Development and application of a multiple nucleotide polymorphism (MNP)-based molecular identification system for...

Pear is one of the most popular and widely cultivated fruits globally, with rich cultivar diversity. Pyrus species are characterized by self-incompatibility and the absence of reproductive barriers between species, leading to extensive gene flow and genetic recombination among different types of cultivars. As a result, cultivar identification technologies have considerable practical significance in pear production. Multiple nucleotide polymorphism (MNP) technology, which combines multiplex PCR amplification and high-throughput sequencing, offers high efficiency and accuracy in pear cultivar identification, and meets the needs of cultivar innovation and industry development.

We applied MNP technology to pear cultivar identification for the first time, establishing fingerprints for major pear cultivars and exploring the feasibility of MNP markers for pear population structure analysis. Based on genomic resequencing data from 143 pear accessions, 558 marker loci were initially developed, and 310 core markers were retained after screening. The 310 MNP markers showed high polymorphism, with an average of 18.14 alleles per marker locus and PIC values ranging from 0.57 to 0.99. Validation using 76 representative pear cultivars demonstrated reproducibility and accuracy rates exceeding 99% for the 310 MNP markers. A total of 2,850 pairwise comparisons among the 76 cultivars showed an average genetic differentiation of 90.89%. Population structure analyses based on MNP markers effectively reflected the classification relationships among the 76 cultivars, clearly distinguishing European from Asian pears.

In summary, the developed pear MNP markers possess high polymorphism, stability, and cultivar-discrimination capability, promising extensive future applications in pear cultivar identification and population genetic research.