Hirsutum Genome Research Articles

Exploring the plasmodesmata callose-binding protein gene family in upland cotton: unraveling insights for enhancing fiber length.

Plasmodesmata are transmembrane channels embedded within the cell wall that can facilitate the intercellular communication in plants. Plasmodesmata callose-binding (PDCB) protein that associates with the plasmodesmata contributes to cell wall extension. Given that the elongation of cotton fiber cells correlates with the dynamics of the cell wall, this protein can be related to the cotton fiber elongation. This study sought to identify PDCB family members within the Gossypium. hirsutum genome and to elucidate their expression profiles. A total of 45 distinct family members were observed through the identification and screening processes. The analysis of their physicochemical properties revealed the similarity in the amino acid composition and molecular weight across most members. The phylogenetic analysis facilitated the construction of an evolutionary tree, categorizing these members into five groups mainly distributed on 20 chromosomes. The fine mapping results facilitated a tissue-specific examination of group V, revealing that the expression level of GhPDCB9 peaked five days after flowering. The VIGS experiments resulted in a marked decrease in the gene expression level and a significant reduction in the mature fiber length, averaging a shortening of 1.43-4.77 mm. The results indicated that GhPDCB9 played a pivotal role in the cotton fiber development and served as a candidate for enhancing cotton yield.

Assessment of Allelopathic Potential of Cotton Chromosome Substitution Lines.

Weed interference consistently poses a significant agronomic challenge in cotton production, leading to unfavorable direct and indirect consequences. Consequently, the predominant strategy employed to manage weeds is the application of synthetic herbicides. However, this extensive reliance has resulted in the development of herbicide-resistant weed populations due to the prolonged use of a single herbicide and the lack of rotation. This project focused on identifying weed-suppressive cotton chromosome substitution (CS) lines. These CS lines closely resemble the parent TM-1, an upland cotton derivative (Gossypium hirsutum). Each CS line carries a single chromosome or chromosome arm exchanged from G. barbadense, G. tomentosum, or G. mustelinum within the TM-1 background. In a greenhouse experiment utilizing a stepwise approach, five CS lines, along with two conventional varieties (Enlist and UA48) and the parent line (TM1), were assessed to determine their potential for suppressing Palmer amaranth growth. The plant height was measured 7, 14, and 21 days after establishment, and the chlorophyll content was measured 21 days after establishment. The results revealed varying levels of chlorophyll reduction in Palmer amaranth, with the Enlist variety displaying the lowest reduction (32%) and TM-1 exhibiting the highest (78%). Within 14 days of establishment, the CS lines T26lo, BNTN 1-15, and T11sh demonstrated substantial suppression of Palmer amaranth height, with reductions of 79, 70, and 71%, respectively. Conversely, Enlist displayed the least effective performance among the CS lines. Moreover, CS22, CS49, CS50, CS34, UA48, and CS23 displayed a decreasing trend in reducing Palmer amaranth height from 14 to 21 days after establishment. This research demonstrates the inherent herbicidal attributes within cotton CS lines against Palmer amaranth. In light of the versatile applications of cotton fibers and the unique characteristics of the G. hirsutum genome, this study investigates the potential of specific cotton lines in enhancing weed management practices. By elucidating the implications of our findings, we aim to contribute to promoting sustainability and developing alternatives to synthetic herbicides in agricultural systems.

Gh4CL20/20A involved in flavonoid biosynthesis is essential for male fertility in cotton (Gossypium hirsutum L.)

