Exploration And Analysis Of Chilling Tolerance Genes In Gossypium Thurberi

Cotton(Gossypium spp.),as the most important fibre crop for the textile industries and ranks highly among the edible crop oil producing crops,plays an important role in our national economy.Chilling stress is one of major abiotic stress factors limiting global cotton production.Cotton originates from the tropical and subtropical climate,can not to acclimatize to extreme temperatures and are therefore sensitive to chilling stress.Human selection of cultivated cotton varieties has narrowed their genetic background.Wild species of Gossypium under natural selection have abundant genetic diversity and many excellent characteristics,therefore,they are of great significance to widen the genetic basis of cultivated cotton.In the present study,transcriptomics analyses,combined with genomic sequences of cotton as well as the elucidated aboitic response and regulation pathways in model plants,were used to identify the candidate genes associated with chilling-tolerance.Furthermore,comparative transcriptomics analyses were utilized to investigate the genetic diversity,molecular evolution and expression patterns of interspecific orthologous genes related with abiotic stress under natural selection.Meanwhile,weighted gene co-expression network analysis(WGCNA)was applied to identify the co-expressed genes responsive to cold and salt stresses in G.thurberi.Our results may be provide theoretical basis for improving abiotic stress tolerance in cultivated cotton by exploiting the genetic resources of wild cotton.The main results are as follows:(1)Identification and evaluation of chilling tolerance of G.thurberiAfter 48 h of cold stress treatment,G.thurberi leave only showed symptoms of wilting but were not necrotic.Variations of the physiological and biochemical indices among the three cotton species,G.thurberi,ZLZ33 and CRI50 seedlings,were investigated after cold stress treatment.Compare to the XLZ33,which is sensitive to chilling stress,G.thurberi had lower level of relative electrolyte leakage and MDA content,but higher level of proline and soluble sugars contents.Moreover,G.thurberi exhibited significantly increased level of SOD and POD activity.These results well explain the reasons that G.thurberi has better cold tolerance from the physiological and biochemical level.(2)Whole genome transcription analysis of G.thurberi under cold stress and functional verification of cold tolerance candidate genes CBF4 and ICE2To understand molecular mechanisms of the cold tolerance in G.thurberi,we compared transcription changes in leaves of G.thurberi under cold stress by high-throughput transcriptome sequencing.A total of 4226 differentially expressed genes(DEGs)were identified from the transcriptome data of G.thurberi under cold stress treatment.GO classification analyses showed that the majority of DEGs belonged to categories of biological process,cellular component and molecular function.KEGG enrichment pathway analysis showed that the plant hormone signal transduction(ko04075)pathway was the most significant enrichment.The expression of several cold responsive genes such as ICE1,CBF4,RAP2-7 and ABA biosynthesis genes involved in different signaling pathways were obviously induced after G.thurberi seedlings were exposed to low temperature.The q R-PCR results also indicated that all these genes may be involved in response to cold stress in G.thurberi.To further investigate the functions of candidate genes,CBF4 and ICE2 genes in G.thurberi seedlings were knocked out through Virus-induced Gene Silencing(VIGS)method.We found that the infected plants become more sensitive to cold stress and exhibited a higher concentration level of MDA and a significant reduction in the levels of proline and SOD compared to the wild types.All these results showed that CBF4 and ICE2 genes played an important role in cold stress response of G.thurberi.They can be as cold tolerance candidate genes for subsequent genetic improvement of cold tolerance traits in cotton.(3)Evolution analysis of stress resistance genes in four diploid wild cotton species.Comparative transcriptome analysis was performed based on RNA-seq data of four wild cotton species under cold and salt stresses A phylogenic tree based on transcriptomic SNP data showed a quite similar topology to the tree based on the unigenes,indicating a solid phylogeny for four D-genome diploid species.D-subgenomes of G.hirsutum and G.barbadense formed a monophyly with G.raimondii,proving that G.raimondii may be as the same donor for the D subgenome of allotetraploid AD1 and AD2 genomes.Hundreds of positive selected genes were founded in four wild cotton species by gene evolutionary analysis,and most of PSGs were enrichment response to stimulation.Twenty-four datasets of leaves without stress treatment were used to detect gene expression divergence and conservation of four species.A total of 29512 DEGs were clustered eight profiles based on gene expression pattern use k-means clustering method.Profile 1,2 and 3,with a large number of DEGs were enriched in carbon metabolism and carbon fixation,plant circadian rhythm and biosynthesis of unsaturated fatty acids pathways,display similar expression pattern of four species.Profiles 4-8 were observed a considerable species-specific expression pattern.(4)Identification of four wild cotton stress-related gene co-expression modules by WGCNATo grasp gene co-expression network conservation and divergence,all of 40 datasets of leaves were used to construct co-expression network by weighted gene co-expression network analysis(WGCNA).A total of 33 modules were identified with significant genome-specific and/or abiotic stress regulated co-expression patterns.We found skyblue3 module displayed significantly negative correlation with Share.T.S,Cold and Salt group.One hub gene,RALF(rapid alkalinization factor)-like,was identified in this module,and can be as an important stress-resistance candidate gene for subsequent research.