPD analyses did not clearly differentiate Northern Spotted Owls from California Spotted Owls. Among Northern Spotted Owls, estimates of population differentiation (FST) ranged from 0.27 among breeding areas to 0.11 among regions. Concordantly, within-group agreement values estimated via multi-response permutation procedures of Jaccarda??s distances ranged from 0.22 among local sites to 0.11 among regions. Pairwise comparisons of FST and geographic distance within regions suggested only the Klamath region was in equilibrium with respect to gene flow and genetic drift. Merging nuclear data with recent mitochondrial data provides support for designation of an Evolutionary Significant Unit for Mexican Spotted Owls and two overlapping Management Units for Northern and California Spotted Owls.

Disease Burden of Invasive Listeriosis and Molecular Characterization of Clinical Isolates in Taiwan, 2000-2013

PubMed Central

Huang, Yu-Tsung; Ko, Wen-Chien; Chan, Yu-Jiun; Lu, Jang-Jih; Tsai, Hsih-Yeh; Liao, Chun-Hsing; Sheng, Wang-Huei; Teng, Lee-Jene; Hsueh, Po-Ren

2015-01-01

The information about disease burden and epidemiology of invasive listeriosis in Asia is scarce. From 2000 to 2013, a total of 338 patients with invasive listeriosis (bacteremia, meningitis, and peritonitis) were treated at four medical centers in Taiwan. The incidence (per 10,000 admissions) of invasive listeriosis increased significantly during the 14-year period among the four centers (0.15 in 2000 and >1.25 during 2010–2012) and at each of the four medical centers. Among these patients, 45.9% were elderly (>65 years old) and 3.3% were less than one year of age. More than one-third (36.7%) of the patients acquired invasive listeriosis in the spring (April to June). Among the 132 preserved Listeria monocytogenes isolates analyzed, the most frequently isolated PCR serogroup-sequence type (ST) was IIb-ST87 (23.5%), followed by IIa-ST378 (19.7%) and IIa-ST155 (12.1%). Isolation of PCR serogroups IIb and IVb increased significantly with year, with a predominance of IIb-ST87 isolates (23.5%) and IIb-ST 228 isolates emerging in 2013. A total of 12 different randomly amplified polymorphic DNA (RAPD) patterns (Patterns I to XII) were identified among the 112 L. monocytogenes isolates belonging to eight main PCR serogroup-STs. Identical RAPD patterns were found among the isolates exhibiting the same PCR serogroup-ST. In conclusion, our study revealed that during 2000–2013, listeriosis at four medical centers in Taiwan was caused by heterogeneous strains and that the upsurge in incidence beginning in 2005 was caused by at least two predominant clones. PMID:26555445

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.

Density and Molecular Epidemiology of Aspergillus in Air and Relationship to Outbreaks of Aspergillus Infection

PubMed Central

Leenders, Alexander C. A. P.; van Belkum, Alex; Behrendt, Myra; Luijendijk, Ad; Verbrugh, Henri A.

1999-01-01

After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Knowledge of the epidemiology of Aspergillus species is necessary for the development of strategies to prevent invasive aspergillosis. RAPD fingerprinting of Aspergillus isolates can help to determine the cause of an outbreak of invasive aspergillosis. PMID:10325319

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

Chemoraces and Habitat Specialization of Claviceps purpurea Populations

PubMed Central

Pažoutová, Sylvie; Olšovská, Jana; Linka, Marek; Kolínská, Renata; Flieger, Miroslav

2000-01-01

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift. PMID:11097923

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.

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

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico.

PubMed

Taylor, Maria Lucia; Chávez-Tapia, Catalina B; Rojas-Martínez, Alberto; del Rocio Reyes-Montes, Maria; del Valle, Mirian Bobadilla; Zúñiga, Gerardo

2005-09-01

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.

Random pulse generator

NASA Technical Reports Server (NTRS)

Lindsey, R. S., Jr. (Inventor)

1975-01-01

An exemplary embodiment of the present invention provides a source of random width and random spaced rectangular voltage pulses whose mean or average frequency of operation is controllable within prescribed limits of about 10 hertz to 1 megahertz. A pair of thin-film metal resistors are used to provide a differential white noise voltage pulse source. Pulse shaping and amplification circuitry provide relatively short duration pulses of constant amplitude which are applied to anti-bounce logic circuitry to prevent ringing effects. The pulse outputs from the anti-bounce circuits are then used to control two one-shot multivibrators whose output comprises the random length and random spaced rectangular pulses. Means are provided for monitoring, calibrating and evaluating the relative randomness of the generator.

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.

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.

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.

[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.

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.

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.

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

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.

Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis.

PubMed

Wu, Jieying; Gao, Weimin; Zhang, Weiwen; Meldrum, Deirdre R

2011-01-01

Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities. Copyright © 2010 Elsevier B.V. All rights reserved.

Isolation and characterization of ethanol tolerant yeast strains

PubMed Central

Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

2013-01-01

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092

Microbial diversity of landslide soils assessed by RFLP and SSCP fingerprints.