Flavonoids have been shown to play an essential role in plant growth and fertility. 4-Coumarate CoA ligase (4CL) is one of the indispensable enzymes involved in the biosynthesis of flavonoids. However, the role of 4CL and flavonoids in impact on cotton fertility is still unknown. In this study, on the basis of identification of an additional Gh4CL gene, Gh4CL20A, by using an updated G. hirsutum genome, we found that Gh4CL20A and its homologous Gh4CL20 were preferentially expressed in petals and stamens. The petals of the loss-of-function Gh4CL20/Gh4CL20A mutant generated by CRISPR/Cas9 gene editing remained white until wilting. Notably, the mutant showed indehiscent anthers, reduced number of pollen grains and pollen viability, leading to male sterility. Histological analysis revealed that abnormal degradation of anther tapetum at the tetrad stage and abnormal pollen grain development at the mature stage caused male sterility of the gene editing mutant. Analysis of the anther transcriptome identified a total of 10574 and 11962 genes up- and down-regulated in the mutant, respectively, compared to the wild-type. GO, KEGG, and WGCNA analyses linked the abnormality of the mutant anthers to the defective flavonoid biosynthetic pathway, leading to decreased activity of 4CL and chalcone isomerase (CHI) and reduced accumulation of flavonoids in the mutant. These results imply a role of Gh4CL20/Gh4CL20A in assuring proper development of cotton anthers by regulating flavonoid metabolism. This study elucidates a molecular mechanism underlying cotton anther development and provides candidate genes for creating cotton male sterile germplasm that has the potential to be used in production of hybrid seeds.

Identification and function analysis of GABA branch three gene families in the cotton related to abiotic stresses.

γ -aminobutyric acid (GABA) is closely related to the growth, development and stress resistance of plants. Combined with the previous study of GABA to promote the cotton against abiotic stresses, the characteristics and expression patterns of GABA branch gene family laid the foundation for further explaining its role in cotton stress mechanism. Members of GAD, GAB-T and SSADH (three gene families of GABA branch) were identified from the Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii genome. The GABA branch genes were 10 GAD genes, 4 GABA-T genes and 2 SSADH genes. The promoter sequences of genes mainly contains response-related elements such as light, hormone and environment.Phylogenetic analysis shows that GAD indicating that even in the same species, the homologous sequences in the family. The GABA-T gene of each cotton genus was in sum the family had gene loss in the process of dicotyledon evolution. SSADH families Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii were closely related to the dicot plants.GABA gene is involved in the regulation of salt stress and high temperature in Gossypium hirsutum.GABA attenuated part of the abiotic stress damage by increasing leaf protective enzyme activity and reducing reactive oxygen species production.This lays the foundation for a thorough analysis of the mechanism of GABA in cotton stress resistance.

Genome-wide identification of the Gossypium hirsutum CAD gene family and functional study of GhiCAD23 under drought stress.

Cinnamyl alcohol dehydrogenase (CAD) is a crucial enzyme in the final stage of lignin monomer biosynthesis. This study focuses on the CAD gene family within Gossypium hirsutum. Through comprehensive genomic analysis, we identified 29 GhiCAD genes within the Gossypium hirsutum genome using a bioinformatics approach. Phylogenetic analysis revealed that the GhiCAD family can be categorized into four subgroups, which are closest to the evolutionary relationship with Arabidopsis thaliana. There are multiple cis-acting elements on the promoters of GhiCAD genes associated with abiotic stress responses. Some GhiCAD genes demonstrated high expression in various tissues like root, leaf, and sepal, as well as in fiber and ovule at different developmental stages (10 days post anthesis (DPA), 15 DPA, 20 DPA, 25 DPA). The transcript levels of GhiCAD23 were notably elevated when exposed to PEG treatment and drought stress (DS). GhiCAD23 is also co-expressed with many known drought response genes, suggesting its involvement in the plant's reaction to DS. Employing virus-induced gene silencing (VIGS) technology to silence the GhiCAD23 gene, it was found that silencing GhiCAD23 reduced the tolerance of cotton to DS. Under DS, the relative leaf water content, superoxide dismutase (SOD), and catalase (CAT) enzyme activities of the GhiCAD23-silenced cotton plants were decreased by 31.84%, 30.22% and 14.19%, respectively, while malondialdehyde (MDA) was increased by 72.16% compared with the control cohort. Drought promotes the accumulation of lignin, and it was found that silencing the GhiCAD23 reduces lignin accumulation in cotton under DS. The analysis of phenotypic and physiological indicators indicates that GhiCAD23 is vital in cotton's resistance to DS. This investigation provides an important reference for future comprehensive exploration of the GhiCAD23 gene's function in cotton's DS response mechanism.

