Deepening genomic sequences of 1081 Gossypium hirsutum accessions reveals novel SNPs and haplotypes relevant for practical breeding utility.

Fiber quality is a major breeding goal in cotton, but phenotypically direct selection is often hindered. In this study, we identified fiber quality and yield related loci using GWAS based on 2.97 million SNPs obtained from 10.65× resequencing data of 1081 accessions. The results showed that 585 novel fiber loci, including two novel stable SNP peaks associated with fiber length on chromosomes At12 and Dt05 and one novel genome regions linked with fiber strength on chromosome Dt12 were identified. Furthermore, by means of gene expression analysis, GhM_A12G0090, GhM_D05G1692, GhM_D12G3135 were identified and GhM_D11G2208 function was identified in Arabidopsis. Additionally, 14 consistent and stable superior haplotypes were identified, and 25 accessions were detected as possessing these 14 superior haplotype in breeding. This study providing fundamental insight relevant to identification of genes associated with fiber quality and yield will enhance future efforts toward improvement of upland cotton.

Transcriptome, Ectopic Expression and Genetic Population Analysis Identify Candidate Genes for Fiber Quality Improvement in Cotton.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I-IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I-V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

A stable QTL qSalt-A04-1 contributes to salt tolerance in the cotton seed germination stage.

KEY MESSAGE: A stable QTL qSalt-A04-1 for salt tolerance in the cotton seed germination stage, and two candidate genes, GhGASA1 and GhADC2, that play negative roles by modulating the GA and PA signalling pathways, respectively, were identified. The successful transition of a seed into a seedling is a prerequisite for plant propagation and crop yield. Germination is a vulnerable stage in a plant's life cycle that is strongly affected by environmental conditions, such as salinity. In this study, we identified a novel quantitative trait locus (QTL) qRGR-A04-1 associated with the relative germination rate (RGR) after salt stress treatment based on a high-density genetic map under phytotron and field conditions, with LOD values that ranged from 6.65 to 16.83 and 6.11-12.63% phenotypic variations in all five environmental tests. Two candidate genes with significantly differential expression between the two parents were finally identified through RNA-seq and qRT-PCR analyses. Further functional analyses showed that GhGASA1- and GhADC2-overexpression lines were more sensitive to salt stress than wild-type Arabidopsis based on the regulation of the transcript levels of gibberellic acid (GA)- and polyamine (PA)- related genes in GA and PA biosynthesis and the reduction in the accumulation of GA and PA, respectively, under salt stress. Virus-induced gene silencing analysis showed that TRV:GASA1 and TRV:ADC2 were more tolerant to salt stress than TRV:00 based on the increased expression of GA synthesis genes and decreased H2O2 content, respectively. Taken together, our results suggested that QTL qRGR-A04-1 and its two harboured genes, GhGASA1 and GhADC2, are promising candidates for salt tolerance improvement in cotton.

A high-density genetic map and multiple environmental tests reveal novel quantitative trait loci and candidate genes for fibre quality and yield in cotton.

KEY MESSAGE: A high-density linkage map of an intraspecific RIL population was constructed using 6187 bins to identify QTLs for fibre quality- and yield-related traits in upland cotton by whole-genome resequencing. Good fibre quality and high yield are important production goals in cotton (Gossypium hirsutum L.), which is a leading natural fibre crop worldwide. However, a greater understanding of the genetic variants underlying fibre quality- and yield-related traits is still required. In this study, a large-scale population including 588 F7 recombinant inbred lines, derived from an intraspecific cross between the upland cotton cv. Nongdamian13, which exhibits high quality, and Nongda601, which exhibits a high yield, was genotyped by using 232,946 polymorphic single-nucleotide polymorphisms obtained via a whole-genome resequencing strategy with 4.3-fold genome coverage. We constructed a high-density bin linkage map containing 6187 bin markers spanning 4478.98 cM with an average distance of 0.72 cM. We identified 58 individual quantitative trait loci (QTLs) and 25 QTL clusters harbouring 94 QTLs, and 119 previously undescribed QTLs controlling 13 fibre quality and yield traits across eight environments. Importantly, the QTL counts for fibre quality in the Dt subgenome were more than two times that in the At subgenome, and chromosome D02 harboured the greatest number of QTLs and clusters. Furthermore, we discovered 24 stable QTLs for fibre quality and 12 stable QTLs for yield traits. Four novel major stable QTLs related to fibre length, fibre strength and lint percentage, and seven previously unreported candidate genes with significantly differential expression between the two parents were identified and validated by RNA-seq. Our research provides valuable information for improving the fibre quality and yield in cotton breeding.

A genome-wide association study uncovers novel genomic regions and candidate genes of yield-related traits in upland cotton.

