| The salt tolerance genetic mechanism is one of the most important subjects in plant science.As a complex quantitative trait,salt tolerance is controlled by multiple genes and involves a variety of physiological and biochemical metabolic pathways in cotton.In addition,the expression of each gene is sensitive to external environment.In the current study,the main conventional breeding approaches for development of salt tolerant varieties are screening and collecting salt tolerant germplasm resources,then transferring elite loci by hybridization,composite hybridization and backcrossing methods.The progress of conventional breeding of salt tolerance in upland cotton is impeded due to the lack of high salt tolerant resources,low heritability,genetic complexity,and the difficulties in phenotyping.The development of molecular markers linked to causal genes for a trait has provided an effective and efficient approach for improving quantitative traits.Once identified,markers linked to a quantitative trait locus（QTL）such as for salt tolerance can then serve as a selection tool for rapid and efficient marker-assisted selection（MAS）.Herein,we reported the genome-wide association study（GWAS）of salt tolerant QTLs during the seedling stage performed over two years of phenotyping on 217 representative upland cotton cultivars through single nucleotide polymorphisms（SNPs）covering the whole cotton genome identified by Genotyping-by-sequencing（GBS）.Factor analysis was employed to evaluate salt tolerance of upland cotton germplasm.Combined with GWAS and transcriptome analysis results,candidate genes were screened within confidence interval of LD decay of associations for salt tolerance traits in Upland cotton,and their functions were preliminarily verified based on the virus-mediated gene silencing（VIGS）method.1.Phenotypic analysis of salt tolerance related traits in upland cotton:In order to evaluate the phenotypic variations of salt tolerance in the GWAS population with 217 upland cotton cultivars,three traits related to salt tolerance including relative plant height（RPH）,relative shoot fresh matter weight（RSFW）and relative shoot dry matter weight（RSDW）were determined.Great differences of the coefficients of variation were found for the 3 salt tolerance related traits.Overall,the upland cotton cultivars in this GWAS panel clearly exhibited considerable natural variations in the three traits related to salt tolerance and displayed very high genetic diversity.Through factor and cluster analysis,204 upland cotton quilts were divided into 4 categories.The scores of common factor of upland cotton germplasm resources in each ecological area were calculated,therefore,we can predict the salt tolerance levels of upland cotton in the four regions:YRR,Yt RR,USA and NIR.2.Genetic diversity analysis:GBS identified 53,321 high-quality polymorphic single nucleotide polymorphisms（SNPs）among the 217 upland cotton cultivars,with 47,133SNPs located in the intergenic intervals and 6,188 SNPs in the coding regions.Of these,95.8%of the loci（51,060 SNPs）were mapped onto 26 chromosomes of the cotton genome,and were selected for the GWAS analysis.These SNPs were not evenly distributed with an average of 1,964 SNPs per chromosome.The average density was 49.0 kb/SNP.The genetic diversity index of this population was 0.303,and the average polymorphism information content（PIC）was 0.248.The above results indicated a relatively large span in genetic diversity index and PIC in the cotton genome.3.Population structure and kinship analysis:The clustering value was calculated from K=1to 10 by STRUCTURE for five repetitions,suggesting that the population could be divided into 3 subgroups,named Group1,Group2 and Group3.The 3 subgroups contained 117,62and 38 materials respectively.The genetic backgrounds of the three subgroups are clearly distinguished,and all the three subgroups have interpenetrating genetic backgrounds.The relationship between upland cotton materials and the structure of subgroups was analyzed,and the three subgroups each contained materials from different geographical sources.The three subgroups were distributed in each geographical group,and the distribution proportion did not change obviously.Materials from Yt RR,YRR,NSEMR and AUS were mainly distributed in Group1,while materials from USA were mainly