EST-SSR Development and Validation in Azalea Breeding Populations from Whole Transcriptome Sequencing

The lack of published genomic information, linkage maps or marker datasets for a given species is no longer as large an obstacle for population genetic studies or molecular breeding as it once was.  High throughput sequencing technologies and computer programs have enabled DNA to be sequenced and analyzed for features of practical interest, such as marker loci, in a short time.  In this study, we performed Illumina whole transcriptome sequencing on vegetative and floral bud tissue of Rhododendron viscosum, a North American native azalea with ornamental appeal occurring across a wide geographic range.  This resulted in a transcriptome consisting of ~35 million paired-end reads that were assembled and analyzed for expressed simple sequence repeat (EST-SSR) motifs and primer sites using WebSat.  Unmapped reads were mined also for EST-SSRs through open source perl scripts and Primer3, respectively, to capture additional EST-SSR loci not mapped to contigs. This allowed for the selection of 48 potential EST-SSR markers within the R. viscosum transcriptome.  Marker primer pairs were tested against six individuals from distinct wild R. viscosum populations to determine rates of successful amplification, the number of alleles per loci, and amplicon length.  This approach showcases a contemporary method for rapid development of new molecular breeding tools in a novel species.