Studies On The Mechanisms Of Grafting-induced Epigenetic Variations And Their Maintenance In The Progeny Of Chimera Of Brassica Juncea And B.oleracea

In the present study,the asexual progeny of Brassicajuncea[Brassica juncea(L.)Czern.et Coss.var.tumida Tsen et Lee]and B.oleracea(B.oleracea L.var.capitata L.)grafting chimera were obtained by inducing adventitious shoots from stem segments in vitro.The molecular basis of grafting-induced variation and its maintenance was investigated by using these materials.The genetic background of asexual progeny was identified with morlphological characteristics and molecular markers.Then the global DNA methylation and the presence small interfering RNA(siRNA)in the asexual progeny and the corresponding parents were screened and compared,to explore the genomic perturbation in the hetero-grafted progeny and clarify the relationship between the phenotypic alteration and DNA methylation modification.Furthermore,an association analysis between grafting-induced specific siRNAs and the methylation changes was performed to determine if these specific siRNAs could direct the modifications in DNA methylation.Moreover,the expression patterns of siRNAs and DNA methyltransferase were analyzed to elucidate the mechanisms of maintaining grafting-acquired variations during cell propagation and regeneration.The main results were as follows:(1)The stem segments of B.juncea and B.oleracea grafting chimera TCC[arranged as layer 1(L?,the outmost),layer 2(L?,the middle)and layer 3(L?,the innermost)of the shoot apical meristem(SAM);'T' and'C'represent the cell lineage from tuber mustard and red cabbage,respectively.I.e.,layer 1 cells are from the tuber mustard,and layers 2 and 3 cells from the red cabbage]were used to induce adventitious shoots.The frequency of formation of adventitious shoots was increased with increasing concentrations of 6-BA.PCR amplifications with B.oleracea-or B.juncea-specific primers demonstrated that the stem base-induced adventitious shoots were derived from the C cell lineage of the TCC chimera(L? and/or L?);the regenerated adventitious shoots were designated as r-CCC(r = regenerated).To investigate the maintenance of grafting variations during cell propagation and regeneration,different generations of asexual progeny(r-CCCn,n = generation)were established through successive regeneration of axillary shoots from r-CCC.The asexual progeny had a similar appearance to the parent red cabbage,with smooth purple leaves and no epidermal hair,which was distinct from the parent tuber mustard.Intriguingly,the asexual progeny of TCC had a lower wax content compared with that of the corresponding red cabbage parent,even after several asexual generations(r-CCC4,n = generation).Amplified fragment length polymorphism analysis showed there was no amplification product of tuber mustard parent from r-CCC,whereas there were 0.76%differential fragments between r-CCC and r-s-CCC(s = self-grafted)and were mainly located in intergenic regions.(2)The fourth generation of r-CCC(r-CCC4)was selected to perform whole genome bisulfite sequencing for comparative analysis of hetero-grafting induced global methylation changes relative to r-s-CCC4(s = self-grafting).Notably,increased CHH methylation levels and proportions were observed in r-CCC4,with substantial changes occurring in the repeat elements.To characterize the functions of differentially methylated genes(DMGs)induced by hetero-grafting,a Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of DMGs was conducted.Interestingly,13 enriched DMGs associated with the cutin,suberin and wax biosynthesis pathway,and most of these DMGs had higher CG methylation in r-CCC4,which was supported by the bisulfite sequencing of four DMGs(LOC106335792,LOC106298662,LOC106311786 and LOC106333935)with notable changes in methylation levels.Bisulfite sequencing validation of the genes demonstrated that the whole genome bisulfite sequencing data was reliable.Then qRT-PCR results showed that the expression of LOC106335792 and LOC106311786 decreased significantly,which may account for the decreased wax content in r-CCC4 relative to that of r-s-CCC4.(3)Analysis of DNA methylation patterns of phenotypic variation associated DMGs in different generations indicated that modifications in DNA methylation could be transmitted to the asexual progeny.Transcription analysis showed that MET1 expression was substantially higher in r-CCC4,which might contribute to the hypermethylation of the CG context.And the decreased expression levels of CMT3 and increased expression levels of MET1 might be responsible for the slight reduction in CHG methylation.Additionally,bisulfite sequencing of DNA methylation patterns of these DMGs in the pollens from TCC suggested that grafting-induced DNA methylation modification could be maintained through meiosis progress.(4)To identify small RNA(sRNA)alterations caused by hetero-grafting,high-throughput sRNA sequencing of r-CCCn,and the corresponding r-s-TTT and r-s-CCC parents was performed.As a result,the majority of the differentially expressed siRNAs showed substantially higher expression in r-CCC than in r-s-CCC.Interestingly,compared with r-s-CCC,the proportion of sRNAs that mapped to the repeat-genic region was higher in r-CCC and the subsequent asexual progeny r-CCC2 and r-CCC4.Small RNA sequencing revealed 1,135 specific siRNA tags that were typically expressed in r-CCC,r-CCC2 and r-CCC4,and 159 specific siRNA tags were also found to be transcribed in r-s-TTT.In addition,22 novel miRNAs were expressed significantly different in r-s-CCC and r-CCCn.Potential targets of these novel miRNAs were found to be involved in coding functional proteins,including zinc finger protein CONSTANS-LIKE 4,ABC transporter G family member 39 and disease resistance protein TAO1-like.Moreover,a known miRNA,bol-miR157a,was remarkably suppressed in r-CCCn compared with in r-s-CCC.As the predicted target of bol-miR157a was involved in regulating the circadian clock,the down-regulated transcriptional level of bol-miRl 57a might be associated with the flowering time variation of r-CCCn.(5)Per the association analysis of DNA methylation variation and siRNA alteration,65%of these specific siRNAs were associated with repeat elements,termed RE siRNAs.Subsequent analysis revealed the CHH methylation of RE siRNAs overlapping regions were mainly hypermethylated in r-CCC4.These RE siRNAs showed constitutive expression through multiple rounds of cell division and regeneration,and therefore might have an important role in maintaining genome stability and grafting-induced variation.