KEY MESSAGE: A total of 62 SNPs associated with yield-related traits were identified by a GWAS. Based on significant SNPs, two candidate genes pleiotropically increase lint yield. Improved fibre yield is considered a constant goal of upland cotton (Gossypium hirsutum) breeding worldwide, but the understanding of the genetic basis controlling yield-related traits remains limited. To better decipher the molecular mechanism underlying these traits, we conducted a genome-wide association study to determine candidate loci associated with six yield-related traits in a population of 719 upland cotton germplasm accessions; to accomplish this, we used 10,511 single-nucleotide polymorphisms (SNPs) genotyped by an Illumina CottonSNP63K array. Six traits, including the boll number, boll weight, lint percentage, fruit branch number, seed index and lint index, were assessed in multiple environments; large variation in all phenotypes was detected across accessions. We identified 62 SNP loci that were significantly associated with different traits on chromosomes A07, D03, D05, D09, D10 and D12. A total of 689 candidate genes were screened, and 27 of them contained at least one significant SNP. Furthermore, two genes (Gh_D03G1064 and Gh_D12G2354) that pleiotropically increase lint yield were identified. These identified SNPs and candidate genes provide important insights into the genetic control underlying high yields in G. hirsutum, ultimately facilitating breeding programmes of high-yielding cotton.

Genome-wide association study discovered genetic variation and candidate genes of fibre quality traits in Gossypium hirsutum L.

Genetic improvement of fibre quality is one of the main breeding goals for the upland cotton, Gossypium hirsutum, but there are difficulties with precise selection of traits. Therefore, it is important to improve the understanding of the genetic basis of phenotypic variation. In this study, we conducted phenotyping and genetic variation analyses of 719 diverse accessions of upland cotton based on multiple environment tests and a recently developed Cotton 63K Illumina Infinium SNP array and performed a genome-wide association study (GWAS) of fibre quality traits. A total of 10 511 polymorphic SNPs distributed in 26 chromosomes were screened across the cotton germplasms, and forty-six significant SNPs associated with five fibre quality traits were detected. These significant SNPs were scattered over 15 chromosomes and were involved in 612 unique candidate genes, many related to polysaccharide biosynthesis, signal transduction and protein translocation. Two major haplotypes for fibre length and strength were identified on chromosomes Dt11 and At07. Furthermore, by combining GWAS and transcriptome analysis, we identified 163 and 120 fibre developmental genes related to length and strength, respectively, of which a number of novel genes and 19 promising genes were screened. These results provide new insight into the genetic basis of fibre quality in G. hirsutum and provide candidate SNPs and genes to accelerate the improvement of upland cotton.

Genome-Wide Identification of the Alfin-like Gene Family in Cotton (Gossypium hirsutum) and the GhAL19 Gene Negatively Regulated Drought and Salt Tolerance.

Alfin-like (AL) is a small plant-specific gene family characterized by a PHD-finger-like structural domain at the C-terminus and a DUF3594 structural domain at the N-terminus, and these genes play prominent roles in plant development and abiotic stress response. In this study, we conducted genome-wide identification and analyzed the AL protein family in Gossypium hirsutum cv. NDM8 to assess their response to various abiotic stresses for the first time. A total of 26 AL genes were identified in NDM8 and classified into four groups based on a phylogenetic tree. Moreover, cis-acting element analysis revealed that multiple phytohormone response and abiotic stress response elements were highly prevalent in AL gene promoters. Further, we discovered that the GhAL19 gene could negatively regulate drought and salt stresses via physiological and biochemical changes, gene expression, and the VIGS assay. The study found there was a significant increase in POD and SOD activity, as well as a significant change in MDA in VIGS-NaCl and VIGS-PEG plants. Transcriptome analysis demonstrated that the expression levels of the ABA biosynthesis gene (GhNCED1), signaling genes (GhABI1, GhABI2, and GhABI5), responsive genes (GhCOR47, GhRD22, and GhERFs), and the stress-related marker gene GhLEA14 were regulated in VIGS lines under drought and NaCl treatment. In summary, GhAL19 as an AL TF may negatively regulate tolerance to drought and salt by regulating the antioxidant capacity and ABA-mediated pathway.

Identification and Expression Analysis of EPSPS and BAR Families in Cotton.

Weeds seriously affect the yield and quality of crops. Because manual weeding is time-consuming and laborious, the use of herbicides becomes an effective way to solve the harm caused by weeds in fields. Both 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS) and acetyltransferase genes (bialaphos resistance, BAR) are widely used to improve crop resistance to herbicides. However, cotton, as the most important natural fiber crop, is not tolerant to herbicides in China, and the EPSPS and BAR family genes have not yet been characterized in cotton. Therefore, we explore the genes of these two families to provide candidate genes for the study of herbicide resistance mechanisms. In this study, 8, 8, 4, and 5 EPSPS genes and 6, 6, 5, and 5 BAR genes were identified in allotetraploid Gossypium hirsutum and Gossypium barbadense, diploid Gossypium arboreum and Gossypium raimondii, respectively. Members of the EPSPS and BAR families were classified into three subgroups based on the distribution of phylogenetic trees, conserved motifs, and gene structures. In addition, the promoter sequences of EPSPS and BAR family members included growth and development, stress, and hormone-related cis-elements. Based on the expression analysis, the family members showed tissue-specific expression and differed significantly in response to abiotic stresses. Finally, qRT-PCR analysis revealed that the expression levels of GhEPSPS3, GhEPSPS4, and GhBAR1 were significantly upregulated after exogenous spraying of herbicides. Overall, we characterized the EPSPS and BAR gene families of cotton at the genome-wide level, which will provide a basis for further studying the functions of EPSPS and BAR genes during growth and development and herbicide stress.

