Characterization of EST-derived microsatellites in the wheat genome and development of eSSR markers

Abstract

EST-derived microsatellites or simple sequence repeats (eSSR) occur in expressed sequence tags (EST). Here we report characteristics of eSSRs in the wheat genome, construction of consensus chromosome bin maps of SSR-containing ESTs ((SSR)ESTs), and development of eSSR markers for the 21 wheat chromosomes. A Perl script known as MISA was used to identify eSSRs in wheat ESTs available in the database http://wheat.pw.usda.gov/cgi-bin/ace/search/wEST ). Among 492,832 ESTs from the database, 36,520 (7.41%) contained 43,598 eSSRs. This is equivalent to 1 eSSR per 5.46 kb EST sequence. About 60% of the eSSRs were trinucleotides, 19.7% were mononucleotide, 16.7% were dinucleotides, and the remaining approximately 3% consisted of tetra-, penta-, and hexanucleotides. Among the identified eSSRs, (CCG/CGG)n is the most frequent (20.5%) followed by (A/T)n at 13.6%, (AAC/GTT)n at 11.7%, and (AG/CT)n at 8.7%. Among ESTs previously mapped to wheat chromosome bins, a total of 1,010 eSSR loci were derived from 341 (SSR)ESTs. Consensus chromosome bin maps showing the chromosome locations of (SSR)ESTs, SSR sequence motif, and cDNA library were constructed. A chi(2) test indicated that the distribution pattern of eSSR loci was generally similar to that of the original mapped ESTs in the wheat genome. Forty-eight (SSR)ESTs were converted into PCR-based eSSR markers, and 266 eSSR loci were mapped to specific chromosome arms using wheat cytogenetic stocks. The average polymorphism information content (0.45+/-0.16) of eSSR markers was lower than that reported for genomic SSRs (0.54+/-0.19), but higher than RFLPs (0.30+/-0.27). The eSSR markers were transferable among related Triticeae species, Triticum aestivum, T. durum, T. dicoccoides, Hordeum spontaneum, H. vulgare, and Secale cereale. The results confirm the presence of SSRs in expressed genes of wheat and demonstrate another application of ESTs in genomics research. eSSRs will be useful for gene tagging, gene cloning, and comparative genomics studies of cereal crops.