distributed in Group2and materials from NIR were mainly distributed in Group3.Most of the germplasm materials in this study were weakly related to each other.4.LD and GWAS analysis:LD decay was estimated by TASSEL in this study.The LD decay distance which was not evenly distributed among chromosomes was 1120 kb when the r2 dropped to 0.1.The average distance of At subgroup was 1246.1 kb,while that of Dt subgroup was 994.0 kb,which was significantly higher in At subgroup than that in Dt subgroup.In order to explore the genetic factors underlying salt tolerance,the mixed linear models（MLMs）were performed by simultaneously accounting for population structure and relative kinship matrix to conduct a GWAS analysis.A total of 23 significant associations(-log10p>4)located on chromosomes A05,A07,A08,A09,A10,A11,A12,A13,D02,D03,D06 and D09 were detected for the 3 salt tolerance related traits in the 2016 and 2017dataset.There were 14 associations detected in 2016 and 14 associations in 2017,repectively.In addition,five associations（SNP＿A13＿1869056,SNP＿D08＿49014753,SNP＿A07＿90682411,SNP＿A08＿6254890 and SNP＿A13＿42992196）were detected in two envrioments.The phenotypic variance explained（PVE）by each SNP ranged from 1.29%to7.00%.5.Transcriptome analysis of upland cotton under salt stress:Illumina Paired double-end transcriptome sequencing was performed on roots and leaves of salt tolerant variety Miscott7913-83 and salt sensitive variety Su 12 during seedling stage,and a large number of differentially expressed genes under salt stress were obtained.Functional annotation revealed that DEGs were mainly involved in oxidoreductase and peroxidase activities,metabolism,transport and photosynthesis.There were 6,037 DEGs in salt tolerant variety Miscott7913-83,including 2,878 DEGs in roots and 3,933 DEGs in leaves.The GO enrichment analysis shows that transporter activity was significantly enriched only at the early stage（12 h）in the roots.Transport-like gene products transport water,sugars,cations,and other molecules through plasma membranes and vacuoles,helping cells adapt to changes in osmotic pressure.And at the later stage（72 h）of salt stress,the differentially expressed genes such as oxidoreductase and peroxidase activities were significantly enriched.Plants would activate the active oxygen scavenging system,improve the peroxidase activity,and maintain a high antioxidant level.The GO items that were significantly enriched at the early and later stage of salt treatment in leaves were both photosynthesises,but not enriched at other GO items that might be related to salt tolerance.There were 6,261 DEGs in salt-sensitive variety Su 12,including 4,708 DEGs in roots and2,561 DEGs in leaves.Different with the salt-tolerant variety Miscott7913-83,less oxidoreductase and peroxidase activities enriched in the early stage in salt-sensitive variety Su 12,but not significantly enriched in the later stage,leading to its low antioxidant level and insufficient scavenging capacity of reactive oxygen species.The mechanism of salt tolerance in leaves is similar to that of salt tolerant varieties Miscott7913-83.6.Identification and function verification of candidate genes:We extracted potential candidate genes within 100 kb of flanking significant markers on the basis of the published upland cotton genome sequencing database.There were 156 genes located in these intervals.Of the 156 genes,12 were differentially expressed between Miscott7913-83 and Su 12according to the previous transcriptome sequencing results.The three promising genes（GH＿A08G0488,GH＿A10G1620 and GH＿A13G0171）were selected for preliminary function verification of salt tolerance in cotton.To confirm the function of GH＿A08G0488,GH＿A10G1620 and GH＿A13G0171 genes under salt stress,VIGS assay was used to repress expression of these genes in salt tolerant variety Miscott7913-83 plants.The increase of GH＿A13G0171-silenced plants in salt related traits under salt stress indicated its negative function in regulating the salt stress response.The oxidase contents of GH＿A13G0171 silenced plants and their control plants p TRV2::00 were determined,revealing that silencing of GH＿A13G0171 under salt stress may activate the activities of CAT,POD and SOD,thus scavenging the oxides produced by plants under salt stress and making plants resistant to salt injury. |
