Transformation of Acinetobacter sp. Strain BD413(pFG4ΔnptII) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants

PubMed Central

Nielsen, Kaare M.; van Elsas, Jan D.; Smalla, Kornelia

2000-01-01

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 109 CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro. PMID:10698801

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance.

PubMed

Ghanem, S

2011-01-01

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.

Effect of promoter driving selectable marker on corn transformation.

PubMed

Prakash, N Shiva; Prasad, V; Chidambram, Thillai P; Cherian, Shoba; Jayaprakash, T L; Dasgupta, Santanu; Wang, Qi; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S

2008-08-01

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.

Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids.

PubMed

Yao, Jianhong; Pang, Yongzhen; Qi, Huaxiong; Wan, Bingliang; Zhao, Xiuyun; Kong, Weiwen; Sun, Xiaofen; Tang, Kexuan

2003-12-01

Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.

[Examination of processed vegetable foods for the presence of common DNA sequences of genetically modified tomatoes].

PubMed

Kitagawa, Mamiko; Nakamura, Kosuke; Kondo, Kazunari; Ubukata, Shoji; Akiyama, Hiroshi

2014-01-01

The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes.

Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

PubMed Central

Cheavegatti-Gianotto, Adriana; Gentile, Agustina; Oldemburgo, Danielle Angeloni; Merheb, Graciela do Amaral; Sereno, Maria Lorena; Lirette, Ron Peter; Ferreira, Thais Helena Silva; de Oliveira, Wladecir Salles

2018-01-01

Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis). The event also expresses neomycin-phosphotransferase type II (NptII) protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as “pure substances, chemically defined,” by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as “GMO derivatives” and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein and DNA are removed and/or degraded during processing. In conclusion, the processing of conventional sugarcane and CTC175-A Bt event results in downstream products with no detectable concentrations of heterologous DNA or new protein. These results help in the classification of sugar and ethanol derived from CTC175-A event as pure, chemically defined substances in Brazil and may relieve regulatory burdens in countries that import Brazilian sugar. PMID:29637070

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

PubMed

Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

2015-06-01

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

REPRESSOR OF SILENCING5 Encodes a Member of the Small Heat Shock Protein Family and Is Required for DNA Demethylation in Arabidopsis[C][W

PubMed Central

Zhao, Yusheng; Xie, Shaojun; Li, Xiaojie; Wang, Chunlei; Chen, Zhongzhou; Lai, Jinsheng; Gong, Zhizhong

2014-01-01

In Arabidopsis thaliana, active DNA demethylation is initiated by the DNA glycosylase REPRESSOR OF SILENCING1 (ROS1) and its paralogs DEMETER, DEMETER-LIKE2 (DML2), and DML3. How these demethylation enzymes are regulated, however, is poorly understood. Here, using a transgenic Arabidopsis line harboring the stress-inducible RESPONSIVE TO DEHYDRATION29A (RD29A) promoter–LUCIFERASE (LUC) reporter gene and the cauliflower mosaic virus 35S promoter (35S)–NEOMYCIN PHOSPHOTRANSFERASE II (NPTII) antibiotic resistance marker gene, we characterize a ROS locus, ROS5, that encodes a protein in the small heat shock protein family. ROS5 mutations lead to the silencing of the 35S-NPTII transgene due to DNA hypermethylation but do not affect the expression of the RD29A-LUC transgene. ROS5 physically interacts with the histone acetyltransferase ROS4/INCREASED DNA METHYLATION1 (IDM1) and is required to prevent the DNA hypermethylation of some genes that are also regulated by ROS1 and IDM1. We propose that ROS5 regulates DNA demethylation by interacting with IDM1, thereby creating a chromatin environment that facilitates the binding of ROS1 to erase DNA methylation. PMID:24920332

Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

PubMed

Ah-Fong, Audrey M V; Judelson, Howard S

2011-09-01

Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

PubMed Central

Li, Xiaojie; Qian, Weiqiang; Zhao, Yusheng; Wang, Chunlei; Shen, Jie; Zhu, Jian-Kang; Gong, Zhizhong

2012-01-01

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression. PMID:22733760

Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton

PubMed Central

Bajwa, Kamran S.; Shahid, Ahmad A.; Rao, Abdul Q.; Bashir, Aftab; Aftab, Asia; Husnain, Tayyab

2015-01-01

Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification. PMID:26583018

Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580--a high triglyceride producer.

PubMed

Muto, Masaki; Fukuda, Yorikane; Nemoto, Michiko; Yoshino, Tomoko; Matsunaga, Tadashi; Tanaka, Tsuyoshi

2013-02-01

A genetic transformation system for the marine pennate diatom, Fistulifera sp. JPCC DA0580, was established using microparticle bombardment methods. Strain JPCC DA0580 has been recently identified as the highest triglyceride (60 % w/w) producer from a culture collection of 1,393 strains of marine microalgae, and it is expected to be a feasible source of biodiesel fuel. The transformation conditions for strain JPCC DA0580 were optimised using the green fluorescent protein gene (gfp) and the gene encoding neomycin phosphotransferase II (nptII). The most efficient rate of transformation was attained when tungsten particles (0.6 μm in diameter) were used for microparticle bombardment. The effect of endogenous and exogenous promoters on the expression of nptII was examined. Endogenous promoters were more efficient for obtaining transformants compared with exogenous promoters. Southern hybridisation analysis suggested that nptII integrated into the nuclear genome. This genetic manipulation technique should allow us to understand the mechanisms of high triglyceride accumulation in this strain, thereby contributing to improving BDF production.

DEVELOPMENT OF GENOMIC AND GENETIC TOOLS FOR FOXTAIL MILLET, AND USE OF THESE TOOLS IN THE IMPROVEMENT OF BIOMASS PRODUCTION FOR BIOENERGY CROPS

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

Chen, Xinlu; Zale, Janice; Chen, Feng

2013-01-22

Foxtail millet (Setaria italica L.) is a warm-season, C4 annual crop commonly grown for grain and forage worldwide. It has a relatively short generation time, yet produces hundreds of seeds per inflorescence. The crop is inbred and it has a small-size genome (~500 Mb). These features make foxtail millet an attractive grass model, especially for bioenergy crops. While a number of genomic tools have been established for foxtail millet, including a fully sequenced genome and molecular markers, the objectives of this project were to develop a tissue culture system, determine the best explant(s) for tissue culture, optimize transient gene expression,more » and establish a stable transformation system for foxtail millet cultivar Yugu1. In optimizing a tissue culture medium for the induction of calli and somatic embryos from immature inflorescences and mature seed explants, Murashige and Skoog medium containing 2.5 mg l-1 2,4-dichlorophenoxyacetic acid and 0.6 mg l-1 6- benzylaminopurine was determined to be optimal for callus induction of foxtail millet. The efficiency of callus induction from explants of immature inflorescences was significantly higher at 76% compared to that of callus induction from mature seed explants at 68%. The calli induced from this medium were regenerated into plants at high frequency (~100%) using 0.2 mg l-1 kinetin in the regeneration media. For performing transient gene expression, immature embryos were first isolated from inflorescences. Transient expression of the GUS reporter gene in immature embryos was significantly increased after sonication, a vacuum treatment, centrifugation and the addition of L-cysteine and dithiothreitol, which led to the efficiency of transient expression at levels greater than 70% after Agrobacterium inoculation. Inoculation with Agrobacterium was also tested with germinated seeds. The radicals of germinated seeds were pierced with needles and dipped into Agrobacterium solution. This method achieved a 10% transient expression efficiency. Throughout these analyses, using plasmids with the hygromycin selectable marker, it was determined that 1.5 mg l-1 hygromycin was the optimal dose for genetic transformation of foxtail millet. In contrast, the nptII selectable marker appeared to yield many escapes. Three methods of transformation were employed in an attempt to produce stable transformants. An in planta transformation experiment, similar to the floral dip method used in Arabidopsis, which utilized a red fluorescent protein pporRFP from coral Porites porites and the hygromycin selectable marker, was tested using immature inflorescences. Although several plants were PCR positive using endpoint and Real-Time PCR and there was transient expression using pporRFP and GUS reporters, no plants were positive on Southern blot. Dipping in Agrobacterium may damage the anther or the pistil because seed production was significantly reduced. Agrobacterium transformation using embryogenic calli was also tested. Although hundreds of plants were regenerated from selection, none were positive using PCR. The third method was to wound germinated seeds with an Agrobacterium coated needle, but none of the plants were PCR positive. Although the Yugu1 genotype was recalcitrant to genetic transformation, several avenues of future research should be considered for foxtail millet. Calli from different foxtail millet genotypes should be screened and selected for regeneration potential, and some genotypes may be more amenable to transformation. Additional selectable markers should also be tested as hygromycin appears to be too stringent and there are too many escapes with nptII. This project has provided training for the following personnel: Dr. Xinlu Chen (postdoc), Xiaomei Liu (postdoc), Jayashree Desai (postdoc) and Kyle Berk (Undergraduate researcher). Conference presentations and peer-reviewed journal articles partly supported by this grant includes the following: 1. Baxter H., Equi R., Chen X, Berk K. and Zale J. Establishing Efficient in vitro Protocols For Foxtail Millet (Setaria italica L. cv. Yugi 1). Plant & Animal Genomes XVIII Conference XVIII, San Diego, California, January 2010 2. Chen X, Zale J and Chen F. The Regeneration and Transformation of Foxtail Millet (Setaria italica), A Model Biofuel Crop. Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, Crystal City, Virginia, April 2011 3. Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011) The family of terpene synthases in plants: A mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 66: 212-229.« less

Survival and DNA Damage in Plant Seeds Exposed for 558 and 682 Days outside the International Space Station

NASA Astrophysics Data System (ADS)

Tepfer, David; Leach, Sydney

2017-03-01

For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV<200nm, and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV254nm, ranging as high as 2420 MJ m-2. Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel.

Faithful transcription initiation from a mitochondrial promoter in transgenic plastids

PubMed Central

Bohne, Alexandra-Viola; Ruf, Stephanie; Börner, Thomas; Bock, Ralph

2007-01-01

The transcriptional machineries of plastids and mitochondria in higher plants exhibit striking similarities. All mitochondrial genes and part of the plastid genes are transcribed by related phage-type RNA polymerases. Furthermore, the majority of mitochondrial promoters and a subset of plastid promoters show a similar structural organization. We show here that the plant mitochondrial atpA promoter is recognized by plastid RNA polymerases in vitro and in vivo. The Arabidopsis phage-type RNA polymerase RpoTp, an enzyme localized exclusively to plastids, was found to recognize the mitochondrial atpA promoter in in vitro assays suggesting the possibility that mitochondrial promoters might function as well in plastids. We have, therefore, generated transplastomic tobacco plants harboring in their chloroplast genome the atpA promoter fused to the coding region of the bacterial nptII gene. The chimeric nptII gene was found to be efficiently transcribed in chloroplasts. Mapping of the 5′ ends of the nptII transcripts revealed accurate recognition of the atpA promoter by the chloroplast transcription machinery. We show further that the 5′ untranslated region (UTR) of the mitochondrial atpA transcript is capable of mediating translation in chloroplasts. The functional and evolutionary implications of these findings as well as possible applications in chloroplast genome engineering are discussed. PMID:17959651

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.

PubMed

Michelmore, R; Marsh, E; Seely, S; Landry, B

1987-12-01

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

PubMed

Tyč, Dimitrij; Nocarová, Eva; Sikorová, Lenka; Fischer, Lukáš

2017-08-01

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing; Sink, K C

2004-12-01

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.

PubMed

Ntui, Valentine Otang; Thirukkumaran, Gunaratnam; Azadi, Pejman; Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2010-09-01

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.

Walnut (Juglans).

PubMed

Leslie, Charles A; Walawage, Sriema L; Uratsu, Sandra L; McGranahan, Gale; Dandekar, Abhaya M

2015-01-01

Walnut species are important nut and timber producers in temperate regions of Europe, Asia, South America, and North America. Trees can be impacted by Phytophthora, crown gall, nematodes, Armillaria, and cherry leaf roll virus; nuts can be severely damaged by codling moth, husk fly, and Xanthomonas blight. The long generation time of walnuts and an absence of identified natural resistance for most of these problems suggest biotechnological approaches to crop improvement. Described here is a somatic embryo-based transformation protocol that has been used to successfully insert horticulturally useful traits into walnut. Selection is based on the combined use of the selectable neomycin phosphotransferase (nptII) gene and the scorable uidA gene. Transformed embryos can be germinated or micropropagated and rooted for plant production. The method described has been used to establish field trials of mature trees.

Heterologous expression of VHb can improve the yield and quality of biocontrol fungus Paecilomyces lilacinus, during submerged fermentation.

PubMed

Zhang, Shumeng; Wang, Jieping; Wei, Yale; Tang, Qing; Ali, Maria Kanwal; He, Jin

2014-10-10

Paecilomyces lilacinus is an egg-parasitic fungus which is effective against plant-parasitic nematodes and it has been successfully commercialized for the control of many plant-parasitic nematodes. However, during the large-scale industrial fermentation process of the filamentous fungus, the dissolved oxygen supply is a limiting factor, which influences yield, product quality and production cost. To solve this problem, we intended to heterologously express VHb in P. lilacinus ACSS. After optimizing the vgb gene, we fused it with a selection marker gene nptII, a promoter PgpdA and a terminator TtrpC. The complete expression cassette PgpdA-nptII-vgb-TtrpC was transferred into P. lilacinus ACSS by Agrobacterium tumefaciens-mediated transformation. Consequently, we successfully screened an applicable fungus strain PNVT8 which efficiently expressed VHb. The submerged fermentation experiments demonstrated that the expression of VHb not only increased the production traits of P. lilacinus such as biomass and spore production, but also improved the beneficial product quality and application value, due to the secretion of more protease and chitinase. It can be speculated that the recombinant strain harboring vgb gene will have a growth advantage over the original strain under anaerobic conditions in soil and therefore will possess higher biocontrol efficiency against plant-parasitic nematodes. Copyright © 2014 Elsevier B.V. All rights reserved.

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.

PubMed

Cho, H J; Farrand, S K; Noel, G R; Widholm, J M

2000-01-01

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

Survival and DNA Damage in Plant Seeds Exposed for 558 and 682 Days outside the International Space Station.

PubMed

Tepfer, David; Leach, Sydney

2017-03-01

For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV <200nm , and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV 254nm , ranging as high as 2420 MJ m -2 . Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel. Key Words: UV light-Flavonoids-Sinapate-DNA degradation-Arabidopsis-Tobacco-Seeds-Space-International Space Station-EXPOSE-E-EXPOSE-R. Astrobiology 17, 205-215.

Survival and DNA Damage in Plant Seeds Exposed for 558 and 682 Days outside the International Space Station

PubMed Central

Leach, Sydney

2017-01-01

Abstract For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV<200nm, and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV254nm, ranging as high as 2420 MJ m−2. Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel. Key Words: UV light—Flavonoids—Sinapate—DNA degradation—Arabidopsis—Tobacco—Seeds—Space—International Space Station—EXPOSE-E—EXPOSE-R. Astrobiology 17, 205–215. PMID:28263676

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts.

PubMed

Richter, Lubna V; Yang, Huijun; Yazdani, Mohammad; Hanson, Maureen R; Ahner, Beth A

2018-01-01

We investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N -terminally fused to the coding region of cel6A , an endoglucanase from Thermobifida fusca . We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5'UTR, previously reported to enhance protein expression, to regulate the expression of the TetC- cel6A gene. We further investigated the accumulation of TetC-Cel6A under N -deplete growth conditions. Both of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII- cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N -deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N -replete medium. The DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

Treesearch

Ningxia Du; Paula M. Pijut

2009-01-01

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...

Folate Polyglutamylation Is Involved in Chromatin Silencing by Maintaining Global DNA Methylation and Histone H3K9 Dimethylation in Arabidopsis[C][W

PubMed Central

Zhou, Hao-Ran; Zhang, Fang-Fang; Ma, Ze-Yang; Huang, Huan-Wei; Jiang, Ling; Cai, Tao; Zhu, Jian-Kang; Zhang, Chuyi; He, Xin-Jian

2013-01-01

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation. PMID:23881414

Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

PubMed

Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N

2007-01-01

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

PubMed

Dong, Niu; Montanez, Belen; Creelman, Robert A; Cornish, Katrina

2006-02-01

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

PubMed

Rawat, Preeti; Singh, Amarjeet Kumar; Ray, Krishna; Chaudhary, Bhupendra; Kumar, Sanjeev; Gautam, Taru; Kanoria, Shaveta; Kaur, Gurpreet; Kumar, Paritosh; Pental, Deepak; Burma, Pradeep Kumar

2011-06-01

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

PubMed Central

2011-01-01

Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event. PMID:21995716

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

PubMed

Singh, Chandra K; Ojha, Abhishek; Bhatanagar, Raj K; Kachru, Devendra N

2008-01-01

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

Establishment of Withania somnifera hairy root cultures for the production of withanolide A.

PubMed

Murthy, Hosakatte N; Dijkstra, Camelia; Anthony, Paul; White, Daniel A; Davey, Mike R; Power, J Brian; Hahn, Eun J; Paek, Kee Y

2008-08-01

Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 microg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 microg/g dry weight).

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.

PubMed

Sriskandarajah, Sridevy; Frello, Stefan; Jørgensen, Kirsten; Serek, Margrethe

2004-08-01

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.

Development and application of a general plasmid reference material for GMO screening.

PubMed

Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).

PubMed

Cui, Cuiju; Song, Fei; Tan, Yi; Zhou, Xuan; Zhao, Wen; Ma, Fengyun; Liu, Yunyi; Hussain, Javeed; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2011-04-01

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.

Coupling two mercury resistance genes in Eastern cottonwood enhances the processing of organomercury.

PubMed

Lyyra, Satu; Meagher, Richard B; Kim, Tehryung; Heaton, Andrew; Montello, Paul; Balish, Rebecca S; Merkle, Scott A

2007-03-01

Eastern cottonwood (Populus deltoides Bartr. ex Marsh.) trees were engineered to express merA (mercuric ion reductase) and merB (organomercury lyase) transgenes in order to be used for the phytoremediation of mercury-contaminated soils. Earlier studies with Arabidopsis thaliana and Nicotiana tabacum showed that this gene combination resulted in more efficient detoxification of organomercurial compounds than did merB alone, but neither species is optimal for long-term field applications. Leaf discs from in vitro-grown merA, nptII (neomycin phosphotransferase) transgenic cottonwood plantlets were inoculated with Agrobacterium tumefaciens strain C58 carrying the merB and hygromycin resistance (hptII) genes. Polymerase chain reaction of shoots regenerated from the leaf discs under selection indicated an overall transformation frequency of 20%. Western blotting of leaves showed that MerA and MerB proteins were produced. In vitro-grown merA/merB plants were highly resistant to phenylmercuric acetate, and detoxified organic mercury compounds two to three times more rapidly than did controls, as shown by mercury volatilization assay. This indicates that these cottonwood trees are reasonable candidates for the remediation of organomercury-contaminated sites.

Survival of plant seeds, their UV screens, and nptII DNA for 18 months outside the International Space Station.

PubMed

Tepfer, David; Zalar, Andreja; Leach, Sydney

2012-05-01

The plausibility that life was imported to Earth from elsewhere can be tested by subjecting life-forms to space travel. Ultraviolet light is the major liability in short-term exposures (Horneck et al., 2001 ), and plant seeds, tardigrades, and lichens-but not microorganisms and their spores-are candidates for long-term survival (Anikeeva et al., 1990 ; Sancho et al., 2007 ; Jönsson et al., 2008 ; de la Torre et al., 2010 ). In the present study, plant seeds germinated after 1.5 years of exposure to solar UV, solar and galactic cosmic radiation, temperature fluctuations, and space vacuum outside the International Space Station. Of the 2100 exposed wild-type Arabidopsis thaliana and Nicotiana tabacum (tobacco) seeds, 23% produced viable plants after return to Earth. Survival was lower in the Arabidopsis Wassilewskija ecotype and in mutants (tt4-8 and fah1-2) lacking UV screens. The highest survival occurred in tobacco (44%). Germination was delayed in seeds shielded from solar light, yet full survival was attained, which indicates that longer space travel would be possible for seeds embedded in an opaque matrix. We conclude that a naked, seed-like entity could have survived exposure to solar UV radiation during a hypothetical transfer from Mars to Earth. Chemical samples of seed flavonoid UV screens were degraded by UV, but their overall capacity to absorb UV was retained. Naked DNA encoding the nptII gene (kanamycin resistance) was also degraded by UV. A fragment, however, was detected by the polymerase chain reaction, and the gene survived in space when protected from UV. Even if seeds do not survive, components (e.g., their DNA) might survive transfer over cosmic distances.

Spiked GBS: A unified, open platform for single marker genotyping and whole-genome profiling

USDA-ARS?s Scientific Manuscript database

In plant breeding, there are two primary applications for DNA markers in selection: 1) selection of known genes using a single marker assay (marker-assisted selection; MAS); and 2) whole-genome profiling and prediction (genomic selection; GS). Typically, marker platforms have addressed only one of t...

Genomic selection in plant breeding.

PubMed

Newell, Mark A; Jannink, Jean-Luc

2014-01-01

Genomic selection (GS) is a method to predict the genetic value of selection candidates based on the genomic estimated breeding value (GEBV) predicted from high-density markers positioned throughout the genome. Unlike marker-assisted selection, the GEBV is based on all markers including both minor and major marker effects. Thus, the GEBV may capture more of the genetic variation for the particular trait under selection.

Comparing genomic selection and marker-assisted selection for Fusarium head blight resistance in wheat (Triticum aestivum L.)

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) and marker-assisted selection (MAS) rely on marker-trait associations and are both routinely used for breeding purposes. Although similar, these two approaches differ in their applications and how markers are used to estimate breeding values. In this study, GS and MAS were com...

Optimal selection of markers for validation or replication from genome-wide association studies.

PubMed

Greenwood, Celia M T; Rangrej, Jagadish; Sun, Lei

2007-07-01

With reductions in genotyping costs and the fast pace of improvements in genotyping technology, it is not uncommon for the individuals in a single study to undergo genotyping using several different platforms, where each platform may contain different numbers of markers selected via different criteria. For example, a set of cases and controls may be genotyped at markers in a small set of carefully selected candidate genes, and shortly thereafter, the same cases and controls may be used for a genome-wide single nucleotide polymorphism (SNP) association study. After such initial investigations, often, a subset of "interesting" markers is selected for validation or replication. Specifically, by validation, we refer to the investigation of associations between the selected subset of markers and the disease in independent data. However, it is not obvious how to choose the best set of markers for this validation. There may be a prior expectation that some sets of genotyping data are more likely to contain real associations. For example, it may be more likely for markers in plausible candidate genes to show disease associations than markers in a genome-wide scan. Hence, it would be desirable to select proportionally more markers from the candidate gene set. When a fixed number of markers are selected for validation, we propose an approach for identifying an optimal marker-selection configuration by basing the approach on minimizing the stratified false discovery rate. We illustrate this approach using a case-control study of colorectal cancer from Ontario, Canada, and we show that this approach leads to substantial reductions in the estimated false discovery rates in the Ontario dataset for the selected markers, as well as reductions in the expected false discovery rates for the proposed validation dataset. Copyright 2007 Wiley-Liss, Inc.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

PubMed Central

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

Comparing the predictive abilities of phenotypic and marker-assisted selection methods in a biparental lettuce population

USDA-ARS?s Scientific Manuscript database

Breeding and selection for the traits with polygenic inheritance is a challenging task that can be done by phenotypic selection, by marker-assisted selection or by genome wide selection. We tested predictive ability of four selection models in a biparental population genotyped with 95 SNP markers an...

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

PubMed

Guarna, M Marta; Hoover, Shelley E; Huxter, Elizabeth; Higo, Heather; Moon, Kyung-Mee; Domanski, Dominik; Bixby, Miriam E F; Melathopoulos, Andony P; Ibrahim, Abdullah; Peirson, Michael; Desai, Suresh; Micholson, Derek; White, Rick; Borchers, Christoph H; Currie, Robert W; Pernal, Stephen F; Foster, Leonard J

2017-08-21

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

Genomic selection in plant breeding

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) is a method to predict the genetic value of selection candidates based on the genomic estimated breeding value (GEBV) predicted from high-density markers positioned throughout the genome. Unlike marker-assisted selection, the GEBV is based on all markers including both minor ...

A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle

PubMed Central

Tong, Qi; Liu, Xu; Su, Feng; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2013-01-01

Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is no longer necessary but raises public concerns regarding biological safety. The aim of this study was to prepare competent antibiotic selectable marker free transgenic cells for somatic cell nuclear transfer (SCNT). PhiC31 intergrase was used to insert a transgene cassette into a “safe harbor” in the bovine genome. Then, Cre recombinase was employed to excise the selectable marker under the monitoring of a fluorescent double reporter. By visually tracking the phenotypic switch from red to green fluorescence, antibiotic selectable marker free cells were easily detected and sorted by fluorescence-activated cell sorting. For safety, we used phiC31 mRNA and cell-permeant Cre protein in this study. When used as donor nuclei for SCNT, these safe harbor integrated marker-free transgenic cells supported a similar developmental competence of SCNT embryos compared with that of non-transgenic cells. After embryo transfer, antibiotic selectable marker free transgenic cattle were generated and anti-bacterial recombinant human β-defensin-3 in milk was detected during their lactation period. Thus, this approach offers a rapid and safe alternative to produce antibiotic selectable marker free transgenic farm animals, thereby making it a valuable tool to promote the healthy development and welfare of transgenic farm animals. PMID:23658729

Comparison of two alternative dominant selectable markers for wine yeast transformation.

PubMed

Cebollero, Eduardo; Gonzalez, Ramon

2004-12-01

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.

Bridging the gap between marker-assisted and genomic selection of heading time and plant height in hybrid wheat.

PubMed

Zhao, Y; Mette, M F; Gowda, M; Longin, C F H; Reif, J C

2014-06-01

Based on data from field trials with a large collection of 135 elite winter wheat inbred lines and 1604 F1 hybrids derived from them, we compared the accuracy of prediction of marker-assisted selection and current genomic selection approaches for the model traits heading time and plant height in a cross-validation approach. For heading time, the high accuracy seen with marker-assisted selection severely dropped with genomic selection approaches RR-BLUP (ridge regression best linear unbiased prediction) and BayesCπ, whereas for plant height, accuracy was low with marker-assisted selection as well as RR-BLUP and BayesCπ. Differences in the linkage disequilibrium structure of the functional and single-nucleotide polymorphism markers relevant for the two traits were identified in a simulation study as a likely explanation for the different trends in accuracies of prediction. A new genomic selection approach, weighted best linear unbiased prediction (W-BLUP), designed to treat the effects of known functional markers more appropriately, proved to increase the accuracy of prediction for both traits and thus closes the gap between marker-assisted and genomic selection.

Bridging the gap between marker-assisted and genomic selection of heading time and plant height in hybrid wheat

PubMed Central

Zhao, Y; Mette, M F; Gowda, M; Longin, C F H; Reif, J C

2014-01-01

Based on data from field trials with a large collection of 135 elite winter wheat inbred lines and 1604 F1 hybrids derived from them, we compared the accuracy of prediction of marker-assisted selection and current genomic selection approaches for the model traits heading time and plant height in a cross-validation approach. For heading time, the high accuracy seen with marker-assisted selection severely dropped with genomic selection approaches RR-BLUP (ridge regression best linear unbiased prediction) and BayesCπ, whereas for plant height, accuracy was low with marker-assisted selection as well as RR-BLUP and BayesCπ. Differences in the linkage disequilibrium structure of the functional and single-nucleotide polymorphism markers relevant for the two traits were identified in a simulation study as a likely explanation for the different trends in accuracies of prediction. A new genomic selection approach, weighted best linear unbiased prediction (W-BLUP), designed to treat the effects of known functional markers more appropriately, proved to increase the accuracy of prediction for both traits and thus closes the gap between marker-assisted and genomic selection. PMID:24518889

Modeling of genetic gain for single traits from marker-assisted seedling selection in clonally propagated crops

PubMed Central

Ru, Sushan; Hardner, Craig; Carter, Patrick A; Evans, Kate; Main, Dorrie; Peace, Cameron

2016-01-01

Seedling selection identifies superior seedlings as candidate cultivars based on predicted genetic potential for traits of interest. Traditionally, genetic potential is determined by phenotypic evaluation. With the availability of DNA tests for some agronomically important traits, breeders have the opportunity to include DNA information in their seedling selection operations—known as marker-assisted seedling selection. A major challenge in deploying marker-assisted seedling selection in clonally propagated crops is a lack of knowledge in genetic gain achievable from alternative strategies. Existing models based on additive effects considering seed-propagated crops are not directly relevant for seedling selection of clonally propagated crops, as clonal propagation captures all genetic effects, not just additive. This study modeled genetic gain from traditional and various marker-based seedling selection strategies on a single trait basis through analytical derivation and stochastic simulation, based on a generalized seedling selection scheme of clonally propagated crops. Various trait-test scenarios with a range of broad-sense heritability and proportion of genotypic variance explained by DNA markers were simulated for two populations with different segregation patterns. Both derived and simulated results indicated that marker-based strategies tended to achieve higher genetic gain than phenotypic seedling selection for a trait where the proportion of genotypic variance explained by marker information was greater than the broad-sense heritability. Results from this study provides guidance in optimizing genetic gain from seedling selection for single traits where DNA tests providing marker information are available. PMID:27148453

Development of DArT-based PCR markers for selecting drought-tolerant spring barley.

PubMed

Fiust, Anna; Rapacz, Marcin; Wójcik-Jagła, Magdalena; Tyrka, Mirosław

2015-08-01

The tolerance of spring barley (Hordeum vulgare L.) cultivars to spring drought is an important agronomic trait affecting crop yield and quality in Poland. Therefore, breeders require new molecular markers to select plants with lower spring drought susceptibility. With the advent of genomic selection technology, simple molecular tools may still be applicable to screen material for markers of the most important traits and in-depth genome scanning. In previous studies, diversity arrays technology (DArT)-based genetic maps were constructed for F2 populations of Polish fodder and malt barley elite breeding lines, and 15 and 18 quantitative trait loci (QTLs) related to spring drought tolerance were identified, respectively. In this paper, we show the results of a conversion of 30 DArT markers corresponding to 11 QTLs into simple sequence repeat (SSR) and sequence tagged site (STS) markers. Twenty-two polymorphic markers were obtained, including 13 DArT-based SSRs. Additionally, 31 SSR markers, located in close proximity to the DArT markers, were selected from the GrainGenes database and tested. Further analyses of 24 advanced breeding lines with different drought tolerances confirmed that five out of the 30 converted markers, as well as three out of the 31 additional SSR markers, were effective in marker-assisted selection for drought tolerance. The possible function of clones related to these markers in drought tolerance is discussed.

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: A case study for marker assisted selection in grapevine

USDA-ARS?s Scientific Manuscript database

Marker assisted selection (MAS) has become widely used in perennial crop breeding programs to accelerate and enhance cultivar development via selection during the juvenile phase and parental selection prior to crossing. Next generation sequencing (NGS) has been widely used for whole genome molecular...

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker assisted selection in grapevine

USDA-ARS?s Scientific Manuscript database

Marker assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next generation sequencing (NGS) and its derivative technologies have been used for genome-wid...

Paternity and Nested-within-Family Marker Assisted Selection in Space Planted Red Clover Nurseries

USDA-ARS?s Scientific Manuscript database

Presented is a cost effective marker assisted selection methodology that utilizes individual plant phenotypes, seed production based knowledge of maternity, molecular marker determined paternity, and nested within halfsib family linkage relationships. Combining all above listed components, selection...

Genome-wide association analysis of bacterial cold water disease resistance in rainbow trout reveals the potential of a hybrid approach between genomic selection and marker assisted selection

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) simultaneously incorporates dense SNP marker genotypes with phenotypic data from related animals to predict animal-specific genomic breeding value (GEBV), which circumvents the need to measure the disease phenotype in potential breeders. Marker assisted selection (MAS) involv...

A Novel Yeast Genomics Method for Identifying New Breast Cancer Susceptibility

DTIC Science & Technology

2005-05-01

selectable marker and tracing this marker through several passages in nonselective medium. The selectable marker will be the hygromycin phosphotransferase ... hygromycin and sensitivity to (32), thereby providing both positive and negative selectivity. The assay involved measurement of the frequency of gancyclovir

Improving selection of markers in nutrition research: evaluation of the criteria proposed by the ILSI Europe Marker Validation Initiative.

PubMed

Calder, Philip C; Boobis, Alan; Braun, Deborah; Champ, Claire L; Dye, Louise; Einöther, Suzanne; Greyling, Arno; Matthys, Christophe; Putz, Peter; Wopereis, Suzan; Woodside, Jayne V; Antoine, Jean-Michel

2017-06-01

The conduct of high-quality nutrition research requires the selection of appropriate markers as outcomes, for example as indicators of food or nutrient intake, nutritional status, health status or disease risk. Such selection requires detailed knowledge of the markers, and consideration of the factors that may influence their measurement, other than the effects of nutritional change. A framework to guide selection of markers within nutrition research studies would be a valuable tool for researchers. A multidisciplinary Expert Group set out to test criteria designed to aid the evaluation of candidate markers for their usefulness in nutrition research and subsequently to develop a scoring system for markers. The proposed criteria were tested using thirteen markers selected from a broad range of nutrition research fields. The result of this testing was a modified list of criteria and a template for evaluating a potential marker against the criteria. Subsequently, a semi-quantitative system for scoring a marker and an associated template were developed. This system will enable the evaluation and comparison of different candidate markers within the same field of nutrition research in order to identify their relative usefulness. The ranking criteria of proven, strong, medium or low are likely to vary according to research setting, research field and the type of tool used to assess the marker and therefore the considerations for scoring need to be determined in a setting-, field- and tool-specific manner. A database of such markers, their interpretation and range of possible values would be valuable to nutrition researchers.

Mapping QTL for popping expansion volume in popcorn with simple sequence repeat markers.

PubMed

Lu, H-J; Bernardo, R; Ohm, H W

2003-02-01

Popping expansion volume is the most important quality trait in popcorn ( Zea mays L.), but its genetics is not well understood. The objectives of this study were to map quantitative trait loci (QTLs) responsible for popping expansion volume in a popcorn x dent corn cross, and to compare the predicted efficiencies of phenotypic selection, marker-based selection, and marker-assisted selection for popping expansion volume. Of 259 simple sequence repeat (SSR) primer pairs screened, 83 pairs were polymorphic between the H123 (dent corn) and AG19 (popcorn) parental inbreds. Popping test data were obtained for 160 S(1) families developed from the [AG19(H123 x AG19)] BC(1) population. The heritability ( h(2)) for popping expansion volume on an S(1) family mean basis was 0.73. The presence of the gametophyte factor Ga1(s) in popcorn complicates the analysis of popcorn x dent corn crosses. But, from a practical perspective, the linkage between a favorable QTL allele and Ga1(s) in popcorn will lead to selection for the favorable QTL allele. Four QTLs, on chromosomes 1S, 3S, 5S and 5L, jointly explained 45% of the phenotypic variation. Marker-based selection for popping expansion volume would require less time and work than phenotypic selection. But due to the high h(2) of popping expansion volume, marker-based selection was predicted to be only 92% as efficient as phenotypic selection. Marker-assisted selection, which comprises index selection on phenotypic and marker scores, was predicted to be 106% as efficient as phenotypic selection. Overall, our results suggest that phenotypic selection will remain the preferred method for selection in popcorn x dent corn crosses.

Recent patents on biosafety strategies of selectable marker genes in genetically modified crops.

PubMed

Jiang, Yiming; Hu, Xiaoning; Huang, Haiying

2014-01-01

Genetically modified crops (GMCs) have been planted world wide since 1990s, but the potential insecurity of selectable marker genes raises the questions about GMC safety. Therefore, several researches have been conducted on marker gene safety issues and recently several patents have been issued on this subject. There are two main approaches to achieve this goal: seeking the biosafety selectable marker and eliminating these insecure marker genes after transformation. Results show that these two systems are quite effective. Recent patents on the two ways are discussed in this review.

Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.

PubMed

Yang, Feng; Njire, Moses M; Liu, Jia; Wu, Tian; Wang, Bangxing; Liu, Tianzhou; Cao, Yuanyuan; Liu, Zhiyong; Wan, Junting; Tu, Zhengchao; Tan, Yaoju; Tan, Shouyong; Zhang, Tianyu

2015-01-01

In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.

Potential and limits to unravel the genetic architecture and predict the variation of Fusarium head blight resistance in European winter wheat (Triticum aestivum L.).

PubMed

Jiang, Y; Zhao, Y; Rodemann, B; Plieske, J; Kollers, S; Korzun, V; Ebmeyer, E; Argillier, O; Hinze, M; Ling, J; Röder, M S; Ganal, M W; Mette, M F; Reif, J C

2015-03-01

Genome-wide mapping approaches in diverse populations are powerful tools to unravel the genetic architecture of complex traits. The main goals of our study were to investigate the potential and limits to unravel the genetic architecture and to identify the factors determining the accuracy of prediction of the genotypic variation of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum L.) based on data collected with a diverse panel of 372 European varieties. The wheat lines were phenotyped in multi-location field trials for FHB resistance and genotyped with 782 simple sequence repeat (SSR) markers, and 9k and 90k single-nucleotide polymorphism (SNP) arrays. We applied genome-wide association mapping in combination with fivefold cross-validations and observed surprisingly high accuracies of prediction for marker-assisted selection based on the detected quantitative trait loci (QTLs). Using a random sample of markers not selected for marker-trait associations revealed only a slight decrease in prediction accuracy compared with marker-based selection exploiting the QTL information. The same picture was confirmed in a simulation study, suggesting that relatedness is a main driver of the accuracy of prediction in marker-assisted selection of FHB resistance. When the accuracy of prediction of three genomic selection models was contrasted for the three marker data sets, no significant differences in accuracies among marker platforms and genomic selection models were observed. Marker density impacted the accuracy of prediction only marginally. Consequently, genomic selection of FHB resistance can be implemented most cost-efficiently based on low- to medium-density SNP arrays.

Optimum location of external markers using feature selection algorithms for real‐time tumor tracking in external‐beam radiotherapy: a virtual phantom study

PubMed Central

Nankali, Saber; Miandoab, Payam Samadi; Baghizadeh, Amin

2016-01-01

In external‐beam radiotherapy, using external markers is one of the most reliable tools to predict tumor position, in clinical applications. The main challenge in this approach is tumor motion tracking with highest accuracy that depends heavily on external markers location, and this issue is the objective of this study. Four commercially available feature selection algorithms entitled 1) Correlation‐based Feature Selection, 2) Classifier, 3) Principal Components, and 4) Relief were proposed to find optimum location of external markers in combination with two “Genetic” and “Ranker” searching procedures. The performance of these algorithms has been evaluated using four‐dimensional extended cardiac‐torso anthropomorphic phantom. Six tumors in lung, three tumors in liver, and 49 points on the thorax surface were taken into account to simulate internal and external motions, respectively. The root mean square error of an adaptive neuro‐fuzzy inference system (ANFIS) as prediction model was considered as metric for quantitatively evaluating the performance of proposed feature selection algorithms. To do this, the thorax surface region was divided into nine smaller segments and predefined tumors motion was predicted by ANFIS using external motion data of given markers at each small segment, separately. Our comparative results showed that all feature selection algorithms can reasonably select specific external markers from those segments where the root mean square error of the ANFIS model is minimum. Moreover, the performance accuracy of proposed feature selection algorithms was compared, separately. For this, each tumor motion was predicted using motion data of those external markers selected by each feature selection algorithm. Duncan statistical test, followed by F‐test, on final results reflected that all proposed feature selection algorithms have the same performance accuracy for lung tumors. But for liver tumors, a correlation‐based feature selection algorithm, in combination with a genetic search algorithm, proved to yield best performance accuracy for selecting optimum markers. PACS numbers: 87.55.km, 87.56.Fc PMID:26894358

Optimum location of external markers using feature selection algorithms for real-time tumor tracking in external-beam radiotherapy: a virtual phantom study.

PubMed

Nankali, Saber; Torshabi, Ahmad Esmaili; Miandoab, Payam Samadi; Baghizadeh, Amin

2016-01-08

In external-beam radiotherapy, using external markers is one of the most reliable tools to predict tumor position, in clinical applications. The main challenge in this approach is tumor motion tracking with highest accuracy that depends heavily on external markers location, and this issue is the objective of this study. Four commercially available feature selection algorithms entitled 1) Correlation-based Feature Selection, 2) Classifier, 3) Principal Components, and 4) Relief were proposed to find optimum location of external markers in combination with two "Genetic" and "Ranker" searching procedures. The performance of these algorithms has been evaluated using four-dimensional extended cardiac-torso anthropomorphic phantom. Six tumors in lung, three tumors in liver, and 49 points on the thorax surface were taken into account to simulate internal and external motions, respectively. The root mean square error of an adaptive neuro-fuzzy inference system (ANFIS) as prediction model was considered as metric for quantitatively evaluating the performance of proposed feature selection algorithms. To do this, the thorax surface region was divided into nine smaller segments and predefined tumors motion was predicted by ANFIS using external motion data of given markers at each small segment, separately. Our comparative results showed that all feature selection algorithms can reasonably select specific external markers from those segments where the root mean square error of the ANFIS model is minimum. Moreover, the performance accuracy of proposed feature selection algorithms was compared, separately. For this, each tumor motion was predicted using motion data of those external markers selected by each feature selection algorithm. Duncan statistical test, followed by F-test, on final results reflected that all proposed feature selection algorithms have the same performance accuracy for lung tumors. But for liver tumors, a correlation-based feature selection algorithm, in combination with a genetic search algorithm, proved to yield best performance accuracy for selecting optimum markers.

Imputation of unordered markers and the impact on genomic selection accuracy

USDA-ARS?s Scientific Manuscript database

Genomic selection, a breeding method that promises to accelerate rates of genetic gain, requires dense, genome-wide marker data. Genotyping-by-sequencing can generate a large number of de novo markers. However, without a reference genome, these markers are unordered and typically have a large propo...

Demonstrating Cost-Effective Marker Assisted Selection for Biomass Yield in Red Clover (Trifolium pratense L.) – Part 1: Paternity Testing

USDA-ARS?s Scientific Manuscript database

Many methods have been proposed to incorporate molecular markers into breeding programs. Presented is a cost effective marker assisted selection (MAS) methodology that utilizes individual plant phenotypes, seed production-based knowledge of maternity, and molecular marker-determined paternity. Proge...

A Comparison Between Genotyping-by-sequencing and Array-based Scoring of SNPs for Genomic Prediction Accuracy in Winter Wheat

USDA-ARS?s Scientific Manuscript database

The utilization of DNA molecular markers in plant breeding to maximize selection response via marker assisted selection (MAS) and genomic selection (GS) has the potential to revolutionize plant breeding. A key factor affecting GS applicability is the choice of molecular marker platform. Genotypying-...

[A comparison study of hpt and bar as selection marker gene of transgenic rice].

PubMed

Zhang, Chun-Yu; Li, Hong-Yu; Liu, Bin

2012-12-01

The decision of using selection marker is one of the key factors for success of plant genetic transformation and offspring screening. As two commonly used selection markers, hpt and bar genes are widely used in tissue culture-based rice transformation. To experimentally compare their performance, we investigated the efficiency of two transformation systems using Hygromycin and Bialaphos as the selection agents, respectively. The result indicated that the system using hpt gene as the selection marker saved 10 days and had double transformation efficiency and lower transgene copy number in comparison to the system using bar gene. Then, we assessed the feasibility of screening transgenic rice in the field by soaking the wild-type and transgenic seeds in a series of solutions containing step diluted hygromycin for two days. We targeted the suitable concentration for distinguishing the transgenic seeds from WT Kitaake seeds was 167 mg L(-1). However, the cost of screening by hygromycin is still much higher than that of Basta in field test. Therefore, this study experimentally demonstrated the advantages and disadvantages of the hpt and bar gene as the selection markers and thus provided a reference for choose of an appropriate selection marker according to the practical applications.

Continental-scale footprint of balancing and positive selection in a small rodent (Microtus arvalis).

PubMed

Fischer, Martin C; Foll, Matthieu; Heckel, Gerald; Excoffier, Laurent

2014-01-01

Genetic adaptation to different environmental conditions is expected to lead to large differences between populations at selected loci, thus providing a signature of positive selection. Whereas balancing selection can maintain polymorphisms over long evolutionary periods and even geographic scale, thus leads to low levels of divergence between populations at selected loci. However, little is known about the relative importance of these two selective forces in shaping genomic diversity, partly due to difficulties in recognizing balancing selection in species showing low levels of differentiation. Here we address this problem by studying genomic diversity in the European common vole (Microtus arvalis) presenting high levels of differentiation between populations (average F ST = 0.31). We studied 3,839 Amplified Fragment Length Polymorphism (AFLP) markers genotyped in 444 individuals from 21 populations distributed across the European continent and hence over different environmental conditions. Our statistical approach to detect markers under selection is based on a Bayesian method specifically developed for AFLP markers, which treats AFLPs as a nearly codominant marker system, and therefore has increased power to detect selection. The high number of screened populations allowed us to detect the signature of balancing selection across a large geographic area. We detected 33 markers potentially under balancing selection, hence strong evidence of stabilizing selection in 21 populations across Europe. However, our analyses identified four-times more markers (138) being under positive selection, and geographical patterns suggest that some of these markers are probably associated with alpine regions, which seem to have environmental conditions that favour adaptation. We conclude that despite favourable conditions in this study for the detection of balancing selection, this evolutionary force seems to play a relatively minor role in shaping the genomic diversity of the common vole, which is more influenced by positive selection and neutral processes like drift and demographic history.

Optimal marker-assisted selection to increase the effective size of small populations.

PubMed

Wang, J

2001-02-01

An approach to the optimal utilization of marker and pedigree information in minimizing the rates of inbreeding and genetic drift at the average locus of the genome (not just the marked loci) in a small diploid population is proposed, and its efficiency is investigated by stochastic simulations. The approach is based on estimating the expected pedigree of each chromosome by using marker and individual pedigree information and minimizing the average coancestry of selected chromosomes by quadratic integer programming. It is shown that the approach is much more effective and much less computer demanding in implementation than previous ones. For pigs with 10 offspring per mother genotyped for two markers (each with four alleles at equal initial frequency) per chromosome of 100 cM, the approach can increase the average effective size for the whole genome by approximately 40 and 55% if mating ratios (the number of females mated with a male) are 3 and 12, respectively, compared with the corresponding values obtained by optimizing between-family selection using pedigree information only. The efficiency of the marker-assisted selection method increases with increasing amount of marker information (number of markers per chromosome, heterozygosity per marker) and family size, but decreases with increasing genome size. For less prolific species, the approach is still effective if the mating ratio is large so that a high marker-assisted selection pressure on the rarer sex can be maintained.

Estimates of epistatic and pleiotropic effects of casein alpha s1 (CSN1S1) and thyroglobulin (TG) genetic markers on beef heifer performance traits enhanced by selection

USDA-ARS?s Scientific Manuscript database

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC II) was subjected to marker assisted selection for two years to equalize CSN1S1 and TG genetic marker frequencies to evaluate the epista...

Using SCC8, SCF27 and VMC7f2 markers in grapevine breeding for seedlessness via marker assisted selection.

PubMed

Akkurt, M; Çakır, A; Shidfar, M; Çelikkol, B P; Söylemezoğlu, G

2012-08-13

We used molecular markers associated with seedlessness in grapes, namely SCC8, SCF27 and VMC7f2, to improve the efficiency of seedless grapevine breeding via marker assisted selection (MAS). DNA from 372 F₁ hybrid progeny from the cross between seeded "Alphonse Lavallée" and seedless "Sultani" was amplified by PCR using three markers. After digestion of SCC8 marker amplification products by restriction enzyme BgIII, 40 individuals showed homozygous SCC8+/SCC8+ alleles at the seed development inhibitor (SdI) locus. DNA from 80 of the progeny amplified with the SCF27 marker produced bands; 174 individuals had 198-bp alleles of the VMC7f2 marker associated with seedlessness. In the second year, based on MAS, 183 F₁ hybrids were designated as seedless grapevine candidates because they were positive for a minimum of one marker. Twenty individuals were selected as genetic resources for future studies on seedless grapevine breeding because they carried alleles for the three markers associated with seedlessness. The VMC7f2 SSR marker was identified as the marker most associated with seedlessness.

Efficient Breeding by Genomic Mating.

PubMed

Akdemir, Deniz; Sánchez, Julio I

2016-01-01

Selection in breeding programs can be done by using phenotypes (phenotypic selection), pedigree relationship (breeding value selection) or molecular markers (marker assisted selection or genomic selection). All these methods are based on truncation selection, focusing on the best performance of parents before mating. In this article we proposed an approach to breeding, named genomic mating, which focuses on mating instead of truncation selection. Genomic mating uses information in a similar fashion to genomic selection but includes information on complementation of parents to be mated. Following the efficiency frontier surface, genomic mating uses concepts of estimated breeding values, risk (usefulness) and coefficient of ancestry to optimize mating between parents. We used a genetic algorithm to find solutions to this optimization problem and the results from our simulations comparing genomic selection, phenotypic selection and the mating approach indicate that current approach for breeding complex traits is more favorable than phenotypic and genomic selection. Genomic mating is similar to genomic selection in terms of estimating marker effects, but in genomic mating the genetic information and the estimated marker effects are used to decide which genotypes should be crossed to obtain the next breeding population.

Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius).

PubMed

Yang, Huaan; Jian, Jianbo; Li, Xuan; Renshaw, Daniel; Clements, Jonathan; Sweetingham, Mark W; Tan, Cong; Li, Chengdao

2015-09-02

Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding. Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding. We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.

A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

PubMed

Zhang, X J; Wang, L X; Chen, X X; Liu, Y L; Meng, R; Wang, Y J; Zhao, Z Y

2014-10-31

Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs.

Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects.

PubMed

Manimaran, P; Ramkumar, G; Sakthivel, K; Sundaram, R M; Madhav, M S; Balachandran, S M

2011-01-01

Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system.

PubMed

Darwish, Nader Ahmed; Khan, Raham Sher; Ntui, Valentine Otang; Nakamura, Ikuo; Mii, Masahiro

2014-03-01

Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker. Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.

Improving the selection efficiency of the counter-selection marker pheS* for the genetic engineering of Bacillus amyloliquefaciens.

PubMed

Kharchenko, Maria S; Teslya, Petr N; Babaeva, Maria N; Zakataeva, Natalia P

2018-05-01

Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheS BsT255S/A309G , with a two-step replacement recombination procedure provides an effective tool for the genetic engineering of industrially important Bacillus species. Copyright © 2018. Published by Elsevier B.V.

Genomic Selection in Multi-environment Crop Trials.

PubMed

Oakey, Helena; Cullis, Brian; Thompson, Robin; Comadran, Jordi; Halpin, Claire; Waugh, Robbie

2016-05-03

Genomic selection in crop breeding introduces modeling challenges not found in animal studies. These include the need to accommodate replicate plants for each line, consider spatial variation in field trials, address line by environment interactions, and capture nonadditive effects. Here, we propose a flexible single-stage genomic selection approach that resolves these issues. Our linear mixed model incorporates spatial variation through environment-specific terms, and also randomization-based design terms. It considers marker, and marker by environment interactions using ridge regression best linear unbiased prediction to extend genomic selection to multiple environments. Since the approach uses the raw data from line replicates, the line genetic variation is partitioned into marker and nonmarker residual genetic variation (i.e., additive and nonadditive effects). This results in a more precise estimate of marker genetic effects. Using barley height data from trials, in 2 different years, of up to 477 cultivars, we demonstrate that our new genomic selection model improves predictions compared to current models. Analyzing single trials revealed improvements in predictive ability of up to 5.7%. For the multiple environment trial (MET) model, combining both year trials improved predictive ability up to 11.4% compared to a single environment analysis. Benefits were significant even when fewer markers were used. Compared to a single-year standard model run with 3490 markers, our partitioned MET model achieved the same predictive ability using between 500 and 1000 markers depending on the trial. Our approach can be used to increase accuracy and confidence in the selection of the best lines for breeding and/or, to reduce costs by using fewer markers. Copyright © 2016 Oakey et al.

