Genetic diversity analysis and construction of DNA fingerprint for pomegranate (Punica granatum L.) germplasm resources in Shandong Province

【Objective】Pomegranate (Punica granatum L.), distinguished by its exceptional environmental adaptability and extensive cultivation history in China, has emerged as a globally significant economic tree crop. As a primary pomegranate production region within China, Shandong Province faces substantial challenges in germplasm resource management. Being predominantly cross-pollinated, cultivated pomegranates in the region exhibit considerable genetic complexity arising from spontaneous somatic mutations, introductions of germplasm from diverse geographical sources, and extensive varietal exchange. Consequently, this has resulted in pronounced varietal disorganization, ambiguous genetic relationships, and significant lineage confusion, collectively impeding the efficient conservation and utilization of pomegranate germplasm resources. Furthermore, accelerated commercial breeding programs have exacerbated genetic homogenization, rendering conventional morphological identification methods inadequate for precise varietal discrimination and classification. This critical limitation severely constrains the development of elite cultivars and hinders the sustainable advancement of the pomegranate industry. To address these challenges, this study employed Inter- Simple Sequence Repeat (ISSR) molecular markers to investigate the genetic diversity and elucidate the genetic relationships among 76 distinct pomegranate germplasm accessions native to Shandong Province. The primary objective was to construct a robust DNA fingerprinting system, providing scientific foundation for enhancing germplasm preservation, formulating efficient breeding strategies, and achieving precise molecular identification of pomegranate genetic resources.【Methods】Experimental materials comprised 76 authenticated pomegranate (Punica granatum L.) germplasm accessions systematically curated from the China Pomegranate Germplasm Resources Nursery located in Zaozhuang City, Shandong Province. Genomic DNA was extracted from fresh young leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) protocol. Sixteen ISSR primers with clear amplification bands and high polymorphism were selected for PCR amplification of 76 pomegranate germplasm accessions. PopGene software was used to calculate: Mean number of observed alleles per locus (Na), Effective number of alleles per locus (Ne), Nei’s unbiased gene diversity index (He), and Shannon’s information index (I) for population genetic diversity analysis. The genetic similarity coefficient (GS) was calculated using NTSYS clustering software. The genetic relationships between pomegranate germplasm accessions were analyzed by the Unweighted Pair Group Method with Arithmetic Averages (UPGMA). Based on the UPGMA clustering results, the discrimination efficiency of a single primer or primer combinations for germplasm accessions was evaluated. Primers/combinations capable of distinguishing all accessions were selected to construct DNA fingerprints for the germplasm accessions.【Results】A total of 305 loci were detected in 76 accessions using 16 primers, of which 269 were polymorphic loci, with a primer polymorphism ratio of 88.2%. This indicated that the 16 primers exhibited good polymorphism. The results of genetic diversity analysis showed that mean number of observed alleles per locus (Na) was 1.980 7, effective number of alleles per locus (Ne) was 1.651 7, Nei’s unbiased gene diversity index (He) was 0.370 4, and Shannon’s information index (I) was 0.545 1. These metrics collectively demonstrated that moderate to high genetic diversity existed within the sampled Shandong germplasm. UPGMA cluster analysis based on genetic similarity coefficients (GS range: 0.49 to 0.98) clustered all 76 accessions into a hierarchical dendrogram. At a GS threshold of 0.688, four distinct genetic clusters emerged: GroupⅠ (n=40): Encompassed accessions with extensive morphological variation, including red/pink flowers, single/double petals, and both ornamental/edible fruit types. Group Ⅱ (n=27): Contained varieties exhibiting diverse pericarp colors and floral characteristics. Group Ⅲ (n=2): Comprised morphologically distinct accessions. Group Ⅳ (n=7): Included varieties with heterogeneous phenotypic traits. Critically, the molecular classification exhibited incongruence with prior morphological taxonomy across all groups. Accessions in Groups Ⅲ and Ⅳ showed the greatest genetic divergence, making them prime candidates for hybrid breeding programs to exploit heterosis and introgress novel genetic variation. Notably, 67 accessions (88.16% of the collection) coalesced into a single cluster at GS=0.678, indicating constrained genetic diversity within the dominant Shandong germplasm. This finding necessitates strategic introduction of genetically distant elite germplasm to broaden the genetic base for sustained breeding progress. According to the UPGMA clustering results, the variety discrimination efficiency of a single primer was 15.78% to 85.52%. No single primer distinguished all 76 accessions, necessitating combinatorial screening. The primer pair UBC834/UBC835 achieved 97.37% discrimination efficiency, generating unique banding profiles for 74 accessions. Only cultivars No. 75 (Hydrangea Peony) and No. 76 (Purple Skin Sweet) shared identical fragment patterns, suggesting either recent divergence or clonal relationship. Consequently, a comprehensive DNA fingerprinting system was established using polymorphic markers from the UBC834/UBC835 combination, enabling unambiguous molecular identification of 74 accessions. 【Conclusion】The 16 ISSR primers used in this study were able to successfully analyze the genetic variation of pomegranate germplasm resources. The genetic diversity of the pomegranate population in Shandong Province was relatively rich, and the ISSR classification results were not consistent with tra- ditional morphological classification. The DNA fingerprints system of 74 accessions established in this study exhibited cultivar- specific characteristics, offering a scientific basis for the scientific classification, precise identification, and breeding of new pomegranate cultivars of pomegranate germplasm resources.