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.
The inferred population structure shows a strong geographical correspondence with the hypothesized glacial refugia and rules out the migration of the chestnut from Turkey and Greece to Italy. The homogeneous gene pool observed in Italy and Spain could have been originated from common refugia along with human-mediated colonization.
A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers was constructed using the two-way pseudo-testcross strategy. A total of 96 individuals from a F 1 full-sib family was genotyped with 381 molecular markers (311 RAPDs, 65 ISSRs, 5 isozymes). Markers in testcross configuration, segregating 1:1, were used to establish two separate maternal and paternal maps including 187 and 148 markers, respectively. The markers identified 12 linkage groups based on the haploid number of chestnut. The female and male framework maps reached a total length of 720 and 721 cM (Kosambi), respectively, representing a 76% and 68% coverage of the overall genome. A total of 46 markers, found in intercross configuration, segregating 3:1 and 1:2:1, were used to identify homologous linkage groups between parental maps; out of 12 linkage groups 11 could be joined. RAPD and ISSR markers showed a good and comparable reliability, allowing for the first time the establishment of a saturated linkage map for European chestnut. These maps will be a starting point for studies on the structure, evolution and function of the chestnut genome. Identification of QTLs for adaptive traits in chestnut will be the primary target.
The genetic structure of sweet chestnut (Castanea sativa Mill.) across Europe was assessed using 73 intersimple sequence repeat markers to screen 1,768 individuals from 68 stands distributed across 29 sites in five European countries (Italy, France, Spain, Greece, and UK). At each site, trees were sampled from three distinct management types (domestication levels): naturalized stands, managed coppice, and grafted fruit orchards. In more than a third of the orchards, nonlocal genetic material (grafted clones) were evident, showing (as predicted) large differences from the other two domestication levels for most of the withinpopulation genetic diversity parameters estimated. Randomly generated linkage disequilibrium analysis revealed weak though significant differences in two-locus allelic correlations between naturalized stands and coppice, suggesting that long-term management techniques may influence the genetic makeup of the populations. Multivariate analysis revealed the existence of five distinct gene pools across the study area; three were located in Greece, one on the northwestern coast of the Iberian peninsula and a large gene pool covering the rest of the Mediterranean basin. The implications of the results are discussed in relation to developing conservation strategies for chestnut genetic resources in Europe.
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