Genetic diversity analysis and core collection construction of 108 pear germplasm resources

【Objective】This study aimed to systematically evaluate the genetic diversity of 108 pear germplasm resources collected from various regions across China using SSR molecular markers and fruit phenotypic traits, in order to establish an optimal methodology for constructing a core collection that would effectively represent the original germplasm while minimizing genetic redundancy. Pear (Pyrus spp.) is an economically important fruit crop in China with rich germplasm resources. Efficient management and utilization of these resources require comprehensive molecular characterization and systematic core collection development. The development of a scientifically sound core collection is crucial for enhancing the efficiency of germplasm conservation, evaluation, and utilization in breeding programs.【Methods】Young leaf tissues were collected from 108 pear accessions collected from 12 provinces in China, with particular emphasis on materials from Gansu Province (78 accessions). Genomic DNA was extracted using the CTAB method, and the quality of DNA was verified through spectrophotometry and agarose gel electrophoresis. Fifteen SSR primer pairs previously developed for pear genetic studies were selected for amplification. PCR reactions were performed in 25 μL volumes containing 0.2 mmol·L-1 of each dNTP, 0.5 μmol·L-1 of each primer, 1.0 U Taq DNA polymerase, 2.5 μL 10×PCR buffer (with Mg2 + ), and 50 ng template DNA. The amplification protocol consisted of initial denaturation at 94 ℃ for 5 min; 35 cycles of denaturation at 94 ℃ for 30 s, annealing at primer-specific temperatures (58-60 ℃) for 30 s, and extension at 72 ℃ for 30 s; followed by a final extension at 72 ℃ for 10 min. Amplification products were separated using an ABI3730XL sequencer and fragment sizes were determined using GeneMapper software. Genetic diversity parameters including observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon's information index (I), and polymorphism information content (PIC) were calculated using GenAIEx 6.5 and POPGENE version 1.32 software. Genetic relationships among accessions were analyzed through cluster analysis using UPGMA algorithm with three different genetic distance measures (Jaccard, SM, and Nei) implemented in R software. Principal component analysis (PCA) was conducted using SPSS 27.0, and score plots were generated with OriginPro 2022. For core collection construction, we systematically evaluated the effects of different clustering methods (single linkage, complete linkage, and UPGMA), genetic distances (Jaccard, SM, and Nei), sampling strategies (random and preferred sampling), and sampling proportions (5%, 10%, 15%, 20%, 25%, 30%, and 35%) on the representativeness of the resulting core collections. The preferred sampling strategy prioritized accessions with higher numbers of polymorphic loci within each cluster. The representativeness of core collections was assessed by comparing genetic diversity parameters between core and original collections, and through visual examination of PCA score plots.【Results】The 15 SSR primers generated 87 alleles across 108 pear accessions, with an average of 9.4 alleles per locus. The number of alleles per locus ranged from 3 (L14) to 20 (L2). The effective number of alleles (Ne) varied from 1.118 7 to 6.879 4, with a mean of 3.778 1. The average values for Shannon's information index (I), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were 1.443 5, 0.487 0, 0.644 5, and 0.614 5, respectively. Twelve primers showed PIC values higher than 0.5, indicating high polymorphism. Cluster analysis based on UPGMA algorithm with Jaccard distance coefficients grouped the 108 accessions into three major clusters. The cluster Ⅰ contained 84 accessions from 11 provinces, with Gansu (56 accessions, 66.67% ) and Liaoning (10 accessions, 11.40% ) being the most represented. This cluster was further divided into seven subclusters without clear geographical patterns. The cluster Ⅱ comprised 19 accessions, predominantly from Gansu (16 accessions, 84.21%). The cluster Ⅲ included 5 accessions from Shaanxi, Liaoning, and Henan. The genetic distance among accessions ranged from 0 to 0.933, with a mean of 0.592. The accessions 29 (Hongli) and 106 (Lüyu Hongli) showed a genetic distance of 0, suggesting they might represent the same genotype. The PCA results were consistent with the cluster analysis, showing clear separation among the three clusters. For core collection construction, comparative analysis revealed that the combination of Jaccard genetic distance, UPGMA clustering method, and preferred sampling strategy yielded the most representative core collection with the highest values for effective allele number (Ne), Nei's gene diversity (H), Shannon's information index (I), and polymorphism information content (PIC). The evaluation of different sampling proportions demonstrated that the 20% proportion (22 accessions) achieved the optimal balance between representativeness and efficiency, with maximum values for Ne (3.843 2) and H (0.651 0). The core collection retained 67.13% of alleles (Na) from the original germplasm, while showing increases in Ne (110.51%), H (104.97%), I (101.62%), and PIC (105.09%) compared with the original collection. The PCA validation confirmed that the core collection effectively captured the genetic variation present in the original germplasm. The final core collection comprised 22 accessions (numbers 3, 9, 11, 12, 13, 15, 18, 24, 28, 34, 47, 49, 62, 67, 68, 78, 85, 87, 89, 94, 96, and 103 in Table 1).【Conclusion】This study demonstrated substantial genetic diversity among pear germplasm resources in China, which would provide a solid foundation for efficient conservation and utilization. The optimal strategy for constructing a representative core collection involved using Jaccard genetic distance, UPGMA clustering method, preferred sampling strategy, and a 20% sampling proportion. The resulting core collection of 22 accessions effectively captured the genetic diversity of the original 108 accessions while significantly reducing redundancy. This core collection would facilitate more efficient management, evaluation, and utilization of pear genetic resources in future breeding programs and conservation efforts. The methodology established in this study could serve as a reference for core collection development in other perennial fruit crops.