Identification Of The Breakpoints Of 1BL.1RS Deletion Translocation Chromosomes And The Drought-resistant Segment Of 1RS

Rye（Secalecereale L.,RR,2n=2x=14）is an important gene donor for improving wheat.The 1BL.1RS translocation chromosome formed by rye 1RS replacing wheat 1BS has the characteristics of drought resistance,cold tolerance and yield increase.1RS is beneficial to drought resistance and root biomass of wheat,but the drought resistant region of 1RS is still unclear,which limited the improvement of 1BL.1RS chromosome.In this study,a 1BL.1RS translocation line and four 1BL.1RS deletion lines were identified from the progeny of Mianyang 11（MY11）×Rye Kustro.Using ND-FISH,single-copy FISH,PCR,whole genome resequencing,RNA-seq,bisulfite sequencing and bioinformatics methods,we accurately located the chromosome breakpoints,analyzed the sequence characteristics of the breakpoints,investigated the expression of the genes adjacent to the breakpoints,and analyzed the methylation of the promoter region of the altered expression genes.At the same time,the drought resistant functional regions of 1RS were explored using 1BL.1RS deletion lines,and the candidate genes related to drought resistance were identified by RNA-seq technology.The main results are as follows:1.A intact 1BL.1RS translocation line(1BL.1RS^intact),a translocation line with the 1BL arm deletion(1BL^90Mdel.1RS)and three translocation lines with the 1RS arm deletion(1BL.1RS^4Mdel、1BL.1RS^17Mdeland 1BL.1RS^113Mdel)were identified by ND-FISH.These deletions occurred at different locations of the 1BL.1RS chromosomes,resulting in chromosomal segment deletions.2.Using single-copy FISH and PCR techniques,the breakpoints of the chromosomes 1BL.1RS^4Mdel,1BL.1RS^17Mdeland 1BL.1RS^113Mdelwere accurately located in the4061534-4062800 bp,17228344-17242447 bp and 113062805-113070176 bp regions of the 1RS,respectively.It was further verified that there was no reinsertion of deletions in the remaining 1RS segments of the three 1BL.1RS deletion translocation chromosomes.In addition,the breakpoint of the chromosome1BL^90Mdel.1RS was accurately located in the chr1B:599634754-599658412 bp by whole genome resequencing.The characteristics of the breakpoints of the four1BL.1RS deletion chromosomes were analyzed.All of them were enriched with a large number of repeats,of which LTR was the highest,followed by DNA transposons.3.It was confirmed that the drought resistance region was located in the 4.06-17.23 Mb region of the 1RS,and 14 candidate genes related to drought resistance function were screened out.4.It is clear that chromosome breakage has an impact on the expression of genes near the breakpoint.When the breakage occurred in the terminal heterochromatin region,the affected genes were scattered on the chromosome arm.When the breakage occurred in the interstitial regions of chromosomes,the affected genes were mainly distributed near the breakpoints.5.The methylation of promoter regions of 13,5 and 7 genes in 1BL.1RS^17Mdel,1BL.1RS^113Mdeland 1BL^90Mdel.1RS were successfully analyzed,and the methylation change of promoter regions of 7,2 and 3 genes was observed,while the rest had no methylation.These results indicated that chromosome breakage affected the epigenetic condition of the gene promoter region,but the methylation changed may not be the cause of gene expression changed.The results of this study identified functional regions related to drought resistance of1RS and identified relevant candidate genes,which provided theoretical guidance for the improvement of 1BL.1RS translocation chromosome in the future.Chromosome breaking can affect the expression of genes near the breakpoints,the impacting models on genes differs when breakpoints were different.DNA methylation status in promoter regions of genes adjacent to breakpoints will be affected,it could provide a theoretical reference for future researches on the mechanism of gene expression changes caused by chromosomal structural variation.