QTL Mapping And Candidate Gene Identification Of Kernel Oil Content Of Cottonseed In Elite RIL Population Under Various Environments

Cotton is increasingly recognized as a significant source of edible oil in addition to being the most significant natural fiber crop.Malvaceae family member cotton（Gossypium spp.）can be found in Africa,America,and the Indian subcontinent.Gossypium is the largest genus of cotton,with 50 different species,but G.hirsutum L.is the main growing species and accounts for over 5%of global oil output.Cottonseed is made up of the fuzz,hull,and kernel while cottonseed kernels among them provide28.24-44.05%oil characterized as important part of cottonseed.Cottonseed oil is rich in unsaturated fatty acids,the ratio of unsaturated vs saturated fatty acids in it is 65-70%vs 26-30%.Cottonseed fatty acids making it as a vital source of vegetable oil,livestock feed and industrial bio-products include lubricant oil and biodiesel.Demand of cottonseed oil is increasing day by day due to its affordable production process and flavor consistency as compared to other oilseed crops.The oil content of upland cottonseed kernels（KOC）exhibits a complex genetic inheritance that is significantly influenced by various environmental parameters（Gong et al.,2022）,making it challenging to enhance this trait through traditional breeding methods.The technique of Marker Assisted Selection（MAS）,however,has demonstrated its robustness and efficacy as a breeding tool for manipulating quantitative trait loci（QTL）that control quantitative traits.The availability of cotton species genome sequences has facilitated the construction of high-density genetic maps and the identification of QTLs.In recent years,various technologies based on next-generation sequencing（NGS）strategies have been widely employed to build genetic linkage maps and to identify QTLs of target traits.There strategrie include genotyping by sequencing（GBS）,restriction-site associated DNA sequencing（RAD-seq）,and resequencing.In this study,an F_6:8 recombinant inbred line population（RIL）was developed from a cross between Lumianyan28（LMY28）,a commercial transgenic upland cotton cultivar and Xinluzao24（XLZ24）,harbored superior fiber quality traits with long staple.The target trait（KOC）of the recombinant inbred lines（RIL）population was evaluated across six different environments namely 14AY,15AY,16AY,14KEL,15ALE and BLUE.Randomly collected seed kernel weighted 1.1-1.2g were used for KOC through Niumag Imaging and Analyzing System.SLAF-sequence and CHIP based two high density genetic linkage maps were used to genotype this population.Candidate genes harbored in the confidence interval of stable QTLs were identified through comparing these intervals with the upland cotton reference genome（TM-1）.The main results are as follows:1.Phenotypic analysis of KOCAnalysis of variance showed the p-value was less than 0.01,suggesting that significant effect of KOC was found.Correlation analysis revealed that kernel oil content had a significant positive relationship across all environments.HCA and heat mapping explained very deeply and identified the KOC bearing three clusters which exhibited the high to low amount of oil contents under six environmental conditions.The first principal component（PC）out of total six PCs displayed eigenvalue>1and had maximum share to total variability 84.5%.2.QTL mapping for KOCThe high density genetic map（HDGM）based on SLAF-seq strategy contains 4366 SNP markers,covering a genetic distance of 3826.92 c M with an average marker interval of 0.73 c M between markers.The HDGM based on CHIP strategy contains 4851 SNP markers,covering a genetic distance of 2477.99c M with an average marker interval of 0.5 c M between markers.A total of 28 stable QTLs for KOC were detected based on the two HDGMs,among which nineteen QTLs were from SLAF-seq HDGM,explaining 3.20-59.79%of phenotypic variance with LOD values ranging from 1.74 to 20.62.Ten QTLs were from CHIP HDGM,explaining 2.99-11.10%of the total phenotypic variance with LOD values ranging from 1.69 to 5.47.Among these QTLs,ten were localized on A_t sub-genome and eighteen on D_t sub-genome.Comparing with previous studies,5 QTLs were consistence with previous reported ones and 23 QTLs were newly identified ones,of which q OC^slaf-c23-1 and q OC^chip-c23-1 were a major locus simultaneously identified by both strategies.3.Candidate gene identification and expression analysis of KOC related genesA total of 4038 and 1070 genes were found in the confidence interval of stable QTLs identified on the two genetic linkage maps of SLAF-sequence and the CHIP respectively.Using transcriptome data of different ovule developmental stages of standard genetic cotton cultivar TM-1 demonstrated only 162genes were commonly found in Arabidopsis and our studied population.After co-expression analysis,only 40 key genes were identified.The genes in MCODE 1,which participated in monocarboxylic and fatty acids metabolic process and the carbon metabolic process,MCODE 2 containing genes involved in monocarboxylic acids and organophosphate and ADP metabolic process,the genes in MCODE 3,participated in the lipid biosynthetic process,lipid metabolic and signaling process and the genes in MCODE 4,participated in glycolysis process.After that only 8 genes were differentially expressed in ovule developmental stages in TM-1 and Hai7124 germplasms.These genes may regulate lipid accumulation in cottonseeds.