Development of EST markers for evaluation of their use in evergreen azalea analysis

Starting from a cDNA library made from flowers of the Rhododendron simsii hybrid ‘Flamenco’, 200 cDNA fragments were randomly picked and sequenced. The putative functions of the cDNA fragments were determined by comparison of the sequences with EMBL accessions. Reliable homologies 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. In the end, this resulted in 32 polymorphic EST markers. Ten of them were selected and the application of these EST markers to study genetic diversity and relationships in and evergreen azalea gene pool was investigated. Forty species and varieties that most concurred in the origin of the known cultivars and 44 cultivars chosen among distinguishable horticultural groups were genotyped using 3 AFLP primer combinations, 6 microsatellite loci and 10 ESTs (Scariot et al., submitted). EST markers revealed a higher genetic distance detection capacity than AFLPs. Similarity matrices produced for each marker technique showed weak, yet significant, correlations when Mantel’s test was applied. Performing the analysis of molecular variance, for all marker techniques used most of the genetic diversity was attributable to differences among cultivars within horticultural groups. However, EST markers outperformed AFLP and STMS markers concerning Fst values indicating 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 still 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 than non-coding regions.