De Novo Transcriptome Assembly and Development of Novel Microsatellite Markers for the Traditional Chinese Medicinal Herb Veratrilla baillonii Franch (Gentianaceae)

摘要

Veratrilla baillonii Franch is an important Chinese medicinal herb for treating liver-related diseases, which has been over-collected in the recent decades. However, the effective conservation and related population genetic study has been hindered because of the lack of genome sequences and genetic markers in the natural population. We have conducted RNA-seq on V. baillonii. We performed de novo assembly of these data to characterize the V. baillonii transcriptome, resulting in 133,019 contigs with size >200 bp. These contigs were annotated using the NCBI nonredundant database and Gene Ontology (GO) terms. From these contigs, we developed novel microsatellite simple sequence repeat (SSR) markers, identifying a total of 40,885 SSRs. SSRs with repeat motifs of 1–4 bp (mono-, di-, tri-, and tetranucleotides) accounted for 99.8% of all SSRs, with mononucleotide repeats most common, followed by dinucleotide (16.2%) and trinucleotide repeats (14.7%). We selected 151 SSRs for experimental validation, of which 74 were confirmed by polymerase chain reaction. Fourteen SSRs were determined to be polymorphic by screening 40 individuals from six distant populations. The number of alleles per locus ranged from two to four, and the expected heterozygosity varied from 0.2637 to 0.8571, suggesting that these SSR markers are highly polymorphic and effective for further genetic analysis in the nature population. In addition, we explored the genetic structure of V. baillonii using five SSRs in four geographic populations and found that the identified genotypes were clustered into two phylogenetic clades: the Mekong River clade and Jinsha River clade. This result indicates that these two regions may harbor highly divergent genetic lineages and enriched genetic diversity. The de novo transcriptome sequences and new SSR markers discovered by this study provide an initial step for understanding the population genetics of V. baillonii, and a valuable resource for effective conservation management.