CAPN1, CAST, and DGAT1 genetic effects on preweaning performance, carcass quality traits, and residual variance of tenderness in a beef cattle population selected for haplotype and allele equalization

USDA-ARS?s Scientific Manuscript database

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. A stable composite population (MARC III) was subjected to marker assisted selection for multiple years to equalize specific marker frequencies to 1) estimate effect size an...

µ-Calpain, calpastatin, and growth hormone receptor genetic effects on preweaning performance, carcass quality traits, and residual variance of tenderness in Angus cattle selected to increase minor haplotype ... frequencies

USDA-ARS?s Scientific Manuscript database

Genetic marker effects and interactions are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to increase divergent haplotype and minor marker allele frequencies to 1) estimate effect size an...

Criteria for selection and application of molecular markers for clinical studies of osteoarthritis.

PubMed

Otterness, I G; Swindell, A C

2003-03-01

To develop criteria for the selection and application of molecular markers for the study of osteoarthritis (OA). Statistical criteria for marker selection for OA are developed. After studying more than 20 different molecular markers for monitoring OA, procedures for choosing markers for clinical studies have been developed. For a particular study, the process starts with the markers showing 'face-validity' for monitoring OA. They are next required to successfully distinguish OA patients from controls. This necessitates definition of the distribution of marker values in OA patients and controls. So far, they have been consistently log-normal. The difference (Delta) in marker values between OA and controls defines the opportunity for marker improvement. The between-visit standard deviation (S) in patients puts limits on the detection of marker changes. The two variables can be combined to estimate the practicality of a marker using a modified power analysis. The number of patients (N*) required to observe a 50% improvement with an alpha level of P=0.05 and with 80% certainty is estimated as 50(S/Delta)(2). N*, S and Delta should be used to characterize and compare markers. Marker efficiency can be refined by regressing on secondary variables, such as age, sex, BMI, severity, etc. Finally, the use of two or more markers may be required to improve marker prediction of clinical outcome. Correlated markers can be used to reinforce conclusions by essentially adding replicative data. Independent, complementary markers can be used to develop associations with clinical parameters, and perhaps diagnose and monitor disease status, activities that so far have not been possible with single markers.

Genetic transformation of carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

[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.

Molecular plant breeding: methodology and achievements.

PubMed

Varshney, Rajeev K; Hoisington, Dave A; Nayak, Spurthi N; Graner, Andreas

2009-01-01

The progress made in DNA marker technology has been remarkable and exciting in recent years. DNA markers have proved valuable tools in various analyses in plant breeding, for example, early generation selection, enrichment of complex F(1)s, choice of donor parent in backcrossing, recovery of recurrent parent genotype in backcrossing, linkage block analysis and selection. Other main areas of applications of molecular markers in plant breeding include germplasm characterization/fingerprinting, determining seed purity, systematic sampling of germplasm, and phylogenetic analysis. Molecular markers, thus, have proved powerful tools in replacing the bioassays and there are now many examples available to show the efficacy of such markers. We have illustrated some basic concepts and methodology of applying molecular markers for enhancing the selection efficiency in plant breeding. Some successful examples of product developments of molecular breeding have also been presented.

Logistic regression trees for initial selection of interesting loci in case-control studies

PubMed Central

Nickolov, Radoslav Z; Milanov, Valentin B

2007-01-01

Modern genetic epidemiology faces the challenge of dealing with hundreds of thousands of genetic markers. The selection of a small initial subset of interesting markers for further investigation can greatly facilitate genetic studies. In this contribution we suggest the use of a logistic regression tree algorithm known as logistic tree with unbiased selection. Using the simulated data provided for Genetic Analysis Workshop 15, we show how this algorithm, with incorporation of multifactor dimensionality reduction method, can reduce an initial large pool of markers to a small set that includes the interesting markers with high probability. PMID:18466557

Selection enhanced estimates of marker effects on means and variances of beef tenderness

USDA-ARS?s Scientific Manuscript database

Genetic marker associations from surveys of industry cattle populations have low frequencies of rare homozygous animals. Selection for calpain (CAPN1) and calpastatin (CAST) genetic markers was replicated in two cattle populations (Angus and MARC III) at the U.S. Meat Animal Research Center. These...

Selective sweep mapping of genes with large phenotypic effects.

PubMed

Pollinger, John P; Bustamante, Carlos D; Fledel-Alon, Adi; Schmutz, Sheila; Gray, Melissa M; Wayne, Robert K

2005-12-01

Many domestic dog breeds have originated through fixation of discrete mutations by intense artificial selection. As a result of this process, markers in the proximity of genes influencing breed-defining traits will have reduced variation (a selective sweep) and will show divergence in allele frequency. Consequently, low-resolution genomic scans can potentially be used to identify regions containing genes that have a major influence on breed-defining traits. We model the process of breed formation and show that the probability of two or three adjacent marker loci showing a spurious signal of selection within at least one breed (i.e., Type I error or false-positive rate) is low if highly variable and moderately spaced markers are utilized. We also use simulations with selection to demonstrate that even a moderately spaced set of highly polymorphic markers (e.g., one every 0.8 cM) has high power to detect regions targeted by strong artificial selection in dogs. Further, we show that a gene responsible for black coat color in the Large Munsterlander has a 40-Mb region surrounding the gene that is very low in heterozygosity for microsatellite markers. Similarly, we survey 302 microsatellite markers in the Dachshund and find three linked monomorphic microsatellite markers all within a 10-Mb region on chromosome 3. This region contains the FGFR3 gene, which is responsible for achondroplasia in humans, but not in dogs. Consequently, our results suggest that the causative mutation is a gene or regulatory region closely linked to FGFR3.

Algorithms for selecting informative marker panels for population assignment.

PubMed

Rosenberg, Noah A

2005-11-01

Given a set of potential source populations, genotypes of an individual of unknown origin at a collection of markers can be used to predict the correct source population of the individual. For improved efficiency, informative markers can be chosen from a larger set of markers to maximize the accuracy of this prediction. However, selecting the loci that are individually most informative does not necessarily produce the optimal panel. Here, using genotypes from eight species--carp, cat, chicken, dog, fly, grayling, human, and maize--this univariate accumulation procedure is compared to new multivariate "greedy" and "maximin" algorithms for choosing marker panels. The procedures generally suggest similar panels, although the greedy method often recommends inclusion of loci that are not chosen by the other algorithms. In seven of the eight species, when applied to five or more markers, all methods achieve at least 94% assignment accuracy on simulated individuals, with one species--dog--producing this level of accuracy with only three markers, and the eighth species--human--requiring approximately 13-16 markers. The new algorithms produce substantial improvements over use of randomly selected markers; where differences among the methods are noticeable, the greedy algorithm leads to slightly higher probabilities of correct assignment. Although none of the approaches necessarily chooses the panel with optimal performance, the algorithms all likely select panels with performance near enough to the maximum that they all are suitable for practical use.

Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in Rice Marker-Assisted Breeding

PubMed Central

Luo, Zhijing; Chen, Mingjiao; Zhao, Xiangxiang; Zhang, Dabing; Qi, Yiping; Yuan, Zheng

2016-01-01

Rapid and accurate genome-wide marker detection is essential to the marker-assisted breeding and functional genomics studies. In this work, we developed an integrated software, AgroMarker Finder (AMF: http://erp.novelbio.com/AMF), for providing graphical user interface (GUI) to facilitate the recently developed restriction-site associated DNA (RAD) sequencing data analysis in rice. By application of AMF, a total of 90,743 high-quality markers (82,878 SNPs and 7,865 InDels) were detected between rice varieties JP69 and Jiaoyuan5A. The density of the identified markers is 0.2 per Kb for SNP markers, and 0.02 per Kb for InDel markers. Sequencing validation revealed that the accuracy of genome-wide marker detection by AMF is 93%. In addition, a validated subset of 82 SNPs and 31 InDels were found to be closely linked to 117 important agronomic trait genes, providing a basis for subsequent marker-assisted selection (MAS) and variety identification. Furthermore, we selected 12 markers from 31 validated InDel markers to identify seed authenticity of variety Jiaoyuanyou69, and we also identified 10 markers closely linked to the fragrant gene BADH2 to minimize linkage drag for Wuxiang075 (BADH2 donor)/Jiachang1 recombinants selection. Therefore, this software provides an efficient approach for marker identification from RAD-seq data, and it would be a valuable tool for plant MAS and variety protection. PMID:26799713

Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in Rice Marker-Assisted Breeding.

PubMed

Fan, Wei; Zong, Jie; Luo, Zhijing; Chen, Mingjiao; Zhao, Xiangxiang; Zhang, Dabing; Qi, Yiping; Yuan, Zheng

2016-01-01

Rapid and accurate genome-wide marker detection is essential to the marker-assisted breeding and functional genomics studies. In this work, we developed an integrated software, AgroMarker Finder (AMF: http://erp.novelbio.com/AMF), for providing graphical user interface (GUI) to facilitate the recently developed restriction-site associated DNA (RAD) sequencing data analysis in rice. By application of AMF, a total of 90,743 high-quality markers (82,878 SNPs and 7,865 InDels) were detected between rice varieties JP69 and Jiaoyuan5A. The density of the identified markers is 0.2 per Kb for SNP markers, and 0.02 per Kb for InDel markers. Sequencing validation revealed that the accuracy of genome-wide marker detection by AMF is 93%. In addition, a validated subset of 82 SNPs and 31 InDels were found to be closely linked to 117 important agronomic trait genes, providing a basis for subsequent marker-assisted selection (MAS) and variety identification. Furthermore, we selected 12 markers from 31 validated InDel markers to identify seed authenticity of variety Jiaoyuanyou69, and we also identified 10 markers closely linked to the fragrant gene BADH2 to minimize linkage drag for Wuxiang075 (BADH2 donor)/Jiachang1 recombinants selection. Therefore, this software provides an efficient approach for marker identification from RAD-seq data, and it would be a valuable tool for plant MAS and variety protection.

Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction.

PubMed

Brøndum, R F; Su, G; Janss, L; Sahana, G; Guldbrandtsen, B; Boichard, D; Lund, M S

2015-06-01

This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index itself. Depending on the trait's economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage disequilibrium and assaying performance on the array, a total of 1,623 QTL markers were selected for inclusion on the custom chip. Genomic prediction analyses were performed for Nordic and French Holstein and Nordic Red animals using either a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model including the QTL markers in the analysis, reliability was increased by up to 4 percentage points for production traits in Nordic Holstein animals, up to 3 percentage points for Nordic Reds, and up to 5 percentage points for French Holstein. Smaller gains of up to 1 percentage point was observed for mastitis, but only a 0.5 percentage point increase was seen for fertility. When using a Bayesian model accuracies were generally higher with only 54k data compared with the genomic BLUP approach, but increases in reliability were relatively smaller when QTL markers were included. Results from this study indicate that the reliability of genomic prediction can be increased by including markers significant in genome-wide association studies on whole genome sequence data alongside the 54k SNP set. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Charles W. Stuber: Maize geneticist and pioneer of marker-assisted selection

USDA-ARS?s Scientific Manuscript database

Charles W. Stuber is considered a pioneer of quantitative genetic mapping and marker-assisted selection in maize. The achievements of his four decade career in research include the development of genetic marker systems used in maize and adapted in many other crops, the first methods and studies to i...

DNA fingerprinting sets for four southern pines

Treesearch

Craig Echt; Sedley Josserand

2018-01-01

DNA markers can provide valuable genetic information for forest tree research, breeding, conservation, and restoration programs. When properly evaluated, selected sets of DNA markers can be used to efficiently get information about genetic diversity in regions, forests, or stands, or in seed lots and orchards. Selected markers also can be used to determine parentage or...

Selection for avian immune response: a commercial breeding company challenge.

PubMed

Fulton, J E

2004-04-01

Selection for immune function in the commercial breeding environment is a challenging proposition for commercial breeding companies. Immune response is only one of many traits that are under intensive selection, thus selection pressure needs to be carefully balanced across multiple traits. The selection environment (single bird cages, biosecure facilities, controlled environment) is a very different environment than the commercial production facilities (multiple bird cages, potential disease exposure, variable environment) in which birds are to produce. The testing of individual birds is difficult, time consuming, and expensive. It is essential that the results of any tests be relevant to actual disease or environmental challenge in the commercial environment. The use of genetic markers as indicators of immune function is being explored by breeding companies. Use of genetic markers would eliminate many of the limitations in enhancing immune function currently encountered by commercial breeding companies. Information on genetic markers would allow selection to proceed without subjecting breeding stock to disease conditions and could be done before production traits are measured. These markers could be candidate genes with known interaction or involvement with disease pathology or DNA markers that are closely linked to genetic regions that influence the immune response. The current major limitation to this approach is the paucity of mapped chicken immune response genes and the limited number of DNA markers mapped on the chicken genome. These limitations should be eliminated once the chicken genome is sequenced.

A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient selectable marker for plastid transformation.

PubMed

Tabatabaei, Iman; Ruf, Stephanie; Bock, Ralph

2017-02-01

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6')-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

Genome-enabled prediction models for yield related traits in chickpea

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) unlike marker-assisted backcrossing (MABC) predicts breeding values of lines using genome-wide marker profiling and allows selection of lines prior to field-phenotyping, thereby shortening the breeding cycle. A collection of 320 elite breeding lines was selected and phenotyped...

Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality.

PubMed

Li, Li; Tacke, Eckhard; Hofferbert, Hans-Reinhardt; Lübeck, Jens; Strahwald, Josef; Draffehn, Astrid M; Walkemeier, Birgit; Gebhardt, Christiane

2013-04-01

Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were identified that significantly improved average chip quality after cold storage and tuber starch content. In F1 progeny of a single-cross combination, MAS with six markers did not give the expected result. Reasons and implications for MAS in potato are discussed.

A Marker-Based Approach for the Automated Selection of a Single Segmentation from a Hierarchical Set of Image Segmentations

NASA Technical Reports Server (NTRS)

Tarabalka, Y.; Tilton, J. C.; Benediktsson, J. A.; Chanussot, J.

2012-01-01

The Hierarchical SEGmentation (HSEG) algorithm, which combines region object finding with region object clustering, has given good performances for multi- and hyperspectral image analysis. This technique produces at its output a hierarchical set of image segmentations. The automated selection of a single segmentation level is often necessary. We propose and investigate the use of automatically selected markers for this purpose. In this paper, a novel Marker-based HSEG (M-HSEG) method for spectral-spatial classification of hyperspectral images is proposed. Two classification-based approaches for automatic marker selection are adapted and compared for this purpose. Then, a novel constrained marker-based HSEG algorithm is applied, resulting in a spectral-spatial classification map. Three different implementations of the M-HSEG method are proposed and their performances in terms of classification accuracies are compared. The experimental results, presented for three hyperspectral airborne images, demonstrate that the proposed approach yields accurate segmentation and classification maps, and thus is attractive for remote sensing image analysis.

Selecting soybean resistant to the cyst nematode Heterodera glycines using simple sequence repeat (microssatellite) markers.

PubMed

Espindola, S M C G; Hamawaki, O T; Oliveira, A P; Hamawaki, C D L; Hamawaki, R L; Takahashi, L M

2016-03-11

The soybean cyst nematode (SCN) is a major cause of soybean yield reduction. The objective of this study was to evaluate the efficiency of marker-assisted selection to identify genotypes resistant to SCN race 3 infection, using Sat_168 and Sat-141 resistance quantitative trait loci. The experiment was carried out under greenhouse conditions, using soybean populations originated from crosses between susceptible and resistant parent stock: CD-201 (susceptible) and Foster IAC (resistant), Conquista (susceptible) and S83-30 (resistant), La-Suprema (susceptible) and S57-11 (resistant), and Parecis (susceptible) and S65-50 (resistant). Plants were inoculated with SCN and evaluated according to the female index (FI), those with FI < 10% were classified as resistant to nematode infection. Plants were genotyped for SCN resistance using microsatellite markers Sat-141 and Sat_168. Marker selection efficiency was analyzed by a contingency table, taking into account genotypic versus phenotypic evaluations for each line. These markers were shown to be useful tool for selection of SCN race 3.

Genetic diversity and trait genomic prediction in a pea diversity panel.

PubMed

Burstin, Judith; Salloignon, Pauline; Chabert-Martinello, Marianne; Magnin-Robert, Jean-Bernard; Siol, Mathieu; Jacquin, Françoise; Chauveau, Aurélie; Pont, Caroline; Aubert, Grégoire; Delaitre, Catherine; Truntzer, Caroline; Duc, Gérard

2015-02-21

Pea (Pisum sativum L.), a major pulse crop grown for its protein-rich seeds, is an important component of agroecological cropping systems in diverse regions of the world. New breeding challenges imposed by global climate change and new regulations urge pea breeders to undertake more efficient methods of selection and better take advantage of the large genetic diversity present in the Pisum sativum genepool. Diversity studies conducted so far in pea used Simple Sequence Repeat (SSR) and Retrotransposon Based Insertion Polymorphism (RBIP) markers. Recently, SNP marker panels have been developed that will be useful for genetic diversity assessment and marker-assisted selection. A collection of diverse pea accessions, including landraces and cultivars of garden, field or fodder peas as well as wild peas was characterised at the molecular level using newly developed SNP markers, as well as SSR markers and RBIP markers. The three types of markers were used to describe the structure of the collection and revealed different pictures of the genetic diversity among the collection. SSR showed the fastest rate of evolution and RBIP the slowest rate of evolution, pointing to their contrasted mode of evolution. SNP markers were then used to predict phenotypes -the date of flowering (BegFlo), the number of seeds per plant (Nseed) and thousand seed weight (TSW)- that were recorded for the collection. Different statistical methods were tested including the LASSO (Least Absolute Shrinkage ans Selection Operator), PLS (Partial Least Squares), SPLS (Sparse Partial Least Squares), Bayes A, Bayes B and GBLUP (Genomic Best Linear Unbiased Prediction) methods and the structure of the collection was taken into account in the prediction. Despite a limited number of 331 markers used for prediction, TSW was reliably predicted. The development of marker assisted selection has not reached its full potential in pea until now. This paper shows that the high-throughput SNP arrays that are being developed will most probably allow for a more efficient selection in this species.

Energy and Nutrient Intake Monitoring

NASA Technical Reports Server (NTRS)

Luckey, T. D.; Venugopal, B.; Hutcheson, D. P.

1975-01-01

A passive system to determine the in-flight intake of nutrients is developed. Nonabsorbed markers placed in all foods in proportion to the nutrients selected for study are analyzed by neutron activation analysis. Fecal analysis for each market indicates how much of the nutrients were eaten and apparent digestibility. Results of feasibility tests in rats, mice, and monkeys indicate the diurnal variation of several markers, the transit time for markers in the alimentary tract, the recovery of several markers, and satisfactory use of selected markers to provide indirect measurement of apparent digestibility. Recommendations are provided for human feasibility studies.

Olfactory Bulb Deep Short-Axon Cells Mediate Widespread Inhibition of Tufted Cell Apical Dendrites

PubMed Central

LaRocca, Greg

2017-01-01

In the main olfactory bulb (MOB), the first station of sensory processing in the olfactory system, GABAergic interneuron signaling shapes principal neuron activity to regulate olfaction. However, a lack of known selective markers for MOB interneurons has strongly impeded cell-type-selective investigation of interneuron function. Here, we identify the first selective marker of glomerular layer-projecting deep short-axon cells (GL-dSACs) and investigate systematically the structure, abundance, intrinsic physiology, feedforward sensory input, neuromodulation, synaptic output, and functional role of GL-dSACs in the mouse MOB circuit. GL-dSACs are located in the internal plexiform layer, where they integrate centrifugal cholinergic input with highly convergent feedforward sensory input. GL-dSAC axons arborize extensively across the glomerular layer to provide highly divergent yet selective output onto interneurons and principal tufted cells. GL-dSACs are thus capable of shifting the balance of principal tufted versus mitral cell activity across large expanses of the MOB in response to diverse sensory and top-down neuromodulatory input. SIGNIFICANCE STATEMENT The identification of cell-type-selective molecular markers has fostered tremendous insight into how distinct interneurons shape sensory processing and behavior. In the main olfactory bulb (MOB), inhibitory circuits regulate the activity of principal cells precisely to drive olfactory-guided behavior. However, selective markers for MOB interneurons remain largely unknown, limiting mechanistic understanding of olfaction. Here, we identify the first selective marker of a novel population of deep short-axon cell interneurons with superficial axonal projections to the sensory input layer of the MOB. Using this marker, together with immunohistochemistry, acute slice electrophysiology, and optogenetic circuit mapping, we reveal that this novel interneuron population integrates centrifugal cholinergic input with broadly tuned feedforward sensory input to modulate principal cell activity selectively. PMID:28003347

Marker-Based Hierarchical Segmentation and Classification Approach for Hyperspectral Imagery

NASA Technical Reports Server (NTRS)

Tarabalka, Yuliya; Tilton, James C.; Benediktsson, Jon Atli; Chanussot, Jocelyn

2011-01-01

The Hierarchical SEGmentation (HSEG) algorithm, which is a combination of hierarchical step-wise optimization and spectral clustering, has given good performances for hyperspectral image analysis. This technique produces at its output a hierarchical set of image segmentations. The automated selection of a single segmentation level is often necessary. We propose and investigate the use of automatically selected markers for this purpose. In this paper, a novel Marker-based HSEG (M-HSEG) method for spectral-spatial classification of hyperspectral images is proposed. First, pixelwise classification is performed and the most reliably classified pixels are selected as markers, with the corresponding class labels. Then, a novel constrained marker-based HSEG algorithm is applied, resulting in a spectral-spatial classification map. The experimental results show that the proposed approach yields accurate segmentation and classification maps, and thus is attractive for hyperspectral image analysis.

Molecular Markers and Cotton Genetic Improvement: Current Status and Future Prospects

PubMed Central

Malik, Waqas; Iqbal, Muhammad Zaffar; Ali Khan, Asif; Qayyum, Abdul; Ali Abid, Muhammad; Noor, Etrat; Qadir Ahmad, Muhammad; Hasan Abbasi, Ghulam

2014-01-01

Narrow genetic base and complex allotetraploid genome of cotton (Gossypium hirsutum L.) is stimulating efforts to avail required polymorphism for marker based breeding. The availability of draft genome sequence of G. raimondii and G. arboreum and next generation sequencing (NGS) technologies facilitated the development of high-throughput marker technologies in cotton. The concepts of genetic diversity, QTL mapping, and marker assisted selection (MAS) are evolving into more efficient concepts of linkage disequilibrium, association mapping, and genomic selection, respectively. The objective of the current review is to analyze the pace of evolution in the molecular marker technologies in cotton during the last ten years into the following four areas: (i) comparative analysis of low- and high-throughput marker technologies available in cotton, (ii) genetic diversity in the available wild and improved gene pools of cotton, (iii) identification of the genomic regions within cotton genome underlying economic traits, and (iv) marker based selection methodologies. Moreover, the applications of marker technologies to enhance the breeding efficiency in cotton are also summarized. Aforementioned genomic technologies and the integration of several other omics resources are expected to enhance the cotton productivity and meet the global fiber quantity and quality demands. PMID:25401149

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.

PubMed

Chin, Dong Poh; Mishiba, Kei-ichiro; Mii, Masahiro

2007-06-01

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

Investigation of the optimum location of external markers for patient setup accuracy enhancement at external beam radiotherapy

PubMed Central

Torshabi, Ahmad Esmaili; Nankali, Saber

2016-01-01

In external beam radiotherapy, one of the most common and reliable methods for patient geometrical setup and/or predicting the tumor location is use of external markers. In this study, the main challenging issue is increasing the accuracy of patient setup by investigating external markers location. Since the location of each external marker may yield different patient setup accuracy, it is important to assess different locations of external markers using appropriate selective algorithms. To do this, two commercially available algorithms entitled a) canonical correlation analysis (CCA) and b) principal component analysis (PCA) were proposed as input selection algorithms. They work on the basis of maximum correlation coefficient and minimum variance between given datasets. The proposed input selection algorithms work in combination with an adaptive neuro‐fuzzy inference system (ANFIS) as a correlation model to give patient positioning information as output. Our proposed algorithms provide input file of ANFIS correlation model accurately. The required dataset for this study was prepared by means of a NURBS‐based 4D XCAT anthropomorphic phantom that can model the shape and structure of complex organs in human body along with motion information of dynamic organs. Moreover, a database of four real patients undergoing radiation therapy for lung cancers was utilized in this study for validation of proposed strategy. Final analyzed results demonstrate that input selection algorithms can reasonably select specific external markers from those areas of the thorax region where root mean square error (RMSE) of ANFIS model has minimum values at that given area. It is also found that the selected marker locations lie closely in those areas where surface point motion has a large amplitude and a high correlation. PACS number(s): 87.55.km, 87.55.N PMID:27929479

Marker-Assisted Introgression in Backcross Breeding Programs

PubMed Central

Visscher, P. M.; Haley, C. S.; Thompson, R.

1996-01-01

The efficiency of marker-assisted introgression in backcross populations derived from inbred lines was investigated by simulation. Background genotypes were simulated assuming that a genetic model of many genes of small effects in coupling phase explains the observed breed difference and variance in backcross populations. Markers were efficient in introgression backcross programs for simultaneously introgressing an allele and selecting for the desired genomic background. Using a marker spacing of 10-20 cM gave an advantage of one to two backcross generations selection relative to random or phenotypic selection. When the position of the gene to be introgressed is uncertain, for example because its position was estimated from a trait gene mapping experiment, a chromosome segment should be introgressed that is likely to include the allele of interest. Even for relatively precisely mapped quantitative trait loci, flanking markers or marker haplotypes should cover ~10-20 cM around the estimated position of the gene, to ensure that the allele frequency does not decline in later backcross generations. PMID:8978075

Recent advances in development of marker-free transgenic plants: regulation and biosafety concern.

PubMed

Tuteja, Narendra; Verma, Shiv; Sahoo, Ranjan Kumar; Raveendar, Sebastian; Reddy, I N Bheema Lingeshwara

2012-03-01

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B.

PubMed

Hu, Wei; Lagarias, J Clark

2017-01-01

Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis (Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection, we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly sensitive reporters of off-target transgene expression. © 2017 American Society of Plant Biologists. All Rights Reserved.

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B1[OPEN

PubMed Central

2017-01-01

Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis (Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection, we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly sensitive reporters of off-target transgene expression. PMID:27881727

Marker-assisted selection in plant breeding for salinity tolerance.

PubMed

Ashraf, M; Akram, N A; Mehboob-Ur-Rahman; Foolad, M R

2012-01-01

Marker-assisted selection (MAS) is the process of using morphological, biochemical, or DNA markers as indirect selection criteria for selecting agriculturally important traits in crop breeding. This process is used to improve the effectiveness or efficiency of selection for the traits of interest in breeding programs. The significance of MAS as a tool for crop improvement has been extensively investigated in different crop -species and for different traits. The use of MAS for manipulating simple/qualitative traits is straightforward and has been well reported. However, MAS for the improvement of complex/polygenic traits, including plant tolerance/resistance to abiotic stresses, is more complicated, although its usefulness has been recognized. With the recent advances in marker technology, including high-throughput genotyping of plants, together with the development of nested association mapping populations, it is expected that the utility of MAS for breeding for stress tolerance traits will increase. In this chapter, we describe the basic procedure for using MAS in crop breeding for salt tolerance.

Application of marker selection to enhance estimation of genetic effects and gene interaction in cattle

USDA-ARS?s Scientific Manuscript database

Selection on important genetic markers can improve estimates of additive and dominance association effects. A composite population of beef cattle was selected for intermediate frequencies of myostatin (GDF8) F94L and µ-calpain (CAPN1) polymorphisms. Important additive associations of the GDF8 locu...

Financial feasibility of marker-aided selection in Douglas-fir.

Treesearch

G.R. Johnson; N.C. Wheeler; S.H. Strauss

2000-01-01

The land area required for a marker-aided selection (MAS) program to break-even (i.e., have equal costs and benefits) was estimated using computer simulation for coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) in the Pacific Northwestern United States. We compared the selection efficiency obtained when using an index that included the...

Selection of chemical markers for the quality control of medicinal plants of the genus Cecropia.

PubMed

Rivera-Mondragón, Andrés; Ortíz, Orlando O; Bijttebier, Sebastiaan; Vlietinck, Arnold; Apers, Sandra; Pieters, Luc; Caballero-George, Catherina

2017-12-01

Several Cecropia (Cecropiaceae) species are traditionally used in Latin America for the treatment of a variety of diseases including diabetes, arterial hypertension, asthma, bronchitis, anxiety, and inflammation. At present, a number of commercial products based on these plants have been introduced into the market with very little information on methods for guaranteeing their quality and safety. This work proposes potential chemical markers for the quality control of the raw materials of Cecropia obtusifolia Bertol., Cecropia peltata L., Cecropia glaziovii Snethl., Cecropia pachystachya Trécul, and Cecropia hololeuca Miq. The Herbal Chemical Marker Ranking System (Herb MaRS) developed by the National Institute of Complementary Medicine (NICM) at the University of Western Sydney was used for selecting chemical markers for the quality control of selected medicinal species of Cecropia. This review covers the period from 1982 to 2016. Chlorogenic acid, flavonoidal glycosides (orientin, isoorientin, vitexin, isovitexin, and rutin), catechin, epicatechin, procyanidins (B2, B5, and C1), steroids (β-sitosterol), and triterpenoids (α-amyrin, pomolic, tormentic and ursolic acids) were selected as chemical markers for the quality control of the leaves. It is necessary to establish comprehensive standards for guaranteeing quality, safety and efficacy of herbal drugs. The selection of adequate chemical markers for quality control purposes requires a good knowledge about the chemical composition of medicinal plants and their associated biological properties. To the best of our knowledge this review article is the first to address the identification and quantitative determination of the chemical markers for the genus Cecropia.

Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9

PubMed Central

Fernandez, Ronan; Berro, Julien

2017-01-01

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. PMID:27327046

Isolation and characterization of novel microsatellite markers from the sika deer (Cervus nippon) genome.

PubMed

Li, Y M; Bai, C Y; Niu, W P; Yu, H; Yang, R J; Yan, S Q; Zhang, J Y; Zhang, M J; Zhao, Z H

2015-09-28

Microsatellite markers are widely and evenly distributed, and are highly polymorphic. Rapid and convenient detection through automated analysis means that microsatellite markers are widely used in the construction of plant and animal genetic maps, in quantitative trait loci localization, marker-assisted selection, identification of genetic relationships, and genetic diversity and phylogenetic tree construction. However, few microsatellite markers remain to be isolated. We used streptavidin magnetic beads to affinity-capture and construct a (CA)n microsatellite DNA-enriched library from sika deer. We selected sequences containing more than six repeats to design primers. Clear bands were selected, which were amplified using non-specific primers following PCR amplification to screen polymorphisms in a group of 65 unrelated sika deer. The positive clone rate reached 82.9% by constructing the enriched library, and we then selected positive clones for sequencing. There were 395 sequences with CA repeats, and the CA repeat number was 4-105. We selected sequences containing more than six repeats to design primers, of which 297 pairs were designed. We next selected clear bands and used non-specific primers to amplify following PCR amplification. In total, 245 pairs of primers were screened. We then selected 50 pairs of primers to randomly screen for polymorphisms. We detected 47 polymorphic and 3 monomorphic loci in 65 unrelated sika deer. These newly isolated and characterized microsatellite loci can be used to construct genetic maps and for lineage testing in deer. In addition, they can be used for comparative genomics between Cervidae species.

Selective DNA Pooling for Determination of Linkage between a Molecular Marker and a Quantitative Trait Locus

PubMed Central

Darvasi, A.; Soller, M.

1994-01-01

Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: ``selective DNA pooling'') takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F(2) and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions. PMID:7896115

Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

PubMed

Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Toki, Seiichi

2016-01-01

Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud.

PubMed

Tang, Wei; Tian, Yingchuan

2003-02-01

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.

Olfactory Bulb Deep Short-Axon Cells Mediate Widespread Inhibition of Tufted Cell Apical Dendrites.

PubMed

Burton, Shawn D; LaRocca, Greg; Liu, Annie; Cheetham, Claire E J; Urban, Nathaniel N

2017-02-01

In the main olfactory bulb (MOB), the first station of sensory processing in the olfactory system, GABAergic interneuron signaling shapes principal neuron activity to regulate olfaction. However, a lack of known selective markers for MOB interneurons has strongly impeded cell-type-selective investigation of interneuron function. Here, we identify the first selective marker of glomerular layer-projecting deep short-axon cells (GL-dSACs) and investigate systematically the structure, abundance, intrinsic physiology, feedforward sensory input, neuromodulation, synaptic output, and functional role of GL-dSACs in the mouse MOB circuit. GL-dSACs are located in the internal plexiform layer, where they integrate centrifugal cholinergic input with highly convergent feedforward sensory input. GL-dSAC axons arborize extensively across the glomerular layer to provide highly divergent yet selective output onto interneurons and principal tufted cells. GL-dSACs are thus capable of shifting the balance of principal tufted versus mitral cell activity across large expanses of the MOB in response to diverse sensory and top-down neuromodulatory input. The identification of cell-type-selective molecular markers has fostered tremendous insight into how distinct interneurons shape sensory processing and behavior. In the main olfactory bulb (MOB), inhibitory circuits regulate the activity of principal cells precisely to drive olfactory-guided behavior. However, selective markers for MOB interneurons remain largely unknown, limiting mechanistic understanding of olfaction. Here, we identify the first selective marker of a novel population of deep short-axon cell interneurons with superficial axonal projections to the sensory input layer of the MOB. Using this marker, together with immunohistochemistry, acute slice electrophysiology, and optogenetic circuit mapping, we reveal that this novel interneuron population integrates centrifugal cholinergic input with broadly tuned feedforward sensory input to modulate principal cell activity selectively. Copyright © 2017 the authors 0270-6474/17/371117-22$15.00/0.

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker for Agrobacterium tumefaciens-Mediated Transformation of Maize

PubMed Central

Li, Xianggan; Volrath, Sandy L.; Nicholl, David B.G.; Chilcott, Charles E.; Johnson, Marie A.; Ward, Eric R.; Law, Marcus D.

2003-01-01

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil. PMID:12972658

Metabolite and transcript markers for the prediction of potato drought tolerance.

PubMed

Sprenger, Heike; Erban, Alexander; Seddig, Sylvia; Rudack, Katharina; Thalhammer, Anja; Le, Mai Q; Walther, Dirk; Zuther, Ellen; Köhl, Karin I; Kopka, Joachim; Hincha, Dirk K

2018-04-01

Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. Current potato varieties are highly susceptible to drought stress. In view of global climate change, selection of cultivars with improved drought tolerance and high yield potential is of paramount importance. Drought tolerance breeding of potato is currently based on direct selection according to yield and phenotypic traits and requires multiple trials under drought conditions. Marker-assisted selection (MAS) is cheaper, faster and reduces classification errors caused by noncontrolled environmental effects. We analysed 31 potato cultivars grown under optimal and reduced water supply in six independent field trials. Drought tolerance was determined as tuber starch yield. Leaf samples from young plants were screened for preselected transcript and nontargeted metabolite abundance using qRT-PCR and GC-MS profiling, respectively. Transcript marker candidates were selected from a published RNA-Seq data set. A Random Forest machine learning approach extracted metabolite and transcript markers for drought tolerance prediction with low error rates of 6% and 9%, respectively. Moreover, by combining transcript and metabolite markers, the prediction error was reduced to 4.3%. Feature selection from Random Forest models allowed model minimization, yielding a minimal combination of only 20 metabolite and transcript markers that were successfully tested for their reproducibility in 16 independent agronomic field trials. We demonstrate that a minimum combination of transcript and metabolite markers sampled at early cultivation stages predicts potato yield stability under drought largely independent of seasonal and regional agronomic conditions. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Applications of molecular markers in the discrimination of Panax species and Korean ginseng cultivars (Panax ginseng).

PubMed

Jo, Ick Hyun; Kim, Young Chang; Kim, Dong Hwi; Kim, Kee Hong; Hyun, Tae Kyung; Ryu, Hojin; Bang, Kyong Hwan

2017-10-01

The development of molecular markers is one of the most useful methods for molecular breeding and marker-based molecular associated selections. Even though there is less information on the reference genome, molecular markers are indispensable tools for determination of genetic variation and identification of species with high levels of accuracy and reproducibility. The demand for molecular approaches for marker-based breeding and genetic discriminations in Panax species has greatly increased in recent times and has been successfully applied for various purposes. However, owing to the existence of diverse molecular techniques and differences in their principles and applications, there should be careful consideration while selecting appropriate marker types. In this review, we outline the recent status of different molecular marker applications in ginseng research and industrial fields. In addition, we discuss the basic principles, requirements, and advantages and disadvantages of the most widely used molecular markers, including restriction fragment length polymorphism, random amplified polymorphic DNA, sequence tag sites, simple sequence repeats, and single nucleotide polymorphisms.

Combining markers with and without the limit of detection

PubMed Central

Dong, Ting; Liu, Catherine Chunling; Petricoin, Emanuel F.; Tang, Liansheng Larry

2014-01-01

In this paper, we consider the combination of markers with and without the limit of detection (LOD). LOD is often encountered when measuring proteomic markers. Because of the limited detecting ability of an equipment or instrument, it is difficult to measure markers at a relatively low level. Suppose that after some monotonic transformation, the marker values approximately follow multivariate normal distributions. We propose to estimate distribution parameters while taking the LOD into account, and then combine markers using the results from the linear discriminant analysis. Our simulation results show that the ROC curve parameter estimates generated from the proposed method are much closer to the truth than simply using the linear discriminant analysis to combine markers without considering the LOD. In addition, we propose a procedure to select and combine a subset of markers when many candidate markers are available. The procedure based on the correlation among markers is different from a common understanding that a subset of the most accurate markers should be selected for the combination. The simulation studies show that the accuracy of a combined marker can be largely impacted by the correlation of marker measurements. Our methods are applied to a protein pathway dataset to combine proteomic biomarkers to distinguish cancer patients from non-cancer patients. PMID:24132938

Surrogate biochemical markers: precise measurement for strategic drug and biologics development.

PubMed

Lee, J W; Hulse, J D; Colburn, W A

1995-05-01

More efficient drug and biologics development is necessary for future success of pharmaceutical and biotechnology companies. One way to achieve this objective is to use rationally selected surrogate markers to improve the early decision-making process. Using typical clinical chemistry methods to measure biochemical markers may not ensure adequate precision and reproducibility. In contrast, using analytical methods that meet good laboratory practices along with rational selection and validation of biochemical markers can give those who use them a competitive advantage over those who do not by providing meaningful data for earlier decision making.

Development and validation of microsatellite markers for Brachiaria ruziziensis obtained by partial genome assembly of Illumina single-end reads

PubMed Central

2013-01-01

Background Brachiaria ruziziensis is one of the most important forage species planted in the tropics. The application of genomic tools to aid the selection of superior genotypes can provide support to B. ruziziensis breeding programs. However, there is a complete lack of information about the B. ruziziensis genome. Also, the availability of genomic tools, such as molecular markers, to support B. ruziziensis breeding programs is rather limited. Recently, next-generation sequencing technologies have been applied to generate sequence data for the identification of microsatellite regions and primer design. In this study, we present a first validated set of SSR markers for Brachiaria ruziziensis, selected from a de novo partial genome assembly of single-end Illumina reads. Results A total of 85,567 perfect microsatellite loci were detected in contigs with a minimum 10X coverage. We selected a set of 500 microsatellite loci identified in contigs with minimum 100X coverage for primer design and synthesis, and tested a subset of 269 primer pairs, 198 of which were polymorphic on 11 representative B. ruziziensis accessions. Descriptive statistics for these primer pairs are presented, as well as estimates of marker transferability to other relevant brachiaria species. Finally, a set of 11 multiplex panels containing the 30 most informative markers was validated and proposed for B. ruziziensis genetic analysis. Conclusions We show that the detection and development of microsatellite markers from genome assembled Illumina single-end DNA sequences is highly efficient. The developed markers are readily suitable for genetic analysis and marker assisted selection of Brachiaria ruziziensis. The use of this approach for microsatellite marker development is promising for species with limited genomic information, whose breeding programs would benefit from the use of genomic tools. To our knowledge, this is the first set of microsatellite markers developed for this important species. PMID:23324172

Dominant selectable markers for Penicillium spp. transformation and gene function studies

USDA-ARS?s Scientific Manuscript database

Penicillium spp. has been genetically manipulated and gene function studies have utilized single gene deletion strains for phenotypic analysis. Fungal transformation experiments have relied on hygromycin and hygromycin phosphotransferase (hph) as the main dominant selectable marker (DSM) system in P...

Oral cancer prognosis based on clinicopathologic and genomic markers using a hybrid of feature selection and machine learning methods

PubMed Central

2013-01-01

Background Machine learning techniques are becoming useful as an alternative approach to conventional medical diagnosis or prognosis as they are good for handling noisy and incomplete data, and significant results can be attained despite a small sample size. Traditionally, clinicians make prognostic decisions based on clinicopathologic markers. However, it is not easy for the most skilful clinician to come out with an accurate prognosis by using these markers alone. Thus, there is a need to use genomic markers to improve the accuracy of prognosis. The main aim of this research is to apply a hybrid of feature selection and machine learning methods in oral cancer prognosis based on the parameters of the correlation of clinicopathologic and genomic markers. Results In the first stage of this research, five feature selection methods have been proposed and experimented on the oral cancer prognosis dataset. In the second stage, the model with the features selected from each feature selection methods are tested on the proposed classifiers. Four types of classifiers are chosen; these are namely, ANFIS, artificial neural network, support vector machine and logistic regression. A k-fold cross-validation is implemented on all types of classifiers due to the small sample size. The hybrid model of ReliefF-GA-ANFIS with 3-input features of drink, invasion and p63 achieved the best accuracy (accuracy = 93.81%; AUC = 0.90) for the oral cancer prognosis. Conclusions The results revealed that the prognosis is superior with the presence of both clinicopathologic and genomic markers. The selected features can be investigated further to validate the potential of becoming as significant prognostic signature in the oral cancer studies. PMID:23725313

Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination

PubMed Central

2013-01-01

Background Antibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype. Results Allium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding completely marker free plants. Conclusions The present study establishes the efficient expression of the newly introduced insect resistant ASAL gene even after Cre/lox mediated recombination resulting in elimination of selectable marker gene. PMID:24144281

Internal amplification control of PCR for the Glu1-Dx5 allele in wheat.

PubMed

Heim, H N; Vieira, E S N; Polo, L R T; Lima, N K; Silva, G J; Linde, G A; Colauto, N B; Schuster, I

2017-08-17

One of the limiting factors in using dominant markers is the unique amplification of the target fragment. Therefore, failures in polymerase chain reaction (PCR) or non-amplifications can be interpreted as an absence of the allele. The possibility of false negatives implies in reduced efficiency in the selection process in genetic breeding programs besides the loss of valuable genetic material. Thus, this study aimed to evaluate the viability of a microsatellite marker as an internal amplification control with a dominant marker for the wheat Glu1-Dx5 gene. A population of 77 wheat cultivars/breeding lines was analyzed. Fourteen microsatellite markers were analyzed in silico regarding the formation of dimers and clamps. The biplex reaction conditions were optimized, and the Xbarc117 marker was selected as the internal amplification control with a Glu1-Dx5 marker in wheat. It was concluded that the Xbarc117 microsatellite marker was effective in the simultaneous amplification with a dominant Glu1-Dx5 marker, making biplex PCR viable in wheat for the studied markers.

Single-tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A.

PubMed

Zhao, M; Chen, M; Tan, A S C; Cheah, F S H; Mathew, J; Wong, P C; Chong, S S

2017-07-01

Essentials Preimplantation genetic diagnosis (PGD) of severe hemophilia A relies on linkage analysis. Simultaneous multi-marker screening can simplify selection of informative markers in a couple. We developed a single-tube tetradecaplex panel of polymorphic markers for hemophilia A PGD use. Informative markers can be used for linkage analysis alone or combined with mutation detection. Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic microsatellite marker panel simplifies identification and selection of informative markers for linkage-based PGD of HEMA. Informative markers can also be easily co-amplified with mutation-containing F8 amplicons for combined mutation detection and linkage analysis. © 2017 International Society on Thrombosis and Haemostasis.

Avian Disease and Oncology Laboratory (ADOL) research update

USDA-ARS?s Scientific Manuscript database

GENOMICS To meet the growing demands of consumers, the poultry industry will need to continue to improve methods of selection in breeding programs for production and associated traits. One possible solution is genome-wide marker-assisted selection (GWMAS). In brief, evenly-spaced genetic markers s...

Molecular breeding for the development of multiple disease resistance in Basmati rice.

PubMed

Singh, Atul; Singh, Vikas K; Singh, S P; Pandian, R T P; Ellur, Ranjith K; Singh, Devinder; Bhowmick, Prolay K; Gopala Krishnan, S; Nagarajan, M; Vinod, K K; Singh, U D; Prabhu, K V; Sharma, T R; Mohapatra, T; Singh, A K

2012-01-01

Basmati rice grown in the Indian subcontinent is highly valued for its unique culinary qualities. Production is, however, often constrained by diseases such as bacterial blight (BB), blast and sheath blight (ShB). The present study developed Basmati rice with inbuilt resistance to BB, blast and ShB using molecular marker-assisted selection. The rice cultivar 'Improved Pusa Basmati 1' (carrying the BB resistance genes xa13 and Xa21) was used as the recurrent parent and cultivar 'Tetep' (carrying the blast resistance gene Pi54 and ShB resistance quality trait loci (QTL), qSBR11-1) was the donor. Marker-assisted foreground selection was employed to identify plants possessing resistance alleles in the segregating generations along with stringent phenotypic selection for faster recovery of the recurrent parent genome (RPG) and phenome (RPP). Background analysis with molecular markers was used to estimate the recovery of RPG in improved lines. Foreground selection coupled with stringent phenotypic selection identified plants homozygous for xa13, Xa21 and Pi54, which were advanced to BC(2)F(5) through pedigree selection. Marker-assisted selection for qSBR11-1 in BC(2)F(5) using flanking markers identified seven homozygous families. Background analysis revealed that RPG recovery was up to 89.5%. Screening with highly virulent isolates of BB, blast and ShB showed that the improved lines were resistant to all three diseases and were on a par with 'Improved Pusa Basmati 1' for yield, duration and Basmati grain quality. This is the first report of marker-assisted transfer of genes conferring resistance to three different diseases in rice wherein genes xa13 and Xa21 for BB resistance, Pi54 for blast resistance, and a major QTL qSBR11-1 have been combined through marker-assisted backcross breeding. In addition to offering the potential for release as cultivars, the pyramided lines will serve as useful donors of gene(s) for BB, blast and ShB in future Basmati rice breeding programmes.

Development of disease-resistant marker-free tomato by R/RS site-specific recombination.

PubMed

Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2011-06-01

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

DOEpatents

Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

2007-02-27

The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

Searching for an Accurate Marker-Based Prediction of an Individual Quantitative Trait in Molecular Plant Breeding

PubMed Central

Fu, Yong-Bi; Yang, Mo-Hua; Zeng, Fangqin; Biligetu, Bill

2017-01-01

Molecular plant breeding with the aid of molecular markers has played an important role in modern plant breeding over the last two decades. Many marker-based predictions for quantitative traits have been made to enhance parental selection, but the trait prediction accuracy remains generally low, even with the aid of dense, genome-wide SNP markers. To search for more accurate trait-specific prediction with informative SNP markers, we conducted a literature review on the prediction issues in molecular plant breeding and on the applicability of an RNA-Seq technique for developing function-associated specific trait (FAST) SNP markers. To understand whether and how FAST SNP markers could enhance trait prediction, we also performed a theoretical reasoning on the effectiveness of these markers in a trait-specific prediction, and verified the reasoning through computer simulation. To the end, the search yielded an alternative to regular genomic selection with FAST SNP markers that could be explored to achieve more accurate trait-specific prediction. Continuous search for better alternatives is encouraged to enhance marker-based predictions for an individual quantitative trait in molecular plant breeding. PMID:28729875

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Shao, Di; Li, Zhenzhong; Sweetingham, Mark W; Buirchell, Bevan J; Li, Chengdao

2013-02-01

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.

ISSR marker-assisted genetic diversity analysis of Dioscorea hispida and selection of the best variety for sustainable production.

PubMed

Nudin, Nur Fatihah Hasan; Ali, Abdul Manaf; Ngah, Norhayati; Mazlan, Nor Zuhailah; Mat, Nashriyah; Ghani, Mohd Noor Abd; Alias, Nadiawati; Zakaria, Abd Jamil; Jahan, Md Sarwar

2017-08-01

Plant breeding is a way of selection of a particular individual for the production of the progeny by separating or combining desired characteristics. The objective of this study was to justify different characteristics of Dioscorea hispida (Ubi gadong) varieties using molecular techniques to select the best variety for sustainable production at the farmer's level. A total of 160 germplasms of Ubi gadong were collected from different locations at the Terengganu and Kelantan states of Malaysia. Forty eight (48) out of 160 germplasms were selected as "primary" selection based on yield and other qualitative characters. Selected collections were then grown and maintained for ISSR marker-assisted genetic diversity analysis. Overall plant growth and yield of tubers were also determined. A total of 12 ISSR markers were tested to justify the characteristics of Ubi gadong varieties among which three markers showed polymorphic bands and on average 57.3% polymorphism were observed representing the highest variation among germplasms. The ISSR marker based on UPGMA cluster analysis grouped all 48 D. hispida into 10 vital groups that proved a vast genetic variation among germplasm collections. Therefore, hybridization should be made between two distant populations. The D. hispida is already proved as the highest starch content tuber crops and very rich in vitamins with both micro and macro minerals. Considering all these criteria and results from marker-assisted diversity analysis, accessions that are far apart based on their genetic coefficient (like DH27 and DH71; DH30 and DH70; DH43 and DH62; DH45 and DH61; DH77 and DH61; DH78 and DH57) could be selected as parents for further breeding programs. This will bring about greater diversity, which will lead to high productive index in terms of increase in yield and overall quality and for the ultimate target of sustainable Ubi gadong production. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

The Power to Detect Linkage Disequilibrium with Quantitative Traits in Selected Samples

PubMed Central

Abecasis, Gonçalo R.; Cookson, William O. C.; Cardon, Lon R.

2001-01-01

Results from power studies for linkage detection have led to many ongoing and planned collections of phenotypically extreme nuclear families. Given the great expense of collecting these families and the imminent availability of a dense diallelic marker map, the families are likely to be used in allelic-association as well as linkage studies. However, optimal selection strategies for linkage may not be equally powerful for association. We examine the power to detect linkage disequilibrium for quantitative traits after phenotypic selection. The results encompass six selection strategies that are in widespread use, including single selection (two designs), affected sib pairs, concordant and discordant pairs, and the extreme-concordant and -discordant design. Selection of sibships on the basis of one extreme proband with high or low trait scores provides as much power as discordant sib pairs but requires the screening and phenotyping of substantially fewer initial families from which to select. Analysis of the role of allele frequencies within each selection design indicates that common trait alleles generally offer the most power, but similarities between the marker- and trait-allele frequencies are much more important than the trait-locus frequency alone. Some of the most widespread selection designs, such as single selection, yield power gains only when both the marker and quantitative trait loci (QTL) are relatively rare in the population. In contrast, discordant pairs and the extreme-proband design provide power for the broadest range of QTL–marker-allele frequency differences. Overall, proband selection from either tail provides the best balance of power, robustness, and simplicity of ascertainment for family-based association analysis. PMID:11349228

Selectively active markers for solving of the partial occlusion problem in matchmoving and chromakeying workflow

NASA Astrophysics Data System (ADS)

Mazurek, Przemysław

2013-09-01

Matchmoving (Match Moving) is the process used for the estimation of camera movements for further integration of acquired video image with computer graphics. The estimation of movements is possible using pattern recognition, 2D and 3D tracking algorithms. The main problem for the workflow is the partial occlusion of markers by the actor, because manual rotoscoping is necessary for fixing of the chroma-keyed footage. In the paper, the partial occlusion problem is solved using the invented, selectively active electronic markers. The sensor network with multiple infrared links detects occlusion state (no-occlusion, partial, full) and switch LED's based markers.

Validation of consensus quantitative trait loci associated with resistance to multiple foliar pathogens of maize.

