LARGE SCALE DEVELOPMENT OF MULTIPLE REPEAT CLASSES OF MICROSATELLITE MARKERS IN COTTON USING AN EFFICIENT SELECTIVE ENRICHMENT PROCEDURE

Department of Trait Development, Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268

DNA marker analysis is a key component in trait introgression programs aimed at the improvement of crop plants. A primary requisite for such a molecular breeding project is the availability of a large number of polymorphic markers dispersed throughout the genome. Simple sequence repeats (SSR) or microsatellites have proven to be abundant, well distributed and highly polymorphic in many eukaryotic genomes studied so far. Moreover, they are PCR-based, making them amenable to high through-put genotyping analyses. Despite its importance as one of the most important crops, so far, not many microsatellite markers are available in cotton, partly due to its large genome size, tetraploidy, the apparent low level of intra-species polymorphism and the expenditure involved in developing a large number of markers. In order to circumvent the problem of low level of polymorphism and fewer markers within species, populations derived from inter-specific cross are being used to map the available RFLP, SSR or other markers. However, for the crop improvement programs involving cultivars of the same species, this approach is not adequate indicating the need for the development of a large number of polymorphic markers. To generate a large number of markers in G.hirsutum, we have constructed six high quality genomic DNA libraries enriched for multiple types of simple sequence repeat (microsatellite) motifs. Randomly sheared and size selected genomic DNA fragments containing simple sequence repeats were captured by hybridization with biotinylated oligo probes and subsequently cloned and archived in 384-well plates. High density replica filters prepared from the libraries have been screened with end-labeled oligos to identify the positive clones in the libraries. The inserts of these clones have been sequenced and the derived information is utilized to design the primer pairs for PCR amplification of the marker loci. Results and methodology pertaining to the construction and characterization of the microsatellite libraries will be presented.