Application Of Issr To Analyse Genetic Diversity And Relationship Of Tea (Camellia Sinensis (L.) O.Kuntze) Gremplasms Distributed In Wuling Mountainous Area

Artificial breeding for better stocks and asexual reproduction of tea (Camellia sinensis) has recently resulted in simplicity of tea germplasm resources creating a critical need to protect existing tea germplasm resources. Among other areas in China, Wuling mountainous area is rich in tea germplasm resources, thus protecting the local tea germplasm resources and revealing their genetic relationships will play key roles in establishing a tea germplasm resource system. The genetic diversity and relationship of183tea germplasms were studied using ISSR (Inter-Simple Sequence Repeats) technique to provide theoretical basis for tea originating in Wuling mountainous area.Based on earlier research in tea, the concentrations of Mg2+, dNTP, DNA template, Taq DNA polymerase and primer DNA were optimized by orthogonal design in ISSR amplification system of tea plants in the present study. The optimized ISSR-PCR system for tea plants was established as follows:0.75U/μL Taq DNA polymerase,2.0mmol·L-1Mg2+20ng DNA template,0.1mmol·L-1dNTP and0.3μmol·L-1primer were contained in20μL reaction system and the reaction procedure was shown as below:94℃for5min, followed by34cycles of94℃for1min, annealing for40s at50℃-60℃, extending for90s at72℃,and was terminated for7min at72℃, then stored at4℃. The bands amplified by the optimized system were clear, stable, and satisfied conditions for ISSR analysis.Twenty eight ISSR primers with high polymorphism were screened from65primers to amplify183tea germplasms where340bands were acquired. The percentage of polymorphism was96.47%. Seven to eighteen bands were amplified by a single primer, and on average each primer amplified12.14bands. The highest polymorphism was100%and the lowest was87.5%, with the bands ranging from100bp to2800bp.The genetic similarity among one hundred and eighty-three tea ascensions is between0.52-0.93, indicating a wide gene pool and a broad genetic variation of Wuling tea resources. The highest similarity of all materials between Nol and No2was0.93, suggesting small genetic difference.One hundred and eighty-three tea germplasms were divided into two sections at GS=0.64. The first category contained all materials in the tea germplasm garden and was divided into three sub-groups. The second category was divided into seven sub-groups, and two of them formed one separate and distinct group.Following the results of ISSR analysis, one hundred and eighty-three germplasms of tea were tested by UPGMA clustering analysis, and protracting the cluster analysis dendrogram based on ISSR markers, also revealed the genetic diversity and relationship of the tea germplasms from Wuling.This study therefore achieved the objective of molecular identification of183tea germplasm resources using ISSR markers indicating that it can be readily applied to identify tea germplasm resources clearly and rapidly.