Establishment of Start Codon Targeted Polymorphism Molecular Markers System and Genetic Diversity Analysis of Fritillaria thunbergii

After years of selective breeding, numerous cultivars of Fritillaria thunbergii have been cultivated in various regions of Zhejiang Province, showing significant genomic and epigenetic differences. Molecular marker loci developed so far are mainly based on non-coding regions of the genome, and their identification efficiency is limited when applied to these cultivars. Therefore, this study used start codon targeted polymorphism molecular marker to detect PCR amplification of 100 Fritillaria thunbergii samples from various sources. The SCoT-PCR reaction system was optimized using a single-factor and orthogonal testing to determine the optimal conditions: DNA concentration of 4.2 ng • μL−1, primer concentration of 0.88 μmol • L−1, dNTP concentration of 0.35 mmol • L−1, Mg2+ concentration of 2.5 mmol • L−1, Supercharm Fidelity enzyme amount of 1.2 U, and total reaction volume of 25 μL. Moreover, 16 effective primers were identified, including SCoT1, SCoT2, SCoT3, SCoT7, SCoT11, SCoT12, SCoT14, SCoT17, SCoT19, SCoT23, SCoT25, SCoT26, SCoT29, SCoT35, and SCoT37. The annealing temperature for each primer was determined. Among them, SCoT14, SCoT25, SCoT29, SCoT35, and SCoT41 were specific for identifying six main cultivars: “Zhebei 1”, “Zhebei 2”, “Zhebei 3”, “Xuansanzi”, “Sanzi”, and “Duozi”. SCoT7, SCoT14, and SCoT35 were useful in tracking mutations in progeny of Zhebei 3 after radiation treatment, while SCoT29 could distinguish Zhebei 1 from Chunan. In addition, cluster analysis was conducted to simulate the genetic relationship between different cultivars, demonstrating that genetic similarity analysis is practically significant for predicting genetic correlation among different cultivars. The results not only improve the identification ability of Fritillaria thunbergii cultivars, but also help objectively evaluate their genetic stability and environmental adaptability. These achievements provide important scientific evidence for the ecological protection and utilization of germplasm resources of Fritillaria thunbergii and serve as a reference for exploring genetic diversity and evolution of traditional Chinese medicine.