PubMed

Asea, Godfrey; Vivek, Bindiganavile S; Bigirwa, George; Lipps, Patrick E; Pratt, Richard C

2009-05-01

Maize production in sub-Saharan Africa incurs serious losses to epiphytotics of foliar diseases. Quantitative trait loci conditioning partial resistance (rQTL) to infection by causal agents of gray leaf spot (GLS), northern corn leaf blight (NCLB), and maize streak have been reported. Our objectives were to identify simple-sequence repeat (SSR) molecular markers linked to consensus rQTL and one recently identified rQTL associated with GLS, and to determine their suitability as tools for selection of improved host resistance. We conducted evaluations of disease severity phenotypes in separate field nurseries, each containing 410 F2:3 families derived from a cross between maize inbred CML202 (NCLB and maize streak resistant) and VP31 (a GLS-resistant breeding line) that possess complimentary rQTL. F2:3 families were selected for resistance based on genotypic (SSR marker), phenotypic, or combined data and the selected F3:4 families were reevaluated. Phenotypic values associated with SSR markers for consensus rQTL in bins 4.08 for GLS, 5.04 for NCLB, and 1.04 for maize streak significantly reduced disease severity in both generations based on single-factor analysis of variance and marker-interval analysis. These results were consistent with the presence of homozygous resistant parent alleles, except in bin 8.06, where markers were contributed by the NCLB-susceptible parent. Only one marker associated with resistance could be confirmed in bins 2.09 (GLS) and 3.06 (NCLB), illustrating the need for more robust rQTL discovery, fine-mapping, and validation prior to undertaking marker-based selection.

The Development of Quality Control Genotyping Approaches: A Case Study Using Elite Maize Lines.

PubMed

Chen, Jiafa; Zavala, Cristian; Ortega, Noemi; Petroli, Cesar; Franco, Jorge; Burgueño, Juan; Costich, Denise E; Hearne, Sarah J

2016-01-01

Quality control (QC) of germplasm identity and purity is a critical component of breeding and conservation activities. SNP genotyping technologies and increased availability of markers provide the opportunity to employ genotyping as a low-cost and robust component of this QC. In the public sector available low-cost SNP QC genotyping methods have been developed from a very limited panel of markers of 1,000 to 1,500 markers without broad selection of the most informative SNPs. Selection of optimal SNPs and definition of appropriate germplasm sampling in addition to platform section impact on logistical and resource-use considerations for breeding and conservation applications when mainstreaming QC. In order to address these issues, we evaluated the selection and use of SNPs for QC applications from large DArTSeq data sets generated from CIMMYT maize inbred lines (CMLs). Two QC genotyping strategies were developed, the first is a "rapid QC", employing a small number of SNPs to identify potential mislabeling of seed packages or plots, the second is a "broad QC", employing a larger number of SNP, used to identify each germplasm entry and to measure heterogeneity. The optimal marker selection strategies combined the selection of markers with high minor allele frequency, sampling of clustered SNP in proportion to marker cluster distance and selecting markers that maintain a uniform genomic distribution. The rapid and broad QC SNP panels selected using this approach were further validated using blind test assessments of related re-generation samples. The influence of sampling within each line was evaluated. Sampling 192 individuals would result in close to 100% possibility of detecting a 5% contamination in the entry, and approximately a 98% probability to detect a 2% contamination of the line. These results provide a framework for the establishment of QC genotyping. A comparison of financial and time costs for use of these approaches across different platforms is discussed providing a framework for institutions involved in maize conservation and breeding to assess the resource use effectiveness of QC genotyping. Application of these research findings, in combination with existing QC approaches, will ensure the regeneration, distribution and use in breeding of true to type inbred germplasm. These findings also provide an effective approach to optimize SNP selection for QC genotyping in other species.

Molecular markers of neuropsychological functioning and Alzheimer's disease.

PubMed

Edwards, Melissa; Balldin, Valerie Hobson; Hall, James; O'Bryant, Sid

2015-03-01

The current project sought to examine molecular markers of neuropsychological functioning among elders with and without Alzheimer's disease (AD) and determine the predictive ability of combined molecular markers and select neuropsychological tests in detecting disease presence. Data were analyzed from 300 participants (n = 150, AD and n = 150, controls) enrolled in the Texas Alzheimer's Research and Care Consortium. Linear regression models were created to examine the link between the top five molecular markers from our AD blood profile and neuropsychological test scores. Logistical regressions were used to predict AD presence using serum biomarkers in combination with select neuropsychological measures. Using the neuropsychological test with the least amount of variance overlap with the molecular markers, the combined neuropsychological test and molecular markers was highly accurate in detecting AD presence. This work provides the foundation for the generation of a point-of-care device that can be used to screen for AD.

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.

Targeted Proteomics Approach for Precision Plant Breeding.

PubMed

Chawade, Aakash; Alexandersson, Erik; Bengtsson, Therese; Andreasson, Erik; Levander, Fredrik

2016-02-05

Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that enables precise quantitation of hundreds of peptides in a single run. This technique provides new opportunities for multiplexed protein biomarker measurements. For precision plant breeding, DNA-based markers have been used extensively, but the potential of protein biomarkers has not been exploited. In this work, we developed an SRM marker panel with assays for 104 potato (Solanum tuberosum) peptides selected using univariate and multivariate statistics. Thereafter, using random forest classification, the prediction markers were identified for Phytopthora infestans resistance in leaves, P. infestans resistance in tubers, and plant yield in potato leaf secretome samples. The results suggest that the marker panel has the predictive potential for three traits, two of which have no commercial DNA markers so far. Furthermore, the marker panel was also tested and found to be applicable to potato clones not used during the marker development. The proposed workflow is thus a proof-of-concept for targeted proteomics as an efficient readout in accelerated breeding for complex and agronomically important traits.

Paternity testing a non-linkage based marker assisted selection scheme for outbred forage species

USDA-ARS?s Scientific Manuscript database

In many major perennial forage species, genomic tools and infrastructure development has advanced enough that their utilization in marker assisted selection (MAS) can be cheaply explored. This paper presents a paternity testing MAS in diploid red clover (Trifolium pratense L.). Utilizing individual ...

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

A Population Genetics Model of Marker-Assisted Selection

PubMed Central

Luo, Z. W.; Thompson, R.; Woolliams, J. A.

1997-01-01

A deterministic two-loci model was developed to predict genetic response to marker-assisted selection (MAS) in one generation and in multiple generations. Formulas were derived to relate linkage disequilibrium in a population to the proportion of additive genetic variance used by MAS, and in turn to an extra improvement in genetic response over phenotypic selection. Predictions of the response were compared to those predicted by using an infinite-loci model and the factors affecting efficiency of MAS were examined. Theoretical analyses of the present study revealed the nonlinearity between the selection intensity and genetic response in MAS. In addition to the heritability of the trait and the proportion of the marker-associated genetic variance, the frequencies of the selectively favorable alleles at the two loci, one marker and one quantitative trait locus, were found to play an important role in determining both the short- and long-term efficiencies of MAS. The evolution of linkage disequilibrium and thus the genetic response over several generations were predicted theoretically and examined by simulation. MAS dissipated the disequilibrium more quickly than drift alone. In some cases studied, the rate of dissipation was as large as that to be expected in the circumstance where the true recombination fraction was increased by three times and selection was absent. PMID:9215918

Evaluation of the effect and profitability of gene-assisted selection in pig breeding system*

PubMed Central

Li, Ya-lan; Zhang, Qin; Chen, Yao-sheng

2007-01-01

Objective: To evaluate the effect and profitability of using the quantitative trait loci (QTL)-linked direct marker (DR marker) in gene-assisted selection (GAS). Methods: Three populations (100, 200, or 300 sows plus 10 boars within each group) with segregating QTL were simulated stochastically. Five economic traits were investigated, including number of born alive (NBA), average daily gain to 100 kg body weight (ADG), feed conversion ratio (FCR), back fat at 100 kg body weight (BF) and intramuscular fat (IMF). Selection was based on the estimated breeding value (EBV) of each trait. The starting frequencies of the QTL’s favorable allele were 0.1, 0.3 and 0.5, respectively. The economic return was calculated by gene flow method. Results: The selection efficiency was higher than 100% when DR markers were used in GAS for 5 traits. The selection efficiency for NBA was the highest, and the lowest was for ADG whose QTL had the lowest variance. The mixed model applied DR markers and obtained higher extra genetic gain and extra economic returns. We also found that the lower the frequency of the favorable allele of the QTL, the higher the extra return obtained. Conclusion: GAS is an effective selection scheme to increase the genetic gain and the economic returns in pig breeding. PMID:17973344

Evaluation of the effect and profitability of gene-assisted selection in pig breeding system.

PubMed

Li, Ya-Lan; Zhang, Qin; Chen, Yao-Sheng

2007-11-01

To evaluate the effect and profitability of using the quantitative trait loci (QTL)-linked direct marker (DR marker) in gene-assisted selection (GAS). Three populations (100, 200, or 300 sows plus 10 boars within each group) with segregating QTL were simulated stochastically. Five economic traits were investigated, including number of born alive (NBA), average daily gain to 100 kg body weight (ADG), feed conversion ratio (FCR), back fat at 100 kg body weight (BF) and intramuscular fat (IMF). Selection was based on the estimated breeding value (EBV) of each trait. The starting frequencies of the QTL's favorable allele were 0.1, 0.3 and 0.5, respectively. The economic return was calculated by gene flow method. The selection efficiency was higher than 100% when DR markers were used in GAS for 5 traits. The selection efficiency for NBA was the highest, and the lowest was for ADG whose QTL had the lowest variance. The mixed model applied DR markers and obtained higher extra genetic gain and extra economic returns. We also found that the lower the frequency of the favorable allele of the QTL, the higher the extra return obtained. GAS is an effective selection scheme to increase the genetic gain and the economic returns in pig breeding.

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

PubMed

Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2017-04-01

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Tracing Asian Seabass Individuals to Single Fish Farms Using Microsatellites

PubMed Central

Yue, Gen Hua; Xia, Jun Hong; Liu, Peng; Liu, Feng; Sun, Fei; Lin, Grace

2012-01-01

Traceability through physical labels is well established, but it is not highly reliable as physical labels can be easily changed or lost. Application of DNA markers to the traceability of food plays an increasingly important role for consumer protection and confidence building. In this study, we tested the efficiency of 16 polymorphic microsatellites and their combinations for tracing 368 fish to four populations where they originated. Using the maximum likelihood and Bayesian methods, three most efficient microsatellites were required to assign over 95% of fish to the correct populations. Selection of markers based on the assignment score estimated with the software WHICHLOCI was most effective in choosing markers for individual assignment, followed by the selection based on the allele number of individual markers. By combining rapid DNA extraction, and high-throughput genotyping of selected microsatellites, it is possible to conduct routine genetic traceability with high accuracy in Asian seabass. PMID:23285169

Elaeis oleifera Genomic-SSR Markers: Exploitation in Oil Palm Germplasm Diversity and Cross-Amplification in Arecaceae

PubMed Central

Zaki, Noorhariza Mohd; Singh, Rajinder; Rosli, Rozana; Ismail, Ismanizan

2012-01-01

Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (Ho) (0.164) and highly positive fixation indices (Fis) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family. PMID:22605966

Verification of STS markers for leaf rust resistance genes of wheat by seven European laboratories.

PubMed

Błaszczyk, Lidia; Chełkowski, Jerzy; Korzun, Victor; Kraic, Jan; Ordon, Frank; Ovesná, Jaroslava; Purnhauser, Laszlo; Tar, Melinda; Vida, Gyula

2004-01-01

A set of Thatcher near-isogenic lines and two breeding lines were used to examine sequence tagged site (STS) markers linked to leaf rust resistance genes Lr9, Lr10, Lr19, Lr24, Lr28, Lr29, Lr35, and a simple sequenced repeat (SSR) marker for Lr39. The selected STS markers for resistance genes Lr9, Lr10, Lr19, Lr24 and Lr28 were identified in seven accessions by seven European laboratories. Near-isogenic lines of the spring wheat Thatcher were used as positive controls. Markers for resistance genes Lr9, Lr10, Lr19, Lr24 were identified in all seven laboratories as amplification products of 1100 bp, 310 bp, 130 bp and 310 bp, respectively. The STS markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr29, Lr35 and the SSR marker for Lr39 were robust and highly specific for these genes and will be useful in marker-assisted selection in wheat. However, the amplification product of 378 bp that corresponded with resistance gene Lr28 was detected in all accessions including genotypes lacking this gene in all seven laboratories. This marker needs to be improved.

Genome wide selection in Citrus breeding.

PubMed

Gois, I B; Borém, A; Cristofani-Yaly, M; de Resende, M D V; Azevedo, C F; Bastianel, M; Novelli, V M; Machado, M A

2016-10-17

Genome wide selection (GWS) is essential for the genetic improvement of perennial species such as Citrus because of its ability to increase gain per unit time and to enable the efficient selection of characteristics with low heritability. This study assessed GWS efficiency in a population of Citrus and compared it with selection based on phenotypic data. A total of 180 individual trees from a cross between Pera sweet orange (Citrus sinensis Osbeck) and Murcott tangor (Citrus sinensis Osbeck x Citrus reticulata Blanco) were evaluated for 10 characteristics related to fruit quality. The hybrids were genotyped using 5287 DArT_seq TM (diversity arrays technology) molecular markers and their effects on phenotypes were predicted using the random regression - best linear unbiased predictor (rr-BLUP) method. The predictive ability, prediction bias, and accuracy of GWS were estimated to verify its effectiveness for phenotype prediction. The proportion of genetic variance explained by the markers was also computed. The heritability of the traits, as determined by markers, was 16-28%. The predictive ability of these markers ranged from 0.53 to 0.64, and the regression coefficients between predicted and observed phenotypes were close to unity. Over 35% of the genetic variance was accounted for by the markers. Accuracy estimates with GWS were lower than those obtained by phenotypic analysis; however, GWS was superior in terms of genetic gain per unit time. Thus, GWS may be useful for Citrus breeding as it can predict phenotypes early and accurately, and reduce the length of the selection cycle. This study demonstrates the feasibility of genomic selection in Citrus.

Integrative Analysis of High-throughput Cancer Studies with Contrasted Penalization

PubMed Central

Shi, Xingjie; Liu, Jin; Huang, Jian; Zhou, Yong; Shia, BenChang; Ma, Shuangge

2015-01-01

In cancer studies with high-throughput genetic and genomic measurements, integrative analysis provides a way to effectively pool and analyze heterogeneous raw data from multiple independent studies and outperforms “classic” meta-analysis and single-dataset analysis. When marker selection is of interest, the genetic basis of multiple datasets can be described using the homogeneity model or the heterogeneity model. In this study, we consider marker selection under the heterogeneity model, which includes the homogeneity model as a special case and can be more flexible. Penalization methods have been developed in the literature for marker selection. This study advances from the published ones by introducing the contrast penalties, which can accommodate the within- and across-dataset structures of covariates/regression coefficients and, by doing so, further improve marker selection performance. Specifically, we develop a penalization method that accommodates the across-dataset structures by smoothing over regression coefficients. An effective iterative algorithm, which calls an inner coordinate descent iteration, is developed. Simulation shows that the proposed method outperforms the benchmark with more accurate marker identification. The analysis of breast cancer and lung cancer prognosis studies with gene expression measurements shows that the proposed method identifies genes different from those using the benchmark and has better prediction performance. PMID:24395534

Bulk development and stringent selection of microsatellite markers in the western flower thrips Frankliniella occidentalis

PubMed Central

Cao, Li-Jun; Li, Ze-Min; Wang, Ze-Hua; Zhu, Liang; Gong, Ya-Jun; Chen, Min; Wei, Shu-Jun

2016-01-01

Recent improvements in next-generation sequencing technologies have enabled investigation of microsatellites on a genome-wide scale. Faced with a huge amount of candidates, the use of appropriate marker selection criteria is crucial. Here, we used the western flower thrips Frankliniella occidentalis for an empirical microsatellite survey and validation; 132,251 candidate microsatellites were identified, 92,102 of which were perfect. Dinucleotides were the most abundant category, while (AG)n was the most abundant motif. Sixty primer pairs were designed and validated in two natural populations, of which 30 loci were polymorphic, stable, and repeatable, but not all in Hardy–Weinberg equilibrium (HWE) and linkage equilibrium. Four marker panels were constructed to understand effect of marker selection on population genetic analyses: (i) only accept loci with single nucleotide insertions (SNI); (ii) only accept the most polymorphic loci (MP); (iii) only accept loci that did not deviate from HWE, did not show SNIs, and had unambiguous peaks (SS) and (iv) all developed markers (ALL). Although the MP panel resulted in microsatellites of highest genetic diversity followed by the SNI, the SS performed best in individual assignment. Our study proposes stringent criteria for selection of microsatellites from a large-scale number of genomic candidates for population genetic studies. PMID:27197749

Bulk development and stringent selection of microsatellite markers in the western flower thrips Frankliniella occidentalis.

PubMed

Cao, Li-Jun; Li, Ze-Min; Wang, Ze-Hua; Zhu, Liang; Gong, Ya-Jun; Chen, Min; Wei, Shu-Jun

2016-05-20

Recent improvements in next-generation sequencing technologies have enabled investigation of microsatellites on a genome-wide scale. Faced with a huge amount of candidates, the use of appropriate marker selection criteria is crucial. Here, we used the western flower thrips Frankliniella occidentalis for an empirical microsatellite survey and validation; 132,251 candidate microsatellites were identified, 92,102 of which were perfect. Dinucleotides were the most abundant category, while (AG)n was the most abundant motif. Sixty primer pairs were designed and validated in two natural populations, of which 30 loci were polymorphic, stable, and repeatable, but not all in Hardy-Weinberg equilibrium (HWE) and linkage equilibrium. Four marker panels were constructed to understand effect of marker selection on population genetic analyses: (i) only accept loci with single nucleotide insertions (SNI); (ii) only accept the most polymorphic loci (MP); (iii) only accept loci that did not deviate from HWE, did not show SNIs, and had unambiguous peaks (SS) and (iv) all developed markers (ALL). Although the MP panel resulted in microsatellites of highest genetic diversity followed by the SNI, the SS performed best in individual assignment. Our study proposes stringent criteria for selection of microsatellites from a large-scale number of genomic candidates for population genetic studies.

Association Mapping of Disease Resistance Traits in Rainbow Trout Using Restriction Site Associated DNA Sequencing

PubMed Central

Campbell, Nathan R.; LaPatra, Scott E.; Overturf, Ken; Towner, Richard; Narum, Shawn R.

2014-01-01

Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site−associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs. PMID:25354781

Fine Mapping for Identification of Citrus Alternaria Brown Spot Candidate Resistance Genes and Development of New SNP Markers for Marker-Assisted Selection

PubMed Central

Cuenca, Jose; Aleza, Pablo; Garcia-Lor, Andres; Ollitrault, Patrick; Navarro, Luis

2016-01-01

Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level. PMID:28066498

Genetic Structure, Linkage Disequilibrium and Signature of Selection in Sorghum: Lessons from Physically Anchored DArT Markers

PubMed Central

Bouchet, Sophie; Pot, David; Deu, Monique; Rami, Jean-François; Billot, Claire; Perrier, Xavier; Rivallan, Ronan; Gardes, Laëtitia; Xia, Ling; Wenzl, Peter; Kilian, Andrzej; Glaszmann, Jean-Christophe

2012-01-01

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r2 decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod. PMID:22428056

Comparison of Models and Whole-Genome Profiling Approaches for Genomic-Enabled Prediction of Septoria Tritici Blotch, Stagonospora Nodorum Blotch, and Tan Spot Resistance in Wheat.

PubMed

Juliana, Philomin; Singh, Ravi P; Singh, Pawan K; Crossa, Jose; Rutkoski, Jessica E; Poland, Jesse A; Bergstrom, Gary C; Sorrells, Mark E

2017-07-01

The leaf spotting diseases in wheat that include Septoria tritici blotch (STB) caused by , Stagonospora nodorum blotch (SNB) caused by , and tan spot (TS) caused by pose challenges to breeding programs in selecting for resistance. A promising approach that could enable selection prior to phenotyping is genomic selection that uses genome-wide markers to estimate breeding values (BVs) for quantitative traits. To evaluate this approach for seedling and/or adult plant resistance (APR) to STB, SNB, and TS, we compared the predictive ability of least-squares (LS) approach with genomic-enabled prediction models including genomic best linear unbiased predictor (GBLUP), Bayesian ridge regression (BRR), Bayes A (BA), Bayes B (BB), Bayes Cπ (BC), Bayesian least absolute shrinkage and selection operator (BL), and reproducing kernel Hilbert spaces markers (RKHS-M), a pedigree-based model (RKHS-P) and RKHS markers and pedigree (RKHS-MP). We observed that LS gave the lowest prediction accuracies and RKHS-MP, the highest. The genomic-enabled prediction models and RKHS-P gave similar accuracies. The increase in accuracy using genomic prediction models over LS was 48%. The mean genomic prediction accuracies were 0.45 for STB (APR), 0.55 for SNB (seedling), 0.66 for TS (seedling) and 0.48 for TS (APR). We also compared markers from two whole-genome profiling approaches: genotyping by sequencing (GBS) and diversity arrays technology sequencing (DArTseq) for prediction. While, GBS markers performed slightly better than DArTseq, combining markers from the two approaches did not improve accuracies. We conclude that implementing GS in breeding for these diseases would help to achieve higher accuracies and rapid gains from selection. Copyright © 2017 Crop Science Society of America.

Genetic structure, linkage disequilibrium and signature of selection in Sorghum: lessons from physically anchored DArT markers.

PubMed

Bouchet, Sophie; Pot, David; Deu, Monique; Rami, Jean-François; Billot, Claire; Perrier, Xavier; Rivallan, Ronan; Gardes, Laëtitia; Xia, Ling; Wenzl, Peter; Kilian, Andrzej; Glaszmann, Jean-Christophe

2012-01-01

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r(2) decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod.

RNA-Seq identifies SNP markers for growth traits in rainbow trout.

PubMed

Salem, Mohamed; Vallejo, Roger L; Leeds, Timothy D; Palti, Yniv; Liu, Sixin; Sabbagh, Annas; Rexroad, Caird E; Yao, Jianbo

2012-01-01

Fast growth is an important and highly desired trait, which affects the profitability of food animal production, with feed costs accounting for the largest proportion of production costs. Traditional phenotype-based selection is typically used to select for growth traits; however, genetic improvement is slow over generations. Single nucleotide polymorphisms (SNPs) explain 90% of the genetic differences between individuals; therefore, they are most suitable for genetic evaluation and strategies that employ molecular genetics for selective breeding. SNPs found within or near a coding sequence are of particular interest because they are more likely to alter the biological function of a protein. We aimed to use SNPs to identify markers and genes associated with genetic variation in growth. RNA-Seq whole-transcriptome analysis of pooled cDNA samples from a population of rainbow trout selected for improved growth versus unselected genetic cohorts (10 fish from 1 full-sib family each) identified SNP markers associated with growth-rate. The allelic imbalances (the ratio between the allele frequencies of the fast growing sample and that of the slow growing sample) were considered at scores >5.0 as an amplification and <0.2 as loss of heterozygosity. A subset of SNPs (n = 54) were validated and evaluated for association with growth traits in 778 individuals of a three-generation parent/offspring panel representing 40 families. Twenty-two SNP markers and one mitochondrial haplotype were significantly associated with growth traits. Polymorphism of 48 of the markers was confirmed in other commercially important aquaculture stocks. Many markers were clustered into genes of metabolic energy production pathways and are suitable candidates for genetic selection. The study demonstrates that RNA-Seq at low sequence coverage of divergent populations is a fast and effective means of identifying SNPs, with allelic imbalances between phenotypes. This technique is suitable for marker development in non-model species lacking complete and well-annotated genome reference sequences.

Sequence-characterized amplified polymorphism markers for selecting rind stripe pattern in watermelon (Citrullus lanatus var. lanatus)

USDA-ARS?s Scientific Manuscript database

The inheritance of foreground stripe pattern in rind of watermelon fruits [Citrullus lanatus (Thunb.) Matsum. & Nakai] was evaluated and the molecular markers for selecting the JT stripe pattern were developed based on bulked segregant analysis (BSA). Divergence in rind pattern among F2 progeny deri...

Advances and Challenges in Genomic Selection for Disease Resistance.

PubMed

Poland, Jesse; Rutkoski, Jessica

2016-08-04

Breeding for disease resistance is a central focus of plant breeding programs, as any successful variety must have the complete package of high yield, disease resistance, agronomic performance, and end-use quality. With the need to accelerate the development of improved varieties, genomics-assisted breeding is becoming an important tool in breeding programs. With marker-assisted selection, there has been success in breeding for disease resistance; however, much of this work and research has focused on identifying, mapping, and selecting for major resistance genes that tend to be highly effective but vulnerable to breakdown with rapid changes in pathogen races. In contrast, breeding for minor-gene quantitative resistance tends to produce more durable varieties but is a more challenging breeding objective. As the genetic architecture of resistance shifts from single major R genes to a diffused architecture of many minor genes, the best approach for molecular breeding will shift from marker-assisted selection to genomic selection. Genomics-assisted breeding for quantitative resistance will therefore necessitate whole-genome prediction models and selection methodology as implemented for classical complex traits such as yield. Here, we examine multiple case studies testing whole-genome prediction models and genomic selection for disease resistance. In general, whole-genome models for disease resistance can produce prediction accuracy suitable for application in breeding. These models also largely outperform multiple linear regression as would be applied in marker-assisted selection. With the implementation of genomic selection for yield and other agronomic traits, whole-genome marker profiles will be available for the entire set of breeding lines, enabling genomic selection for disease at no additional direct cost. In this context, the scope of implementing genomics selection for disease resistance, and specifically for quantitative resistance and quarantined pathogens, becomes a tractable and powerful approach in breeding programs.

Endogenous enzyme activities and polyamine levels in diverse rice cultivars depend on the genetic background and are not affected by the presence of the hygromycin phosphotransferase selectable marker.

PubMed

Lepri, O.; Bassie, L.; Thu-Hang, P.; Christou, P.; Capell, T.

2002-09-01

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.

Association of RGA-SSCP markers with resistance to downy mildew and anthracnose in grapevines.

PubMed

Tantasawat, P A; Poolsawat, O; Prajongjai, T; Chaowiset, W; Tharapreuksapong, A

2012-07-02

Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.

A family of LRR sequences in the vicinity of the Co-2 locus for anthracnose resistance in Phaseolus vulgaris and its potential use in marker-assisted selection.

PubMed

Geffroy, V; Creusot, F; Falquet, J; Sévignac, M; Adam-Blondon, A F; Bannerot, H; Gepts, P; Dron, M

1998-03-01

Molecular markers offer new opportunities for breeding for disease resistance. Resistance gene pyramiding in a single cultivar, as a strategy for durable resistance, can be facilitated by marker-assisted selection (MAS). A RAPD marker, ROH20(450), linked to the Mesoamerican Co-2 anthracnose resistance gene, was previously transformed into a SCAR marker, SCH20. In the present paper we have further characterized the relevance of the SCH20 SCAR marker in different genetic backgrounds. Since this SCAR marker was found to be useful mainly in the Andean gene pool, we identified a new PCR-based marker (SCAreoli) for indirect scoring of the presence of the Co-2 gene. The SCAreoli SCAR marker is polymorphic in the Mesoamerican as well as in the Andean gene pool and should be useful in MAS. We also report that PvH20, the cloned sequence corresponding to the 450-bp RAPD marker ROH20(450), contains six imperfect leucine-rich repeats, and reveals a family of related sequences in the vicinity of the Co-2 locus. These results are discussed in the context of the recent cloning of some plant resistance genes.

Genomic selection & association mapping in rice: effect of trait genetic architecture, training population composition, marker number & statistical model on accuracy of rice genomic selection in elite, tropical rice breeding

USDA-ARS?s Scientific Manuscript database

Genomic Selection (GS) is a new breeding method in which genome-wide markers are used to predict the breeding value of individuals in a breeding population. GS has been shown to improve breeding efficiency in dairy cattle and several crop plant species, and here we evaluate for the first time its ef...

µ-Calpain (CAPN1), calpastatin (CAST), and growth hormone receptor (GHR) genetic effects on Angus beef heifer performance traits and reproduction

USDA-ARS?s Scientific Manuscript database

Genetic marker effects and type of inheritance are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to equalize CAPN1 haplotypes, CAST, and GHR genetic marker frequencies. The objective was t...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Marker residue and target tissue. 500.86 Section...-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall...) From these data, FDA will select a target tissue and a marker residue and designate the concentration...

21 CFR 500.86 - Marker residue and target tissue.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Marker residue and target tissue. 500.86 Section...-Producing Animals § 500.86 Marker residue and target tissue. (a) For each edible tissue, the sponsor shall...) From these data, FDA will select a target tissue and a marker residue and designate the concentration...

A framework linkage map of perennial ryegrass based on SSR markers

Treesearch

G.P. Gill; P.L. Wilcox; D.J. Whittaker; R.A. Winz; P. Bickerstaff; Craig E. Echt; J. Kent; M.O. Humphreys; K.M. Elborough; R.C. Gardner

2006-01-01

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third ( 124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci...

[DNA marker-assisted selection of medicinal plants (Ⅰ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

PubMed

Dong, Lin-Lin; Chen, Zhong-Jian; Wang, Yong; Wei, Fu-Gang; Zhang, Lian-Juan; Xu, Jiang; Wei, Guang-Fei; Wang, Rui; Yang, Juan; Liu, Wei-Lin; Li, Xi-Wen; Yu, Yu-Qi; Chen, Shi-Lin

2017-01-01

DNA marker-assisted selection of medicinal plants is based on the DNA polymorphism, selects the DNA sequences related to the phenotypes such as high yields, superior quality, stress-resistance and so on according to the technologies of molecular hybridization, polymerase chain reaction and high-throughput sequencing, and assists the breeding of new cultivars. This study bred the first disease-resistant cultivar of notoginseng "Miaoxiang Kangqi 1" using the technology of DNA marker-assisted selection of medicinal plants and systematic breeding. The disease-resistant cultivar of notoginseng contained 12 special SNPs based on the analysis of Restriction-site Associated DNA Sequencing (RAD-Seq). Among the SNP (record_519688) was related to the root rot-resistant characteristics, which indicated this SNP could serve as genetic markers of disease-resistant cultivars and assist the systematic breeding. Compared to the conventional cultivated cultivars, the incidence rate of root-rot and rust-rot in notoginseng seedlings decreased by 83.6% and 71.8%, respectively. The incidence rate of root-rot respectively declined by 43.6% and 62.9% in notoginseng cultivation for 2 and 3 years compared with those of the conventional cultivated cultivars. Additionally, the potential disease-resistant groups were screened based on the relative SNP, and this model enlarged the target groups and advanced the breeding efficiency. DNA marker-assisted selection of medicinal plants accelerated the breeding and promotion of new cultivars, and guaranteed the healthy development of Chinese medicinal materials industry. Copyright© by the Chinese Pharmaceutical Association.

Screening and identification of a microsatellite marker associated with sex in Wami tilapia, Oreochromis urolepis hornorum.

PubMed

Zhu, Huaping; Liu, Zhigang; Lu, Maixin; Gao, Fengying; Ke, Xiaoli; Ma, Dongmei; Huang, Zhanghan; Cao, Jianmeng; Wang, Miao

2016-06-01

In this study, primer pairs of 15 microsatellite markers associated with sex determination of tilapia were selected and amplified in Wami tilapia, Oreochromis urolepis hornorum. While one marker, UNH168, on linkage group 3 (LG3) was associated (P <0.001) with the phenotypic sex in the experimental population, nine genotypes were detected in both sexes. Only 99-bp allele was detected in the female samples, while 141, 149 and 157-bp alleles were present in both male and female samples. UNH168 was localized by fluorescence in situ hybridization (FISH) on the long arm of the largest tilapia chromosome pair (chromosome 1, equivalent to LG3). This sex-linked microsatellite marker could potentially be used for marker-assisted selection in tilapia breeding programmes to produce monosex male tilapia.

CombiROC: an interactive web tool for selecting accurate marker combinations of omics data.

PubMed

Mazzara, Saveria; Rossi, Riccardo L; Grifantini, Renata; Donizetti, Simone; Abrignani, Sergio; Bombaci, Mauro

2017-03-30

Diagnostic accuracy can be improved considerably by combining multiple markers, whose performance in identifying diseased subjects is usually assessed via receiver operating characteristic (ROC) curves. The selection of multimarker signatures is a complicated process that requires integration of data signatures with sophisticated statistical methods. We developed a user-friendly tool, called CombiROC, to help researchers accurately determine optimal markers combinations from diverse omics methods. With CombiROC data from different domains, such as proteomics and transcriptomics, can be analyzed using sensitivity/specificity filters: the number of candidate marker panels rising from combinatorial analysis is easily optimized bypassing limitations imposed by the nature of different experimental approaches. Leaving to the user full control on initial selection stringency, CombiROC computes sensitivity and specificity for all markers combinations, performances of best combinations and ROC curves for automatic comparisons, all visualized in a graphic interface. CombiROC was designed without hard-coded thresholds, allowing a custom fit to each specific data: this dramatically reduces the computational burden and lowers the false negative rates given by fixed thresholds. The application was validated with published data, confirming the marker combination already originally described or even finding new ones. CombiROC is a novel tool for the scientific community freely available at http://CombiROC.eu.

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication.

PubMed

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-06-04

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to "Gopoong" and "K-1" were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information.

[A Method for Selecting Self-Adoptive Chromaticity of the Projected Markers].

PubMed

Zhao, Shou-bo; Zhang, Fu-min; Qu, Xing-hua; Zheng, Shi-wei; Chen, Zhe

2015-04-01

The authors designed a self-adaptive projection system which is composed of color camera, projector and PC. In detail, digital micro-mirror device (DMD) as a spatial light modulator for the projector was introduced in the optical path to modulate the illuminant spectrum based on red, green and blue light emitting diodes (LED). However, the color visibility of active markers is affected by the screen which has unknown reflective spectrum as well. Here active markers are projected spot array. And chromaticity feature of markers is sometimes submerged in similar spectral screen. In order to enhance the color visibility of active markers relative to screen, a method for selecting self-adaptive chromaticity of the projected markers in 3D scanning metrology is described. Color camera with 3 channels limits the accuracy of device characterization. For achieving interconversion of device-independent color space and device-dependent color space, high-dimensional linear model of reflective spectrum was built. Prior training samples provide additional constraints to yield high-dimensional linear model with more than three degrees of freedom. Meanwhile, spectral power distribution of ambient light was estimated. Subsequently, markers' chromaticity in CIE color spaces was selected via maximization principle of Euclidean distance. The setting values of RGB were easily estimated via inverse transform. Finally, we implemented a typical experiment to show the performance of the proposed approach. An 24 Munsell Color Checker was used as projective screen. Color difference in the chromaticity coordinates between the active marker and the color patch was utilized to evaluate the color visibility of active markers relative to the screen. The result comparison between self-adaptive projection system and traditional diode-laser light projector was listed and discussed to highlight advantage of our proposed method.

Molecular mapping and marker development for the Triticum dicoccoides-derived stripe rust resistance gene YrSM139-1B in bread wheat cv. Shaanmai 139.

PubMed

Zhang, Hong; Zhang, Lu; Wang, Changyou; Wang, Yajuan; Zhou, Xinli; Lv, Shikai; Liu, Xinlun; Kang, Zhensheng; Ji, Wanquan

2016-02-01

YrSM139-1B maybe a new gene for effective resistance to stripe rust and useful flanking markers for marker-assisted selection were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. Two dominant stripe rust resistant genes YrSM139-1B and YrSM139-2D were pyramided in bread wheat cultivar Shaanmai 139; one from wild emmer and the other from Thinopyrum intermedium. Three near-isogenic F7:8 line pairs (contrasting RILs), N122-1013R/S, N122-185R/S, and N122-1812R/S, independently derived from different F2 plants and differing at the YrSM139-1B locus were generated from the cross Shaanmai 139 × Hu 901-19 through marker-assisted selection. A large F2:3 population from cross N122-1013R × N122-1013S tested for stripe rust response and subjected to analysis with markers in the 1BS10-0.5 bin region using SSR expressed sequence tags (EST) and site-specific sequence markers developed from the 90 K Illumina iSelect SNP array. Five EST-STS markers and four allele-specific PCR markers were mapped to the YrSM139-1B region. The 30.5 cM genetic map for YrSM139-1B consisted of nine markers, two of which were closer to YrSM139-1B than Xgwm273, which was used in producing the contrasting RIL pairs. Race response data and allelism tests showed that YrSM139-1B is different from Yr10, Yr15, and Yr24/26/CH42.

Pathogenic bacteria and microbial-source tracking markers in Brandywine Creek Basin, Pennsylvania and Delaware, 2009-10

USGS Publications Warehouse

Duris, Joseph W.; Reif, Andrew G.; Olson, Leif E.; Johnson, Heather E.

2011-01-01

The City of Wilmington, Delaware, is in the downstream part of the Brandywine Creek Basin, on the main stem of Brandywine Creek. Wilmington uses this stream, which drains a mixed-land-use area upstream, for its main drinking-water supply. Because the stream is used for drinking water, Wilmington is in need of information about the occurrence and distribution of specific fecally derived pathogenic bacteria (disease-causing bacteria) and their relations to commonly measured fecal-indicator bacteria (FIB), as well as information regarding the potential sources of the fecal pollution and pathogens in the basin. This study focused on five routinely sampled sites within the basin, one each on the West Branch and the East Branch of Brandywine Creek and at three on the main stem below the confluence of the West and East Branches. These sites were sampled monthly for 1 year. Targeted event samples were collected on two occasions during high flow and two occasions during normal flow. On the basis of this study, high flows in the Brandywine Creek Basin were related to increases in FIB densities, and in the frequency of selected pathogen and source markers, in the West Branch and main stem of Brandywine Creek, but not in the East Branch. Water exceeding the moderate fullbody-contact single-sample recreational water-quality criteria (RWQC) for Escherichia coli (E. coli) was more likely to contain selected markers for pathogenic E. coli (eaeA,stx1, and rfbO157 gene markers) and bovine fecal sources (E. hirae and LTIIa gene markers), whereas samples exceeding the enterococci RWQC were more likely to contain the same pathogenic markers but also were more likely to carry a marker indicative of human source (esp gene marker). On four sample dates, during high flow between October and March, the West Branch was the only observed potential contributor of selected pathogen and bovine source markers to the main stem of Brandywine Creek. Indeed, the stx2 marker, which indicates a highly virulent type of pathogenic E. coli, was found only in the West Branch and main stem at high flow but was not found in the East Branch under similar conditions. However, it must be noted that throughout the entire year of sampling there were occasions, during both high and normal flows, when both the East and West Branches were potential contributors of pathogen and microbial-source tracking markers to the main stem. Therefore, this study indicates that under selected conditions (high flow, October through March), West Branch Brandywine Creek Basin was the most likely source of elevated FIB densities in the main stem. These elevated densities are associated with more frequent detection of selected pathogenic E. coli markers (rfbO157 stx1) and are associated with MST markers of bovine source. However, during other times of the year, both the West Branch and East Branch Basins are acting as potential sources of FIB and fecally derived pathogens.

A 48 SNP set for grapevine cultivar identification

PubMed Central

2011-01-01

Background Rapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification. Results We analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification. Conclusions We have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable. PMID:22060012

Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA

USDA-ARS?s Scientific Manuscript database

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise ...

Development of next-generation mapping populations: Multi-parent Advanced Generation Inter-Cross (MAGIC) and Marker-Assisted Recurrent Selection (MARS) populations in peanut

USDA-ARS?s Scientific Manuscript database

Generation Inter-Cross (MAGIC) and Marker-Assisted Recurrent Selection (MARS) have been proposed and used in many crops to dissect complex traits or QTL. MAGIC allows for dissecting genomic structure, and for improving breeding populations by integrating multiple alleles from different parents. MAR...

Accuracy and training population design for genomic selection in elite north american oats

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) is a method to estimate the breeding values of individuals by using markers throughout the genome. We evaluated the accuracies of GS using data from five traits on 446 oat lines genotyped with 1005 Diversity Array Technology (DArT) markers and two GS methods (RR-BLUP and Bayes...

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao.

PubMed

Lima, L S; Gramacho, K P; Carels, N; Novais, R; Gaiotto, F A; Lopes, U V; Gesteira, A S; Zaidan, H A; Cascardo, J C M; Pires, J L; Micheli, F

2009-07-14

In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Maximization of Markers Linked in Coupling for Tetraploid Potatoes via Monoparental Haploids

PubMed Central

Bartkiewicz, Annette M.; Chilla, Friederike; Terefe-Ayana, Diro; Lübeck, Jens; Strahwald, Josef; Tacke, Eckhard; Hofferbert, Hans-Reinhard; Linde, Marcus; Debener, Thomas

2018-01-01

Haploid potato populations derived from a single tetraploid donor constitute an efficient strategy to analyze markers segregating from a single donor genotype. Analysis of marker segregation in populations derived from crosses between polysomic tetraploids is complicated by a maximum of eight segregating alleles, multiple dosages of the markers and problems related to linkage analysis of marker segregation in repulsion. Here, we present data on two monoparental haploid populations generated by prickle pollination of two tetraploid cultivars with Solanum phureja and genotyped with the 12.8 k SolCAP single nucleotide polymorphism (SNP) array. We show that in a population of monoparental haploids, the number of biallelic SNP markers segregating in linkage to loci from the tetraploid donor genotype is much larger than in putative crosses of this genotype to a diverse selection of 125 tetraploid cultivars. Although this strategy is more laborious than conventional breeding, the generation of haploid progeny for efficient marker analysis is straightforward if morphological markers and flow cytometry are utilized to select true haploid progeny. The level of introgressed fragments from S. phureja, the haploid inducer, is very low, supporting its suitability for genetic analysis. Mapping with single-dose markers allowed the analysis of quantitative trait loci (QTL) for four phenotypic traits. PMID:29868076

[Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen].

PubMed

Rukavtsova, E B; Gaiazova, A R; Chebotareva, E N; Bur'ianova, Ia I

2009-08-01

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

Empirical Selection of Informative Microsatellite Markers within Co-ancestry Pig Populations Is Required for Improving the Individual Assignment Efficiency

PubMed Central

Li, Y. H.; Chu, H. P.; Jiang, Y. N.; Lin, C. Y.; Li, S. H.; Li, K. T.; Weng, G. J.; Cheng, C. C.; Lu, D. J.; Ju, Y. T.

2014-01-01

The Lanyu is a miniature pig breed indigenous to Lanyu Island, Taiwan. It is distantly related to Asian and European pig breeds. It has been inbred to generate two breeds and crossed with Landrace and Duroc to produce two hybrids for laboratory use. Selecting sets of informative genetic markers to track the genetic qualities of laboratory animals and stud stock is an important function of genetic databases. For more than two decades, Lanyu derived breeds of common ancestry and crossbreeds have been used to examine the effectiveness of genetic marker selection and optimal approaches for individual assignment. In this paper, these pigs and the following breeds: Berkshire, Duroc, Landrace and Yorkshire, Meishan and Taoyuan, TLRI Black Pig No. 1, and Kaohsiung Animal Propagation Station Black pig are studied to build a genetic reference database. Nineteen microsatellite markers (loci) provide information on genetic variation and differentiation among studied breeds. High differentiation index (FST) and Cavalli-Sforza chord distances give genetic differentiation among breeds, including Lanyu’s inbred populations. Inbreeding values (FIS) show that Lanyu and its derived inbred breeds have significant loss of heterozygosity. Individual assignment testing of 352 animals was done with different numbers of microsatellite markers in this study. The testing assigned 99% of the animals successfully into their correct reference populations based on 9 to 14 markers ranking D-scores, allelic number, expected heterozygosity (HE) or FST, respectively. All miss-assigned individuals came from close lineage Lanyu breeds. To improve individual assignment among close lineage breeds, microsatellite markers selected from Lanyu populations with high polymorphic, heterozygosity, FST and D-scores were used. Only 6 to 8 markers ranking HE, FST or allelic number were required to obtain 99% assignment accuracy. This result suggests empirical examination of assignment-error rates is required if discernible levels of co-ancestry exist. In the reference group, optimum assignment accuracy was achievable achieved through a combination of different markers by ranking the heterozygosity, FST and allelic number of close lineage populations. PMID:25049996

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes.

PubMed

Krak, Karol; Alvarez, Inés; Caklová, Petra; Costa, Andrea; Chrtek, Jindrich; Fehrer, Judith

2012-02-01

The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology(1.).

PubMed

Robarts, Daniel W H; Wolfe, Andrea D

2014-07-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance.

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology1

PubMed Central

Robarts, Daniel W. H.; Wolfe, Andrea D.

2014-01-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance. PMID:25202637

Mapping Late Leaf Spot Resistance in Peanut (Arachis hypogaea) Using QTL-seq Reveals Markers for Marker-Assisted Selection.

PubMed

Clevenger, Josh; Chu, Ye; Chavarro, Carolina; Botton, Stephanie; Culbreath, Albert; Isleib, Thomas G; Holbrook, C C; Ozias-Akins, Peggy

2018-01-01

Late leaf spot (LLS; Cercosporidium personatum ) is a major fungal disease of cultivated peanut ( Arachis hypogaea ). A recombinant inbred line population segregating for quantitative field resistance was used to identify quantitative trait loci (QTL) using QTL-seq. High rates of false positive SNP calls using established methods in this allotetraploid crop obscured significant QTLs. To resolve this problem, robust parental SNPs were first identified using polyploid-specific SNP identification pipelines, leading to discovery of significant QTLs for LLS resistance. These QTLs were confirmed over 4 years of field data. Selection with markers linked to these QTLs resulted in a significant increase in resistance, showing that these markers can be immediately applied in breeding programs. This study demonstrates that QTL-seq can be used to rapidly identify QTLs controlling highly quantitative traits in polyploid crops with complex genomes. Markers identified can then be deployed in breeding programs, increasing the efficiency of selection using molecular tools. Key Message: Field resistance to late leaf spot is a quantitative trait controlled by many QTLs. Using polyploid-specific methods, QTL-seq is faster and more cost effective than QTL mapping.

Mapping Late Leaf Spot Resistance in Peanut (Arachis hypogaea) Using QTL-seq Reveals Markers for Marker-Assisted Selection

PubMed Central

Clevenger, Josh; Chu, Ye; Chavarro, Carolina; Botton, Stephanie; Culbreath, Albert; Isleib, Thomas G.; Holbrook, C. C.; Ozias-Akins, Peggy

2018-01-01

Late leaf spot (LLS; Cercosporidium personatum) is a major fungal disease of cultivated peanut (Arachis hypogaea). A recombinant inbred line population segregating for quantitative field resistance was used to identify quantitative trait loci (QTL) using QTL-seq. High rates of false positive SNP calls using established methods in this allotetraploid crop obscured significant QTLs. To resolve this problem, robust parental SNPs were first identified using polyploid-specific SNP identification pipelines, leading to discovery of significant QTLs for LLS resistance. These QTLs were confirmed over 4 years of field data. Selection with markers linked to these QTLs resulted in a significant increase in resistance, showing that these markers can be immediately applied in breeding programs. This study demonstrates that QTL-seq can be used to rapidly identify QTLs controlling highly quantitative traits in polyploid crops with complex genomes. Markers identified can then be deployed in breeding programs, increasing the efficiency of selection using molecular tools. Key Message: Field resistance to late leaf spot is a quantitative trait controlled by many QTLs. Using polyploid-specific methods, QTL-seq is faster and more cost effective than QTL mapping. PMID:29459876

On prediction of genetic values in marker-assisted selection.

PubMed Central

Lange, C; Whittaker, J C

2001-01-01

We suggest a new approximation for the prediction of genetic values in marker-assisted selection. The new approximation is compared to the standard approach. It is shown that the new approach will often provide substantially better prediction of genetic values; furthermore the new approximation avoids some of the known statistical problems of the standard approach. The advantages of the new approach are illustrated by a simulation study in which the new approximation outperforms both the standard approach and phenotypic selection. PMID:11729177

Selection of recombinant MVA by rescue of the essential D4R gene.

PubMed

Ricci, Patricia S; Schäfer, Birgit; Kreil, Thomas R; Falkner, Falko G; Holzer, Georg W

2011-12-12

Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.

Saturation of an Intra-Gene Pool Linkage Map: Towards a Unified Consensus Linkage Map for Fine Mapping and Synteny Analysis in Common Bean

PubMed Central

Galeano, Carlos H.; Fernandez, Andrea C.; Franco-Herrera, Natalia; Cichy, Karen A.; McClean, Phillip E.; Vanderleyden, Jos; Blair, Matthew W.

2011-01-01

Map-based cloning and fine mapping to find genes of interest and marker assisted selection (MAS) requires good genetic maps with reproducible markers. In this study, we saturated the linkage map of the intra-gene pool population of common bean DOR364×BAT477 (DB) by evaluating 2,706 molecular markers including SSR, SNP, and gene-based markers. On average the polymorphism rate was 7.7% due to the narrow genetic base between the parents. The DB linkage map consisted of 291 markers with a total map length of 1,788 cM. A consensus map was built using the core mapping populations derived from inter-gene pool crosses: DOR364×G19833 (DG) and BAT93×JALO EEP558 (BJ). The consensus map consisted of a total of 1,010 markers mapped, with a total map length of 2,041 cM across 11 linkage groups. On average, each linkage group on the consensus map contained 91 markers of which 83% were single copy markers. Finally, a synteny analysis was carried out using our highly saturated consensus maps compared with the soybean pseudo-chromosome assembly. A total of 772 marker sequences were compared with the soybean genome. A total of 44 syntenic blocks were identified. The linkage group Pv6 presented the most diverse pattern of synteny with seven syntenic blocks, and Pv9 showed the most consistent relations with soybean with just two syntenic blocks. Additionally, a co-linear analysis using common bean transcript map information against soybean coding sequences (CDS) revealed the relationship with 787 soybean genes. The common bean consensus map has allowed us to map a larger number of markers, to obtain a more complete coverage of the common bean genome. Our results, combined with synteny relationships provide tools to increase marker density in selected genomic regions to identify closely linked polymorphic markers for indirect selection, fine mapping or for positional cloning. PMID:22174773

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.

Genome-Wide Association Study for Identification and Validation of Novel SNP Markers for Sr6 Stem Rust Resistance Gene in Bread Wheat.

PubMed

Mourad, Amira M I; Sallam, Ahmed; Belamkar, Vikas; Wegulo, Stephen; Bowden, Robert; Jin, Yue; Mahdy, Ezzat; Bakheit, Bahy; El-Wafaa, Atif A; Poland, Jesse; Baenziger, Peter S

2018-01-01

Stem rust (caused by Puccinia graminis f. sp. tritici Erikss. & E. Henn.), is a major disease in wheat ( Triticum aestivium L.). However, in recent years it occurs rarely in Nebraska due to weather and the effective selection and gene pyramiding of resistance genes. To understand the genetic basis of stem rust resistance in Nebraska winter wheat, we applied genome-wide association study (GWAS) on a set of 270 winter wheat genotypes (A-set). Genotyping was carried out using genotyping-by-sequencing and ∼35,000 high-quality SNPs were identified. The tested genotypes were evaluated for their resistance to the common stem rust race in Nebraska (QFCSC) in two replications. Marker-trait association identified 32 SNP markers, which were significantly (Bonferroni corrected P < 0.05) associated with the resistance on chromosome 2D. The chromosomal location of the significant SNPs (chromosome 2D) matched the location of Sr6 gene which was expected in these genotypes based on pedigree information. A highly significant linkage disequilibrium (LD, r 2 ) was found between the significant SNPs and the specific SSR marker for the Sr6 gene ( Xcfd43 ). This suggests the significant SNP markers are tagging Sr6 gene. Out of the 32 significant SNPs, eight SNPs were in six genes that are annotated as being linked to disease resistance in the IWGSC RefSeq v1.0. The 32 significant SNP markers were located in nine haplotype blocks. All the 32 significant SNPs were validated in a set of 60 different genotypes (V-set) using single marker analysis. SNP markers identified in this study can be used in marker-assisted selection, genomic selection, and to develop KASP (Kompetitive Allele Specific PCR) marker for the Sr6 gene. Novel SNPs for Sr6 gene, an important stem rust resistant gene, were identified and validated in this study. These SNPs can be used to improve stem rust resistance in wheat.

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.

Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach

USDA-ARS?s Scientific Manuscript database

Expressed sequence tag (EST) simple sequence repeats (SSRs) in Prunus were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability. A total of 12,618 contigs were assembled from 84,727 ESTs, along with 34...

