Alterations Of The Methylation Patterns Around The Transposable Elements In The First Four Generations Of The Synthetic Brassica Napus Allotetraploid

Polyploidization is a common phenomenon in nature, which plays an important role in the evolution of plants, but the generation of polyploidy and the mechanism of evolution has not yet been thoroughly researched clearly. Brassica napus is a polyploidy that obtained by genome doubling after crossing Brassica. rapa and Brassica. oleracea under natural conditions, is also one of the typical research object of polyploidization. The synthetic B. napus allotetraploid not only can be used to broaden the genetic diversity of B. napus, provide new germplasm resources for rapeseed breeding, but also can be used to research the specific changes in the process of evolution by artificially recreating the heterologous hybridization process. Large-scale changes in genomic structure and the gene expression level will happen in the forming process of synthetic B. napus allotetraploid, with a large number of epigenetics changes at the same time. Some research results indicated that transposons may play a central role in reconstruction of allopolyploidization and epigenetic regulatory mechanisms, but the understand about its specific function and regulation mechanism is not so much. DNA methylation as one of the earlier found epigenetic phenomena, plays a primary and important role in epigenetic regulatory. The activity of transposon is related to the degree of methylation closely.By now there is little research for alterations of the methylation patterns around the transposable elements in the early generations of the synthetic B. napus allotetraploid. In order to investigate the alterations of the methylation patterns around the transposon in the early generations of allotetraploids and the role of transposons in B. napus allotetraploid formation, this research detected the methylation status around transposons in the first four generations (So-S3) of a newly synthetic B. napus allotetraploid and the parents (B. rapa and B. oleracea) by MSAP method. The main results were obtained as follows.In this work, we detected the state of methylation at CCGG site flanking the transposon of synthetic B. napus allotetraploid (So-S3) and the parents in a total of36samples. All together221bands were obtained by16pairs of primers (6pairs of LTR,1pair of LINE,2pairs of SINE,1pair of Harbinger transposons,2pairs of hAT,1pair of Tel-Mariner,1pair of PiggyBac and2pairs of certromete region), a total in which136locus could be recorded clearly. The retrotransposon primers detected76bands and DNA transposon primers detected60bands.Extensive methylation changes happened at CCGG sites flanking transposon in allotetraploid synthetic B. napus (So) compared to the diploid parents. The rate of change bands for LTR, LINE、SINE、Harbinger、hAT、Tcl-Mariner、PiggyBac and Centromere region transposons were36.54%,37.50%,43.75%,62.50%,43.75%,37.50%,100%and27.78%respectively. Change types included hypomethylation and hypermethylation. As to LTR, LINE, SINE, Harbinger, hAT, Tcl-Mariner, and Centromere region transposons the hypermethylation was predominant, while hypomethylation was predominant for PiggyBac. P1parental specific hypomethylation change was detected in LTR, hAT, PiggyBac and Centromere region transposons. No hypomethlation change was detected in P2-specific bands and in bands from both P1and P2. The hypermethylation could happen at both P1parental specific loci (LTR, LINE, SINE, Harbinger, hAT, PiggyBac and Centromere region transposons) and P2specific loci, besides, locus from both P1and P2parents could suffer hypermethylation change at the same time.There was a big difference about the transposon methylation patterns among different lines of the same generation for inbred offsprings of the synthetic B. napus (So). In fact, considering all the testing sites, there were not any2or more lines with the same transposon methylation patterns in all four generations.Compared with So, the S1, S2and S3had a considerable proportion of the transposon flanking CCGG loci subjected to methylation status alterations. In S3generation, changes of methylation status could be found at all the detected locus of the transposons except LINE and SINE. From S1to S2and then S3, for LTR, LINE, SINE and PiggyBac methylation changes performanced down-up feature in the proportion, for Harbinger, hAT, Tcl-Mariner and Centromere region the transposon methylation changes showed a continuous going up feature in the proportion. The loci with stable methylation status among So, S1, S2and S3generations were analyzed. LTR, SINE, Harbinger, hAT, Tcl-Mariner and Centromere region transposons had a small amount of loci with satable methylation status in the first three generations, but changes in the methylation status happened in S3generation so that there were no steady-state loci in all the4generations. There were two loci in all four generations whose methylation status remained stable among the eight loci of LINE transposons which accounting for25%. For PiggyBac there was not a locus whose methylation status remained stable between any2of the4generations.The methylation levels around all kinds of transposons showed a certain regularity variation among generations of the synthetic B. napus (So) and its inbreeding progenies. From S0to S1to S2and then S3, around retrotransposons LTR, LINE and SINE, unmethylation levels performanced down-up-down features. Around DNA transposons Harbinger and Tcl-Mariner, unmethylation levels showed up-up-down features.Compared with synthetic B. napus (So), there were both hypomethylation and hypermethylation changes at CCGG sites flanking the transposons in the first three S generations, with a trend from predominant hypermethylation change to predominant hypomethylation change.These results indicated that methylation patterns around transposons had enormously massive alterations in the first four generations of the synthetic B. napus allotetraploid. The alterations included both hypoemethylation and hypermethylation. In So compared with the parents and S1compared with S0. the methylation changes around tansposons were hypermethylation predominantly. Subsequently hypomethylation was the predominant alterations in S2and S3. The methylation pattern changes near transposon sequences might alter the activity of transposons, so that the genomic structure and expression of genesand even plant phenotypes would be affected.