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Bambara groundnut (Vigna subterranea (L.) Verdc.) is an indigenous underutilized legume that has the potential to improve food security in semi-arid Africa. So far, there are a lack of reports [...]

Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries. Key words: DOR364, genomic microsatellites, plasmid clones, simple sequence repeats. Les microsatellites constituent des outils genetiques utiles pour une grande gamme d'analyses genomiques bien que leur developpement prenne du temps et necessite l'identification de sequences simples repetees (SSR) au sein des sequences genomiques. Le criblage de banques a inserts courts sans enrichissement prealable represente une methode efficace pour isoler des SSR, laquelle peut livrer un porkrait de la frequency des motifs qui soft sans biais. Dans ce travail, les auteurs adaptent des protocoles a haut debit pour robotiser le prelevement des colonies et la preparation des membranes au tours du criblage de banques de plasmides. Un jeu de sept banques genomiques non-enrichies preparees a partir de l'ADN genomique du haricot fragmente par sonication ou encore digere avec quatre enzymes coupant frequemment ou une double digestion avec ces enzymes et une enzyme coupant moms souvent. La quality de banques a ete comparee et trois des banques a inserts courts ont ete retenues pour analyse ulterieure. Chaque banque a ete etalee et les colonies disposees dans des plaques a 384 puns, lesquelles ont servi a preparer les membranes a haute density comprenant plus de 18 000 clones. Ces membranes ont ete ensuite criblees avec des sondes oligonucleotidiques pour les differents motifs SSR. Il a ete observe que les clones positifs presentaient une faible redundancy et 100 marqueurs SSR ont ete mis au point dont 80 ont ete testes pour le polymorphisme sur un jeu de parents. Ces microsatellites derives de banques non-enrichies devraient s'averer un ajout utile aux marqueurs developpes precedemment a partir de banques enrichies. Mots-cles : DOR364, microsatellites genomiques, clones plasmidiques, sequences simples repetees. [Traduit par la Redaction], Introduction Microsatellites are PCR-based markers that target simple sequence repeat (SSR) loci based on various repeat motifs that are evaluated for size polymorphisms with different electrophoretic systems (Powell et al. [...]

Expressed sequence tag (EST)-derived simple sequence repeat (eSSR) markers are important resources for gene discovery and comparative mapping aimed at crop improvement. In this study, we developed eSSR markers for Medicago truncatula and assessed their cross-species transferability. We detected 36847 non-redundant sequences ('unigenes') from 198 642 M. truncatula EST sequences. Mining of microsatellites from the 36 847 unigene sequences (representing ti 25.8 Mb) revealed 14 637 eSSRs in 11750 SSR-containing ESTs, and primer pairs were successfully designed for 4 636 (39.5%). Of the 14637 eSSRs, 82.6% were mononucleotide repeats and the rest (in descending order of abundance) were tri-, di-, penta-, and tetranucleotide repeats. When less stringent SSR detection criteria were used, the frequency of dinucleotide repeat motifs increased more than twofold, and the frequencies of di- (11%) and trinucleotide motifs (10.6%) were almost equal. This demonstrates that the eSSR frequency and distribution were related to the choice of search criteria. Forty-one randomly selected primer pairs were validated, and their transferability in three leguminous and three nonleguminous species was assessed. The markers showed a high level of transferability in the leguminous (53%-71%) and non-leguminous (33%-44%) species. The validation studies thus demonstrate the utility of the Medicago eSSRs in assessing genomic relationships in both leguminous and non-leguminous species. Key words: Medicago truncatula, unigene, EST-derived simple sequence repeats (eSSRs), transferability. Des microsatellites identifies au sein d'etiquettes de sequences exprimees (EST), connus sous le nom de marqueurs eSSR, sont des resources importantes en vue du clonage de genes et de la cartographie comparee visant l'amelioration genetique. Dans ce travail, les auteurs ont mis au point des marqueurs eSSR chez le Medicago truncatula et determine leur portabilite chez d' autres especes. Les auteurs ont detecte 36 847 unigenes au sein de 198 642 sequences EST provenant du M. truncatula. L'examen des 36847 unigenes, totalisant 25,8 Mb, a permis d'identifier 14637 (39,7 %) eSSR au sein de 11750 EST qui contenaient au moms un SSR. Il a ete possible de synthetiser des paires d'amorces pour 4636 (39,5 %) de ces eSSR. Des 14637 eSSR, 82,6 % etaient des suites mononucleotidiques, les autres etant (en ordee decroissant) des suites tri-, di-, penta- et tetranucleotidiques. L'emploi de criteres moms stricts pour la detection des SSR a permis de plus que doubler la frequence des suites dinucleotidiques et de rendre presque egales les proportions de motifs di- (11 %) et trinucleotidiques (10,6 %). Cela montre que la frequence et la distribution des eSSR soot liees au choix des criteres d'interrogation des banques. Au total, 41 paires d'amorces choisies au hasard ont ete validees et leur portabilite a ete examinee chez trois especes de legumineuses ainsi que chez trois autres especes. Les marqueurs ont affiche une grande portabilite tant chez les legumineuses (53-71 %) que chez les autres especes (33-44 %). Cette validation illustre l'utilite des eSSR de Medicago pour etudier les relations genomiques au sein des legumineuses et d'autres especes. Mots-cles : Medicago truncatula, unigene, microsatellites derives d'un EST (eSSR), portabilite. [Traduit par la Redaction], Introduction The legume family is the second most important food and forage source after the grass family. Within the legume family, one of the most studied species is Medicago truncatula, [...]

