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

Starting from 2 cDNA libraries made from flowers of the Rhododendron simsii hybrid ‘Flamenco’ and the species Rhododendron luteum, 323 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 31 % of all fragments. Primers were developed on 127 cDNA fragments and used for PCR amplification on 6 different azalea cultivars and species. PAGE was used to separate the fragments and after UV staining, the presence of polymorphic bands was evaluated. In the end, this resulted in 45 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. EST markers revealed a higher genetic distance detection capacity than AFLPs and outperformed AFLP and STMS markers concerning Fst values indicating a low but significant differentiation among horticultural groups. Although ESTs appear to be 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.