Molecular-genetic and cytogenetic analyses of cotton chromosome introgression from Gossypium barbadense L. into the genome of G. hirsutum L. in BC2F1 hybrids.

Substitution lines of the cotton Gossypium hirsutum L. involving chromosomes of the tetraploid species G. barbadense L., G. tomentosum Nutt. ex Seem., and G. mustelinum Miers ex Watt. are a valuable source for breeding, increasing the genetic diversity of G. hirsutum. The substitution of certain G. hirsutum L. chromosomes with G. barbadense chromosomes affect fibre elongation, fibre yield, fibre strength, and micronaire. To increase the efficiency of creating lines, it is necessary to study the nature of the introgression of alien chromosomes into the G. hirsutum L. genome. As a result of molecular genetic analysis of BC2F1 hybrids obtained from crossing monosomic lines of the cotton G. hirsutum from the cytogenetic collection of Uzbekistan with monosomic backcross hybrids BC1F1 G. hirsutum × G. barbadense on the same chromosomes, genetic differences between the hybrids in the profile of chromosome-specific microsatellite SSR markers were found. The predominant introgression of chromosomes 4, 6 and 12 of the At-subgenome and 22 of the Dt-subgenome of G. barbadense was revealed, while chromosomes 2 and 7 of the At-subgenome and 18 of the Dt- subgenome of G. barbadense were characterized by elimination. Among them, chromosomes 7 of the At- subgenome and 18 of the Dt-subgenome of G. barbadense were eliminated in the first backcross generation. In this work, two lines, CS- B06 and CS-B07, from the American cytogenetic collection with a putative substitution involving chromosomes 6 and 7 of the At-subgenome were analysed. The presence of only polymorphic alleles from the species G. hirsutum and the absence of polymorphic alleles from the species G. barbadense were revealed, which showed the absence of substitution involving these chromosomes. BC2F1 hybrids with monosomy for both G. barbadense and G. hirsutum chromosomes were characterized by regular pairing of chromosomes and high meiotic indexes. However, many hybrids were characterized by a decrease in pollen fertility. Two hybrids with monosomy for chromosome 7 of the At-subgenome of G. hirsutum and chromosome 6 of the At-subgenome of G. barbadense had the greatest reduction in pollen viability (70.09 ± 1.57 and 75.00 ± 1.66 %, respectively). Thus, this work shows a specific feature in the introgression of individual chromosomes of the cotton species G. barbadense into the cotton G. hirsutum genome.

Activation of Gossypium hirsutum ACS6 Facilitates Fiber Development by Improving Sucrose Metabolism and Transport.

Cotton fiber yield depends on the density of fiber cell initials that form on the ovule epidermis. Fiber initiation is triggered by MYB-MIXTA-like transcription factors (GhMMLs) and requires a sucrose supply. Ethylene or its precursor ACC (1-aminocyclopropane-1-carboxylic acid) is suggested to affect fiber yield. The Gossypium hirsutum (L.) genome contains 35 ACS genes (GhACS) encoding ACC synthases. Here, we explored the role of a GhACS family member in the regulation of fiber initiation. Expression analyses showed that the GhACS6.3 gene pair was specifically expressed in the ovules during fiber initiation (3 days before anthesis to 5 days post anthesis, -3 to 5 DPA), especially at -3 DPA, whereas other GhACS genes were expressed at very low or undetectable levels. The expression profile of GhACS6.3 during fiber initial development was confirmed by qRT-PCR analysis. Transgenic lines overexpressing GhACS6.3 (GhACS6.3-OE) showed increased ACC accumulation in ovules, which promoted the formation of fiber initials and fiber yield components. This was accompanied by increased transcript levels of GhMML3 and increased transcript levels of genes encoding sucrose transporters and sucrose synthase. These findings imply that GhACS6.3 activation is required for fiber initial development. Our results lay the foundation for further research on increasing cotton fiber production.

