Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.

BACKGROUND: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. RESULTS: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1L86P and GhTFL1K53G+S78G mutations, we find that the GhTFL1L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. CONCLUSIONS: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

Genome-Wide Identification and Analysis of the WNK Kinase Gene Family in Upland Cotton.

With-No-Lysine (WNK) kinases are a subfamily of serine/threonine protein kinases. WNKs are involved in plant abiotic stress response and circadian rhythms. However, members of the WNK subfamily and their responses to abiotic and biotic stresses in Gossypium hirsutum have not been reported. In this study, 26 GhWNKs were identified in G. hirsutum. The gene structure, conserved motifs, and upstream open reading frames (uORFs) of GhWNKs were identified. Moreover, GhWNKs regulation is predicted to be regulated by cis-acting elements, such as ABA responsive element (ABRE), MBS, and MYC. Furthermore, transcription factors including MIKC_MADS, C2H2, TALE, bZIP, Dof, MYB, bHLH, and HD-ZIP are projected to play a regulatory role in GhWNKs. The expression patterns of GhWNKs under normal conditions and biotic and abiotic stresses were evaluated, and their expression was found to vary. The expression patterns of several GhWNKs were induced by infiltration with Verticillium dahliae, suggesting that several GhWNKs may play important roles in the response of cotton to V. dahliae. Interestingly, a homoeologous expression bias within the GhWNKs was uncovered in upland cotton. Homoeologous expression bias within GhWNKs provides a framework to assist researchers and breeders in developing strategies to improve cotton traits by manipulating individual or multiple homeologs.

Cotton 4-coumarate-CoA ligase 3 enhanced plant resistance to Verticillium dahliae by promoting jasmonic acid signaling-mediated vascular lignification and metabolic flux.

Lignins and their antimicrobial-related polymers cooperatively enhance plant resistance to pathogens. Several isoforms of 4-coumarate-coenzyme A ligases (4CLs) have been identified as indispensable enzymes involved in lignin and flavonoid biosynthetic pathways. However, their roles in plant-pathogen interaction are still poorly understood. This study uncovers the role of Gh4CL3 in cotton resistance to the vascular pathogen Verticillium dahliae. The cotton 4CL3-CRISPR/Cas9 mutant (CR4cl) exhibited high susceptibility to V. dahliae. This susceptibility was most probably due to the reduction in the total lignin content and the biosynthesis of several phenolic metabolites, e.g., rutin, catechin, scopoletin glucoside, and chlorogenic acid, along with jasmonic acid (JA) attenuation. These changes were coupled with a significant reduction in 4CL activity toward p-coumaric acid substrate, and it is likely that recombinant Gh4CL3 could specifically catalyze p-coumaric acid to form p-coumaroyl-coenzyme A. Thus, overexpression of Gh4CL3 (OE4CL) showed increasing 4CL activity that augmented phenolic precursors, cinnamic, p-coumaric, and sinapic acids, channeling into lignin and flavonoid biosyntheses and enhanced resistance to V. dahliae. Besides, Gh4CL3 overexpression activated JA signaling that instantly stimulated lignin deposition and metabolic flux in response to pathogen, which all established an efficient plant defense response system, and inhibited V. dahliae mycelium growth. Our results propose that Gh4CL3 acts as a positive regulator for cotton resistance against V. dahliae by promoting JA signaling-mediated enhanced cell wall rigidity and metabolic flux.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

N6 -methyladenosine (m6 A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+ -related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6 A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

Genome and haplotype provide insights into the population differentiation and breeding improvement of Gossypium barbadense.

INTRODUCTION: Sea-island cotton (Gossypium barbadense, Gb) is one of the major sources of high-grade natural fiber. Besides the common annual Gb cotton, perennial Gb cotton is also cultivated, but studies on perennial Gb cotton are rare. OBJECTIVES: We aimed to make a systematic analysis of perennial sea-island cotton and lay a foundation for its utilization in breeding, and try to identify the representative structural variations (SVs) in sea-island cotton, and to reveal the population differentiation and adaptive improvement of sea-island cotton. METHODS: Through genome assembly of one perennial Gb cotton accession (named Gb_M210936) and comparative genome analysis, variations during Gb cotton domestication were identified by comparing Gb_M210936 with annual Gb accession 3-79 and with wild allotetraploid cotton G. darwinii. Six perennial Gb accessions combining with the resequenced 1,129 cotton accessions were used to conduct population and genetic analysis. Large haplotype blocks (haploblocks), generated from interspecific introgressions and intraspecific inversions, were identified and were used to analyze their effects on population differentiation and agronomic traits of sea-island cotton. RESULTS: One reference genome of perennial sea-island cotton was assembled. Representative SVs in sea-island cotton were identified, and 31 SVs were found to be associated with agronomic traits. Perennial Gb cotton had a closer kinship with the wild-to-landrace continuum Gb cotton from south America where Gb cotton is originally domesticated. Haploblocks were associated with agronomic traits improvement of sea-island cotton, promoted sea-island cotton differentiation into three subgroups, were suffered from breeding selection, and may drive Gb cotton to be adapted to central Asian. CONCLUSION: Our study made up the lack of perennial Gb cotton genome, and clarified that exotic introgressions improved the traits of sea-island cotton, promoted the population differentiation, and drove sea-island cotton adaptive to central Asia, which will provide new insights for the genetic breeding improvement of sea-island cottons.

