Genetic Diversity And Relationship Of30Tea Plant Main Germplasm In Sichuan Revealed By SRAP Marker

Sichuan has a very long history of cultivating tea plant and is the core of tea plant originated. The natural environment in Sichuan is very complex and diverse and suitable for the growth of various types of tea varieties. So Sichuan has very rich germplasm resource in which the Gulin Rhododendron chrysanthum Pallas、Yingjing Loquat tea、 Chongzhou Loquat tea and Nanjiang large leaf tea is particularly well-known. Although domestic and foreign scholars have preliminary investigationn on botanical characteristics, biological characteristics, main component index, tea quality and resistance some of these varieties of resources, botany Preliminary study of botanical characteristics, biological characteristics, indicators of the main component of tea quality and resistance of these resources, but the genetic diversity of these varieties of resources has not been reported. In this research, the main local groups of Sichuan tea germplasm had been researched and levels of diversity and variability of these resources had systematically evaluated and the genetic distance among these resources had been analyzed, which has provided a theoretical basis for the further protection and development of good, specific tea materials and varieties breeding of Sichuan tea germplasm resources.Using SRAP marker method, we chose30local groups of tea resources as materials, which from Sichuan main tea-growing areas such as Muchuan, Chongzhou Yingjing, Nam Giang, Gulin, Mingshan, etc.Their genomic DNA were extracted by modified SDS method. To optimize amplification procedure of SRAP-PCR and reaction system, we designed8renaturation temperature gradient,6template concentration gradient,6primer concentration gradient,6dNTPs concentration gradient,6Mg2+concentration gradient,6Taq DNA polymerase concentration gradient.6forward primers and8reverse primer by combination of two formed48primer combinations from which better amplification primer combinations were selected to amplify all samples. The PCR products by polyacrylamide gel electrophoresis were separated and were stained by silver staining and were recorded by photograph. Through the artificial method counting DNA bands, a0/1matrix was built and DNA bands were counted, and with POPGENE and NTSYS software to analyzes data, the genetic similarity coefficients for test material were obtained.And with cluster analysis method, the molecular tree analyzing the genetic relationship between the tested materials was constructed. The main results of this test are as follows:1. The optimum reaction system (25ul) of SRAP markers are as follows:30,0ng of DNA template,0.8umol/L of primer,0.3mmol/L of dNTPs,3.0mmol/L of Mg2+,2.0U of TaqDNA polymerase,2.5ul of10xBuffer (Mg2+free), taking ddH2O as complementarity.2.11primer combinations with better amplification effect selected from48primer combinations were used to amply30test materials, in SRAP-PCR, and there were2744amplified loci, detected, in which2474loci were polymorphic, accounting for90.16%of the total number of loci. The percentage of polymorphic loci is between75.14%-100.00%with an average of92.43%. And polymorphism information index is between0.22-0.36with an average of0.30. The total number of every material’s amplified bands is between55-118with an average of91.47. The number of polymorphic bands is between46-109with an average of82.47. The percentage of polymorphic bands is between83.64%-92.31%with an average of89.62%.3. Genetic diversity index is between0.3421-0.5455with an average of0.4227, and shannon index is between0.2043-0.3710with an average of0.2711, which suggests that the genetic diversity of the30tested materials is higher.30copies of the test material is divided into A, B, two categories.At the genetic similarity coefficient of0.66,30samples can be divided into A, B two categories, and at the genetic similarity coefficient of0.72, A can be divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ four subclasses,and at the genetic similarity coefficient of0.78, B can be divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ four subclasses.