Studies On The Molecular Mechanism Of Heritable Variation Of Leaf Morphology In The Progeny Of Chimeras Of Brassica Juncea And B. Oleracea And Germplasm Innovation

The phenotypic variation progenies of periclinal chimeras obtained by apical grafting in vitro between tuber mustard (Brassica juncea (L.) Czern. et Coss. var. tumida Tsen et Lee) and red cabbage (B. oleracea L. var. capitata L.), were used as the materials in the present study. The effects of grafting on gene expression at transcriptional level were studied with RNA-Seq, and then the relevancy between the occurrence of variation and change in expression of genes were confirmed. Furthermore, MSAP, small RNA sequencing were performed to analyze the change of DNA methylation and small RNA, and explore the role of epigenetic modification in variation; Moreover, the allohexaploid was synthesized between tuber mustard and red cabbage through sexual hybridzation. The main results were as follows:(1) In situ hybridization technique was used to identify the SAM constitution of chimera on molecular level. The chimera named TTC, which identified by the structure of leaves, were analysed by hybridization using different specific probes. We found that, hybridization signals were only captured in L1when using the genome DNA of Brassica nigra genome DNA probe to do the hybridization with the SAM of chimera. But when using the specific atpA segments probe, hybridization signals were observed both in L2and L3. According to the origin of cell of SAM, the chimera was named TCC. It was concluded that the identification result of chimera through the molecular way was consistent with the leaf structure way.(2) The remarkable phenotypic variations were observed in the progenies of chimeras. The GS1was obtained by the self-crossing of the chimera TTC, and through several self-crossing GS5generation was got. In addition, by the adventitious bud induction we obtained the asexual progeny rTTT. With the self-crossing of rTTT, the RS2was obtained. In the offsprings of two kinds of chimeras, character of leaf variation was similar and found to be inherited steadily. In our opinion, the variation induced by grafting offers new expansion for the germplasm resource.(3) We performed transcriptome assembly and digital gene expression profiling analyses of TTT and GS3using the RNA-Seq technology.63,973Unigenes (mean length=662bp) were obtained through de novo transcriptome assembly. The unigenes were further assigned with GO and KEGG to find their function. Furthermore, different gene expression analysis between TTT and GS3were examined. Compared with TTT,1187genes were significant different expressed in GS3, including676and511genes were up-and down-regulated respectively. These genes were found taking part in a broad range of biology process. Two candidate genes (ARP、CKO)related to the development of leaf margin were identified. The results showed that the occurance of leaf variations was caused by the change of gene expression.(4) The progenies of TTC and TCC shown remarkable phenotypic variations. The MSAP was performed to analyse the DNA methylation among different materials. The results showed that compared with TTT, the level of DNA methylation were decreased and patterns of DNA methylation were changed in progenies. Moreover, the stable variant DNA methylation patterns were screened out, and cloning sequencing. The results indicate that one of these genes may be associated with development of leaf, and involved in gibberellin regulation pathway. Therefore, changes in DNA methylation may be one of the significant reasons of variations induced by grafting.(5) In this study, the asexual progeny of TCC (rTTT) and TTT were used, and the effect of grating on small RNA was systematically studied using small RNA sequencing. Compared with TTT,26miRNAs were expressed significantly different in rTTT, including miRNA156, miRNA319, miRNA172associated with leaf shape. Furthermore, the target genes associated with different expressed miRNA were analysed on transcription level, and the results showed that the change trend of expression of target genes consistent with the change in the miRNA expression. In addition, some of the different expressed miRNAs were involved in other processes such as plant metabolites process and response to stresses. We can conclude that grafting can affect the expression of miRNA, and then regulates the growth and development of plants.(6) The triploid (ABC, n=27) and allohexaploid (AABBCC,2n=54) interspecific hybrid were obtained through interspecific hybridization between tuber mustard(AABB,2n=36) and red cabbage(CC,2n=18). In this study, the embryo cultures were performed to save the ovules with different days after pollination (DAP), and the embryo cultured at14DAP have the highest seed survival rate about6.67%. One triploid plant was confirmed through ploidy analysis, RAPD and morphology. Furthermore, the allohexaploid plants were obtained by the chromosome double treat using liquid medium adding colchicines, and some unique traits were observed in hybrid when compared with parents.