Genetic Evolution And Formation Mechanism Of Pigment Gland And Gossypol Of Gossypium Bickii

Cotton is one of the most critical cash crops worldwide with valuable main and by-products.The high-quality oil and superior protein in cottonseed can meet the needs of about 500 million people in the world.However,the pigment glands,distributed in whole cotton plants,contain a large part of gossypol and its derivatives.The utilization of cottonseed was limitted by the gossypol,which are toxic to human and monogastric animals.At present,some cultivars with low content of gossypol in cottonseed could be consumed directly,meanwhile it drastically lose the resistance to pests and diseases.The wild diploid Australian cotton species,such as Gossypium bickii（G₁）,have the trait of delayed formation of cotyledon pigment glands-The cottonseeds were pigment glandless and extremely low in the gossypol content,and only formates of pigment glands and accumulates of gossypol after germination.It is an important trait for creating a new type of high-quality pigment-glandless cultivars.Therefore,the study of the evolutionary characteristics and genetic basis of G.bickii will provide insight into searching for related functional genes,as well as breeding work.In this study,Gossypium bicki（G₁）was sequenced by a number of technologies,including whole genome sequencing,chloroplast sequencing and genome resequencing.Then comparative genomics research was used to reveal the genetic background and evolutionary law of G.bickii.In addition,functional genomics and transcriptomics were carried out to explore the genetic mechanism of delayed formation of pigment glands and gossypol.The research contents and main results were listed as follows:1.The whole genome of G.bickii was sequenced and assembled by Illumina sequencing,Pac Bio sequencing and Hi-C assisted assembly technology,generating1,224.21 Gb of raw data,with a contig N50 of 4.62 Mb and a scaffold N50 of 133.90Mb.The genome size was 1,776.06 Mb and the mapping rate reaches to 96.51%.The genome was assembled with high integrity and continuity,reaching the quality standard of reference genome.2.By analyzing the phylogenetic tree constructed by G.bickii and 9 other Malvaceae plants,the genome of G.bickii was closest to Gossypium australe（G₂）,followed by A-genome and D-genome.In addition,it was found that transposon amplification happened in the evolutionary process of Gossypium by analyzing the repeat sequences and transposon elements,and G.bickii showed more similar transposons with G.australe than that of other species.3.Chromosome collinearity analysis was carried out by comparing G.bickii with other cotton species.Two translocations unique on chromosome 5 and 13 of G-genome were identified,which only existed between G-genome（G.bickii and G.australe）and other genomes of cotton species,rather than within the two species of G-genome.The evidence showed that these two translocations appeared before the divergence of G-genome and was supposed to be an important factor for the divergence of G-genome and other genome group.In addition,two inversions particular in G.bickii and G.australe were also found on chromosome 12 and 13,indicating that they appeared after the formation of G-genome,and might be related to the independent divergence of G.bickii and G.australe themselves within G-genome.Furthermore,some genes related to gossypol biosynthesis and fiber formation were identified in the area of inversions and translocations,which might play an important role in the formation of G-genome and the divergence of G.bickii and G.australe.4.There was a whole genome duplication（WGD）event identified in the evolution of Gossypium genome.All cotton species had experienced a common duplication event at 13～20 MYA,which did not happen in Theobroma cacao.This unique duplication event might initiate the divergence of Gossypium from other species in Malvaceae,rather than the divergence within Gossypium.5.The chloroplast genomes of G.bickii,G.australe,Gossypium nelsonii（G₃）and Gossypium sturtianum（C₁）,were sequenced and assembled completely.The chloroplast genomes were highly conserved and the genome size were 159.54,159.57,159.58 and 159.46 Kb,respectively.These genomes were composed of 132 functional genes.The phylogenetic tree showed that chloroplast genome of G.bickii was closer to that of G.sturtianum（C₁）in C-genome,while genome resequencing results showed that the nuclear genome of G.bickii was closer to that of G.australe and Gossypium nelsonii（G₃）in G-genome.It was inferred that G.bickii was evolved by the hybridization of ancestor of C-genome as the female parent,and the ancestors of G-genome as the male parent.6.The phenotypic observation,and gene expression analysis were carried out in different periods of ovule development and seed germination among G.bickii,Gossypium herbaceum（A₁）and Gossypium arboreum（A₂）.Our study showed that pigment glands and gossypol did not appear in G.bickii at ovule development period until seed germinated,while they visibly apperared in G.herbaceum（A₁）and G.arboreum（A₂）at 20 days after anthesis（dpa）,which comfirmed the delayed formation of pigment glands and gossypol in cotyledons of G.bickii.In addition,the expression level of pigment glands and gossypol related genes in G.bickii were low in the ovules and only up-regulated after seed germination.While related genes in G.herbaceum and G.arboreum were up-regulated at 20 dpa of ovule development,indicating that those genes might took part in the process of pigment glands and gossypol delayed formation.7.89 differentially expressed genes were screened among G.bickii,G.herbaceum,and G.arboreum by different expression analysis.KEGG analysis showed that those genes were enriched in the terpene biosynthesis pathway,which was speculated to be related to gossypol biosynthesis.According to weight gene co-expression network analysis（WGCNA）,3 candidate genes were mapped and screened with high relationship with pigment gland gene Go PGF and gossypol biosynthesis genes.It was found that one cytochrome P450 related candidate gene Gbi CYP76B6 could reduce the gossypol content on stems and leaves by about 50%,based on virus induced gene silencing（VIGS）.It was inferred that CYP76B6 was downstream of Go PGF,and regulated by Go PGF.In summary,our study revealed the origin and evolutionary characteristics of Gossypium bickii by whole genome sequencing and comparative genomics analysis.The unique inversions and translocations in G₁-genome of G.bickii were identified.It was found that transposon amplification and WGD events unique existed in the process of evolution.Those results of chloroplast genome sequencing and genome resequencing showed that G.bickii originated from the ancestral species of G and C-genomes.On the basis of transcriptome sequencing,we identified a candidate gene named Gbi CYP76B6,which related to gossypol biosynthesis.