Comprehensive analysis of mulberry genetic diversity based on 1-DNJ content and SNP markers

<jats:p>Mulberry trees have high economic and ecological value, and a robust molecular marker system plus germplasm genetic diversity analysis is critical for innovative utilization of high-quality medicinal and economic mulberry germplasm. Here, 51 mulberry samples were used to develop SNP primers via genome resequencing, with the SNP-PCR system optimized by single-factor and orthogonal assays. The phenotypic diversity and SNP molecular marker genetic diversity of 1-deoxynojirimycin (1-DNJ) in mulberry leaves were analyzed respectively, and the genetic correlation between molecular markers and phenotypic traits was evaluated by Mantel test. Tested germplasm showed marked 1-DNJ variation (0.4805-2.5300 mg/g, CV=0.4241), reflecting rich genetic diversity. The optimal SNP-PCR system included Buffer (containing Mg²+) 2.2 μL, 2.5 mM dNTP 0.4 μL, forward and reverse primers (10μmol·μL-1) totaling 2.75 μL, Taq DNA polymerase (5 U·μL-1) 0.3 μL, DNA (50 ng·μL-1) 1.1 μL, and ddH2O 13.65 μL. 23 highly polymorphic ones amplified 91 loci (81 polymorphic, 89.10% polymorphism rate). Genetic diversity analysis showed that the average genetic distance was 0.3010, and the average expected heterozygosity (H) and Shannon information index (I) reached 0.4667 and 0.3104 respectively, indicating that the genetic differentiation among the tested mulberry germplasms was significant and the population had a moderate to upper level of genetic diversity. UPGMA clustering divided 51 germplasms into 6 major groups at a genetic similarity coefficient of about 0.7, while phenotypic clustering based on 1-DNJ content divided them into 2 major categories and 4 subcategories with high 1-DNJ germplasm clustered independently. Mantel correlation analysis showed that 6 SNP sites were significantly weakly correlated with 1-DNJ content (r &lt; 0.3, p &lt; 0.05), and can be used as candidate molecular markers for subsequent genetic analysis of 1-DNJ content.This study established a stable mulberry SNP-PCR system, Analyze the molecular genetic characteristics of mulberry germplasm and DNJ phenotypic variation rules respectively, and provide basic data for cluster comparison. and provided a scientific basis for marker database improvement, germplasm identification and molecular-assisted breeding.</jats:p>