Genome-wide Association Study Reveal The Genetic And Biochemical Bases Of Metabolites In Cotton

Plants produce a large number of metabolites with diversity structures and functions,some of which are closely related to the formation of quality or resistance traits in crops.Therefore,it is important to investigate the genetic and biochemical bases of metabolites for revealing the underlying regulatory mechanism of complex traits.In this study,we performed metabolomic analysis and genome-wide association study to provide new insight into metabolism in cotton.1.Development of an untargeted method for metabolomics study in cottonIn order to analyze the metabolites of cotton stablely with high throughput,we developed an untargeted method based on UPLC-QTOF-MS platform for separation and detection of semi polar metabolites in cotton.By using computer simulation and manual search,we finally annotated or identified 125 metabolites,10 of them were verified by commercial standards.2.Metabolic changes in cotton induced by oral secretion from insectsTo verify the effectiveness of the developed untargeted method,we investigated the effects of oral secretion of insect on cotton metabolism as an example.We analyzed the metabolic changes of cotton leaves treated by wounding plus oral secretion and wounding plus water,the latter is a control.Our results indicated insect oral secretions can significantly reshaped the metabolic profiling of cotton under wounding treatment.In addition,we have also found that mechanical damage to cotton can induced the accumulation of gossypol,a compound with anti-insect activity,where it can be significantly inhibited after the insect's oral secretion treatment.This result could also be supported by the expression analysis of genes on the gossypol biosynthesis pathway.3.Comparative and evolutionary analysis of metabolism in cottonTo explore the divergence of metabolism across different cotton species,we conducted metabolic profiling analysis on 25 representative Upland cotton cultivars,6races and other species of cotton.Our results showed there was a significant metabolic differentiation between cultivars and wild species.Further evolutionary analysis showed that among the 1,775 metabolic features we detected,595 of them including flavonoids and glutathione metabolism related metabolites showed potential domestication signal between cultivars and race population.4.Metabolic genome-wide associated study in natural population of cottonTo investigate the genetic bases of metabolites in Upland cotton,we analyzed a comprehensive metabolic profiling of a natural population consisted of 267 Upland cotton cultivars and detected 996(Wuhan,2016),828(Ezhou,2016)and 776(Ezhou,2017)almost no redundant metabolites in different environments.Subsequently,by using ?2.7million high-quality SNP and metabolic traits,we performed genome-wide association study for each metabolite and detected 1877(Wuhan,2016),1419(Ezhou,2016)and 898(Ezhou,2017)m QTL respectively.Finally,14 candidate genes that may affect the accumulation of metabolites were identified.5.A ?5kb insertion is responsible for the content variation of multiple metabolitesTo estimate the homogeneity of the distribution of m QTL,we performed hotspot analysis and observed a region located on chromosome A12 significantly associated with multiple metabolites.Further analysis showed the gland formation related gene,Ghir?A12G025340 was included in this hotspot.After silencing the expression of this gene,more than 20% of the metabolites were observed to differently accumulate,the gossypol content decreased by more than 80%.Ghir?A12G025340 overexpressed callus showed higher accumulation of gossypol but stopped differentiation.Sequence analysis showed a ? 5kb transposon was observed to insert into the coding region of Ghir?A12G025340 in low-gossypol accessions,which is responsible for the content variation of gossypol and other metabolites.6.Multi-omics analysis promote candidate gene identificationTo effectively identify candidate gene,we performed e QTL for candidate genes and correlation analysis between metabolites and genes to construct the network based on SNP,genes and metabolites.Among them,Ghir?D13g004710 was identified as a gene encoding acyltransferase.The expression of Ghir?D13g004710 was positively correlated with the content of target metabolite,and the cis-e QTL was observed to overlap with m QTL of target metabolite.Silencing the expression of Ghir?D13g004710 could significantly decrease the content of target metabolite.Similarly,Ghir?A12G019280which encoding flavonoid 3'-monooxygenase was identified to regulate the accumulation of kaempferol and its derivatives in cotton.7.Glycosyltransferase regulates the biosynthesis of ABA-GE and confers cotton drought resistanceA significant association between the content of ABA-GE and the glycosyltransferase gene Ghir?D02G002230 was observed in our study.Expression pattern analysis showed this gene could be significantly down regulated by drought stress and has lower expression level in drought tolerant line compared with sensitive ones.In vitro analysis was performed,the recombinant protein of Ghir?D02G002230 showed the activity of ABA glycosyltransferase.Silencing the gene expression of Ghir?D02G002230in cotton significantly reduced the accumulation of ABA-GE and enhanced the resistance of cotton plants to drought stress.8.Identification of metabolites related to insect resistanceSecondary metabolites play important roles in plant defense against insects.To identify metabolites which provide insect resistance to cotton,co-expression network analysis was conducted based on metabolites.Further analysis identified a metabolic module including an unknow metabolite which positively correlated with cotton insect resistance and significant associated with SNP on chromosome A06.