SRAP Molecular Markers Linked To Heat-tolerence Gene In Chinese Cabbage

In this study, different ecotypes of Chinese cabbage heat-tolerence inbred lines'413'and heat-sensitive inbred lines'96'were used as the parent hybrid combinations, in order to get F2 populations. Parents and F2 populations were used as experimental materials. SRAP molecular markers were used firstly as a method, which researched the heat-resistant characteristics of Chinese cabbage.304 pairs of SRAP primer combinations were screened initially by parental gene pools.The SRAP primer combinations obtained by the initial screening were screened further using the heat-tolerence gene pool and heat-sensitive gene pool.The primer combinations obtained by the further screening were verified using the plants of the heat-tolerence gene pool and heat-sensitive gene pool. At last, the research obtained a SRAP marker,which linked the heat-resistant Chinese cabbage gene, and that SRAP markers were sequenced. The Blast online tool of NCBI were used, and in the nucleic acid database the sequences were compared. At last, the results of sequence alignment were analysised.The results of the experiment were as follows:1. F2 population of Chinese cabbage seedlings heat injury level survey data were analyzed,and the survey data was consonant with normal distribution, indicating that heat-resistant trait is the number of genetic traits and controlled by multiple genes.2. Established the best Chinese cabbage SRAP-PCR reaction system (20μl):dNTPs 0.25mM, Mg2+1.25 mM, Primers 0.5μM, Taq enzyme 2.5U, template 25ng.3.304 SRAP primer combinations were initially screened by parental gene pools, the result showed that 10 pair primers can not be amplified; that the rest can obtain good bands; that 160 pair primers can be amplified and discrepant bands can be obtained.4. According to the F2 segregating population of Chinese cabbage of heat damage surveys, builded the heat-tolerence gene pool and heat-sensitive gene pool. Using heat and thermal pools to further screen the 160 primer combinations, obtained 36 primers combinations/which can produce different bands.36 pairs of these primer combinations were verified by the plants of the heat-tolerence and heat-sensitive gene pools. As a result, only the primer combination ME13EM2 verified and obtained a SRAP marker. The exchange value between the marker and the heat gene is 16.7%, which indicated that the linkage between the two is close.5. Specific fragments was recovered and sequenced,as a result,obtaining a sequence of 186bp length. Online Blast tools at the NCBI was used to compare results.the result is that the maximum sequence similarity.between the fragment and the sequence of Chinese cabbage chloroplast, chloroplast DNA of Brassica napusβsubunit of ATP synthase (atpB) gene sequences, the carrot and cucumber chloroplast ATP synthaseβsubunit (atpB) of the mRNA sequences are all 96%.