Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons.

Allotetraploid cotton (Gossypium) species represents a model system for the study of plant polyploidy, molecular evolution, and domestication. Here, chromosome-scale genome sequences were obtained and assembled for two recently described wild species of tetraploid cotton, Gossypium ekmanianum [(AD)6, Ge] and Gossypium stephensii [(AD)7, Gs], and one early form of domesticated Gossypium hirsutum, race punctatum [(AD)1, Ghp]. Based on phylogenomic analysis, we provide a dated whole-genome level perspective for the evolution of the tetraploid Gossypium clade and resolved the evolutionary relationships of Gs, Ge, and domesticated G. hirsutum. We describe genomic structural variation that arose during Gossypium evolution and describe its correlates-including phenotypic differentiation, genetic isolation, and genetic convergence-that contributed to cotton biodiversity and cotton domestication. Presence/absence variation is prominent in causing cotton genomic structural variations. A presence/absence variation-derived gene encoding a phosphopeptide-binding protein is implicated in increasing fiber length during cotton domestication. The relatively unimproved Ghp offers the potential for gene discovery related to adaptation to environmental challenges. Expanded gene families enoyl-CoA δ isomerase 3 and RAP2-7 may have contributed to abiotic stress tolerance, possibly by targeting plant hormone-associated biochemical pathways. Our results generate a genomic context for a better understanding of cotton evolution and for agriculture.

Genome resequencing-based high-density genetic map and QTL detection for yield and fiber quality traits in diploid Asiatic cotton (Gossypium arboreum).

Cotton is the most important fiber crop in the world. Asiatic cotton (Gossypium arboreum, genome A2) is a diploid cotton species producing spinnable fibers and important germplasm for cotton breeding and a significant model for fiber biology. However, the genetic map of Asiatic cotton has been lagging behind tetraploid cottons, as well as other stable crops. This study aimed to construct a high-density SNP genetic map and to map QTLs for important yield and fiber quality traits. Using a recombinant inbred line (RIL) population and genome resequencing technology, we constructed a high-density genetic map that covered 1980.17 cM with an average distance of 0.61 cM between adjacent markers. QTL analysis revealed a total of 297 QTLs for 13 yield and fiber quality traits in three environments, explaining 5.0-37.4% of the phenotypic variance, among which 75 were stably detected in two or three environments. Besides, 47 QTL clusters, comprising 131 QTLs for representative traits, were identified. Our works laid solid foundation for fine mapping and cloning of QTL for yield and fiber quality traits in Asiatic cotton.

MPK homolog GhNTF6 was involved in cotton against Verticillium wilt by interacted with VdEPG1.

Mitogen-activated protein kinases (MPKs) are important in regulating plant development and stress response. Rapid activation of MPKs in plants usually depends on its phosphorylated. In view of this situation, a phosphorylated GhNTF6 belonged to MPKs family was screened in cotton roots under Verticillium dahliae challenge by phosphoproteomics analysis. Expression of GhNTF6 in cotton plants was did not induce by V. dahliae infection, while, silencing GhNTF6 results to enhance cotton plants susceptibility to V. dahliae, overexpression - GhNTF6 enhance Arabidopsis plants survivability to V. dahliae. Moreover, the mutation of GhNTF6 at site Thr195 and Thy197 with the phosphorylation decreased the plant resistance to V. dahliae. Therefore, GhNTF6 phosphorylation is important in plants against V. dahliae. Further analysis demonstrated that GhNTF6 interacted with a V. dahliae endopolygalacturonase (VdEPG1) on the cell nucleus. We propose that GhNTF6 is a potential molecular target for improving resistance to Verticillium wilt in cotton.

Identification of Raf-Like Kinases B Subfamily Genes in Gossypium Species Revealed GhRAF42 Enhanced Salt Tolerance in Cotton.

