Genetic Diversity Of Flowering Chinese Cabbage Revealed By ISSR Molecular Markers

As a variation of Chinese cabbage subspecies in Brassicas of cruciferae and a special localized vegetable in South of China,flowering Chinese cabbage(Brassica campestris L.ssp.chinensis Var.utilis Tssen.et Lee.)having a long growing history more than a century.ISSR analysis was employed to detect genetic diversity of 27 varieties,the genetic divergence among varieties were discussed through cluster analysis.The results were as following:1,Genemic DNA extraction form flowering Chinese cabbageThe modified CTAB method has been employed to extract the genomic DNA from flowering Chinese cabbage which contains abundant polysacchrides.The DNA samples were suitable for ISSR analysis.The tatios of OD₂₆₀and OD₂₈₀determined by UV spectrometers were about 1.8 and the tatios of OD₂₆₀and OD₂₃₀were about 2.0. Moreover,the results of electrophoresis were all single bright with clean lines.2,Establishment of ISSR reaction conditionsFactors which affected the ISSR-PCR reaction of flowering Chinese cabbages were studied by two methods.One of the optimal ISSR-PCR reaction systems for flowering Chinese cabbage was determined:in a total volume of 25μl consisting of 2.5μl 10×buffer,2.0mmol/L Mg²⁺,0.5 units of Taq DNA polymerase,0.24mmol/L dNTPs,0.50μmol/L primer and 30ng template DNA.The reaction program fits to ISSR-PCR of flowering Chinese cabbage was devised as following:initial 3min at 94℃, 40 cycles of 1min at 94℃,1min at 53.8℃(different primer has different ananealing temperature),45sec at 72℃,and a final 5 min extension at 72℃. 3,Screening polymorphic primers12 primers that could produce polymorphic bands in 10 typical flowering Chinese cabbage cultivars were chosen form 50 primers.4,The analysis of genetic diversity of flowering Chinese cabbageIn total 103 loci,there are 58 of which were polymorphic ones were detected using 12 ISSR primers.Total percentage of polymorphic loci(P)%was 56.3%,while Shannon's information index(I)was 0.229 and Nei's gene diversity(H)was 0.144,the observed number of aUeles(Na)was 1.563 and the effective number of alleles(Ne)was 1.225 respectively,ranging the genetic distance(GD)form 0.029 to 0.344.The result indicted that the genetic diversity of flowering Chinese cabbage was relatively low.27 cultivars were divided into six groups based on UPGMA cluster analysis.It was not accorded with the traditional classification based on the response of Chinese cabbage on temperature and cultivation season.