Transcriptome Analysis Of Wild Cotton Of The D Genome Infected By Verticillium Dahliae

Verticillium wilt is a vascular fungal disease transmitted by soil and seeds.Most pathogenic bacteria are Verticillium dahliae.Verticillium wilt seriously affects the yield and fiber quality of cotton,which poses a great threat to the cotton industry.Breeding high-quality and high-resistant cotton varieties resistant to Verticillium wilt is one of the effective ways to control the spread of Verticillium wilt.After long-term artificial orientation selection,the agronomic traits and economic traits of cultivated cotton varieties are continuously optimized,but the narrow genetic basis and the lack of new excellent genes have become important reasons for limiting the progress of disease-resistant breeding.The growth environment of wild cotton is harsh,many pests and diseases occur and form reproductive isolation from cultivated cotton.Previous studies have confirmed that a variety of wild cotton have excellent disease resistance traits.Therefore,it is of great theoretical and practical value to excavate the potential genetic resources of wild cotton against Verticillium wilt.In this study,the resistance of cotton species of the D genome G.raimondii?D₅?,G.trilobum?D₈?and G.thurberi?D₁?to Verticillium wilt at seedling stage was identified.The root,stem and leaf materials of three cotton species infected by Verticillium dahliae at0h?control?,12h and 48h were obtained.A total of 81 libraries were constructed and sequenced by transcriptome.This work lays the foundation for excavating disease resistance genes in wild cotton of the D genome.The main results are as follows:1.Phenotypic analysis of the three wild cotton species of the D genome,G.raimondii,G.trilobum and G.thurberi grown in soil highly infected with Verticillium dahliae.The identification of resistance to Verticillium wilt among the three cotton varieties showed significant differences in their level of resistance to Verticillium wilt.G.thurberi showed the highest level of disease resistance while G.raimondii exhibited higher disease index compared to both G.thurberi and G.tribolum.The degree of browning of the vascular bundles was further verified by longitudinally transection of the stem of the three cotton varieties infected by Verticillium dahliae.The vascular bundle of G.raimondii was severely infected as shown by dense browning of the region.Moreover,the cotton seedlings were treated with Verticillium dahliae infused with the green fluorescent protein?GFP?,the root tissues were examined under microscope and the degree of V.dahliae infection was monitored by the intensity of the GFP at 3h,6h and 12h.The infection rate was significantly higher in the roots of G.raimondii compared to G.thurberi and G.trilobum,but G.thurberi showed the lowest level of infection more so after longer period of exposure to GFP-infused with the V.dahliae.The result showed that G.thurberi was highly tolerant to Verticillium wilt compared to the other two wild cotton species of the D genome.2.Transcriptome sequencing analysis of wild cotton material in response to Verticillium dahliae infection:The physiological and biochemical indicators related to oxidation showed that the expression of intrinsic proteins in the three cotton species was different under pathogen treatment.Therefore,we used the Illumina Hiseq^TM 4000platform to transcribe the transcriptome of the root,stem and leaf materials of G.raimondii,G.trilobum and G.thurberi for 0h,12h and 48h.Bioinformatics analysis was performed using the G.raimondii genome sequence as a reference genome,and 37,223 genes were found to be expressed in all the samples.The analysis of differentially expressed genes?DEGs?showed that the roots of G.raimondii,G.trilobum and G.thurberi had 1,707,3,145 and 2,435 DEGs,respectively.The number of DEGs in G.trilobum and G.thurberi were much higher than that in G.raimondii.Moreover,Gene Ontology?GO?functional enrichment analysis found that DEGs were mostly enriched in the oxidation-reduction process,so we used physiological and biochemical indicators related to oxidation as an important indicator.The KEGG enrichment analysis was the most abundant in the carbohydrate metabolism pathway,indicating that the immune response of plant is energy demanding.Among the DEGS,66%were found to be involved in the membrane transport pathway,and are principally modulated by the proteins encoded by the ABC gene families that are dependent on ATP for transport function.3.Identification and analysis of the D genome ABC gene family:166 ABC genes were identified from the G.raimondii genome,of which 165 were randomly distributed on 13chromosomes and 1 was only located on the Scaffold.The sequencing rate reached 96.4%in transcriptome sequencing.These genes possess a variety of physicochemical properties,but the total average hydrophilicity is less than one.Subcellular localization predicted that141 genes encoded proteins were localized to the plasma membrane.Based on the transcriptome gene expression level and phylogenetic tree analysis,44 genes were screened from 160 genes for fluorescence quantitative analysis,and the expression level was consistent with the identification results of three cotton species.The resistance gene candidate Gorai.007G244600 was further screened by transcriptome data and fluorescence quantitative data,and the homologous gene Gh_D11G3432 with similarity of 99.71%was silenced in upland cotton.The phenotype and physiological and biochemical indexes of Verticillium dahliae were found to have certain effects in cotton against Verticillium wilt.4.Identification and analysis of the D genome Bet v 1 gene family:Through the functional domain PF00407,we identified 59 Bet v 1 genes in G.raimondii;except for 4genes located in scaffold,and the remaining 55 Bet v 1 genes.Eight of the 13chromosomes distributed in the diploid?D₅?cotton genome.Physical and chemical analysis showed that Bet v 1 protein has a small range of protein length,and most Bet v 1proteins are hydrophilic proteins.Phylogenetic analysis indicated that the Bet v 1 protein is highly conserved in nature.RNA-seq combined with fluorescence quantitative analysis revealed that the expression pattern of the Bet v 1 gene of the three cotton species was consistent with the resistance of the three cotton species.A resistance candidate gene Gorai.012G129000?BET V 1-54?was screened from the D genome Bet v 1 and the homologous gene Gh_D04G1399 with similarity of 99.58%in terrestrial cotton was silenced by VIGS technique.From the phenotype,this gene has a certain function in cotton against Verticillium wilt.