Dissection of Hyperspectral Reflectance to Estimate Photosynthetic Characteristics in Upland Cotton (Gossypium hirsutum L.) under Different Nitrogen Fertilizer Application Based on Machine Learning Algorithms.

Hyperspectral technology has enabled rapid and efficient nitrogen monitoring in crops. However, most approaches involve direct monitoring of nitrogen content or physiological and biochemical indicators directly related to nitrogen, which cannot reflect the overall plant nutritional status. Two important photosynthetic traits, the fraction of absorbed photosynthetically active radiation (FAPAR) and the net photosynthetic rate (Pn), were previously shown to respond positively to nitrogen changes. Here, Pn and FAPAR were used for correlation analysis with hyperspectral data to establish a relationship between nitrogen status and hyperspectral characteristics through photosynthetic traits. Using principal component and band autocorrelation analyses of the original spectral reflectance, two band positions (350-450 and 600-750 nm) sensitive to nitrogen changes were obtained. The performances of four machine learning algorithm models based on six forms of hyperspectral transformations showed that the light gradient boosting machine (LightGBM) model based on the hyperspectral first derivative could better invert the Pn of function-leaves in cotton, and the random forest (RF) model based on hyperspectral first derivative could better invert the FAPAR of the cotton canopy. These results provide advanced metrics for non-destructive tracking of cotton nitrogen status, which can be used to diagnose nitrogen nutrition and cotton growth status in large farms.

CottonMD: a multi-omics database for cotton biological study.

Cotton is an important economic crop, and many loci for important traits have been identified, but it remains challenging and time-consuming to identify candidate or causal genes/variants and clarify their roles in phenotype formation and regulation. Here, we first collected and integrated the multi-omics datasets including 25 genomes, transcriptomes in 76 tissue samples, epigenome data of five species and metabolome data of 768 metabolites from four tissues, and genetic variation, trait and transcriptome datasets from 4180 cotton accessions. Then, a cotton multi-omics database (CottonMD, http://yanglab.hzau.edu.cn/CottonMD/) was constructed. In CottonMD, multiple statistical methods were applied to identify the associations between variations and phenotypes, and many easy-to-use analysis tools were provided to help researchers quickly acquire the related omics information and perform multi-omics data analysis. Two case studies demonstrated the power of CottonMD for identifying and analyzing the candidate genes, as well as the great potential of integrating multi-omics data for cotton genetic breeding and functional genomics research.

Genome-wide association study and transcriptome analysis reveal key genes controlling fruit branch angle in cotton.

Fruit branch angle (FBA), a pivotal component of cotton plant architecture, is vital for field and mechanical harvesting. However, the molecular mechanism of FBA formation is poorly understood in cotton. To uncover the genetic basis for FBA formation in cotton, we performed a genome-wide association study (GWAS) of 163 cotton accessions with re-sequencing data. A total of 55 SNPs and 18 candidate genes were significantly associated with FBA trait. By combining GWAS and transcriptome analysis, four genes underlying FBA were identified. An FBA-associated candidate gene Ghi_A09G08736, which is homologous to SAUR46 in Arabidopsis thaliana, was detected in our study. In addition, transcriptomic evidence was provided to show that gravity and light were implicated in the FBA formation. This study provides new insights into the genetic architecture of FBA that informs architecture breeding in cotton.

Genetic Mapping and Analysis of a Compact Plant Architecture and Precocious Mutant in Upland Cotton.