PubMed

Guida, Marco; Cannavacciuolo, Paolo Losanno; Cesarano, Mara; Borra, Marco; Biffali, Elio; D'Alessandro, Raffaella; De Felice, Bruna

2014-08-01

Landslides are a significant component of natural disasters in most countries around the world. Understanding these destructive phenomena through the analysis of possible correlations between microbial communities and the alteration of the soil responsible for landslides is important in order to reduce their negative consequences. To address this issue, bacterial and fungal communities in soils triggering landslides in Termini-Nerano and Massa Lubrense-Nerano (Naples, Italy) were analysed by genetic profiling techniques. Fingerprints were generated by single-strand conformation polymorphisms (SSCP) and random amplified polymorphic DNA (RAPD). The microbial community in both soil types was enriched in species which could contribute to the degradation process occurring during landslides, forming biofilms and leading to the transformation or the formation of minerals. Indeed, some of the identified bacteria were found to favour the transformation of clay minerals. These findings suggest a possible relationship between bacterial and fungal community-colonising soils and the occurrence of landslides.

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

PubMed

Atia, Mohamed A M; Sakr, Mahmoud M; Adawy, Sami S

2017-01-01

Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.).

PubMed

Peredo, Elena L; Arroyo-García, Rosa; Reed, Barbara M; Revilla, M Angeles

2008-12-01

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.

Genetic diversity of Brazilian natural populations of Anthonomus grandis Boheman (Coleoptera: Curculionidae), the major cotton pest in the New World.

PubMed

Martins, W F S; Ayres, C F J; Lucena, W A

2007-01-27

Twenty-five RAPD loci and 6 isozyme loci were studied to characterize the genetic variability of natural populations of Anthonomus grandis from two agroecosystems of Brazil. The random-amplified polymorphic DNA data disclosed a polymorphism that varied from 52 to 84% and a heterozygosity of 0.189 to 0.347. The index of genetic differentiation (GST) among the six populations was 0.258. The analysis of isozymes showed a polymorphism and a heterozygosity ranging from 25 to 100% and 0.174 to 0.277, respectively. The genetic differentiation (FST) among the populations obtained by isozyme data was 0.544. It was possible to observe rare alleles in the populations from the Northeast region. The markers examined allowed us to distinguish populations from large-scale, intensive farming region (cotton belts) versus populations from areas of small-scale farming

Recurrent recovery of Pseudomonas oryzihabitans strains in a karstified chalk aquifer.

PubMed

Dussart-Baptista, L; Bodilis, J; Barray, S; Frébourg, N; Fournier, M; Dupont, J-P; Jouenne, T

2007-01-01

Pseudomonas oryzihabitans is an uncommon pathogen that may cause catheter-associated infections, particularly in immunocompromised patients. Although it has been isolated from environment, the source of human infection is not well documented. In the present study, 14 isolates of P. oryzihabitans were recovered over a 28-month period from a karstified chalk aquifer, allowing to advance that distributed natural water could be a source of contamination. Microbiological analyses showed that the bacterium was mainly associated with suspended particulate matters. To investigate the clonality of P. oryzihabitans environmental isolates, 16S rRNA gene sequencing, antibiogram and randomly amplified polymorphic DNA (RAPD) typings were performed. Results demonstrated (i) the presence of at least three clones within the aquifer and (ii) that the presence of the bacterium in groundwater is not only the result of a biofilm bloom but also of an exogenous contamination.

Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates.

PubMed

Sun, Qinghui; Ba, Zhaofen; Wu, Guoying; Wang, Wei; Lin, Shuxiang; Yang, Hongjiang

2016-05-01

Carbapenem resistance mechanisms were investigated in 32 imipenem-resistant Pseudomonas aeruginosa clinical isolates recovered from hospitalised children. Sequence analysis revealed that 31 of the isolates had an insertion sequence element ISRP10 disrupting the porin gene oprD, demonstrating that ISRP10 inactivation of oprD conferred imipenem resistance in the majority of the isolates. Multilocus sequence typing (MLST) was used to discriminate the isolates. In total, 11 sequence types (STs) were identified including 3 novel STs, and 68.3% (28/41) of the tested strains were characterised as clone ST253. In combination with random amplified polymorphic DNA (RAPD) analysis, the imipenem-resistant isolates displayed a relatively high degree of genetic variability and were unlikely associated with nosocomial infections. Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion.

PubMed

Fu, Yusi; Chen, He; Liu, Lu; Huang, Yanyi

2016-11-15

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-seq", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 × 10 5 aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing.

Inverse four-wave-mixing and self-parametric amplification effect in optical fibre

PubMed Central

Turitsyn, Sergei K.; Bednyakova, Anastasia E.; Fedoruk, Mikhail P.; Papernyi, Serguei B.; Clements, Wallace R.L.

2015-01-01

An important group of nonlinear processes in optical fibre involves the mixing of four waves due to the intensity dependence of the refractive index. It is customary to distinguish between nonlinear effects that require external/pumping waves (cross-phase modulation and parametric processes such as four-wave mixing) and self-action of the propagating optical field (self-phase modulation and modulation instability). Here, we present a new nonlinear self-action effect, self-parametric amplification (SPA), which manifests itself as optical spectrum narrowing in normal dispersion fibre, leading to very stable propagation with a distinctive spectral distribution. The narrowing results from an inverse four-wave mixing, resembling an effective parametric amplification of the central part of the spectrum by energy transfer from the spectral tails. SPA and the observed stable nonlinear spectral propagation with random temporal waveform can find applications in optical communications and high power fibre lasers with nonlinear intra-cavity dynamics. PMID:26345290