Genome-wide identification of GhRLCK-VII subfamily genes in Gossypium hirsutum and investigation of their functions in resistance to Verticillium wilt

BackgroundThe receptor-like cytoplasmic kinases subfamily VII (RLCK-VII) is critical in regulating plant growth, development, and pattern-triggered immunity. However, a comprehensive exploration of these genes in the allotetraploid Gossypium hirsutum is still lacking. This study aimed to identify RLCK-VII genes in G. hirsutum and investigate their evolutionary history, structural features, expression patterns, and role in plant defense.ResultsSeventy-two RLCK-VII genes in the G. hirsutum genome were unveiled and classified into nine groups following their phylogenetic analysis with Arabidopsis thaliana. Group VII-1 was the largest, accounting for 28%, while Groups VII-2 and VII-3 had only one member each. The analysis using MCScanX revealed that these 72 genes formed 166 collinear gene pairs and were resided on 26 chromosomes of G. hirsutum, suggesting that they were derived from whole genome segmental duplication events. Their calculated Ka/Ks values were below one, implying the occurrence of purification selection during the evolution and inhibition of gene function differentiation/loss. All members of the RLCK-VII subfamily possessed two conserved domains, PKinase-Tyr and PKinase, and several conserved PBS1 kinase subdomains, individually included in one of the ten motifs identified using MEME. The RNA-Seq results showed that RLCK-VII genes exhibited different spatiotemporal expression, indicating their involvement in cotton growth, development, and defense responses to Verticillium dahliae. The transcription patterns of RLCK-VII genes found by RNA-Seq were further validated using qRT-PCR assays after inoculating “20B12” (cotton cultivar) with “V991” (V. dahliae). The virus-induced gene silencing (VIGS) assays uncovered that two RLCK-VII genes (Gohir.A13G227248 and Gohir.A10G219900) were essential to G. hirsutum resistance to Verticillium wilt.ConclusionsThese observations offer valuable insight into the attributes and roles of RLCK-VII genes in G. hirsutum, potentially enable the breeding of new cotton cultivars with enhanced resistance to Verticillium wilt.

A recessive LRR-RLK gene causes hybrid breakdown in cotton.

An LRR-RLK gene causing interspecific hybrid breakdown between Gossypium. anomalum and G. hirsutum was identified by deploying a map-based cloning strategy. The self-destructing symptoms of hybrid incompatibility in most cases are attributed to autoimmunity. The cloning of genes responsible for hybrid incompatibility in cotton is helpful to clarify the mechanisms underlying hybrid incompatibility and can break the barriers in distant hybridization. In this study, a temperature-dependent lethality was identified in CSSL11-9 (chromosome segment substitution line) with Gossypium anomalum chromosome segment on chromosome A11. Transcriptome analysis showed the differentially expressed genes related to autoimmune responses were highly enriched, suggesting that expression of CSSL11-9 plant lethal gene activated autoimmunity in the absence of any pathogen or external stimulus, inducing programmed cell death (PCD) and causing a lethal phenotype. The lethal phenotype was controlled by a pair of recessive genes and then fine mapped between JAAS3191-JAAS3050 interval, which covered 63.87 kb in G. hirsutum genome and 98.66 kb in G. anomalum. We demonstrated that an LRR-RLK gene designated as hybrid breakdown 1 (GoanoHBD1) was the causal gene underlying this locus for interspecific hybrid incompatibility between G. anomalum and G. hirsutum. Silencing this LRR-RLK gene could restore CSSL11-9 plants from a lethal to a normal phenotype. Our findings provide new insights into reproductive isolation and may benefit cotton breeding.

Identification of salt stress-tolerant candidate genes in the BC2F2 population at the seedling stages of G. hirsutum and G. darwinii using NGS-based bulked segregant analysis.