Salinity is a critical abiotic factor that significantly reduces agricultural production. Cotton is an important fiber crop and a pioneer on saline soil, hence genetic architecture that underpins salt tolerance should be thoroughly investigated. The Raf-like kinase B-subfamily (RAF) genes were discovered to regulate the salt stress response in cotton plants. However, understanding the RAFs in cotton, such as Enhanced Disease Resistance 1 and Constitutive Triple Response 1 kinase, remains a mystery. This study obtained 29, 28, 56, and 54 RAF genes from G. arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. The RAF gene family described allopolyploidy and hybridization events in allotetraploid cotton evolutionary connections. Ka/Ks analysis advocates that cotton evolution was subjected to an intense purifying selection of the RAF gene family. Interestingly, integrated analysis of synteny and gene collinearity suggested dispersed and segmental duplication events involved in the extension of RAFs in cotton. Transcriptome studies, functional validation, and virus-induced gene silencing on salt treatments revealed that GhRAF42 is engaged in salt tolerance in upland cotton. This research might lead to a better understanding of the role of RAFs in plants and the identification of suitable candidate salt-tolerant genes for cotton breeding.

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.

Evolutionary and Characteristic Analysis of RING-DUF1117 E3 Ubiquitin Ligase Genes in Gossypium Discerning the Role of GhRDUF4D in Verticillium dahliae Resistance.

Verticillium wilt, primarily induced by the soil-borne fungus Verticillium dahliae, is a serious threat to cotton fiber production. There are a large number of really interesting new gene (RING) domain-containing E3 ubiquitin ligases in Arabidopsis, of which three (At2g39720 (AtRHC2A), At3g46620 (AtRDUF1), and At5g59550 (AtRDUF2)) have a domain of unknown function (DUF) 1117 domain in their C-terminal regions. This study aimed to detect and characterize the RDUF members in cotton, to gain an insight into their roles in cotton's adaptation to environmental stressors. In this study, a total of 6, 7, 14, and 14 RDUF (RING-DUF1117) genes were detected in Gossypium arboretum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These RDUF genes were classified into three groups. The genes in each group were highly conserved based on gene structure and domain analysis. Gene duplication analysis revealed that segmental duplication occurred during cotton evolution. Expression analysis revealed that the GhRDUF genes were widely expressed during cotton growth and under abiotic stresses. Many cis-elements related to hormone response and environment stressors were identified in GhRDUF promoters. The predicted target miRNAs and transcription factors implied that GhRDUFs might be regulated by gra-miR482c, as well as by transcription factors, including MYB, C2H2, and Dof. The GhRDUF genes responded to cold, drought, and salt stress and were sensitive to jasmonic acid, salicylic acid, and ethylene signals. Meanwhile, GhRDUF4D expression levels were enhanced after V. dahliae infection. Subsequently, GhRDUF4D was verified by overexpression in Arabidopsis and virus-induced gene silencing treatment in upland cotton. We observed that V. dahliae resistance was significantly enhanced in transgenic Arabidopsis, and weakened in GhRDUF4D silenced plants. This study conducted a comprehensive analysis of the RDUF genes in Gossypium, hereby providing basic information for further functional studies.

Ethnobotanical and antimicrobial activities of the Gossypium (Cotton) genus: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: The Malvaceae family, an important group of plants that have the Gossypium (cotton) genus has been used in folk medicine to treat microbial diseases and symptoms. AIMS OF THE STUDY: This article aims to understand its ethnobotany expression in communities and scientific elucidation of antimicrobial activities of this genus through literature review. MATERIALS AND METHODS: The bibliographic survey was carried out from 1999 to 2019 with keywords combinations such as "Gossypium + ethnobotanical", " Gossypium + medicinal ", "Gossypium + the biological activity" in scientific databases as Pubmed, Scopus, Web of Science, DOAJ, Scielo, Bireme. RESULTS: After data analysis, we found that the Gossypium genus, specifically Gossypium hirsutum, G. barbadense, G. herbaceum, G. arboreum are the species most cited in the treatment of microbial diseases and symptoms in communities all over the world. In light of scientific elucidation of biological activities, the Gossypium genus has been used to treat protozoal, bacterial, fungal, and viral diseases. CONCLUSIONS: The review demonstrated that the Gossypium genus is a promising source of biological activities against microbial diseases, especially in the treatment of protozoal diseases like malaria.

Genomic insight into the divergence and adaptive potential of a forgotten landrace G. hirsutum L. purpurascens.

