Six Express Sequence Tag–Simple Sequence Repeat Primers Reveal Genetic Diversity in the Cultivars of Three Zanthoxylum Species

Zanthoxylum (Sichuan pepper), with its rich cultivars, has long been widely cultivated in China for its unique seasoning and medicinal uses, but most of its cultivars have similar morphological characteristics. Therefore, we hypothesized that the genetic diversity of Zanthoxylum cultivars is low because of their apomixis and long cultivation history. In this study, we aimed to investigate the genetic diversity of three Zanthoxylum species on the cultivar level based on express sequence tag–simple sequence repeat (EST-SSR) primers. In total, 121 samples of three Zanthoxylum species (Z. bungeanum, Z. armatum and Z. piperitum) were collected from different areas in China for genetic diversity analysis. A total of six specificity and polymorphism EST-SSR primers, which we selected from among 120 primers based on two transcriptomes (Z. bungeanum, Z. armatum) in our earlier study, were used to evaluate genetic diversity based on capillary electrophoresis technology. The results of our analysis using the unweighted pair group method with arithmetic mean (UPGMA) indicated that most of the samples are clustered in one clade in the UPGMA dendrogram, and the average genetic distance was 0.6409. Principal component analysis (PCA) showed that Z. piperitum may have a closer genetic relationship with Z. bungeanum than with Z. armatum. An analysis of molecular variation (AMOVA) showed that the genetic variation mainly stemmed from individuals within populations; the genetic differentiation coefficient (PhiPT) was 0.429, the gene flow (Nm) between populations was 0.333, and the differences among populations were not significant (p > 0.001). For the intraspecific populations of ZB, the percentage of genetic variation was 53% among populations and 47% within populations, with non-significant differences between populations (p > 0.001). The genetic differentiation coefficient (PhiT) was 0.529, and the gene flow (Nm) was 0.223. For the intraspecific populations of ZA, the results indicated that the percentage of genetic variation was 29% among populations and 71% within populations, with non-significant differences between populations (p > 0.001); the genetic differentiation coefficient (PhiPT) was 0.293, and the gene flow (Nm) was 0.223. Through genetic structure analysis (GSA), we predicted that these 121 samples belonged to two optimal subgroups, which means that all the samples probably originated from two gene pools. Above all, this indicated that the genetic diversity of the 121 Zanthoxylum samples was relatively low at both the species and cultivar levels, a finding which was consistent with our initial assumptions. This study provides a reference, with molecular-level data, for the further identification of Zanthoxylum species.