Rapid Indentification For The Genetic Specificity And Diversity Of Brassica Main Vegetables

In this study,32 Brassica germplasm were selected as material, including11copies of Brassica oleracea,11 copies of Brassica rapa,10 copies of Brassica juncea. Using three methods to extract DNA of Brassica seed, trying to explore a simple,fast,efficient DNA extraction method, which is applicable to SSR markers; Identification and analyses for Brassica germplasm with 31 pairs of SSR primers, trying to find the specific fragment from the three kind of Brassica vegetable, and define the genetic relationship of Brassica germplasm; Finally, cloning and sequencing the specific fragments, then analyzed their sequences. the main results are as follows:1. By comparing the DNA quality of 22 Brassica vegetable seed, which came from three different methods (SDS method, CTAB method, NaOH method), The results showed:SDS method could extract DNA for the best quality and high integrity, OD260/ OD 280 average was 1.873, As good as the DNA quality of leaves DNA extracted with CTAB method (OD260/OD 280 ratio of 1.802-1.906); CTAB method was the second, which OD260/OD 280 average was 1.600; DNA quality extracted with NaOH method was worst, OD260/OD 280 values were lower than 1.3.2. Analysis for the inter-specific specificity,intraspecific polymorphism and genetic relationship of the three kind of Brassica vegetables (Brassica oleracea, Brassica rapa, Brassica juncea) with SSR marker.21 pairs of polymorphic primers were screened out of 31 pairs of primers, which amplified 113 clear and stable polymorphic bands. Primer MF4, SSRRAS46, P004 and NA3 had specific bands respectively in B. Oleracea, B. Rapa, B. Juncea, and the specific bands were verified by 10 material verification. The average PIC value was 0.604 from 21 pairs of polymorphic primers, primer B01 could distinguish five varieties of B. oleracea germplasm, eight varieties of B. rapa germplasm, JU5 could distinguish five varieties of B juncea germplasm. The cluster analysis divided the germplasm into 2 groups:one group of B. Oleracea, B. rapa and B. junce as class. The Bootstrap values were up to 100%. B. rapa and B. juncea were clustered into two subgroups when the similarity coefficient was 0.29. The results of principal coordinate analysis were consistent with those of cluster analysis.3. Cloning and sequencing the specific fragments, then analyzed their sequences. The length of specific fragment was 140bp in B. oleracea, The length of specific fragment was 194bp in B. rapa, NA3 and P004 in B. junce amplified specific fragment lengh respectively was 207bp and 213bp. By BLAST alignment, the homology from specific sequence comparison with the original sequence was 98%,100%. The homology comparison with other sequences was also higher, reaching 85%-96%.