Salinity is a major threat to the yield and productivity of cotton seedlings. In the present study, we developed a BC2F2 population of cotton plants from Gossypium darwinii (5-7) and Gossypium hirsutum (CCRI 12-4) salt-susceptible parents to identify salt-resistant candidate genes. The Illumina HiSeq™ strategy was used with bulked segregant analysis. Salt-resistant and salt-susceptible DNA bulks were pooled by using 30 plants from a BC2F2 population. Next-generation sequencing (NGS) technology was used for the sequencing of parents and both bulks. Four significant genomic regions were identified: the first genomic region was located on chromosome 18 (1.86 Mb), the second and third genomic regions were on chromosome 25 (1.06 Mb and 1.94 Mb, respectively), and the fourth was on chromosome 8 (1.41 Mb). The reads of bulk1 and bulk2 were aligned to the G. darwinii and G. hirsutum genomes, respectively, leading to the identification of 20,664,007 single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels). After the screening, 6,573 polymorphic markers were obtained after filtration of the candidate regions. The SNP indices in resistant and susceptible bulks and Δ(SNP-index) values of resistant and susceptible bulks were measured. Based on the higher Δ(SNP-index) value, six effective polymorphic SNPs were selected in a different chromosome. Six effective SNPs were linked to five candidate genes in four genomic regions. Further validation of these five candidate genes was carried out using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), resulting in an expression profile that showed two highly upregulated genes in the salt-tolerant species G. darwinii, i.e., Gohir.D05G367800 and Gohir.D12G239100; however, the opposite was shown in G. hirsutum, for which all genes, except one, showed partial expression. The results indicated that Gohir.D05G367800 and Gohir.D12G239100 may be salt-tolerant genes. We are confident that this study could be helpful for the cloning, transformation, and development of salt-resistant cotton varieties.

Identification of MYB gene family and functional analysis of GhMYB4 in cotton (Gossypium spp.).

Myeloblastosis (MYB) transcription factors (TFs) form a large gene family involved in a variety of biological processes in plants. Little is known about their roles in the development of cotton pigment glands. In this study, 646 MYB members were identified in Gossypium hirsutum genome and phylogenetic classification was analyzed. Evolution analysis revealed assymetric evolution of GhMYBs during polyploidization and sequence divergence of MYBs in G. hirustum was preferentially happend in D sub-genome. WGCNA (weighted gene co-expression network analysis) showed that four modules had potential relationship with gland development or gossypol biosynthesis in cotton. Eight differentially expressed GhMYB genes were identified by screening transcriptome data of three pairs of glanded and glandless cotton lines. Of these, four were selected as candidate genes for cotton pigment gland formation or gossypol biosynthesis by qRT-PCR assay. Silencing of GH_A11G1361 (GhMYB4) downregulated expression of multiple genes in gossypol biosynthesis pathway, indicating it could be involved in gossypol biosynthesis. The potential protein interaction network suggests that several MYBs may have indirect interaction with GhMYC2-like, a key regulator of pigment gland formation. Our study was the systematic analysis of MYB genes in cotton pigment gland development, providing candidate genes for further study on the roles of cotton MYB genes in pigment gland formation, gossypol biosynthesis and future crop plant improvement.

Genome-wide identification and expression reveal the involvement of the FCS-like zinc finger (FLZ) gene family in Gossypium hirsutum at low temperature.

FCS-like zinc finger (FLZ) is a plant-specific gene family that plays an important regulatory role in plant growth and development and its response to stress. However, studies on the characteristics and functions of cotton FLZ family genes are still lacking. This study systematically identified members of the cotton FLZ gene family based on cotton genome data. The cotton FLZ family genes were systematically analyzed by bioinformatics, and their expression patterns in different tissues and under low-temperature stress were analyzed by transcriptome and qRT-PCR. The G. hirsutum genome contains 56 FLZ genes distributed on 20 chromosomes, and most of them are located in the nucleus. According to the number and evolution analysis of FLZ family genes, FLZ family genes can be divided into five subgroups in cotton. The G. hirsutum FLZ gene has a wide range of tissue expression types, among which the expression is generally higher in roots, stems, leaves, receptacles and calyx. Through promoter analysis, it was found that it contained the most cis-acting elements related to methyl jasmonate (MeJA) and abscisic acid (ABA). Combined with the promoter and qRT-PCR results, it was speculated that GhFLZ11, GhFLZ25, GhFLZ44 and GhFLZ55 were involved in the response of cotton to low-temperature stress. Taken together, our findings suggest an important role for the FLZ gene family in the cotton response to cold stress. This study provides an important theoretical basis for further research on the function of the FLZ gene family and the molecular mechanism of the cotton response to low temperature.

