Molecular Mechanism And Gene Function Identification Of Premature Senescence In Early-Maturing Cotton

Although early-maturing breeding is one of the main breeding goals for cotton,early-maturing traits usually appear with premature senescence,which seriously affects the yield and fiber of cotton fiber.Therefore,revealing the genetic basis and regulatory network of cotton senescence and the genetic basis of premature senescence in early-maturing cotton will be beneficial to the understanding of cotton senescence mechanism and cotton breeding work.In this study,genome-wide association analysis was used to reveal the genetic basis of cotton senescence,and multi-omics technologies,such as transcriptome and metabolome,were used to identify specific metabolites and regulatory networks associated with precocious senescence.The main conclusions are as follows:(1)Based on SLAF-seq,multi-locus GWAS was used to perform association analysis on eighty senescence-related traits/indicators of 185 upland cotton materials,and 50 senescence-related genomic regions were identified.Considering significance and repeatability,we focused on three stable and reliable genomic regions,D03＿37952328＿38393621,A02＿105891088＿107196428 and D13＿59408561＿60730103.In D03＿37952328＿38393621 region,a candidate gene GhCDF1(cell growth defect factor 1)was found.The q RT-PCR analysis showed that the expression level of GhCDF1 was significantly down-regulated in old cotton leaves.By VIGS,the silencing of endogenous GhCDF1 resulted in chlorosis,decreased SPAD value and up-regulation of senescence marker genes in leaves of silenced plants,indicating that GhCDF1 is a negative regulator of upland senescence.In addition,candidate genes,the wall-associated receptor kinase-like 14(GhWAKL14)and the protein phosphatase2 C family gene(GhABI2),were identified in genomic regions A02＿105891088＿107196428 and D13＿59408561＿60730103,respectively.Using transcriptome data,the expression level of GhWAKL14 showed different degrees of increase under different stress conditions,but no significant difference detected in young and old cotton leaves.The expression level of GhABI2 was down-regulated in the stress-treated transcriptome data,and significantly up-regulated in senescent leaves by q RT-PCR.(2)Using the resequencing data,GWAS analysis was performed on the chlorophyll content and the rate of change of chlorophyll at FBP stage and BOP stage in 355 upland cottons.Finally,we identified 54 senescence-related genomic regions,distributed on all At subgenomes and 8 Dt subgenome chromosomes,and harbored 939 candidate genes.Using the information of functional annotation,we focused on a MAPKs family gene,MAP kinase kinase 9(GhMKK9),located in the A12＿108514660＿108934586 genomic region.There is a non-synonymous mutation site A12＿108859102 and a synonymous mutation site A12＿108860059 in the exon region of GhMKK9,forming two haplotypes,Hap1(TG)and Hap2(GA).Hap1 had higher chlorophyll content at BOP stage and lower rate of chlorophyll degradation.So Hap1 is a favorable haplotype for delayed leaf senescence.Using the q RT-PCR,the expression level of GhMKK9 was significantly increased in old cotton leaves.And overexpression of GhMKK9 in Arabidopsis could promote Arabidopsis senescence.Meanwhile,silencing the expression of GhMKK9 gene by VIGS could improve the drought tolerance of silenced plants.And in contrary,overexpressing GhMKK9 in Arabidopsis showed a more sensitive response to drought.In conclusion,GhMKK9 positively regulates leaf senescence and negatively regulates drought tolerance in Upland cotton and Arabidopsis.(3)Using transcriptomic and metabolomic techniques,we preformed omics analysis of leaves at different senescence stages of early-maturing with no-premature senescence cotton varieties and earlymaturing with premature senescence cotton varieties.We identified 21 specific metabolites related to premature senescence in early-maturity cotton,including flavonoids,lipids,phenolic acids,lignans,and alkaloids.Using K-means clustering,these 21 metabolites were divided into four distinct clusters.Transcriptome data were analyzed using the WGCNA method,and 33 co-expression modules were obtained.Integrating the results of GWAS,metabolome,and transcriptome,three co-expression modules associated with Cluster 1 were identified,including brown,blue and floralwhite,which may regulate the specific metabolites of premature senescence in the early-maturing cotton.Filtering with the thresholds of KME| > 0.8 & |GS| > 0.6,47 key genes involved in the regulatory network of premature senescence in early-maturing cotton were revealed.