Fine Mapping And Allelic Variation Analysis Of Candidate Genes For QFS7 Associated With Cotton Fiber Strength

Cotton is a major source of natural fiber and an important cash crop in the world.Fiber quality is an important economic property of cotton,including fiber strength,fiber length and fiber fineness.Fiber strength is one of the key fiber quality parameters to determine the quality of spinning.Therefore,improving fiber strength is always an important research direction for cotton breeders.As is known to all,there are linkage dragging between various traits of fiber quality and yield,which cause some difficulties in fiber quality breeding and improvement.As a result,limited progresses have been made in cotton fiber strength improvement,suggesting it is very difficult to improve fiber strength only using traditional breeding methods.In recent years,the rapid development of molecular marker technology provides a new technical means for us to use genetic engineering or molecular marker to break the linkage to improve the cotton fiber strength through fine mapping and developing SNP marker.In our previous study,an F2 population was constructed by backcrossing SL-7 with LMY22 to map fiber strength QTL（qFS7）within an interval of 1.189 Mb.In this study,SNP markers were further developed to fine map qFS7,and candidate genes were identified.Further,the allelic variation of candidate gene was analyzed by natural population to verify the function of candidate gene.The main results are as follows:1.Fine mapping of fiber strength QTL and identification of candidate genesIn order to fine mapping qFS7,217 pairs of SNP molecular markers were developed within the candidate interval of 1.189 Mb,and a total of 17 pairs were identified as polymorphic.A total of 13 exchange types were identified from the finely mapping population using the molecular markers.Finally,the candidate range was narrowed down to a 174 kb interval between SNP-254 and SNP-271.There are only two genes in this region,Gh_A07G1730 and Gh_A07G1731.The results of transcriptome sequencing showed that the expression of Gh_A07G1731,rather than Gh_A07G1730,was significantly different between the two parents,so Gh_A07G1731 was identified as candidate gene.2.Bioinformatics analysis and comparison of gene structure of Gh_A07G1731 between parentsGene function annotation of candidate gene shows that the homologous gene of Gh_A07G1731 in Arabidopsis thaliana（L.）is AT3G13130,which is related to transmembrane protein.And the function of AT3G13130 has not been reported.The gDNA sequence and promoter sequence of Gh_A0761731 of LMY22 and SL-7 was cloned and sequenced.One nonsynonymous SNP variation was detected in exon 1 of Gh_A07G1731 and named as SNP₉1.In the promoter region,there was a 279 bp insertion,one Indel and 11 SNPs and they were named as Insert_-1690,Indel_-264,SNP_-579,SNP_-757,SNP_-850,SNP_-1040,SNP_-1146,SNP_-1233,SNP_-1235,SNP_-1386,SNP_-2267,SNP_-2274,and SNP_-2634,respectively.Using the promoter element prediction software,Insert_-1690,Indel_-264,SNP_-579,SNP_-757,SNP_-1040,SNP_-1233,SNP_-1235,SNP_-2267,and SNP_-2634 were detected variation of promoter elements.The 279 bp insertion sequence（Insert_-1690）was compared with the genome of Gossypium spp.including G.hirsutum.G.barbadense、G.arboretum、G.raimondii,and the result showed that the 279 bp insertion was homologous with G.harbadense.According to the prediction analysis,the Gh_A07G1731 was a transmembrane protein and the amino acid conversion caused by SNP₉1 in CDS was located in the transmembrane region.3.Allelic variation analysis of Gh_A07G1731Allelic variation of Gh_A07G1731 was analyzed using natural population comprising 208 cotton accessions,and the gene and promotor of Gh_A07G1731 cloned from the 208 accessions were sequenced and analyzed.According to the allelic variation of Gh_A07G1731,the 208 accessions were divided into 8 haplotypes,and the fiber strength of Hap-2 whose genotype is the same as the parent SL-7 genotype with excellent fiber strength was higher than that of other haplotypes.Correlation analysis was performed between the Gh_A07G1731 sequences and fiber strength traits of the natural population.The general linear model（GLM）,mixed linear model（MLM）and general linear model（GLM+Q）using population structure matrix as covariable were used as the analysis model.MLM is not related to the SNP,and GLM and GLM+Q are related to 5 SNP,namely SNP_-2634,Insert_-1690_-23,SNP_-1386,SNP_-850 and SNP₉1.Insert_-1690₂3 was located in Insert_-1690.It was concluded that SNP_-2634,Insert_-1690,SNP_-1386,SNP_-850 and SNP₉1 were correlated with fiber strength.4.Transient expression of Gh_A07G1731 promoterTo identify the effect of allelic variation on promoter activity,the promoter sequence within 3000 bp were divided into five segments,and were cloned from Hap-1,Hap-2 and Hap-3 haplotypes respectively to construct the instantaneous expression vector.The GUS activity quantitative detection results showed that the high expression of candidate gene Gh_A07G1731 was affected by Insert_-1690.According to the high GUS activity of Hap-3 promoter and low fiber strength of Hap-3 natural population,the correlation between SNP₉1 in CDS and fiber strength was stronger.However,it cannot be excluded that high expression of Gh_A07G1731 is related to fiber strength.