Genome-wide analysis of zinc finger-homeodomain (ZF-HD) transcription factors in diploid and tetraploid cotton.

ZF-HD (zinc finger-homeodomain) gene family plays important roles in plant growth, development, and various stress responses. In the present study, 49, 50, 22, and 32 ZF-HD genes were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, and Gossypium raimondii genomes, respectively. According to their phylogenetic features, the ZF-HD genes were classified into six groups. Segmental duplication, whole genome duplication, and transposable elements provides major forces for the expansion of cotton ZF-HD gene family during the divergence of Gossypium species and the divergence between monocots and dicots. The Ka/Ks ratios of the ZF-HD segmental duplication pairs were mainly distributed around 0.12, which indicated that they have experienced strong purifying selective pressure during evolution. Transcriptome analysis showed that 6 Gossypium hirsutum and 4 Gossypium barbadense ZF-HD genes were expressed in all tested tissues. Further, expression profiles under abiotic stress exhibited that the ZF-HD genes were differentially regulated in response to various stresses. Taken together, our findings provide a valuable information on the characterization of ZF-HD gene family and lay foundation for their further function investigations in cotton.

Association analysis of germination level cold stress tolerance and candidate gene identification in Upland cotton (Gossypium hirsutum L.).

Cotton originated from ancestors in the Gossypium genus that grew in semi-desert habitats. As a result, it is adversely affected by low temperatures especially during germination and the first weeks of growth. Despite this, there are relatively few molecular studies on cold stress in cotton. This limitation may present a future breeding handicap, as recent years have witnessed increased low temperature damage to cotton production. Cold tolerance is a sustainable approach to obtain good production in case of extreme cold. In the present study, 110 Upland cotton (Gossypium hirsutum) genotypes were evaluated for cold tolerance at the germination stage. We identified vigorous genotypes with cold-related parameters that outperformed the panel's average performance ( = 76.9% CG, 83.9% CSI, 167.5 CWVI). Molecular genetic diversity analysis with 101 simple sequence repeat (SSR) markers yielding 416 loci was used to select tolerant genotypes that could be important materials for breeding this trait. A total of 16 marker-cold tolerance trait associations (p < 0.005) were identified with 10 of them having major effects (PVE > 10%). Based on the positions of these markers, candidate genes for cold tolerance in the G. hirsutum genome were identified. Three of these markers (BNL0569, CIR081 and CIR202) are important candidates for use in marker-assisted breeding for cold tolerance because they mapped to genes previously associated with cold tolerance in other plant species such as Arabidopsis thaliana, rice and tomato. The online version contains supplementary material available at 10.1007/s12298-022-01184-6.

Genome-wide identification and characterization of terpene synthase genes in Gossypium hirsutum

Terpenoids are widely distributed in plants and play important roles in the regulation of plant growth and development and in the interactions between plants and both the environment and other organisms. However, terpene synthase (TPS) genes have not been systematically investigated in the tetraploid Gossypium hirsutum. In this study, whole genome identification and characterization of the TPS family from G. hirsutum were carried out. Eighty-five TPS genes, including 47 previously unidentified genes, were identified in the G. hirsutum genome and classified into 5 subfamilies according to protein sequence similarities, as follows: 43 GhTPS-a, 29 GhTPS-b, 4 GhTPS-c, 7 GhTPS-e/f, and 2 GhTPS-g members. These 85 TPS genes were mapped onto 19 chromosomes of the G. hirsutum genome. Segmental duplications and tandem duplications contributed greatly to the expansion of TPS genes in G. hirsutum and were followed by intense purifying selection during evolution. Indentification of cis-acting regulatory elements suggest that the expression of TPS genes is regulated by a variety of hormones. RNA sequencing (RNA-seq) expression profile analysis revealed that the TPS genes had distinct spatiotemporal expression patterns, and several genes were highly and preferentially expressed in the leaves of cotton with gossypol glands (glanded cotton) versus a glandless strain. Virus-induced gene silencing (VIGS) of three TPS genes yielded plants characterized by fewer, smaller, and lighter gossypol glands, which indicated that these three genes were responsible for gland activity. Taken together, our results provide a solid basis for further elucidation of the biological functions of TPS genes in relation to gland activity and gossypol biosynthesis to develop cotton cultivars with low cottonseed gossypol contents.