Wild progenitors are an excellent source for strengthening the genetic basis and accumulation of desirable variation lost because of directional selection and adaptation in modern cultivars. Here, we re-evaluate a landrace of Gossypium hirsutum, formerly known as Gossypium purpurascens. Our study seeks to understand the genomic structure, variation, and breeding potential of this landrace, providing potential insights into the biogeographic history and genomic changes likely associated with domestication. A core set of accessions, including current varieties, obsolete accessions, G. purpurascens, and other geographical landraces, are subjected to genotyping along with multilocation phenotyping. Population fixation statistics suggests a marked differentiation between G. purpurascens and three other groups, emphasizing the divergent genomic behavior of G. purpurascens. Phylogenetic analysis establishes the primitive nature of G. purpurascens, identifying it as a vital source of functional variation, the inclusion of which in the upland cotton (cultivated G. hirsutum) gene pool may broaden the genetic basis of modern cultivars. Genome-wide association results indicate multiple loci associated with domestication regions corresponding to flowering and fiber quality. Moreover, the conserved nature of G. purpurascens can also provide insights into the evolutionary process of G. hirsutum.

Novel Structural Variation and Evolutionary Characteristics of Chloroplast tRNA in Gossypium Plants.

Cotton is one of the most important fiber and oil crops in the world. Chloroplast genomes harbor their own genetic materials and are considered to be highly conserved. Transfer RNAs (tRNAs) act as "bridges" in protein synthesis by carrying amino acids. Currently, the variation and evolutionary characteristics of tRNAs in the cotton chloroplast genome are poorly understood. Here, we analyzed the structural variation and evolution of chloroplast tRNA (cp tRNA) based on eight diploid and two allotetraploid cotton species. We also investigated the nucleotide evolution of chloroplast genomes in cotton species. We found that cp tRNAs in cotton encoded 36 or 37 tRNAs, and 28 or 29 anti-codon types with lengths ranging from 60 to 93 nucleotides. Cotton chloroplast tRNA sequences possessed specific conservation and, in particular, the Ψ-loop contained the conserved U-U-C-X3-U. The cp tRNAs of Gossypium L. contained introns, and cp tRNAIle contained the anti-codon (C-A-U), which was generally the anti-codon of tRNAMet. The transition and transversion analyses showed that cp tRNAs in cotton species were iso-acceptor specific and had undergone unequal rates of evolution. The intergenic region was more variable than coding regions, and non-synonymous mutations have been fixed in cotton cp genomes. On the other hand, phylogeny analyses indicated that cp tRNAs of cotton were derived from several inferred ancestors with greater gene duplications. This study provides new insights into the structural variation and evolution of chloroplast tRNAs in cotton plants. Our findings could contribute to understanding the detailed characteristics and evolutionary variation of the tRNA family.

Genome wide analysis of IQD gene family in diploid and tetraploid species of cotton (Gossypium spp.).

Calmodulin (CaM) is considered as the most significant Ca2+ signaling messenger that mediate various biochemical and physiological reactions. IQ domain (IQD) proteins are plant specific CML/CaM calcium binding which are characterized by domains of 67 amino acids. 50, 50, 94, and 99 IQD genes were detected from G. arboreum (A2), G. raimondii (D5), G. barbadense (AD2) and G. hirsutum (AD1) respectively. Existence of more orthologous genes in cotton species than Arabidopsis, advocated that polyploidization produced new cotton specific orthologous gene clusters. Duplication of gene events depicts that IQD gene family of cotton evolution was under strong purifying selection. G. hirsutum exhibited high level synteny. GarIQD25 exhibited high expression in stem, root, flower, ovule and fiber in G. arboreum. In G. raimondii, GraIQD03 demonstrated upregulation across stem, ovule, fiber and seed. GbaIQD11 and GbaIQD62 exhibited upregulation in fiber development in G. barbadense. GhiIQD69 recognized as main candidate genes for plant parts, floral tissues, fiber and ovule development. Promotor analysis identified cis-regulatory elements were involved in plant growth and development. Overwhelmingly, present study paves the way to better understand the evolution of cotton IQD genes and lays a foundation for future investigation of IQD in cotton.

The cellulose synthase (CesA) gene family in four Gossypium species: phylogenetics, sequence variation and gene expression in relation to fiber quality in Upland cotton.

