De Novo Assembly of Transcriptome and Development of Novel EST-SSR Markers in Rhododendron rex Lévl. through Illumina Sequencing

Abstract

Transcriptome sequences generated by next-generation sequencing (NGS) technologies can be utilized to rapidly detect and characterize a large number of gene-based microsatellites from different plants. <i>Rhododendron rex</i> Lévl. is a perennial woody species from the family Ericaceae and an endangered plant with high ornamental value endemic to Southwestern China. Nevertheless, the genetic and genomic information of <i>R. rex</i> remain unknown. In this study, we performed transcriptome sequencing for <i>R. rex</i> leaf samples, and generated large transcript sequences for functional characterization and development gene-associated SSR markers. A total of 164,242 unigenes were assembled and 115,089 (70.07%) unigenes were successfully annotated in public databases. In addition, a total of 15,314 potential EST-SSRs were identified, and the frequency of SSRs in the <i>R. rex</i> unigenes was 9.32%, with an average of one EST-SSR per 5.65 kb. The most abundant type was repeated di-nucleotide (54.63%), followed by mono- (26.03%) and tri-nucleotide (18.51%) repeats. Based on the SSR-containing sequence, 100 primer pairs were randomly selected and synthesized and used for assessment of the polymorphism. Thirty-six primer pairs were polymorphic and revealed polymorphism among 20 individuals from four <i>R. rex</i> populations. A total of 197 alleles were identified, with an average of 5.472 alleles per locus. The Polymorphism Information Content ranged from 0.154 to 0.870, with a mean of 0.482. The newly developed EST-SSR markers exhibited high transferability (58.33-83.33%) among the six subgenera. Thus, these novel EST-SSR markers developed would provide valuable sequence resources for population structure, genetic diversity analysis, and genetic resource assessments of <i>R. rex</i> and its related species.