EST markers in evergreen azalea: development and use for classifications compared to AFLP, microsatellite and morphological data.

摘要

Starting from a cDNA library made from flowers of the Rhododendron simsii hybrid 'Flamenco' 200 cDNA fragments were randomly selected and sequenced. Putative functions compared to EMBL accessions were found for 30% of the fragments. Primers were developed on 87 cDNA fragments and used for PCR amplification on 6 different azalea cultivars and species. Polyacrylamide gel electrophoresis was used to separate the fragments and after UV staining the presence of polymorphic bands was evaluated. In those cases that no polymorphisms could be detected new primers were developed and the procedure was repeated once. This resulted in 32 polymorphic EST markers. Of them 10 were selected and the application of these EST markers to study genetic diversity and relationships in and evergreen azalea gene pool was investigated: 40 species and varieties, ancestors of known cultivars, and 44 cultivars chosen among distinguishable horticultural groups were genotyped using 3 AFLP primer combinations 6 microsatellite loci and 10 ESTs. EST markers revealed a higher genetic distance detection capacity than AFLPs. Similarity matrices produced for each marker technique showed weak yet significant correlations. Analysis of molecular variance showed that most of the genetic diversity was attributable to differences among cultivars within horticultural groups. EST markers outperformed AFLP and STMS markers representing Fst values. This indicated a low but significant differentiation among horticultural groups. Although ESTs and STMSs appear to be the most appropriate markers for paternity analysis and assessment of narrow genetic relationships, AFLP remains the best technique for phylogenetic studies. EST markers could be particularly useful for QTL mapping using a candidate gene approach because they target expressed genes, and for comparative mapping studies because they are derived from gene coding regions which are more likely to be conserved across populations and species.