Comparison of genetic diversity and population structure of Pacific Coast whitebark pine across multiple markers

Treesearch

Andrew D. Bower; Bryce A. Richardson; Valerie Hipkins; Regina Rochefort; Carol Aubry

2011-01-01

Analysis of "neutral" molecular markers and "adaptive" quantitative traits are common methods of assessing genetic diversity and population structure. Molecular markers typically reflect the effects of demographic and stochastic processes but are generally assumed to not reflect natural selection. Conversely, quantitative (or "adaptive")...

Development of a gene-centered ssr atlas as a resource for papaya (Carica papaya) marker-assisted selection and population genetic studies.

PubMed

Vidal, Newton Medeiros; Grazziotin, Ana Laura; Ramos, Helaine Christine Cancela; Pereira, Messias Gonzaga; Venancio, Thiago Motta

2014-01-01

Carica papaya (papaya) is an economically important tropical fruit. Molecular marker-assisted selection is an inexpensive and reliable tool that has been widely used to improve fruit quality traits and resistance against diseases. In the present study we report the development and validation of an atlas of papaya simple sequence repeat (SSR) markers. We integrated gene predictions and functional annotations to provide a gene-centered perspective for marker-assisted selection studies. Our atlas comprises 160,318 SSRs, from which 21,231 were located in genic regions (i.e. inside exons, exon-intron junctions or introns). A total of 116,453 (72.6%) of all identified repeats were successfully mapped to one of the nine papaya linkage groups. Primer pairs were designed for markers from 9,594 genes (34.5% of the papaya gene complement). Using papaya-tomato orthology assessments, we assembled a list of 300 genes (comprising 785 SSRs) potentially involved in fruit ripening. We validated our atlas by screening 73 SSR markers (including 25 fruit ripening genes), achieving 100% amplification rate and uncovering 26% polymorphism rate between the parental genotypes (Sekati and JS12). The SSR atlas presented here is the first comprehensive gene-centered collection of annotated and genome positioned papaya SSRs. These features combined with thousands of high-quality primer pairs make the atlas an important resource for the papaya research community.

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication

PubMed Central

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-01-01

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to “Gopoong” and “K-1” were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information. PMID:27271615

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

PubMed

Ma, G J; Song, Q J; Markell, S G; Qi, L L

2018-07-01

A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F 2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

Solar Radiation-Associated Adaptive SNP Genetic Differentiation in Wild Emmer Wheat, Triticum dicoccoides.

PubMed

Ren, Jing; Chen, Liang; Jin, Xiaoli; Zhang, Miaomiao; You, Frank M; Wang, Jirui; Frenkel, Vladimir; Yin, Xuegui; Nevo, Eviatar; Sun, Dongfa; Luo, Ming-Cheng; Peng, Junhua

2017-01-01

Whole-genome scans with large number of genetic markers provide the opportunity to investigate local adaptation in natural populations and identify candidate genes under positive selection. In the present study, adaptation genetic differentiation associated with solar radiation was investigated using 695 polymorphic SNP markers in wild emmer wheat originated in a micro-site at Yehudiyya, Israel. The test involved two solar radiation niches: (1) sun, in-between trees; and (2) shade, under tree canopy, separated apart by a distance of 2-4 m. Analysis of molecular variance showed a small (0.53%) but significant portion of overall variation between the sun and shade micro-niches, indicating a non-ignorable genetic differentiation between sun and shade habitats. Fifty SNP markers showed a medium (0.05 ≤ F ST ≤ 0.15) or high genetic differentiation ( F ST > 0.15). A total of 21 outlier loci under positive selection were identified by using four different F ST -outlier testing algorithms. The markers and genome locations under positive selection are consistent with the known patterns of selection. These results suggested that genetic differentiation between sun and shade habitats is substantial, radiation-associated, and therefore ecologically determined. Hence, the results of this study reflected effects of natural selection through solar radiation on EST-related SNP genetic diversity, resulting presumably in different adaptive complexes at a micro-scale divergence. The present work highlights the evolutionary theory and application significance of solar radiation-driven natural selection in wheat improvement.

Balancing Selection and Its Effects on Sequences in Nearby Genome Regions

PubMed Central

Charlesworth, Deborah

2006-01-01

Our understanding of balancing selection is currently becoming greatly clarified by new sequence data being gathered from genes in which polymorphisms are known to be maintained by selection. The data can be interpreted in conjunction with results from population genetics models that include recombination between selected sites and nearby neutral marker variants. This understanding is making possible tests for balancing selection using molecular evolutionary approaches. Such tests do not necessarily require knowledge of the functional types of the different alleles at a locus, but such information, as well as information about the geographic distribution of alleles and markers near the genes, can potentially help towards understanding what form of balancing selection is acting, and how long alleles have been maintained. PMID:16683038

Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.).

PubMed

Elkot, Ahmed Fawzy Abdelnaby; Chhuneja, Parveen; Kaur, Satinder; Saluja, Manny; Keller, Beat; Singh, Kuldeep

2015-01-01

Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F1 of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F1. The resulting F1s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC1F1. Marker assisted selection was carried both in BC1F1 and the BC2F1 generations. Introgression of alien chromatin in BC2F1 plants varied from 15.4-62.9 percent. Out of more than 110 BC2F1 plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC2F2 plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC2F1 plants. Thus, in addition to PM resistance, these progeny might also carry resistance to stem rust race Ug99.

The GCP molecular marker toolkit, an instrument for use in breeding food security crops.

PubMed

Van Damme, Veerle; Gómez-Paniagua, Humberto; de Vicente, M Carmen

2011-12-01

Crop genetic resources carry variation useful for overcoming the challenges of modern agriculture. Molecular markers can facilitate the selection of agronomically important traits. The pervasiveness of genomics research has led to an overwhelming number of publications and databases, which are, nevertheless, scattered and hence often difficult for plant breeders to access, particularly those in developing countries. This situation separates them from developed countries, which have better endowed programs for developing varieties. To close this growing knowledge gap, we conducted an intensive literature review and consulted with more than 150 crop experts on the use of molecular markers in the breeding program of 19 food security crops. The result was a list of effectively used and highly reproducible sequence tagged site (STS), simple sequence repeat (SSR), single nucleotide polymorphism (SNP), and sequence characterized amplified region (SCAR) markers. However, only 12 food crops had molecular markers suitable for improvement. That is, marker-assisted selection is not yet used for Musa spp., coconut, lentils, millets, pigeonpea, sweet potato, and yam. For the other 12 crops, 214 molecular markers were found to be effectively used in association with 74 different traits. Results were compiled as the GCP Molecular Marker Toolkit, a free online tool that aims to promote the adoption of molecular approaches in breeding activities.

[Prognostic and predictive molecular markers for urologic cancers].

PubMed

Hartmann, A; Schlomm, T; Bertz, S; Heinzelmann, J; Hölters, S; Simon, R; Stoehr, R; Junker, K

2014-04-01

Molecular prognostic factors and genetic alterations as predictive markers for cancer-specific targeted therapies are used today in the clinic for many malignancies. In recent years, many molecular markers for urogenital cancers have also been identified. However, these markers are not clinically used yet. In prostate cancer, novel next-generation sequencing methods revealed a detailed picture of the molecular changes. There is growing evidence that a combination of classical histopathological and validated molecular markers could lead to a more precise estimation of prognosis, thus, resulting in an increasing number of patients with active surveillance as a possible treatment option. In patients with urothelial carcinoma, histopathological factors but also the proliferation of the tumor, mutations in oncogenes leading to an increasing proliferation rate and changes in genes responsible for invasion and metastasis are important. In addition, gene expression profiles which could distinguish aggressive tumors with high risk of metastasis from nonmetastasizing tumors have been recently identified. In the future, this could potentially allow better selection of patients needing systemic perioperative treatment. In renal cell carcinoma, many molecular markers that are associated with metastasis and survival have been identified. Some of these markers were also validated as independent prognostic markers. Selection of patients with primarily organ-confined tumors and increased risk of metastasis for adjuvant systemic therapy could be clinically relevant in the future.

CmMDb: a versatile database for Cucumis melo microsatellite markers and other horticulture crop research.

PubMed

Bhawna; Chaduvula, Pavan K; Bonthala, Venkata S; Manjusha, Verma; Siddiq, Ebrahimali A; Polumetla, Ananda K; Prasad, Gajula M N V

2015-01-01

Cucumis melo L. that belongs to Cucurbitaceae family ranks among one of the highest valued horticulture crops being cultivated across the globe. Besides its economical and medicinal importance, Cucumis melo L. is a valuable resource and model system for the evolutionary studies of cucurbit family. However, very limited numbers of molecular markers were reported for Cucumis melo L. so far that limits the pace of functional genomic research in melon and other similar horticulture crops. We developed the first whole genome based microsatellite DNA marker database of Cucumis melo L. and comprehensive web resource that aids in variety identification and physical mapping of Cucurbitaceae family. The Cucumis melo L. microsatellite database (CmMDb: http://65.181.125.102/cmmdb2/index.html) encompasses 39,072 SSR markers along with its motif repeat, motif length, motif sequence, marker ID, motif type and chromosomal locations. The database is featured with novel automated primer designing facility to meet the needs of wet lab researchers. CmMDb is a freely available web resource that facilitates the researchers to select the most appropriate markers for marker-assisted selection in melons and to improve breeding strategies.

Comparison of Marker-Based Genomic Estimated Breeding Values and Phenotypic Evaluation for Selection of Bacterial Spot Resistance in Tomato.

PubMed

Liabeuf, Debora; Sim, Sung-Chur; Francis, David M

2018-03-01

Bacterial spot affects tomato crops (Solanum lycopersicum) grown under humid conditions. Major genes and quantitative trait loci (QTL) for resistance have been described, and multiple loci from diverse sources need to be combined to improve disease control. We investigated genomic selection (GS) prediction models for resistance to Xanthomonas euvesicatoria and experimentally evaluated the accuracy of these models. The training population consisted of 109 families combining resistance from four sources and directionally selected from a population of 1,100 individuals. The families were evaluated on a plot basis in replicated inoculated trials and genotyped with single nucleotide polymorphisms (SNP). We compared the prediction ability of models developed with 14 to 387 SNP. Genomic estimated breeding values (GEBV) were derived using Bayesian least absolute shrinkage and selection operator regression (BL) and ridge regression (RR). Evaluations were based on leave-one-out cross validation and on empirical observations in replicated field trials using the next generation of inbred progeny and a hybrid population resulting from selections in the training population. Prediction ability was evaluated based on correlations between GEBV and phenotypes (r g ), percentage of coselection between genomic and phenotypic selection, and relative efficiency of selection (r g /r p ). Results were similar with BL and RR models. Models using only markers previously identified as significantly associated with resistance but weighted based on GEBV and mixed models with markers associated with resistance treated as fixed effects and markers distributed in the genome treated as random effects offered greater accuracy and a high percentage of coselection. The accuracy of these models to predict the performance of progeny and hybrids exceeded the accuracy of phenotypic selection.

Selection enhanced estimates of µ-calpain, calpastatin, and dacylglycerol O-acyltransferase 1 genetic effects on pre-weaning performance, carcass quality traits, and residual variance of tenderness in composite ... cattle

USDA-ARS?s Scientific Manuscript database

Selection of the composite MARC III population for markers allowed better estimates of effects and inheritance of markers for targeted carcass quality traits (n=254) and nontargeted traits and an evaluation of SNP specific residual variance models for tenderness. Genotypic effects of CAPN1 haplotyp...

Identification of body fluid-specific DNA methylation markers for use in forensic science.

PubMed

Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

2014-11-01

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed Central

2012-01-01

Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. Conclusions We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species. PMID:22805587

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Li, Chengdao; Sweetingham, Mark W; Howieson, John G

2012-07-17

In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.

Marker development for rice blast resistance gene Pi66(t) and application in USDA rice mini-core collection

USDA-ARS?s Scientific Manuscript database

Molecular markers are useful for the identification of critical genes controlling agricultural traits of interest in crop germplasm and for the utilization of these genes in crop improvement using marker assisted selection (MAS). The improvement of blast disease resistance of rice varieties is one o...

Microsatellite markers reveal the below ground distribution of genets in two species of Rhizopogon forming tuberculate ectomycorrhizas on Douglas-fir.

Treesearch

Annette M. Kretzer; Susie Dunham; Randy Molina; Joseph W. Spatafora

2003-01-01

We have developed microsatellite markers for two sister species of Rhizopogon, R. vesiculosus and R. vinicolor (Boletales, Basidiomycota), and used selected markers to investigate genet size and distribution from ectomycorrhizal samples. Both species form ectomycorrhizas with tuberculate morphology on Douglas-fir (...

Genome wide association study (GWAS) for grain yield in rice cultivated under water deficit.

PubMed

Pantalião, Gabriel Feresin; Narciso, Marcelo; Guimarães, Cléber; Castro, Adriano; Colombari, José Manoel; Breseghello, Flavio; Rodrigues, Luana; Vianello, Rosana Pereira; Borba, Tereza Oliveira; Brondani, Claudio

2016-12-01

The identification of rice drought tolerant materials is crucial for the development of best performing cultivars for the upland cultivation system. This study aimed to identify markers and candidate genes associated with drought tolerance by Genome Wide Association Study analysis, in order to develop tools for use in rice breeding programs. This analysis was made with 175 upland rice accessions (Oryza sativa), evaluated in experiments with and without water restriction, and 150,325 SNPs. Thirteen SNP markers associated with yield under drought conditions were identified. Through stepwise regression analysis, eight SNP markers were selected and validated in silico, and when tested by PCR, two out of the eight SNP markers were able to identify a group of rice genotypes with higher productivity under drought. These results are encouraging for deriving markers for the routine analysis of marker assisted selection. From the drought experiment, including the genes inherited in linkage blocks, 50 genes were identified, from which 30 were annotated, and 10 were previously related to drought and/or abiotic stress tolerance, such as the transcription factors WRKY and Apetala2, and protein kinases.

Estimation by simulation of the efficiency of the French marker-assisted selection program in dairy cattle (Open Access publication)

PubMed Central

Guillaume, François; Fritz, Sébastien; Boichard, Didier; Druet, Tom

2008-01-01

The efficiency of the French marker-assisted selection (MAS) was estimated by a simulation study. The data files of two different time periods were used: April 2004 and 2006. The simulation method used the structure of the existing French MAS: same pedigree, same marker genotypes and same animals with records. The program simulated breeding values and new records based on this existing structure and knowledge on the QTL used in MAS (variance and frequency). Reliabilities of genetic values of young animals (less than one year old) obtained with and without marker information were compared to assess the efficiency of MAS for evaluation of milk, fat and protein yields and fat and protein contents. Mean gains of reliability ranged from 0.015 to 0.094 and from 0.038 to 0.114 in 2004 and 2006, respectively. The larger number of animals genotyped and the use of a new set of genetic markers can explain the improvement of MAS reliability from 2004 to 2006. This improvement was also observed by analysis of information content for young candidates. The gain of MAS reliability with respect to classical selection was larger for sons of sires with genotyped progeny daughters with records. Finally, it was shown that when superiority of MAS over classical selection was estimated with daughter yield deviations obtained after progeny test instead of true breeding values, the gain was underestimated. PMID:18096117

Stochastic dynamics of adaptive trait and neutral marker driven by eco-evolutionary feedbacks.

PubMed

Billiard, Sylvain; Ferrière, Régis; Méléard, Sylvie; Tran, Viet Chi

2015-11-01

How the neutral diversity is affected by selection and adaptation is investigated in an eco-evolutionary framework. In our model, we study a finite population in continuous time, where each individual is characterized by a trait under selection and a completely linked neutral marker. Population dynamics are driven by births and deaths, mutations at birth, and competition between individuals. Trait values influence ecological processes (demographic events, competition), and competition generates selection on trait variation, thus closing the eco-evolutionary feedback loop. The demographic effects of the trait are also expected to influence the generation and maintenance of neutral variation. We consider a large population limit with rare mutation, under the assumption that the neutral marker mutates faster than the trait under selection. We prove the convergence of the stochastic individual-based process to a new measure-valued diffusive process with jumps that we call Substitution Fleming-Viot Process (SFVP). When restricted to the trait space this process is the Trait Substitution Sequence first introduced by Metz et al. (1996). During the invasion of a favorable mutation, a genetical bottleneck occurs and the marker associated with this favorable mutant is hitchhiked. By rigorously analysing the hitchhiking effect and how the neutral diversity is restored afterwards, we obtain the condition for a time-scale separation; under this condition, we show that the marker distribution is approximated by a Fleming-Viot distribution between two trait substitutions. We discuss the implications of the SFVP for our understanding of the dynamics of neutral variation under eco-evolutionary feedbacks and illustrate the main phenomena with simulations. Our results highlight the joint importance of mutations, ecological parameters, and trait values in the restoration of neutral diversity after a selective sweep.

FIFS: A data mining method for informative marker selection in high dimensional population genomic data.

PubMed

Kavakiotis, Ioannis; Samaras, Patroklos; Triantafyllidis, Alexandros; Vlahavas, Ioannis

2017-11-01

Single Nucleotide Polymorphism (SNPs) are, nowadays, becoming the marker of choice for biological analyses involving a wide range of applications with great medical, biological, economic and environmental interest. Classification tasks i.e. the assignment of individuals to groups of origin based on their (multi-locus) genotypes, are performed in many fields such as forensic investigations, discrimination between wild and/or farmed populations and others. Τhese tasks, should be performed with a small number of loci, for computational as well as biological reasons. Thus, feature selection should precede classification tasks, especially for Single Nucleotide Polymorphism (SNP) datasets, where the number of features can amount to hundreds of thousands or millions. In this paper, we present a novel data mining approach, called FIFS - Frequent Item Feature Selection, based on the use of frequent items for selection of the most informative markers from population genomic data. It is a modular method, consisting of two main components. The first one identifies the most frequent and unique genotypes for each sampled population. The second one selects the most appropriate among them, in order to create the informative SNP subsets to be returned. The proposed method (FIFS) was tested on a real dataset, which comprised of a comprehensive coverage of pig breed types present in Britain. This dataset consisted of 446 individuals divided in 14 sub-populations, genotyped at 59,436 SNPs. Our method outperforms the state-of-the-art and baseline methods in every case. More specifically, our method surpassed the assignment accuracy threshold of 95% needing only half the number of SNPs selected by other methods (FIFS: 28 SNPs, Delta: 70 SNPs Pairwise FST: 70 SNPs, In: 100 SNPs.) CONCLUSION: Our approach successfully deals with the problem of informative marker selection in high dimensional genomic datasets. It offers better results compared to existing approaches and can aid biologists in selecting the most informative markers with maximum discrimination power for optimization of cost-effective panels with applications related to e.g. species identification, wildlife management, and forensics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification and mapping of Sr46 from Aegilops tauschii accession CIae 25 conferring resistance to race TTKSK (Ug99) of wheat stem rust pathogen.

PubMed

Yu, Guotai; Zhang, Qijun; Friesen, Timothy L; Rouse, Matthew N; Jin, Yue; Zhong, Shaobin; Rasmussen, Jack B; Lagudah, Evans S; Xu, Steven S

2015-03-01

Mapping studies confirm that resistance to Ug99 race of stem rust pathogen in Aegilops tauschii accession Clae 25 is conditioned by Sr46 and markers linked to the gene were developed for marker-assisted selection. The race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal pathogen for wheat stem rust, is considered as a major threat to global wheat production. To address this threat, researchers across the world have been devoted to identifying TTKSK-resistant genes. Here, we report the identification and mapping of a stem rust resistance gene in Aegilops tauschii accession CIae 25 that confers resistance to TTKSK and the development of molecular markers for the gene. An F2 population of 710 plants from an Ae. tauschii cross CIae 25 × AL8/78 were first evaluated against race TPMKC. A set of 14 resistant and 116 susceptible F2:3 families from the F2 plants were then evaluated for their reactions to TTKSK. Based on the tests, 179 homozygous susceptible F2 plants were selected as the mapping population to identify the simple sequence repeat (SSR) and sequence tagged site (STS) markers linked to the gene by bulk segregant analysis. A dominant stem rust resistance gene was identified and mapped with 16 SSR and five new STS markers to the deletion bin 2DS5-0.47-1.00 of chromosome arm 2DS in which Sr46 was located. Molecular marker and stem rust tests on CIae 25 and two Ae. tauschii accessions carrying Sr46 confirmed that the gene in CIae 25 is Sr46. This study also demonstrated that Sr46 is temperature-sensitive being less effective at low temperatures. The marker validation indicated that two closely linked markers Xgwm210 and Xwmc111 can be used for marker-assisted selection of Sr46 in wheat breeding programs.

Evaluating the Influence of the Microsatellite Marker Set on the Genetic Structure Inferred in Pyrus communis L.

PubMed Central

Urrestarazu, Jorge; Royo, José B.; Santesteban, Luis G.; Miranda, Carlos

2015-01-01

Fingerprinting information can be used to elucidate in a robust manner the genetic structure of germplasm collections, allowing a more rational and fine assessment of genetic resources. Bayesian model-based approaches are nowadays majorly preferred to infer genetic structure, but it is still largely unresolved how marker sets should be built in order to obtain a robust inference. The objective was to evaluate, in Pyrus germplasm collections, the influence of the SSR marker set size on the genetic structure inferred, also evaluating the influence of the criterion used to select those markers. Inferences were performed considering an increasing number of SSR markers that ranged from just two up to 25, incorporated one at a time into the analysis. The influence of the number of SSR markers used was evaluated comparing the number of populations and the strength of the signal detected, and also the similarity of the genotype assignments to populations between analyses. In order to test if those results were influenced by the criterion used to select the SSRs, several choosing scenarios based on the discrimination power or the fixation index values of the SSRs were tested. Our results indicate that population structure could be inferred accurately once a certain SSR number threshold was reached, which depended on the underlying structure within the genotypes, but the method used to select the markers included on each set appeared not to be very relevant. The minimum number of SSRs required to provide robust structure inferences and adequate measurements of the differentiation, even when low differentiation levels exist within populations, was proved similar to that of the complete list of recommended markers for fingerprinting. When a SSR set size similar to the minimum marker sets recommended for fingerprinting it is used, only major divisions or moderate (F ST>0.05) differentiation of the germplasm are detected. PMID:26382618

Development of a Plant Transformation Selection System Based on Expression of Genes Encoding Gentamicin Acetyltransferases

PubMed Central

Hayford, Maria B.; Medford, June I.; Hoffman, Nancy L.; Rogers, Stephen G.; Klee, Harry J.

1988-01-01

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666057

Introduction of new genetic markers on human chromosomes

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

Satoh, Hitoshi; Barrett, J.C.; Oshimura, Mitsuo

1991-03-01

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter{yields}3p12::Xq26{yields}Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. These results demonstrate that microcell chromosome transfer can bemore » used to select chromosomes containing multiple markers.« less

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

PubMed

Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

1992-03-15

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

PubMed Central

Hsiao, Edward C.; Yoshinaga, Yuko; Nguyen, Trieu D.; Musone, Stacy L.; Kim, Judy E.; Swinton, Paul; Espineda, Isidro; Manalac, Carlota; deJong, Pieter J.; Conklin, Bruce R.

2008-01-01

Background Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available. Methodology Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice. Conclusions Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library. PMID:18596956

Power analysis of QTL detection in half-sib families using selective DNA pooling

PubMed Central

Baro, Jesús Á; Carleos, Carlos; Corral, Norberto; López, Teresa; Cañón, Javier

2001-01-01

Individual loci of economic importance (QTL) can be detected by comparing the inheritance of a trait and the inheritance of loci with alleles readily identifiable by laboratory methods (genetic markers). Data on allele segregation at the individual level are costly and alternatives have been proposed that make use of allele frequencies among progeny, rather than individual genotypes. Among the factors that may affect the power of the set up, the most important are those intrinsic to the QTL: the additive effect of the QTL, and its dominance, and distance between markers and QTL. Other factors are relative to the choice of animals and markers, such as the frequency of the QTL and marker alleles among dams and sires. Data collection may affect the detection power through the size of half-sib families, selection rate within families, and the technical error incurred when estimating genetic frequencies. We present results for a sensitivity analysis for QTL detection using pools of DNA from selected half-sibs. Simulations showed that conclusive detection may be achieved with families of at least 500 half-sibs if sires are chosen on the criteria that most of their marker alleles are either both missing, or one is fixed, among dams. PMID:11403746

Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

NASA Astrophysics Data System (ADS)

Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

2014-02-01

PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

Marker-assisted combination of major genes for pathogen resistance in potato.

PubMed

Gebhardt, C; Bellin, D; Henselewski, H; Lehmann, W; Schwarzfischer, J; Valkonen, J P T

2006-05-01

Closely linked PCR-based markers facilitate the tracing and combining of resistance factors that have been introgressed previously into cultivated potato from different sources. Crosses were performed to combine the Ry ( adg ) gene for extreme resistance to Potato virus Y (PVY) with the Gro1 gene for resistance to the root cyst nematode Globodera rostochiensis and the Rx1 gene for extreme resistance to Potato virus X (PVX), or with resistance to potato wart (Synchytrium endobioticum). Marker-assisted selection (MAS) using four PCR-based diagnostic assays was applied to 110 F1 hybrids resulting from four 2x by 4x cross-combinations. Thirty tetraploid plants having the appropriate marker combinations were selected and tested for presence of the corresponding resistance traits. All plants tested showed the expected resistant phenotype. Unexpectedly, the plants segregated for additional resistance to pathotypes 1, 2 and 6 of S. endobioticum, which was subsequently shown to be inherited from the PVY resistant parents of the crosses. The selected plants can be used as sources of multiple resistance traits in pedigree breeding and are available from a potato germplasm bank.

Additive Genetic Variability and the Bayesian Alphabet

PubMed Central

Gianola, Daniel; de los Campos, Gustavo; Hill, William G.; Manfredi, Eduardo; Fernando, Rohan

2009-01-01

The use of all available molecular markers in statistical models for prediction of quantitative traits has led to what could be termed a genomic-assisted selection paradigm in animal and plant breeding. This article provides a critical review of some theoretical and statistical concepts in the context of genomic-assisted genetic evaluation of animals and crops. First, relationships between the (Bayesian) variance of marker effects in some regression models and additive genetic variance are examined under standard assumptions. Second, the connection between marker genotypes and resemblance between relatives is explored, and linkages between a marker-based model and the infinitesimal model are reviewed. Third, issues associated with the use of Bayesian models for marker-assisted selection, with a focus on the role of the priors, are examined from a theoretical angle. The sensitivity of a Bayesian specification that has been proposed (called “Bayes A”) with respect to priors is illustrated with a simulation. Methods that can solve potential shortcomings of some of these Bayesian regression procedures are discussed briefly. PMID:19620397

A discrimination index for selecting markers of tumor growth dynamic across multiple cancer studies with a cure fraction.

PubMed

Rouam, Sigrid; Broët, Philippe

2013-08-01

To identify genomic markers with consistent effect on tumor dynamics across multiple cancer series, discrimination indices based on proportional hazards models can be used since they do not depend heavily on the sample size. However, the underlying assumption of proportionality of the hazards does not always hold, especially when the studied population is a mixture of cured and uncured patients, like in early-stage cancers. We propose a novel index that quantifies the capability of a genomic marker to separate uncured patients, according to their time-to-event outcomes. It allows to identify genomic markers characterizing tumor growth dynamic across multiple studies. Simulation results show that our index performs better than classical indices based on the Cox model. It is neither affected by the sample size nor the cure rate fraction. In a cross-study of early-stage breast cancers, the index allows to select genomic markers with a potential consistent effect on tumor growth dynamics. Copyright © 2013 Elsevier Inc. All rights reserved.

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

PubMed

Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

2013-12-01

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

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).

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed

Blochlinger, K; Diggelmann, H

1984-12-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells.

PubMed Central

Blochlinger, K; Diggelmann, H

1984-01-01

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells. Images PMID:6098829

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis.

PubMed

Zhang, Xiao-Zhou; Yan, Xin; Cui, Zhong-Li; Hong, Qing; Li, Shun-Peng

2006-05-19

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.

Development of a Gene-Centered SSR Atlas as a Resource for Papaya (Carica papaya) Marker-Assisted Selection and Population Genetic Studies

PubMed Central

Vidal, Newton Medeiros; Grazziotin, Ana Laura; Ramos, Helaine Christine Cancela; Pereira, Messias Gonzaga; Venancio, Thiago Motta

2014-01-01

Carica papaya (papaya) is an economically important tropical fruit. Molecular marker-assisted selection is an inexpensive and reliable tool that has been widely used to improve fruit quality traits and resistance against diseases. In the present study we report the development and validation of an atlas of papaya simple sequence repeat (SSR) markers. We integrated gene predictions and functional annotations to provide a gene-centered perspective for marker-assisted selection studies. Our atlas comprises 160,318 SSRs, from which 21,231 were located in genic regions (i.e. inside exons, exon-intron junctions or introns). A total of 116,453 (72.6%) of all identified repeats were successfully mapped to one of the nine papaya linkage groups. Primer pairs were designed for markers from 9,594 genes (34.5% of the papaya gene complement). Using papaya-tomato orthology assessments, we assembled a list of 300 genes (comprising 785 SSRs) potentially involved in fruit ripening. We validated our atlas by screening 73 SSR markers (including 25 fruit ripening genes), achieving 100% amplification rate and uncovering 26% polymorphism rate between the parental genotypes (Sekati and JS12). The SSR atlas presented here is the first comprehensive gene-centered collection of annotated and genome positioned papaya SSRs. These features combined with thousands of high-quality primer pairs make the atlas an important resource for the papaya research community. PMID:25393538

Genomic selection for slaughter age in pigs using the Cox frailty model.

PubMed

Santos, V S; Martins Filho, S; Resende, M D V; Azevedo, C F; Lopes, P S; Guimarães, S E F; Glória, L S; Silva, F F

2015-10-19

The aim of this study was to compare genomic selection methodologies using a linear mixed model and the Cox survival model. We used data from an F2 population of pigs, in which the response variable was the time in days from birth to the culling of the animal and the covariates were 238 markers [237 single nucleotide polymorphism (SNP) plus the halothane gene]. The data were corrected for fixed effects, and the accuracy of the method was determined based on the correlation of the ranks of predicted genomic breeding values (GBVs) in both models with the corrected phenotypic values. The analysis was repeated with a subset of SNP markers with largest absolute effects. The results were in agreement with the GBV prediction and the estimation of marker effects for both models for uncensored data and for normality. However, when considering censored data, the Cox model with a normal random effect (S1) was more appropriate. Since there was no agreement between the linear mixed model and the imputed data (L2) for the prediction of genomic values and the estimation of marker effects, the model S1 was considered superior as it took into account the latent variable and the censored data. Marker selection increased correlations between the ranks of predicted GBVs by the linear and Cox frailty models and the corrected phenotypic values, and 120 markers were required to increase the predictive ability for the characteristic analyzed.

Comprehensive evaluation of untargeted metabolomics data processing software in feature detection, quantification and discriminating marker selection.

PubMed

Li, Zhucui; Lu, Yan; Guo, Yufeng; Cao, Haijie; Wang, Qinhong; Shui, Wenqing

2018-10-31

Data analysis represents a key challenge for untargeted metabolomics studies and it commonly requires extensive processing of more than thousands of metabolite peaks included in raw high-resolution MS data. Although a number of software packages have been developed to facilitate untargeted data processing, they have not been comprehensively scrutinized in the capability of feature detection, quantification and marker selection using a well-defined benchmark sample set. In this study, we acquired a benchmark dataset from standard mixtures consisting of 1100 compounds with specified concentration ratios including 130 compounds with significant variation of concentrations. Five software evaluated here (MS-Dial, MZmine 2, XCMS, MarkerView, and Compound Discoverer) showed similar performance in detection of true features derived from compounds in the mixtures. However, significant differences between untargeted metabolomics software were observed in relative quantification of true features in the benchmark dataset. MZmine 2 outperformed the other software in terms of quantification accuracy and it reported the most true discriminating markers together with the fewest false markers. Furthermore, we assessed selection of discriminating markers by different software using both the benchmark dataset and a real-case metabolomics dataset to propose combined usage of two software for increasing confidence of biomarker identification. Our findings from comprehensive evaluation of untargeted metabolomics software would help guide future improvements of these widely used bioinformatics tools and enable users to properly interpret their metabolomics results. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and utilization of InDel markers to identify peanut (Arachis hypogaea) disease resistance

USDA-ARS?s Scientific Manuscript database

To date, nearly 10,000 SSR-based markers have been identified by various research groups around the world, but less than 14.5% showed polymorphism in peanut and only 6.4% were mapped. Low levels of polymorphism limit the application of marker assisted selection (MAS) in peanut breeding programs. I...

Selection for genetic markers in beef cattle reveals complex associations of thyroglobulin and casein1-S1 with carcass and meat traits

USDA-ARS?s Scientific Manuscript database

Genetic markers in casein (CSN1S1) and thyroglobulin (TG) genes have previously been associated with fat distribution in cattle. Determining the nature of these genetic associations (additive, recessive, or dominant) has been difficult because both markers have small minor allele frequencies in mos...

Does genomic selection have a future in plant breeding?

PubMed

Jonas, Elisabeth; de Koning, Dirk-Jan

2013-09-01

Plant breeding largely depends on phenotypic selection in plots and only for some, often disease-resistance-related traits, uses genetic markers. The more recently developed concept of genomic selection, using a black box approach with no need of prior knowledge about the effect or function of individual markers, has also been proposed as a great opportunity for plant breeding. Several empirical and theoretical studies have focused on the possibility to implement this as a novel molecular method across various species. Although we do not question the potential of genomic selection in general, in this Opinion, we emphasize that genomic selection approaches from dairy cattle breeding cannot be easily applied to complex plant breeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Selected Tools and Techniques for Physical and Biological Monitoring of Aquatic Dredged Material Disposal Sites

DTIC Science & Technology

1990-09-01

expanded in a specific direction if movement is indicated. Controlled dumping at precise coordinates or at marker buoys may reduce the required survey area...of the meters or theft of the marker buoys. Subsurface markers using acoustic releases prevent vandalism and loss of marker buoys, but they...data from field studies such as impact investigations ’Underwood 1981; Heck and Horowitz 1984; Hurlbert 1984; Millard and Lettenmaier 1986; Stewart

Fast internal marker tracking algorithm for onboard MV and kV imaging systems

PubMed Central

Mao, W.; Wiersma, R. D.; Xing, L.

2008-01-01

Intrafraction organ motion can limit the advantage of highly conformal dose techniques such as intensity modulated radiation therapy (IMRT) due to target position uncertainty. To ensure high accuracy in beam targeting, real-time knowledge of the target location is highly desired throughout the beam delivery process. This knowledge can be gained through imaging of internally implanted radio-opaque markers with fluoroscopic or electronic portal imaging devices (EPID). In the case of MV based images, marker detection can be problematic due to the significantly lower contrast between different materials in comparison to their kV-based counterparts. This work presents a fully automated algorithm capable of detecting implanted metallic markers in both kV and MV images with high consistency. Using prior CT information, the algorithm predefines the volumetric search space without manual region-of-interest (ROI) selection by the user. Depending on the template selected, both spherical and cylindrical markers can be detected. Multiple markers can be simultaneously tracked without indexing confusion. Phantom studies show detection success rates of 100% for both kV and MV image data. In addition, application of the algorithm to real patient image data results in successful detection of all implanted markers for MV images. Near real-time operational speeds of ∼10 frames∕sec for the detection of five markers in a 1024×768 image are accomplished using an ordinary PC workstation. PMID:18561670

Risk Classification with an Adaptive Naive Bayes Kernel Machine Model.

PubMed

Minnier, Jessica; Yuan, Ming; Liu, Jun S; Cai, Tianxi

2015-04-22

Genetic studies of complex traits have uncovered only a small number of risk markers explaining a small fraction of heritability and adding little improvement to disease risk prediction. Standard single marker methods may lack power in selecting informative markers or estimating effects. Most existing methods also typically do not account for non-linearity. Identifying markers with weak signals and estimating their joint effects among many non-informative markers remains challenging. One potential approach is to group markers based on biological knowledge such as gene structure. If markers in a group tend to have similar effects, proper usage of the group structure could improve power and efficiency in estimation. We propose a two-stage method relating markers to disease risk by taking advantage of known gene-set structures. Imposing a naive bayes kernel machine (KM) model, we estimate gene-set specific risk models that relate each gene-set to the outcome in stage I. The KM framework efficiently models potentially non-linear effects of predictors without requiring explicit specification of functional forms. In stage II, we aggregate information across gene-sets via a regularization procedure. Estimation and computational efficiency is further improved with kernel principle component analysis. Asymptotic results for model estimation and gene set selection are derived and numerical studies suggest that the proposed procedure could outperform existing procedures for constructing genetic risk models.

Genetic Linkage Mapping of Economically Important Traits in Cultivated Tetraploid Potato (Solanum tuberosum L.).

PubMed

Massa, Alicia N; Manrique-Carpintero, Norma C; Coombs, Joseph J; Zarka, Daniel G; Boone, Anne E; Kirk, William W; Hackett, Christine A; Bryan, Glenn J; Douches, David S

2015-09-14

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosum L.). The 156 F1 progeny and parents of a cross (MSL603) between "Jacqueline Lee" and "MSG227-2" were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype of Phytophthora infestans (Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in "Jacqueline Lee." The best SNP marker mapped ~0.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped ~0.25 Mb from the StCDF1 gene, which is a candidate gene for the maturity trait. The identification of markers for P. infestans resistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications. Copyright © 2015 Massa et al.

Genetic Linkage Mapping of Economically Important Traits in Cultivated Tetraploid Potato (Solanum tuberosum L.)

PubMed Central

Massa, Alicia N.; Manrique-Carpintero, Norma C.; Coombs, Joseph J.; Zarka, Daniel G.; Boone, Anne E.; Kirk, William W.; Hackett, Christine A.; Bryan, Glenn J.; Douches, David S.

2015-01-01

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosum L.). The 156 F1 progeny and parents of a cross (MSL603) between “Jacqueline Lee” and “MSG227-2” were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype of Phytophthora infestans (Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in “Jacqueline Lee.” The best SNP marker mapped ∼0.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped ∼0.25 Mb from the StCDF1 gene, which is a candidate gene for the maturity trait. The identification of markers for P. infestans resistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications. PMID:26374597

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes.

PubMed

Shimizu, Tokurou; Kitajima, Akira; Nonaka, Keisuke; Yoshioka, Terutaka; Ohta, Satoshi; Goto, Shingo; Toyoda, Atsushi; Fujiyama, Asao; Mochizuki, Takako; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2016-01-01

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes

PubMed Central

Kitajima, Akira; Nonaka, Keisuke; Yoshioka, Terutaka; Ohta, Satoshi; Goto, Shingo; Toyoda, Atsushi; Fujiyama, Asao; Mochizuki, Takako; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2016-01-01

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy–Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies. PMID:27902727

Germplasm-regression-combined (GRC) marker-trait association identification in plant breeding: a challenge for plant biotechnological breeding under soil water deficit conditions.

PubMed

Ruan, Cheng-Jiang; Xu, Xue-Xuan; Shao, Hong-Bo; Jaleel, Cheruth Abdul

2010-09-01

In the past 20 years, the major effort in plant breeding has changed from quantitative to molecular genetics with emphasis on quantitative trait loci (QTL) identification and marker assisted selection (MAS). However, results have been modest. This has been due to several factors including absence of tight linkage QTL, non-availability of mapping populations, and substantial time needed to develop such populations. To overcome these limitations, and as an alternative to planned populations, molecular marker-trait associations have been identified by the combination between germplasm and the regression technique. In the present preview, the authors (1) survey the successful applications of germplasm-regression-combined (GRC) molecular marker-trait association identification in plants; (2) describe how to do the GRC analysis and its differences from mapping QTL based on a linkage map reconstructed from the planned populations; (3) consider the factors that affect the GRC association identification, including selections of optimal germplasm and molecular markers and testing of identification efficiency of markers associated with traits; and (4) finally discuss the future prospects of GRC marker-trait association analysis used in plant MAS/QTL breeding programs, especially in long-juvenile woody plants when no other genetic information such as linkage maps and QTL are available.

Automatic rice crop height measurement using a field server and digital image processing.

PubMed

Sritarapipat, Tanakorn; Rakwatin, Preesan; Kasetkasem, Teerasit

2014-01-07

Rice crop height is an important agronomic trait linked to plant type and yield potential. This research developed an automatic image processing technique to detect rice crop height based on images taken by a digital camera attached to a field server. The camera acquires rice paddy images daily at a consistent time of day. The images include the rice plants and a marker bar used to provide a height reference. The rice crop height can be indirectly measured from the images by measuring the height of the marker bar compared to the height of the initial marker bar. Four digital image processing steps are employed to automatically measure the rice crop height: band selection, filtering, thresholding, and height measurement. Band selection is used to remove redundant features. Filtering extracts significant features of the marker bar. The thresholding method is applied to separate objects and boundaries of the marker bar versus other areas. The marker bar is detected and compared with the initial marker bar to measure the rice crop height. Our experiment used a field server with a digital camera to continuously monitor a rice field located in Suphanburi Province, Thailand. The experimental results show that the proposed method measures rice crop height effectively, with no human intervention required.

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.

A temperature-tolerant multiplex elements and genes screening system for genetically modified organisms based on dual priming oligonucleotide primers and capillary electrophoresis.

PubMed

Fu, Wei; Wei, Shuang; Wang, Chenguang; Du, Zhixin; Zhu, Pengyu; Wu, Xiyang; Wu, Gang; Zhu, Shuifang

2017-08-15

High throughput screening systems are the preferred solution to meet the urgent requirement of increasing number of genetically modified organisms (GMOs). In this study, we have successfully developed a multiplex GMO element screening system with dual priming oligonucleotide (DPO) primers. This system can detect the cauliflower mosaic virus 35S (CaMV 35S), terminator of nopaline synthase gene (NOS), figwort mosaic virus 35S (FMV 35S) promoter, neomycin phosphotransferaseII (NPTII), Bt Cry 1Ab, phosphinothricin acetyltransferase genes (bar) and Streptomyces viridochromogenes (pat) simultaneously, which covers more than 90% of all authorized GMO species worldwide. This system exhibits a high tolerance to annealing temperatures, high specificity and a limit of detection equal to conventional PCR. A total of 214 samples from markets, national entry-exit agencies, the Institute for Reference Materials and Measurement (IRMM) and the American Oil Chemists' Society (AOCS) were also tested for applicability. This screening system is therefore suitable for GMO screening. Copyright © 2017 Elsevier Ltd. All rights reserved.

Marker-assisted introgression of broad-spectrum blast resistance genes into the cultivated MR219 rice variety.

PubMed

Miah, Gous; Rafii, Mohd Y; Ismail, Mohd R; Puteh, Adam B; Rahim, Harun A; Latif, Mohammad A

2017-07-01

The rice cultivar MR219 is famous for its better yield and long and fine grain quality; however, it is susceptible to blast disease. The main objective of this study was to introgress blast resistance genes into MR219 through marker-assisted selection (MAS). The rice cultivar MR219 was used as the recurrent parent, and Pongsu Seribu 1 was used as the donor. Marker-assisted foreground selection was performed using RM6836 and RM8225 to identify plants possessing blast resistance genes. Seventy microsatellite markers were used to estimate recurrent parent genome (RPG) recovery. Our analysis led to the development of 13 improved blast resistant lines with Piz, Pi2 and Pi9 broad-spectrum blast resistance genes and an MR219 genetic background. The RPG recovery of the selected improved lines was up to 97.70% with an average value of 95.98%. Selected improved lines showed a resistance response against the most virulent blast pathogen pathotype, P7.2. The selected improved lines did not express any negative effect on agronomic traits in comparison with MR219. The research findings of this study will be a conducive approach for the application of different molecular techniques that may result in accelerating the development of new disease-resistant rice varieties, which in turn will match rising demand and food security worldwide. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

High degree of genetic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus).

PubMed

Defaveri, Jacquelin; Shikano, Takahito; Shimada, Yukinori; Merilä, Juha

2013-09-01

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene-based genome-scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection-as determined with several outlier detection methods-was low (F(ST) = 0.033 or 0.011, respectively), whereas average FST for directionally selected markers was significantly higher (F(ST) = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes. © 2013 John Wiley & Sons Ltd.

Optimizing trial design in pharmacogenetics research: comparing a fixed parallel group, group sequential, and adaptive selection design on sample size requirements.

PubMed

Boessen, Ruud; van der Baan, Frederieke; Groenwold, Rolf; Egberts, Antoine; Klungel, Olaf; Grobbee, Diederick; Knol, Mirjam; Roes, Kit

2013-01-01

Two-stage clinical trial designs may be efficient in pharmacogenetics research when there is some but inconclusive evidence of effect modification by a genomic marker. Two-stage designs allow to stop early for efficacy or futility and can offer the additional opportunity to enrich the study population to a specific patient subgroup after an interim analysis. This study compared sample size requirements for fixed parallel group, group sequential, and adaptive selection designs with equal overall power and control of the family-wise type I error rate. The designs were evaluated across scenarios that defined the effect sizes in the marker positive and marker negative subgroups and the prevalence of marker positive patients in the overall study population. Effect sizes were chosen to reflect realistic planning scenarios, where at least some effect is present in the marker negative subgroup. In addition, scenarios were considered in which the assumed 'true' subgroup effects (i.e., the postulated effects) differed from those hypothesized at the planning stage. As expected, both two-stage designs generally required fewer patients than a fixed parallel group design, and the advantage increased as the difference between subgroups increased. The adaptive selection design added little further reduction in sample size, as compared with the group sequential design, when the postulated effect sizes were equal to those hypothesized at the planning stage. However, when the postulated effects deviated strongly in favor of enrichment, the comparative advantage of the adaptive selection design increased, which precisely reflects the adaptive nature of the design. Copyright © 2013 John Wiley & Sons, Ltd.

Comparative genome analysis of a large Dutch Legionella pneumophila strain collection identifies five markers highly correlated with clinical strains

PubMed Central

2010-01-01

Background Discrimination between clinical and environmental strains within many bacterial species is currently underexplored. Genomic analyses have clearly shown the enormous variability in genome composition between different strains of a bacterial species. In this study we have used Legionella pneumophila, the causative agent of Legionnaire's disease, to search for genomic markers related to pathogenicity. During a large surveillance study in The Netherlands well-characterized patient-derived strains and environmental strains were collected. We have used a mixed-genome microarray to perform comparative-genome analysis of 257 strains from this collection. Results Microarray analysis indicated that 480 DNA markers (out of in total 3360 markers) showed clear variation in presence between individual strains and these were therefore selected for further analysis. Unsupervised statistical analysis of these markers showed the enormous genomic variation within the species but did not show any correlation with a pathogenic phenotype. We therefore used supervised statistical analysis to identify discriminating markers. Genetic programming was used both to identify predictive markers and to define their interrelationships. A model consisting of five markers was developed that together correctly predicted 100% of the clinical strains and 69% of the environmental strains. Conclusions A novel approach for identifying predictive markers enabling discrimination between clinical and environmental isolates of L. pneumophila is presented. Out of over 3000 possible markers, five were selected that together enabled correct prediction of all the clinical strains included in this study. This novel approach for identifying predictive markers can be applied to all bacterial species, allowing for better discrimination between strains well equipped to cause human disease and relatively harmless strains. PMID:20630115

Stem Cell Therapy for Healing Wounded Skin and Soft Tissues

DTIC Science & Technology

2012-07-01

changes of ASC surface markers due to repetitive in vitro sub-culturing. ASCs were harvested, washed in PBS to remove cell culture medium, and resuspended...Our in vitro and in vivo studies suggest that ASC and BM-MSC are not identical, though they have similar surface markers . We found that topically...ofpolybrene. Transduced cells were selected by treating 10 J.!g/rnl ofblasticidin. GFP expressing cells were further selected by flow cytometry using

The pht4;1-3 mutant line contains a loss of function allele in the Fatty Acid Desaturase 7 gene caused by a remnant inactivated selection marker-a cautionary tale.

PubMed

Nilsson, Anders K; Andersson, Mats X

2017-01-01

A striking and unexpected biochemical phenotype was found in an insertion mutant line in the model plant Arabidopsis thaliana . One of two investigated insertion mutant lines in the gene encoding the phosphate transporter PHT4;1 demonstrated a prominent loss of trienoic fatty acids, whereas the other insertion line was indistinguishable from wild type in this aspect. We demonstrate that the loss of trienoic fatty acids was due to a remnant inactive negative selection marker gene in this particular transposon tagged line, pht4;1-3 . This constitutes a cautionary tale that warns of the importance to confirm the loss of this type of selection markers and the importance of verifying the relationship between a phenotype and genotype by more than one independent mutant line or alternatively genetic complementation.

Marker Assisted Transfer of Two Powdery Mildew Resistance Genes PmTb7A.1 and PmTb7A.2 from Triticum boeoticum (Boiss.) to Triticum aestivum (L.)

PubMed Central

Elkot, Ahmed Fawzy Abdelnaby; Chhuneja, Parveen; Kaur, Satinder; Saluja, Manny; Keller, Beat; Singh, Kuldeep

2015-01-01

Powdery mildew (PM), caused by Blumeria graminis f. sp. tritici, is one of the important wheat diseases, worldwide. Two PM resistance genes, designated as PmTb7A.1 and PmTb7A.2, were identified in T. boeoticum acc. pau5088 and mapped on chromosome 7AL approximately 48cM apart. Two resistance gene analogue (RGA)-STS markers Ta7AL-4556232 and 7AL-4426363 were identified to be linked to the PmTb7A.1 and PmTb7A.2, at a distance of 0.6cM and 6.0cM, respectively. In the present study, following marker assisted selection (MAS), the two genes were transferred to T. aestivum using T. durum as bridging species. As many as 12,317 florets of F1 of the cross T. durum /T. boeoticum were pollinated with T. aestivum lines PBW343-IL and PBW621 to produce 61 and 65 seeds, respectively, of three-way F1. The resulting F1s of the cross T. durum/T. boeoticum//T. aestivum were screened with marker flanking both the PM resistance genes PmTb7A.1 and PmTb7A.2 (foreground selection) and the selected plants were backcrossed to generate BC1F1. Marker assisted selection was carried both in BC1F1 and the BC2F1 generations. Introgression of alien chromatin in BC2F1 plants varied from 15.4 - 62.9 percent. Out of more than 110 BC2F1 plants showing introgression for markers linked to the two PM resistance genes, 40 agronomically desirable plants were selected for background selection for the carrier chromosome to identify the plants with minimum of the alien introgression. Cytological analysis showed that most plants have chromosome number ranging from 40-42. The BC2F2 plants homozygous for the two genes have been identified. These will be crossed to generate lines combining both the PM resistance genes but with minimal of the alien introgression. The PM resistance gene PmTb7A.1 maps in a region very close to Sr22, a stem rust resistance gene effective against the race Ug99. Analysis of selected plants with markers linked to Sr22 showed introgression of Sr22 from T. boeoticum in several BC2F1 plants. Thus, in addition to PM resistance, these progeny might also carry resistance to stem rust race Ug99. PMID:26066332

Sequencing consolidates molecular markers with plant breeding practice.

PubMed

Yang, Huaan; Li, Chengdao; Lam, Hon-Ming; Clements, Jonathan; Yan, Guijun; Zhao, Shancen

2015-05-01

Plenty of molecular markers have been developed by contemporary sequencing technologies, whereas few of them are successfully applied in breeding, thus we present a review on how sequencing can facilitate marker-assisted selection in plant breeding. The growing global population and shrinking arable land area require efficient plant breeding. Novel strategies assisted by certain markers have proven effective for genetic gains. Fortunately, cutting-edge sequencing technologies bring us a deluge of genomes and genetic variations, enlightening the potential of marker development. However, a large gap still exists between the potential of molecular markers and actual plant breeding practices. In this review, we discuss marker-assisted breeding from a historical perspective, describe the road from crop sequencing to breeding, and highlight how sequencing facilitates the application of markers in breeding practice.

