Development of EST-SSR markers based on transcriptome for genetic analysis in Radix Ardisia

Radix Ardisia is a commonly used medicine for the Miao nationality distributed over Guizhou and used to treat laryngeal diseases. The medicinal materials of the Radix Ardisia come from various sources, including Bailiangjin (Ardisia crispa (Thunb.) A. DC.), Zhushagen (Ardisia crenata Sims), Hongliangsan (Ardisia crenata Sims var. bicolor (Walker) C. Y. Wu et C. Chen), Xibingbailiangjin (Ardisia crispa (Thunb.) A. DC. var. dielsii (Levl.) Walker) and Dayebailiangjin (Ardisia crispa (Thunb.) A. DC. var. amplifolia Walker). With the continuous improvement of the medicinal value of the Miao medicine and the increasing scarcity of wild resources, it is of great practical significance to solve the problems of effective identification and genetic structure analysis of medicinal materials and adulterants, for the protection of its germplasm resources and the protection of clinical drug safety. The development of expressed sequence tag-simple sequence repeat (EST-SSR) based on transcriptome strategy is considered to be a very effective means. In this study, 51,237 sequences of A. crenata were retrieved, with a total length of 71.4 MB. A total of 32,827 SSR loci were detected, averaging one SSR locus per 2.1 KB. The distribution of primers was detected, and 28,322 SSR loci were unigene SSR. 32,827 pairs of EST-SSR molecular markers were developed for the whole genome, with an average of 0.64 pairs of SSR primers for each unigene, and the sequence coverage was high. The statistical analysis showed that six types of SSR nucleotides could be detected, but the number and frequency of EST-SSR in different primitive types were significantly different. Mononucleotide and dinucleotide were the main repeat types, accounting for 90.51% of the total SSR of A. crenata. Sixty pairs of primers were randomly selected and applied to genetic research. Among them, 51 pairs could amplify 200 polymorphic bands. Genetic analysis was carried out on 46 mixed species of Radix Ardisia. The results showed that the original plants of Radix Ardisia showed high genetic diversity and could be divided into two populations. The results of systematic clustering showed that the EST-SSR used could well distinguish Radix Ardisia and its easily mixed species; it can be applied to the identification of Radix Ardisia, as well as the molecular identification between the original Bailiangjin, Zhushagen and Hongliangsan. This study can provide a reference for the genetic analysis of the Radix Ardisia.