Identification and stress function verification of the HAK/KUP/KT family in Gossypium hirsutum.

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and play a crucial role in plant growth and development. The members of the family play an important role in the response of plants to abiotic stress by maintaining osmotic balance. However, the function of the family in cotton is unclear. In this study, whole genome identification and characterization of the HAK/KUP/KT family from upland cotton (Gossypium hirsutum) were carried out. Bioinformatics methods were used to identify HAK/KUP/KT family members from the G. hirsutum genome and to analyse the physical and chemical properties, basic characteristics, phylogeny, chromosome location and expression of HAK/KUP/KT family members. A total of 41 HAK/KUP/KT family members were identified in the G. hirsutum genome. Phylogenetic analysis grouped these genes into four clusters (I, II, III, IV), containing 6, 10, 3 and 22 genes, respectively. Chromosomal distribution, gene structure and conserved motif analyses of the 41 GhHAK genes were subsequently performed. The RNA-seq data and qRT-PCR results showed that the family had a wide range of tissue expression patterns, and they responded to certain drought stresses. Through expression analysis, seven HAK/KUP/KT genes involved in drought stress were screened, and four genes with obvious phenotypes under drought stress were obtained by VIGS verification, which laid a theoretical foundation for the function of the cotton HAK/KUP/KT family.

Genome-Wide Identification and Evolutionary Analysis of Gossypium Tubby-Like Protein (TLP) Gene Family and Expression Analyses During Salt and Drought Stress.

Tubby-like proteins (TLPs) possess a highly conserved closed β barrel tubby domain at C-terminal and N-terminal F-box. The role of TLP gene family members has been widely discussed in numerous organisms; however, the detailed genome-wide study of this gene family in Gossypium species has not been reported till date. Here, we systematically identified 105 TLP gene family members in cotton (Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense) genomes and classified them into eight phylogenetic groups. Cotton TLP12 gene family members clustered into two groups, 4 and 8. They experienced higher evolutionary pressure in comparison to others, indicating the faster evolution in both diploid as well as in tetraploid cotton. Cotton TLP gene family members expanded mainly due to segmental duplication, while only one pair of tandem duplication was found in cotton TLPs paralogous gene pairs. Subsequent qRT-PCR validation of seven putative key candidate genes of GhTLPs indicated that GhTLP11A and GhTLP12A.1 genes were highly sensitive to salt and drought stress. The co-expression network, pathways, and cis-regulatory elements of GhTLP11A and GhTLP12A.1 genes confirmed their functional importance in salt and drought stress responses. This study proposes the significance of GhTLP11A and GhTLP12A.1 genes in exerting control over salt and drought stress responses in G. hirsutum and also provides a reference for future research, elaborating the biological roles of G. hirsutum TLPs in both stress responses.

Genome-Wide Comparative Analysis of Heat Shock Transcription Factors Provides Novel Insights for Evolutionary History and Expression Characterization in Cotton Diploid and Tetraploid Genomes.

