| Tea plant [Camellia sinensis (L.).O.Kuntze] originated from China, and is a perennially and economically important plant. Tea plant is widely distributed in China, easily adapted to different ecological conditions, and hybridized both naturally and artificially, which have formed considerably abundant tea germplasm resources. In this study, using EST-SSR and ISSR techniques, we have worked on tea germplasms from Yunnan Province, which provided more theoretical evidences for classification, identification, breeding and germplasm conservation of Yunnan tea resources. The main results are summarized as followings:1. Thirty SSR primer pairs with better polymorphism in this study, including fifteen new primer pairs, were used to assess genetic diversity and relationship of 134 tea germplasms from Yunnan Province. The analysis results showed a high level of genetic polymorphism and a broad genetic variation in Yunnan tea resources, but the genetic variation between wild tea germplasms and traditional cultivars was unconspicuous. A total of 127 putative alleles were generated, with a mean of 4.23 for putative alleles per locus. Totally 263 Genotypes were detected in all materials, with a mean of 8.8 for each polymorphism primer pairs. The polymorphism information content (PIC) varied from 0.014 to 0.736, with a higher average of 0.501 than pertinent research results. These results showed a high level of genetic polymorphism in Yunnan tea resources. The average genetic distance and the average similarity coefficient among 134 tea varieties were 0.413 and 0.597, respectively. These results suggested that the materials used in the experiment possessed a broad genetic variation. Based on the similarity coefficient, four major groups were generated from all the accessions tested by UPGMA clustering analysis. The principal components analysis (PCA) results suggested that the first three components of PCA accounted for 27.93% of total variation. The Clustering and the principal components analysis showed the complicated genetic relationships among tested tea resources. The average PIC and the mean similarity coefficient analyses indicated that the genetic diversity was considerably abundant whether within traditional cultivars or wild resources, but the genetic variation between wild tea germplasms and traditional cultivars was unconspicuous.2. Two different DNA-based techniques, inter-simple sequence repeats (ISSR) and (expressed sequence tags) ESTs-derived SSRs (simple sequence repeats), were used for detecting genetic diversity of 33 tea accessions. The analysis indicated that both DNA-based techniques were able to amplify all of the genotypes, showing a high genetic diversity and a good consistency among tea resources tested, but ISSRs presented greater information content. Using 12 ISSR primers, 248 discernible DNA fragments were generated with 228 (91.94%) being polymorphic, the mean PIC value of each ISSR primer was 0.318. Thirty EST-SSR primers produced an average of 4.9 alleles /locus and an average PIC value of 0.499. Both DNA-based techniques were able to amplify all of the genotypes, showing a high genetic diversity among tea resources tested. ISSRs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 33 tea genotypes, and ISSRs presented a greater information content. The mean genetic similarity coefficients among all tea genotypes ascribed by ISSR and EST-SSR matrices were closely 0.709 and 0.743, respectively. Some differences in clustering results were observed. The correlation coefficients of similarity were statistically significant for both marker systems used (r=0.817, p﹤0.01). It could be inferred that the two molecular fingerprinting techniques both were suitable for tea genetic diversity research.3. In this study, inter-simple sequence repeats (ISSR) was used to determine genetic relationships among 134 tea germplasms from the China National Germplasm Tea Repositories (CNGTR). The results indicated that these tea resources in Yunnan possessed a high level of genetic polymorphism, a broad genetic variation, and a good consistency of Principal Component analysis and cluster analysis, and were divided into three main groups, but genetic diversity of different populations existed variation. Eighteen reliable ISSR primers were able to separate or distinguish all of the 134 tea germplasms based on a total of 470 polymorphic bands (98.9%) among the test accessions. The mean genetic distance and the average Jaccard similarity coefficient among the tested genotypes were 0.465 and 0.512, respectively, indicating a wide gene pool of Yunnan tea resources. The 134 tea germplasms were divided into three main groups on the basis of unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis. The dendrogram did not indicate clear division among tested germplasms based on their geographical origin. Principal Component analysis (PCA) for ISSR data supported UPGMA clustering result, but showed more explicit relationships among the test accessions with different lays and orientation. The genetic similarity coefficient among 8 populations ranged from 0.850 to 0.987, with a 0.92 average, indicating that genetic diversity of different populations existed variation.4. Molecular identification of 134 tea germplasm resources using ISSR markers was conducted in the study. There were 3 independen ways to identify tea germplasms:a) unique ISSR markers;b) unique band patterns and c) a combination of the band patterns provided by different primers.The result showed that the presence of 10 unique ISSR markers and the absence of 15 unique markers obtained from 12 primers made it possible to identify 21 tea germplasms. Using 54 unique band patterns of primer UBC811 could identify 35 tea germplasms. It was of much importance using minimum primers to obtain the maximum identification ability.A1l the 134 tea germplasms could be entirely identified by the band patterns combination of primer UBC811,UBC835,ISSR2 and ISSR3,which was successfully used to construct the ISSR fingerprinting for discriminating 134 tea germplasms from Yunnan Province.5. Parentage identification of filial generation tea plants, which derived from tissue culture of immature embryos of'Yunnan Dalicha'and'Fuding Dabaicha'by artificial hybrid method, was carried out with ISSR method, and the technology of identificating the filial generations was established. There were more 96% bands in three filial generations, which were same as their male parent or female parent from 15 primers. There were not significant diference among bands between 1F1(2F2 or 3F1)and their parents in the same position byχ2 test. The clustering result and the genetic similarity coefficient clearly showed the genetic relationships among five tested tea materials. ISSR analysis suggested that they were really hybrids. |
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