Establishment of SCoT-PCR reaction system and primer screening of Guangxi Zhuang-Yao medicine Uvaria kweichowensis

【Objective】The purpose of the study was to establish the optimal SCoT-PCR reaction system for Uvaria kweichowensis，and to screen out the suitable SCoT primers，so as to provide theoretical references for germplasm identification and classification，genetic relationship analysis and genetic diversity analysis of U. kweichowensis.【Method】 Four wild germplasms of U. kweichowensis in Hechi， Baise， Liuzhou and Nanning of Guangxi were used as test materials， and the three factors of the SCoT-PCR reaction system of U. kweichowensis， namely， DNA template dosage， primer dosage and Mix premix dosage were optimized by single factor test and orthogonal test，and the optimized SCoT-PCR reaction system was used for SCoT primer screening. The optimal SCoT-PCR reaction system and screened primers were used to analyze the genetic diversity of U. kweichowensis germplasm.【Result】The results of single factor test showed that the amplification effect was better at the dosages of 40-70 ng of DNA template，1.0-1.4 μL of primer（10 μmoL/L），and 9.0-12.0 μL of Mix premix. The optimal SCoT-PCR reaction system（20.0 μL） for U. kichowensis was determined by L₁₆ （4³）orthogonal test：1.0 μL of DNA template（40 ng/μL），1.2 μL of primer（10 μmoL/L），10.0 μL of Mix premix，and 7.8 μL of ddH₂O. The 26 SCoT primers suitable for U. kweichowensis were screened using the optimal reaction system， and the optimal annealing temperature of each primer was determined. A total of 305 gene loci were amplified by 26 SCoT primers in four U. kweichowensis materials，of which 204 were polymorphic loci，wit a polymorphism rate of 66.89%. On average， 11.7 gene loci were amplified by each primer，among which there were 8.7 polymorphic loci. The average allele number（Na）of the four U. kweichowensis germplasms was 1.7377，the average effective number of alleles（Ne） was 1.5344，the Nei’s gene diversity index（H'）was 0.3053，and Shannon index（I） was 0.4449. The genetic similarity coefficients among the four U. kweichowensis ranged from 0.5443 to 0.6492，and the genetic distances ranged from 0.4320 to 0.6083，indicating that there was a large genetic variation among the four U. kweichowensis germplasms，and the level of genetic diversity was high.【Conclusion】 The established SCoT-PCR optimal reaction system and the screened 26 SCoT marker primers have good amplification effects，which can be used for germplasm identification and classification， genetic relationship analysis， and genetic diversity analysis of germplasm resources of U. kweichowensis.