PCR-RFLP Analysis Chloroplast DNA Among 30 Tea (Camellia Sinensis(L.)O.K Untze) Cultivars In Sichuan, China

Sichuan is rich in tea resource and has plenty of introduced and breeding tea varieties. At present, the common means of evaluation and utilization of tea varieties are traditional breeding methods. In this study, 30 tea cultivars, including those origins in Sichuan and introduced from Fujian, Zhejiang, Guizhou,Hunan, Guangzhou into sichuan tea areas, as materials.This article analysed the polymorphism of the cpDNA of 30 tea cultivars by PCR-RFLP and maked the cpDNA molecular dendrogram. Not only provided a favorable theoretical basis for hybridization and cultivation of new tea varieties,but asol in favor of the collation and research in tea varieties. The main results were briefly summarized as follows:1. Optimized the PCR-RFLP conditions through two orthogonal designs.The optimal PCR system for PCR-RFLP analysis of tea was as follows: 3U Taq polymerase, 1×buffer , 1.5 mmol/ L MgCl2,200μmol/ L dNTPs , 50ng cpDNA primer, 100ng DNA template in 25μl reaction solution. The optimal digestion system for PCR-RFLP analysis was as follows: 6μL amplification products,2U Endonuclease, 1×Endonuclease buffer in digestion solution , digestion time was 6h at 37℃.2. A total of 135 bands were detected in 53 cpDNA PCR-RFLP marker/enzyme combinations of 7 cpDNA PCR markers,among which 99 bands ( 73.3% ) were polymorphic.The cpDNA PCR-RFLP-based genetic diversity (GD) among 30 tea cultivars ranged from 0 to 0.071, with the mean of 0.049. The results indicated that the genetic diversity of nDNA was much higher than cpDNA in tea plant, and the extent of variation was about 8 times of cpDNA.3. All 30 tea cultivars could not be distinguished by 7 cpDNA PCR markers, the genetic diversity of 9 materials were 0, these 9 materials could not be distinguished effectively. Based on genetic diversity, the UPGMA dendrogram of 30 tea cultivars was made by NTSYS cluster analysis. At genetic diversity of 0.073, all the accessions could be clustered into three groups.6 cultivars from Sichuan and Guizhou(Mengshan9, Mengshan11, Mengshan23, Shuyong307, Qianmei419, Juhuaxiang) were classified into group1; 17 cultivars origins from Zhejiang and Fujian(Anjibaicha, Wuniuzao, Longjing43, Longjingchangye, Zhe'nong113, Zhe'nong117, Chunbolv, Fuding, Yingshuang, Jingfeng, Yuanxiaocha, Zhenghe, Meizhan, Fudingdahaocha, Fuxuan9, Pingyangtezao, Fujianshuixian) were classified into group2;others (Donghuzao, Zhuyeqi, Qianmei303, Yinghong1, Yinghong2, Hainandaye, Huangyeshuixian) were classified into group3.