Analysis Of Sites And Functional Genes In Cotton Resistance To Verticillium Wilt Based On CSSLs Of Gh × Gb

Object: Verticillium wilt(VW),known as the ‘cancer’ of cotton,which seriously affects the yield and quality of cotton.Sea-island cotton has excellent resistance to VW than upland cotton.However,due to the low recombination,large segregation and aberrations,the progress of disease resistance gene transfer in island cotton has been slow.Because it can minimize the interference of genetic background,chromosome segments substitution lines(CSSLs)also an ideal material for QTL/gene fine mapping and cloning.In the early stage,our research group introduced a set of cotton chromosome segment substitution lines and successfully located loci and candidate functional genes related to verticillium wilt.The research results are of great significance to improve the resistance of upland cotton to verticillium wilt by means of genetic engineering.Methods:(1)The research strategy of genome-wide association analysis(GWAS)is conducive to making full use of the historical recombination events in the natural population.The population is rich in genetic variation and has higher resolution for the location of resistance sites.Based on the phenotypic data and genotype data of 318 sea and land chromosome segment substitution lines in different environments,this study used the software Fast LMM to carry out association analysis through mixed linear model(MLM),and drew Manhattan map to display the marker sites that were significantly related to the target traits detected by association analysis and screen candidate genes.(2)In this study,transcriptome sequencing technology was used to mine the differentially expressed genes between the verticillium wilt resistant varieties M20,M34 and the verticillium wilt susceptible varieties Emian 22 in the sea-land chromosome segment substitution lines,and the related pathways involved in the genes induced by verticillium wilt were annotated,and the resistance candidate genes were screened by the weighted gene co-expression network analysis(WGCNA).(3)The role of Gh PP2C52 in cotton resistance to verticillium wilt was revealed by real-time fluorescence quantitative PCR(q RT-PCR)and virus-induced gene silencing(VIGS).Results and Conclusion:(1)In this study,we utilized the quantitative trait locus(QTL)mapping approaches and 318 CSSLs materials constructed from Emian-22(G.hirsutum)and 3-79(G.barbadense)to discover quantitative genetic factors responsible for VW resistance,superior fiber quality,and high yield.Phenotypic data were obtained from a 3-year investigation in one field with two replications per year.We identified 78 QTLs relevant to the VW disease index(DI)based on a high-resolution recombination bin map for uncovering genetic loci determining the phenotypic variance between Gh and Gb.Further genomewide association analysis(GWAS)in combination with genome re-sequencing identified 1,303,535 single nucleotide polymorphisms(SNPs)in these CSSL populations.Among them,12 SNPs were simultaneously associated with different environments in CSSL populations.Surprisingly,the Manhattan plot showed that SNPs on chromosomes A01 and D12 were associated with VW resistance in four environments over the three years.Annotation identified nine genes on chromosome A01 and nine genes on chromosome D12 as candidate genes responsible for VW resistance.Among them,the candidate genes Ghir＿A01G021860,Ghir＿A01G021790,Ghir＿A01G021870,Ghir＿A01G021760,Ghir＿D12G018010,Ghir＿D12G018180,and Ghir＿D12G018300 were associated with the VW resistance with non-synonymous mutations in the exons encoding amino acids.Our findings revealed the genetic and molecular mechanisms of VW resistance and laid a solid foundation for further fine mapping,gene cloning,and molecular designing in breeding programs for resistant cotton varieties.Our findings provide insights for studying the interspecific inheritance of disease resistance traits and guidance for utilizing Gb alleles in interspecific breeding.(2)In this study,a comparative transcriptome analysis was performed to identify DEGs at three time points after V.dahliae infection using two VW-resistant CSSLs(M20 and M34)derived from an interspecific cross(Gh x Gb)and the VW-susceptible parent(Emina22)used in the generation of the CSSLs.A total of8,027,5,238 and 11,354 DEGs were identified in Emian22,M20 and M34,respectively.According to the gene expression trend analysis,the DEGs identified in Emian22,M20 and M34 at the three time points could be clustered into 8,8,7 profiles,respectively.WGCNA analysis identified gene modules and hub genes highly related to the response of cotton plants to V.dahliae infection.Two genes(Ghi＿D02G10111and Ghi＿A03G10581)encoding probable protein phosphatase 2 were considered as good candidates for further functional characterization.(3)We identified 4,2,2 and 4 PP2C52 genes in G.hirsutum,Gossypium raimondii,Gossypium arboreum and Gossypium barbadense,respectively.The phylogenetic analysis suggested that Gh PP2C52 is a homology of At PP2C52.Each Gh PP2C52 contained at least one hormone responsive cis-element in its promoter.Among them,the abscisic acid(ABRE)cis responsive element is present in the promoter regions of all PP2C52 genes.VIGS was employed to knock down the expression of Gh PP2C52 in V.dahliae resistant cultivars(HR2).Observation of the expected yellowing leaf phenotype in TRV:: Gh CHLI plants and notable reduction of Gh PP2C52 in the corresponding VIGS plants suggested success of the VIGS experiment.At 12 days after V.dahliae inoculation,Gh PP2C52-silenced plants displayed severe leaf yellowing and wilting.Gh PP2C52-silenced plants also showed obvious vascular browning.In contrast,the control plants(TRV:00)were much healthier and did not show obvious vascular browning.The Gh PP2C52-silenced plants had significantly(P < 0.05)raise disease indices(32.08 ± 5.05 and 56.25 ±6.25%,respectively)than the control plants(9.58 ± 1.91% and 26.25 ± 2.50%,respectively)at 12 and 18 dpi in V.dahliae resistant cultivar HR2.Compared to the control plants,the Gh PP2C52-silenced plants accumulated more fungal biomass in their stems and had a upper fungal recovery from the stem sections collected from the inoculated plants.As a result,we confirmed that down regulation of Gh PP2C52 was able to weaken cotton’s resistance to V.dahliae.