Use of Both Cumulus Cells’ Transcriptomic Markers and Zona Pellucida Birefringence to Select Developmentally Competent Oocytes in Human Assisted Reproductive Technologies

PubMed Central

2015-01-01

Background Selection of the best oocyte for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in human infertility treatment procedure. Oocyte selection using morphological criteria mainly Zona pellucida (ZP) has been the gold standard method in assisted reproductive technologies (ART) clinics, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs-based approaches have allowed the identification of key molecular markers that quantitatively and non-invasively predict the oocyte quality for higher pregnancy rates and efficient infertility treatment. These biomarkers are a valuable reinforcement of the morphological selection criteria widely used in in vitro fertilization (IVF) clinics. In this context, this study was designed to investigate the relationship between transcriptomic predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality mainly the ZP birefringence. Results Microarray data revealed that 48 and 27 differentially expressed candidate genes in cumulus cells (CCs) were respectively overexpressed and underexpressed in the ZGP (Zona Good Pregnant) versus ZBNP (Zona Bad Non Pregnant) groups. More than 70% of previously reported transcriptomic biomarkers of oocyte developmental competence were confirmed in this study. The analysis of possible association between ZP birefringence versus molecular markers approach showed an absence of correlation between them using the current set of markers. Conclusions This study suggested a new integrative approach that matches morphological and molecular approaches used to select developmentally competent oocytes able to lead to successful pregnancy and the delivery of healthy baby. For each ZP birefringence score, oocytes displayed a particular CCs' gene expression pattern. However, no correlations were found between the 7 gene biomarkers of oocyte developmental potential and the ZP birefringence score. Further studies using larger lists of candidate markers are required to identify suitable genes that are highly correlated with the morphological criteria, and therefore able to reinforce the accuracy of oocyte selection and the effectiveness of infertility treatment. PMID:25923296

Saturation of an intra-gene pool linkage map: toward unified consensus linkage map in common bean

USDA-ARS?s Scientific Manuscript database

Map-based cloning to find genes of interest and marker assisted selection (MAS) requires good genetic maps with high reproducible markers. In this study, we saturated the linkage map of the intra-gene pool population of common bean DOR364×BAT477 (DB) by evaluating 2,706 molecular markers in includin...

Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.).

PubMed

Sun, Lidan; Zhang, Qixiang; Xu, Zongda; Yang, Weiru; Guo, Yu; Lu, Jiuxing; Pan, Huitang; Cheng, Tangren; Cai, Ming

2013-10-06

Mei (Prunus mume Sieb. et Zucc.) is a famous ornamental plant and fruit crop grown in East Asian countries. Limited genetic resources, especially molecular markers, have hindered the progress of mei breeding projects. Here, we performed low-depth whole-genome sequencing of Prunus mume 'Fenban' and Prunus mume 'Kouzi Yudie' to identify high-quality polymorphic markers between the two cultivars on a large scale. A total of 1464.1 Mb and 1422.1 Mb of 'Fenban' and 'Kouzi Yudie' sequencing data were uniquely mapped to the mei reference genome with about 6-fold coverage, respectively. We detected a large number of putative polymorphic markers from the 196.9 Mb of sequencing data shared by the two cultivars, which together contained 200,627 SNPs, 4,900 InDels, and 7,063 SSRs. Among these markers, 38,773 SNPs, 174 InDels, and 418 SSRs were distributed in the 22.4 Mb CDS region, and 63.0% of these marker-containing CDS sequences were assigned to GO terms. Subsequently, 670 selected SNPs were validated using an Agilent's SureSelect solution phase hybridization assay. A subset of 599 SNPs was used to assess the genetic similarity of a panel of mei germplasm samples and a plum (P. salicina) cultivar, producing a set of informative diversity data. We also analyzed the frequency and distribution of detected InDels and SSRs in mei genome and validated their usefulness as DNA markers. These markers were successfully amplified in the cultivars and in their segregating progeny. A large set of high-quality polymorphic SNPs, InDels, and SSRs were identified in parallel between 'Fenban' and 'Kouzi Yudie' using low-depth whole-genome sequencing. The study presents extensive data on these polymorphic markers, which can be useful for constructing high-resolution genetic maps, performing genome-wide association studies, and designing genomic selection strategies in mei.

Masked Selection: A Straightforward and Flexible Approach for the Selection of Binders Against Specific Epitopes and Differentially Expressed Proteins by Phage Display*

PubMed Central

Even-Desrumeaux, Klervi; Nevoltris, Damien; Lavaut, Marie Noelle; Alim, Karima; Borg, Jean-Paul; Audebert, Stéphane; Kerfelec, Brigitte; Baty, Daniel; Chames, Patrick

2014-01-01

Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers. PMID:24361863

Selection for genetic markers in beef cattle reveals complex associations of thyroglobulin and casein1-s1 with carcass and meat traits.

PubMed

Bennett, G L; Shackelford, S D; Wheeler, T L; King, D A; Casas, E; Smith, T P L

2013-02-01

Genetic markers in casein (CSN1S1) and thyroglobulin (TG) genes have previously been associated with fat distribution in cattle. Determining the nature of these genetic associations (additive, recessive, or dominant) has been difficult, because both markers have small minor allele frequencies in most beef cattle populations. This results in few animals homozygous for the minor alleles. selection to increase the frequencies of the minor alleles for 2 SNP markers in these genes was undertaken in a composite population. The objective was to obtain better estimates of genetic effects associated with these markers and determine if there were epistatic interactions. Selection increased the frequencies of minor alleles for both SNP from <0.30 to 0.45. Bulls (n = 24) heterozygous for both SNP were used in 3 yr to produce 204 steer progeny harvested at an average age of 474 d. The combined effect of the 9 CSN1S1 × TG genotypes was associated with carcass-adjusted fat thickness (P < 0.06) and meat tenderness predicted at the abattoir by visible and near-infrared reflectance spectroscopy (P < 0.04). Genotype did not affect BW from birth through harvest, ribeye area, marbling score, slice shear force, or image-based yield grade (P > 0.10). Additive, dominance, and epistatic SNP association effects were estimated from genotypic effects for adjusted fat thickness and predicted meat tenderness. Adjusted fat thickness showed a dominance association with TG SNP (P < 0.06) and an epistatic additive CSN1S1 × additive TG association (P < 0.03). For predicted meat tenderness, heterozygous TG meat was more tender than meat from either homozygote (P < 0.002). Dominance and epistatic associations can result in different SNP allele substitution effects in populations where SNP have the same linkage disequilibrium with causal mutations but have different frequencies. Although the complex associations estimated in this study would contribute little to within-population selection response, they could be important for marker-assisted management or reciprocal selection schemes.

Genomic Selection in Commercial Perennial Crops: Applicability and Improvement in Oil Palm (Elaeis guineensis Jacq.).

PubMed

Kwong, Qi Bin; Ong, Ai Ling; Teh, Chee Keng; Chew, Fook Tim; Tammi, Martti; Mayes, Sean; Kulaveerasingam, Harikrishna; Yeoh, Suat Hui; Harikrishna, Jennifer Ann; Appleton, David Ross

2017-06-06

Genomic selection (GS) uses genome-wide markers to select individuals with the desired overall combination of breeding traits. A total of 1,218 individuals from a commercial population of Ulu Remis x AVROS (UR x AVROS) were genotyped using the OP200K array. The traits of interest included: shell-to-fruit ratio (S/F, %), mesocarp-to-fruit ratio (M/F, %), kernel-to-fruit ratio (K/F, %), fruit per bunch (F/B, %), oil per bunch (O/B, %) and oil per palm (O/P, kg/palm/year). Genomic heritabilities of these traits were estimated to be in the range of 0.40 to 0.80. GS methods assessed were RR-BLUP, Bayes A (BA), Cπ (BC), Lasso (BL) and Ridge Regression (BRR). All methods resulted in almost equal prediction accuracy. The accuracy achieved ranged from 0.40 to 0.70, correlating with the heritability of traits. By selecting the most important markers, RR-BLUP B has the potential to outperform other methods. The marker density for certain traits can be further reduced based on the linkage disequilibrium (LD). Together with in silico breeding, GS is now being used in oil palm breeding programs to hasten parental palm selection.

P21, COX-2, and E-cadherin are potential prognostic factors for esophageal squamous cell carcinoma.

PubMed

Lin, Yao; Shen, Lu-Yan; Fu, Hao; Dong, Bin; Yang, He-Li; Yan, Wan-Pu; Kang, Xiao-Zheng; Dai, Liang; Zhou, Hai-Tao; Yang, Yong-Bo; Liang, Zhen; Chen, Ke-Neng

2017-02-01

Much research effort has been devoted to identifying prognostic factors for esophageal squamous cell carcinoma (ESCC) by immunohistochemistry; however, no conclusive findings have been reached thus far. We hypothesized that certain molecules identified in previous studies might serve as useful prognostic markers for ESCC. Therefore, the aim of the current study was to validate the most relevant markers showing potential for ESCC prognosis in our prospective esophageal cancer database. A literature search was performed using the PubMed database for papers published between 1980 and 2015 using the following key words: 'esophageal cancer,' 'prognosis,' and 'immunohistochemistry.' Literature selection criteria were established to identify the most widely studied markers, and we further validated the selected markers in a cohort from our single-surgeon team, including 153 esophageal cancer patients treated from 2000 to 2010. A total of 1799 articles were identified, 82 of which met the selection criteria. Twelve markers were found to be the most widely studied, and the validation results indicated that only P21, COX-2, and E-cadherin were independent prognostic factors for ESCC patients in this series. The systemic review and cohort validation suggest that P21, COX-2, and E-cadherin are potential prognostic factors for ESCC, paving the way for more targeted prospective validation in the future. © 2016 International Society for Diseases of the Esophagus.

Prediction of Cacao (Theobroma cacao) Resistance to Moniliophthora spp. Diseases via Genome-Wide Association Analysis and Genomic Selection.

PubMed

McElroy, Michel S; Navarro, Alberto J R; Mustiga, Guiliana; Stack, Conrad; Gezan, Salvador; Peña, Geover; Sarabia, Widem; Saquicela, Diego; Sotomayor, Ignacio; Douglas, Gavin M; Migicovsky, Zoë; Amores, Freddy; Tarqui, Omar; Myles, Sean; Motamayor, Juan C

2018-01-01

Cacao ( Theobroma cacao ) is a globally important crop, and its yield is severely restricted by disease. Two of the most damaging diseases, witches' broom disease (WBD) and frosty pod rot disease (FPRD), are caused by a pair of related fungi: Moniliophthora perniciosa and Moniliophthora roreri , respectively. Resistant cultivars are the most effective long-term strategy to address Moniliophthora diseases, but efficiently generating resistant and productive new cultivars will require robust methods for screening germplasm before field testing. Marker-assisted selection (MAS) and genomic selection (GS) provide two potential avenues for predicting the performance of new genotypes, potentially increasing the selection gain per unit time. To test the effectiveness of these two approaches, we performed a genome-wide association study (GWAS) and GS on three related populations of cacao in Ecuador genotyped with a 15K single nucleotide polymorphism (SNP) microarray for three measures of WBD infection (vegetative broom, cushion broom, and chirimoya pod), one of FPRD (monilia pod) and two productivity traits (total fresh weight of pods and % healthy pods produced). GWAS yielded several SNPs associated with disease resistance in each population, but none were significantly correlated with the same trait in other populations. Genomic selection, using one population as a training set to estimate the phenotypes of the remaining two (composed of different families), varied among traits, from a mean prediction accuracy of 0.46 (vegetative broom) to 0.15 (monilia pod), and varied between training populations. Simulations demonstrated that selecting seedlings using GWAS markers alone generates no improvement over selecting at random, but that GS improves the selection process significantly. Our results suggest that the GWAS markers discovered here are not sufficiently predictive across diverse germplasm to be useful for MAS, but that using all markers in a GS framework holds substantial promise in accelerating disease-resistance in cacao.

Marker-assisted breeding for introgression of opaque-2 allele into elite maize inbred line BML-7.

PubMed

Krishna, M S R; Sokka Reddy, S; Satyanarayana, Sadam D V

2017-07-01

Improvement of quality protein maize (QPM) along with high content of lysine and tryptophan had foremost importance in maize breeding program. The efficient and easiest way of developing QPM hybrids was by backcross breeding in marker aided selection. Hence, the present investigation aimed at conversion of elite maize inbred line BML-7 into QPM line. CML-186 was identified to be a donor variety as it revealed high-quality polymorphism with BML-7 for opaque-2 gene specific marker umc1066. Non-QPM inbred line BML-7 was crossed with QPM donor CML-186 and produced F 1 followed by the development of BC 1 F 1 and BC 2 F 1 population. Foreground selection was carried out with umc1066 in F 1 , and selected plants were used for BC 1 F 1 and BC 2 F 1 populations. Two hundred plants were screened in both BC 1 F 1 and BC 2 F 1 population with umc1066 for foreground selection amino acid modifiers. Foreground selected plants for both opaque-2 and amino acid modifiers were screened for background selection for BML-7 genome. Recurrent parent genome (RPG) was calculated for BC 2 F 1 population plants. Two plants have shown with RPG 90-93% in two generation with back cross population. Two BC 2 F 2 populations resulted from marker recognized BC 2 F 1 individuals subjected toward foreground selection followed by tryptophan estimation. The tryptophan and lysine concentration was improved in all the plants. BC 2 F 2 lines developed from hard endosperm kernels were selfed for BC 2 F 2 lines and finest line was selected to illustrate the QPM version of BML-7, with 0.97% of tryptophan and 4.04% of lysine concentration in protein. Therefore, the QPM version of BML-7 line can be used for the development of single cross hybrid QPM maize version.

Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of indica rice.

PubMed

Steele, Katherine A; Quinton-Tulloch, Mark J; Amgai, Resham B; Dhakal, Rajeev; Khatiwada, Shambhu P; Vyas, Darshna; Heine, Martin; Witcombe, John R

2018-01-01

Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

Breeding Jatropha curcas by genomic selection: A pilot assessment of the accuracy of predictive models.

PubMed

Azevedo Peixoto, Leonardo de; Laviola, Bruno Galvêas; Alves, Alexandre Alonso; Rosado, Tatiana Barbosa; Bhering, Leonardo Lopes

2017-01-01

Genomic wide selection is a promising approach for improving the selection accuracy in plant breeding, particularly in species with long life cycles, such as Jatropha. Therefore, the objectives of this study were to estimate the genetic parameters for grain yield (GY) and the weight of 100 seeds (W100S) using restricted maximum likelihood (REML); to compare the performance of GWS methods to predict GY and W100S; and to estimate how many markers are needed to train the GWS model to obtain the maximum accuracy. Eight GWS models were compared in terms of predictive ability. The impact that the marker density had on the predictive ability was investigated using a varying number of markers, from 2 to 1,248. Because the genetic variance between evaluated genotypes was significant, it was possible to obtain selection gain. All of the GWS methods tested in this study can be used to predict GY and W100S in Jatropha. A training model fitted using 1,000 and 800 markers is sufficient to capture the maximum genetic variance and, consequently, maximum prediction ability of GY and W100S, respectively. This study demonstrated the applicability of genome-wide prediction to identify useful genetic sources of GY and W100S for Jatropha breeding. Further research is needed to confirm the applicability of the proposed approach to other complex traits.

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

PubMed

Liang, Lisa; Aiken, Christopher; McClelland, Robyn; Morrison, Ludivine Coudière; Tatari, Nazanin; Remke, Marc; Ramaswamy, Vijay; Issaivanan, Magimairajan; Ryken, Timothy; Del Bigio, Marc R; Taylor, Michael D; Werbowetski-Ogilvie, Tamra E

2015-11-17

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

Genomic selection and association mapping in rice (Oryza sativa): effect of trait genetic architecture, training population composition, marker number and statistical model on accuracy of rice genomic selection in elite, tropical rice breeding lines.

PubMed

Spindel, Jennifer; Begum, Hasina; Akdemir, Deniz; Virk, Parminder; Collard, Bertrand; Redoña, Edilberto; Atlin, Gary; Jannink, Jean-Luc; McCouch, Susan R

2015-02-01

Genomic Selection (GS) is a new breeding method in which genome-wide markers are used to predict the breeding value of individuals in a breeding population. GS has been shown to improve breeding efficiency in dairy cattle and several crop plant species, and here we evaluate for the first time its efficacy for breeding inbred lines of rice. We performed a genome-wide association study (GWAS) in conjunction with five-fold GS cross-validation on a population of 363 elite breeding lines from the International Rice Research Institute's (IRRI) irrigated rice breeding program and herein report the GS results. The population was genotyped with 73,147 markers using genotyping-by-sequencing. The training population, statistical method used to build the GS model, number of markers, and trait were varied to determine their effect on prediction accuracy. For all three traits, genomic prediction models outperformed prediction based on pedigree records alone. Prediction accuracies ranged from 0.31 and 0.34 for grain yield and plant height to 0.63 for flowering time. Analyses using subsets of the full marker set suggest that using one marker every 0.2 cM is sufficient for genomic selection in this collection of rice breeding materials. RR-BLUP was the best performing statistical method for grain yield where no large effect QTL were detected by GWAS, while for flowering time, where a single very large effect QTL was detected, the non-GS multiple linear regression method outperformed GS models. For plant height, in which four mid-sized QTL were identified by GWAS, random forest produced the most consistently accurate GS models. Our results suggest that GS, informed by GWAS interpretations of genetic architecture and population structure, could become an effective tool for increasing the efficiency of rice breeding as the costs of genotyping continue to decline.

Cancer diagnosis marker extraction for soft tissue sarcomas based on gene expression profiling data by using projective adaptive resonance theory (PART) filtering method

PubMed Central

Takahashi, Hiro; Nemoto, Takeshi; Yoshida, Teruhiko; Honda, Hiroyuki; Hasegawa, Tadashi

2006-01-01

Background Recent advances in genome technologies have provided an excellent opportunity to determine the complete biological characteristics of neoplastic tissues, resulting in improved diagnosis and selection of treatment. To accomplish this objective, it is important to establish a sophisticated algorithm that can deal with large quantities of data such as gene expression profiles obtained by DNA microarray analysis. Results Previously, we developed the projective adaptive resonance theory (PART) filtering method as a gene filtering method. This is one of the clustering methods that can select specific genes for each subtype. In this study, we applied the PART filtering method to analyze microarray data that were obtained from soft tissue sarcoma (STS) patients for the extraction of subtype-specific genes. The performance of the filtering method was evaluated by comparison with other widely used methods, such as signal-to-noise, significance analysis of microarrays, and nearest shrunken centroids. In addition, various combinations of filtering and modeling methods were used to extract essential subtype-specific genes. The combination of the PART filtering method and boosting – the PART-BFCS method – showed the highest accuracy. Seven genes among the 15 genes that are frequently selected by this method – MIF, CYFIP2, HSPCB, TIMP3, LDHA, ABR, and RGS3 – are known prognostic marker genes for other tumors. These genes are candidate marker genes for the diagnosis of STS. Correlation analysis was performed to extract marker genes that were not selected by PART-BFCS. Sixteen genes among those extracted are also known prognostic marker genes for other tumors, and they could be candidate marker genes for the diagnosis of STS. Conclusion The procedure that consisted of two steps, such as the PART-BFCS and the correlation analysis, was proposed. The results suggest that novel diagnostic and therapeutic targets for STS can be extracted by a procedure that includes the PART filtering method. PMID:16948864

Genomic Selection and Association Mapping in Rice (Oryza sativa): Effect of Trait Genetic Architecture, Training Population Composition, Marker Number and Statistical Model on Accuracy of Rice Genomic Selection in Elite, Tropical Rice Breeding Lines

PubMed Central

Spindel, Jennifer; Begum, Hasina; Akdemir, Deniz; Virk, Parminder; Collard, Bertrand; Redoña, Edilberto; Atlin, Gary; Jannink, Jean-Luc; McCouch, Susan R.

2015-01-01

Genomic Selection (GS) is a new breeding method in which genome-wide markers are used to predict the breeding value of individuals in a breeding population. GS has been shown to improve breeding efficiency in dairy cattle and several crop plant species, and here we evaluate for the first time its efficacy for breeding inbred lines of rice. We performed a genome-wide association study (GWAS) in conjunction with five-fold GS cross-validation on a population of 363 elite breeding lines from the International Rice Research Institute's (IRRI) irrigated rice breeding program and herein report the GS results. The population was genotyped with 73,147 markers using genotyping-by-sequencing. The training population, statistical method used to build the GS model, number of markers, and trait were varied to determine their effect on prediction accuracy. For all three traits, genomic prediction models outperformed prediction based on pedigree records alone. Prediction accuracies ranged from 0.31 and 0.34 for grain yield and plant height to 0.63 for flowering time. Analyses using subsets of the full marker set suggest that using one marker every 0.2 cM is sufficient for genomic selection in this collection of rice breeding materials. RR-BLUP was the best performing statistical method for grain yield where no large effect QTL were detected by GWAS, while for flowering time, where a single very large effect QTL was detected, the non-GS multiple linear regression method outperformed GS models. For plant height, in which four mid-sized QTL were identified by GWAS, random forest produced the most consistently accurate GS models. Our results suggest that GS, informed by GWAS interpretations of genetic architecture and population structure, could become an effective tool for increasing the efficiency of rice breeding as the costs of genotyping continue to decline. PMID:25689273

High MHC diversity maintained by balancing selection in an otherwise genetically monomorphic mammal

PubMed Central

Aguilar, Andres; Roemer, Gary; Debenham, Sally; Binns, Matthew; Garcelon, David; Wayne, Robert K.

2004-01-01

The San Nicolas Island fox (Urocyon littoralis dickeyi) is genetically the most monomorphic sexually reproducing animal population yet reported and has no variation in hypervariable genetic markers. Such low levels of variation imply lower resistance to pathogens, reduced fitness, and problems in distinguishing kin from non-kin. In vertebrates, the MHC contains genes that influence disease resistance and kin recognition and may be under intense balancing selection in some populations. Hence, genetic variation at the MHC might persist despite the extreme monomorphism shown by neutral markers. We examine variation of five loci within the MHC of San Nicolas Island foxes and find remarkably high levels of variation. Further, we show by simulation that genetic monomorphism at neutral loci and high MHC variation could arise only through an extreme population bottleneck of <10 individuals, ≈10–20 generations ago, accompanied by unprecedented selection coefficients of >0.5 on MHC loci. These results support the importance of balancing selection as a mechanism to maintain variation in natural populations and expose the difficulty of using neutral markers as surrogates for variation in fitness-related loci. PMID:14990802

The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and is a selectable marker for Bacillus subtilis.

PubMed

Geraskina, Natalia V; Butov, Ivan A; Yomantas, Yurgis A V; Stoynova, Nataliya V

2015-02-01

Genetically engineered microbes are of high practical importance due to their cost-effective production of valuable metabolites and enzymes, and the search for new selectable markers for genetic manipulation is of particular interest. Here, we revealed that the soil bacterium Bacillus amyloliquefaciens A50 is tolerant to the non-canonical amino acid D-tyrosine (D-Tyr), in contrast to the closely related Bacillus strain B. subtilis 168, which is a widely used "domesticated" laboratory strain. The gene responsible for resistance to D-Tyr was identified. The resistance was associated with the activity of a potential D-tyrosyl-tRNA(Tyr) deacylase. Orthologs of this enzyme are capable of hydrolyzing the ester bond and recycling misacetylated D-aminoacyl-tRNA molecules into free tRNAs and D-amino acids. This gene, yrvI (dtd), is applicable as a convenient, small selectable marker for non-antibiotic resistance selection in experiments aimed at genome editing of D-Tyr-sensitive microorganisms. Copyright © 2014 Elsevier GmbH. All rights reserved.

Prediction of genetic values of quantitative traits in plant breeding using pedigree and molecular markers.

PubMed

Crossa, José; Campos, Gustavo de Los; Pérez, Paulino; Gianola, Daniel; Burgueño, Juan; Araus, José Luis; Makumbi, Dan; Singh, Ravi P; Dreisigacker, Susanne; Yan, Jianbing; Arief, Vivi; Banziger, Marianne; Braun, Hans-Joachim

2010-10-01

The availability of dense molecular markers has made possible the use of genomic selection (GS) for plant breeding. However, the evaluation of models for GS in real plant populations is very limited. This article evaluates the performance of parametric and semiparametric models for GS using wheat (Triticum aestivum L.) and maize (Zea mays) data in which different traits were measured in several environmental conditions. The findings, based on extensive cross-validations, indicate that models including marker information had higher predictive ability than pedigree-based models. In the wheat data set, and relative to a pedigree model, gains in predictive ability due to inclusion of markers ranged from 7.7 to 35.7%. Correlation between observed and predictive values in the maize data set achieved values up to 0.79. Estimates of marker effects were different across environmental conditions, indicating that genotype × environment interaction is an important component of genetic variability. These results indicate that GS in plant breeding can be an effective strategy for selecting among lines whose phenotypes have yet to be observed.

Identification and Validation of SNP Markers Linked to Dwarf Traits Using SLAF-Seq Technology in Lagerstroemia

PubMed Central

Ju, Yiqian; Jiao, Yao; Feng, Lu; Pan, Huitang; Cheng, Tangren; Zhang, Qixiang

2016-01-01

The genetic control of plant architecture is a promising approach to breed desirable cultivars, particularly in ornamental flowers. In this study, the F1 population (142 seedlings) derived from Lagerstroemia fauriei (non-dwarf) × L. indica ‘Pocomoke’ (dwarf) was phenotyped for six traits (plant height (PH), internode length (IL), internode number, primary lateral branch height (PLBH), secondary lateral branch height and primary branch number), and the IL and PLBH traits were positively correlated with the PH trait and considered representative indexes of PH. Fifty non-dwarf and dwarf seedlings were pooled and subjected to a specific-locus amplified fragment sequencing (SLAF-seq) method, which screened 1221 polymorphic markers. A total of 3 markers segregating between bulks were validated in the F1 population, with the M16337 and M38412 markers highly correlated with the IL trait and the M25207 marker highly correlated with the PLBH trait. These markers provide a predictability of approximately 80% using a single marker (M25207) and a predictability of 90% using marker combinations (M16337 + M25207) in the F1 population, which revealed that the IL and the PLBH traits, especially the PLBH, were the decisive elements for PH in terms of molecular regulation. Further validation was performed in the BC1 population and a set of 28 Lagerstroemia stocks using allele-specific PCR (AS-PCR) technology, and the results showed the stability and reliability of the SNP markers and the co-determination of PH by multiple genes. Our findings provide an important theoretical and practical basis for the early prediction and indirect selection of PH using the IL and the PLBH, and the detected SNPs may be useful for marker-assisted selection (MAS) in crape myrtle. PMID:27404662

Cell-surface marker discovery for lung cancer

PubMed Central

Cohen, Allison S.; Khalil, Farah K.; Welsh, Eric A.; Schabath, Matthew B.; Enkemann, Steven A.; Davis, Andrea; Zhou, Jun-Min; Boulware, David C.; Kim, Jongphil; Haura, Eric B.; Morse, David L.

2017-01-01

Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan–Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients. PMID:29371917

Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers.

PubMed

Gao, Chunsheng; Xin, Pengfei; Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining

2014-01-01

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.

Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers

PubMed Central

Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining

2014-01-01

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis. PMID:25329551

Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

PubMed

Koyuncu, Can Fahrettin; Akhan, Ece; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2016-04-01

Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

The value of using DNA markers for beef bull selection in the seedstock sector.

PubMed

Van Eenennaam, A L; van der Werf, J H J; Goddard, M E

2011-02-01

The objective of this study was to estimate the value derived from using DNA information to increase the accuracy of beef sire selection in a closed seedstock herd. Breeding objectives for commercial production systems targeting 2 diverse markets were examined using multiple-trait selection indexes developed for the Australian cattle industry. Indexes included those for both maternal (self-replacing) and terminal herds targeting either a domestic market, where steers are finished on pasture, or the export market, where steers are finished on concentrate rations in feedlots and marbling has a large value. Selection index theory was used to predict the response to conventional selection based on phenotypic performance records, and this was compared with including information from 2 hypothetical marker panels. In 1 case the marker panel explained a percentage of additive genetic variance equal to the heritability for all traits in the breeding objective and selection criteria, and in the other case to one-half of this amount. Discounted gene flow methodology was used to calculate the value derived from the use of superior bulls selected using DNA test information and performance recording over that derived from conventional selection using performance recording alone. Results were ultimately calculated as discounted returns per DNA test purchased by the seedstock operator. The DNA testing using these hypothetical marker panels increased the selection response between 29 to 158%. The value of this improvement above that obtained using traditional performance recording ranged from $89 to 565 per commercial bull, and $5,332 to 27,910 per stud bull. Assuming that the entire bull calf crop was tested to achieve these gains, the value of the genetic gain derived from DNA testing ranged from $204 to 1,119 per test. All values assumed that the benefits derived from using superior bulls were efficiently transferred along the production chain to the seedstock producer incurring the costs of genotyping. These results suggest that the development of greater-accuracy DNA tests for beef cattle selection could be beneficial from an industry-wide perspective, but the commercial viability will strongly depend on price signaling throughout the production chain.

Sunflower Hybrid Breeding: From Markers to Genomic Selection

PubMed Central

Dimitrijevic, Aleksandra; Horn, Renate

2018-01-01

In sunflower, molecular markers for simple traits as, e.g., fertility restoration, high oleic acid content, herbicide tolerance or resistances to Plasmopara halstedii, Puccinia helianthi, or Orobanche cumana have been successfully used in marker-assisted breeding programs for years. However, agronomically important complex quantitative traits like yield, heterosis, drought tolerance, oil content or selection for disease resistance, e.g., against Sclerotinia sclerotiorum have been challenging and will require genome-wide approaches. Plant genetic resources for sunflower are being collected and conserved worldwide that represent valuable resources to study complex traits. Sunflower association panels provide the basis for genome-wide association studies, overcoming disadvantages of biparental populations. Advances in technologies and the availability of the sunflower genome sequence made novel approaches on the whole genome level possible. Genotype-by-sequencing, and whole genome sequencing based on next generation sequencing technologies facilitated the production of large amounts of SNP markers for high density maps as well as SNP arrays and allowed genome-wide association studies and genomic selection in sunflower. Genome wide or candidate gene based association studies have been performed for traits like branching, flowering time, resistance to Sclerotinia head and stalk rot. First steps in genomic selection with regard to hybrid performance and hybrid oil content have shown that genomic selection can successfully address complex quantitative traits in sunflower and will help to speed up sunflower breeding programs in the future. To make sunflower more competitive toward other oil crops higher levels of resistance against pathogens and better yield performance are required. In addition, optimizing plant architecture toward a more complex growth type for higher plant densities has the potential to considerably increase yields per hectare. Integrative approaches combining omic technologies (genomics, transcriptomics, proteomics, metabolomics and phenomics) using bioinformatic tools will facilitate the identification of target genes and markers for complex traits and will give a better insight into the mechanisms behind the traits. PMID:29387071

Rapid fixation of non-native alleles revealed by genome-wide SNP analysis of hybrid tiger salamanders.

PubMed

Fitzpatrick, Benjamin M; Johnson, Jarrett R; Kump, D Kevin; Shaffer, H Bradley; Smith, Jeramiah J; Voss, S Randal

2009-07-24

Hybrid zones represent valuable opportunities to observe evolution in systems that are unusually dynamic and where the potential for the origin of novelty and rapid adaptation co-occur with the potential for dysfunction. Recently initiated hybrid zones are particularly exciting evolutionary experiments because ongoing natural selection on novel genetic combinations can be studied in ecological time. Moreover, when hybrid zones involve native and introduced species, complex genetic patterns present important challenges for conservation policy. To assess variation of admixture dynamics, we scored a large panel of markers in five wild hybrid populations formed when Barred Tiger Salamanders were introduced into the range of California Tiger Salamanders. At three of 64 markers, introduced alleles have largely displaced native alleles within the hybrid populations. Another marker (GNAT1) showed consistent heterozygote deficits in the wild, and this marker was associated with embryonic mortality in laboratory F2's. Other deviations from equilibrium expectations were idiosyncratic among breeding ponds, consistent with highly stochastic demographic effects. While most markers retain native and introduced alleles in expected proportions, strong selection appears to be eliminating native alleles at a smaller set of loci. Such rapid fixation of alleles is detectable only in recently formed hybrid zones, though it might be representative of dynamics that frequently occur in nature. These results underscore the variable and mosaic nature of hybrid genomes and illustrate the potency of recombination and selection in promoting variable, and often unpredictable genetic outcomes. Introgression of a few, strongly selected introduced alleles should not necessarily affect the conservation status of California Tiger Salamanders, but suggests that genetically pure populations of this endangered species will be difficult to maintain.

Rapid fixation of non-native alleles revealed by genome-wide SNP analysis of hybrid tiger salamanders

PubMed Central

Fitzpatrick, Benjamin M; Johnson, Jarrett R; Kump, D Kevin; Shaffer, H Bradley; Smith, Jeramiah J; Voss, S Randal

2009-01-01

Background Hybrid zones represent valuable opportunities to observe evolution in systems that are unusually dynamic and where the potential for the origin of novelty and rapid adaptation co-occur with the potential for dysfunction. Recently initiated hybrid zones are particularly exciting evolutionary experiments because ongoing natural selection on novel genetic combinations can be studied in ecological time. Moreover, when hybrid zones involve native and introduced species, complex genetic patterns present important challenges for conservation policy. To assess variation of admixture dynamics, we scored a large panel of markers in five wild hybrid populations formed when Barred Tiger Salamanders were introduced into the range of California Tiger Salamanders. Results At three of 64 markers, introduced alleles have largely displaced native alleles within the hybrid populations. Another marker (GNAT1) showed consistent heterozygote deficits in the wild, and this marker was associated with embryonic mortality in laboratory F2's. Other deviations from equilibrium expectations were idiosyncratic among breeding ponds, consistent with highly stochastic demographic effects. Conclusion While most markers retain native and introduced alleles in expected proportions, strong selection appears to be eliminating native alleles at a smaller set of loci. Such rapid fixation of alleles is detectable only in recently formed hybrid zones, though it might be representative of dynamics that frequently occur in nature. These results underscore the variable and mosaic nature of hybrid genomes and illustrate the potency of recombination and selection in promoting variable, and often unpredictable genetic outcomes. Introgression of a few, strongly selected introduced alleles should not necessarily affect the conservation status of California Tiger Salamanders, but suggests that genetically pure populations of this endangered species will be difficult to maintain. PMID:19630983

Construction of a genetic linkage map and analysis of quantitative trait loci associated with the agronomically important traits of Pleurotus eryngii

Treesearch

Chak Han Im; Young-Hoon Park; Kenneth E. Hammel; Bokyung Park; Soon Wook Kwon; Hojin Ryu; Jae-San Ryu

2016-01-01

Breeding new strains with improved traits is a long-standing goal of mushroom breeders that can be expedited by marker-assisted selection (MAS). We constructed a genetic linkage map of Pleurotus eryngii based on segregation analysis of markers in postmeiotic monokaryons from KNR2312. In total, 256 loci comprising 226 simple sequence-repeat (SSR) markers, 2 mating-type...

Genetic evolutionary taboo search for optimal marker placement in infrared patient setup

NASA Astrophysics Data System (ADS)

Riboldi, M.; Baroni, G.; Spadea, M. F.; Tagaste, B.; Garibaldi, C.; Cambria, R.; Orecchia, R.; Pedotti, A.

2007-09-01

In infrared patient setup adequate selection of the external fiducial configuration is required for compensating inner target displacements (target registration error, TRE). Genetic algorithms (GA) and taboo search (TS) were applied in a newly designed approach to optimal marker placement: the genetic evolutionary taboo search (GETS) algorithm. In the GETS paradigm, multiple solutions are simultaneously tested in a stochastic evolutionary scheme, where taboo-based decision making and adaptive memory guide the optimization process. The GETS algorithm was tested on a group of ten prostate patients, to be compared to standard optimization and to randomly selected configurations. The changes in the optimal marker configuration, when TRE is minimized for OARs, were specifically examined. Optimal GETS configurations ensured a 26.5% mean decrease in the TRE value, versus 19.4% for conventional quasi-Newton optimization. Common features in GETS marker configurations were highlighted in the dataset of ten patients, even when multiple runs of the stochastic algorithm were performed. Including OARs in TRE minimization did not considerably affect the spatial distribution of GETS marker configurations. In conclusion, the GETS algorithm proved to be highly effective in solving the optimal marker placement problem. Further work is needed to embed site-specific deformation models in the optimization process.

Measurement Marker Recognition In A Time Sequence Of Infrared Images For Biomedical Applications

NASA Astrophysics Data System (ADS)

Fiorini, A. R.; Fumero, R.; Marchesi, R.

1986-03-01

In thermographic measurements, quantitative surface temperature evaluation is often uncertain. The main reason is in the lack of available reference points in transient conditions. Reflective markers were used for automatic marker recognition and pixel coordinate computations. An algorithm selects marker icons to match marker references where particular luminance conditions are satisfied. Automatic marker recognition allows luminance compensation and temperature calibration of recorded infrared images. A biomedical application is presented: the dynamic behaviour of the surface temperature distributions is investigated in order to study the performance of two different pumping systems for extracorporeal circulation. Sequences of images are compared and results are discussed. Finally, the algorithm allows to monitor the experimental environment and to alert for the presence of unusual experimental conditions.

Isolation and characterization of microsatellite markers for Jasminum sambac (Oleaceae) using Illumina shotgun sequencing.

PubMed

Li, Yong; Zhang, Weirui

2015-10-01

Microsatellite markers of Jasminum sambac (Oleaceae) were isolated to investigate wild germplasm resources and provide markers for breeding. Illumina sequencing was used to isolate microsatellite markers from the transcriptome of J. sambac. A total of 1322 microsatellites were identified from 49,772 assembled unigenes. One hundred primer pairs were randomly selected to verify primer amplification efficiency. Out of these tested primer pairs, 31 were successfully amplified: 18 primer pairs yielded a single allele, seven exhibited fixed heterozygosity with two alleles, and only six displayed polymorphisms. This study obtained the first set of microsatellite markers for J. sambac, which will be helpful for the assessment of wild germplasm resources and the development of molecular marker-assisted breeding.

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.

Transport of sewage molecular markers through saturated soil column and effect of easily biodegradable primary substrate on their removal.

PubMed

Foolad, Mahsa; Ong, Say Leong; Hu, Jiangyong

2015-11-01

Pharmaceutical and personal care products (PPCPs) and artificial sweeteners (ASs) are emerging organic contaminants (EOCs) in the aquatic environment. The presence of PPCPs and ASs in water bodies has an ecologic potential risk and health concern. Therefore, it is needed to detect the pollution sources by understanding the transport behavior of sewage molecular markers in a subsurface area. The aim of this study was to evaluate transport of nine selected molecular markers through saturated soil column experiments. The selected sewage molecular markers in this study were six PPCPs including acetaminophen (ACT), carbamazepine (CBZ), caffeine (CF), crotamiton (CTMT), diethyltoluamide (DEET), salicylic acid (SA) and three ASs including acesulfame (ACF), cyclamate (CYC), and saccharine (SAC). Results confirmed that ACF, CBZ, CTMT, CYC and SAC were suitable to be used as sewage molecular markers since they were almost stable against sorption and biodegradation process during soil column experiments. In contrast, transport of ACT, CF and DEET were limited by both sorption and biodegradation processes and 100% removal efficiency was achieved in the biotic column. Moreover, in this study the effect of different acetate concentration (0-100mg/L) as an easily biodegradable primary substrate on a removal of PPCPs and ASs was also studied. Results showed a negative correlation (r(2)>0.75) between the removal of some selected sewage chemical markers including ACF, CF, ACT, CYC, SAC and acetate concentration. CTMT also decreased with the addition of acetate, but increasing acetate concentration did not affect on its removal. CBZ and DEET removal were not dependent on the presence of acetate. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exploiting rice-sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map.

PubMed

Ramu, P; Kassahun, B; Senthilvel, S; Ashok Kumar, C; Jayashree, B; Folkertsma, R T; Reddy, L Ananda; Kuruvinashetti, M S; Haussmann, B I G; Hash, C T

2009-11-01

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 x E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice-sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.

Toward Genomics-Based Breeding in C3 Cool-Season Perennial Grasses.

PubMed

Talukder, Shyamal K; Saha, Malay C

2017-01-01

Most important food and feed crops in the world belong to the C3 grass family. The future of food security is highly reliant on achieving genetic gains of those grasses. Conventional breeding methods have already reached a plateau for improving major crops. Genomics tools and resources have opened an avenue to explore genome-wide variability and make use of the variation for enhancing genetic gains in breeding programs. Major C3 annual cereal breeding programs are well equipped with genomic tools; however, genomic research of C3 cool-season perennial grasses is lagging behind. In this review, we discuss the currently available genomics tools and approaches useful for C3 cool-season perennial grass breeding. Along with a general review, we emphasize the discussion focusing on forage grasses that were considered orphan and have little or no genetic information available. Transcriptome sequencing and genotype-by-sequencing technology for genome-wide marker detection using next-generation sequencing (NGS) are very promising as genomics tools. Most C3 cool-season perennial grass members have no prior genetic information; thus NGS technology will enhance collinear study with other C3 model grasses like Brachypodium and rice. Transcriptomics data can be used for identification of functional genes and molecular markers, i.e., polymorphism markers and simple sequence repeats (SSRs). Genome-wide association study with NGS-based markers will facilitate marker identification for marker-assisted selection. With limited genetic information, genomic selection holds great promise to breeders for attaining maximum genetic gain of the cool-season C3 perennial grasses. Application of all these tools can ensure better genetic gains, reduce length of selection cycles, and facilitate cultivar development to meet the future demand for food and fodder.

HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

PubMed

Fiedler, Markus R M; Gensheimer, Tarek; Kubisch, Christin; Meyer, Vera

2017-03-08

For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers.

PubMed

Sengupta, Subhadipa; Chakraborti, Dipankar; Mondal, Hossain A; Das, Sampa

2010-03-01

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T(0) plants with ASAL- lox-hpt-lox T-DNA and three single-copy T(0) plants with cre-bar T-DNA. Marker gene excisions were detected in T(1) hybrids through hygromycin sensitivity assay. Molecular analysis of T(1) plants exhibited 27.4% recombination efficiency. T(2) progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T(2) progeny plants. In planta bioassay of GLH and BPH performed on these T(2) progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

ABCG2 Is a Selectable Marker for Enhanced Multilineage Differentiation Potential in Periodontal Ligament Stem Cells

PubMed Central

Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Várady, György; Szabó, Gyula; Uher, Ferenc; Sarkadi, Balázs

2015-01-01

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth. PMID:25101689

Regularized quantile regression for SNP marker estimation of pig growth curves.

PubMed

Barroso, L M A; Nascimento, M; Nascimento, A C C; Silva, F F; Serão, N V L; Cruz, C D; Resende, M D V; Silva, F L; Azevedo, C F; Lopes, P S; Guimarães, S E F

2017-01-01

Genomic growth curves are generally defined only in terms of population mean; an alternative approach that has not yet been exploited in genomic analyses of growth curves is the Quantile Regression (QR). This methodology allows for the estimation of marker effects at different levels of the variable of interest. We aimed to propose and evaluate a regularized quantile regression for SNP marker effect estimation of pig growth curves, as well as to identify the chromosome regions of the most relevant markers and to estimate the genetic individual weight trajectory over time (genomic growth curve) under different quantiles (levels). The regularized quantile regression (RQR) enabled the discovery, at different levels of interest (quantiles), of the most relevant markers allowing for the identification of QTL regions. We found the same relevant markers simultaneously affecting different growth curve parameters (mature weight and maturity rate): two (ALGA0096701 and ALGA0029483) for RQR(0.2), one (ALGA0096701) for RQR(0.5), and one (ALGA0003761) for RQR(0.8). Three average genomic growth curves were obtained and the behavior was explained by the curve in quantile 0.2, which differed from the others. RQR allowed for the construction of genomic growth curves, which is the key to identifying and selecting the most desirable animals for breeding purposes. Furthermore, the proposed model enabled us to find, at different levels of interest (quantiles), the most relevant markers for each trait (growth curve parameter estimates) and their respective chromosomal positions (identification of new QTL regions for growth curves in pigs). These markers can be exploited under the context of marker assisted selection while aiming to change the shape of pig growth curves.

Using case-control designs for genome-wide screening for associations between genetic markers and disease susceptibility loci.

PubMed

Yang, Q; Khoury, M J; Atkinson, M; Sun, F; Cheng, R; Flanders, W D

1999-01-01

We used a case-control design to scan the genome for any associations between genetic markers and disease susceptibility loci using the first two replicates of the Mycenaean population from the GAW11 (Problem 2) data. Using a case-control approach, we constructed a series of 2-by-3 tables for each allele of every marker on all six chromosomes. Odds ratios (ORs) and 95% confidence intervals (95% CI) were estimated for all alleles of every marker. We selected the one allele for which the estimated OR had the minimum p-value to plot in the graph. Among these selected ORs, we calculated 95% CI for those that had a p-value < or = adjusted alpha level. Significantly high ORs were taken to indicate an association between a marker locus and a suspected disease-susceptibility gene. For the Mycenaean population, the case-control design identified allele number 1 of marker 24 on chromosome 1 to be associated with a disease susceptibility gene, OR = 2.10 (95% CI 1.66-2.62). Our approach failed to show any other significant association between case-control status and genetic markers. Stratified analysis on the environmental risk factor (E1) provided no further evidence of significant association other than allele 1 of marker 24 on chromosome 1. These data indicate the absence of linkage disequilibrium for markers flanking loci A, B, and C. Finally, we examined the effect of gene x environment (G x E) interaction for the identified allele. Our results provided no evidence of G x E interaction, but suggested that the environmental exposure alone was a risk factor for the disease.

Exploration of the Hypothalamic-Pituitary-Adrenal Axis to Improve Animal Welfare by Means of Genetic Selection: Lessons from the South African Merino.

PubMed

Hough, Denise; Swart, Pieter; Cloete, Schalk

2013-05-17

It is a difficult task to improve animal production by means of genetic selection, if the environment does not allow full expression of the animal's genetic potential. This concept may well be the future for animal welfare, because it highlights the need to incorporate traits related to production and robustness, simultaneously, to reach sustainable breeding goals. This review explores the identification of potential genetic markers for robustness within the hypothalamic-pituitary-adrenal axis (HPAA), since this axis plays a vital role in the stress response. If genetic selection for superior HPAA responses to stress is possible, then it ought to be possible to breed robust and easily managed genotypes that might be able to adapt to a wide range of environmental conditions whilst expressing a high production potential. This approach is explored in this review by means of lessons learnt from research on Merino sheep, which were divergently selected for their multiple rearing ability. These two selection lines have shown marked differences in reproduction, production and welfare, which makes this breeding programme ideal to investigate potential genetic markers of robustness. The HPAA function is explored in detail to elucidate where such genetic markers are likely to be found.

Prediction of hepatocellular carcinoma biological behavior in patient selection for liver transplantation

PubMed Central

Cillo, Umberto; Giuliani, Tommaso; Polacco, Marina; Herrero Manley, Luz Maria; Crivellari, Gino; Vitale, Alessandro

2016-01-01

Morphological criteria have always been considered the benchmark for selecting hepatocellular carcinoma (HCC) patients for liver transplantation (LT). These criteria, which are often inappropriate to express the tumor’s biological behavior and aggressiveness, offer only a static view of the disease burden and are frequently unable to correctly stratify the tumor recurrence risk after LT. Alpha-fetoprotein (AFP) and its progression as well as AFP-mRNA, AFP-L3%, des-γ-carboxyprothrombin, inflammatory markers and other serological tests appear to be correlated with post-transplant outcomes. Several other markers for patient selection including functional imaging studies such as 18F-FDG-PET imaging, histological evaluation of tumor grade, tissue-specific biomarkers, and molecular signatures have been outlined in the literature. HCC growth rate and response to pre-transplant therapies can further contribute to the transplant evaluation process of HCC patients. While AFP, its progression, and HCC response to pre-transplant therapy have already been used as a part of an integrated prognostic model for selecting patients, the utility of other markers in the transplant setting is still under investigation. This article intends to review the data in the literature concerning predictors that could be included in an integrated LT selection model and to evaluate the importance of biological aggressiveness in the evaluation process of these patients. PMID:26755873

Applications of random forest feature selection for fine-scale genetic population assignment.

PubMed

Sylvester, Emma V A; Bentzen, Paul; Bradbury, Ian R; Clément, Marie; Pearce, Jon; Horne, John; Beiko, Robert G

2018-02-01

Genetic population assignment used to inform wildlife management and conservation efforts requires panels of highly informative genetic markers and sensitive assignment tests. We explored the utility of machine-learning algorithms (random forest, regularized random forest and guided regularized random forest) compared with F ST ranking for selection of single nucleotide polymorphisms (SNP) for fine-scale population assignment. We applied these methods to an unpublished SNP data set for Atlantic salmon ( Salmo salar ) and a published SNP data set for Alaskan Chinook salmon ( Oncorhynchus tshawytscha ). In each species, we identified the minimum panel size required to obtain a self-assignment accuracy of at least 90% using each method to create panels of 50-700 markers Panels of SNPs identified using random forest-based methods performed up to 7.8 and 11.2 percentage points better than F ST -selected panels of similar size for the Atlantic salmon and Chinook salmon data, respectively. Self-assignment accuracy ≥90% was obtained with panels of 670 and 384 SNPs for each data set, respectively, a level of accuracy never reached for these species using F ST -selected panels. Our results demonstrate a role for machine-learning approaches in marker selection across large genomic data sets to improve assignment for management and conservation of exploited populations.

Disease resistance breeding in rose: current status and potential of biotechnological tools.

PubMed

Debener, Thomas; Byrne, David H

2014-11-01

The cultivated rose is a multispecies complex for which a high level of disease protection is needed due to the low tolerance of blemishes in ornamental plants. The most important fungal diseases are black spot, powdery mildew, botrytis and downy mildew. Rose rosette, a lethal viral pathogen, is emerging as a devastating disease in North America. Currently rose breeders use a recurrent phenotypic selection approach and perform selection for disease resistance for most pathogen issues in a 2-3 year field trial. Marker assisted selection could accelerate this breeding process. Thus far markers have been identified for resistance to black spot (Rdrs) and powdery mildew and with the ability of genotyping by sequencing to generate 1000s of markers our ability to identify markers useful in plant improvement should increase exponentially. Transgenic rose lines with various fungal resistance genes inserted have shown limited success and RNAi technology has potential to provide virus resistance. Roses, as do other plants, have sequences homologous to characterized R-genes in their genomes, some which have been related to specific disease resistance. With improving next generation sequencing technology, our ability to do genomic and transcriptomic studies of the resistance related genes in both the rose and the pathogens to reveal novel gene targets to develop resistant roses will accelerate. Finally, the development of designer nucleases opens up a potentially non-GMO approach to directly modify a rose's DNA to create a disease resistant rose. Although there is much potential, at present rose breeders are not using marker assisted breeding primarily because a good suite of marker/trait associations (MTA) that would ensure a path to stable disease resistance is not available. As our genomic analytical tools improve, so will our ability to identify useful genes and linked markers. Once these MTAs are available, it will be the cost savings, both in time and money, that will convince the breeders to use the technology. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Recurrent parent genome recovery analysis in a marker-assisted backcrossing program of rice (Oryza sativa L.).

PubMed

Miah, Gous; Rafii, Mohd Y; Ismail, Mohd R; Puteh, Adam B; Rahim, Harun A; Latif, Mohammad A

2015-02-01

Backcross breeding is the most commonly used method for incorporating a blast resistance gene into a rice cultivar. Linkage between the resistance gene and undesirable units can persist for many generations of backcrossing. Marker-assisted backcrossing (MABC) along with marker-assisted selection (MAS) contributes immensely to overcome the main limitation of the conventional breeding and accelerates recurrent parent genome (RPG) recovery. The MABC approach was employed to incorporate (a) blast resistance gene(s) from the donor parent Pongsu Seribu 1, the blast-resistant local variety in Malaysia, into the genetic background of MR219, a popular high-yielding rice variety that is blast susceptible, to develop a blast-resistant MR219 improved variety. In this perspective, the recurrent parent genome recovery was analyzed in early generations of backcrossing using simple sequence repeat (SSR) markers. Out of 375 SSR markers, 70 markers were found polymorphic between the parents, and these markers were used to evaluate the plants in subsequent generations. Background analysis revealed that the extent of RPG recovery ranged from 75.40% to 91.3% and from 80.40% to 96.70% in BC1F1 and BC2F1 generations, respectively. In this study, the recurrent parent genome content in the selected BC2F2 lines ranged from 92.7% to 97.7%. The average proportion of the recurrent parent in the selected improved line was 95.98%. MAS allowed identification of the plants that are more similar to the recurrent parent for the loci evaluated in backcross generations. The application of MAS with the MABC breeding program accelerated the recovery of the RP genome, reducing the number of generations and the time for incorporating resistance against rice blast. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

A preliminary study for identification of candidate AFLP markers under artificial selection for shell color in pearl oyster Pinctada fucata.

