Analyses On Genomic MSAP And Gene Expression In Chinese Cabbage After Continuous Seedling Breeding

Vernalization is the key process of bolting and flowering in Chinese cabbage [Brassica campestris L. ssp. pekinensis(Lour.) Olsson).Plants can turns vegetative growth into reproductive growth come with flowering only on condition that going through a low temperature induce stage. If Chinese cabbage have bolted in the early age, and because the bolting phenomenon occurred before the vegetative body fully grown, the cabbage will lose lose its edible value and lead to economic benefits greatly reduced. We observed that continuous seedlingbreeding will lead the plants easier bolting and flowering and reduce the heading of the cabbage.In this study, we use two inbred lines (A161A105) as experiment material. The two lines were subjected to vernalization and formed four types of seeds, seedling breeding once, seedling breeding twice, seedling breeding thrice and normal type, comparing the difference of plant phenotype after planting.Their genes were analyzed by MSAP technology to precede DNA methylation analysis. The difference fragments of which were cloned and analyzed, by fluorescence qPCR. Experimental results demonstrate that seedling breeding thrice plants have a loosen heading leaves and it’s center column elongated and were easier to bolting and flowering.suggest that continuous seedling breeding have a weaker winterness.Along with the continuous seedling breeding,the genome methylation level decreased.In this study,we’ve had4gene identification,they are BraAPC1、 BraEMP3、BraUBC26and BraAL5. Fluorescent qPCR analysis showed that expressiones of four genes changed in different modes of reproduction and different vernalization time.which means these genes involved in development and regulation of bolting and flowering of the plants. Our study preliminary analyzed the molecular mechanism that continuous seedling breeding could result in weaker winterness and has an important guiding significance for Chinese cabbage breeding.Main results are as follows:1According to the survey to seedling of A161and A105(the normal, vernalization once, vernalization twice, vernalization thrice), We observed that continuous seedling breeding will lead the plants more easier bolting and flowering and reduce the heading of the cabbage along with the weaker winterness.2MSAP and genomic DNA analyze of A161and A105(the normal, vernalization once, vernalization twice, vernalization thrice), we found that the two type of cabbage are similar in that genome methylation, the methylation and semi methylation level reduced with the increasing generations of continuous vernalization. 3According to the genomic DNA analyze of A161and A105with32primer, we have found that methylation bands is more than semi-methylation bands. Methylation polymorphism ratio varied from1.8%to15.6%, the average methylation polymorphism ratio is6.4%, suggest that the32primers have differences in methylation polymorphism.4We got17different bands in MSAP analyze,12of which are non-methylation bands and the other are methylation bands.5We performed recycling cloning and sequencing to the bands, and got4specific genes regulating methylation in Chinese cabbage, they were BraAL5, BraAPC1, BraEMP3and BraUBC26.6Expression analysis of the differentially methylated genes in different Vernalization generations indicate that BraAPCl, BraEMP3, BraUBC26, which expression quantity are higher than that of the normal type while the is opposite, and it’s expression quantity is decreased along with the generation increased.7Expression analysis of the differentially methylated genes in different time of vernalization suggest that BraAPC1, BraEMP3, BraUBC26, which expression quantity is higher after one week’s vernalization,and decreased along with the time of vernalization increased.BraAL5’s expression quantity is losing all the time.after three weeks of vernalization it’s expression was significantly lower than non-vernalization.