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

WU Zhiping; YANG Yang; LUO Hua; MA Li

doi:10.13925/j.cnki.gsxb.20250317

Abstract：[Objective]Pomegranate(Punica granatum L.),distinguished by its exceptional environmen-tal adaptability and extensive cultivation history in China,has emerged as a globally significant econom-ic tree crop.As a primary pomegranate production region within China,Shandong Province faces sub-stantial challenges in germplasm resource management.Being predominantly cross-pollinated,cultivat-ed pomegranates in the region exhibit considerable genetic complexity arising from spontaneous somat-ic mutations,introductions of germplasm from diverse geographical sources,and extensive varietal ex-change.Consequently,this has resulted in pronounced varietal disorganization,ambiguous genetic rela-tionships,and significant lineage confusion,collectively impeding the efficient conservation and utiliza-tion of pomegranate germplasm resources.Furthermore,accelerated commercial breeding programs have exacerbated genetic homogenization,rendering conventional morphological identification meth-ods inadequate for precise varietal discrimination and classification.This critical limitation severely constrains the development of elite cultivars and hinders the sustainable advancement of the pomegran-ate 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 pome-granate(Punica granatum L.)germplasm accessions systematically curated from the China Pomegran-ate Germplasm Resources Nursery located in Zaozhuang City,Shandong Province.Genomic DNA was extracted from fresh young leaf tissue using a modified cetyltrimethylammonium bromide(CTAB)pro-tocol.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 unbi-ased 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 eval-uated.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 analy-sis showed that mean number of observed alleles per locus(Na)was 1.980 7,effective number of al-leles 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 genet-ic 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 dendro-gram.At a GS threshold of 0.688,four distinct genetic clusters emerged:Group Ⅰ(n=40):Encom-passed accessions with extensive morphological variation,including red/pink flowers,single/double pet-als,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 acces-sions.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 di-versity within the dominant Shandong germplasm.This finding necessitates strategic introduction of ge-netically 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.Conse-quently,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 vari-ation 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 classifica-tion,precise identification,and breeding of new pomegranate cultivars of pomegranate germplasm re-sources.