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sur le développement des zones arides et semi-arides

Titre : Development and applications of EST-SSR markers in sorghum.

Auteur : Ramu, P

Etablissement de soutenance : Osmania University

Grade : Doctor of Philosophy (PhD) 2009

Résumé partiel
Microsatellites or simple sequence repeat (SSR) markers are playing a significant role in molecular breeding programs. But, conventional methods of SSR markers development are laborious and expensive. The alternative way is to explore the existing databases for the development of new markers. Complete genome sequencing projects depositing numerous amounts of genomic and genic (expressed sequence tag, EST) sequence in the public databases. This facilitates to explore for the development of new marker systems including SSRs, SNPs, etc. In the present study, 600 EST-SSR markers were developed in sorghum by mining the J. Craig Research Institute [formerly, The Institute for Genome Research (TIGR)] database. Non-redundant EST sequences which are having the homology with rice genome were selected for the designing these 600 markers (by exploiting rice-sorghum synteny). PCR conditions for these primer pairs were optimized on four parental pairs of sorghum genotypes constituting two mapping populations. About 23% of markers were found polymorphic in parents of shootfly resistance mapping population (BTx623  IS18551). These polymorphic markers were screened against the shootfly RIL mapping population. A total of 82 EST-SSR markers were mapped onto the previously existing skeleton map of the sorghum using shootfly resistance mapping population. After adding these 82 new EST-SSR markers, the map distance was extended to 2966.4 cM. This map distance was 250 cM larger than the previously reported longest linkage map in sorghum. These EST-SSR markers, however, filled gaps or marker-rare regions in the existing linkage map of sorghum targeted based on rice-sorghum sequence similarity. 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. During the course of this study, complete sorghum genome sequence is available. Aligned sorghum genome sequence was downloaded (Sorbi v1.0) (http://genome.jgipsf.org/Sorbi1/Sor...) and made as a database. SSR markers (available and unavailable for public use) were used to develop in-silico sequence-based physical maps for sorghum for all linkage groups. The order of EST-SSR markers from the in-silico mapping agrees with the linkage map positions for all ten sorghum linkage groups. The markers which had conflicting linkage and physical mapping positions can be attributed to the presence of duplicate loci or the presence of best hit for forward and reverse primer sequences on the different chromosomes. Upon integration of sorghum physical and linkage maps, it was found that 1 cM was equal to 223 kb (in other words, 1Mb = 4.47 cM). This indicates that if recombination rates are similar across the genome (which is not likely), then if 4–5 markers are present in each bin (1 Mb) the map can be considered as saturated, which is essential for map-based cloning. Integration of physical maps and genetic maps provides the bridge to isolate the genes through positional or map-based cloning. This physical map facilitated the researchers to select more number of markers in a given genomic region for effective transfer of QTLs from donor parent to locally improved varieties through markers-assisted selection (MAS).

Présentation : ICRISAT

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