With the promotion and popularization of machine cotton-picking, more and more attention has been paid to the selection of early-maturity varieties with compact plant architecture. The type of fruit branch is one of the most important factors affecting plant architecture and early maturity of cotton. Heredity analysis of the cotton fruit branch is beneficial to the breeding of machine-picked cotton. Phenotype analysis showed that the types of fruit branches in cotton are controlled by a single recessive gene. Using an F2 population crossed with Huaxin102 (normal branch) and 04N-11 (nulliplex branch), BSA (Bulked Segregant Analysis) resequencing analysis and GhNB gene cloning in 04N-11, and allelic testing, showed that fruit branch type was controlled by the GhNB gene, located on chromosome D07. Ghnb5, a new recessive genotype of GhNB, was found in 04N-11. Through candidate gene association analysis, SNP 20_15811516_SNV was found to be associated with plant architecture and early maturity in the Xinjiang natural population. The GhNB gene, which is related to early maturity and the plant architecture of cotton, is a branch-type gene of cotton. The 20_15811516_SNV marker, obtained from the Xinjiang natural population, was used for the assisted breeding of machine-picked cotton varieties.

ß-Sitosterol differentially regulates key metabolites for growth improvement and stress tolerance in rice plants during prolonged UV-B stress.

BACKGROUND: Elevated ultraviolet-B (UV-B) radiation is potentially deleterious to many organisms specifically crop plants and has become a global challenge. Rice is an exceptionally important staple food which is grown worldwide, and many efforts have been done recently to improve rice varieties against UV-B stress. This current study aims to investigate the effects of exogenous application of ß-sitosterol (ßSito) on growth improvement and tolerance level of rice plants against prolonged UV-B stress. The physiological and metabolic responses were evaluated in rice plants not supplemented with ßSito (Nß) and those supplemented with ßSito (Sß). RESULTS: The Nß and Sß plants were grown under non-stress (ns) and under prolonged UV-B stress (uvs) conditions and termed as Nßns, Sßns and Nßuvs, Sßuvs, respectively. The application of ßSito contributes positively under non-stress and specifically to UV-B stress in terms of improving numerous physiological parameters associated with growth and development such as shoot and root length, RWC, whole plant biomass, chlorophyll pigments, and photosynthetic-related parameters (Pn, Gs, Tr, WUEi, Fv/Fm, and NPQ) in Sß compared with Nß plants. Moreover, enhanced oxidative stress tolerance of Sßuvs vs. Nßuvs plants under stress was attributed to low levels of ROS and substantial trigger in activities of antioxidant enzymes (SOD, POD, CAT, and APX). Metabolic analysis was performed using GC-TOFMS, which revealed higher accumulation of several key metabolites including organic acids, sugars, amino acids, and others in Sßuvs vs. Nßuvs plants, which were mainly reduced in Nß plants under stress vs. non-stress conditions. CONCLUSION: These results provide useful data regarding the important role of ßSito on growth maintenance and modulation of several metabolites associated with osmotic and redox adjustments during UV-B stress tolerance in rice plants. Importantly, ßSito-regulated plasticity could further be explored specifically in relation to different environmental stresses in other economically useful crop plants.

A Single-Nucleotide Mutation in a GLUTAMATE RECEPTOR-LIKE Gene Confers Resistance to Fusarium Wilt in Gossypium hirsutum.

Fusarium wilt (FW) disease of cotton, caused by the fungus Fusarium oxysporum f. sp. vasinfectum (Fov), causes severe losses in cotton production worldwide. Though significant advancements have been made in development of FW-resistant Upland cotton (Gossypium hirsutum) in resistance screening programs, the precise resistance genes and the corresponding molecular mechanisms for resistance to Fov remain unclear. Herein it is reported that Fov7, a gene unlike canonical plant disease-resistance (R) genes, putatively encoding a GLUTAMATE RECEPTOR-LIKE (GLR) protein, confers resistance to Fov race 7 in Upland cotton. A single nucleotide polymorphism (SNP) (C/A) in GhGLR4.8, resulting in an amino acid change (L/I), is associated with Fov resistance. A PCR-based DNA marker (GhGLR4.8SNP(A/C) ) is developed and shown to cosegregate with the Fov resistance. CRISPR/Cas9-mediated knockout of Fov7 results in cotton lines extremely susceptible to Fov race 7 with a loss of the ability to induce calcium influx in response to total secreted proteins (SEPs) of Fov. Furthermore, coinfiltration of SEPs with GhGLR4.8A results in a hypersensitive response. This first report of a GLR-encoding gene that functions as an R gene provides a new insight into plant-pathogen interactions and a new handle to develop cotton cultivars with resistance to Fov race 7.