Heat shock transcription factors (HSFs) are involved in environmental stress response and plant development, such as heat stress and flowering development. According to the structural characteristics of the HSF gene family, HSF genes were classified into three major types (HSFA, HSFB, and HSFC) in plants. Using conserved domains of HSF genes, we identified 621 HSF genes among 13 cotton genomes, consisting of eight diploid and five tetraploid genomes. Phylogenetic analysis indicated that HSF genes among 13 cotton genomes were grouped into two different clusters: one cluster contained all HSF genes of HSFA and HSFC, and the other cluster contained all HSF genes of HSFB. Comparative analysis of HSF genes in Arabidopsis thaliana, Gossypium herbaceum (A1), Gossypium arboreum (A2), Gossypium raimondii (D5), and Gossypium hirsutum (AD1) genomes demonstrated that four HSF genes were inherited from a common ancestor, A0, of all existing cotton A genomes. Members of the HSF gene family in G. herbaceum (A1) genome indicated a significant loss compared with those in G. arboretum (A2) and G. hirsutum (AD1) A genomes. However, HSF genes in G. raimondii (D5) showed relative loss compared with those in G. hirsutum (AD1) D genome. Analysis of tandem duplication (TD) events of HSF genes revealed that protein-coding genes among different cotton genomes have experienced TD events, but only the two-gene tandem array was detected in Gossypium thurberi (D1) genome. The expression analysis of HSF genes in G. hirsutum (AD1) and Gossypium barbadense (AD2) genomes indicated that the expressed HSF genes were divided into two different groups, respectively, and the expressed HSF orthologous genes between the two genomes showed totally different expression patterns despite the implementation of the same abiotic stresses. This work will provide novel insights for the study of evolutionary history and expression characterization of HSF genes in different cotton genomes and a widespread application model for the study of HSF gene families in plants.

Molecular Traits and Functional Analysis of the CLAVATA3/Endosperm Surrounding Region-Related Small Signaling Peptides in Three Species of Gossypium Genus.

The CLAVATA3/endosperm surrounding region-related (CLE) small peptides are a group of C-terminally encoded and post-translationally modified signal molecules involved in regulating the growth and development of various plants. However, the function and evolution of these peptides have so far remained elusive in cotton. In this study, 55, 56, and 86 CLE genes were identified in the Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum genomes, respectively, and all members were divided into seven groups. These groups were distinctly different in their protein characteristics, gene structures, conserved motifs, and multiple sequence alignment. Whole genome or segmental duplications played a significant role in the expansion of the CLE family in cotton, and experienced purifying selection during the long evolutionary process in cotton. Cis-acting regulatory elements and transcript profiling revealed that the CLE genes of cotton exist in different tissues, developmental stages, and respond to abiotic stresses. Protein properties, structure prediction, protein interaction network prediction of GhCLE2, GhCLE33.2, and GhCLE28.1 peptides were, respectively, analyzed. In addition, the overexpression of GhCLE2, GhCLE33.2, or GhCLE28.1 in Arabidopsis, respectively, resulted in a distinctive shrub-like dwarf plant, slightly purple leaves, large rosettes with large malformed leaves, and lack of reproductive growth. This study provides important insights into the evolution of cotton CLEs and delineates the functional conservatism and divergence of CLE genes in the growth and development of cotton.

Cotton pan-genome retrieves the lost sequences and genes during domestication and selection

BackgroundMillennia of directional human selection has reshaped the genomic architecture of cultivated cotton relative to wild counterparts, but we have limited understanding of the selective retention and fractionation of genomic components.ResultsWe construct a comprehensive genomic variome based on 1961 cottons and identify 456 Mb and 357 Mb of sequence with domestication and improvement selection signals and 162 loci, 84 of which are novel, including 47 loci associated with 16 agronomic traits. Using pan-genome analyses, we identify 32,569 and 8851 non-reference genes lost from Gossypium hirsutum and Gossypium barbadense reference genomes respectively, of which 38.2% (39,278) and 14.2% (11,359) of genes exhibit presence/absence variation (PAV). We document the landscape of PAV selection accompanied by asymmetric gene gain and loss and identify 124 PAVs linked to favorable fiber quality and yield loci.ConclusionsThis variation repertoire points to genomic divergence during cotton domestication and improvement, which informs the characterization of favorable gene alleles for improved breeding practice using a pan-genome-based approach.