Lysine 2-Hydroxyisobutyrylation- and Succinylation-Based Pathways Act Inside Chloroplasts to Modulate Plant Photosynthesis and Immunity.

Crops must efficiently allocate their limited energy resources to survival, growth and reproduction, including balancing growth and defense. Thus, investigating the underlying molecular mechanism of crop under stress is crucial for breeding. Chloroplasts immunity is an important facet involving in plant resistance and growth, however, whether and how crop immunity modulated by chloroplast is influenced by epigenetic regulation remains unclear. Here, the cotton lysine 2-hydroxyisobutyrylation (Khib) and succinylation (Ksuc) modifications are firstly identified and characterized, and discover that the chloroplast proteins are hit most. Both modifications are strongly associated with plant resistance to Verticillium dahliae, reflected by Khib specifically modulating PR and salicylic acid (SA) signal pathway and the identified GhHDA15 and GhSRT1 negatively regulating Verticillium wilt (VW) resistance via removing Khib and Ksuc. Further investigation uncovers that photosystem repair protein GhPSB27 situates in the core hub of both Khib- and Ksuc-modified proteins network. The acylated GhPSB27 regulated by GhHDA15 and GhSRT1 can raise the D1 protein content, further enhancing plant biomass- and seed-yield and disease resistance via increasing photosynthesis and by-products of chloroplast-derived reactive oxygen species (cROS). Therefore, this study reveals a mechanism balancing high disease resistance and high yield through epigenetic regulation of chloroplast protein, providing a novel strategy to crop improvements.

A Genome Wide Association Study Revealed Key Single Nucleotide Polymorphisms/Genes Associated With Seed Germination in Gossypium hirsutum L.

Fast and uniform seed germination is essential to stabilize crop yields in agricultural production. It is important to understand the genetic basis of seed germination for improving the vigor of crop seeds. However, little is known about the genetic basis of seed vigor in cotton. In this study, we evaluated four seed germination-related traits of a core collection consisting of 419 cotton accessions, and performed a genome-wide association study (GWAS) to explore important loci associated with seed vigor using 3.66 million high-quality single nucleotide polymorphisms (SNPs). The results showed that four traits, including germination potential, germination rate, germination index, and vigor index, exhibited broad variations and high correlations. A total of 92 significantly associated SNPs located within or near 723 genes were identified for these traits, of which 13 SNPs could be detected in multiple traits. Among these candidate genes, 294 genes were expressed at seed germination stage. Further function validation of the two genes of higher expression showed that Gh_A11G0176 encoding Hsp70-Hsp90 organizing protein negatively regulated Arabidopsis seed germination, while Gh_A09G1509 encoding glutathione transferase played a positive role in regulating tobacco seed germination and seedling growth. Furthermore, Gh_A09G1509 might promote seed germination and seedling establishment through regulating glutathione metabolism in the imbibitional seeds. Our findings provide unprecedented information for deciphering the genetic basis of seed germination and performing molecular breeding to improve field emergence through genomic selection in cotton.

High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement.

Cotton produces natural fiber for the textile industry. The genetic effects of genomic structural variations underlying agronomic traits remain unclear. Here, we generate two high-quality genomes of Gossypium hirsutum cv. NDM8 and Gossypium barbadense acc. Pima90, and identify large-scale structural variations in the two species and 1,081 G. hirsutum accessions. The density of structural variations is higher in the D-subgenome than in the A-subgenome, indicating that the D-subgenome undergoes stronger selection during species formation and variety development. Many structural variations in genes and/or regulatory regions potentially influencing agronomic traits were discovered. Of 446 significantly associated structural variations, those for fiber quality and Verticillium wilt resistance are located mainly in the D-subgenome and those for yield mainly in the A-subgenome. Our research provides insight into the role of structural variations in genotype-to-phenotype relationships and their potential utility in crop improvement.

Resequencing a core collection of upland cotton identifies genomic variation and loci influencing fiber quality and yield.

Upland cotton is the most important natural-fiber crop. The genomic variation of diverse germplasms and alleles underpinning fiber quality and yield should be extensively explored. Here, we resequenced a core collection comprising 419 accessions with 6.55-fold coverage depth and identified approximately 3.66 million SNPs for evaluating the genomic variation. We performed phenotyping across 12 environments and conducted genome-wide association study of 13 fiber-related traits. 7,383 unique SNPs were significantly associated with these traits and were located within or near 4,820 genes; more associated loci were detected for fiber quality than fiber yield, and more fiber genes were detected in the D than the A subgenome. Several previously undescribed causal genes for days to flowering, fiber length, and fiber strength were identified. Phenotypic selection for these traits increased the frequency of elite alleles during domestication and breeding. These results provide targets for molecular selection and genetic manipulation in cotton improvement.