Identification of SSR markers using soybean (Glycine max) ESTs from globular stage embryos

Microsatellites, or simple sequence repeats (SSRs), in expressed sequence tags (ESTs) provide an opportunity for low cost SSR development. We looked for EST-SSRs in 403,511 ESTs (generated by 454 sequencing and representing 70,654 contigs and 52,082 singletons) from soybean globular stage embryos. Among 122,736 unique ESTs, 3,729 contained one or more SSRs. In total, 3,989 SSRs were identified including 304 mono, 1,374 di, 2,208 tri, 70 tetra, 13 penta and 20 hexanucleotide SSRs. Thirty three EST-SSRs were selected for primer design and polymorphism analysis using twenty soybean cultivars and one wild-type soybean. Successful amplification was obtained using 21 of these primer pairs, 11 of which detected polymorphisms in these soybean cultivars. These results demonstrated that 454 high throughput sequencing is a powerful tool for molecular marker development. From the 3,989 identified SSRs we expect to obtain a large number of makers with polymorphism among different soybean cultivars, which would be useful for analysis of genetic diversity and maker assisted selection in the soybean breeding programs.

EST sequencing and SSR marker development from cultivated peanut (Arachis hypogaea L)

Making use of the gene resources of wild type peanuts is a way to increase the genetic diversity of the cultivars. Marker assisted selection (MAS) could shorten the process of inter-specific hybridization and provide a possible way to remove the undesirable traits. However, the limited number of molecular markers available in peanut retarded its MAS process. We started a peanut ESTs (Expressed Sequence Tags) project aiming at cloning genes with agronomic importance and developing molecular markers. In this study we found 610 ESTs that contained one or more SSRs from 12,000 peanut ESTs. The most abundant SSRs in peanut are trinucleotides (66.3 percent) SSRs and followed by dinucleotide (28.8 percent) SSRs. AG/TC (10.7 percent) repeat was the most abundant and followed by CT/GA (9.0 percent), CTT/GAA (7.4 percent), and AAG/TTC (7.3 percent) repeats. Ninety-four SSR containing ESTs were randomly selected for primer design and synthesis, of which 33 pairs could generate good amplification and were used for polymorphism assessment. Results showed that polymorphism was very low in cultivars, while high level of polymorphism was revealed in wild type peanuts.