A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study

Abstract

BackgroundExpressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).ResultsA catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.ConclusionWe have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

A linkage disequilibrium perspective on the genetic mosaic of speciation in two hybridizing Mediterranean white oaks

“…The EST-SSRs markers used in this study were selected from available European white oaks linkage (Bodénès et al, 2012) and bin maps (Durand et al, 2010;), whereas the nSSRs were selected from multilocus scans previously carried out in Q. petraea and Q. robur (Scotti- Saintagne et al, 2004b;Goicoechea et al, 2012). Part of our data set was used in Bodénès et al (2012), with the only intention of providing a proof of concept on the subject of the EST-SSRs transferability to the Mediterranean white oaks.…”

“…Positive selection outliers were plotted on the corresponding LGs from the bin maps developed for Q. robur (Durand et al, 2010). Furthermore, most markers (with the exception of six EST-SSRs, three of them outliers) were placed in a composite linkage map constructed in this study (see next section).…”

Genomics of Fagaceae

“…While triSSRs were more frequently found in coding regions, diSSRs were more abundant in noncoding regions. Durand et al (2010) designed primer pairs for a subset of 748 UE. Overall, 37.7 % (283) were found to amplify a single polymorphic locus in the reference mapping population of Q. robur.…”

“…The bin set consisted of 14 highly informative offspring selected based on the number and position of crossover sites. A total of 256 EST-SSRs were finally assigned to bins and their map position was validated by linkage mapping using an extended set of 48 to 96 offspring (Durand et al 2010). The fact that SSRs were easy to handle and once developed were cost-effective in genotyping hundreds of individuals (also because of multiplexing possibilities) led the EVOLTREE consortium (http://www.evoltree.eu/) to genotype four additional mapping populations including intraspecific crosses between Q. robur and Q. petraea genotypes as well as an interspecific cross between these two species.…”

Patterns of genetic diversity in European beech (Fagus sylvatica L.) at the eastern margins of its distribution range

“…Seven gSSRs originally developed for F. sylvatica (FS3-04, FS4-46, Mfs11 -Pastorelli et al 2003, Vornam et al 2004) and F. crenata (Sfc0018, Sfc0161, Sfc1063 and Sfc1143 -Asuka et al 2004) and one EST-SSRs (Fir065) originally developed for Quercus spp. (Durand et al 2010) were used. FS4-46 was excluded from further analysis because of some ambiguities in its interpretation and due to the presence of a large number of null alleles.…”