Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.)

Abstract

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.

Characterization and development of EST-derived SSR markers in cultivated sweetpotato (Ipomoea batatas)

“…Of these, 897 primer pairs (84.6%) yielded amplicons in the two parents of our mapping population. This result was similar to EST-SSR amplification rate in sweetpotato [26,28] and many other studies in which a success rate of 60-90% amplification has also been reported [37,40-43]. In those studies (except [26]), they also reported a similar success rate of amplification for both genomic SSRs and EST-SSRs.…”

“…The previous studies of arabidopsis [33] and soybean [35] also suggested that the tri-nucleotide AAG motif may be common motif in dicots. In contrast, the most frequent tri-nucleotide repeat motifs were (AAC/TTG)n in wheat, (AGG/TCC)n in rice, and (CCG/GGC)n in maize, barley and sorghum [31,36,37]. The abundance of the tri-nucleotide CCG repeat motif was favored overwhelmingly in cereal species [31,36,38] and also considered as a specific feature of monocot genome, which may be due to the high GC content and consequent codon usage bias [5,39].…”

Development of EST-based new SSR markers in seabuckthorn

“…The resulting sequences were clustered using CAP3 (Huang and Madan 1999). Microsatellites were identified in ESTs using MISA (Thiel et al 2003) with its default parameters except that mononucleotides were not included in the search parameters. The primers were designed using Primer 3 (Rozen and Skaletsky 2000).…”

Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers

“…SSRs were mined in the genome sequence using the MIcroSAtellite (MISA) 52 program, with the following parameters: at least ten repeats for mono-, six repeats for di-, and five repeats for tri-, tetra-, penta-and hexa-nucleotide for simple SSRs. The Primer3 program 53 was used for designing the primer pairs for identified SSRs based on the following criteria: (i) annealing temperature (Tm) between 50-65 °C with 60 °C as optimum; (ii) product size ranging from 100 bp to 350 bp; (iii) primer length ranging from 18 bp to 24 bp with an optimum of 20 bp; (iv) GC % content in the range of 40-60%.…”