Identification Of Cotton Verticillium Wilt Resistance Germplasm And Expression Analysis On Related Resistance Genes

Upland cotton(Gossypium hirsutum L.) is one of the most important crops worldwide. Its highly fiber and oil productivity dominates cotton commerce worldwide. Verticillium wilt is a widespread and destructive cotton disease caused by the soil-borne fungus pathogen Verticillium dahliae. And to breed plant resistant varieties is the most effective mean to mitigate the hazards of verticillium wilt. So it is of great significance to screen disease-resistant Upland cotton germplasm and to identify R genes having critical roles during various stages of disease development in cotton plants.In this study,107cotton germplasm were identified in a verticillium wilt disease nursery with three years. At last,8resistant varieties (Ji2658,04-123, Aizaofeng, Ji1316, Ziyou, Ji228, Ji79, Jimian958) were screened with all hybrids from distant hybridization between upland cotton, Sea island cotton and wild relatives. The resistant varietie Ji79, one of the8lines, showed the resistance mechanism inherited partially by dominant effect and additive effect, while the varietie TM-1were susceptible to the disease. Using28genes related to disease resistance (R genes) as candidates, we designed corresponding primers to amplify and sequence these genes, found nucleotide variations occurred among three genes:EREB (ethylene responsive element binding protein), GER (vdrgl9GASA-like protein), GST (glutathione S-transferase) between Ji79and TM-1. Hereafter, restriction endonuclease cleavage sites were analyzed between these genes. The results show that2(Rsal, Taql) restriction sites differed in EREB sequences, and3(MseI, Sau3AI, MspI) restriction site differences in GST sequences, and no restriction site differences existed in GER sequences between the two varieties.The transcriptional profiles of resistant variety Ji79and susceptible variety TM-1for4d inoculation with V. dahliae at the six leaves stages was performed. The expression regulation number of unigene of TM-1inoculated by V. dahliae was significantly higher than that of Ji79inoculated by V. dahlia. Following which large-scale differentially expressed transcripts in response to V. dahliae were explored, KEGG analysis of Ji79inoculated or not, revealed that these unigenes were significantly enriched in various known resistance relevant metabolic or signaling pathways, such as Galactose metabolism, Aminobenzoate degradation, Phenylpropanoid biosynthesis, Glycerolipid metabolism, Flavonoid biosynthesis, Riboflavin metabolism, Diterpenoid biosynthesis, Limonene and pinene degradation. Dirigent-like protein gene were induced by V. dahliae infection, increase in the synthesis of lignins as a defence response. We acquired12Dirigent-like protein gene family members through RNA-Seq sequencing technology using Illumina Hiseq2000sequencing platform. These gene members allocated to three different phylogenetic groups with four conserved domains, the homologous genes were scattered in G. raimondii D5genome chromosomes repeatedly, mainly in chromosome No.2,3,6,7,9,10, and13.Subsequently, specific quantitative real-time PCR primers were designed to analyze the expression differences of this gene family, whose DEGs (Differentially Expressed Genes) between resistant variety Ji79and susceptible cultivar TM-1, were validated before inoculation (0h) and at1h,8h,24h,48h,72h and96h after inoculation. The comparative2-△△Ct method of quantification was used with cotton GhACTIN as the reference.In resistant variety Ji79,6dirigent-like protein genes (GhDIR4, GhDIR6, GhDIR7, GhDIR9, GhDIR10, GhDIR11) were up-regulated at1hour after inoculation with GhDIR9up to6.5-fold at most,2genes (GhDIR4, GhDIR7) were up-regulated at8h,1gene (GhDIR4) were up-regulated at24h,4genes (GhDIR1, GhDIR4, GhDIR7, GhDIRll) were up-regulated at48h,1gene (GhDIR9) were up-regulated at72h and1gene (GhDIR4) were up-regulated at96h.In susceptible cultivar TM-1,1dirigent-like protein gene (GhDIR7) were up-regulated at1h after inoculation,2genes (GhDIR7, GhDIR10) were up-regulated at8h with GhDIR7up to325-fold,6genes (GhDIR4, GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10) were all up-regulated at24and48h,2genes (GhDIR4, GhDIR7) were up-regulated at72h and6genes (GhDIR5, GhDIR6, GhDIR7, GhDIR9, GhDIR10, GhDIR11) maintained high expression levels at96h after inoculation.So the results showed that the expression of GhDIR in response to infection by V. dahlia in resistant variety Ji79was earlier than in susceptible variety TM-1. The expression of GhDIR members in susceptible variety became stronger after8h vaccination by V. dahliae and continuously went up after96h inoculation. Significant up-regulation of GhDIR members GhDIR4, as well as GhDIR7, GhDIR9and GhDIR11, were observed in resistant variety, GhDIR7and3other members (GhDIR5, GhDIR6and GhDIR10) expressed in susceptible variety, which indicates that dirigent-like proteins play critical roles during various infection stages of disease development in cotton plants. Otherwise, nucleotide polymorphism of6GhDIR genes (GhDIR1, GhDIR3, GhDIR5, GhDIR8, GhDIR10and GhDIRl2) was detected between resistant variety Ji79and susceptible variety TM-1.DET2is a gene that encodes a key enzyme in the biosynthesis pathway of brassinosteorid which has essential roles in cell elongation, guard cell development, vascular development, reproductive processes, and tolerance to various stresses. Checking for DET2mutations in the coding region among Upland cotton, Sea Island cotton, G. raimondii and other cotton species,25SNP mutations occurred in13species, including8involved in amino acid variations. There were in total8kinds of ligand binding domains for its encoded proteins, which is consistent with the SNP combinations types between species; analysis for trait correlation revealed that DET2was associated with the formation of cotton fiber, however nothing to do with disease resistance and fertility.