Studies On The Molecular Mechanism Of Phenotypic Variations In The Progenies Of Chimeras Produced By In Vitro Grafting Between Brassica Juncea And B.Oleracea

Phenotypic variations induced by grafting have been observed in production practice and scientific research.Research works on variations induced by grafting have been on highlighted in the world.In this paper,GSn(GSn = grafting-selfing,n=generations)were obtained by selfing of chimera produced by in vitro grafting between tuber mustard(Brassica juncea(L.)Czern.et Coss.var.tumida Tsen et Lee)and red cabbage(B.oleracea L.var.capitata L.)to explore the variation and heredity characters of grafting-induced phenotypic variations;RNA-seq was per-formed to explore the differentially expressed genes which may be related to phenotypic varia-tions,and their inheritance law after grafting;MSAP(methylation-sensitive amplified polymor-phism)was used to explore the DNA methylation variation induced by grafting and its relationship with phenotypic variation;small RNA sequencing was performed to analyze the relationship be-tween transformed siRNAs and DNA methylation induced by grafting;F2 population were pro-duced to map the leaf margin with serration gene.The main results of this paper are as follows:(1)GS1 was obtained by selfing of TTC[L1-L2-L3,L1=outer layer of shoot apical meristem(SAM),L2 = middle layer,L3 = inner layer,T = tuber mustard,C = red cabbage]which was produced by in vitro grafting between tuber mustard(TTT)and red cabbage(CCC).Pheno-typic variations including SAM variation and leaf shape variation.The leaf shape variation can inherit stably from GS1 to GSs,but the SAM variation reverts to its original station over generations.(2)The result of RNA-seq showed that 413 genes expressed differentially between GS1 and TTT.There were 59 differentially expressed genes(DEGs)which kept the same changing pattern from GSi to GS5,including auxin-responsive protein IAA18 and auxin-induced protein 15A,and there were 94 DEGs which kept the different changing pattern from GS1 to GSs,including protein YLS9-like and WRKY22.In addition,two methylation related genes:Factor of DNA methylation 1 and methyltransferase-like protein 7A changed their expression in GSn after grafting.(3)Subsequent measurement of DNA methylation by MSAP in GS1 revealed 5.29%-6.59%methylation changed compared with TTT,and 31.58%of their changes were stably transmit-ted to GS5,but the remainder reverted to the original status over generations,suggesting graft-ing-induced DNA methylation changes could be heritable and reversible.Sequence analysis of differentially methylated fragments(DMFs)revealed methylation mainly changed within transposons and exon regions,which further affected the expression of genes,including flow-ering time and gibberellin response-related genes.(4)The small RNA sequencing results showed that 6704 siRNAs changed their expression in GS1 when compared with TTT.A total of 82 differentially expressed siRNAs were mapped to 3 DMFs,including 17 24-nt siRNAs which were all mapped to uDMF9.Then the bisulfite se-quencing revealed that the CHH methylation in GSn was lower than that in TTT,which is consistent with the expression of siRNA,indicating the siRNAs may direct the DNA methyl-ation induced by grafting through RdDM(RNA directed DNA methylation).(5)The F2 epigenetic population were produced by GS6XTTT.In F2 population,the segregation of leaf margin with serious serration,leaf margin with slight serration,smooth leaf margin conformed to Mendelian inheritance.512 pairs of MSAP primers were used to screen poly-morphic primers between two pools,and a total of 70 pairs of MSAP polymorphic primers were obtained.The further selection were performed between 10 plants with seriously serrated leaves and 10 plants with slight serrated leaves,and one marker 31-31H were significantly associated with leaf margin variation with 12%exchange rate.Therefore,31-31H was located at 25.95 M of chromosome A2,and the genetic distance between target gene and 31-31H was 12.21 cM.This paper showed that as.one important reason for grafting-acquired phenotypic varia-tion,DNA methylation could stable inherit and revert simultaneously over generations,which may be maintained by siRNAs variation induced by grafting.Our results revealed the molec-ular mechanism of "grafting-acquired variation",which is very important for genetic improve-ment and new variety creation of horticultural plants.