Effects of cotton-maize rotation on soil microbiome structure.

Verticillium wilt is a disastrous disease in cotton-growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton-maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long-term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton-maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control.

Phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating jasmonic acid biosynthesis and phenylpropanoid pathway.

Living in natural environment, plants often suffer from various biotic and abiotic stresses. Phosphate deficiency is a common factor affecting crop production in field, while pathogen invasion is another serious problem. Here we report that Pi-deficient cotton plants exhibit enhanced resistance to Verticillium dahliae. Transcriptomic and histochemical analysis revealed that cotton phenylpropanoid pathway was activated under phosphate deficiency, including lignin and flavonoid biosynthesis. Metabolomic data showed that Pi-deficient cotton accumulates many flavonoids metabolites and displays obvious anti-fungi activity in terms of methanolic extract. Additionally, JA biosynthesis was activated under phosphate deficiency and the Pi-deficiency induced disease resistance was significantly attenuated in GhAOS knock down plants. Taken together, our study demonstrated that phosphate deficiency enhanced cotton resistance to V. dahliae through activating phenylpropanoid pathway and JA biosynthesis, providing new insights into how phosphate deficiency affects plant disease resistance.

Genetic dissection of an allotetraploid interspecific CSSLs guides interspecific genetics and breeding in cotton.

BACKGROUND: The low genetic diversity of Upland cotton limits the potential for genetic improvement. Making full use of the genetic resources of Sea-island cotton will facilitate genetic improvement of widely cultivated Upland cotton varieties. The chromosome segments substitution lines (CSSLs) provide an ideal strategy for mapping quantitative trait loci (QTL) in interspecific hybridization. RESULTS: In this study, a CSSL population was developed by PCR-based markers assisted selection (MAS), derived from the crossing and backcrossing of Gossypium hirsutum (Gh) and G. barbadense (Gb), firstly. Then, by whole genome re-sequencing, 11,653,661 high-quality single nucleotide polymorphisms (SNPs) were identified which ultimately constructed 1211 recombination chromosome introgression segments from Gb. The sequencing-based physical map provided more accurate introgressions than the PCR-based markers. By exploiting CSSLs with mutant morphological traits, the genes responding for leaf shape and fuzz-less mutation in the Gb were identified. Based on a high-resolution recombination bin map to uncover genetic loci determining the phenotypic variance between Gh and Gb, 64 QTLs were identified for 14 agronomic traits with an interval length of 158 kb to 27 Mb. Surprisingly, multiple alleles of Gb showed extremely high value in enhancing cottonseed oil content (SOC). CONCLUSIONS: This study provides guidance for studying interspecific inheritance, especially breeding researchers, for future studies using the traditional PCR-based molecular markers and high-throughput re-sequencing technology in the study of CSSLs. Available resources include candidate position for controlling cotton quality and quantitative traits, and excellent breeding materials. Collectively, our results provide insights into the genetic effects of Gb alleles on the Gh, and provide guidance for the utilization of Gb alleles in interspecific breeding.

High-density genetic variation maps reveal the correlation between asymmetric interspecific introgressions and improvement of agronomic traits in Upland and Pima cotton varieties developed in Xinjiang, China.

The two new world tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, are cultivated worldwide and are characterised by a high yield and superior fibre quality, respectively. Historical genetic introgression has been reported between them; however, the existence of introgression and its genetic effects on agronomic traits remain unclear with regard to independent breeding of G. hirsutum (Upland cotton) and G. barbadense (Pima cotton) elite cultivars. We collected 159 G. hirsutum and 70 G. barbadense cultivars developed in Xinjiang, China, along with 30 semi-wild accessions of G. hirsutum, to perform interspecific introgression tests, intraspecific selection analyses and genome-wide association studies (GWAS) with fibre quality and yield component traits in multiple environments. In total, we identified seven interspecific introgression events and 52 selective sweep loci in G. hirsutum, as well as 17 interspecific introgression events and 19 selective sweep loci in G. barbadense. Correlation tests between agronomic traits and introgressions showed that introgression loci were mutually beneficial for the improvement of fibre quality and yield traits in both species. In addition, the phenotypic effects of four interspecific introgression events could be detected by intraspecific GWAS, with Gb_INT13 significantly improving fibre yield in G. barbadense. The present study describes the landscape of genetic introgression and selection between the two species, and highlights the genetic effects of introgression among populations, which can be used for future improvement of fibre yield and quality in G. barbadense and G. hirsutum, respectively.

Effects of Long-term Cotton Continuous Cropping on Soil Microbiome.

