Multi-Omics Data Reveal Genetic Regulation And Key Metabolites In The Transition Stage During Cotton Fiber Development

Cotton is an important cash crop and fiber is the main product.The diversity of fiber quality is determined by a variety of genetic variations.This study presented an analysis of fiber transcriptomes and metabolomes from natural cotton accessions.We revealed the key genetic regulators and metabolites of the temporal modulation of secondary cell wall synthesis during fiber cell elongation.Flavonoid metabolites were found to be significantly correlated with cotton fiber quality.The function of two genes in flavonoid biosynthesis pathway,GhCHS and GhDFR,in fiber development was analysed.The main findings are:1.Combined GWAS and eQTL analysis uncovers a genetic regulatory network orchestrating the initiation of secondary cell wall development in cotton fiber.In this study,a genome-wide association study(GWAS)was performed on 251 cotton accessions(Gossypium hirsutum),and 28 genetic loci associated with fiber quality in allotetraploid cotton were identified.To investigate the regulatory roles of these loci,RNA-seq was performed for the 15 days post anthesis(15 DPA)fiber for transcriptome analysis of these cotton accessions and identified 15,330 expression quantitative trait loci(eQTL).Analysis of local eQTL and GWAS data prioritized 13 likely causal genes for differential fiber quality in a transcriptome-wide association study(TWAS).Characterization of distal eQTL revealed unequal genetic regulation patterns between two subgenomes,highlighted by an eQTL hotspot(Hot216)in the D subgenome that establishes a genome-wide genetic network regulating the expression of 962 genes.The primary regulatory role of Hot216,and specifically the gene encoding a KIP-related protein,was found to be the transcriptional regulation of genes responsible for cell wall synthesis,which contributes to fiber length by modulating the developmental transition from rapid cell elongation to secondary cell wall synthesis.This study uncovers the genetic regulation of fiber-cell development and reveals the molecular basis of the temporal modulation of secondary cell wall synthesis during plant cell elongation.2.Metabolites significantly correlated with fiber quality were identified by metabolomics analysis.To investigate the relationship between metabolites and cotton fiber quality,a MS~2spectral tag(MS2T)library of cotton 15 DPA fiber with 961 metabolites was constructed by using fibers at 5 different development stages of 18 cotton accessions with genetic and fiber quality variation,and 272 metabolites in MS2T library were annotated.Then the MS2T database was used to determine the metabolites contents of a natural cotton populations with 279 accessions.Variation analysis of cotton metabolome showed that abundant metabolic variation existed in fiber.Cluster analysis showed that diploid cotton species and tetraploid cotton species were divided into two clusters,upland cotton(G.hirsutum)species and island cotton(G.barbadense)species were divided into two clusters.The correlation analysis of annotated metabolites showed that the correlation between metabolites was very complex,but the correlation between the same-type metabolites was stronger.The analysis of the correlation between 272 annotated metabolites and fiber quality-related traits showed that amino acids and amino acid derivatives,saccharides and lipids were positively correlated with fiber quality and yield traits(boll weight and lint percent).Analysis of 961 metabolites and fiber quality found that 97 metabolites were significantly correlated with fiber macronaire value,130 metabolites were significantly correlated with fiber strength,and 239 metabolites were significantly correlated with fiber length.The coumarins and flavonoids were correlated with fiber strength and fiber length.This study revealed the metabolite-related network in the fiber during the transition of rapid fiber elongation to secondary cell wall synthesis,and identified metabolites significantly related to fiber quality.3.Down-regulation of flavonoids biosynthesis genes GhCHS and GhDFR inhibites fiber development.The gene families of CHS and DFR,two key genes in flavonoid biosynthesis pathway,in two tetraploid cultivated cotton(G.hirsutum TM-1 and G.barbadense 3-79)genome were identified.The distribution of CHS on these two cotton chromosomes was asymmetric.GhCHS and GhDFR RNAi vectors driven by Ca MV35S promoter were constructed and transformed into G.hirsutum YZ1.The GhCHS-RNAi lines had shorter fiber length,higher micronaire values,decreased fiber strength and uniformity.The GhDFR-RNAi lines had significantly decreased micronaire values(decreased from control 5.6 to 3.0),increased fiber strength and decreased fiber uniformity.Among the yield traits,boll weight,seed index and lint index of the GhDFR-RNAi lines were also decreased significantly.Flavonoid histochemical staining found that the flavonoids contents in GhCHS and GhDFR RNAi lines were significantly reduced.LC-MS analysis of flavonoid contents in cotton fibers found that the content of naringin in the GhCHS-RNAi lines were significantly decreased,while the content of quercetin and kaempferol were accumulated in the GhDFR-RNAi lines.The GhCHS and GhDFR RNAi segments were transferred into a brown cotton background via cross.In F1 plant,lighter colored cotton fiber was observed in plants containing GhCHS-RNAi segment,while darker brown cotton fiber was observed in plants containing GhDFR-RNAi segment.These results indicated that a complex flavonoid metabolism in cotton fiber existed,and GhCHS and GhDFR affected the pigment formation and deposition in brown cotton fiber possibly in a contrary direction.