Development of polymorphic EST-SSR markers and their applicability in genetic diversity evaluation in Rhododendron arboreum

The genus Rhododendron, known for large impressive flowers is widely distributed throughout the world. Rhododendrons have limited genetic information, despite of comprising high species diversity, morphological overlap and weak genetic barrier. In present study, expressed sequence tag (EST) data from Rhododendron catawbiense Michx (Subgenus Hymenanthes, Section Ponticum) and Rhododendron mucronatum var. ripense (Makino) E.H. Wilson (Subgenus Tsutsusi, Section Tsutsusi) were utilized for mining and identification of the SSRs for genetic diversity analysis of R. arboreum Smith (Subgenus Tsutsusi, Section Tsutsusi). A total of 249 SSRs were developed from 1767 contigs. Di-nucleotide was found to be most abundant repeat followed by tri- and tetra-nucleotide repeats. The motif AG/CT was most common di-nucleotide motif (31.73%), whereas, AAC/GTT (8.43%), ACG/CGT (8.03%), AAG/CTT (7.23%) and AGG/CCT (6.43%) were most abundant tri-nucleotide repeat motif. Among these SSRs, 168 sequences were only fit into the criteria to design flanking primer pairs. A total of 30 randomly selected primer pairs were utilized for validation and genetic diversity study in 36 genotypes of R. arboreum collected from western Himalayan region. In aggregate, 26 SSR markers (86.66%) produced good and repeatable amplifications. Expected heterozygosity (H-E) ranged from 0.322 to 0.841 and observed heterozygosity (H-O) ranged from 0.327 to 1.000 and PIC value ranged from 0.008 to 0.786. These primers were able to distinguish the geographic differences of occurrence based on cluster analysis. These developed EST-SSRs can be useful in future population genetics analysis and micro-evolutionary studies in Rhododendron species.