PubMed

Zou, Keshu; Zhang, Dianchang; Guo, Huayang; Zhu, Caiyan; Li, Min; Jiang, Shigui

2014-05-25

Pearl oyster Pinctada fucata is widely cultured to produce seawater pearl in South China, and the quality of pearl is significantly affected by its shell color. Thus the Pearl Oyster Selective Breeding Program (POSBP) was carried out for the shell color and growth traits. The black (B), gold (G), red (R) and white (W) shell strains with fast growth trait were achieved after five successive generation selection. In this study, AFLP technique was used to scan genome of four strains with different shell colors to identify the candidate markers under artificial selection. Eight AFLP primer combinations were screened and yielded 688 loci, 676 (98.26%) of which were polymorphic. In black, gold, red and white strains, the percentage of polymorphic loci was 90.41%, 87.79%, 93.60% and 93.31%, respectively, Nei's gene diversity was 0.3225, 0.2829, 0.3221 and 0.3292, Shannon's information index was 0.4801, 0.4271, 0.4825 and 0.4923, and the value of FST was 0.1805. These results suggested that the four different shell color strains had high genetic diversity and great genetic differentiation among strains, which had been subjected to the continuous selective pressures during the artificial selective breeding. Furthermore, six outlier loci were considered as the candidate markers under artificial selection for shell color. This study provides a molecular evidence for the inheritance of shell color of P. fucata. Copyright © 2014 Elsevier B.V. All rights reserved.

Identifying molecular markers associated with stigma characteristics in rice

USDA-ARS?s Scientific Manuscript database

Stigma characteristics play essential roles in hybrid seed production of rice and marker-assisted breeding plays essential role because they are quantitatively inherited with single-flowered perfect spikelet. Ninety four accessions originated from 47 countries were selected from the USDA rice core c...

Genotyping variability of computationally categorized peach microsatellite markers

USDA-ARS?s Scientific Manuscript database

Numerous expressed sequence tag (EST) simple sequence repeat (SSR) primers can be easily mined out. The obstacle to develop them into usable markers is how to optimally select downsized subsets of the primers for genotyping, which accordingly reduces amplification failure and monomorphism often occu...

Integrative Analysis of Cancer Diagnosis Studies with Composite Penalization

PubMed Central

Liu, Jin; Huang, Jian; Ma, Shuangge

2013-01-01

Summary In cancer diagnosis studies, high-throughput gene profiling has been extensively conducted, searching for genes whose expressions may serve as markers. Data generated from such studies have the “large d, small n” feature, with the number of genes profiled much larger than the sample size. Penalization has been extensively adopted for simultaneous estimation and marker selection. Because of small sample sizes, markers identified from the analysis of single datasets can be unsatisfactory. A cost-effective remedy is to conduct integrative analysis of multiple heterogeneous datasets. In this article, we investigate composite penalization methods for estimation and marker selection in integrative analysis. The proposed methods use the minimax concave penalty (MCP) as the outer penalty. Under the homogeneity model, the ridge penalty is adopted as the inner penalty. Under the heterogeneity model, the Lasso penalty and MCP are adopted as the inner penalty. Effective computational algorithms based on coordinate descent are developed. Numerical studies, including simulation and analysis of practical cancer datasets, show satisfactory performance of the proposed methods. PMID:24578589

Marker-assisted selection: an approach for precision plant breeding in the twenty-first century.

PubMed

Collard, Bertrand C Y; Mackill, David J

2008-02-12

DNA markers have enormous potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection (MAS). The large number of quantitative trait loci (QTLs) mapping studies for diverse crops species have provided an abundance of DNA marker-trait associations. In this review, we present an overview of the advantages of MAS and its most widely used applications in plant breeding, providing examples from cereal crops. We also consider reasons why MAS has had only a small impact on plant breeding so far and suggest ways in which the potential of MAS can be realized. Finally, we discuss reasons why the greater adoption of MAS in the future is inevitable, although the extent of its use will depend on available resources, especially for orphan crops, and may be delayed in less-developed countries. Achieving a substantial impact on crop improvement by MAS represents the great challenge for agricultural scientists in the next few decades.

Genetic considerations in human sex-mate selection: partners share human leukocyte antigen but not short-tandem-repeat identity markers.

PubMed

Israeli, Moshe; Kristt, Don; Nardi, Yuval; Klein, Tirza

2014-05-01

Previous studies support a role for MHC on mating preference, yet it remains unsettled as to whether mating occurs preferentially between individuals sharing human leukocyte antigen (HLA) determinants or not. Investigating sex-mate preferences in the contemporary Israeli population is of further curiosity being a population with distinct genetic characteristics, where multifaceted cultural considerations influence mate selection. Pairs of male-female sex partners were evaluated in three groups. Two groups represented unmarried (n = 1002) or married (n = 308) couples and a control group of fictitious male-female couples. HLA and short-tandem-repeat (STR) genetic identification markers were assessed for the frequency of shared antigens and alleles. Human leukocyte antigen results showed that Class I and/ or Class II single antigen as well as double antigen sharing was more common in sex partners than in control group couples (P < 0.001). Married versus unmarried pairs were not distinguishable. In contrast, STR-DNA markers failed to differentiate between sex-mates and controls (P = 0.78). Sex partnerships shared HLA determinants more frequently than randomly constituted male-female pairs. The observed phenomenon does not reflect a syngenetic background between sex-mates as STR markers were not selectively shared. Thus, sex-mate selection in man may contravene the evolutionary pressure for genetic diversity in regard to HLA. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

AFLPs and Mitochondrial Haplotypes Reveal Local Adaptation to Extreme Thermal Environments in a Freshwater Gastropod

PubMed Central

Quintela, María; Johansson, Magnus P.; Kristjánsson, Bjarni K.; Barreiro, Rodolfo; Laurila, Anssi

2014-01-01

The way environmental variation shapes neutral and adaptive genetic variation in natural populations is a key issue in evolutionary biology. Genome scans allow the identification of the genetic basis of local adaptation without previous knowledge of genetic variation or traits under selection. Candidate loci for divergent adaptation are expected to show higher FST than neutral loci influenced solely by random genetic drift, migration and mutation. The comparison of spatial patterns of neutral markers and loci under selection may help disentangle the effects of gene flow, genetic drift and selection among populations living in contrasting environments. Using the gastropod Radix balthica as a system, we analyzed 376 AFLP markers and 25 mtDNA COI haplotypes for candidate loci and associations with local adaptation among contrasting thermal environments in Lake Mývatn, a volcanic lake in northern Iceland. We found that 2% of the analysed AFLP markers were under directional selection and 12% of the mitochondrial haplotypes correlated with differing thermal habitats. The genetic networks were concordant for AFLP markers and mitochondrial haplotypes, depicting distinct topologies at neutral and candidate loci. Neutral topologies were characterized by intense gene flow revealed by dense nets with edges connecting contrasting thermal habitats, whereas the connections at candidate loci were mostly restricted to populations within each thermal habitat and the number of edges decreased with temperature. Our results suggest microgeographic adaptation within Lake Mývatn and highlight the utility of genome scans in detecting adaptive divergence. PMID:25007329

A vision-based automated guided vehicle system with marker recognition for indoor use.

PubMed

Lee, Jeisung; Hyun, Chang-Ho; Park, Mignon

2013-08-07

We propose an intelligent vision-based Automated Guided Vehicle (AGV) system using fiduciary markers. In this paper, we explore a low-cost, efficient vehicle guiding method using a consumer grade web camera and fiduciary markers. In the proposed method, the system uses fiduciary markers with a capital letter or triangle indicating direction in it. The markers are very easy to produce, manipulate, and maintain. The marker information is used to guide a vehicle. We use hue and saturation values in the image to extract marker candidates. When the known size fiduciary marker is detected by using a bird's eye view and Hough transform, the positional relation between the marker and the vehicle can be calculated. To recognize the character in the marker, a distance transform is used. The probability of feature matching was calculated by using a distance transform, and a feature having high probability is selected as a captured marker. Four directional signals and 10 alphabet features are defined and used as markers. A 98.87% recognition rate was achieved in the testing phase. The experimental results with the fiduciary marker show that the proposed method is a solution for an indoor AGV system.

Space-based visual attention: a marker of immature selective attention in toddlers?

PubMed

Rivière, James; Brisson, Julie

2014-11-01

Various studies suggested that attentional difficulties cause toddlers' failure in some spatial search tasks. However, attention is not a unitary construct and this study investigated two attentional mechanisms: location selection (space-based attention) and object selection (object-based attention). We investigated how toddlers' attention is distributed in the visual field during a manual search task for objects moving out of sight, namely the moving boxes task. Results show that 2.5-year-olds who failed this task allocated more attention to the location of the relevant object than to the object itself. These findings suggest that in some manual search tasks the primacy of space-based attention over object-based attention could be a marker of immature selective attention in toddlers. © 2014 Wiley Periodicals, Inc.

Molecular Engineering of Vector-Based Oncolytic and Imaging Approaches for Advanced Prostate Cancer

DTIC Science & Technology

2007-02-01

has begun to unravel, through the discovery of specific markers for lymphatic endothelial cells and their selective growth factors and receptors.13...Paraffin-embedded sections (5 lm) were stained for a vascular endothelial marker (biotinylated rat anti- mouse CD31 1:100, BD Biosciences, San Jose...CA), lymphatic endothelial markers (rabbit anti-LYVE-1 1:300, RELIATech, Braunschweig, Germany; rabbit anti-Prox1, 1:100, Novus Biologi- cals

Microsatellite markers associated with resistance to Marek's disease in commercial layer chickens.

PubMed

McElroy, J P; Dekkers, J C M; Fulton, J E; O'Sullivan, N P; Soller, M; Lipkin, E; Zhang, W; Koehler, K J; Lamont, S J; Cheng, H H

2005-11-01

The objective of the current study was to identify QTL conferring resistance to Marek's disease (MD) in commercial layer chickens. To generate the resource population, 2 partially inbred lines that differed in MD-caused mortality were intermated to produce 5 backcross families. Vaccinated chicks were challenged with very virulent plus (vv+) MD virus strain 648A at 6 d and monitored for MD symptoms. A recent field isolate of the MD virus was used because the lines were resistant to commonly used older laboratory strains. Selective genotyping was employed using 81 microsatellites selected based on prior results with selective DNA pooling. Linear regression and Cox proportional hazard models were used to detect associations between marker genotypes and survival. Significance thresholds were validated by simulation. Seven and 6 markers were significant based on proportion of false positive and false discovery rate thresholds less than 0.2, respectively. Seventeen markers were associated with MD survival considering a comparison-wise error rate of 0.10, which is about twice the number expected by chance, indicating that at least some of the associations represent true effects. Thus, the present study shows that loci affecting MD resistance can be mapped in commercial layer lines. More comprehensive studies are under way to confirm and extend these results.

Progress in the molecular and genetic modification breeding of beef cattle in China.

PubMed

Tong, Bin; Zhang, Li; Li, Guang-Peng

2017-11-20

The studies of beef cattle breeding in China have been greatly improved with the rapid development of the international beef cattle industrialization. The beef cattle breeding technologies have rapidly transformed from traditional breeding to molecular marker-assisted breeding, genomic selection and genetic modification breeding. Hundreds of candidate genes and molecular markers associated with growth, meat quality, reproduction performance and diseases resistance have been identified, and some of them have already been used in cattle breeding. Genes and molecular markers associated with growth and development are focused on the growth hormone, muscle regulatory factors, myostatin and insulin-like growth factors. Meat quality is mediated by fatty acid transport and deposition related signals, calpains and calpain system, muscle regulatory factors and muscle growth regulation pathways. Reproduction performance is regulated by GnRH-FSH-LH, growth differentiation factor 9, prolactin receptor and forkhead box protein O1. Disease resistance is modulated by the major histocompatibility complex gene family, toll-like receptors, mannose-binding lectin and interferon gene signals. In this review, we summarize the most recent progress in beef cattle breeding in marker-assisted selection, genome-wide selection and genetic modification breeding, aiming to provide a reference for further genetic breeding research of beef cattle in China.

A novel light-dependent selection marker system in plants.

PubMed

Koh, Serry; Kim, Hongsup; Kim, Jinwoo; Goo, Eunhye; Kim, Yun-Jung; Choi, Okhee; Jwa, Nam-Soo; Ma, Jun; Nagamatsu, Tomohisa; Moon, Jae Sun; Hwang, Ingyu

2011-04-01

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

A theory of modern cultural shifts and meltdowns.

PubMed

Hochberg, Michael E

2004-08-07

Many cultural attributes such as adornment, language slang, mannerisms and rituals are thought to have little or no influence on individual survival and reproduction, functioning rather as markers of cultural identity that promote group cohesion. Here, I show that if cultural markers are under weak selection and subject to loss or substitution, then the breakdown of cultural cohesiveness may proceed without stabilizing reactions until many or most of a culture's identifiers are forever lost. This may culminate in a 'cultural meltdown', whereby the culture is caught in a vortex of ever-decreasing membership and insufficient selection against the accumulation of unfamiliar markers. In progressively altering the topology of communication from diffusion to broadcasting, globalization may be both accelerating the erosion of cultural identities and amplifying dominance behaviours above their normal adaptive levels.

Speed congenics: accelerated genome recovery using genetic markers.

PubMed

Visscher, P M

1999-08-01

Genetic markers throughout the genome can be used to speed up 'recovery' of the recipient genome in the backcrossing phase of the construction of a congenic strain. The prediction of the genomic proportion during backcrossing depends on the assumptions regarding the distribution of chromosome segments, the population structure, the marker spacing and the selection strategy. In this study simulation was used to investigate the rate of recovery of the recipient genome for a mouse, Drosophila and Arabidopsis genome. It was shown that an incorrect assumption of a binomial distribution of chromosome segments, and failing to take account of a reduction in variance in genomic proportion due to selection, can lead to a downward bias of up to two generations in the estimation of the number of generations required for the formation of a congenic strain.

Developing biochemical and molecular markers for cyanobacterial inoculants.

PubMed

Prasanna, R; Madhan, K; Singh, R N; Chauhan, A K; Nain, L

2010-09-01

Markers for evaluating the establishment of cyanobacteria based on their sensitivity or resistance to antibiotics, saccharide utilization patterns and PCR generated fingerprints were developed. Four selected strains (isolates from rhizosphere soils of diverse agro-ecosystems) have shown potential as diazotrophs and exhibited plant growth promoting abilities. Different responses were obtained on screening against 40 antibiotics, which aided in developing selectable antibiotic markers for each strain. Biochemical profiles generated using standardized chromogenic identification system (including saccharide utilization tests) revealed that 53 % of the saccharides tested were not utilized by any strain, while some strains exhibited unique ability for utilization of saccharides such as melibiose, cellobiose, maltose and glucosamine. PCR based amplification profiles developed using a number of primers based on repeat sequences revealed the utility of 3 primers in providing unique fingerprints for the strains.

A theory of modern cultural shifts and meltdowns.

PubMed Central

Hochberg, Michael E

2004-01-01

Many cultural attributes such as adornment, language slang, mannerisms and rituals are thought to have little or no influence on individual survival and reproduction, functioning rather as markers of cultural identity that promote group cohesion. Here, I show that if cultural markers are under weak selection and subject to loss or substitution, then the breakdown of cultural cohesiveness may proceed without stabilizing reactions until many or most of a culture's identifiers are forever lost. This may culminate in a 'cultural meltdown', whereby the culture is caught in a vortex of ever-decreasing membership and insufficient selection against the accumulation of unfamiliar markers. In progressively altering the topology of communication from diffusion to broadcasting, globalization may be both accelerating the erosion of cultural identities and amplifying dominance behaviours above their normal adaptive levels. PMID:15504004

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai (Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB).

PubMed

Usman, Magaji G; Rafii, Mohd Y; Martini, Mohammad Y; Yusuff, Oladosu A; Ismail, Mohd R; Miah, Gous

2018-03-01

Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F 1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC 1 F 1 , BC 2 F 1 and BC 3 F 1 . The average recipient allele of the selected four BC 1 F 1 plants was 80.75% which were used to produce the BC 2 F 1 generation. BC 1 -P 7 was the best BC 1 F 1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC 3 F 1 was 95.37%. Hsp gene expression analysis was carried out on BC 1 F 1 , BC 2 F 1 and BC 3 F 1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Characterization and mapping of LanrBo: a locus conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.).

PubMed

Fischer, Kristin; Dieterich, Regine; Nelson, Matthew N; Kamphuis, Lars G; Singh, Karam B; Rotter, Björn; Krezdorn, Nicolas; Winter, Peter; Wehling, Peter; Ruge-Wehling, Brigitte

2015-10-01

A novel and highly effective source of anthracnose resistance in narrow-leafed lupin was identified. Resistance was shown to be governed by a single dominant locus. Molecular markers have been developed, which can be used for selecting resistant genotypes in lupin breeding. A screening for anthracnose resistance of a set of plant genetic resources of narrow-leafed lupin (Lupinus angustifolius L.) identified the breeding line Bo7212 as being highly resistant to anthracnose (Colletotrichum lupini). Segregation analysis indicated that the resistance of Bo7212 is inherited by a single dominant locus. The corresponding resistance gene was given the designation LanrBo. Previously published molecular anchor markers allowed us to locate LanrBo on linkage group NLL-11 of narrow-leafed lupin. Using information from RNAseq data obtained with inoculated resistant vs. susceptible lupin entries as well as EST-sequence information from the model genome Lotus japonicus, additional SNP and EST markers linked to LanrBo were derived. A bracket of two LanrBo-flanking markers allows for precise marker-assisted selection of the novel resistance gene in narrow-leafed lupin breeding programs.

The Prognostic Role of Cancer Stem Cell Markers for Long-term Outcome After Resection of Colonic Liver Metastases.

PubMed

Spelt, Lidewij; Sasor, Agata; Ansari, Daniel; Hilmersson, Katarzyna Said; Andersson, Roland

2018-01-01

To assess the expression of cancer stem cell (CSC) markers CD44, CD133 and CD24 in colon cancer liver metastases and analyse their predictive value for overall survival (OS) and disease-free survival (DFS) after liver resection. Patients operated on for colon cancer liver metastases were included. CSC marker expression was determined through immunohistochemistry analysis. OS and DFS were compared between marker-positive and marker-negative patients. Multivariate analysis was performed to select predictive variables for OS and DFS. CD133-positive patients had a worse DFS than CD133-negative patients, with a median DFS of 12 and 25 months (p=0.051). Multivariate analysis selected CD133 expression as a significant predictor for DFS. CD44 and CD24 were not found to predict OS or DFS. CD133 expression in colonic liver metastases is a negative prognostic factor for DFS after liver resection. In the future, CD133 could be used as a biomarker for risk stratification, and possibly for developing novel targeted therapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

High genetic load in the Pacific oyster Crassostrea gigas.

PubMed Central

Launey, S; Hedgecock, D

2001-01-01

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena. PMID:11560902

Development and validation of 697 novel polymorphic genomic and EST-SSR markers in the American cranberry (Vaccinium macrocarpon Ait.).

PubMed

Schlautman, Brandon; Fajardo, Diego; Bougie, Tierney; Wiesman, Eric; Polashock, James; Vorsa, Nicholi; Steffan, Shawn; Zalapa, Juan

2015-01-27

The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the p < 0.05 level, and contained moderate to high levels of polymorphism with a polymorphic information content >0.25. This comprehensive collection of developed and validated microsatellite loci represents a substantial addition to the molecular tools available for geneticists, genomicists, and breeders in cranberry and Vaccinium.

[Medicinal plant DNA marker assisted breeding (Ⅱ) the assistant identification of SNPs assisted identification and breeding research of high yield Perilla frutescens new variety].

PubMed

Shen, Qi; Zhang, Dong; Sun, Wei; Zhang, Yu-Jun; Shang, Zhi-Wei; Chen, Shi-Lin

2017-05-01

Perilla frutescens is one of 60 kinds of food and medicine plants in the initial directory announced by health ministry of China. With the development of Perilla domain in recent , the breeding and application of good varieties has become the main bottleneck of its development. This study reported that applied to the system selection, add to marker-assisted method to breed perilla varieties. Through the whole genome sequencing and consistency matching, annotated the mutation locus according to genome data, and comparison analysis with Perilla common variants database, finally selected 30 non-synonymous mutation SNPs used as characteristic markers of Zhongyan Feishu No.1. those SNP marker were used as chosen standard of Perilla varieties. Finally breeding new perilla variety Zhongyan Feishu No.1, which possess to characters of the leaf and seed dual-used, high yield, high resistance, and could used to green fertilizer. The Zhongyan Feishu No.1 acquired the plant new varieties identification of Beijing city , the identification numbers is 2016054. Marker assisted identification guide new varieties breeding in plants, which can provide a new reference for breeding of medicinal plants. Copyright© by the Chinese Pharmaceutical Association.

An efficient empirical Bayes method for genomewide association studies.

PubMed

Wang, Q; Wei, J; Pan, Y; Xu, S

2016-08-01

Linear mixed model (LMM) is one of the most popular methods for genomewide association studies (GWAS). Numerous forms of LMM have been developed; however, there are two major issues in GWAS that have not been fully addressed before. The two issues are (i) the genomic background noise and (ii) low statistical power after Bonferroni correction. We proposed an empirical Bayes (EB) method by assigning each marker effect a normal prior distribution, resulting in shrinkage estimates of marker effects. We found that such a shrinkage approach can selectively shrink marker effects and reduce the noise level to zero for majority of non-associated markers. In the meantime, the EB method allows us to use an 'effective number of tests' to perform Bonferroni correction for multiple tests. Simulation studies for both human and pig data showed that EB method can significantly increase statistical power compared with the widely used exact GWAS methods, such as GEMMA and FaST-LMM-Select. Real data analyses in human breast cancer identified improved detection signals for markers previously known to be associated with breast cancer. We therefore believe that EB method is a valuable tool for identifying the genetic basis of complex traits. © 2015 Blackwell Verlag GmbH.

Probability genotype imputation method and integrated weighted lasso for QTL identification.

PubMed

Demetrashvili, Nino; Van den Heuvel, Edwin R; Wit, Ernst C

2013-12-30

Many QTL studies have two common features: (1) often there is missing marker information, (2) among many markers involved in the biological process only a few are causal. In statistics, the second issue falls under the headings "sparsity" and "causal inference". The goal of this work is to develop a two-step statistical methodology for QTL mapping for markers with binary genotypes. The first step introduces a novel imputation method for missing genotypes. Outcomes of the proposed imputation method are probabilities which serve as weights to the second step, namely in weighted lasso. The sparse phenotype inference is employed to select a set of predictive markers for the trait of interest. Simulation studies validate the proposed methodology under a wide range of realistic settings. Furthermore, the methodology outperforms alternative imputation and variable selection methods in such studies. The methodology was applied to an Arabidopsis experiment, containing 69 markers for 165 recombinant inbred lines of a F8 generation. The results confirm previously identified regions, however several new markers are also found. On the basis of the inferred ROC behavior these markers show good potential for being real, especially for the germination trait Gmax. Our imputation method shows higher accuracy in terms of sensitivity and specificity compared to alternative imputation method. Also, the proposed weighted lasso outperforms commonly practiced multiple regression as well as the traditional lasso and adaptive lasso with three weighting schemes. This means that under realistic missing data settings this methodology can be used for QTL identification.

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass.

PubMed

Jespersen, David; Belanger, Faith C; Huang, Bingru

2017-01-01

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection.

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

PubMed

Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M; McClean, Phillip E

2014-01-01

Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate.

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

PubMed Central

Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M.; McClean, Phillip E.

2013-01-01

Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate. PMID:24860578

Candidate genes and molecular markers associated with heat tolerance in colonial Bentgrass

PubMed Central

Jespersen, David; Belanger, Faith C.; Huang, Bingru

2017-01-01

Elevated temperature is a major abiotic stress limiting the growth of cool-season grasses during the summer months. The objectives of this study were to determine the genetic variation in the expression patterns of selected genes involved in several major metabolic pathways regulating heat tolerance for two genotypes contrasting in heat tolerance to confirm their status as potential candidate genes, and to identify PCR-based markers associated with candidate genes related to heat tolerance in a colonial (Agrostis capillaris L.) x creeping bentgrass (Agrostis stolonifera L.) hybrid backcross population. Plants were subjected to heat stress in controlled-environmental growth chambers for phenotypic evaluation and determination of genetic variation in candidate gene expression. Molecular markers were developed for genes involved in protein degradation (cysteine protease), antioxidant defense (catalase and glutathione-S-transferase), energy metabolism (glyceraldehyde-3-phosphate dehydrogenase), cell expansion (expansin), and stress protection (heat shock proteins HSP26, HSP70, and HSP101). Kruskal-Wallis analysis, a commonly used non-parametric test used to compare population individuals with or without the gene marker, found the physiological traits of chlorophyll content, electrolyte leakage, normalized difference vegetative index, and turf quality were associated with all candidate gene markers with the exception of HSP101. Differential gene expression was frequently found for the tested candidate genes. The development of candidate gene markers for important heat tolerance genes may allow for the development of new cultivars with increased abiotic stress tolerance using marker-assisted selection. PMID:28187136

Accuracy of Genomic Prediction in Switchgrass (Panicum virgatum L.) Improved by Accounting for Linkage Disequilibrium

PubMed Central

Ramstein, Guillaume P.; Evans, Joseph; Kaeppler, Shawn M.; Mitchell, Robert B.; Vogel, Kenneth P.; Buell, C. Robin; Casler, Michael D.

2016-01-01

Switchgrass is a relatively high-yielding and environmentally sustainable biomass crop, but further genetic gains in biomass yield must be achieved to make it an economically viable bioenergy feedstock. Genomic selection (GS) is an attractive technology to generate rapid genetic gains in switchgrass, and meet the goals of a substantial displacement of petroleum use with biofuels in the near future. In this study, we empirically assessed prediction procedures for genomic selection in two different populations, consisting of 137 and 110 half-sib families of switchgrass, tested in two locations in the United States for three agronomic traits: dry matter yield, plant height, and heading date. Marker data were produced for the families’ parents by exome capture sequencing, generating up to 141,030 polymorphic markers with available genomic-location and annotation information. We evaluated prediction procedures that varied not only by learning schemes and prediction models, but also by the way the data were preprocessed to account for redundancy in marker information. More complex genomic prediction procedures were generally not significantly more accurate than the simplest procedure, likely due to limited population sizes. Nevertheless, a highly significant gain in prediction accuracy was achieved by transforming the marker data through a marker correlation matrix. Our results suggest that marker-data transformations and, more generally, the account of linkage disequilibrium among markers, offer valuable opportunities for improving prediction procedures in GS. Some of the achieved prediction accuracies should motivate implementation of GS in switchgrass breeding programs. PMID:26869619

Analysis of facial motion patterns during speech using a matrix factorization algorithm

PubMed Central

Lucero, Jorge C.; Munhall, Kevin G.

2008-01-01

This paper presents an analysis of facial motion during speech to identify linearly independent kinematic regions. The data consists of three-dimensional displacement records of a set of markers located on a subject’s face while producing speech. A QR factorization with column pivoting algorithm selects a subset of markers with independent motion patterns. The subset is used as a basis to fit the motion of the other facial markers, which determines facial regions of influence of each of the linearly independent markers. Those regions constitute kinematic “eigenregions” whose combined motion produces the total motion of the face. Facial animations may be generated by driving the independent markers with collected displacement records. PMID:19062866

Extended Linkage Disequilibrium Surrounding the Hemoglobin E Variant Due to Malarial Selection

PubMed Central

Ohashi, Jun ; Naka, Izumi ; Patarapotikul, Jintana ; Hananantachai, Hathairad ; Brittenham, Gary ; Looareesuwan, Sornchai ; Clark, Andrew G. ; Tokunaga, Katsushi 

2004-01-01

The hemoglobin E variant (HbE; β26Glu→Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240–4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection. PMID:15114532

Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection.

PubMed

Ohashi, Jun; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Brittenham, Gary; Looareesuwan, Sornchai; Clark, Andrew G; Tokunaga, Katsushi

2004-06-01

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

PubMed

Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

2014-01-01

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

Determining Source Impacts Near Roadways using Wind Regression and Organic Source Markers

EPA Science Inventory

Concentrations of 13 organic source markers (10 polycyclic aromatic hydrocarbons and 3 hopanes) are reported from time-integrated samples (24-hr and sub-daily) collected near an highway in Las Vegas, NV. Sample selection for assessing source impacts from the roadway was complete...

An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S.

PubMed

Argov, Tal; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

2017-03-15

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene ( pheS* ). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p -chloro-phenylalanine analog ( p -Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p -Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenes IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a long-standing need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism. Copyright © 2017 American Society for Microbiology.

ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.).

PubMed

Pandeya, Devendra; Campbell, LeAnne M; Nunes, Eugenia; Lopez-Arredondo, Damar L; Janga, Madhusudhana R; Herrera-Estrella, Luis; Rathore, Keerti S

2017-12-01

This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

Genetic association of marbling score with intragenic nucleotide variants at selection signals of the bovine genome.

PubMed

Ryu, J; Lee, C

2016-04-01

Selection signals of Korean cattle might be attributed largely to artificial selection for meat quality. Rapidly increased intragenic markers of newly annotated genes in the bovine genome would help overcome limited findings of genetic markers associated with meat quality at the selection signals in a previous study. The present study examined genetic associations of marbling score (MS) with intragenic nucleotide variants at selection signals of Korean cattle. A total of 39 092 nucleotide variants of 407 Korean cattle were utilized in the association analysis. A total of 129 variants were selected within newly annotated genes in the bovine genome. Their genetic associations were analyzed using the mixed model with random polygenic effects based on identical-by-state genetic relationships among animals in order to control for spurious associations produced by population structure. Genetic associations of MS were found (P<3.88×10-4) with six intragenic nucleotide variants on bovine autosomes 3 (cache domain containing 1, CACHD1), 5 (like-glycosyltransferase, LARGE), 16 (cell division cycle 42 binding protein kinase alpha, CDC42BPA) and 21 (snurportin 1, SNUPN; protein tyrosine phosphatase, non-receptor type 9, PTPN9; chondroitin sulfate proteoglycan 4, CSPG4). In particular, the genetic associations with CDC42BPA and LARGE were confirmed using an independent data set of Korean cattle. The results implied that allele frequencies of functional variants and their proximity variants have been augmented by directional selection for greater MS and remain selection signals in the bovine genome. Further studies of fine mapping would be useful to incorporate favorable alleles in marker-assisted selection for MS of Korean cattle.

Polymorphic microsatellite markers for the endangered fish, the slender shiner Pseudopungtungia tenuicorpa and cross-species amplification across five related species.

PubMed

Kim, K S; Moon, S J; Han, S H; Kim, K Y; Bang, I C

2016-09-02

The slender shiner Pseudopungtungia tenuicorpa (Cypriniformes; Cyprinidae; Gobioninae) is an endangered freshwater fish species endemic to Korea. The current strategies for its conservation involve the study of population genetic characters and identification of management units. These strategies require suitable molecular markers to study genetic diversity and genetic structure. Here, we developed nine polymorphic microsatellite markers for P. tenuicorpa for the first time by applying an enrichment method from a size-selected genomic library. The developed microsatellite markers produced a total of 101 alleles (average 11.2). The observed and expected heterozygosities averaged 0.805 and 0.835, respectively. Among the nine identified markers, five markers showed successful amplification across five related Korean Gobioninae species. Thus, the microsatellite markers developed in this study will be useful to establish conservation strategies for both P. tenuicorpa and other related species.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters.

PubMed

Miyata, Luzia Yuriko; Harakava, Ricardo; Stipp, Liliane Cristina Libório; Mendes, Beatriz Madalena Januzzi; Appezzato-da-Glória, Beatriz; de Assis Alves Mourão Filho, Francisco

2012-11-01

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Single Marker and Haplotype-Based Association Analysis of Semolina and Pasta Colour in Elite Durum Wheat Breeding Lines Using a High-Density Consensus Map.

PubMed

N'Diaye, Amidou; Haile, Jemanesh K; Cory, Aron T; Clarke, Fran R; Clarke, John M; Knox, Ron E; Pozniak, Curtis J

2017-01-01

Association mapping is usually performed by testing the correlation between a single marker and phenotypes. However, because patterns of variation within genomes are inherited as blocks, clustering markers into haplotypes for genome-wide scans could be a worthwhile approach to improve statistical power to detect associations. The availability of high-density molecular data allows the possibility to assess the potential of both approaches to identify marker-trait associations in durum wheat. In the present study, we used single marker- and haplotype-based approaches to identify loci associated with semolina and pasta colour in durum wheat, the main objective being to evaluate the potential benefits of haplotype-based analysis for identifying quantitative trait loci. One hundred sixty-nine durum lines were genotyped using the Illumina 90K Infinium iSelect assay, and 12,234 polymorphic single nucleotide polymorphism (SNP) markers were generated and used to assess the population structure and the linkage disequilibrium (LD) patterns. A total of 8,581 SNPs previously localized to a high-density consensus map were clustered into 406 haplotype blocks based on the average LD distance of 5.3 cM. Combining multiple SNPs into haplotype blocks increased the average polymorphism information content (PIC) from 0.27 per SNP to 0.50 per haplotype. The haplotype-based analysis identified 12 loci associated with grain pigment colour traits, including the five loci identified by the single marker-based analysis. Furthermore, the haplotype-based analysis resulted in an increase of the phenotypic variance explained (50.4% on average) and the allelic effect (33.7% on average) when compared to single marker analysis. The presence of multiple allelic combinations within each haplotype locus offers potential for screening the most favorable haplotype series and may facilitate marker-assisted selection of grain pigment colour in durum wheat. These results suggest a benefit of haplotype-based analysis over single marker analysis to detect loci associated with colour traits in durum wheat.

Gene action analysis by inheritance and QTL mapping of resistance to root-knot nematodes in cotton.

USDA-ARS?s Scientific Manuscript database

Host-plant resistance is highly effective in controlling crop loss from nematode infection. In addition, molecular markers can be powerful tools for marker-assisted selection (MAS), where they reduce laborious greenhouse phenotype evaluation to identify root-knot nematode (RKN) Meloidogyne incognita...

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 ...

The Swift Turbidity Marker

ERIC Educational Resources Information Center

Omar, Ahmad Fairuz; MatJafri, Mohd Zubir

2011-01-01

The Swift Turbidity Marker is an optical instrument developed to measure the level of water turbidity. The components and configuration selected for the system are based on common turbidity meter design concepts but use a simplified methodology to produce rapid turbidity measurements. This work is aimed at high school physics students and is the…

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...

Predictive markers in calpastatin for tenderness in commercial pig populations

USDA-ARS?s Scientific Manuscript database

The identification of predictive DNA markers for pork quality would allow U.S. pork producers and breeders to more quickly and efficiently select genetically superior animals for production of consistent, high quality meat. Genome scans have identified QTL for tenderness on pig chromosome 2 which ha...

EFFECT OF SAMPLING LOCATION ON CONCENTRATION IN LARGE CHAMBER INVESTIGATION OF EMISSIONS FROM MARKERS

EPA Science Inventory

Markers were selected for evaluation in this study because (1) they are widely used in schools, offices, and homes; (2) they are a known source of volatile organic compounds (VOCs) in nonoccupational indoor environments; and (3) according to the Source Ranking Database developed ...

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC MICROBIAL GENETIC MARKERS IN COW FECAL SAMPLES

EPA Science Inventory

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host specific markers. Here, we describe the application of a genome fragment enrichment met...

New Advances in Marker Assisted Selection for Winter Hardiness in Oats.

USDA-ARS?s Scientific Manuscript database

Oat (Avena sativa L.) breeding and genetics research has lagged behind other small grains, such as wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), in the development of PCR based markers and map construction due to fewer oat researchers and reduced research funding. As a result, marke...

Marker-assisted selection for biotic stress resistance in peanut

USDA-ARS?s Scientific Manuscript database

Peanut ranks second to soybean in the world market trade of oilseeds both in area grown and tonnage produced, and is well-suited to contribute significantly to poverty reduction in the developing world. Peanut is a tetrapoid of recent origin, and has shown low levels of molecular marker polymorphis...

COMPETITIVE METAGENOMIC DNA HYBRIDIZATION IDENTIFIES HOST-SPECIFIC GENETIC MARKERS IN CATTLE FECAL SAMPLES - ABSTRACT

EPA Science Inventory

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host specific markers. Here, we describe the application of a genome fragment enrichment met...

An evaluation of the genetic-matched pair study design using genome-wide SNP data from the European population.

PubMed

Lu, Timothy Tehua; Lao, Oscar; Nothnagel, Michael; Junge, Olaf; Freitag-Wolf, Sandra; Caliebe, Amke; Balascakova, Miroslava; Bertranpetit, Jaume; Bindoff, Laurence Albert; Comas, David; Holmlund, Gunilla; Kouvatsi, Anastasia; Macek, Milan; Mollet, Isabelle; Nielsen, Finn; Parson, Walther; Palo, Jukka; Ploski, Rafal; Sajantila, Antti; Tagliabracci, Adriano; Gether, Ulrik; Werge, Thomas; Rivadeneira, Fernando; Hofman, Albert; Uitterlinden, André Gerardus; Gieger, Christian; Wichmann, Heinz-Erich; Ruether, Andreas; Schreiber, Stefan; Becker, Christian; Nürnberg, Peter; Nelson, Matthew Roberts; Kayser, Manfred; Krawczak, Michael

2009-07-01

Genetic matching potentially provides a means to alleviate the effects of incomplete Mendelian randomization in population-based gene-disease association studies. We therefore evaluated the genetic-matched pair study design on the basis of genome-wide SNP data (309,790 markers; Affymetrix GeneChip Human Mapping 500K Array) from 2457 individuals, sampled at 23 different recruitment sites across Europe. Using pair-wise identity-by-state (IBS) as a matching criterion, we tried to derive a subset of markers that would allow identification of the best overall matching (BOM) partner for a given individual, based on the IBS status for the subset alone. However, our results suggest that, by following this approach, the prediction accuracy is only notably improved by the first 20 markers selected, and increases proportionally to the marker number thereafter. Furthermore, in a considerable proportion of cases (76.0%), the BOM of a given individual, based on the complete marker set, came from a different recruitment site than the individual itself. A second marker set, specifically selected for ancestry sensitivity using singular value decomposition, performed even more poorly and was no more capable of predicting the BOM than randomly chosen subsets. This leads us to conclude that, at least in Europe, the utility of the genetic-matched pair study design depends critically on the availability of comprehensive genotype information for both cases and controls.

Selection of broilers for increased innate immune markers: Past strategies and looking ahead

USDA-ARS?s Scientific Manuscript database

Economic efficiency demanded by the poultry industry has pushed selection towards high production with improved feed conversion ratios (FCR) and high yield; however, selection based heavily on growth characteristics and other phenotypic traits has adversely affected immune competence. Despite incre...

Assessing genomic selection prediction accuracy in a dynamic barley breeding

USDA-ARS?s Scientific Manuscript database

Genomic selection is a method to improve quantitative traits in crops and livestock by estimating breeding values of selection candidates using phenotype and genome-wide marker data sets. Prediction accuracy has been evaluated through simulation and cross-validation, however validation based on prog...

Selection of Plasmodium falciparum pfcrt and pfmdr1 polymorphisms after treatment with artesunate-amodiaquine fixed dose combination or artemether-lumefantrine in Liberia.

PubMed

Otienoburu, Sabina Dahlström; Maïga-Ascofaré, Oumou; Schramm, Birgit; Jullien, Vincent; Jones, Joel J; Zolia, Yah M; Houzé, Pascal; Ashley, Elizabeth A; Kiechel, Jean-René; Guérin, Philippe J; Le Bras, Jacques; Houzé, Sandrine

2016-09-05

Plasmodium falciparum uncomplicated malaria can successfully be treated with an artemisinin-based combination therapy (ACT). However resistance is spreading to the different ACT compounds; the artemisinin derivative and the partner drug. Studies of P. falciparum polymorphisms associated with drug resistance can provide a useful tool to track resistance and guide treatment policy as well as an in-depth understanding of the development and spread of resistance. The role of P. falciparum molecular markers in selection of reinfections was assessed in an efficacy trial comparing artesunate-amodiaquine fixed-dose combination with artemether-lumefantrine to treat malaria in Nimba County, Liberia 2008-2009. P. falciparum polymorphisms in pfcrt 76, pfmdr1 86, 184 and 1246, and pfmrp1 876 and 1466 were analysed by PCR-RFLP and pyrosequencing. High baseline prevalence of pfmdr1 1246Y was found in Nimba county (38 %). Pfmdr1 1246Y and pfmdr1 86+184+1246 haplotypes NYY and YYY were selected in reinfections in the artesunate-amodiaquine arm and pfcrt K76, pfmdr1 N86 and pfmdr1 haplotype NFD were selected in artemether-lumefantrine reinfections. Parasites harbouring pfmdr1 1246Y could reinfect earlier after treatment with artesunate-amodiaquine and parasites carrying pfmdr1 N86 could reinfect at higher lumefantrine concentrations in patients treated with artemether-lumefantrine. Although treatment is highly efficacious, selection of molecular markers in reinfections could indicate a decreased sensitivity or tolerance of parasites to the current treatments and the baseline prevalence of molecular markers should be closely monitored. Since individual drug levels and the day of reinfection were demonstrated to be key determinants for selection of reinfections, this data needs to be collected and taken into account for accurate evaluation of molecular markers for anti-malarial treatments. The protocols for the clinical trial was registered with Current Controlled Trials, under the Identifier Number ISRCTN51688713 on 9 October 2008.

Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits

PubMed Central

Pecetti, Luciano; Brummer, E. Charles; Palmonari, Alberto; Tava, Aldo

2017-01-01

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield. PMID:28068350

Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits.

PubMed

Biazzi, Elisa; Nazzicari, Nelson; Pecetti, Luciano; Brummer, E Charles; Palmonari, Alberto; Tava, Aldo; Annicchiarico, Paolo

2017-01-01

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3-0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

Content Variation of Catechin Markers, Total Phenolics and Caffeine in Green Tea Dietary Supplements.

PubMed

Abourashed, Ehab A; Roberson, Cindy Leslie A; Elsharkawy, Nancy

2016-01-01

Green tea (Camellia sinensis) preparations are among the top selling products in the United States dietary supplements market. Numerous manufacturers claim different levels of specific catechin markers in their products while many others use total phenolic concentration instead, or not at all. Limited quality control results have been published for green tea dietary supplements over the past seven years. Thus, the goal of this work was to correlate determined levels of phenolics, catechins, and caffeine with manufacturer label claims for selected dietary supplement products (26 total) purchased in the United States. The Folin-Ciocalteu (FC) method was used to determine the total phenolic content while reversed-phase (RP) HPLC was used to quantify the major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG). The developed HPLC method was validated for accuracy and precision. It utilized a C8 column with gradient elution of acetonitrile in 0.1% aqueous formic acid over 11 min total run time. Peak detection was performed at 280 nm. Caffeine was also included in the HPLC method as another non-phenolic alkaloid marker commonly found in green tea. Both methods showed a good correlation between the content of catechins and polyphenolic compounds in the selected products. The ranges of total catechins and polyphenol concentrations were 3.8-70.2% and 3.6-95.8%, respectively, while that of caffeine was 0.8-11.2%. The selected products displayed a wide range of marker levels. A lack of conformity in disclosing the actual levels of marker compounds was also noticed in the labeling of many products.

Genome-wide association study (GWAS) for growth rate and age at sexual maturation in Atlantic salmon (Salmo salar).

PubMed

Gutierrez, Alejandro P; Yáñez, José M; Fukui, Steve; Swift, Bruce; Davidson, William S

2015-01-01

Early sexual maturation is considered a serious drawback for Atlantic salmon aquaculture as it retards growth, increases production times and affects flesh quality. Although both growth and sexual maturation are thought to be complex processes controlled by several genetic and environmental factors, selection for these traits has been continuously accomplished since the beginning of Atlantic salmon selective breeding programs. In this genome-wide association study (GWAS) we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype ∼ 480 individuals from the Cermaq Canada broodstock program and search for SNPs associated with growth and age at sexual maturation. Using a mixed model approach we identified markers showing a significant association with growth, grilsing (early sexual maturation) and late sexual maturation. The most significant associations were found for grilsing, with markers located in Ssa10, Ssa02, Ssa13, Ssa25 and Ssa12, and for late maturation with markers located in Ssa28, Ssa01 and Ssa21. A lower level of association was detected with growth on Ssa13. Candidate genes, which were linked to these genetic markers, were identified and some of them show a direct relationship with developmental processes, especially for those in association with sexual maturation. However, the relatively low power to detect genetic markers associated with growth (days to 5 kg) in this GWAS indicates the need to use a higher density SNP array in order to overcome the low levels of linkage disequilibrium observed in Atlantic salmon before the information can be incorporated into a selective breeding 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.

Development of Novel SSR Markers for Flax (Linum usitatissimum L.) Using Reduced-Representation Genome Sequencing.

PubMed

Wu, Jianzhong; Zhao, Qian; Wu, Guangwen; Zhang, Shuquan; Jiang, Tingbo

2016-01-01

Flax ( Linum usitatissimum L.) is a major fiber and oil yielding crop grown in northeastern China. Identification of flax molecular markers is a key step toward improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers, which are based on genomic structural variation, are considered the most valuable type of genetic marker for this purpose. In this study, we screened 1574 microsatellites from Linum usitatissimum L. obtained using reduced representation genome sequencing (RRGS) to systematically identify SSR markers. The resulting set of microsatellites consisted mainly of trinucleotide (56.10%) and dinucleotide (35.23%) repeats, with each motif consisting of 5-8 repeats. We then evaluated marker sensitivity and specificity based on samples of 48 flax isolates obtained from northeastern China. Using the new SSR panel, the results demonstrated that fiber flax and oilseed flax varieties clustered into two well separated groups. The novel SSR markers developed in this study show potential value for selection of varieties for use in flax breeding programs.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

PubMed Central

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

High-resolution genetic linkage mapping, high-temperature tolerance and growth-related quantitative trait locus (QTL) identification in Marsupenaeus japonicus.

PubMed

Lu, Xia; Luan, Sheng; Hu, Long Yang; Mao, Yong; Tao, Ye; Zhong, Sheng Ping; Kong, Jie

2016-06-01

The Kuruma prawn, Marsupenaeus japonicus, is one of the most promising marine invertebrates in the industry in Asia, Europe and Australia. However, the increasing global temperatures result in considerable economic losses in M. japonicus farming. In the present study, to select genetically improved animals for the sustainable development of the Kuruma prawn industry, a high-resolution genetic linkage map and quantitative trait locus (QTL) identification were performed using the RAD technology. The maternal map contained 5849 SNP markers and spanned 3127.23 cM, with an average marker interval of 0.535 cM. Instead, the paternal map contained 3927 SNP markers and spanned 3326.19 cM, with an average marker interval of 0.847 cM. The consensus map contained 9289 SNP markers and spanned 3610.90 cM, with an average marker interval of 0.388 cM and coverage of 99.06 % of the genome. The markers were grouped into 41 linkage groups in the maps. Significantly, negative correlation was detected between high-temperature tolerance (UTT) and body weight (BW). The QTL mapping revealed 129 significant QTL loci for UTT and four significant QTL loci for BW at the genome-wide significance threshold. Among these QTLs, 129 overlapped with linked SNPs, and the remaining four were located in regions between contiguous SNPs. They explained the total phenotypic variance ranging from 8.9 to 12.4 %. Because of a significantly negative correlation between growth and high-temperature tolerance, we demonstrate that this high-resolution linkage map and QTLs would be useful for further marker-assisted selection in the genetic improvement of M. japonicus.

Use of luminescent gunshot residues markers in forensic context.

PubMed

Weber, I T; Melo, A J G; Lucena, M A M; Consoli, E F; Rodrigues, M O; de Sá, G F; Maldaner, A O; Talhavini, M; Alves, S

2014-11-01

Chemical evaluation of gunshot residues (GSR) produced by non-toxic lead-free ammunition (NTA) has been a challenge to forensic analyses. Our group developed some luminescent markers specific to the detection of GSR. Here, we evaluated the performance of selected markers in experiments that mimic forensic context and/or routines in which luminescent characteristics would be very useful. We evaluated the influence of markers' addition on the bullet's speed, the rate of shot failure (i.e., when the cartridge case is not fully ejected and/or a new ammunition is not automatically replaced in the gun chamber) as a function of marker percentage, the possibility of collecting luminescent gunshot residue (LGSR) in unconventional locations (e.g. the shooters' nostrils), the LGSR lifetime after hand washing, the transfer of LGSR to objects handled by the shooter, and the dispersion of LGSR at the crime scene and on simulated victims. It was observed that high amounts of marker (10 wt%) cause high rates of failure on pistols, as well as a substantial decrease in bullet speed. However, the use of 2 wt% of marker minimizes these effects and allows LGSR detection, collection and analysis. Moreover, in all conditions tested, markers showed high performance and provided important information for forensic analyses. For instance, the LGSR particles were found on the floor, ranging from 0 to 9.4 m away from the shooter, on the door panel and seats after a car shooting experiment, and were found easily on a pig leg used to simulate a victim. When a selective tagging was done, it was possible to obtain positive or negative correlation between the victim and shooter. Additionally LGSR possesses a fairly long lifetime (9 h) and good resistance to hand washing (up to 16 washes). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Molecular and genetic characterization of the Ry adg locus on chromosome XI from Andigena potatoes conferring extreme resistance to potato virus Y.

PubMed

Herrera, María Del Rosario; Vidalon, Laura Jara; Montenegro, Juan D; Riccio, Cinzia; Guzman, Frank; Bartolini, Ida; Ghislain, Marc

2018-05-31

We have elucidated the Andigena origin of the potato Ry adg gene on chromosome XI of CIP breeding lines and developed two marker assays to facilitate its introgression in potato by marker-assisted selection. Potato virus Y (PVY) is causing yield and quality losses forcing farmers to renew periodically their seeds from clean stocks. Two loci for extreme resistance to PVY, one on chromosome XI and the other on XII, have been identified and used in breeding. The latter corresponds to a well-known source of resistance (Solanum stoloniferum), whereas the one on chromosome XI was reported from S. stoloniferum and S. tuberosum group Andigena as well. To elucidate its taxonomic origin in our breeding lines, we analyzed the nucleotide sequences of tightly linked markers (M45, M6) and screened 251 landraces of S. tuberosum group Andigena for the presence of this gene. Our results indicate that the PVY resistance allele on chromosome XI in our breeding lines originated from S. tuberosum group Andigena. We have developed two marker assays to accelerate the introgression of Ry adg gene into breeding lines by marker-assisted selection (MAS). First, we have multiplexed RYSC3, M6 and M45 DNA markers flanking the Ry adg gene and validated it on potato varieties with known presence/absence of the Ry adg gene and a progeny of 6,521 individuals. Secondly, we developed an allele-dosage assay particularly useful to identify multiplex Ry adg progenitors. The assay based on high-resolution melting analysis at the M6 marker confirmed Ry adg plex level as nulliplex, simplex and duplex progenitors and few triplex progenies. These marker assays have been validated and can be used to facilitate MAS in potato breeding.

Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

PubMed

Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong; Qu, Shaohong

2015-01-01

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. © 2015 American Society of Plant Biologists. All Rights Reserved.

Mechanisms and genetic control of interspecific crossing barriers in Lycopersicon. Final report

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

Mutschler, M.A.