Cellulose synthases (CesAs) are multi-subunit enzymes found on the plasma membrane of plant cells and play a pivotal role in cellulose production. The cotton fiber is mainly composed of cellulose, and the genetic relationships between CesA genes and cotton fiber yield and quality are not fully understood. Through a phylogenetic analysis, the CesA gene family in diploid Gossypium arboreum and Gossypium raimondii, as well as tetraploid Gossypium hirsutum ('TM-1') and Gossypium barbadense ('Hai-7124' and '3-79'), was divided into 6 groups and 15 sub-groups, with each group containing two to five homologous genes. Most CesA genes in the four species are highly collinear. Among the five cotton genomes, 440 and 1929 single nucleotide polymorphisms (SNPs) in the CesA gene family were identified in exons and introns, respectively, including 174 SNPs resulting in amino acid changes. In total, 484 homeologous SNPs between the A and D genomes were identified in diploids, while 142 SNPs were detected between the two tetraploids, with 32 and 82 SNPs existing within G. hirsutum and G. barbadense, respectively. Additionally, 74 quantitative trait loci near 18 GhCesA genes were associated with fiber quality. One to four GhCesA genes were differentially expressed (DE) in ovules at 0 and 3 days post anthesis (DPA) between two backcross inbred lines having different fiber lengths, but no DE genes were identified between these lines in developing fibers at 10 DPA. Twenty-seven SNPs in above DE CesA genes were detected among seven cotton lines, including one SNP in Ghi_A08G03061 that was detected in four G. hirsutum genotypes. This study provides the first comprehensive characterization of the cotton CesA gene family, which may play important roles in determining cotton fiber quality.

Interspecific grafting between Gossypium hirsutum, G. barbadense and G. herbaceum lines.

Seedling grafting could provide additional crop improvement strategies for cotton. However, there existed limited studies on interspecific grafting and approaches. Four different grafting approaches were developed and compared between lines representing three of the four cultivated cotton species G. hirsutum, G. barbadense and G. herbaceum. Grafting approaches of this study focused on the cotyledon node and cotyledon leaves retained on scions, rootstocks, without cotyledon node and cotyledon leaves on scions and rootstocks or halved cotyledon node and single cotyledon leaf on scions and rootstocks. Evaluations of the grafting approaches were made by comparing survival and growth rate during the second and fifth weeks after transplantation, respectively. The formation of any lateral shoots at the grafted sites were studied in two of four grafting approaches in the first and the second year during flowering stage. DNA alterations due to grafting were investigated using microsatellite markers. There were no statistically significant differences between grafts and their control in survival rate and locus specific DNA alteration. Growth rate and lateral shoot formation, on the other hand, were different among grafting types and grafts. We concluded that grafting without cotyledon node and cotyledon leaves on rootstocks, and with cotyledon node but without cotyledon leaves on scions were easy to perform and suitable for interspecific cotton grafting. Results suggested that grafting seedlings and allowing time to heal graft wounds prior to spring transplanting or double cropping is suitable for wheat-cotton intercropping to prevent late or early chilling damage associated with seed sowing or conventional transplanting of susceptible seedlings. Furthermore, the rapid and consistent wound healing in seedling grafts along with lateral shoot formation occurring in two of four grafting approaches make them a suitable approach to investigate possible genetic and epigenetic movement between scions and rootstocks, especially across species.

Extensive haplotypes are associated with population differentiation and environmental adaptability in Upland cotton (Gossypium hirsutum).

KEY MESSAGE: Three extensive eco-haplotypes associated with population differentiation and environmental adaptability in Upland cotton were identified, with A06_85658585, A08_43734499 and A06_113104285 considered the eco-loci for environmental adaptability. Population divergence is suggested to be the primary force driving the evolution of environmental adaptability in various species. Chromosome inversion increases reproductive isolation between subspecies and accelerates population divergence to adapt to new environments. Although modern cultivated Upland cotton (Gossypium hirsutum L.) has spread worldwide, the noticeable phenotypic differences still existed among cultivars grown in different areas. In recent years, the long-distance migration of cotton cultivation areas throughout China has demanded that breeders better understand the genetic basis of environmental adaptability in Upland cotton. Here, we integrated the genotypes of 419 diverse accessions, long-term environment-associated variables (EAVs) and environment-associated traits (EATs) to evaluate subgroup differentiation and identify adaptive loci in Upland cotton. Two highly divergent genomic regions were found on chromosomes A06 and A08, which likely caused by extensive chromosome inversions. The subgroups could be geographically classified based on distinct haplotypes in the divergent regions. A genome-wide association study (GWAS) also confirmed that loci located in these regions were significantly associated with environmental adaptability in Upland cotton. Our study first revealed the cause of population divergence in Upland cotton, as well as the consequences of variation in its environmental adaptability. These findings provide new insights into the genetic basis of environmental adaptability in Upland cotton, which could accelerate the development of molecular markers for adaptation to climate change in future cotton breeding.

