| Tea (Camellia sinensis) is a perennial cross-pollinated plant, with a high degree of heterogeneity. During long-term propagation, a variety of tea cultivars (lines) were selected in different tea area. Most of them were bred from natural hybrids or local groups through the system selection, but their pedigree and genetic relationship was not clear. The knowledge and information on genetic relationships, their diversity is essential for proper utilization and in plant breeding. The present study was undertaken to estimate the genetic variations and relationships of tea cultivars (lines) in order to selection of the cultivar for classification, hybridization program and identity of tea using the ISSR, RAPD and isozyme markers.1.SDS/silica gel adsorption column method, a simple and safe extraction of tea genomic DNA methods was established. DNA was extracted from winter mature leaves by changing the DNA extraction buffer formulations and silica gel adsorption column specific combination of DNA. The A260/A280,size, yield, of extracted DNA was 1.62～1.85,23kb, 36.55μg/g～60.37μ/g, respectively. It was also suitable for downstream PCR amplification. Fresh winter winter mature leaves as selected as material, it is very important for DNA extracted from fresh mature tea throughout the year.2. Genetic variations and relationships of tea cultivars (lines) of different tea areas were analyzed using the ISSR, RAPD and isozyme markers.(1) ①Out of the 200 ISSR primers 22 were chosen for the study. These amplified a total of 204 bands out of which 169 (80.29%) were polymorphic, and their polymorphism information index (PIC) ranged from 0.73 to 0.91. It was also found that there was no clear relationship between total repeat lengthand degree of polymorphism in the present study. ②Out of the 200 RAPD primers 23 were chosen for the study. These amplified a total of 225 bands out of which 199 (88.44%) were polymorphic, and their PIC ranged from 0.63 to 0.91.③Analysis conditions for the peroxidase and esterase were created and optimized, and the zymography with mature leaves under a higher concentration gel electrophoresis was good.16 peroxidase were isolated, and their relative mobility (Rf value) between 0.02 to 0.56,18 esterase were also isolated with Rf value between 0.01 to 0.79.(2)①ISSR analysis showed that number of polymorphic loci (PPL), gene diversity index (H) and shannon information index (Ⅰ) of cultivars (lines) in three tea area were in the range of 74.02% to 75.98%,0.22 to 0.23,0.33 to 0.34. ②RAPD analysis showed that PPL, H and Iof cultivars (lines) were in the range of 77.78% to 80.00%,0.22 to 0.23,0.33 to 0.34. diversity levels whthin tea area were lower than the overall level.(3) ①ISSR analysis showed that genetic similarity coefficient (GS) among cultivars (lines) was in the range of 0.61 to 0.89, with an average of 0.73.②RPD analysis showed that GS was in the range of 0.62 to 0.88, with an average of 0.72.③Peroxidase and esterase analysis showed that GS was in the range of 0.44 to 0.91, with an average of 0.67. These showed that genetic basis of tea cultivars (lines) were relatively narrow.(4) ①ISSR clustering analysis showed that the tea cultivars (lines) were divided into two categories, the first category included cultivars (lines) in southwest tea area, the second category included cultivars (lines) in south China tea area and in jiangnan tea area. ②RAPD cluster analysis showed that they were divided into three categories, the first category included cultivars (lines) in southwest tea area, the second category included cultivars (lines) in southwest tea area, the third category included cultivars (lines) in south China tea area and in jiangnan tea area, ﹑eroxidase and esterase clustering analysis showed that they divided into two categories, the first category included ultivars (lines) in southwest tea area and a cultivar in south China tea areas, the second category included cuultivars (lines) in south China tea area and in jiangnan tea area, and a cultivar in Southwest tea area. The above analysis showed that the clustering results were related with route of transmission, but differences with the botanical classification. It was also found that the clustering results did not support that cultivars were clustered according to chromosome number, germination, or manufacture suitability. This provides a basis for the correct tea cultivars classification.(5) The taxonomic status of Taiwan-dayezhong were C.sinensis var assamica with ISSR, RAPD and isozyme analysis combined with morphological characteristics.(6) ISSR specific markers and RAPD specific markers could be used as molecular basis of identification of specific cultivars of Anjibaicha, Longjing 43, Liannan-dayecha.3. Genetic variations and relationships of tea cultivars(lines) of the same tea areas were analyzed using RAPD markers.(1) 20 primers were chosen for the study. These amplified a total of 265 bands out of which 242 (91.32%) were polymorphic, and their PIC ranged from 0.82 to 0.92.(2) RAPD analysis showed that GS was in the range of 0.62 to 0.96, with an average of 0.70. These showed that genetic basis of tea cultivars (lines) were relatively narrow. It was found that the genetic basis of hybrid cultivars (lines) were slightly wider than conventional cultivars (lines).(3) The cluster analysis showed that cultivars (lines) were divided into six categories, the first category included Shukelian 1, Mabianlv 1, Chuanmu 28, the second category included Mingshan 213, Yucha 2, Chuannong-huangyazao, the third category included Shuyong series, Tianfu 24, Tianfu 28, Shukelian 3, Chengxi 4, Nanjiang 3, the fouth category included Chengxi 4, Mingshan 311, Mingshan 130, the fifth category included Zaobaijian 5, the sixth category included Chongpi 71-1, Yucha 1, but Tianful 1 and Tianfu 36 were outside the free taxa. These showed that the clustering results were related with breeding source. Mingshan 213 and Chuanmu 28 were farther with most of other Chuanyu cultivars(lines) in genetic distances, and both cultivars have high agronomic traits. Both of them will be good cultivars in the future breeding programme. This is of great significance for the expansion of the genetic basis of cultivars in Sichuan and Chongqing, and breeding good cultivars.(4) RAPD specific markers could be used as molecular basis of identification of specific cultivars of Mingshan 213 and Chuannong-huangyazao.4. Genetic variations and relationships of tea cultivars (lines) from the same sexual groups of the same tea areas were analyzed using ISSR, RAPD markers.(1) Leaf characters Mengshan cultivars (lines) of Mengshan tea area were surveyed, and found that they blades were similar appearance. It was difficult to accurately separate them.(2) ①18 ISSR primers were chosen for the study. These amplified a total of 170 bands out of which 134 (76.82%) were polymorphic, and their PIC ranged from 0.74 to 0.92. ②10 RAPD primers were chosen for the study. These amplified a total of 107 bands out of which 84 (78.50%) were polymorphic, and their PIC ranged from 0.83 to 0.90.(3) ③ISSR analysis showed that GS among cultivars (lines) were in the range of 0.59 to 0.70, with an average of 0.64.②RAPD analysis showed that GS were in the range of 0.58 to 0.74, with an average of 0.65. These showed that genetic basis of tea cultivars (lines) were relatively narrow.(4) ①ISSR clustering analysis showed that tea cultivars (lines) were divided into two categories, the first category included Mengshan 9, the second category included the other cultivars (lines). ②RAPD cluster analysis showed that were divided into two categories, the first category included Mengshan 9, the second category included the other cultivars (lines). Mengshan 9 with flowers aroma and high caffeine, were farther with most of other Mengshan cultivars in genetic distances. The cultivar can be as one of the hybrid parents for Mengshan cultivars. This is of great significance for the expansion of the genetic basis of Mengshan cultivars, and breeding good cultivars.(5) ISSR specific markers and RAPD specific markers could be used as molecular basis of identification of Mengshan cultivars. |
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