Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat

Abstract

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivumL.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1-7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.

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.…”

EST-SSRs as a resource for population genetic analyses

“…Despite their potential to cause selectively deleterious frameshift mutations, however, EST-SSRs located in coding regions appear to reveal equivalent levels of polymorphism as compared to those located in UTRs, most likely due to a general trend toward trinucleotide repeats in coding regions. In fact, this trend toward trinucleotide repeats in exons has been observed in a variety of other taxa, including wheat (Gupta et al, 2003) barrel medic , tall fescue (Saha et al, 2004), and pine (Chagne et al, 2004). Regardless of the cause, if this observed tendency toward higher transferability and equivalent levels of polymorphism turns out to be a general feature of EST-SSRs located in proteincoding regions, the targeting of exonic trinucleotide repeat motifs might be the best strategy for developing portable sets of polymorphic EST-SSR markers.…”

“…In contrast, the apricot primers were found to be most useful within the subgenus Prunophora. In the cereals, Gupta et al (2003) demonstrated extensive transferability of Triticum aestivum L. (bread wheat) EST-SSRs to 18 related wild species in the Triticum-Aegilops complex and to five cereal species of barley, oat, rye, rice and maize.…”

“…In fact, the rapid and inexpensive development of SSRs from expressed sequence tag (EST) databases has been shown to be a feasible option for obtaining high-quality nuclear markers (Gupta et al, 2003;Bhat et al, 2005). Moreover, the National Center for Biotechnology Information (NCBI) EST database (dbEST; Boguski et al, 1993) contains an ever-increasing number of these 'single-pass' cDNA sequences, meaning that the resources necessary for the efficient development of large numbers of so-called EST-SSRs already exist for a wide variety of taxa.…”

Methods for Development of Microsatellite Markers: An Overview

“…The generation of SSR markers from EST resources is relatively fast and inexpensive (Thiel et al, 2003;Gupta et al, 2003) and could be achieved rapidly using bioinformatics softwares (Varshney et al, 2005). EST-SSRs reveal variation in the expressed regions of the genome, thereby detecting perfect marker-trait associations (Gupta et al, 2003).…”