Genome Wide Analyses Reveal The Genetic Basis Of Early Maturity And Identification Of Candidate Genes In Upland Cotton

Upland cotton（Gossypium hirsutum L.AADD,2n=52）is the most widely planted economic crop and the leading source of natural fiber worldwide.Since the 1920s,China introduced amount of upland cotton varieties from the United States,including Deltapine,Stoneville,Acala and King.Accompanying the planting and spreading process of upland cotton,breeders have made great efforts to improve their yield,fiber quality and early maturity.In particular,the study of genetic basic of early maturity and development of early maturity cotton,which is seeded directly after wheat or rape harvest,is a remarkable achievement that has helped address the grain-cotton balance caused by population growth and loss of farmland and increase multiple crop indexes in China.Along with this process,abundant early maturity genetic variations were accumulated in upland cotton.In this study,we used a natural population with abundant genetic diversity which contained representative early maturity cotton varieties as research materials by many methods,such as literature research,field investigation and whole genome re-sequencing and verification of gene function in this study to make a comprehensive research on the genetic basis of early maturity in upland cotton.A total of 436 Gossypium hirsutum accessions were collected worldwide for genomic sequence analysis,comprising 32 wild Gossypium hirsutum lines and 404 cultivars.These accessions originated from different countries and have wide geographical distribution in China,representing more than 100 years of upland cotton breeding around the world.The main results as follows:1.We detected 10,118,884 high-quality SNPs and 864,132 Indels based on the resequencing data of 436 upland cotton accessions.Most SNPs（91.06%）were in intergenic regions,with only a small portion（0.66%）located in coding sequences corresponding to 19,318 genes.Of these,42,052 nonsynonymous SNPs were identified among 15,006 genes.2.We detected the selective sweeps and genes altered by strong human selection by using CLR method during the breeding of eary maturity cotton.These genes were functionally related to flowering time control,hormone catabolism,aging,and defense response adaptations to environmental changes.3.From years of 2015 to 2017,seven quantitative traits for early maturity,comprising flowering time（FT）,the period from first flower blooming to first boll opening（FBP）,whole growth period（WGP）,yield percentage before frost（YPBF）,node of the first fruiting branch（NFFB）,height of the node of the first fruiting branch（HNFFB）,and plant height（PH）,were investigated at three locations.Significant variation was observed for seven maturity-related traits among the 355 upland cotton accessions.Pearson’s correlation coefficient analysis showed a significant negative correlation between YPBF and the other traits,all six of which showed significantly positive pairwise correlations.A broad sense heritability analysis was performed,and all traits ranged from 0.67（WGP）to 0.79（FT）.4.We performed GWAS analysis for 7 early maturity related traits and identified 307 significant loci distributed on 9 chromosomes.Of all these significant loci,six（rsD03₃7996318,rsD03₃7952328,rsD03₃8191576,rsD03₃8175272,rsD03₃8370420 and rsD03₃9122594）shared more than four traits,indicating a genetic basis for pleiotropism that made it possible to simultaneously improve multiple early maturity traits during breeding.Notably,two distinct enrichment regions were located on chromosome A05 and chromosome D03,which accounted for more than 88.92%（273）of the loci.5.The SNP（rsD03₃9122594）was located 1,810 bp upstream of the start codon of Ghir_D03G011310,which was found to have the strongest association with FT,WGP,YPBF,and PH（average-log10（P）=6.88）.Varieties carrying the A allele exhibited earlier maturity than carriers of the G allele.The HRM assay showed that the mean WGP in the two RIL lines with the A allele was significantly shorter than that in the lines with the G allele.The RNA-seq data showed that Ghir_D03G011310 had higher expression levels in the early maturity variety "CRI50" than in the late maturity variety "TM-1" compared with the other two genes during flower development from 0 to 20 DPS.We further validated the function of Ghir_D03G011310 through virus-induced gene silencing（VIGS）in early maturity cotton "CRI50".The fruit branches or squares have not been observed in the silenced plants.However,the CK plants have yielded fruit branches with squares at the same growth status.In addition,ESM group had lowest diversity than wild and MLM group and Fst values of ESM versus MLM was significantly higher than the flanking region.Therefore,it is reasonable to postulate that Ghir_D03G011310 is a new candidate gene for influencing early flowering and involved in artificial selection in early maturity cotton breeding.6.Ghir_A05G017390 on A05 chromosome and it’s homologous Ghir_D05G017200 on D05 chromosome was significantly associated with PH and HNFFB.The qRT-PCR results indicated that Ghir_A05G017390 expression was higher in apical buds in early maturity varieties（PH=59.25±5.69 cm）than in those in late maturity varieties（PH=87.96±4.39 cm）.Overexpression of the Ghir_A05G017390 in Arabidopsis resulted in dwarf phenotypes compared with the wild type.Furthermore,the resulting VIGS lines had significantly higher compared to "CRI50" and CLCrVA（empty vector）.From the above results,we inferred that Ghir_A05G017390 has a potential role in controlling cotton stature in the future.7.A high-density linkage map of upland cotton was constructed using genotyping by sequencing（GBS）to discover single nucleotide polymorphism（SNP）markers associated with early maturity in 170 F2 individuals derived from a cross between Lu28 and Zhong213.The high-density genetic map,which was composed of 3,978 SNP markers across the 26 cotton chromosomes,spanned 2,480 cM with an average genetic distance of 0.62 cM.Based on this high-density linkage map,QTL analysis was performed on cotton early maturity traits.A total 47 QTLs for the six traits were detected.A major region controlling early maturity traits in Gossypium hirsutum was identified for FT,FBP,WGP,NFFB and HNFFB on chromosome D03.QTL analyses revealed that phenotypic variation explained（PVE）ranged from 10.42%to 32.57%.Two potential candidate genes,Gh_D03G0885（TOC1）and Gh_D03G0922（AGL8）,were predicted in a stable QTL region and had higher expression levels in the early-maturity variety Zhong213 than in the late-maturity variety Lu28.