Genome-Wide Characterization and Expression Analysis of NHX Gene Family under Salinity Stress in Gossypium barbadense and Its Comparison with Gossypium hirsutum.

Cotton is an important economic crop affected by different abiotic stresses at different developmental stages. Salinity limits the growth and productivity of crops worldwide. Na+/H+ antiporters play a key role during the plant development and in its tolerance to salt stress. The aim of the present study was a genome-wide characterization and expression pattern analysis under the salinity stress of the sodium-proton antiporter (NHX) of Gossypium barbadense in comparison with Gossypium hirsutum. In G. barbadense, 25 NHX genes were identified on the basis of the Na+_H+ exchanger domain. All except one of the G. barbadenseNHX transporters have an Amiloride motif that is a known inhibitor of Na+ ions in plants. A phylogenetic analysis inferred three classes of GbNHX genes-viz., Vac (GbNHX1, 2 and 4), Endo (GbNHX6), and PM (GbNHX7). A high number of the stress-related cis-acting elements observed in promoters show their role in tolerance against abiotic stresses. The Ka/Ks values show that the majority of GbNHX genes are subjected to strong purifying selection under the course of evolution. To study the functional divergence of G. barbadenseNHX transporters, the real-time gene expression was analyzed under salt stress in the root, stem, and leaf tissues. In G. barbadense, the expression was higher in the stem, while in G. hirsutum the leaf and root showed a high expression. Moreover, our results revealed that NHX2 homologues in both species have a high expression under salinity stress at higher time intervals, followed by NHX7. The protein-protein prediction study revealed that GbNHX7 is involved in the CBL-CIPK protein interaction pathway. Our study also provided valuable information explaining the molecular mechanism of Na+ transport for the further functional study of Gossypium NHX genes.

A cotton α1,3-/4-fucosyltransferase-encoding gene, FucT4, plays an important role in cell elongation and is significantly associated with fiber quality.

Fucosylation, one of the key posttranslational modifications, plays an important role in plants. It is involved in the development, signal transduction, reproduction, and disease resistance. α1,3-/4-Fucosyltransferase is responsible for transferring L-fucose from GDP-L-fucose to the N-glycan to exert fucosylational functions. However, the roles of the fucosyltransferase gene in cotton remain unknown. This study provided a comprehensive investigation of its possible functions. A genome-wide analysis identified four, four, eight, and eight FucT genes presented in the four sequenced cotton species, diploid Gossypium raimondii, G. arboreum, tetraploid G. hirsutum acc. TM-1, and G. barbadense cv. H7124, respectively. These FucTs were classified into two groups, with FucT4 homologs alone as a group. We isolated FucT4 in TM-1 and H7124, and named it GhFucT4 and GbFucT4, respectively. Quantitative RT-PCR and transcriptome data demonstrated that GhFucT4 had the highest expression levels in fibers among all GhFucT genes. Association studies and QTL co-localization supported the possible involvement of GhFucT4 in cotton fiber development. GhFucT4 and GbFucT4 shared high sequence identities, and FucT4 had higher expression in H7124 fiber tissues compared with TM-1. Furthermore, ectopic expression of FucT4 in transgenic Arabidopsis promoted root cell elongation, upregulated expression of genes related to cell wall loosening, and led to longer primary root. These results collectively indicate that FucT4 plays an important role in promoting cell elongation and modulating fiber development, which could be utilized to improve fiber quality traits in cotton breeding.

