Genetic diversity analysis and DNA fingerprint construction of Zanthoxylum species based on SSR and iPBS markers

Abstract

Zanthoxylum is a versatile economic tree species utilized for its spice, seasoning, oil, medicinal, and industrial raw material applications, and it has a lengthy history of cultivation and domestication in China. This has led to the development of numerous cultivars. However, the phenomenon of mixed cultivars and confusing names has significantly obstructed the effective utilization of Zanthoxylum resources and industrial development. Consequently, conducting genetic diversity studies and cultivar identification on Zanthoxylum are crucial. This research analyzed the genetic traits of 80 Zanthoxylum cultivars using simple sequence repeat (SSR) and inter-Primer Binding Site (iPBS) molecular markers, leading to the creation of a DNA fingerprint. This study identified 206 and 127 alleles with 32 SSR markers and 10 iPBS markers, respectively, yielding an average of 6.4 and 12.7 alleles (Na) per marker. The average polymorphism information content (PIC) for the SSR and iPBS markers was 0.710 and 0.281, respectively. The genetic similarity coefficients for the 80 Zanthoxylum accessions ranged from 0.0947 to 0.9868 and from 0.2206 to 1.0000, with mean values of 0.3864 and 0.5215, respectively, indicating substantial genetic diversity. Cluster analysis, corroborated by principal coordinate analysis (PCoA), categorized these accessions into three primary groups. Analysis of the genetic differentiation among the three Zanthoxylum (Z. bungeanum, Z. armatum, and Z. piperitum) populations using SSR markers revealed a mean genetic differentiation coefficient (Fst) of 0.335 and a gene flow (Nm) of 0.629, suggesting significant genetic divergence among the populations. Molecular variance analysis (AMOVA) indicated that 65% of the genetic variation occurred within individuals, while 35% occurred among populations. Bayesian model-based analysis of population genetic structure divided all materials into two groups. The combined PI and PIsibs value of the 32 SSR markers were 4.265 × 10^{- 27} and 1.282 × 10^{- 11}, respectively, showing strong fingerprinting power. DNA fingerprints of the 80 cultivars were established using eight pairs of SSR primers, each assigned a unique numerical code. In summary, while both markers were effective at assessing the genetic diversity and relationships of Zanthoxylum species, SSR markers demonstrated superior polymorphism and cultivar discrimination compared to iPBS markers. These findings offer a scientific foundation for the conservation and sustainable use of Zanthoxylum species.

Keywords: Zanthoxylum; DNA fingerprint; Genetic diversity; Genetic structure; SSR markers; iPBS markers.

Fig. 1

UPGMA clustering tree of 80 Zanthoxylum accessions based on SSR markers

Fig. 2

Principal coordinate analysis of 3 populations of Zanthoxylum species based on SSR markers

Fig. 3

Delta K values for different numbers of populations assumed (K) in the STRUCURE analysis

Fig. 4

Population genetic structure of 80 Zanthoxylum accessions. Each rectangular column in the figure represents one accession, and the color and color scale of the columns represent the subpopulation to which it belongs and the proportion of the subpopulation it occupies (Blue represents Pop1, and Orange represents Pop2). The number on the X-axis is the accession number

Fig. 5

Fingerprint information of 80 Zanthoxylum cultivars based on SSR markers

Fig. 6

UPGMA clustering tree of 80 Zanthoxylum accessions based on iPBS markers

Fig. 7

Principal coordinate analysis of 80 Zanthoxylum accessions based on iPBS markers

Fig. 8

UPGMA clustering tree of 80 Zanthoxylum accessions based on SSR + iPBS markers