Establishment of Novel Simple Sequence Repeat (SSR) Markers from Transcriptome Data and Their Application in the Identification of Varieties.

, a member of the Calycanthaceae family, is a unique, traditional, and famous flowering economic tree species in China. Despite the existence of several varieties, only a few cultivars have been formally named. Currently, expression sequence tag-simple sequence repeat (EST-SSR) markers are extensively used to identify different species and varieties; a large number of microsatellites can be identified from transcriptome databases. A total of 162,638 unigenes were assembled using RNA-seq; 82,778 unigenes were annotated using the Nr, Nt, Swiss-Prot, Pfam, GO, KOG, and KEGG databases. In total, 13,556 SSR loci were detected from 11,691 unigenes, with trinucleotide repeat motifs being the most abundant among the six repeat motifs. To develop the markers, 64,440 pairs of SSR primers with polymorphism potential were designed, and 75 pairs of primers were randomly selected for amplification. Among these markers, seven pairs produced amplified fragments of the expected size with high polymorphism. Using these markers, 12 varieties were clustered into two monophyletic clades. Microsatellites in the transcriptome of exhibit rich types, strong specificity, and great polymorphism potential. These EST-SSR markers serve as molecular technical methods for identifying different varieties of and facilitate the exploration of a large number of candidate genes associated with important traits.