Genetic diversity in 20 elite blackgram (Vigna mungo (L.) Hepper) genotypes was studied using microsatellite and AFLP markers. Thirty-six microsatellite markers from azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) were successfully amplified across the 20 blackgram genotypes and 33 microsatellite markers showed polymorphism. A total of 137 microsatellite alleles were generated with an average of 4.1 alleles per locus. The number of alleles ranged from two to nine and the polymorphic information content value for the microsatellite markers varied from 0.10 to 0.87 with an average of 0.49. Microsatellite markers were highly informative and a combination of only three microsatellite markers (CEDG264, CEDG173, and CEDG044) was sufficient to discriminate all 20 blackgram genotypes. In the case of AFLP, 11 primer pairs generated 324 polymorphic marker fragments. The polymorphic information content values for AFLP primer combinations ranged from 0.21 to 0.34 with an average of 0.29. Similarity measures and clustering analyses were made using microsatellite and AFLP data separately. The resulting dendrograms distributed the 20 blackgram genotypes into five main clusters. The dendrograms were comparable with each other with the Mantel test between the cophenetic matrices of microsatellite data and AFLP data showing moderate correlation (r = 0.64). The results of the principal components analysis were well congruent with the dendrograms. In the dendrograms as well as in the principal components analyses, genotype Trombay wild (Vigna mungo var. silvestris) was placed separately from rest of the genotypes. This study demonstrated that the azuki bean microsatellite markers are highly polymorphic and informative and can be successfully used for genome analysis in blackgram. Results indicate that sufficient variability is present in the blackgram genotypes and would be helpful in the selection of suitable parents for breeding purposes and gene mapping studies. Key words: genetic diversity, blackgram, microsatellite markers, AFLP markers, genotype identification. La diversite genetique chez 20 genotypes elites du haricot mung a grains noirs (Vigna mungo (L.) Hepper) a ete etudiee a l'aide de microsatellites et de marqueurs AFLP. Trente-six marqueurs microsatellites provenant du haricot azuki (Vigna angularis (Wild.) Ohwi & Ohashi) ont amplifie avec succes chez les 20 genotypes elites du haricot mung a grains noirs et 33 de ceux-ci etaient polymorphes. Un ensemble de 137 alleles microsatellites ont ete observes pour une moyenne de 4,1 alleles par locus. Le nombre d'alleles variait entre deux et neuf tandis que l'indice de contenu d'information polymorphique pour ces marqueurs variait entre 0,10 et 0,87 pour une moyenne de 0,49. Les marqueurs microsatellites se sont averes tres informatifs et une combinaison de seulement trois marqueurs (CEDG264, CEDG173 et CEDG044) suffisait a distinguer les 20 genotypes. Dans le cas des AFLP, 11 paires d'amorces ont genere 324 fragments polymorphes. Les indices de contenu d'information polymorphique pour les AFLP allaient de 0,21 a 0,34 pour une moyenne de 0,29. Des mesures de similarite et des analyses de groupement ont ete effectuees a l'aide des donnees microsatellites et AFLP separement. Les dendrogrammes obtenus divisaient les 20 genotypes en cinq groupes principaux. Les dendrogrammes etaient semblables sur la base d'un test de Mantel effectue sur les matrices co-phenetiques des donnees microsatellites et AFLP, lequel montrait une correlation moderee (r = 0,64). Les resultats d'une analyse des composantes principales concordaient avec les dendrogrammes. Dans les dendrogrammes tout comme dans les analyses des composantes principales, le genotype Trombay wild (V. mungo var. silvestris) formait un groupe distinct des autres genotypes. Cette etude montre que les microsatellites du haricot azuki sont tres polymorphes et informatifs de sorte qu'ils sont utiles pour l'analyse du genome chez le haricot mung a grains noirs. Les resultats indiquent qu'une variabilite suffisante est presente au sein de ces genotypes et qu'elle serait utile en vue de la selection de parents appropries pour des fins de selection varietale et des etudes de cartographie genetique. Mots-cles: diversite genetique, haricot mung a grains noirs, marqueurs microsatellites, marqueurs AFLP, identification des genotypes. [Traduit par la Redaction], Introduction Grain legumes are among the most important crops in many countries, since they provide one third of the dietary protein for human consumption. Blackgram (Vigna mungo (L.) Hepper) is [...]

Abstract: To facilitate the wider use of genetic resources including newly collected cultivated and wild azuki bean germplasm, the genetic diversity of the azuki bean complex, based on 13 simple [...]

Abstract: Convicilins, a set of seed storage proteins, differ from vicilins, a related group of seed storage proteins, mainly because of the presence of the N-terminal extension, an additional sequence [...]

Abstract: Legumes are members of the family Fabaceae or Leguminosae and include economically important grain legumes, oilseed crops, forage crops, shrubs, and tropical or subtropical trees. Legumes are a rich [...]

Abstract: Sequence data for 2 non-coding regions of the chalcone isomerase gene were analyzed to study the genetic architecture of common bean (Phaseolus vulgaris L.). One region corresponded to the [...]

Abstract: A PCR approach with degenerate primers designed from conserved NBS-LRR (nucleotide binding site--leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from [...]

Abstract: The identification of markers in legume pasture crops, which can be associated with traits such as protein and lipid production, disease resistance, and reduced pod shattering, is generally accepted [...]