Construction and Application of Multiple Nucleotide Polymorphism (MNP)-Based DNA Fingerprinting for Polygonatum cyrtonema Identification

Min Tang^1,2Yuqing Long¹Wei Xiang^2,3Peng Huang²Gang Wang^4*Jianguo Zeng^2,3*

• ¹Yiyang Medical College, Yiyang, China
• ²Yuelushan laboratory, Changsha, China
• ³Hunan Agricultural University, Changsha, China
• ⁴Wuhan Mingliao Biotechnology Co., Ltd, Wuhan, China

Polygonatum cyrtonema Hua is an important economic crop with dual use as food and medicine. Its market demand has been increasing steadily. However, in the current market, widespread cultivar confusion coupled with the lack of efficient and accurate cultivar identification methods has severely hindered the genetic improvement and standardized development of its industry. In this study, we aimed to address the issues of germplasm resource confusion and difficulty in cultivar discrimination of P. cyrtonema while providing a novel technical tool for investigating the genetic diversity of Polygonatum plants. We successfully developed a high-throughput identification system based on multiple nucleotide polymorphism (MNP) markers for P. cyrtonema cultivar identification. Via genome sequencing of 30 representative accessions, we screened 505 core MNP loci with high polymorphism and further optimized and established stable protocols for multiplex polymerase chain reaction amplification and high-throughput sequencing. Validation results revealed that this marker panel had excellent amplification efficiency and polymorphism across all 80 tested accessions. For the MNP markers, the average number of allelic genotypes reached 28.95 ± 15.11. The polymorphism information content was 0.73 ± 0.16. Both metrics were substantially superior to those of traditional identification methods. Both phylogenetic analysis and principal component analysis distinguished all the tested accessions, with an identification accuracy rate of 99.90%. A unique molecular ID code was assigned to each cultivar. The MNP marker system developed in this study combines the advantages of high throughput, high accuracy, and favorable reproducibility. It provides the technical means and solutions for the authenticity identification of P. cyrtonema cultivars, purity detection of seeds and seedlings, conservation of germplasm resources, as well as the conduct of distinctness, uniformity, and stability testing for new plant varieties and the application for new variety protection rights. These findings are expected to be applied for the source authentication of medicinal plants and population genetics research.