Mechanisms Of Pigment Glands Formation And Gossypol Biosynthesis And Their Genome-wide Analysis In Upland Cotton

Cotton is one of the most important economic crops worldwide,it is not only the largest source of natural fibres for the textile industry,but also great souce of high quality oil and protein.Pigment gland is one of the specific biological characteristics in Gossypium and its relative plants,which contains a lot of compounds,and the main material is gossypol.Gossypol is the most important secondary metabolite,which is stored in the cotton pigment glands.It plays a critical role in the process of cotton development,but its toxicity for human and non-ruminant animals greatly limits the comprehensive utilization of cottonseeds.Therefore,research on the pigment gland and gossypol is always the focus of the scientists,including the morphological differences,morphogenesis,distribution and genetic mechanism of pigment glands,and biosynthesis sites,pathway,detection and regulation mechanism of gossypol.They aim to breed the better variety by the precision molecular breeding effectively.Especially the cotton variety with glandless cottonseeds and glanded plant,it not only retains the gossypol to defense the pest and disease,but also makes better use of the cottonseeds.This research chooses the near isogenic lines(NILs)with different types of pigment glands to research on the morphogenesis of pigment glands,biosynthesis of gossypol,the relationship between pigment glands and gossypol,and comparative genomic analysis on NILs of Gossypium hirsutum L.to reveal the molecular mechanism of pigment glands formation and gossypol biosynthesis.The main research contents and results are as follows:(1)Morphogenesis of pigment gland in upland cottonCCRI17,Coker 312,Gl2Gl2gk3gk3,gl2gGl3Gl3 and TM-1 were selected to investigate the pigment morphology,including the size and density.Results indicated that the size and density of pigment glands were significantly different among different organs.Pigment glands in boll shell were the largest,followed by the sepal,and the pigment glands in upper leaves,petal and seed were smaller.Pigment glands density in seed were the largest,followed by the leaves and petal,and the density in boll shell were the smallest.From the perspective of genotypes,Gl2 plays a major role in the size of the pigment glands,and Gl2 and Gl3 togetherly determine the density of the pigment glands.Besides,through continuous paraffin section of pigment glands,we found that some groups of special pigment gland cells with high cytoplasmic density,large nucleus,small intercellular space and deeply stained nuclear appeared at 18 DPA.Two days later,the cells in these groups began to lyse from inside to outside,formed mature cavity and the morphogenesis of pigment glands were completed(2)Gossypol biosynthesis in upland cottonThrough the detection on the dynamic change of gossypol in glanded and glandless cottons at seed germination and seedling stages,grafting between glanded and glandless cottons,grafting between glanded cotton and sunflower,root and rootless seedling culture in vitro,expression profile of genes related to pigment gland and gossypol biosynthesis in different organs at seedling stage,it was indicated that root was the main organ to synthesize gossypol and other organs also had a relative low ability to synthesize gossypol.Both glanded and glandless roots had a strong ability of gossypol biosynthesis.Besides,with analysis of(±)-gossypol,it was figured out that cotton root synthesized the racemic gossypol,while other organs synthesize the optical gossypol(3)Relationship between pigment glands and gossypol in upland cottonThe relationship between pigment glands and gossypol in cotton is independent and closely related.Through correlation analysis between pigment gland and gossypol content,it was figured out that in all organs,the density of pigment glands was extremely significantly correlated with gossypol content,while the size of pigment glands was significantly correlated with gossypol in some organs.Besides,glandless cotton and sunflower rootstock did not affect the expression of pigment glands in glanded scion.Expression profile analysis suggested that expression of genes related to pigment gland and gossypol synthesis were inconsistent,which was indicated that the pathways of pigment glands formation and gossypol biosynthesis were independent.According to the results of gossypol content change in glanded embryos before and after pigment glands formation,differences in gossypol content between glanded and glandless organs,and gossypol dynamic change in root and rootless seedling culture in vitro,it was also indicated that pigment glands and gossypol were tightly related.Pigment glands were the storage tissue of gossypol,and gossypol were the storage material of pigment glands(4)Comparative genomic analysis of two pairs of NILs with different pigment glandsBased on the genome-wide information of upland cotton TM-1,we deeply resequenced(34×)the four accessions,CCRI12,CCRI12W,Coker 312 and Coker 312W.After comparison with TM-1 genome,we identified 2034021,1974262,2371614 and 2046733 SNPs,177324,167971,181706 and 180714 Indels,3963,3771,4208 and 4026 SVs,36388,34765,46765 and 35976 CNVs.We also compared the genomes in NILs,we found 18083 and 14913 genes with SNPs and Indels between Coker 312 and Coker 312W,respectively,and 7172 and 8087 genes with SNPs and Indels between CCRI12 and CCRI12W,respectively.GO enrichment analyses figured out that GO items differed between dominant and recessive NILs.KEGG pathway analyses found that genes with SNPs and Indels in dominant and recessive NILs shared some common significantly enriched pathways,including biosynthesis of secondary metabolite,flavonoid biosynthesis pathways.Genes with Indels in dominant NILs and genes with SNPs and Indels in recessive NILs were all significantly enriched in a common pathway:Sesquiterpenoid and triterpenoid biosynthesis,which is quite related to gossypol biosynthesis.In this pathway,we discovered the previously reported genes,such as TPS1 and cad1-A,and unknown candidate genes.Furthermore,we carried out the expression analysis of these unknown genes,and found the expression patterns of these genes were similar with key genes in gossypol biosynthesis pathwayIn conclusion,through the methods of tissue culture,grafting and paraffin section,this study revealed the difference and correlation between pigment glands and gossypol in different organs,morphogenesis of pigment gland and mechanism of gossypol biosynthesis in upland cotton Besides,by the methods of bioinformatics and comparative genomics,we discovered the important loci related to pigment gland morphogenesis and gossypol biosynthesis,and provided a theoretical basis for breeding new low-gossypol cotton with high gossypol content in plant and low gossypol content in seeds by genetic engineering.