EST Databases as a Source for Molecular Markers: Lessons from Helianthus

Abstract

Expressed sequence tag (EST) databases represent a potentially valuable resource for the development of molecular markers for use in evolutionary studies. Because EST-derived markers come from transcribed regions of the genome, they are likely to be conserved across a broader taxonomic range than are other sorts of markers. This paper describes a case study in which the publicly available cultivated sunflower (Helianthus annuus) EST database was used to develop simple sequence repeat (SSR) markers for use in the genetic analysis of a rare sunflower species, Helianthus verticillatus, as well as the more widespread Helianthus angustifolius. EST-derived SSRs were found to be more than 3 times as transferable across species as compared with anonymous SSRs (73% vs. 21%, respectively). Moreover, EST-SSRs whose primers were located within protein-coding sequence were more readily transferable than those derived from untranslated regions, and the former loci were no less variable than the latter. The utility of existing EST databases as a means for facilitating population genetic analyses in plants was further explored by cross-referencing publicly available EST resources against available lists of rare or invasive flowering plant taxa. This survey revealed that more than one-third of all plant-derived EST collections of sufficient size could conceivably serve as a source of EST-SSRs for the analysis of rare, endangered, or invasive plant species worldwide.

EST-SSRs as a resource for population genetic analyses

“…Finally, high levels transferability and substantial polymorphism were observed among 23 cotton (Gossypium) species (Guo et al, 2006). In general terms, this sort of transferability is unique to EST-SSRs, with anonymous SSRs being significantly less portable (Chagne et al, 2004;Liewlaksaneeyanawin et al, 2004;Gutierrez et al, 2005;Pashley et al, 2006; but see Fitzsimmons et al, 1995;Dayanandan et al, 1997). EST-SSRs have also been shown to produce substantially 'cleaner' data (that is, easier to analyze/interpret amplification profiles) as compared to their anonymous counterparts .…”

“…In fact, an estimated 2-5% of all plant-derived ESTs are thought to harbor SSRs (Kantety et al, 2002), although the actual frequency of SSR-bearing ESTs in any particular analysis is highly dependent on the search parameters (see below). Moreover, a large fraction of EST-SSRs (on the order of 80-90%) are typically found to be polymorphic (Bandopadhyay et al, 2004;Fraser et al, 2004;Pashley et al, 2006). Taking into account typical marker development attrition rates, it therefore seems likely that EST databases containing as few as 1000 sequences could provide enough markers to facilitate population genetic analyses.…”

“…After controlling for inherent differences in variability from one locus to the next, however, it becomes clear that H. verticillatus actually harbors more genetic diversity than does H. angustifolius despite its rarity (Figure 1). Beyond providing more statistical power in paired comparisons, ESTSSRs also produce cleaner results for scoring as there are fewer null alleles (Leigh et al, 2003;Rungis et al, 2004) and fewer stutter bands (Leigh et al, 2003;Woodhead et al, 2003;Eujayl et al, 2004;Pashley et al, 2006). Despite these advantages, however, EST-SSRs are not without their drawbacks.…”

“…In general, EST-SSRs have been found to be significantly more transferable across taxonomic boundaries than are traditional 'anonymous' SSRs (Chagne et al, 2004;Liewlaksaneeyanawin et al, 2004;Gutierrez et al, 2005;Pashley et al, 2006), and reports of EST-SSR transferability have become increasingly common. This is particularly true in plants, where transferability among economically important crop taxa has been demonstrated on a number of occasions (Decroocq et al, 2003;Thiel et al, 2003;Bandopadhyay et al, 2004;Saha et al, 2004;Varshney et al, 2005b).…”

Environmental variables, habitat discontinuity and life history shaping the genetic structure of Pomatoschistus marmoratus

“…We do not favor the hypothesis of null alleles because all F IS estimates were positive and significant; it seems highly improbable that all loci exhibit null alleles with such a constant frequency. The heterozygotes deficiency detected in all populations can be boosted by the use of locus primers designed for other species (cross-amplification) (Rungis et al 2004;Pashley et al 2006;Keever et al 2008). Finally, the selection against heterozygotes cannot be demonstrated from our results, although we have detected a significant link among genetic structure and maximum salinity (implying that selection could be acting on P. marmoratus).…”