Deficiency of Lycopersicon esculentum allele (E) was observed from the RFLP and isozyme data of the F{sub 2} populations derived from the cross L. esculentum x L. pennellii. The genome composition of the F{sub 2} populations containing L. pennellii cytoplasm (F{sub 2}{sup Lp4}) has a lower proportion of the homozygous L. pennellii (PP) genotypes and a higher proportion of heterozygote (EP) genotypes than that of the F{sub 2} populations containing L. esculentum cytoplasm (F{sub 2}{sup Le}). A lower proportion of the L. pennellii alleles (P) was also observed in F{sub 2}{sup Lp4} as compared to F{sub 2}{sup Le} when eachmore » marker locus was tested individually. To study the effects of gametic and zygotic selection on segregation distortion, the expected patterns of segregation at a marker locus were derived for ten selection models with gametic or zygotic selection at a hidden linked locus. Segregation distortion caused by four of the selection models studied can be uniquely identified by the patterns of significance expected for the likelihood ratio tests at the marker loci. Comparison of the chromosomal regions associated with specific selection models across populations (of this experiment and previous publications) indicated that the segregation distortion observed in chromosome 10 is associated with zygotic selection affecting both arms of the chromosome, and cytoplasm substitution has the effect of decreasing the segregation distortion on the long arm of the chromosome.« less

Physicochemical and biochemical characterization of transgenic papaya modified for protection against Papaya ringspot virus.

PubMed

Roberts, Madeen; Minott, Donna A; Pinnock, Simone; Tennant, Paula F; Jackson, Jose C

2014-03-30

Papaya, a nutritious tropical fruit, is consumed both in its fresh form and as a processed product worldwide. Major quality indices which include firmness, acidity, pH, colour and size, are cultivar dependent. Transgenic papayas engineered for resistance to Papaya ringspot virus were evaluated over the ripening period to address physicochemical quality attributes and food safety concerns. With the exception of one transgenic line, no significant differences (P > 0.05) were observed in firmness, acidity and pH. Lightness (L*) and redness (a*) of the pulps of non-transgenic and transgenic papaya were similar but varied over the ripening period (P < 0.05). Fruit mass, though non-uniform (P < 0.05) for some lines, was within the range reported for similar papaya cultivars, as were shape indices of female fruits. Transgene proteins, CP and NPTII, were not detected in fruit pulp at the table-ready stage. The findings suggest that transformation did not produce any major unintended alterations in the physicochemical attributes of the transgenic papayas. Transgene proteins in the edible fruit pulp were low or undetectable. © 2013 Society of Chemical Industry.

Simulating a base population in honey bee for molecular genetic studies

PubMed Central

2012-01-01

Background Over the past years, reports have indicated that honey bee populations are declining and that infestation by an ecto-parasitic mite (Varroa destructor) is one of the main causes. Selective breeding of resistant bees can help to prevent losses due to the parasite, but it requires that a robust breeding program and genetic evaluation are implemented. Genomic selection has emerged as an important tool in animal breeding programs and simulation studies have shown that it yields more accurate breeding value estimates, higher genetic gain and low rates of inbreeding. Since genomic selection relies on marker data, simulations conducted on a genomic dataset are a pre-requisite before selection can be implemented. Although genomic datasets have been simulated in other species undergoing genetic evaluation, simulation of a genomic dataset specific to the honey bee is required since this species has a distinct genetic and reproductive biology. Our software program was aimed at constructing a base population by simulating a random mating honey bee population. A forward-time population simulation approach was applied since it allows modeling of genetic characteristics and reproductive behavior specific to the honey bee. Results Our software program yielded a genomic dataset for a base population in linkage disequilibrium. In addition, information was obtained on (1) the position of markers on each chromosome, (2) allele frequency, (3) χ2 statistics for Hardy-Weinberg equilibrium, (4) a sorted list of markers with a minor allele frequency less than or equal to the input value, (5) average r2 values of linkage disequilibrium between all simulated marker loci pair for all generations and (6) average r2 value of linkage disequilibrium in the last generation for selected markers with the highest minor allele frequency. Conclusion We developed a software program that takes into account the genetic and reproductive biology specific to the honey bee and that can be used to constitute a genomic dataset compatible with the simulation studies necessary to optimize breeding programs. The source code together with an instruction file is freely accessible at http://msproteomics.org/Research/Misc/honeybeepopulationsimulator.html PMID:22520469

Simulating a base population in honey bee for molecular genetic studies.

PubMed

Gupta, Pooja; Conrad, Tim; Spötter, Andreas; Reinsch, Norbert; Bienefeld, Kaspar

2012-06-27

Over the past years, reports have indicated that honey bee populations are declining and that infestation by an ecto-parasitic mite (Varroa destructor) is one of the main causes. Selective breeding of resistant bees can help to prevent losses due to the parasite, but it requires that a robust breeding program and genetic evaluation are implemented. Genomic selection has emerged as an important tool in animal breeding programs and simulation studies have shown that it yields more accurate breeding value estimates, higher genetic gain and low rates of inbreeding. Since genomic selection relies on marker data, simulations conducted on a genomic dataset are a pre-requisite before selection can be implemented. Although genomic datasets have been simulated in other species undergoing genetic evaluation, simulation of a genomic dataset specific to the honey bee is required since this species has a distinct genetic and reproductive biology. Our software program was aimed at constructing a base population by simulating a random mating honey bee population. A forward-time population simulation approach was applied since it allows modeling of genetic characteristics and reproductive behavior specific to the honey bee. Our software program yielded a genomic dataset for a base population in linkage disequilibrium. In addition, information was obtained on (1) the position of markers on each chromosome, (2) allele frequency, (3) χ(2) statistics for Hardy-Weinberg equilibrium, (4) a sorted list of markers with a minor allele frequency less than or equal to the input value, (5) average r(2) values of linkage disequilibrium between all simulated marker loci pair for all generations and (6) average r2 value of linkage disequilibrium in the last generation for selected markers with the highest minor allele frequency. We developed a software program that takes into account the genetic and reproductive biology specific to the honey bee and that can be used to constitute a genomic dataset compatible with the simulation studies necessary to optimize breeding programs. The source code together with an instruction file is freely accessible at http://msproteomics.org/Research/Misc/honeybeepopulationsimulator.html.

Potential Signals of Natural Selection in the Top Risk Loci for Coronary Artery Disease: 9p21 and 10q11

PubMed Central

Zanetti, Daniela; Carreras-Torres, Robert; Esteban, Esther

2015-01-01

Background Coronary artery disease (CAD) is a complex disease and the leading cause of death in the world. Populations of different ancestry do not always share the same risk markers. Natural selective processes may be the cause of some of the population differences detected for specific risk mutations. Objective In this study, 384 single nucleotide polymorphisms (SNPs) located in four genomic regions associated with CAD (1p13, 1q41, 9p21 and 10q11) are analysed in a set of 19 populations from Europe, Middle East and North Africa and also in Asian and African samples from the 1000 Genomes Project. The aim of this survey is to explore for the first time whether the genetic variability in these genomic regions is better explained by demography or by natural selection. Results The results indicate significant differences in the structure of genetic variation and in the LD patterns among populations that probably explain the population disparities found in markers of susceptibility to CAD. Conclusions The results are consistent with potential signature of positive selection in the 9p21 region and of balancing selection in the 9p21 and 10q11. Specifically, in Europe three CAD risk markers in the 9p21 region (rs9632884, rs1537371 and rs1333042) show consistent signals of positive selection. The results of this study are consistent with a potential selective role of CAD in the configuration of genetic diversity in current human populations. PMID:26252781

Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

PubMed

Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

2006-01-01

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

Marker optimization for facial motion acquisition and deformation.

PubMed

Le, Binh H; Zhu, Mingyang; Deng, Zhigang

2013-11-01

A long-standing problem in marker-based facial motion capture is what are the optimal facial mocap marker layouts. Despite its wide range of potential applications, this problem has not yet been systematically explored to date. This paper describes an approach to compute optimized marker layouts for facial motion acquisition as optimization of characteristic control points from a set of high-resolution, ground-truth facial mesh sequences. Specifically, the thin-shell linear deformation model is imposed onto the example pose reconstruction process via optional hard constraints such as symmetry and multiresolution constraints. Through our experiments and comparisons, we validate the effectiveness, robustness, and accuracy of our approach. Besides guiding minimal yet effective placement of facial mocap markers, we also describe and demonstrate its two selected applications: marker-based facial mesh skinning and multiresolution facial performance capture.

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

PubMed

Lee, Wonsik; Do, Truc; Zhang, Ge; Kahne, Daniel; Meredith, Timothy C; Walker, Suzanne

2018-06-08

Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

Identification and characterization of dinucleotide repeat (CA)[sub n] markers for genetic mapping in dog

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

Ostrander, E.A.; Sprague, G.F. Jr.; Rine, J.

1993-04-01

A large block of simple sequence repeat (SSR) polymorphisms for the dog genome has been isolated and characterized. Screening of primary libraries by conventional hybridization methods as well as by screening of enriched marker-selected libraries led to the isolation of a large number of genomic clones that contained (CA)[sub n] repeats. The sequences of 101 clones showed that the size and complexity of (CA)[sub n] repeats in the dog genome were similar to those reported for these markers in the human genome. Detailed analysis of a representative subset of these markers revealed that most markers were moderately to highly polymorphic,more » with PIC values exceeding 0.70 for 33% of the markers tested. An association between higher PIC values and markers containing longer (CA)[sub n] repeats was observed in these studies, as previously noted for similar markers in the human genome. A list of primer sequences that tag each characterized marker is provided, and a comprehensive system of nomenclature for the dog genome is suggested. 28 refs., 4 figs., 2 tabs.« less

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice.

PubMed

Baliyan, Nikita; Malik, Rekha; Rani, Reema; Mehta, Kirti; Vashisth, Urvashi; Dhillon, Santosh; Boora, Khazan Singh

2018-01-01

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC 3 F 1 three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC 3 F 2 generation. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

An Efficient Method for High-Fidelity BAC/PAC Retrofitting with a Selectable Marker for Mammalian Cell Transfection

PubMed Central

Wang, Zunde; Engler, Peter; Longacre, Angelika; Storb, Ursula

2001-01-01

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination. PMID:11156622

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Effect of pulse sequence parameter selection on signal strength in positive-contrast MRI markers for MRI-based prostate postimplant assessment

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

Lim, Tze Yee

Purpose: For postimplant dosimetric assessment, computed tomography (CT) is commonly used to identify prostate brachytherapy seeds, at the expense of accurate anatomical contouring. Magnetic resonance imaging (MRI) is superior to CT for anatomical delineation, but identification of the negative-contrast seeds is challenging. Positive-contrast MRI markers were proposed to replace spacers to assist seed localization on MRI images. Visualization of these markers under varying scan parameters was investigated. Methods: To simulate a clinical scenario, a prostate phantom was implanted with 66 markers and 86 seeds, and imaged on a 3.0T MRI scanner using a 3D fast radiofrequency-spoiled gradient recalled echo acquisitionmore » with various combinations of scan parameters. Scan parameters, including flip angle, number of excitations, bandwidth, field-of-view, slice thickness, and encoding steps were systematically varied to study their effects on signal, noise, scan time, image resolution, and artifacts. Results: The effects of pulse sequence parameter selection on the marker signal strength and image noise were characterized. The authors also examined the tradeoff between signal-to-noise ratio, scan time, and image artifacts, such as the wraparound artifact, susceptibility artifact, chemical shift artifact, and partial volume averaging artifact. Given reasonable scan time and managable artifacts, the authors recommended scan parameter combinations that can provide robust visualization of the MRI markers. Conclusions: The recommended MRI pulse sequence protocol allows for consistent visualization of the markers to assist seed localization, potentially enabling MRI-only prostate postimplant dosimetry.« less

Accuracy of genomic prediction in switchgrass ( Panicum virgatum L.) improved by accounting for linkage disequilibrium

DOE PAGES

Ramstein, Guillaume P.; Evans, Joseph; Kaeppler, Shawn M.; ...

2016-02-11

Switchgrass is a relatively high-yielding and environmentally sustainable biomass crop, but further genetic gains in biomass yield must be achieved to make it an economically viable bioenergy feedstock. Genomic selection (GS) is an attractive technology to generate rapid genetic gains in switchgrass, and meet the goals of a substantial displacement of petroleum use with biofuels in the near future. In this study, we empirically assessed prediction procedures for genomic selection in two different populations, consisting of 137 and 110 half-sib families of switchgrass, tested in two locations in the United States for three agronomic traits: dry matter yield, plant height,more » and heading date. Marker data were produced for the families’ parents by exome capture sequencing, generating up to 141,030 polymorphic markers with available genomic-location and annotation information. We evaluated prediction procedures that varied not only by learning schemes and prediction models, but also by the way the data were preprocessed to account for redundancy in marker information. More complex genomic prediction procedures were generally not significantly more accurate than the simplest procedure, likely due to limited population sizes. Nevertheless, a highly significant gain in prediction accuracy was achieved by transforming the marker data through a marker correlation matrix. Our results suggest that marker-data transformations and, more generally, the account of linkage disequilibrium among markers, offer valuable opportunities for improving prediction procedures in GS. Furthermore, some of the achieved prediction accuracies should motivate implementation of GS in switchgrass breeding programs.« less

Genetic variability in selected date palm (Phoenix dactylifera L.) cultivars of United Arab Emirates using ISSR and DAMD markers.

PubMed

Purayil, Fayas T; Robert, Gabriel A; Gothandam, Kodiveri M; Kurup, Shyam S; Subramaniam, Sreeramanan; Cheruth, Abdul Jaleel

2018-02-01

Nine (9) different date palm ( Phoenix dactylifera L.) cultivars from UAE, which differ in their flower timings were selected to determine the polymorphism and genetic relationship between these cultivars. Hereditary differences and interrelationships were assessed utilizing inter-simple sequence repeat (ISSR) and directed amplification of minisatellite DNA region (DAMD) primers. Analysis on eight DAMD and five ISSR markers produced total of 113 amplicon including 99 polymorphic and 14 monomorphic alleles with a polymorphic percentage of 85.45. The average polymorphic information content for the two-marker system was almost similar (DAMD, 0.445 and ISSR, 0.459). UPGMA based clustering of DAMD and ISSR revealed that mid-season cultivars, Mkh (Khlas) and MB (Barhee) grouped together to form a subcluster in both the marker systems. The genetic similarity analysis followed by clustering of the cumulative data from the DAMD and ISSR resulted in two major clusters with two early-season cultivars (ENg and Ekn), two mid-season cultivars (MKh and MB) and one late-season cultivar (Lkhs) in cluster 1, cluster 2 includes two late-season cultivars, one early-season cultivar and one mid-season cultivar. The cluster analysis of both DAMD and ISSR marker revealed that, the patterns of variation between some of the tested cultivars were similar in both DNA marker systems. Hence, the present study signifies the applicability of DAMD and ISSR marker system in detecting genetic diversity of date palm cultivars flowering at different seasons. This may facilitate the conservation and improvement of date palm cultivars in the future.

Accuracy of genomic prediction in switchgrass ( Panicum virgatum L.) improved by accounting for linkage disequilibrium

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

Ramstein, Guillaume P.; Evans, Joseph; Kaeppler, Shawn M.

Switchgrass is a relatively high-yielding and environmentally sustainable biomass crop, but further genetic gains in biomass yield must be achieved to make it an economically viable bioenergy feedstock. Genomic selection (GS) is an attractive technology to generate rapid genetic gains in switchgrass, and meet the goals of a substantial displacement of petroleum use with biofuels in the near future. In this study, we empirically assessed prediction procedures for genomic selection in two different populations, consisting of 137 and 110 half-sib families of switchgrass, tested in two locations in the United States for three agronomic traits: dry matter yield, plant height,more » and heading date. Marker data were produced for the families’ parents by exome capture sequencing, generating up to 141,030 polymorphic markers with available genomic-location and annotation information. We evaluated prediction procedures that varied not only by learning schemes and prediction models, but also by the way the data were preprocessed to account for redundancy in marker information. More complex genomic prediction procedures were generally not significantly more accurate than the simplest procedure, likely due to limited population sizes. Nevertheless, a highly significant gain in prediction accuracy was achieved by transforming the marker data through a marker correlation matrix. Our results suggest that marker-data transformations and, more generally, the account of linkage disequilibrium among markers, offer valuable opportunities for improving prediction procedures in GS. Furthermore, some of the achieved prediction accuracies should motivate implementation of GS in switchgrass breeding programs.« less

Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum▿

PubMed Central

Smulian, A. George; Gibbons, Reta S.; Demland, Jeffery A.; Spaulding, Deborah T.; Deepe, George S.

2007-01-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene. PMID:17873086

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

PubMed

Smulian, A George; Gibbons, Reta S; Demland, Jeffery A; Spaulding, Deborah T; Deepe, George S

2007-11-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

Using rice genome-wide association studies to identify DNA markers for marker-assisted selection

USDA-ARS?s Scientific Manuscript database

Rice association mapping panels are collections of rice (Oryza sativa L.) accessions developed for genome-wide association (GWA) studies. One of these panels, the Rice Diversity Panel 1 (RDP1) was phenotyped by various research groups for several traits of interest, and more recently, genotyped with...

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangiferaindica) transcriptome for mapping and estimation of genetic diversity

USDA-ARS?s Scientific Manuscript database

The development of resources for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here a first step in developing such resources, our identification of thousands una...

Isolation and characterization of microsatellite markers in Fraser fir (Abies fraseri)

Treesearch

S.A. Josserand; K.M. Potter; G. Johnson; J.A. Bowen; J. Frampton; C.D. Nelson

2006-01-01

We describe the isolation and characterization of 14 microsatellite loci from Fraser fir (Abies fraseri). These markers originated from cloned inserts enriched for DNA sequences containing tandem di- and tri-nucleotide repeats. In total, 36 clones were selected, sequenced and evaluated. Polymerase chain reaction (PCR) primers for 14 of these...

Genetic markers that influence feed efficiency phenotypes also affect cattle temperament as measured by flight speed

USDA-ARS?s Scientific Manuscript database

The measure of flight speed for cattle has been shown to be a predictive indicator of temperament and has also been associated with feed efficiency phenotypes, thus, genetic markers associated with both traits may assist with the selection of animals with calmer disposition and economic value. Chrom...

Mapping and QTL analysis of drought tolerance in a spring wheat population using AFLP and DArt markers

USDA-ARS?s Scientific Manuscript database

Water availability is commonly the most limiting factor to crop production. This study was conducted to map quantitative trait loci (QTL) involved in drought tolerance in wheat (Triticum aestivum L.) to enable their use for marker assisted selection (MAS) in breeding. Using amplified fragment leng...

A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

USDA-ARS?s Scientific Manuscript database

In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

Evaluation of genetic markers for prediction of pre-harvest sprouting tolerance in hard white winter wheats

USDA-ARS?s Scientific Manuscript database

Pre-harvest sprouting of wheat results in significant financial loss at all steps in the production and marketing chain. Due to its intermittent nature in many wheat growing regions, direct selection for tolerance to pre-harvest sprouting is difficult. DNA markers linked to genes conditioning tole...

Isolation of plasma membrane fractions from the intestinal epithelial model T84.

PubMed

Kaoutzani, P; Parkos, C A; Delp-Archer, C; Madara, J L

1993-05-01

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

Identification and validation of single nucleotide polymorphic markers linked to Ug99 stem rust resistance in spring wheat

PubMed Central

Chao, Shiaoman; Singh, Ravi P.; Sorrells, Mark E.

2017-01-01

Wheat stem rust (Puccinia graminis f. sp. tritici Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of Puccinia graminis f. sp. tritici race TTKST, the Sr24-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes SrND643 and Sr37, respectively. Markers identified on 7DL were collocated with Sr25. Additional significant markers were located in the regions where no Sr gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the Sr25-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat. PMID:28241006

Increased prediction accuracy in wheat breeding trials using a marker x environment interaction genomic selection model

USDA-ARS?s Scientific Manuscript database

Genomic selection (GS) models use genome-wide genetic information to predict genetic values of candidates for selection. Originally these models were developed without considering genotype ' environment interaction (GE). Several authors have proposed extensions of the cannonical GS model that accomm...

A resource of large-scale molecular markers for monitoring Agropyron cristatum chromatin introgression in wheat background based on transcriptome sequences.

PubMed

Zhang, Jinpeng; Liu, Weihua; Lu, Yuqing; Liu, Qunxing; Yang, Xinming; Li, Xiuquan; Li, Lihui

2017-09-20

Agropyron cristatum is a wild grass of the tribe Triticeae and serves as a gene donor for wheat improvement. However, very few markers can be used to monitor A. cristatum chromatin introgressions in wheat. Here, we reported a resource of large-scale molecular markers for tracking alien introgressions in wheat based on transcriptome sequences. By aligning A. cristatum unigenes with the Chinese Spring reference genome sequences, we designed 9602 A. cristatum expressed sequence tag-sequence-tagged site (EST-STS) markers for PCR amplification and experimental screening. As a result, 6063 polymorphic EST-STS markers were specific for the A. cristatum P genome in the single-receipt wheat background. A total of 4956 randomly selected polymorphic EST-STS markers were further tested in eight wheat variety backgrounds, and 3070 markers displaying stable and polymorphic amplification were validated. These markers covered more than 98% of the A. cristatum genome, and the marker distribution density was approximately 1.28 cM. An application case of all EST-STS markers was validated on the A. cristatum 6 P chromosome. These markers were successfully applied in the tracking of alien A. cristatum chromatin. Altogether, this study provided a universal method of large-scale molecular marker development to monitor wild relative chromatin in wheat.

A new series of yeast shuttle vectors for the recovery and identification of multiple plasmids from Saccharomyces cerevisiae.

PubMed

Frazer, LilyAnn Novak; O'Keefe, Raymond T

2007-09-01

The availability of Saccharomyces cerevisiae yeast strains with multiple auxotrophic markers allows the stable introduction and selection of more than one yeast shuttle vector containing marker genes that complement the auxotrophic markers. In certain experimental situations there is a need to recover more than one shuttle vector from yeast. To facilitate the recovery and identification of multiple plasmids from S. cerevisiae, we have constructed a series of plasmids based on the pRS series of yeast shuttle vectors. Bacterial antibiotic resistance genes to chloramphenicol, kanamycin and zeocin have been combined with the yeast centromere sequence (CEN6), the autonomously replicating sequence (ARSH4) and one of the four yeast selectable marker genes (HIS3, TRP1, LEU2 or URA3) from the pRS series of vectors. The 12 plasmids produced differ in antibiotic resistance and yeast marker gene within the backbone of the multipurpose plasmid pBluescript II. The newly constructed vectors show similar mitotic stability to the original pRS vectors. In combination with the ampicillin-resistant pRS series of yeast shuttle vectors, these plasmids now allow the recovery and identification in bacteria of up to four different vectors from S. cerevisiae. Copyright (c) 2007 John Wiley & Sons, Ltd.

Incorporating thyroid markers in Down syndrome screening protocols.

PubMed

Dhaifalah, Ishraq; Salek, Tomas; Langova, Dagmar; Cuckle, Howard

2017-05-01

The article aimed to assess the benefit of incorporating maternal serum thyroid disease marker levels (thyroid-stimulating hormone and free thyroxine) into first trimester Down syndrome screening protocols. Statistical modelling was used to predict performance with and without the thyroid markers. Two protocols were considered: the combined test and the contingent cell-free DNA (cfDNA) test, where 15-40% women are selected for cfDNA because of increased risk based on combined test results. Published parameters were used for the combined test, cfDNA and the Down syndrome means for thyroid-stimulating hormone and free thyroxine; other parameters were derived from a series of 5230 women screened for both thyroid disease and Down syndrome. Combined test: For a fixed 85% detection rate, the predicted false positive rate was reduced from 5.3% to 3.6% with the addition of the thyroid markers. Contingent cfDNA test: For a fixed 95% detection rate, the proportion of women selected for cfDNA was reduced from 25.6% to 20.2%. When screening simultaneously for maternal thyroid disease and Down syndrome, thyroid marker levels should be used in the calculation of Down syndrome risk. The benefit is modest but can be achieved with no additional cost. © 2017 John Wiley & Sons, Ltd. © 2017 John Wiley & Sons, Ltd.

Fecal markers of inflammation, protein loss, and microbial changes in dogs with the acute hemorrhagic diarrhea syndrome (AHDS).

PubMed

Heilmann, Romy M; Guard, Melissa M; Steiner, Jörg M; Suchodolski, Jan S; Unterer, Stefan

2017-09-01

Idiopathic acute hemorrhagic diarrhea syndrome (AHDS) is characterized by acute onset of bloody diarrhea, severe dehydration, and increased vascular and intestinal mucosal permeability. Markers of gastrointestinal inflammation, protein loss, and changes in the intestinal microbiota have not been studied extensively in dogs with AHDS. For 3 consecutive days, feces were collected from dogs with AHDS, and assayed for calprotectin and S100A12 (both markers of inflammation), α 1 -proteinase inhibitor (a marker of gastrointestinal protein loss), and the presence of selected species of bacteria. Concentrations of all assayed markers were significantly greater than the institutional reference intervals at the time of presentation, and all decreased significantly by Day 3 of treatment. Abundances of selected bacterial groups (Ruminococcaceae, Faecalibacterium spp., Bifidobacterium spp., and Proteobacteria) at the time of diagnosis were consistent with an intestinal bacterial dysbiosis. No differences in the abundance of bacterial groups over time was seen, except for a mild but significant decrease in Ruminococcaceae at Day 3. These results suggest that canine AHDS is associated with a significant but transient gastrointestinal loss of protein and intestinal inflammation. The intestinal bacterial dysbiosis appears to outlast the protein loss and inflammation. © Veterinary Emergency and Critical Care Society 2017.

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

Discovery of Genome-Wide Microsatellite Markers in Scombridae: A Pilot Study on Albacore Tuna

PubMed Central

Nikolic, Natacha; Duthoy, Stéphanie; Destombes, Antoine; Bodin, Nathalie; West, Wendy; Puech, Alexis; Bourjea, Jérôme

2015-01-01

Recent developments in sequencing technologies and bioinformatics analysis provide a greater amount of DNA sequencing reads at a low cost. Microsatellites are the markers of choice for a variety of population genetic studies, and high quality markers can be discovered in non-model organisms, such as tuna, with these recent developments. Here, we use a high-throughput method to isolate microsatellite markers in albacore tuna, Thunnus alalunga, based on coupling multiplex enrichment and next-generation sequencing on 454 GS-FLX Titanium pyrosequencing. The crucial minimum number of polymorphic markers to infer evolutionary and ecological processes for this species has been described for the first time. We provide 1670 microsatellite design primer pairs, and technical and molecular genetics selection resulting in 43 polymorphic microsatellite markers. On this panel, we characterized 34 random and selectively neutral markers («neutral») and 9 «non-neutral» markers. The variability of «neutral» markers was screened with 136 individuals of albacore tuna from southwest Indian Ocean (42), northwest Indian Ocean (31), South Africa (31), and southeast Atlantic Ocean (32). Power analysis demonstrated that the panel of genetic markers can be applied in diversity and population genetics studies. Global genetic diversity for albacore was high with a mean number of alleles at 16.94; observed heterozygosity 66% and expected heterozygosity 77%. The number of individuals was insufficient to provide accurate results on differentiation. Of the 9 «non-neutral» markers, 3 were linked to a sequence of known function. The one is located to a sequence having an immunity function (ThuAla-Tcell-01) and the other to a sequence having energy allocation function (ThuAla-Hki-01). These two markers were genotyped on the 136 individuals and presented different diversity levels. ThuAla-Tcell-01 has a high number of alleles (20), heterozygosity (87–90%), and assignment index. ThuAla-Hki-01 has a lower number of alleles (9), low heterozygosity (24–27%), low assignment index and significant inbreeding. Finally, the 34 «neutral» and 3 «non-neutral» microsatellites markers were tested on four economically important Scombridae species—Thunnus albacares, Thunnus thynnus, Thunnus obesus, and Acanthocybium solandri. PMID:26544051

Selection of discrimination marker from various propolis for mapping and identify anti Candida albicans activity

NASA Astrophysics Data System (ADS)

Mahadewi, Alfiani Guntari; Christina, Daisy; Hermansyah, Heri; Wijanarko, Anondho; Farida, Siti; Adawiyah, Robiatul; Rohmatin, Etin; Sahlan, Muhamad

2018-02-01

The increase in fungal resistance against antifungal drugs available in the market will reduce the effectiveness of treatment for Candidiasis. Propolis contains various compounds with antifungal properties Candida albicans, but the content of each type is very diverse. The sample used was Sulawesi propolis type smooth (taken from inside the nest), rough (taken from outside the hive) and mix (a combination of both). Anti-C. albicans molecule marker is a marker compound for selecting propolis with the ability to overcome Candidiasis. The initial step was to test the levels of flavonoids and phenolic by using UV-Vis spectrometry method. It was founded that each sample was not always superior to any substance, so propolis cannot be directly selected. In Phenolic content, mix propolis has the highest value than other 5.109%. In Flavonoid content, propolis smooth has the highest value than other, 16.38%. Furthermore, propolis selected by antifungal activity test with good diffusion method at the concentration propolis 5% either 7%, the inhibitory diameter zone propolis smooth and rough has same value 10 mm. Propolis mix has an advantage while propolis smooth and rough have the same capability range 12 mm and 13 mm. In this study, the phenolic content plays a major role in antifungal cases.

Q-marker based strategy for CMC research of Chinese medicine: A case study of Panax Notoginseng saponins.

PubMed

Zhong, Yi; Zhu, Jieqiang; Yang, Zhenzhong; Shao, Qing; Fan, Xiaohui; Cheng, Yiyu

2018-01-31

To ensure pharmaceutical quality, chemistry, manufacturing and control (CMC) research is essential. However, due to the inherent complexity of Chinese medicine (CM), CMC study of CM remains a great challenge for academia, industry, and regulatory agencies. Recently, quality-marker (Q-marker) was proposed to establish quality standards or quality analysis approaches of Chinese medicine, which sheds a light on Chinese medicine's CMC study. Here manufacture processes of Panax Notoginseng Saponins (PNS) is taken as a case study and the present work is to establish a Q-marker based research strategy for CMC of Chinese medicine. The Q-markers of Panax Notoginseng Saponins (PNS) is selected and established by integrating chemical profile with pharmacological activities. Then, the key processes of PNS manufacturing are identified by material flow analysis. Furthermore, modeling algorithms are employed to explore the relationship between Q-markers and critical process parameters (CPPs) of the key processes. At last, CPPs of the key processes are optimized in order to improving the process efficiency. Among the 97 identified compounds, Notoginsenoside R 1 , ginsenoside Rg 1 , Re, Rb 1 and Rd are selected as the Q-markers of PNS. Our analysis on PNS manufacturing show the extraction process and column chromatography process are the key processes. With the CPPs of each process as the inputs and Q-markers' contents as the outputs, two process prediction models are built separately for the extraction process and column chromatography process of Panax notoginseng, which both possess good prediction ability. Based on the efficiency models of extraction process and column chromatography process we constructed, the optimal CPPs of both processes are calculated. Our results show that the Q-markers derived from CMC research strategy can be applied to analyze the manufacturing processes of Chinese medicine to assure product's quality and promote key processes' efficiency simultaneously. Copyright © 2018 Elsevier GmbH. All rights reserved.

Predicting Quantitative Traits With Regression Models for Dense Molecular Markers and Pedigree

PubMed Central

de los Campos, Gustavo; Naya, Hugo; Gianola, Daniel; Crossa, José; Legarra, Andrés; Manfredi, Eduardo; Weigel, Kent; Cotes, José Miguel

2009-01-01

The availability of genomewide dense markers brings opportunities and challenges to breeding programs. An important question concerns the ways in which dense markers and pedigrees, together with phenotypic records, should be used to arrive at predictions of genetic values for complex traits. If a large number of markers are included in a regression model, marker-specific shrinkage of regression coefficients may be needed. For this reason, the Bayesian least absolute shrinkage and selection operator (LASSO) (BL) appears to be an interesting approach for fitting marker effects in a regression model. This article adapts the BL to arrive at a regression model where markers, pedigrees, and covariates other than markers are considered jointly. Connections between BL and other marker-based regression models are discussed, and the sensitivity of BL with respect to the choice of prior distributions assigned to key parameters is evaluated using simulation. The proposed model was fitted to two data sets from wheat and mouse populations, and evaluated using cross-validation methods. Results indicate that inclusion of markers in the regression further improved the predictive ability of models. An R program that implements the proposed model is freely available. PMID:19293140

Development of Cymbidium ensifolium genic-SSR markers and their utility in genetic diversity and population structure analysis in cymbidiums.

PubMed

Li, Xiaobai; Jin, Feng; Jin, Liang; Jackson, Aaron; Huang, Cheng; Li, Kehu; Shu, Xiaoli

2014-12-05

Cymbidium is a genus of 68 species in the orchid family, with extremely high ornamental value. Marker-assisted selection has proven to be an effective strategy in accelerating plant breeding for many plant species. Analysis of cymbidiums genetic background by molecular markers can be of great value in assisting parental selection and breeding strategy design, however, in plants such as cymbidiums limited genomic resources exist. In order to obtain efficient markers, we deep sequenced the C. ensifolium transcriptome to identify simple sequence repeats derived from gene regions (genic-SSR). The 7,936 genic-SSR markers were identified. A total of 80 genic-SSRs were selected, and primers were designed according to their flanking sequences. Of the 80 genic-SSR primer sets, 62 were amplified in C. ensifolium successfully, and 55 showed polymorphism when cross-tested among 9 Cymbidium species comprising 59 accessions. Unigenes containing the 62 genic-SSRs were searched against Non-redundant (Nr), Gene Ontology database (GO), eukaryotic orthologous groups (KOGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The search resulted in 53 matching Nr sequences, of which 39 had GO terms, 18 were assigned to KOGs, and 15 were annotated with KEGG. Genetic diversity and population structure were analyzed based on 55 polymorphic genic-SSR data among 59 accessions. The genetic distance averaged 0.3911, ranging from 0.016 to 0.618. The polymorphic index content (PIC) of 55 polymorphic markers averaged 0.407, ranging from 0.033 to 0.863. A model-based clustering analysis revealed that five genetic groups existed in the collection. Accessions from the same species were typically grouped together; however, C. goeringii accessions did not always form a separate cluster, suggesting that C. goeringii accessions were polyphyletic. The genic-SSR identified in this study constitute a set of markers that can be applied across multiple Cymbidium species and used for the evaluation of genetic relationships as well as qualitative and quantitative trait mapping studies. Genic-SSR's coupled with the functional annotations provided by the unigenes will aid in mapping candidate genes of specific function.

Precise mapping of a locus affecting grain protein content in durum wheat.

PubMed

Olmos, S; Distelfeld, A; Chicaiza, O; Schlatter, A R; Fahima, T; Echenique, V; Dubcovsky, J

2003-11-01

Grain protein content (GPC) is an important factor in pasta and breadmaking quality, and in human nutrition. It is also an important trait for wheat growers because premium prices are frequently paid for wheat with high GPC. A promising source for alleles to increase GPC was detected on chromosome 6B of Triticum turgidum var. dicoccoides accession FA-15-3 (DIC). Two previous quantitative trait locus (QTL) studies found that the positive effect of DIC-6B was associated to a single locus located between the centromere and the Nor-B2 locus on the short arm of chromosome 6B. Microsatellite markers Xgwm508 and Xgwm193 flanking the QTL region were used in this study to develop 20 new homozygous recombinant substitution lines (RSLs) with crossovers between these markers. These 20 RSLs, plus nine RSLs developed in previous studies were characterized with four new RFLP markers located within this chromosome segment. Grain protein content was determined in three field experiments organized as randomized complete block designs with ten replications each. The QTL peaks for protein content were located in the central region of a 2.7-cM interval between RFLP markers Xcdo365 and Xucw67 in the three experiments. Statistical analyses showed that almost all lines could be classified unequivocally within low- and high- protein groups, facilitating the mapping of this trait as a single Mendelian locus designated Gpc-6B1. The Gpc-6B1 locus was mapped 1.5-cM proximal to Xcdo365 and 1.2-cM distal to Xucw67. These new markers can be used to reduce the size of the DIC chromosome segment selected in marker-assisted selection programs. Markers Nor-B2 and Xucw66 flanking the previous two markers can be used to select against the DIC segment and reduce the linkage drag during the transfer of Gpc-6B1 into commercial bread and pasta wheat varieties. The precise mapping of the high GPC gene, the high frequency of recombinants recovered in the targeted region, and the recent development of a tetraploid BAC library including the Gpc-6B1 DIC allele are the first steps towards the map-based cloning of this gene.

An integrated genetic map based on four mapping populations and quantitative trait loci associated with economically important traits in watermelon (Citrullus lanatus)

PubMed Central

2014-01-01

Background Modern watermelon (Citrullus lanatus L.) cultivars share a narrow genetic base due to many years of selection for desirable horticultural qualities. Wild subspecies within C. lanatus are important potential sources of novel alleles for watermelon breeding, but successful trait introgression into elite cultivars has had limited success. The application of marker assisted selection (MAS) in watermelon is yet to be realized, mainly due to the past lack of high quality genetic maps. Recently, a number of useful maps have become available, however these maps have few common markers, and were constructed using different marker sets, thus, making integration and comparative analysis among maps difficult. The objective of this research was to use single-nucleotide polymorphism (SNP) anchor markers to construct an integrated genetic map for C. lanatus. Results Under the framework of the high density genetic map, an integrated genetic map was constructed by merging data from four independent mapping experiments using a genetically diverse array of parental lines, which included three subspecies of watermelon. The 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel), 36 structure variation (SV) and 386 SNP markers from the four maps were used to construct an integrated map. This integrated map contained 1339 markers, spanning 798 cM with an average marker interval of 0.6 cM. Fifty-eight previously reported quantitative trait loci (QTL) for 12 traits in these populations were also integrated into the map. In addition, new QTL identified for brix, fructose, glucose and sucrose were added. Some QTL associated with economically important traits detected in different genetic backgrounds mapped to similar genomic regions of the integrated map, suggesting that such QTL are responsible for the phenotypic variability observed in a broad array of watermelon germplasm. Conclusions The integrated map described herein enhances the utility of genomic tools over previous watermelon genetic maps. A large proportion of the markers in the integrated map are SSRs, InDels and SNPs, which are easily transferable across laboratories. Moreover, the populations used to construct the integrated map include all three watermelon subspecies, making this integrated map useful for the selection of breeding traits, identification of QTL, MAS, analysis of germplasm and commercial hybrid seed detection. PMID:24443961

Development and Molecular Characterization of Novel Polymorphic Genomic DNA SSR Markers in Lentinula edodes.

PubMed

Moon, Suyun; Lee, Hwa-Yong; Shim, Donghwan; Kim, Myungkil; Ka, Kang-Hyeon; Ryoo, Rhim; Ko, Han-Gyu; Koo, Chang-Duck; Chung, Jong-Wook; Ryu, Hojin

2017-06-01

Sixteen genomic DNA simple sequence repeat (SSR) markers of Lentinula edodes were developed from 205 SSR motifs present in 46.1-Mb long L. edodes genome sequences. The number of alleles ranged from 3-14 and the major allele frequency was distributed from 0.17-0.96. The values of observed and expected heterozygosity ranged from 0.00-0.76 and 0.07-0.90, respectively. The polymorphic information content value ranged from 0.07-0.89. A dendrogram, based on 16 SSR markers clustered by the paired hierarchical clustering' method, showed that 33 shiitake cultivars could be divided into three major groups and successfully identified. These SSR markers will contribute to the efficient breeding of this species by providing diversity in shiitake varieties. Furthermore, the genomic information covered by the markers can provide a valuable resource for genetic linkage map construction, molecular mapping, and marker-assisted selection in the shiitake mushroom.

Vision based object pose estimation for mobile robots

NASA Technical Reports Server (NTRS)

Wu, Annie; Bidlack, Clint; Katkere, Arun; Feague, Roy; Weymouth, Terry

1994-01-01

Mobile robot navigation using visual sensors requires that a robot be able to detect landmarks and obtain pose information from a camera image. This paper presents a vision system for finding man-made markers of known size and calculating the pose of these markers. The algorithm detects and identifies the markers using a weighted pattern matching template. Geometric constraints are then used to calculate the position of the markers relative to the robot. The selection of geometric constraints comes from the typical pose of most man-made signs, such as the sign standing vertical and the dimensions of known size. This system has been tested successfully on a wide range of real images. Marker detection is reliable, even in cluttered environments, and under certain marker orientations, estimation of the orientation has proven accurate to within 2 degrees, and distance estimation to within 0.3 meters.

Phylogenetic relationships of chrysanthemums in Korea based on novel SSR markers.

PubMed

Khaing, A A; Moe, K T; Hong, W J; Park, C S; Yeon, K H; Park, H S; Kim, D C; Choi, B J; Jung, J Y; Chae, S C; Lee, K M; Park, Y J

2013-11-07

Chrysanthemums are well known for their esthetic and medicinal values. Characterization of chrysanthemums is vital for their conservation and management as well as for understanding their genetic relationships. We found 12 simple sequence repeat markers (SSRs) of 100 designed primers to be polymorphic. These novel SSR markers were used to evaluate 95 accessions of chrysanthemums (3 indigenous and 92 cultivated accessions). Two hundred alleles were identified, with an average of 16.7 alleles per locus. KNUCRY-77 gave the highest polymorphic information content value (0.879), while KNUCRY-10 gave the lowest (0.218). Similar patterns of grouping were observed with a distance-based dendrogram developed using PowerMarker and model-based clustering with Structure. Three clusters with some admixtures were identified by model-based clustering. These newly developed SSR markers will be useful for further studies of chrysanthemums, such as taxonomy and marker-assisted selection breeding.

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.

Island-Model Genomic Selection for Long-Term Genetic Improvement of Autogamous Crops.

PubMed

Yabe, Shiori; Yamasaki, Masanori; Ebana, Kaworu; Hayashi, Takeshi; Iwata, Hiroyoshi

2016-01-01

Acceleration of genetic improvement of autogamous crops such as wheat and rice is necessary to increase cereal production in response to the global food crisis. Population and pedigree methods of breeding, which are based on inbred line selection, are used commonly in the genetic improvement of autogamous crops. These methods, however, produce a few novel combinations of genes in a breeding population. Recurrent selection promotes recombination among genes and produces novel combinations of genes in a breeding population, but it requires inaccurate single-plant evaluation for selection. Genomic selection (GS), which can predict genetic potential of individuals based on their marker genotype, might have high reliability of single-plant evaluation and might be effective in recurrent selection. To evaluate the efficiency of recurrent selection with GS, we conducted simulations using real marker genotype data of rice cultivars. Additionally, we introduced the concept of an "island model" inspired by evolutionary algorithms that might be useful to maintain genetic variation through the breeding process. We conducted GS simulations using real marker genotype data of rice cultivars to evaluate the efficiency of recurrent selection and the island model in an autogamous species. Results demonstrated the importance of producing novel combinations of genes through recurrent selection. An initial population derived from admixture of multiple bi-parental crosses showed larger genetic gains than a population derived from a single bi-parental cross in whole cycles, suggesting the importance of genetic variation in an initial population. The island-model GS better maintained genetic improvement in later generations than the other GS methods, suggesting that the island-model GS can utilize genetic variation in breeding and can retain alleles with small effects in the breeding population. The island-model GS will become a new breeding method that enhances the potential of genomic selection in autogamous crops, especially bringing long-term improvement.

Island-Model Genomic Selection for Long-Term Genetic Improvement of Autogamous Crops

PubMed Central

Yabe, Shiori; Yamasaki, Masanori; Ebana, Kaworu; Hayashi, Takeshi; Iwata, Hiroyoshi

2016-01-01

Acceleration of genetic improvement of autogamous crops such as wheat and rice is necessary to increase cereal production in response to the global food crisis. Population and pedigree methods of breeding, which are based on inbred line selection, are used commonly in the genetic improvement of autogamous crops. These methods, however, produce a few novel combinations of genes in a breeding population. Recurrent selection promotes recombination among genes and produces novel combinations of genes in a breeding population, but it requires inaccurate single-plant evaluation for selection. Genomic selection (GS), which can predict genetic potential of individuals based on their marker genotype, might have high reliability of single-plant evaluation and might be effective in recurrent selection. To evaluate the efficiency of recurrent selection with GS, we conducted simulations using real marker genotype data of rice cultivars. Additionally, we introduced the concept of an “island model” inspired by evolutionary algorithms that might be useful to maintain genetic variation through the breeding process. We conducted GS simulations using real marker genotype data of rice cultivars to evaluate the efficiency of recurrent selection and the island model in an autogamous species. Results demonstrated the importance of producing novel combinations of genes through recurrent selection. An initial population derived from admixture of multiple bi-parental crosses showed larger genetic gains than a population derived from a single bi-parental cross in whole cycles, suggesting the importance of genetic variation in an initial population. The island-model GS better maintained genetic improvement in later generations than the other GS methods, suggesting that the island-model GS can utilize genetic variation in breeding and can retain alleles with small effects in the breeding population. The island-model GS will become a new breeding method that enhances the potential of genomic selection in autogamous crops, especially bringing long-term improvement. PMID:27115872

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

PubMed Central

Lipscomb, Gina L.; Conway, Jonathan M.; Blumer-Schuette, Sara E.; Kelly, Robert M.

2016-01-01

ABSTRACT Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-type C. bescii was sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCE Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism. PMID:27208106

Isolation and characterization of microsatellite markers for Jasminum sambac (Oleaceae) using Illumina shotgun sequencing1

PubMed Central

Li, Yong; Zhang, Weirui

2015-01-01

Premise of the study: Microsatellite markers of Jasminum sambac (Oleaceae) were isolated to investigate wild germplasm resources and provide markers for breeding. Methods and Results: Illumina sequencing was used to isolate microsatellite markers from the transcriptome of J. sambac. A total of 1322 microsatellites were identified from 49,772 assembled unigenes. One hundred primer pairs were randomly selected to verify primer amplification efficiency. Out of these tested primer pairs, 31 were successfully amplified: 18 primer pairs yielded a single allele, seven exhibited fixed heterozygosity with two alleles, and only six displayed polymorphisms. Conclusions: This study obtained the first set of microsatellite markers for J. sambac, which will be helpful for the assessment of wild germplasm resources and the development of molecular marker–assisted breeding. PMID:26504683

Quantitative trait loci affecting lactose and total solids on chromosome 6 in Brazilian Gir dairy cattle.

PubMed

Silva, A A; Azevedo, A L S; Gasparini, K; Verneque, R S; Peixoto, M G C D; Panetto, B R; Guimarães, S E F; Machado, M A

2011-10-31

Fourteen Brazilian Gir sire families with 657 daughters were analyzed for quantitative trait loci (QTL) on chromosome 6 affecting lactose and total solids. Cows and sires were genotyped with 27 microsatellites with a mean spacing between markers of 4.9 cM. We used a 1% chromosome-wide threshold for QTL qualification. A QTL for lactose yield was found close to marker MNB66 in three families. A QTL for total solid yield was identified close to marker BMS2508 in three families. A QTL for lactose percentage, close to marker DIK1182, was identified in two families. A QTL for total solid percentage, close to marker MNB208, was identified in four families. These QTLs could be used for selection of animals in dairy production systems.

New polymorphic markers in the vicinity of the pearl locus on mouse chromosome 13.

PubMed

Xu, H P; Yanak, B L; Wigler, M H; Gorin, M B

1996-01-01

We have used a Mus domesticus/-Mus spretus congenic animal that was selected for retention of Mus spretus DNA around the pearl locus to create a highly polymorphic region suitable for screening new markers. Representation difference analysis (RDA) was performed with either DNA from the congenic animal or C57BL/6J as the driver for subtraction. Four clones were identified, characterized, and converted to PCR-based polymorphic markers. Three of the four markers equally subdivide a 10-cM interval containing the pearl locus, with the fourth located centromeric to it. These markers have been placed on the mouse genetic map by use of an interspecific backcross panel between Mus domesticus (C57BL/6J) and Mus spretus generated by The Jackson Laboratory.

Development of 12 genic microsatellite loci for a biofuel grass, Miscanthus sinensis (Poaceae).

PubMed

Ho, Chuan-Wen; Wu, Tai-Han; Hsu, Tsai-Wen; Huang, Jao-Ching; Huang, Chi-Chun; Chiang, Tzen-Yuh

2011-08-01

Miscanthus, a nonfood plant with high potential as a biofuel, has been used in Europe and the United States. The selection of a cultivar with high biomass, photosynthetic efficiency, and stress resistance from wild populations has become an important issue. New genic microsatellite markers will aid the assessment of genetic diversity for different strains. Twelve polymorphic microsatellite markers derived from the transcriptome of Miscanthus sinensis fo. glaber were identified and screened on 80 individuals of M. sinensis. The number of alleles per locus ranged from 6 to 12, and the mean expected heterozygosity was 0.75. Cross-taxa transferability revealed that all loci can be applied to all varieties of M. sinensis, as well as the closely related species M. floridulus. These new genic microsatellite markers are useful for characterizing different traits in breeding programs or to select genes useful for biofuel.

Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

PubMed

Niklitschek, Mauricio; Baeza, Marcelo; Fernández-Lobato, María; Cifuentes, Víctor

2012-01-01

Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms.

Highly Discriminatory Variable-Number Tandem-Repeat Markers for Genotyping of Trichophyton interdigitale Strains

PubMed Central

Drira, Ines; Hadrich, Ines; Neji, Sourour; Mahfouth, Nedia; Trabelsi, Houaida; Sellami, Hayet; Makni, Fattouma

2014-01-01

Trichophyton interdigitale is the second most frequent cause of superficial fungal infections of various parts of the human body. Studying the population structure and genotype differentiation of T. interdigitale strains may lead to significant improvements in clinical practice. The present study aimed to develop and select suitable variable-number tandem-repeat (VNTR) markers for 92 clinical strains of T. interdigitale. On the basis of an analysis of four VNTR markers, four to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a D value of 0.802. The combination of all four markers yielded a D value of 0.969 with 29 distinct multilocus genotypes. VNTR typing revealed the genetic diversity of the strains, identifying three populations according to their colonization sites. A correlation between phenotypic characteristics and multilocus genotypes was observed. Seven patients harbored T. interdigitale strains with different genotypes. Typing of clinical T. interdigitale samples by VNTR markers displayed excellent discriminatory power and 100% reproducibility. PMID:24989614

Bone orientation and position estimation errors using Cosserat point elements and least squares methods: Application to gait.

PubMed

Solav, Dana; Camomilla, Valentina; Cereatti, Andrea; Barré, Arnaud; Aminian, Kamiar; Wolf, Alon

2017-09-06

The aim of this study was to analyze the accuracy of bone pose estimation based on sub-clusters of three skin-markers characterized by triangular Cosserat point elements (TCPEs) and to evaluate the capability of four instantaneous physical parameters, which can be measured non-invasively in vivo, to identify the most accurate TCPEs. Moreover, TCPE pose estimations were compared with the estimations of two least squares minimization methods applied to the cluster of all markers, using rigid body (RBLS) and homogeneous deformation (HDLS) assumptions. Analysis was performed on previously collected in vivo treadmill gait data composed of simultaneous measurements of the gold-standard bone pose by bi-plane fluoroscopy tracking the subjects' knee prosthesis and a stereophotogrammetric system tracking skin-markers affected by soft tissue artifact. Femur orientation and position errors estimated from skin-marker clusters were computed for 18 subjects using clusters of up to 35 markers. Results based on gold-standard data revealed that instantaneous subsets of TCPEs exist which estimate the femur pose with reasonable accuracy (median root mean square error during stance/swing: 1.4/2.8deg for orientation, 1.5/4.2mm for position). A non-invasive and instantaneous criteria to select accurate TCPEs for pose estimation (4.8/7.3deg, 5.8/12.3mm), was compared with RBLS (4.3/6.6deg, 6.9/16.6mm) and HDLS (4.6/7.6deg, 6.7/12.5mm). Accounting for homogeneous deformation, using HDLS or selected TCPEs, yielded more accurate position estimations than RBLS method, which, conversely, yielded more accurate orientation estimations. Further investigation is required to devise effective criteria for cluster selection that could represent a significant improvement in bone pose estimation accuracy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Automated recognition of stratigraphic marker shales from geophysical logs in iron ore deposits

NASA Astrophysics Data System (ADS)

Silversides, Katherine; Melkumyan, Arman; Wyman, Derek; Hatherly, Peter

2015-04-01

The mining of stratiform ore deposits requires a means of determining the location of stratigraphic boundaries. A variety of geophysical logs may provide the required data but, in the case of banded iron formation hosted iron ore deposits in the Hamersley Ranges of Western Australia, only one geophysical log type (natural gamma) is collected for this purpose. The information from these logs is currently processed by slow manual interpretation. In this paper we present an alternative method of automatically identifying recurring stratigraphic markers in natural gamma logs from multiple drill holes. Our approach is demonstrated using natural gamma geophysical logs that contain features corresponding to the presence of stratigraphically important marker shales. The host stratigraphic sequence is highly consistent throughout the Hamersley and the marker shales can therefore be used to identify the stratigraphic location of the banded iron formation (BIF) or BIF hosted ore. The marker shales are identified using Gaussian Processes (GP) trained by either manual or active learning methods and the results are compared to the existing geological interpretation. The manual method involves the user selecting the signatures for improving the library, whereas the active learning method uses the measure of uncertainty provided by the GP to select specific examples for the user to consider for addition. The results demonstrate that both GP methods can identify a feature, but the active learning approach has several benefits over the manual method. These benefits include greater accuracy in the identified signatures, faster library building, and an objective approach for selecting signatures that includes the full range of signatures across a deposit in the library. When using the active learning method, it was found that the current manual interpretation could be replaced in 78.4% of the holes with an accuracy of 95.7%.

