Genome-Wide Association Studies Reveal Elite Allelic Variation And Candidate Genes Related To Drought Stress And Yield Components Traits In Cotton (Gossypium Hirsutum L.)

Cotton(Gossypium spp.)is the main source of high-quality natural fiber and an important economic crop.Cotton production mainly relies on upland cotton(Gossypium hirsutum L.),which accounts for about 95%of the world’s cotton planting area.Yield is the most important goal in the cotton breeding and production.The main factors that constitute cotton fiber yield are complex quantitative traits controlled by multiple genes,and the planting area and climate have a great influence on cotton yield.Due to the deterioration of the ecological environment and the competition for land between food crops and cotton,cotton production faces the challenge of planting in drought and semi-drought areas,cotton breeding for drought tolerance has become more urgent.Cotton is a relatively drought-tolerant crop,but its growth and fiber yield and quality will still be significantly affected under high-intensity drought stress.The reasonable evaluation of the drought tolerance of cotton accessions from different sources,and the discovery of loci and candidate genes related to cotton drought tolerance traits are important to cotton drought tolerance breeding.In this study,a population containing 319 accessions of upland cotton was treated with drought stress,the drought tolerance capacity of upland cotton was evaluated and some advanced drought-tolerant accessions were selected.The genetic diversity and population structure of the upland cotton population were analyzed by high-density markers from Cotton SNP80K array.The genome-wide association analysis between drought-tolerant traits and high-density SNP markers was used to reveal candidate genes related to drought.At the same time,this study investigated the yield-related traits of242 upland cotton accessions at multi-environments,and explored the relationships between different yield-related traits and cotton yield.Genome-wide association analysis discovered some important QTLs and candidate genes related to cotton yield traits.Our study provides important materials,candidate loci and genes for drought tolerance and high-yield breeding of cotton.1.Genome-wide association analysis of traits related to cotton drought-toleranceIn this study,319 upland cotton accessions from different years,eco-regions and countries were selected to evaluate the phenotypic variation under drought stress in the natural population.We used PEG to manufacturing drought stress.Traits related to drought tolerance including plant height(PH),shoot dry matter(SDM),root dry matter(RDM),proline content(PC),superoxide dismutase activities(SOD),malonaldehyde content(MDA),soluble sugar content(SS),hypocotyl length(HL)and germination percentage(GP).PC had the largest coefficients of variation(CV)under drought treatment(95.89%),and HL had the smallest coefficients of variation under drought treatment(11.77%).Correlation analysis indicated that relative PH(RPH),relative SDM(RSDM)and relative RDM(RRDM)showed significant and positive correlation to each other.Relative PC(RPC),relative SOD(RSOD),relative MDA(RMDA)and relative SS(RSS)showed significant and positive correlation to each other.Relative HL(RHL)showed significant and positive correlation with RSDM and relative GP(RGP).Both RHL and RSDM showed a significant and negative correlation with RPC,RMDA,and RSS,respectively.Based on factor and cluster analyses 319 upland cotton accessions were divided into four groups rely on drought-tolerance capacity,including 16 advanced accessions such as Lumianyan27,Xinqiu4 and Yinshan8,75 medium accessions,207 sensitive accessions and 21 extremely sensitive accessions.Population structure analysis and LD analysis were performed using55,060 high-quality SNPs filtered from Cotton SNP80K array.As a result,the 319accessions could be clustered into four groups and the LD rate declining to half its maximum value was 980 Kb.Genome-wide association analysis between drought-tolerant traits and high quality SNPs were conducted by mr MLM model,and identified 20 SNPs significantly associated with six drought-tolerance traits.Based on these significantly related SNPs,we found 1,326 drought-related candidate genes.Through transcriptome analysis and gene ontology(GO)analysis,46 elite drought-related candidate genes were further screened such as RD2、HAT22、PIP2 and PP2C.Four candidate genes up-regulated under drought stress,including RD2,were identified by VIGS.After the candidate genes were silenced,the cotton plants showed a more obviously drought stress phenotype,and the drought related traits also indicated that the candidate gene silenced plants’drought-tolerance capacity were significantly reduced.2.Genome-wide association analysis of traits related to cotton yieldIn this study,242 accessions from upland cotton natural population were planted in 13different environments,yield-related traits including boll number(BN),boll weight(BW),lint percentage(LP)and seed index(SI).BN had the largest coefficients of variation(CV),ranging from 13.13%to 24.26%,while LP with the smallest CV ranging from 7.19%to10.53%.Analysis of variance indicated that accessions have significant differences of these traits,and the genotype and environment interacted significantly.The broad-sense heritability(h~2)of BN,BW,PL and SI is 72.92%,86.09%,94.97%and 92.08%,respectively.Correlation analysis indicated that BN and LP,BW and SI showed significant and positive correlation,SI showed significant and negative correlation with BN and LP,respectively.Yield-related traits and its BLUP value of 242 upland cotton were performed genome-wide association analysis with 56,010 high-quality SNPs filtered from Cotton SNP80K array.A total of 360 QTLs were identified,and 95 candidate QTLs associated with each yield component within more than two environments were further selected,including 57 novel QTLs.Candidate genes with expression more than three transcripts per million(TPM)in cotton tissues were extracted.In total,1490 candidate genes were identified.GO analysis for each trait was individually conducted and candidate genes from QTLs region of different traits were predominately enriched in different biological processes.For BN,the candidate genes were involved in tryptophan catabolic process and floral organ abscission.For BW,the function of genes were mainly enriched in energy metabolism such as ADP metabolic process.For LP,the enriched processes contained glycol-metabolism related processes and fiber development processes,such as carbohydrate biosynthetic process and secondary cell wall biogenesis.For SI,embryonic development and flowering related processes were enriched,such as post-embryonic development regulation and flowering.There were 12 QTLs detected repeatedly in more than six environments,and found a stable QTL hotspot(A08:10.7-14.6 Mb)including three novel QTLs,qtl24 and qtl25 associated with both LP and SI,and qlt26 with LP.Based on tissue and organ transcriptome profiling,there were 15 genes located on the stable QTL hotspot and mainly expressed in ovule or fiber during fiber development.Of them,two NET orthologous genes which highly expressed in the process of fiber development were identified in qtl24 and qtl25,respectively.Furthermore,qtl19(A07:25.4-26.5 Mb)was identified to be associated with SI and detected 17 candidate genes highly expressed from-3 DPA to 1 DPA during fiber development,including a TPR gene related to short fiber phenotype.A QTL qtl83(D09:43.2-44.0 Mb)which was detected in nine different environments,were simultaneously associated with BW,LP and SI.There were 28candidate genes in this QTL region and most of them were involved in fiber development,such as EXPA4,AGD7 and ARF16.Through the evaluation of the yield of upland cotton in different planting areas,combination with QTLs related to yield components,21 widely suitable and high-yielding accessions including Jimian 958 were selected.