Optimization of InDel molecular marker system and analysis of genetic diversity in mulberry

【Objective】By developing species- specific molecular markers for mulberry, we systematically dissected the genetic diversity of mulberry germplasm resources. This approach would provide a solid foundation for accurate germplasm identification, analysis of genetic relationships, and selection of core germplasm collections. Ultimately, it facilitates the efficient utilization of breeding materials, supports germplasm innovation, and thus accelerates the development of elite mulberry varieties. 【Methods】In this study, 46 mulberry germplasm resources were used as test materials, and Indel primers were designed and screened based on mulberry genome resequencing data. The InDel-PCR reaction system was optimized via single-factor and orthogonal experiments to ensure the accuracy of PCR results. Subsequently, genetic diversity analysis was conducted on the test materials using the optimized system and the selected primers. The experimental procedure involved collecting 46 representative mulberry germplasm resources, extracting genomic DNA, and performing genome resequencing. Indel primers were designed from resequencing data. Single- factor experiments were conducted to explore the optimal concentration ranges of PCR components: (Buffer with Mg2 + , dNTPs, primers, Taq DNA polymerase, template, and ddH2O. Orthogonal experiments were then performed to determine the optimal combination of these components. Finally, the optimized reaction system and polymorphic primers were used to amplify the genomic DNA of 46 mulberry germplasm resources, which enabled genetic diversity analysis based on the amplification results.【Results】The final optimal InDel-PCR reaction system was determined to consist of 2.0 μL of Buffer (containing Mg2+ ), 0.4 μL of dNTPs, 0.8 μL each of upstream and downstream primers (10 μmol· μL-1 ), 0.25 μL of Taq DNA polymerase, 1 μL of DNA (50 ng·μL-1 ), and 14.75 μL of ddH2O. The amplification program was set as follows: initial pre-denaturation at 95 ℃ for 3 min, followed by 30 cycles of denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 60 s, and extension at 72 ℃ for 60 s, with a final extension at 72 ℃ for 10 min, and the reaction product was stored at 4 ℃. This reaction system and amplification protocol demonstrated good stability and repeatability, ensuring the smooth conduct of subsequent experiments. From the 42 pairs of designed primers, 17 pairs with polymorphism and clear bands were screened out. These screened primers could effectively distinguish different mulberry germplasm resources and provide reliable tools for genetic diversity analysis. Using these 17 pairs of primers, a total of 37 polymorphic loci were detected. For each locus, the observed number of alleles (Na) was 2.0, the effective number of alleles (Ne) averaged 1.524, and Shannon's information index (I) ranged from 0.112 5 to 0.692 0, with an average of 0.470 1. Nei's genetic diversity (h) ranged between 0.023 7 and 0.499 7, with an average of 0.374 1, and the average observed heterozygosity (Ho) and expected heterozygosity (He) were 0 and 0.308 8, respectively. These data indicated that the tested mulberry germplasm resources exhibited rich genetic diversity, providing a solid genetic foundation for germplasm innovation and variety improvement.【Conclusion】Using the optimized InDel-PCR reaction system for Morus (mulberry trees) and the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis, the tested mulberry materials were classified into six major groups. The results revealed the complex geographic genetic relationships among different mulberry germplasm resources. These research findings can provide a scientific basis for improving the mulberry molecular marker database, identifying mulberry germplasm resources, and conducting molecular-assisted breeding for mulberry.