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

PubMed

Pradhan, Subrata; Chakraborty, Anirban; Sikdar, Narattam; Chakraborty, Saikat; Bhattacharyya, Jagannath; Mitra, Joy; Manna, Anulina; Dutta Gupta, Snehasish; Sen, Soumitra Kumar

2016-10-01

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.

Identifying fecal sources in a selected catchment reach using multiple source-tracking tools

USGS Publications Warehouse

Vogel, J.R.; Stoeckel, D.M.; Lamendella, R.; Zelt, R.B.; Santo, Domingo J.W.; Walker, S.R.; Oerther, D.B.

2007-01-01

Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools - Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers - were evaluated in a selected reach of Plum Creek in sooth-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to lowing surface water. ?? ASA, CSSA, SSSA.

Using plant wax markers to estimate the diet composition of grazing Holstein dairy cows.

PubMed

Heublein, C; Südekum, K-H; Gill, F L; Dohme-Meier, F; Schori, F

2017-02-01

The objective of this study was to test whether diet selection of dairy cows under grazing conditions could be estimated using plant wax markers. Furthermore, differences between 2 cow strains and the effect of concentrate supplementation on plant species selection were investigated. The experiment was a study with a crossover design performed on an organic farm with 12 Swiss Holstein cows and 12 New Zealand Holstein cows. Both experimental periods consisted of a 21-d adaptation and a 7-d measurement period. All cows grazed full time in a rotational stocking system and received either no concentrate or 6 kg/d of a commercial cereal-grain mix. Representative herbage samples of each grazed paddock were taken and botanical composition of subsamples was manually determined. The average proportions of the plant species were 27.8% Lolium perenne, 6.1% Dactylis glomerata, 10.4% Trifolium repens, and 9.0% Taraxacum officinale. Other grass species were merged as "other grass" (38.2%) and other forb species as "other forbs" (8.5%). n-Alkanes, long-chain fatty acids, and long-chain alcohols (LCOH) were analyzed in the samples of plant species, concentrate, and feces from each cow. A linear discriminant analysis indicated that diet components were differentiated best with LCOH (96%) and worst with the combination of all marker groups together (12%). For each marker, the fecal marker recovery (FR) relative to dosed ytterbium was determined in 2 ways. Estimation of diet composition was performed with the software "EatWhat," and results were compared with botanical composition with the Aitchison distance. The results indicate that the diet composition of grazing dairy cows can be estimated using plant wax markers. Additionally, the calculation of FR led to mostly reliable results, yet this approach needs further validation. The most accurate estimation was achieved with the marker combination of n-alkanes and LCOH with a correction for FR. Less accurate estimations were achieved with long-chain fatty acids alone or in combination with n-alkanes. No difference relating to diet selection between the 2 cow strains was recorded, but supplemented cows apparently ingested higher proportions of T. repens than nonsupplemented cows. Awareness that supplementation influences selection behavior of grazing dairy cows may lead to adaptations in botanical composition of the pasture according to the demand of the animals. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Lessons from mouse chimaera experiments with a reiterated transgene marker: revised marker criteria and a review of chimaera markers.

PubMed

Keighren, Margaret A; Flockhart, Jean; Hodson, Benjamin A; Shen, Guan-Yi; Birtley, James R; Notarnicola-Harwood, Antonio; West, John D

2015-08-01

Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral.

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Development of Novel SSR Markers for Flax (Linum usitatissimum L.) Using Reduced-Representation Genome Sequencing

PubMed Central

Wu, Jianzhong; Zhao, Qian; Wu, Guangwen; Zhang, Shuquan; Jiang, Tingbo

2017-01-01

Flax (Linum usitatissimum L.) is a major fiber and oil yielding crop grown in northeastern China. Identification of flax molecular markers is a key step toward improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers, which are based on genomic structural variation, are considered the most valuable type of genetic marker for this purpose. In this study, we screened 1574 microsatellites from Linum usitatissimum L. obtained using reduced representation genome sequencing (RRGS) to systematically identify SSR markers. The resulting set of microsatellites consisted mainly of trinucleotide (56.10%) and dinucleotide (35.23%) repeats, with each motif consisting of 5–8 repeats. We then evaluated marker sensitivity and specificity based on samples of 48 flax isolates obtained from northeastern China. Using the new SSR panel, the results demonstrated that fiber flax and oilseed flax varieties clustered into two well separated groups. The novel SSR markers developed in this study show potential value for selection of varieties for use in flax breeding programs. PMID:28133461

Evaluation of the innate immune response of Angus heifers with genetic marker variation for intramuscular fat deposition following a lipopolysaccharide challenge

USDA-ARS?s Scientific Manuscript database

This study evaluated the effect of genetic selection for markers related to marbling deposition in Angus heifers on the immune response following a lipopolysaccharide (LPS) challenge. Fall-born heifers (n = 19; ~7 months of age, 274 +/- 24 kg) with genetic variation for marbling were utilized inclu...

Cognitive Impairment among the Aging Population in a Community in Southwest Nigeria

ERIC Educational Resources Information Center

Adebiyi, Akindele O.; Ogunniyi, Adesola; Adediran, Babatunde A.; Olakehinde, Olaide O.; Siwoku, Akeem A.

2016-01-01

Background: Vascular risk models can be quite informative in assisting the clinician to make a prediction of an individual's risk of cognitive impairment. Thus, a simple marker is a priority for low-capacity settings. This study examines the association of selected simple to deploy vascular markers with cognitive impairment in an elderly…

A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight

USDA-ARS?s Scientific Manuscript database

The use of genetic markers to aid in selection decisions to improve carcass and growth characteristics is of great interest to the beef industry. However, it is important to examine potential antagonistic interactions with fertility in the cows before widespread application of marker-assisted selec...

[Methods of hygromycin B phosphotransferase activity assay in transgenic plant].

PubMed

Zhuo, Qin; Yang, Xiaoguang

2004-07-01

Hygromycin B phosphotransferase (HPT) is a widely used selectable marker protein of transgenic plant. Detection of its activity is critical to studies on the development of various transgenic plants, silence of inserted gene, marker-free system development and safety assessment of transgenic food. In this paper, several methods for detecting the activity of this enzyme were reviewed.

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...

Validation and Implementation of Marker-Assisted Selection (MAS) for PVY Resistance (Ryadg gene) in a Tetraploid Potato Breeding Program

USDA-ARS?s Scientific Manuscript database

The gene Ryadg from S. tuberosum ssp. andigena provides extreme resistance to PVY. This gene has been mapped to chromosome XI and linked PCR-based DNA markers have been identified. Advanced tetraploid russeted potato clones developed by the U.S. Pacific Northwest Potato Breeding Program with Ryadg P...

Rindsel: an R package for phenotypic and molecular selection indices used in plant breeding.

PubMed

Perez-Elizalde, Sergío; Cerón-Rojas, Jesús J; Crossa, José; Fleury, Delphine; Alvarado, Gregorio

2014-01-01

Selection indices are estimates of the net genetic merit of the individual candidates for selection and are calculated based on phenotyping and molecular marker information collected on plants under selection in a breeding program. They reflect the breeding value of the plants and help breeders to choose the best ones for next generation. Rindsel is an R package that calculates phenotypic and molecular selection indices.

Hoxb2 and hoxb4 act together to specify ventral body wall formation.

PubMed

Manley, N R; Barrow, J R; Zhang, T; Capecchi, M R

2001-09-01

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contain insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlated with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those previously reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4(PolII), homozygous mutants died either in utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4(PolII) mutants. Hoxb4(loxp) mutants in which the neo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4(PolII) and the previously described Hoxb2(PolII) alleles that have body wall defects have been shown to disrupt the expression of both Hoxb2 and Hoxb4 in cell types that contribute to body wall formation. Our results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4. Copyright 2001 Academic Press.

Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum.

PubMed

Woods, J P; Heinecke, E L; Goldman, W E

1998-04-01

We developed an efficient electrotransformation system for the pathogenic fungus Histoplasma capsulatum and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant Escherichia coli genes, hph and lacZ. Linearized DNA and plasmids containing Histoplasma telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable URA5 marker (native Histoplasma gene or a heterologous Podospora anserina gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the E. coli hph gene flanked by Aspergillus nidulans promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in H. capsulatum, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing Histoplasma URA5 with E. coli lacZ, resulting in expression of functional beta-galactosidase in H. capsulatum. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of H. capsulatum, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.

Unlocking Diversity in Germplasm Collections via Genomic Selection: A Case Study Based on Quantitative Adult Plant Resistance to Stripe Rust in Spring Wheat.

PubMed

Muleta, Kebede T; Bulli, Peter; Zhang, Zhiwu; Chen, Xianming; Pumphrey, Michael

2017-11-01

Harnessing diversity from germplasm collections is more feasible today because of the development of lower-cost and higher-throughput genotyping methods. However, the cost of phenotyping is still generally high, so efficient methods of sampling and exploiting useful diversity are needed. Genomic selection (GS) has the potential to enhance the use of desirable genetic variation in germplasm collections through predicting the genomic estimated breeding values (GEBVs) for all traits that have been measured. Here, we evaluated the effects of various scenarios of population genetic properties and marker density on the accuracy of GEBVs in the context of applying GS for wheat ( L.) germplasm use. Empirical data for adult plant resistance to stripe rust ( f. sp. ) collected on 1163 spring wheat accessions and genotypic data based on the wheat 9K single nucleotide polymorphism (SNP) iSelect assay were used for various genomic prediction tests. Unsurprisingly, the results of the cross-validation tests demonstrated that prediction accuracy increased with an increase in training population size and marker density. It was evident that using all the available markers (5619) was unnecessary for capturing the trait variation in the germplasm collection, with no further gain in prediction accuracy beyond 1 SNP per 3.2 cM (∼1850 markers), which is close to the linkage disequilibrium decay rate in this population. Collectively, our results suggest that larger germplasm collections may be efficiently sampled via lower-density genotyping methods, whereas genetic relationships between the training and validation populations remain critical when exploiting GS to select from germplasm collections. Copyright © 2017 Crop Science Society of America.

Social-Cognitive Processes in Preschoolers' Selective Trust: Three Cultures Compared

ERIC Educational Resources Information Center

Lucas, Amanda J.; Lewis, Charlie; Pala, F. Cansu; Wong, Katie; Berridge, Damon

2013-01-01

Research on preschoolers' selective learning has mostly been conducted in English-speaking countries. We compared the performance of Turkish preschoolers (who are exposed to a language with evidential markers), Chinese preschoolers (known to be advanced in executive skills), and English preschoolers on an extended selective trust task (N = 144).…

IVF or IUI as first-line treatment in unexplained subfertility: the conundrum of treatment selection markers.

PubMed

Tjon-Kon-Fat, R I; Tajik, P; Zafarmand, M H; Bensdorp, A J; Bossuyt, P M M; Oosterhuis, G J E; van Golde, R; Repping, S; Lambers, M D A; Slappendel, E; Perquin, D; Pelinck, M J; Gianotten, J; Maas, J W M; Eijkemans, M J C; van der Veen, F; Mol, B W; van Wely, M

2017-05-01

Are there treatment selection markers that could aid in identifying couples, with unexplained or mild male subfertility, who would have better chances of a healthy child with IVF with single embryo transfer (IVF-SET) than with IUI with ovarian stimulation (IUI-OS)? We did not find any treatment selection markers that were associated with better chances of a healthy child with IVF-SET instead of IUI-OS in couples with unexplained or mild male subfertility. A recent trial, comparing IVF-SET to IUI-OS, found no evidence of a difference between live birth rates and multiple pregnancy rates. It was suggested that IUI-OS should remain the first-line treatment instead of IVF-SET in couples with unexplained or mild male subfertility and female age between 18 and 38 years. The question remains whether there are some couples that may have higher pregnancy chances if treated with IVF-SET instead of IUI. We performed our analyses on data from the INeS trial, where couples with unexplained or mild male subfertility and an unfavourable prognosis for natural conception were randomly allocated to IVF-SET, IVF in a modified natural cycle or IUI-OS. In view of the aim of this study, we only used data of the comparison between IVF-SET (201 couples) and IUI-OS (207 couples). We pre-defined the following baseline characteristics as potential treatment selection markers: female age, ethnicity, smoking status, type of subfertility (primary/secondary), duration of subfertility, BMI, pre-wash total motile count and Hunault prediction score. For each potential treatment selection marker, we explored the association with the chances of a healthy child after IVF-SET and IUI-OS and tested if there was an interaction with treatment. Given the exploratory nature of our analysis, we used a P-value of 0.1. None of the markers were associated with higher chances of a healthy child from IVF-SET compared to IUI-OS (P-value for interaction >0.10). Since this is the first large study that looked at potential treatment selection markers for IVF-SET compared to IUI-OS, we had no data on which to base a power calculation. The sample size was limited, making it difficult to detect any smaller associations. We could not identify couples with unexplained or mild male subfertility who would have had higher chances of a healthy child from immediate IVF-SET than from IUI-OS. As in the original trial IUI-OS had similar effectiveness and was less costly compared to IVF-SET, IUI-OS should remain the preferred first-line treatment in these couples. The study was supported by a grant from the Netherlands Organization for Health Research and Development, and a grant from the Netherlands' association of health care insurers. There are no conflicts of interest. The trial was registered at the Dutch trial registry (NTR939). © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.).

PubMed

Wang, Jiping; Li, Runzhi; Mao, Xinguo; Jing, Ruilian

2017-01-01

Calreticulin (CRT), an endoplasmic reticulum (ER)-localized Ca 2+ -binding/buffering protein, is highly conserved and extensively expressed in animal and plant cells. To understand the function of CRTs in wheat ( Triticum aestivum L.), particularly their roles in stress tolerance, we cloned the full-length genomic sequence of the TaCRT-D isoform from D genome of common hexaploid wheat, and characterized its function by transgenic Arabidopsis system. TaCRT-D exhibited different expression patterns in wheat seedling under different abiotic stresses. Transgenic Arabidopsis plants overexpressing ORF of TaCRT-D displayed more tolerance to drought, cold, salt, mannitol, and other abiotic stresses at both seed germination and seedling stages, compared with the wild-type controls. Furthermore, DNA polymorphism analysis and gene mapping were employed to develop the functional markers of this gene for marker-assistant selection in wheat breeding program. One SNP, S440 (T→C) was detected at the TaCRT-D locus by genotyping a wheat recombinant inbred line (RIL) population (114 lines) developed from Opata 85 × W7984. The TaCRT-D was then fine mapped between markers Xgwm645 and Xgwm664 on chromosome 3DL, corresponding to genetic distances of 3.5 and 4.4 cM, respectively, using the RIL population and Chinese Spring nulli-tetrasomic lines. Finally, the genome-specific and allele-specific markers were developed for the TaCRT-D gene. These findings indicate that TaCRT-D function importantly in plant stress responses, providing a gene target for genetic engineering to increase plant stress tolerance and the functional markers of TaCRT-D for marker-assistant selection in wheat breeding.

Functional Analysis and Marker Development of TaCRT-D Gene in Common Wheat (Triticum aestivum L.)

PubMed Central

Wang, Jiping; Li, Runzhi; Mao, Xinguo; Jing, Ruilian

2017-01-01

Calreticulin (CRT), an endoplasmic reticulum (ER)-localized Ca2+-binding/buffering protein, is highly conserved and extensively expressed in animal and plant cells. To understand the function of CRTs in wheat (Triticum aestivum L.), particularly their roles in stress tolerance, we cloned the full-length genomic sequence of the TaCRT-D isoform from D genome of common hexaploid wheat, and characterized its function by transgenic Arabidopsis system. TaCRT-D exhibited different expression patterns in wheat seedling under different abiotic stresses. Transgenic Arabidopsis plants overexpressing ORF of TaCRT-D displayed more tolerance to drought, cold, salt, mannitol, and other abiotic stresses at both seed germination and seedling stages, compared with the wild-type controls. Furthermore, DNA polymorphism analysis and gene mapping were employed to develop the functional markers of this gene for marker-assistant selection in wheat breeding program. One SNP, S440 (T→C) was detected at the TaCRT-D locus by genotyping a wheat recombinant inbred line (RIL) population (114 lines) developed from Opata 85 × W7984. The TaCRT-D was then fine mapped between markers Xgwm645 and Xgwm664 on chromosome 3DL, corresponding to genetic distances of 3.5 and 4.4 cM, respectively, using the RIL population and Chinese Spring nulli-tetrasomic lines. Finally, the genome-specific and allele-specific markers were developed for the TaCRT-D gene. These findings indicate that TaCRT-D function importantly in plant stress responses, providing a gene target for genetic engineering to increase plant stress tolerance and the functional markers of TaCRT-D for marker-assistant selection in wheat breeding. PMID:28955354

Genome wide association analysis of cold tolerance at germination in temperate japonica rice (Oryza sativa L.) varieties.

PubMed

Sales, Ester; Viruel, Juan; Domingo, Concha; Marqués, Luis

2017-01-01

A pool of 200 traditional, landraces and modern elite and old cultivars of rice, mainly japonica varieties adapted to temperate regions, have been used to perform a genome wide association study to detect chromosome regions associated to low temperature germination (LTG) regulation using a panel of 1672 SNP markers. Phenotyping was performed by determining growth rates when seeds were germinated at 25° and 15°C in order to separate the germination vigorousness from cold tolerance effects. As expected, the ability to produce viable seedlings varied widely among rice cultivars and also depended greatly on temperature. Furthermore, we observed a differential response during seed germination and in coleoptile elongation. Faster development at 15°C was observed in seeds from varieties traditionally used as cold tolerant parents by breeders, along with other potentially useful cultivars, mainly of Italian origin. When phenotypic data were combined with the panel of SNPs for japonica rice cultivars, significant associations were detected for 31 markers: 7 were related to growth rate at 25°C and 24 to growth rates at 15°. Among the latter, some chromosome regions were associated to LTG while others were related to coleoptile elongation. Individual effects of the associated markers were low, but by combining favourable alleles in a linear regression model we estimated that 27 loci significantly explained the observed phenotypic variation. From these, a core panel of 13 markers was selected and, furthermore, two wide regions of chromosomes 3 and 6 were consistently associated to rice LTG. Varieties with higher numbers of favourable alleles for the panels of associated markers significantly correlated with increased phenotypic values at both temperatures, thus corroborating the utility of the tagged markers for marker assisted selection (MAS) when breeding japonica rice for LTG.

Whole-genome single-nucleotide polymorphism (SNP) marker discovery and association analysis with the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in Larimichthys crocea

PubMed Central

Xiao, Shijun; Wang, Panpan; Dong, Linsong; Zhang, Yaguang; Han, Zhaofang; Wang, Qiurong

2016-01-01

Whole-genome single-nucleotide polymorphism (SNP) markers are valuable genetic resources for the association and conservation studies. Genome-wide SNP development in many teleost species are still challenging because of the genome complexity and the cost of re-sequencing. Genotyping-By-Sequencing (GBS) provided an efficient reduced representative method to squeeze cost for SNP detection; however, most of recent GBS applications were reported on plant organisms. In this work, we used an EcoRI-NlaIII based GBS protocol to teleost large yellow croaker, an important commercial fish in China and East-Asia, and reported the first whole-genome SNP development for the species. 69,845 high quality SNP markers that evenly distributed along genome were detected in at least 80% of 500 individuals. Nearly 95% randomly selected genotypes were successfully validated by Sequenom MassARRAY assay. The association studies with the muscle eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content discovered 39 significant SNP markers, contributing as high up to ∼63% genetic variance that explained by all markers. Functional genes that involved in fat digestion and absorption pathway were identified, such as APOB, CRAT and OSBPL10. Notably, PPT2 Gene, previously identified in the association study of the plasma n-3 and n-6 polyunsaturated fatty acid level in human, was re-discovered in large yellow croaker. Our study verified that EcoRI-NlaIII based GBS could produce quality SNP markers in a cost-efficient manner in teleost genome. The developed SNP markers and the EPA and DHA associated SNP loci provided invaluable resources for the population structure, conservation genetics and genomic selection of large yellow croaker and other fish organisms. PMID:28028455

Genome wide association analysis of cold tolerance at germination in temperate japonica rice (Oryza sativa L.) varieties

PubMed Central

Viruel, Juan; Domingo, Concha; Marqués, Luis

2017-01-01

A pool of 200 traditional, landraces and modern elite and old cultivars of rice, mainly japonica varieties adapted to temperate regions, have been used to perform a genome wide association study to detect chromosome regions associated to low temperature germination (LTG) regulation using a panel of 1672 SNP markers. Phenotyping was performed by determining growth rates when seeds were germinated at 25° and 15°C in order to separate the germination vigorousness from cold tolerance effects. As expected, the ability to produce viable seedlings varied widely among rice cultivars and also depended greatly on temperature. Furthermore, we observed a differential response during seed germination and in coleoptile elongation. Faster development at 15°C was observed in seeds from varieties traditionally used as cold tolerant parents by breeders, along with other potentially useful cultivars, mainly of Italian origin. When phenotypic data were combined with the panel of SNPs for japonica rice cultivars, significant associations were detected for 31 markers: 7 were related to growth rate at 25°C and 24 to growth rates at 15°. Among the latter, some chromosome regions were associated to LTG while others were related to coleoptile elongation. Individual effects of the associated markers were low, but by combining favourable alleles in a linear regression model we estimated that 27 loci significantly explained the observed phenotypic variation. From these, a core panel of 13 markers was selected and, furthermore, two wide regions of chromosomes 3 and 6 were consistently associated to rice LTG. Varieties with higher numbers of favourable alleles for the panels of associated markers significantly correlated with increased phenotypic values at both temperatures, thus corroborating the utility of the tagged markers for marker assisted selection (MAS) when breeding japonica rice for LTG. PMID:28817683

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.).

PubMed

Zhao, Yongli; Williams, Roxanne; Prakash, C S; He, Guohao

2012-12-15

Date palm (Phoenix dactylifera L.) is an important tree in the Middle East and North Africa due to the nutritional value of its fruit. Molecular Breeding would accelerate genetic improvement of fruit tree through marker assisted selection. However, the lack of molecular markers in date palm restricts the application of molecular breeding. In this study, we analyzed 28,889 EST sequences from the date palm genome database to identify simple-sequence repeats (SSRs) and to develop gene-based markers, i.e. expressed sequence tag-SSRs (EST-SSRs). We identified 4,609 ESTs as containing SSRs, among which, trinucleotide motifs (69.7%) were the most common, followed by tetranucleotide (10.4%) and dinucleotide motifs (9.6%). The motif AG (85.7%) was most abundant in dinucleotides, while motifs AGG (26.8%), AAG (19.3%), and AGC (16.1%) were most common among trinucleotides. A total of 4,967 primer pairs were designed for EST-SSR markers from the computational data. In a follow up laboratory study, we tested a sample of 20 random selected primer pairs for amplification and polymorphism detection using genomic DNA from date palm cultivars. Nearly one-third of these primer pairs detected DNA polymorphism to differentiate the twelve date palm cultivars used. Functional categorization of EST sequences containing SSRs revealed that 3,108 (67.4%) of such ESTs had homology with known proteins. Date palm EST sequences exhibits a good resource for developing gene-based markers. These genic markers identified in our study may provide a valuable genetic and genomic tool for further genetic research and varietal development in date palm, such as diversity study, QTL mapping, and molecular breeding.

PIPEMicroDB: microsatellite database and primer generation tool for pigeonpea genome.

PubMed

Sarika; Arora, Vasu; Iquebal, M A; Rai, Anil; Kumar, Dinesh

2013-01-01

Molecular markers play a significant role for crop improvement in desirable characteristics, such as high yield, resistance to disease and others that will benefit the crop in long term. Pigeonpea (Cajanus cajan L.) is the recently sequenced legume by global consortium led by ICRISAT (Hyderabad, India) and been analysed for gene prediction, synteny maps, markers, etc. We present PIgeonPEa Microsatellite DataBase (PIPEMicroDB) with an automated primer designing tool for pigeonpea genome, based on chromosome wise as well as location wise search of primers. Total of 123 387 Short Tandem Repeats (STRs) were extracted from pigeonpea genome, available in public domain using MIcroSAtellite tool (MISA). The database is an online relational database based on 'three-tier architecture' that catalogues information of microsatellites in MySQL and user-friendly interface is developed using PHP. Search for STRs may be customized by limiting their location on chromosome as well as number of markers in that range. This is a novel approach and is not been implemented in any of the existing marker database. This database has been further appended with Primer3 for primer designing of selected markers with left and right flankings of size up to 500 bp. This will enable researchers to select markers of choice at desired interval over the chromosome. Furthermore, one can use individual STRs of a targeted region over chromosome to narrow down location of gene of interest or linked Quantitative Trait Loci (QTLs). Although it is an in silico approach, markers' search based on characteristics and location of STRs is expected to be beneficial for researchers. Database URL: http://cabindb.iasri.res.in/pigeonpea/

Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding.

PubMed

Ellur, Ranjith K; Khanna, Apurva; Yadav, Ashutosh; Pathania, Sandeep; Rajashekara, H; Singh, Vikas K; Gopala Krishnan, S; Bhowmick, Prolay K; Nagarajan, M; Vinod, K K; Prakash, G; Mondal, Kalyan K; Singh, Nagendra K; Vinod Prabhu, K; Singh, Ashok K

2016-01-01

Marker assisted backcross breeding was employed to incorporate the blast resistance genes, Pi2 and Pi54 and bacterial blight (BB) resistance genes xa13 and Xa21 into the genetic background of Pusa Basmati 1121 (PB1121) and Pusa Basmati 6. Foreground selection for target gene(s) was followed by arduous phenotypic and background selection which fast-tracked the recovery of recurrent parent genome (RPG) to an extent of 95.8% in one of the near-isogenic lines (NILs) namely, Pusa 1728-23-33-31-56, which also showed high degree of resemblance to recurrent parent, PB6 in phenotype. The phenotypic selection prior to background selection provided an additional opportunity for identifying the novel recombinants viz., Pusa 1884-9-12-14 and Pusa 1884-3-9-175, superior to parental lines in terms of early maturity, higher yield and improved quality parameters. There was no significant difference between the RPG recovery estimated based on SSR or SNP markers, however, the panel of SNPs markers was considered as the better choice for background selection as it provided better genome coverage and included SNPs in the genic regions. Multi-location evaluation of NILs depicted their stable and high mean performance in comparison to the respective recurrent parents. The Pi2+Pi54 carrying NILs were effective in combating a pan-India panel of Magnaporthe oryzae isolates with high level of field resistance in northern, eastern and southern parts of India. Alongside, the PB1121-NILs and PB6-NILs carrying BB resistance genes xa13+Xa21 were resistant against Xanthomonas oryzae pv. oryzae races of north-western, southern and eastern parts of the country. Three of NILs developed in this study, have been promoted to final stage of testing during the ​Kharif 2015 in the Indian National Basmati Trial. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Exploration of the Hypothalamic-Pituitary-Adrenal Axis to Improve Animal Welfare by Means of Genetic Selection: Lessons from the South African Merino

PubMed Central

Hough, Denise; Swart, Pieter; Cloete, Schalk

2013-01-01

Simple Summary Breeding sheep that are robust and easily managed may be beneficial for both animal welfare and production. Sheep that are more readily able to adapt to stressful situations and a wide variety of environmental conditions are likely to have more resources available for a higher expression of their production potential. This review explores the utilization of one of the stress response pathways, namely the hypothalamic-pituitary-adrenal axis, to locate potential sites where genetic markers might be identified that contribute to sheep robustness. A South African Merino breeding programme is used to demonstrate the potential benefits of this approach. Abstract It is a difficult task to improve animal production by means of genetic selection, if the environment does not allow full expression of the animal’s genetic potential. This concept may well be the future for animal welfare, because it highlights the need to incorporate traits related to production and robustness, simultaneously, to reach sustainable breeding goals. This review explores the identification of potential genetic markers for robustness within the hypothalamic-pituitary-adrenal axis (HPAA), since this axis plays a vital role in the stress response. If genetic selection for superior HPAA responses to stress is possible, then it ought to be possible to breed robust and easily managed genotypes that might be able to adapt to a wide range of environmental conditions whilst expressing a high production potential. This approach is explored in this review by means of lessons learnt from research on Merino sheep, which were divergently selected for their multiple rearing ability. These two selection lines have shown marked differences in reproduction, production and welfare, which makes this breeding programme ideal to investigate potential genetic markers of robustness. The HPAA function is explored in detail to elucidate where such genetic markers are likely to be found. PMID:26487412

Single Marker and Haplotype-Based Association Analysis of Semolina and Pasta Colour in Elite Durum Wheat Breeding Lines Using a High-Density Consensus Map

PubMed Central

Haile, Jemanesh K.; Cory, Aron T.; Clarke, Fran R.; Clarke, John M.; Knox, Ron E.; Pozniak, Curtis J.

2017-01-01

Association mapping is usually performed by testing the correlation between a single marker and phenotypes. However, because patterns of variation within genomes are inherited as blocks, clustering markers into haplotypes for genome-wide scans could be a worthwhile approach to improve statistical power to detect associations. The availability of high-density molecular data allows the possibility to assess the potential of both approaches to identify marker-trait associations in durum wheat. In the present study, we used single marker- and haplotype-based approaches to identify loci associated with semolina and pasta colour in durum wheat, the main objective being to evaluate the potential benefits of haplotype-based analysis for identifying quantitative trait loci. One hundred sixty-nine durum lines were genotyped using the Illumina 90K Infinium iSelect assay, and 12,234 polymorphic single nucleotide polymorphism (SNP) markers were generated and used to assess the population structure and the linkage disequilibrium (LD) patterns. A total of 8,581 SNPs previously localized to a high-density consensus map were clustered into 406 haplotype blocks based on the average LD distance of 5.3 cM. Combining multiple SNPs into haplotype blocks increased the average polymorphism information content (PIC) from 0.27 per SNP to 0.50 per haplotype. The haplotype-based analysis identified 12 loci associated with grain pigment colour traits, including the five loci identified by the single marker-based analysis. Furthermore, the haplotype-based analysis resulted in an increase of the phenotypic variance explained (50.4% on average) and the allelic effect (33.7% on average) when compared to single marker analysis. The presence of multiple allelic combinations within each haplotype locus offers potential for screening the most favorable haplotype series and may facilitate marker-assisted selection of grain pigment colour in durum wheat. These results suggest a benefit of haplotype-based analysis over single marker analysis to detect loci associated with colour traits in durum wheat. PMID:28135299

Mapping Quantitative Trait Loci in Crosses between Outbred Lines Using Least Squares

PubMed Central

Haley, C. S.; Knott, S. A.; Elsen, J. M.

1994-01-01

The use of genetic maps based upon molecular markers has allowed the dissection of some of the factors underlying quantitative variation in crosses between inbred lines. For many species crossing inbred lines is not a practical proposition, although crosses between genetically very different outbred lines are possible. Here we develop a least squares method for the analysis of crosses between outbred lines which simultaneously uses information from multiple linked markers. The method is suitable for crosses where the lines may be segregating at marker loci but can be assumed to be fixed for alternative alleles at the major quantitative trait loci (QTLs) affecting the traits under analysis (e.g., crosses between divergent selection lines or breeds with different selection histories). The simultaneous use of multiple markers from a linkage group increases the sensitivity of the test statistic, and thus the power for the detection of QTLs, compared to the use of single markers or markers flanking an interval. The gain is greater for more closely spaced markers and for markers of lower information content. Use of multiple markers can also remove the bias in the estimated position and effect of a QTL which may result when different markers in a linkage group vary in their heterozygosity in the F(1) (and thus in their information content) and are considered only singly or a pair at a time. The method is relatively simple to apply so that more complex models can be fitted than is currently possible by maximum likelihood. Thus fixed effects and effects of background genotype can be fitted simultaneously with the exploration of a single linkage group which will increase the power to detect QTLs by reducing the residual variance. More complex models with several QTLs in the same linkage group and two-locus interactions between QTLs can similarly be examined. Thus least squares provides a powerful tool to extend the range of crosses from which QTLs can be dissected whilst at the same time allowing flexible and realistic models to be explored. PMID:8005424

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don

PubMed Central

2012-01-01

Background High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. Results We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. Conclusions Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity. PMID:22424262

A new leaf rust resistance gene Lr79 mapped in chromosome 3BL from the durum wheat landrace Aus26582.

PubMed

Qureshi, Naeela; Bariana, Harbans; Kumran, Vikas Venu; Muruga, Sivasamy; Forrest, Kerrie L; Hayden, Mathew J; Bansal, Urmil

2018-05-01

A new leaf rust resistance gene Lr79 has been mapped in the long arm of chromosome 3B and a linked marker was identified for marker-assisted selection. Aus26582, a durum wheat landrace from the A. E. Watkins Collection, showed seedling resistance against durum-specific and common wheat-specific Puccinia triticina (Pt) pathotypes. Genetic analysis using a recombinant inbred line (RIL) population developed from a cross between Aus26582 and the susceptible parent Bansi with Australian Pt pathotype showed digenic inheritance and the underlying loci were temporarily named LrAW2 and LrAW3. LrAW2 was located in chromosome 6BS and this study focused on characterisation of LrAW3 using RILs lacking LrAW2. LrAW3 was incorporated into the DArTseq map of Aus26582/Bansi and was located in chromosome 3BL. Markers linked with LrAW3 were developed from the chromosome survey sequence contig 3B_10474240 in which closely-linked DArTseq markers 1128708 and 3948563 were located. Although bulk segregant analysis (BSA) with the 90 K Infinium array identified 51 SNPs associated with LrAW3, only one SNP-derived KASP marker mapped close to the locus. Deletion bin mapping of LrAW3-linked markers located LrAW3 between bins 3BL11-0.85-0.90 and 3BL7-0.63. Since no other all stage leaf rust resistance gene is located in chromosome 3BL, LrAW3 represented a new locus and was designated Lr79. Marker sun786 mapped 1.8 cM distal to Lr79 and Aus26582 was null for this locus. However, the marker can be reliably scored as it also amplifies a monomorphic fragment that serves as an internal control to differentiate the null status of Aus26582 from reaction failure. This marker was validated among a set of durum and common wheat cultivars and was shown to be useful for marker-assisted selection of Lr79 at both ploidy levels.

Evaluating surrogate endpoints, prognostic markers, and predictive markers: Some simple themes.

PubMed

Baker, Stuart G; Kramer, Barnett S

2015-08-01

A surrogate endpoint is an endpoint observed earlier than the true endpoint (a health outcome) that is used to draw conclusions about the effect of treatment on the unobserved true endpoint. A prognostic marker is a marker for predicting the risk of an event given a control treatment; it informs treatment decisions when there is information on anticipated benefits and harms of a new treatment applied to persons at high risk. A predictive marker is a marker for predicting the effect of treatment on outcome in a subgroup of patients or study participants; it provides more rigorous information for treatment selection than a prognostic marker when it is based on estimated treatment effects in a randomized trial. We organized our discussion around a different theme for each topic. "Fundamentally an extrapolation" refers to the non-statistical considerations and assumptions needed when using surrogate endpoints to evaluate a new treatment. "Decision analysis to the rescue" refers to use the use of decision analysis to evaluate an additional prognostic marker because it is not possible to choose between purely statistical measures of marker performance. "The appeal of simplicity" refers to a straightforward and efficient use of a single randomized trial to evaluate overall treatment effect and treatment effect within subgroups using predictive markers. The simple themes provide a general guideline for evaluation of surrogate endpoints, prognostic markers, and predictive markers. © The Author(s) 2014.

Association Genetics of Wood Physical Traits in the Conifer White Spruce and Relationships With Gene Expression

PubMed Central

Beaulieu, Jean; Doerksen, Trevor; Boyle, Brian; Clément, Sébastien; Deslauriers, Marie; Beauseigle, Stéphanie; Blais, Sylvie; Poulin, Pier-Luc; Lenz, Patrick; Caron, Sébastien; Rigault, Philippe; Bicho, Paul; Bousquet, Jean; MacKay, John

2011-01-01

Marker-assisted selection holds promise for highly influencing tree breeding, especially for wood traits, by considerably reducing breeding cycles and increasing selection accuracy. In this study, we used a candidate gene approach to test for associations between 944 single-nucleotide polymorphism markers from 549 candidate genes and 25 wood quality traits in white spruce. A mixed-linear model approach, including a weak but nonsignificant population structure, was implemented for each marker–trait combination. Relatedness among individuals was controlled using a kinship matrix estimated either from the known half-sib structure or from the markers. Both additive and dominance effect models were tested. Between 8 and 21 single-nucleotide polymorphisms (SNPs) were found to be significantly associated (P ≤ 0.01) with each of earlywood, latewood, or total wood traits. After controlling for multiple testing (Q ≤ 0.10), 13 SNPs were still significant across as many genes belonging to different families, each accounting for between 3 and 5% of the phenotypic variance in 10 wood characters. Transcript accumulation was determined for genes containing SNPs associated with these traits. Significantly different transcript levels (P ≤ 0.05) were found among the SNP genotypes of a 1-aminocyclopropane-1-carboxylate oxidase, a β-tonoplast intrinsic protein, and a long-chain acyl-CoA synthetase 9. These results should contribute toward the development of efficient marker-assisted selection in an economically important tree species. PMID:21385726

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.).

PubMed

Allen, Alexandra M; Barker, Gary L A; Berry, Simon T; Coghill, Jane A; Gwilliam, Rhian; Kirby, Susan; Robinson, Phil; Brenchley, Rachel C; D'Amore, Rosalinda; McKenzie, Neil; Waite, Darren; Hall, Anthony; Bevan, Michael; Hall, Neil; Edwards, Keith J

2011-12-01

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker-assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single-nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single-nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next-generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single-nucleotide polymorphisms in hexaploid bread wheat using competitive allele-specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross-section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single-nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Penetration enhancing effects of selected natural oils utilized in topical dosage forms.

PubMed

Viljoen, Joe M; Cowley, Amé; du Preez, Jan; Gerber, Minja; du Plessis, Jeanetta

2015-01-01

Various natural products, including oils, have been utilized as penetration enhancers due to their "safety profiles". These oils contain fatty acids promoting skin permeability through lipid fluidization within the stratum corneum; and might therefore be able to effectively enhance transdermal drug delivery. We investigated possible penetration enhancing properties of selected oils, utilizing flurbiprofen as marker compound in emulgel formulations. The formulations were compared to a liquid paraffin emulgel and a hydrogel to establish any significant penetration enhancing effects. Gas chromatographic analysis of the natural oils was performed at ambient temperature to determine the fatty acid composition in each selected natural oils. Franz cell diffusion studies and tape stripping methods were employed to study delivery of the marker into, and through the skin. The following rank order for the emulgel flux-values was obtained: Hydrogel > olive oil > liquid paraffin > coconut oil > grape seed oil > Avocado oil ≥ Crocodile oil > Emu oil. Results suggested that oils containing predominantly mono-unsaturated oleic acid, on average increased the flux of the marker to a larger extent than oils containing an almost even mixture of both mono- and poly-unsaturated fatty acids. Oils comprising saturated fatty acids (SFAs) with alkyl chains between C12 and C14, increased the marker flux to a higher extent than oils containing C16-C18 SFAs. Effects observed for branched fatty acids, however, did not vary significantly from effects for unbranched fatty acids with the same carbon chain length. Natural oils possess penetration enhancing effects.

Short Communication: Genetic linkage map of Cucurbita maxima with molecular and morphological markers.

PubMed

Ge, Y; Li, X; Yang, X X; Cui, C S; Qu, S P

2015-05-22

Cucurbita maxima is one of the most widely cultivated vegetables in China and exhibits distinct morphological characteristics. In this study, genetic linkage analysis with 57 simple-sequence repeats, 21 amplified fragment length polymorphisms, 3 random-amplified polymorphic DNA, and one morphological marker revealed 20 genetic linkage groups of C. maxima covering a genetic distance of 991.5 cM with an average of 12.1 cM between adjacent markers. Genetic linkage analysis identified the simple-sequence repeat marker 'PU078072' 5.9 cM away from the locus 'Rc', which controls rind color. The genetic map in the present study will be useful for better mapping, tagging, and cloning of quantitative trait loci/gene(s) affecting economically important traits and for breeding new varieties of C. maxima through marker-assisted selection.

Capturing pair-wise epistatic effects associated with three agronomic traits in barley.

PubMed

Xu, Yi; Wu, Yajun; Wu, Jixiang

2018-04-01

Genetic association mapping has been widely applied to determine genetic markers favorably associated with a trait of interest and provide information for marker-assisted selection. Many association mapping studies commonly focus on main effects due to intolerable computing intensity. This study aims to select several sets of DNA markers with potential epistasis to maximize genetic variations of some key agronomic traits in barley. By doing so, we integrated a MDR (multifactor dimensionality reduction) method with a forward variable selection approach. This integrated approach was used to determine single nucleotide polymorphism pairs with epistasis effects associated with three agronomic traits: heading date, plant height, and grain yield in barley from the barley Coordinated Agricultural Project. Our results showed that four, seven, and five SNP pairs accounted for 51.06, 45.66 and 40.42% for heading date, plant height, and grain yield, respectively with epistasis being considered, while corresponding contributions to these three traits were 45.32, 31.39, 31.31%, respectively without epistasis being included. The results suggested that epistasis model was more effective than non-epistasis model in this study and can be more preferred for other applications.

Prospective evaluation of 64 serum autoantibodies as biomarkers for early detection of colorectal cancer in a true screening setting.

PubMed

Chen, Hongda; Werner, Simone; Butt, Julia; Zörnig, Inka; Knebel, Phillip; Michel, Angelika; Eichmüller, Stefan B; Jäger, Dirk; Waterboer, Tim; Pawlita, Michael; Brenner, Hermann

2016-03-29

Novel blood-based screening tests are strongly desirable for early detection of colorectal cancer (CRC). We aimed to identify and evaluate autoantibodies against tumor-associated antigens as biomarkers for early detection of CRC. 380 clinically identified CRC patients and samples of participants with selected findings from a cohort of screening colonoscopy participants in 2005-2013 (N=6826) were included in this analysis. Sixty-four serum autoantibody markers were measured by multiplex bead-based serological assays. A two-step approach with selection of biomarkers in a training set, and validation of findings in a validation set, the latter exclusively including participants from the screening setting, was applied. Anti-MAGEA4 exhibited the highest sensitivity for detecting early stage CRC and advanced adenoma. Multi-marker combinations substantially increased sensitivity at the price of a moderate loss of specificity. Anti-TP53, anti-IMPDH2, anti-MDM2 and anti-MAGEA4 were consistently included in the best-performing 4-, 5-, and 6-marker combinations. This four-marker panel yielded a sensitivity of 26% (95% CI, 13-45%) for early stage CRC at a specificity of 90% (95% CI, 83-94%) in the validation set. Notably, it also detected 20% (95% CI, 13-29%) of advanced adenomas. Taken together, the identified biomarkers could contribute to the development of a useful multi-marker blood-based test for CRC early detection.

High-Throughput Development of SSR Markers from Pea (Pisum sativum L.) Based on Next Generation Sequencing of a Purified Chinese Commercial Variety

PubMed Central

Zhang, Xiaoyan; Hu, Jinguo; Bao, Shiying; Hao, Junjie; Li, Ling; He, Yuhua; Jiang, Junye; Wang, Fang; Tian, Shufang; Zong, Xuxiao

2015-01-01

Pea (Pisum sativum L.) is an important food legume globally, and is the plant species that J.G. Mendel used to lay the foundation of modern genetics. However, genomics resources of pea are limited comparing to other crop species. Application of marker assisted selection (MAS) in pea breeding has lagged behind many other crops. Development of a large number of novel and reliable SSR (simple sequence repeat) or microsatellite markers will help both basic and applied genomics research of this crop. The Illumina HiSeq 2500 System was used to uncover 8,899 putative SSR containing sequences, and 3,275 non-redundant primers were designed to amplify these SSRs. Among the 1,644 SSRs that were randomly selected for primer validation, 841 yielded reliable amplifications of detectable polymorphisms among 24 genotypes of cultivated pea (Pisum sativum L.) and wild relatives (P. fulvum Sm.) originated from diverse geographical locations. The dataset indicated that the allele number per locus ranged from 2 to 10, and that the polymorphism information content (PIC) ranged from 0.08 to 0.82 with an average of 0.38. These 1,644 novel SSR markers were also tested for polymorphism between genotypes G0003973 and G0005527. Finally, 33 polymorphic SSR markers were anchored on the genetic linkage map of G0003973 × G0005527 F2 population. PMID:26440522

Dynamics of shoot vs. root C assessed by natural 13C abundance of their biomarkers

NASA Astrophysics Data System (ADS)

Mendez-Millan, Mercedes; Dignac, Marie-France; Rumpel, Cornelia; Rasse, Daniel P.; Derenne, Sylvie

2010-05-01

Cutins and suberins are biopolyesters that have been suggested to significantly contribute to the stable pool of soil organic matter (SOM). They might be used as tracers for the above- or belowground origin of plant material. The aim of this study was to evaluate the dynamics of shoot and root-derived biomarkers in soils using a wheat/maize (C3/C4) chronosequence. Our results suggest that α,?-alkanedioic acids can be considered as root specific markers and mid-chain hydroxy acids as shoot specific markers of wheat and maize in this agricultural soil. The changes of the 13C isotopic signatures of these markers with years of maize cropping after wheat evidenced their contrasted behaviour in soil. After 12 years of maize cropping, shoot markers present in soils probably originated from old C3 vegetation suggesting that new maize cutin added to soils was mostly degraded within a year. The reasons for long-term stabilisation of shoot biomarkers remain unclear. By contrast, maize root markers were highly incorporated into SOM during the first six years of maize crop, which suggested a selective preservation of root biomass when compared to shoots, possibly due to physical protection. The contrasting distribution of the plant-specific monomers in plants and soils might be explained by different chemical mechanisms leading to selective degradation or stabilization of some biomarkers.

Selected oxidative stress markers in a South American crocodilian species.

PubMed

Furtado-Filho, Orlando V; Polcheira, Cássia; Machado, Daniel P; Mourão, Guilherme; Hermes-Lima, Marcelo

2007-01-01

Crocodilians and other diving vertebrates experience hypoperfusion and hypoxia of several internal organs during long dives. At the end of a dive, reperfusion of aerated blood may cause a physiologically relevant oxidative stress. In this study, we analyzed selected markers of oxidative stress in eight organs of normoxic Paraguayan caiman (Caiman yacare) captured in the Brazilian Pantanal wetlands during the winter of 2001 (six mature-adult males and eight young-adult males; AD-1 and YA-1 groups, respectively), and during the summer of 2002 (six young-adult males (YA-2 group), ten hatchlings and five embryos). Lipid peroxidation products determined by three different assays were generally highest in brain, liver and kidney (in comparison with all other organs), and lowest in white muscles from the tail and hind legs. Liver and kidney showed the highest levels of carbonyl protein, while brain showed low levels. Intermediate levels of oxidative stress markers were mostly found in the heart ventricles and lung. Differences in oxidative stress markers between AD-1 and YA-1 were organ-specific, showing no age-related correlation. However, most oxidative stress markers in YA-2 organs were either higher than (by 1.4- to 3.7-fold) or not significantly different from respective values in hatchlings organs. This pattern (hatchlings versus young-adults) was confirmed using correlation analysis of individual caiman size versus levels of oxidative damage markers in four organs. The higher level of oxidative stress markers in young-adults possibly relates to the fast growth rate (and thus, increased oxidative metabolic rate) of C. yacare in the first years of life. Differences in oxidative stress markers between YA-1 and YA-2 were also observed and were ascribed to seasonal changes in free radical metabolism. These results in normoxic C. yacare represent the first step towards understanding the age-related physiological oxidative stress of a diving reptile from a seasonally changing wetland environment.

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

Optimal tumor sampling for immunostaining of biomarkers in breast carcinoma

PubMed Central

2011-01-01

Introduction Biomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidence-based standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau. Methods Two collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set. Results The optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement. Conclusions The optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis. PMID:21592345

Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection.

PubMed

Wang, Caihong; Wu, Caisheng; Zhang, Jinlan; Jin, Ying

2015-04-15

Prenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites. This study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWH-derived prenylflavonoid monomers in rats. The study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers. Major plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma. The pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for ∼99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)(0-96 h) values for total icaritin (24.1 vs. 32.0 mg/L h). Icaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates. Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.

A second generation genetic linkage map of Japanese flounder (Paralichthys olivaceus)

PubMed Central

2010-01-01

Background Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine species in Northeast Asia. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. Commercial production of Japanese flounder could be increased by developing disease-resistant fish and improving commercially important traits. Previous maps have been constructed with AFLP markers and a limited number of microsatellite markers. In this study, improved genetic linkage maps are presented. In contrast with previous studies, these maps were built mainly with a large number of codominant markers so they can potentially be used to analyze different families and populations. Results Sex-specific genetic linkage maps were constructed for the Japanese flounder including a total of 1,375 markers [1,268 microsatellites, 105 single nucleotide polymorphisms (SNPs) and two genes]; 1,167 markers are linked to the male map and 1,067 markers are linked to the female map. The lengths of the male and female maps are 1,147.7 cM and 833.8 cM, respectively. Based on estimations of map lengths, the female and male maps covered 79 and 82% of the genome, respectively. Recombination ratio in the new maps revealed F:M of 1:0.7. All linkage groups in the maps presented large differences in the location of sex-specific recombination hot-spots. Conclusions The improved genetic linkage maps are very useful for QTL analyses and marker-assisted selection (MAS) breeding programs for economically important traits in Japanese flounder. In addition, SNP flanking sequences were blasted against Tetraodon nigroviridis (puffer fish) and Danio rerio (zebrafish), and synteny analysis has been carried out. The ability to detect synteny among species or genera based on homology analysis of SNP flanking sequences may provide opportunities to complement initial QTL experiments with candidate gene approaches from homologous chromosomal locations identified in related model organisms. PMID:20937088

The effects of genetic markers on BTA6 significant for steer feed intake and gain traits on carcass and meat traits

USDA-ARS?s Scientific Manuscript database

Genetic selection for animals that require less feed while still achieving acceptable levels of production could result in substantial cost savings for cattle producers. We have identified DNA markers on bovine chromosome 6 located between 38.16 - 38.38 Mb that were significant for both feed intake ...

Discourse Markers in Composition Writings: The Case of Iranian Learners of English as a Foreign Language

ERIC Educational Resources Information Center

Jalilifar, Alireza

2008-01-01

The aim of this study was to investigate discourse markers in descriptive compositions of 90 Iranian students who were selected from two universities. Without any instruction, they were given a topic to write a descriptive composition per week for 8 weeks. 598 compositions were collected, and they were analyzed qualitatively and quantitatively by…

Development of molecular markers closely linked to the potato leafroll virus resistance gene, Rlretb, for use in marker-assisted selection

USDA-ARS?s Scientific Manuscript database

Potato leafroll virus (PLRV) is a major pathogen of potato with worldwide impact on seed and commercial production of potato. In North America, the primary varieties grown by industry are not resistant to PLRV and require the application of insecticides to control the aphid vector of PLRV, so as to...

Marker-assisted selection for elevated concentrations of the a' subunit of B-conglycinin and its influence on agronomic and seed traits of soybean

USDA-ARS?s Scientific Manuscript database

Soybean [Glycine max (L.) Merr.] cultivars with elevated concentrations of the a' subunit of ß-conglycinin (BC) may provide health benefits to soy protein consumers. Two Monsanto single nucleotide polymorphism markers were used to classify F2 plants in four segregating populations as having elevate...