QTL Analysis For Non-heading Chinese Cabbage Heat Tolerance And DNA Methylome Analysis Under High Temperature Stress

Non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino;NHCC)is an important vegetable crop of Brassica(Cruciferae)plants.With the increasing of global temperature,high frequency of high temperature weather in summer and autumn influence the production and supplies of non-heading Chinese cabbage.Non-heading Chinese cabbage is rich in genetic resources,and has significant differences in heat resistance between different varieties.In addition,with the development of sequencing technology and the reduction of cost,it is convenient to study the QTL for heat tolerance and heat-resistant genes for non-heading Chinese cabbage.In this study,the genomic re-sequencing of two parental materials(' suzhouqing' and 'aijiaohuang')with different heat tolerance and F2 population were used to construct a high-density linkage map of non-heading Chinese cabbage,and the QTLs for heat tolerance were mapped.Two non-heading Chinese cabbage cultivars(' suzhouqing' and 'aijiaohuang')were RNA sequenced under normal and heat stress conditions.The differential expression genes and involved pathways under heat stress response were analyzed.The normal growth and heat treatment of'suzhouqing' were used to whole genome methylation sequencing.The DNA methylation patterns and the methylation changes under heat stress were analysised,and the effect of DNA methylation on the gene expression was analyzed.The main findings are as follows:1.two parents material of heat-resistant 'suzhouqing' and heat-sensitive 'aijiaohuang'were used to construct F2 separation group.Using the genome re-sequencing technique,the two parent materials were subjected to high-depth sequencing,and the 'aijiaohuang' was given a total of 13.09 Gbp clean data,with 45.97 X depth of reference genome,and'suzhouqing' was given a total of 13.46 Gbp of clean data,depth of 47.26 X.At the same time,312 F2 plants were also re-sequenced5 the total data obtained was 882.56 Gbp,the average depth is 9.93 X,Q30 were more than 80%.A total of 1,034,222 SNPs were detected between the two parents,of which 686,888 SNP markers were not less than 10X depth and suitable for F2 genotype,and the number of bin was 3,608.The total figure distance of 10 linkage groups is 1,156.88 cM,the average distance is 0.32 cM.Two QTLs for heat tolerance were identified on the two chromosomes of A01 and A07 by the method of composite interval mapping.The contribution rate of QTL to A01 was 6.101%.According to the annotation information of the reference genome,this region contains a total of 57 candidate genes.According to the expression level of these genes,there are two NAC-encoded transcription factors,which were up-regulated in the heat sensitive material and were stable in the heat-resistant material.In Arabidopsis,these two genes are positively regulating leaf aging and chlorophyll degradation,which will provide theoretical basis for revealing the heat tolerance in NHCC.2.RNA-seq were performed under three conditions(normal growth condition at 22?as control,4 and 14 hours at 43?)for heat-resistant 'suzhouqing' and heat-sensitive'aijiaohuang'.A total of 18 sequencing libraries were constructed,and 85.19 Gb Clean Data was obtained.After comparison with the reference genome,the efficiency of the mapping was from 72.65%to 81.33%.Differences in gene expression among four groups showed that a total of 8265 differential expressed genes were obtained.In addition,under the condition of 43? high temperature treatment,the heat-sensitive 'aijiaohuang' has more differentially expressed genes than the heat-resistant 'suzhouqing'.The expression patterns of these differentially expressed genes are also analyzed,and the different regulatory mechanisms of these genes in heat stress response are also shown.Transcription factor gene differential expression analysis showed that 'aijiaohuang' had 78 specific up-regulated transcription factors,far more than 'suzouqing' which only containing 16 specific up-regulated transcription factors.According to the results of QTL mapping,we also analyzed the expression of NAC transcription factors in these two materials,and obtained a class of NAC genes,which were up-regulated in 'aijiaohuang' and relatively stable and low expression level in 'suzouqing'.Therefore,the EIN-NAC regulation model of non-heading Chinese cabbage heat tolerance was summarized.3.The genomic DNA methylation of the heat-resistant 'suzhouqing' was carried out,and the whole genome methylation map of non-heading Chinese cabbage was drawn.The results showed that the DNA methylation levels of CG,CHG and CHH in non-heading Chinese cabbage were 39.3%,15.38%and 5.24%,respectively.We found that the methylation pattern was similar to that of other dicotyledonous plants,but had a higher CHH methylation level.Further comparative analysis showed that the change pattern of the three sequence contexts(mCG,mCHG and mCHH)under heat stress showed a context sequence and a position-dependent methylation regulation mechanism.DNA methylation near TSS and TES may be closely related to methylation-dependent transcriptional silencing.The association analysis of differential methylation and differential expression genes showed a significant difference in DNA methylation modalities in the early and late stages of heat stress.Systematic characterization of dynamic DNA methylation under heat stress will raise our understanding of epigenetic regulation mechanism.