Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer "stutter bands" as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar 'Chinese Spring' were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA)(n), (GGA/CCT)(n), (TAA/ATT)(n), (CAA/GTT)(n), (GGT/CCA)(n), (CAT/GTA)(n), (CGA/GCT)(n), (CTA/GAT)(n), and (CGT/GCA)(n) repeats were estimated to be 5.4x10(4), 3.5x10(4), 3.2x10(4), 1.2x10(4), 6.3x10(3), 4.9x10(3), 4.5x10(3), 4.5x10(3) and 3.6x10(3), i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT)(n), 30 (43%) (CTT/GAA)(n), 16 (59%) (CAA/GTT)(n), 3 (27%) (CAT/GTA)(n), and 2 (4%) (GGA/CCT)(n) clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT)(n), (CTT/GAA)(n), and (CAA/GTT)(n) microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT)(n), four (14.8%) to (CTT/GAA)(n), and two (12.5%) to (CAA/GTT)(n), resulted in polymorphic markers. The results indicated that (TAA/ATT)(n) microsatellites would provide the most abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat.