Plant & Animal Genome VI Conference

P66

SIMPLE SEQUENCE REPEAT LOCI IN COTTON

Crop Biotechnology Center and Dept. of Soil and Crop Sciences, Texas A&M University, College Station, Texas, 77843-2123

SSRs, also referred to as"microsatellites", are tandemly repeating short nucleotide units, and are relatively abundant in plants. SSR unit sizes are highly variable, and easily detectable by PCR. In species such as Gossypium hirsutum, with low level of polymorphism, SSRs would be a useful supplement to other marker systems. Cotton SSR enriched libraries were constructed using 5'-biotinylated oligonucleotide probes. These probes were hybridized to adapter-ligated restriction endonuclease digested, size fractionated Gossypium hirsutum genomic DNA. Selection of SSRs were then accomplished via streptavidin-bound magnetic beads. To facilitate our efforts to develop a microsatellite map of cotton, we have constructed 6 different SSR enriched libraries (GA, CA, TA, AGA, ACA, CAT) and were screened with the respective oligonucleotide repeat probes. Initial results on enrichment procedure is very encouraging. For example, analysis of [GA]₁₅ probed filters revealed 224 positive clones. Most of the clones that are sequenced contained SSR units ranged from 16 to 60 with an average of 22. Duplicate sequences were not common; only one of the 12 GA repeat containing sequences was found more than once. SSRs were trimmed for vector sequences and exported to a local database. PCR primers flanking SSR were designed and synthesized. A few selected SSRs are being evaluated for polymorphism information content (PIC) and to integrate into the existing genetic maps. Results of our enrichment procedures, PIC values and mapping of a few SSRs will be presented.