Cloning And Functional Analysis Of Aphid Resistance QTL-qRAD04.1 In Upland Cotton

[Objective]Aphis gossypii Glover,as a highly harmful global cotton pest,occurs in most cotton producing areas in China,posing a serious threat to the yield and quality of cotton in China.At present,there is relatively little research on the mining of cotton aphid resistance genes in cotton,which seriously affects the development of cotton insect resistance breeding in China.Therefore,exploring genes related to cotton aphid resistance and analyzing their molecular mechanisms is of great significance for cotton insect resistance breeding and improving cotton yield and quality.[Methods]In this study,we first identified two accessions exhibiting extreme phenotypes against gossypii aphids,Z50（highly susceptible ZS）and Z61（highly resistant HR）.An F₂ segregating population with 600valid individuals was constructed from Z50 and Z61.Using the number of insects as the main resistance index,83 extreme materials（ZS:41,HR:42）were selected from the F₂ isolate population,which showed differences in resistance to cotton aphid.Simplified single genome sequencing（dd RAD）was performed on83 extreme materials and their two parents.Linkage analysis was performed using the mixed linear model in GEMMA（Version 0.94.1）software.Subsequently,the parental materials were infested with aphids and subjected to transcriptome analysis.Combined with the results of QTL mapping and transcriptome sequencing,candidate genes were screened,and their functions were verified by virus-induced gene silencing（VIGS）.[Results]（1）Mapping QTL for aphid resistance in cotton based on dd RAD-seq genome-wide linkage analysis A total of four Quantitative Trait Locus（QTL）were obtained by mixed linear model calculation,which were located on chromosomes Chr A09,Chr D04,Chr D07 and Chr D10.Therefore,we named them q RAA09.1,q RAD04.1,q RAD07.1 and q RAD10.1,respectively.Among them,a major QTL was identified on chromosome Chr D04 with a candidate interval from 40.67 to 50.14Mb and at LOD score 5.99,explained 27%of the phenotypic variation.BLASTn in the reference genome data,a total of 460 coding genes were identified in this region.（2）Identification of candidate genes related with aphid resistance in upland cotton based on transcriptome analysis and In Del markersTranscriptome analysis combined with QTL mapping results,We found that a total of 18 genes were significantly differentially expressed in the two extreme materials in the candidate interval.The expression levels of GH＿D04G1280,GH＿D04G1369,GH＿D04G1126,GH＿D04G1418 and GH＿D04G1459 is higher in Z61 than Z50.In addition,re-sequencing analysis of the five candidate genes revealed a 10bp insertion in the fourth exon of GH＿D04G1418 gene in the Z50,which caused a mutation at amino acid 280 and premature translation termination at amino acid 300 in the protein sequence.GH＿D04G1418 clustered together with At LAC4 and Gh LAC4,a member of the Arabidopsis laccase gene family.These results indicated that GH＿D04G1418 was a laccase gene in cotton.Therefore,we named it Gh LAC4-3.（3）VIGS validation and functional analysis of aphid resistance candidate gene Gh LAC4-3To verify the above results,VIGS used to silenceing the Gh LAC4-3 gene,and aphid selection and non-selective feeding experiments were performed.The results showed that the silenced plants were more sensitive to aphids,indicating that the Gh LAC4-3 gene is involved in the process of cotton resistance to aphids.According to previous research progress,plant laccase genes were mainly involved in the accumulation of lignin in the cell wall.Therefore,we further performed tissue staining and determination of lignin content in Gh LAC4-3 gene-silenced plants and control plants.we found that the lignin tissue staining of silenced plants was significantly lighter than that of control plants,and the amount of lignin in leaves and stems were significantly reduced.Based on these results,q RT-PCR analysis of the six key lignin synthesis genes showed that the expression levels of six key lignin synthesis genes were significantly decreased in Gh LAC4-3 silenced plants compared with the control plants.[Conclusion]In this study,four QTLs（q RAA09.1,q RAD04.1,q RAD07.1 and q RAD10.1）resistant to cotton aphid were identified by dd RAD-seq and mixed linear models.The key candidate gene Gh LAC4-3 was screened by RNA-seq,amino acid variation and functional annotation.The VIGS test showed that Gh LAC4-3 could affect the resistance of cotton to cotton aphid by regulating the accumulation of lignin in the cell wall of cotton.The results of this study provide important candidate genes and theoretical basis for the directed cultivation and breeding of insect-resistant cotton flowers,and also enrich the functional research of laccase family and its importance in plant insect resistance.