Verticillium wilt is a severe disease of cotton crops in Xinjiang and affecting yields and quality, due to the continuous cotton cropping in the past decades. The relationship between continuous cropping and the changes induced on soil microbiome remains unclear to date. In this study, the culture types of 15 isolates from Bole (5F), Kuitun (7F), and Shihezi (8F) of north Xinjiang were sclerotium type. Only isolates from field 5F belonged to nondefoliating pathotype, the others belonged to defoliating pathotype. The isolates showed pathogenicity differentiation in cotton. Fungal and bacterial communities in soils had some difference in alpha-diversity, relative abundance, structure and taxonomic composition, but microbial groups showed similarity in the same habitat, despite different sampling sites. The fungal phyla Ascomycota, and the bacterial phyla Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes were strongly enriched. Verticillium abundance was significantly and positively correlated with AN, but negatively correlated with soil OM, AK and pH. Moreover, Verticillium was correlated in abundances with 5 fungal and 6 bacterial genera. Overall, we demonstrate that soil microbiome communities have similar responses to long-term continuous cotton cropping, providing new insights into the effects of continuous cotton cropping on soil microbial communities.

GhFAD2-3 is required for anther development in Gossypium hirsutum.

BACKGROUND: In higher plants, the FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for the biosynthesis of the polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, in the biosynthesis of the polyunsaturated fatty acids (PUFAs) found in the cell membrane and cell wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development. RESULTS: We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all analyzed tissues, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of the wild type (WT), the content of C18:1 was 41.48%, which increased by 5 fold in the fad2-3 anther at the pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid was 5.43 in fad2-3 anther, which was much higher than that of the WT (only 0.39). Through compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content. CONCLUSIONS: Our work demonstrated the importance of C18:2 and/or C18:3 in the development of the pollen exine and anther cuticle in cotton and provided clue for further investigation of the physiological significance of the fatty acid composition for plant growth and development.

GhUMC1, a blue copper-binding protein, regulates lignin synthesis and cotton immune response.

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae is a serious problem for the sustainable production of cotton. The mechanism of cotton resistance to V. dahliae is unclear, which makes it is difficult to improve cotton resistance breeding. In this study, we characterized an umecyanin-like gene GhUMC1 in cotton, which is homologous to the AtBCB gene in Arabidopsis. It is predominantly expressed in roots and responds to pathogen infection. Knock-down of GhUMC1 increases plant susceptibility to V. dahliae. Expression levels of genes in the JA and SA signaling pathways in roots were down-regulated in GhUMC1-silenced plants. The transcripts of lignin synthesis genes, such as C4H, HCT, CCoAOMT and CAD, were also decreased in GhUMC1 knock-down seedlings, as was lignin content. Interestingly, knock-down of the GhUMC1 also decreased the contents of H202 compared with the control. Our results suggest that GhUMC1 is involved in cotton resistance to V. dahliae by the regulation of the JA signaling pathway and lignin metabolism.

Population structure and genetic basis of the agronomic traits of upland cotton in China revealed by a genome-wide association study using high-density SNPs.

Gossypium hirsutum L. represents the largest source of textile fibre, and China is one of the largest cotton-producing and cotton-consuming countries in the world. To investigate the genetic architecture of the agronomic traits of upland cotton in China, a diverse and nationwide population containing 503 G. hirsutum accessions was collected for a genome-wide association study (GWAS) on 16 agronomic traits. The accessions were planted in four places from 2012 to 2013 for phenotyping. The CottonSNP63K array and a published high-density map based on this array were used for genotyping. The 503 G. hirsutum accessions were divided into three subpopulations based on 11 975 quantified polymorphic single-nucleotide polymorphisms (SNPs). By comparing the genetic structure and phenotypic variation among three genetic subpopulations, seven geographic distributions and four breeding periods, we found that geographic distribution and breeding period were not the determinants of genetic structure. In addition, no obvious phenotypic differentiations were found among the three subpopulations, even though they had different genetic backgrounds. A total of 324 SNPs and 160 candidate quantitative trait loci (QTL) regions were identified as significantly associated with the 16 agronomic traits. A network was established for multieffects in QTLs and interassociations among traits. Thirty-eight associated regions had pleiotropic effects controlling more than one trait. One candidate gene, Gh_D08G2376, was speculated to control the lint percentage (LP). This GWAS is the first report using high-resolution SNPs in upland cotton in China to comprehensively investigate agronomic traits, and it provides a fundamental resource for cotton genetic research and breeding.