Gene Mining And Functional Verification Of Candidate Genes Related Resistance To High Temperature In Gossypium Hirsutum

Cotton is the most important economic crop in China and is highly sensitive to high temperature stress.As the largest cotton-producing region in China,Xinjiang experiences high temperatures during the cotton flowering period in July and August,which leads to a decrease in pollen vitality,withered and dropped young cotton bolls,and a significant reduction in the number of bolls on the upper fruit branches.This seriously affects cotton yield and quality.High temperature has become an important problem limiting cotton production in Xinjiang,and breeding heat-tolerant cotton varieties is a fundamental measure to address future high temperatures and break through the bottleneck of low yield.In this study,13 phenotypic traits of 273 different heat-tolerant upland cotton germplasm resources from18 geographical regions were identified from 2017 to 2019.Based on the SLAF-seq technology,genome-wide association analysis was performed on seven of these traits.Moreover,using heat-tolerant cotton variety "Xinluzao 36" and sensitive variety "Che61-72" as research materials,ONT transcriptome sequencing analysis was conducted to explore heat-tolerance-related genes in upland cotton and verify the biological function of candidate genes.The study provides a theoretical basis and material support for genetic improvement of heat tolerance in upland cotton.The main research results are as follows:1.Phenotypic analysis revealed that all phenotypic traits followed a normal distribution,with coefficient of variation ranging from 8.23% to 33.24%.By using principal component analysis,13 traits were reduced to 5 principal components,with a cumulative contribution rate of 62.96%.Correlation analysis indicated a highly significant positive correlation between plant height and initial internode height,as well as between initial internode height and number of primary branches.Inverted fruit branch fruit set showed a highly significant negative correlation with inverted fruit branch shedding.Fruit branch number was significantly positively correlated with plant height and number of primary branches.Additionally,heat-tolerant germplasm exhibited a significant positive correlation between plant height and leaf area,as well as between fruit branch number and number of primary branches.A total of 23 heat-tolerant germplasm accessions were identified from the population.2.Genome-wide association analysis was performed on seven important agronomic traits,including leaf area,chlorophyll,pollen vitality,dry boll number,bolls on upper fruit branches,the rate of bolls dropping on upper fruit branches,and the number of fruit branches.A total of 36 QTL loci were detected,associated with 552 genes.Through expression profile analysis and gene annotation information,10 genes that were highly expressed in the stem,leaf,and flower organs of cotton and may be related to heat tolerance were initially screened as candidate genes based on previous studies.3.ONT transcriptome sequencing analysis produced 120,605 non-redundant sequences from the raw data and annotated 78,601 genes.A total of 19,600 differentially expressed genes were screened through differential gene expression analysis,which were involved in ribosomes,heat shock proteins,auxin and ethylene signaling transduction,and photosynthetic pathways,and may be related to heat tolerance in cotton varieties.A total of 5,118 lnc RNAs and 24,462 corresponding target genes were predicted.The analysis of target genes showed that the expression of some ribosome,heat shock response,auxin and ethylene signaling transduction-related proteins,and photosynthetic proteins may be regulated by lnc RNAs and further participate in heat tolerance in cotton.4.Through joint analysis of GWAS and ONT transcriptome data,GH＿D07G0997 and GH＿D12G1432were finally screened as candidate genes,and biological function verification was performed using VIGS technology.It was found that the plants silenced with the two genes had better stress resistance than the control.GH＿D12G1432 may respond to high-temperature stress by regulating ROS levels.GH＿D07G0997mainly responds to stress by affecting plant water and malondialdehyde content,and does not participate in the biosynthesis pathway of proline.