Casparian strip membrane domain proteins in Gossypium arboreum: genome-wide identification and negative regulation of lateral root growth.

BACKGROUND: Root systems are critical for plant growth and development. The Casparian strip in root systems is involved in stress resistance and maintaining homeostasis. Casparian strip membrane domain proteins (CASPs) are responsible for the formation of Casparian strips. RESULTS: To investigate the function of CASPs in cotton, we identified and characterized 48, 54, 91 and 94 CASPs from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively, at the genome-wide level. However, only 29 common homologous CASP genes were detected in the four Gossypium species. A collinearity analysis revealed that whole genome duplication (WGD) was the primary reason for the expansion of the genes of the CASP family in the four cotton species. However, dispersed duplication could also contribute to the expansion of the GaCASPs gene family in the ancestors of G. arboreum. Phylogenetic analysis was used to cluster a total of 85 CASP genes from G. arboreum and Arabidopsis into six distinct groups, while the genetic structure and motifs of CASPs were conserved in the same group. Most GaCASPs were expressed in diverse tissues, with the exception of that five GaCASPs (Ga08G0113, Ga08G0114, Ga08G0116, Ga08G0117 and Ga08G0118) that were highly expressed in root tissues. Analyses of the tissue and subcellular localization suggested that GaCASP27 genes (Ga08G0117) are membrane protein genes located in the root. In the GaCASP27 silenced plants and the Arabidopsis mutants, the lateral root number significantly increased. Furthermore, GaMYB36, which is related to root development was found to regulate lateral root growth by targeting GaCASP27. CONCLUSIONS: This study provides a fundamental understanding of the CASP gene family in cotton and demonstrates the regulatory role of GaCASP27 on lateral root growth and development.

Comprehensive characterization and gene expression patterns of LBD gene family in Gossypium.

MAIN CONCLUSION: A comprehensive account of the LBD gene family of Gossypium was provided in this work. Expression analysis and functional characterization revealed that LBD genes might play different roles in G. hirsutum and G. barbadense. The Lateral Organ Boundaries Domain (LBD) proteins comprise a plant-specific transcription factor family, which plays crucial roles in physiological processes of plant growth, development, and stress tolerance. In the present work, a systematical analysis of LBD gene family from two allotetraploid cotton species, G. hirsutum and G. barbadense, together with their genomic donor species, G. arboreum and G. raimondii, was conducted. There were 131, 128, 62, and 68 LBDs identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. The LBD proteins could be classified into two main classes, class I and class II, based on the structure of their lateral organ boundaries domain and traits of phylogenetic tree, and class I was further divided into five subgroups. The gene structure and motif composition analyses conducted in both G. hirsutum and G. barbadense revealed that LBD genes kept relatively conserved within the subfamilies. Synteny analysis suggested that segmental duplication acted as an important mechanism in expansion of the cotton LBD gene family. Cis-element analysis predicated the possible functions of LBD genes. Public RNA-seq data were investigated to analyze the expression patterns of cotton LBD genes in various tissues as well as gene expression under abiotic stress treatments. Furthermore, RT-qPCR results found that GhLBDs had various expression regulation under MeJA treatments. Expression analysis indicated the differential functions of cotton LBD genes in response to abiotic stress and hormones.

Genome-wide identification and analyses of the AHL gene family in cotton (Gossypium).

