Molecular Mechanism Of Cotton In Response To Whitefly Ifestation And Epigenomics Basis Of Cotton Somatic Embryogenesis

Cotton is one of the three major transgenic crops in the world,however,the fiber quality and yield of cotton are severely affected by several insect pests species.With the widespread popularization of Bt cotton,lepidopteran pests such as Helicoverpa armigera and Pectinophora gossypiella have been successfully controlled,but their insect resistance function has been losing gradually due to the emerging of the resistant insect population.The whitefly(Bemisia tabaci),a sucking pest species,causes tremendous damage to cotton production worldwide.However,limited information is available for the question how cotton perceive and defend themselves from whitefly infestation.In this study,to investigate the interaction between cotton and Bemisia tabaci,RNA-Seq and sRNA-Seq transcriptome were performed to detect cotton endogenous resistance genes,and to construct the regulation co-expression network of cotton response to whitefly infestation.Meanwhile,Genome-Wide Association Study(GWAS)was used to identify insect resistance loci on the genome-wide scale.Secondly,a high regeneration ability cotton cultivar was developed and the epigenetic mechanism of cotton regeneration and somatic embryogenesis were systematically analyzed to accelerate the process of transgenic breeding.Therefore,there has two chapters in this thesis,one is to explore the cotton endogenous insect resistance and the other is to investigate the epigenomic mechanism of cotton plant regeneration.The main findings are: 1.Transcriptome analysis of cotton-whitefly interactionRNA-Seq was used to compare the transcriptome differences of Bemisia tabaci surface resistant cultivar(HR)and sensitive cultivar(ZS)at different infection time points.Functional enrichment analysis indicated that the cotton transcriptional response to whitefly infestation involves genes encoding protein kinases,transcription factors,metabolite synthesis,and phytohormone signalling.In combination with GO and KEGG enrichment analysis,resistance genes with different transcriptional level were detected after whitefly infection between HR and ZS.A weighted gene co-expression network constructed from RNA-Seq datasets showed that WRKY40 and copper transport protein were hub genes that might regulate cotton defenses to whitefly infestation.Silencing GhMPK3 by virus-induced gene silencing(VIGS)resulted in suppression of the MPK-WRKY-JA pathway and led to enhanced whitefly susceptibility.This study provides comprehensive insight into the cotton defense system to whitefly infestation and has identified several candidate genes for control of phloem-feeding pests.2.Identification of non-coding RNAs and their potential function in cotton response to whitefly infestationWe performed small RNA and degradome sequencing between resistant and susceptible cotton cultivars following infestation with the whitefly.In total,260 miRNA families and 241 targets were identified.Quantitative-PCR analysis revealed that several miRNAs and their corresponding targets exhibited dynamic spatio-temporal expression patterns.The 2,365 long intergenic non-coding RNAs(lincRNAs)was identified in the RNA-Seq dataset.Furthermore,17 miRNA precursors were generated from 29 lincRNA transcripts.Genome-wide analysis also led to the identification of 85 phased small interfering RNA(phasiRNA)loci,nine PHAS genes were triggered by six miRNAs,including those encoding a leucine-rich repeat(LRR)disease resistance protein,an auxin response factor(ARF),and MYB.Through hypothesis model and experimental data,we explored and expanded the miR390-tasiARF cascade during the cotton response to whitefly.VIGS of ARF8 in whitefly-susceptible cotton plants increased auxin and JA accumulation,resulting in increased tolerance to whitefly infestation.A comprehensive analysis of diverse non-coding RNAs provides a useful transcriptomic resource for plant-herbivore interaction.3.GWAS analysis to detect potential insect resistance loci in cotton genomeWe collected a core collection 269 accessions and 2.88 million SNPs for population phylogenetic-tree,population structure,PCA and Linkage disequilibrium(LD)analyses.The four-pest resistance index were investigated in three environment using 269 cotton accessions,namely Leaf Damage Rate(LDR),Plant Damage Rate(PDR),Susceptible Resistant Level(SRL)and Mirid Index(MI).We identified 7 candidate loci for LDR,PDR and SRL in three environment,one loci co-located.Combined with LD analysis and transcriptome data,we identified a candidate insect resistant gene,copper transporter CRC1.4.Multi-omics data reveal epigenomics basis for cotton somatic embryogenesis and regenerated cotton plantsThe elite cotton Jin668,with a high regeneration ability,was developed from its maternal inbred Y668 cultivar using a Successive Regeneration Acclimation(SRA)strategy.To reveal the underlying mechanism of SRA,we constructed genome-wide single-base resolution methylation maps for non-embryogenic calluses(NECs),ECs,somatic embryos(SEs)during the somatic embryogenesis development and the leaves of regenerated offspring plants.Jin668(R4)regenerated plants were CHH hypomethylated compared with the R0 regenerated plants of SRA process.The results showed that Jin668 showed hypo DNA methylation,and the overall methylation decreased trend in the regenerated offspring.The increase of CHH methylation from NEC to EC were demonstrated to be associated with the RNA-dependent DNA methylation(RdDM)and the H3K9me2-dependent pathway.The decrease of DNA methylation from EC to SE is not associated with RdDM and the H3K9me2 pathway.Further analysis revealed that DNA demethylase ROS1 and DME were significantly up-regulated from EC to SE,which suggested a possibility of active demethylation.By Integration of RNA-Seq and differentially methylated regions(DMRs),the hypomethylated CHH-DMRs in promoter regions activated auxin and WUSCHEL-related genes during the somatic embryogenesis process.Inhibiting DNA methylation using zebularine treatment in NEC increased the number of embryos.Our multi-omics data provide new insights into the dynamics of DNA methylation during the plant tissue culture and regenerated offspring plants.This study also reveals that induced hypomethylation may faciliate the higher plant regeneration ability and optimize maternal genetic cultivar.