Development and characterization of SSR markers in Phoebe zhennan

Phoebe zhennan S. K. Lee & F. N. Wei is a valuable endemic species in China, widely recognized for its high-quality timber and ornamental value. It has been classified as an endangered species under national protection, with its primary distribution areas including Guizhou, Sichuan, and Hunan. Investigating the genetic diversity (GD) of this species plays a crucial role in developing effective strategies for its conservation and sustainable utilization. Nevertheless, the progress in molecular marker development has been constrained by the scarcity of reported reference genomes for P. zhennan. In this study, the genomic gap of the endangered and precious tree species P. zhennan was filled. The simple sequence repeat (SSR) markers of the P. zhennan genome were analyzed using magnetic bead enrichment technology. A total of 794,128 SSR loci were identified. From a random selection of 108 primer pairs, 20 primer pairs with high polymorphism and excellent stability were screened and subsequently utilized to assess the GD of 24 Phoebe populations comprising 174 individuals primarily from Guizhou Province. The results demonstrated that the SSR loci were predominantly dinucleotide repeats (accounting for 54.31%), among which the AG/CT motif exhibited the highest frequency. The polymorphism information content (PIC) of the 20 SSR primer pairs ranged from 0.777 to 0.903, with an average value of 0.859, reflecting their substantial polymorphism. The Shannon information index of the Guizhou P. zhennan population ranged from 1.196 to 1.928, with an average value of 1.518. The Nei's genetic diversity index (H) varied between 0.661 and 0.832, averaging 0.743. The SN population exhibited relatively high genetic diversity, whereas the MT population demonstrated relatively low genetic diversity. Further investigation revealed that the populations in Gulin, Sichuan; Zigong, Sichuan; Tongnan, Chongqing; and Baijia, Hunan possessed high and stable genetic diversity values. Genetic differentiation analysis indicated that the genetic variation within the Guizhou P. zhennan population primarily stemmed from intra-population variation. Additionally, frequent gene flow was observed among populations, with alleles being widely distributed across populations. Based on the phylogenetic tree, the species Phoebe bournei, Phoebe sheareri, and Phoebe chekiangensis, as well as the populations of P. zhennan from different regions, were clearly differentiated. The SSR molecular markers developed in this study confirmed at the molecular level that these species represent distinct evolutionary lineages. These findings provide valuable SSR molecular markers and a robust scientific foundation for breeding programs, species identification, and genetic diversity studies of germplasm resources within the genus Phoebe.