BACKGROUND: Members of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family are involved in various plant biological processes via protein-DNA and protein-protein interaction. However, no the systematic identification and analysis of AHL gene family have been reported in cotton. RESULTS: To investigate the potential functions of AHLs in cotton, genome-wide identification, expressions and structure analysis of the AHL gene family were performed in this study. 48, 51 and 99 AHL genes were identified from the G.raimondii, G.arboreum and G.hirsutum genome, respectively. Phylogenetic analysis revealed that the AHLs in cotton evolved into 2 clades, Clade-A with 4-5 introns and Clade-B with intronless (excluding AHL20-2). Based on the composition of the AT-hook motif(s) and PPC/DUF 296 domain, AHL proteins were classified into three types (Type-I/-II/-III), with Type-I AHLs forming Clade-B, and the other two types together diversifying in Clade-A. The detection of synteny and collinearity showed that the AHLs expanded with the specific WGD in cotton, and the sequence structure of AHL20-2 showed the tendency of increasing intron in three different Gossypium spp. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of orthologous gene pairs revealed that the AHL genes of G.hirsutum had undergone through various selection pressures, purifying selection mainly in A-subgenome and positive selection mainly in D-subgenome. Examination of their expression patterns showed most of AHLs of Clade-B expressed predominantly in stem, while those of Clade-A in ovules, suggesting that the AHLs within each clade shared similar expression patterns with each other. qRT-PCR analysis further confirmed that some GhAHLs higher expression in stems and ovules. CONCLUSION: In this study, 48, 51 and 99 AHL genes were identified from three cotton genomes respectively. AHLs in cotton were classified into two clades by phylogenetic relationship and three types based on the composition of motif and domain. The AHLs expanded with segmental duplication, not tandem duplication. The expression profiles of GhAHLs revealed abundant differences in expression levels in various tissues and at different stages of ovules development. Our study provided significant insights into the potential functions of AHLs in regulating the growth and development in cotton.

Genetic regulatory networks for salt-alkali stress in Gossypium hirsutum with differing morphological characteristics.

BACKGROUND: Cotton grows in altering environments that are often unfavorable or stressful for its growth and development. Consequently, the plant must cope with abiotic stresses such as soil salinity, drought, and excessive temperatures. Alkali-salt stress response remains a cumbersome biological process and is regulated via a multifaceted transcriptional regulatory network in cotton. RESULTS: To discover the molecular mechanisms of alkali-salt stress response in cotton, a comprehensive transcriptome analysis was carried out after alkali-salt stress treatment in three accessions of Gossypium hirsutum with contrasting phenotype. Expression level analysis proved that alkali-salt stress response presented significant stage-specific and tissue-specific. GO enrichment analysis typically suggested that signal transduction process involved in salt-alkali stress response at SS3 and SS12 stages in leaf; carbohydrate metabolic process and oxidation-reduction process involved in SS48 stages in leaf; the oxidation-reduction process involved at all three phases in the root. The Co-expression analysis suggested a potential GhSOS3/GhCBL10-SOS2 network was involved in salt-alkali stress response. Furthermore, Salt-alkali sensitivity was increased in GhSOS3 and GhCBL10 Virus-induced Gene Silencing (VIGS) plants. CONCLUSION: The findings may facilitate to elucidate the underlying mechanisms of alkali-salt stress response and provide an available resource to scrutinize the role of candidate genes and signaling pathway governing alkali-salt stress response.

Drought-induced disturbance of carbohydrate metabolism in anthers and male abortion of two Gossypium hirsutum cultivars differing in drought tolerance.

KEY MESSAGE: Cotton pollen abortion, under drought stress, was closely associated with changes in anther carbohydrate metabolism, and pollen abortion rate due to drought was higher in drought-sensitive cultivars than drought-tolerant cultivars. Cotton reproductive failure under drought stress is intrinsically connected with altered male fertility, however, studies investigating the effect of drought stress on cotton male fertility are nonexistent. Thus, a drought stress experiment was conducted with two cotton cultivars, differing in drought tolerance, to study pollen fertility and anthers' physiology. Results indicated that drought stress reduced pollen fertility of both cultivars due to decreases in anther starch and adenosine triphosphate (ATP) synthesis. Lower assimilate supply capacity in conjunction with impaired activities of ADP-glucose pyrophosphorylase and soluble starch synthase were the main reasons for the decreased starch levels in drought-stressed anthers. The decreased activities of sucrose synthetase and acid invertase were responsible for the higher sucrose level in drought-stressed anthers than well-watered anthers and the changing trend of sucrose was intensified by the decreased expressions of sucrose synthase genes (GhSusA, GhSusB, GhSusD) and acid invertase genes (GhINV1, GhINV2). However, despite sucrose degradation being limited in drought-stressed anthers, glucose level was higher in droughted anthers than well-watered ones, and that might be attributed to the down-regulated respiration since decreased anther ATP levels were detected in drought-stressed plants. Furthermore, compared to the drought-tolerant cultivar, pollen fertility was more suppressed by drought stress for the drought-sensitive cultivar, and that was attributed to the larger decrease in starch and ATP contents.