Preliminary Study Of Germplasm Genetic Diversity In Non-heading Chinese Cabbage With Molecular Marker

The non-heading Chinese cabbage (Brassic campestris L. ssp. Chinensis(L.)Makino) is an important vegetable crop and is native to China, with numerous and complex germplasms. However, with the application of some high yield varieties in large area, many local varieties were eliminated, and variety singleness and genetic frangibility were increasingly serious. Researches on evaluation and classification of non-heading Chinese cabbage germplasm with molecular markers play an important role in germplasm innovation and new varieties breeding. In order to exploit the genetic potential, widen genetic foundation and make full use of non-heading Chinese cabbage germplasm, the genetic diversity of non-heading Chinese cabbage germplasm at molecular level were analyzed in this study. In the experiment, the best ISSR and SRAP reaction system was established by combination of single factor tests and orthogonal design. 96 germplasms were used in this study to analyze their genetic diversity with ISSR, RAPD and SRAP markers. Genetic similarity coefficients were calculated by Jaccard method and clustering was conducted for 96 germplasms by UPGMA, and genetic differentiation was analyzed among non-heading Chinese cabbage. Two hybrids and their parents of non-heading Chinese cabbage were analyzed by using RAPD, ISSR and SRAP methods, and the DNA fingerprinting of hybrids was constructed preliminarily in two crosses. The main results were as follows:1. The best concentration of four factors, such as Taq DNA polymerase, dNTP, primer and Mg2+, was identified by single factor tests in the ISSR and SRAP amplification system on non-heading Chinese cabbage. And then orthogonal scheme in four factors at three levels was designed based on the result of above single factor tests to optimize ISSR and SRAP amplification system. A suitable ISSR reaction system was established that included 20μL reaction volume containing Taq DNA polymerase1U, dNTP 0.25mmol/L, primer 0.25μmol/L, 1×PCR buffer, Mg2+ 2.5mmol/L, and template DNA 30ng. An ideal SRAP reaction system was also established that included 20μL reaction volume containing Taq DNA polymerase1∪, dNTP 0.15mmol/L, forward primer 0.35μmol/L, reverse primer 0.35μmol/L, Mg2+ 3.5mmol/L.The optimal annealing temperature and cycling numbers for ISSR-PCR reaction were confirmed by gradient PCR and the gradient treatment of cycling numbers. Seven species of Brassic campestris L could be identified effectively by the optimized reaction system. It might provide a groundwork for germplasm classification, genetic map construction and important gene mapping in non-heading Chinese cabbage with ISSR and SRAP markers.2. 96 germplasms were used in this study to analyze their genetic diversity with ISSR, RAPD and SRAP markers. The results showed that SRAP has the highest polymorphic among three markers, followed by RAPD and ISSR. With 11 SRAP primers, 14 RAPD primers and 15 ISSR primers, 346, 98 and 185 bands were amplified respectively in 96 germplasms of non-heading Chinese cabbage, and 293 (84.68%) bands, 71 (72.45%) bands and 127(68.65%) bands were presented polymorphic respectively. The Shannon information index of B. c. var. communis was the highest among different types, especially the autumn-winter Chinese cabbage. Clustering results showed that the different types could not be separated absolutely from each other, and especially in the clustering result with RAPD method. On the whole, the"Xiangqingcai"germplasms in B. c. var. communis were rather specific, the majority of them were clustering together and distinguished from the others of B. c. var. communis. The analysis of genetic divisity showed that the coefficient of genetic differentiation among different types of non-heading Chinese cabbage was lower, most of genetic variation existed in same types. The analysis of gene flow showed that the gene flowed more among types, and this indicated that the degree of genetic differentiation was rather lower . In addition, it was found that the use of combination markers can effectively increase the veracity of the diversity research. Results with three markers methods were not only mutual authentication but also complementarity. Mantel-test revealed that the correlation of results between three markers was lower, and this indicated that they can reflect the genetic background of non-heading Chinese cabbage in different ways. It was more authoritative and objective to analyze the genetic diversity and relationship among non-heading Chinese cabbage germplasm by integrating test results of three markers. In this paper, combination marker had shown its superiority, especially in clustering, testing results with combination markers were regular more than with single marker. 9 specific markers of 6 germplasms had been found in this study, through cloning and sequencing the specific fragment, one specific marker(UBC834-206) was successfully converted into the SCAR marker(SU834) which can quickly identify the specific germplasm(206).3. Two hybrids and their parents of non-heading Chinese cabbage were analyzed by using three molecular markers RAPD, ISSR and SRAP. The results indicated that three RAPD primers and three ISSR primers and three pairs of SRAP primers could amplify parental complementary characteristic bands for the"Xinxiaqing 2"; three ISSR primers and two pairs of SRAP primers could amplify parental complementary characteristic bands for the"Xinlu". The molecular fingerprint of two hybrids of non-heading Chinese cabbage was established, and then converted into digital fingerprints. It provided a new technology and an important means for identification of seed purity and was good for protection of intellectual property of new varieties.