Development of De Novo Reference Transcriptome Assembly and Large-Scale Identification of Simple Sequence Repeat Markers in Stevia rebaudiana

Stevia rebaudiana is a small perennial shrub used for centuries as a bio-sweetener and for its medicinal properties. Stevia accumulates high concentrations of diterpene steviol glycosides which contribute to its high-potency sweetening with no calorific value. The development of molecular markers linked to glycoside and agronomic traits will facilitate early selection for breeding improved varieties. We developed a reference transcriptome from RNA seq data. A total of 194,965,048 paired-end reads of 100bp length were obtained from a bulked RNA sample of diverse tissue types. After processing, 171,503,922 reads were used to construct a reference transcriptome. This de novo assembly was then mined for simple-sequence repeat (SSR) markers. A single locus isoform was selected and the remaining 48,238 transcripts were used to identify SSRs using the MISA software package. A total of 5,876 SSRs were identified in 4,879 transcripts. Primers were designed for 4,193 SSR. In silico PCR was performed on these primers to remove primers that may produce multiple amplicons. The remaining 3,396 SSR primers were then compared to two other de novo assemblies. Those present in all three assemblies (1,063) were selected for evaluation. Additional SSRs were mined from publicly available ESTs. We tested 148 primers pairs on stevia DNA and found over 95% successful amplicon production.  These markers were used to evaluate the level of genetic variation in our germplasm and will be utilized in future genetic mapping efforts.