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.
Highlights
Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies
Because ESTs correspond to coding DNA, the flanking sequences of EST-SSRs are located in well-conserved regions across phylogenetically related species, making them markers of choice for comparative mapping and relevant functional and positional candidate genes to study their colocation with quantitative trait loci (QTLs)
Despite a lower rate of polymorphisms compared to genomic SSRs, EST-SSRs offer a number of advantages over genomic SSRs [2]: (i) their development requires no investment in de novo sequencing; (ii) they detect variation in the expressed portion of the genome; (iii) the conservation of primer sites makes them readily transferable across closely related species as illustrated here between oak and chestnut; and (iv) in most cases they can be exploited for population genetic analysis [1]
Summary
Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. Catalogues of Expressed Sequence Tags (ESTs) are developed from cDNA libraries to obtain expressional sequence information in contrasting environmental conditions or across developmental stages When available, they offer an inexpensive source of gene-based DNA markers, in particular SSRs [1]. The optimal bin set of a given size presents the maximum number of breaking points evenly spaced throughout the map, ideally resulting in a number of bins that is close to the number of framework marker intervals This approach has been used successfully in peach [5], melon [6], strawberry [7] and apple [8,9]. We use this approach for the first time in a forest tree species: oak
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