Genome-wide association study identifies GhSAL1 affects cold tolerance at the seedling emergence stage in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Genomic analysis of upland cotton revealed that cold tolerance was associated with ecological distribution. GhSAL1 on chromosome D09 negatively regulated cold tolerance of upland cotton. Cotton can undergo low-temperature stress at the seedling emergence stage, which adversely affects growth and yield; however, the regulatory mechanism underlying cold tolerance remains nebulous. Here, we analyze the phenotypic and physiological parameters in 200 accessions from 5 ecological distributions under constant chilling (CC) and diurnal variation of chilling (DVC) stresses at the seedling emergence stage. All accessions were clustered into four groups, of which Group IV, with most germplasms from the northwest inland region (NIR), had better phenotypes than Groups I-III under the two kinds of chilling stresses. A total of 575 significantly associated single-nucleotide polymorphism (SNP) were identified, and 35 stable genetic quantitative trait loci (QTL) were obtained, of which 5 were associated with traits under CC and DVC stress, respectively, while the remaining 25 were co-associated. The accumulation of dry weight (DW) of seedling was associated with the flavonoid biosynthesis process regulated by Gh_A10G0500. The emergence rate (ER), DW, and total length of seedling (TL) under CC stress were associated with the SNPs variation of Gh_D09G0189 (GhSAL1). GhSAL1HapB was the elite haplotype, which increased ER, DW, and TL by 19.04%, 11.26%, and 7.69%, respectively, compared with that of GhSAL1HapA. The results of virus-induced gene silencing (VIGS) experiment and determination of metabolic substrate content preliminarily illustrated that GhSAL1 negatively regulated cotton cold tolerance through IP3-Ca2+ signaling pathway. The elite haplotypes and candidate genes identified in this study could be used to improve cold tolerance at the seedling emergence stage in future upland cotton breeding.

Genome-wide association study identifies variants of GhSAD1 conferring cold tolerance in cotton.

Cold stress is a major environmental factor affecting plant growth and development. Although some plants have developed resistance to cold stress, the molecular mechanisms underlying this process are poorly understood. Using genome-wide association mapping with 200 cotton accessions collected from different regions, we identified variations in the short chain alcohol dehydrogenase gene, GhSAD1, that responds to cold stress. Virus-induced gene silencing and overexpression in Arabidopsis revealed that GhSAD1 fulfils important roles in cold stress responses. Ectopic expression of a haploid genotype of GhSAD1 (GhSAD1HapB) in Arabidopsis increased cold tolerance. Silencing of GhSAD1HapB resulted in a decrease in abscisic acid (ABA) content. Conversely, overexpression of GhSAD1HapB increased ABA content. GhSAD1HapB regulates cold stress responses in cotton through modulation of C-repeat binding factor activity, which regulates ABA signalling. GhSAD1HapB induces the expression of COLD-REGULATED (COR) genes and increases the amount of metabolites associated with cold stress tolerance. Overexpression of GhSAD1HapB partially complements the phenotype of the Arabidopsis ABA2 mutant, aba2-1. Collectively, these findings increase our understanding of the mechanisms underlying GhSAD1-mediated cold stress responses in cotton.

Genome-wide association study reveals that GhTRL1 and GhPIN8 affect cotton root development.

KEY MESSAGE: Two regions located at chromosome A05 and D04 were found to be significantly associated with 0-0.5 mm and 0.5-2 mm diameter roots, respectively, and two candidate genes related to root development were identified. Roots absorb water and nutrients, and play an important role in plant growth. However, there are few genetic developmental studies on cotton root structural traits. In this study, we used 200 upland cotton (Gossypium hirsutum L.) varieties to analyze the phenotypic variation of 43 traits. A total of 2001 related single-nucleotide polymorphism (SNP) sites located within or near 1046 genes were detected through a genome-wide association study (GWAS). The 32 root traits were linked to SNPs that corresponded to 317 nonrepetitive genes. For SNPs associated with root length and 0-0.5 mm diameter root traits, a significant peak appeared on chromosome A05 (between 21.91 and 22.24 Mb). For SNPs associated with root surface area, root volume and 0.5-2 mm diameter root traits, a significant peak appeared on chromosome D04 (between 7.35 and 7.70 Mb). Within these two key regions, SNPs were detected in the promoter and coding regions of two candidate genes, GhTRL1-A05 and GhPIN8-D04. The expression levels of these two genes also changed significantly according to transcriptome sequencing and quantitative real-time PCR (qRT-PCR). After silencing the GhTRL1 and GhPIN8 genes via virus-induced gene silencing (VIGS), we found that the plants expressing TRV2::GhTRL1 and TRV2::GhPIN8 had a reduced root length, surface area. Moreover, the contents of cis-12-oxo-phytodienoic acid (cis-OPDA), isopentenyl adenosine (iPR) and cis-zeatin (cZ) in the roots of the plants expressing TRV2::GhTRL1 decreased. This study contributes to the cultivation and improvement of cotton varieties.

Comparative Transcriptome Analysis Provides Insights into the Seed Germination in Cotton in Response to Chilling Stress.

Gossypium hirsutum L., is a widely cultivated cotton species around the world, but its production is seriously threatened by its susceptibility to chilling stress. Low temperature affects its germination, and the underlying molecular mechanisms are rarely known, particularly from a transcriptional perspective. In this study, transcriptomic profiles were analyzed and compared between two cotton varieties, the cold-tolerant variety KN27-3 and susceptible variety XLZ38. A total of 7535 differentially expressed genes (DEGs) were identified. Among them, the transcripts involved in energy metabolism were significantly enriched during germination based on analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as glycolysis/gluconeogenesis, tricarboxylic acid cycle (TCA cycle), and glyoxylate cycle (GAC). Results from further GO enrichment analysis show the earlier appearance of DNA integration, meristem growth, cotyledon morphogenesis, and other biological processes in KN27-3 compared with XLZ38 under chilling conditions. The synthesis of asparagine, GDP-mannose, and trehalose and the catabolic process of raffinose were activated. DEGs encoding antioxidants (spermidine) and antioxidase (CAT1, GPX4, DHAR2, and APX1) were much more up-regulated in embryos of KN27-3. The content of auxin (IAA), cis-zeatin riboside (cZR), and trans-zeatin riboside (tZR) in KN27-3 are higher than that in XLZ38 at five stages (from 12 h to 54 h). GA3 was expressed at a higher level in KN27-3 from 18 h to 54 h post imbibition compared to that in XLZ38. And abscisic acid (ABA) content of KN27-3 is lower than that in XLZ38 at five stages. Results from hormone content measurements and the related gene expression analysis indicated that IAA, CTK, and GA3 may promote germination of the cold-tolerant variety, while ABA inhibits it. These results expand the understanding of cottonseed germination and physiological regulations under chilling conditions by multiple pathways.

Comprehensive analyses of ZFP gene family and characterization of expression profiles during plant hormone response in cotton.

BACKGROUND: Zinc finger proteins (ZFPs) containing only a single zinc finger domain play important roles in the regulation of plant growth and development, as well as in biotic and abiotic stress responses. To date, the evolutionary history and functions of the ZFP gene family have not been identified in cotton. RESULTS: In this paper, we identified 29 ZFP genes in Gossypium hirsutum. This gene family was divided into seven subfamilies, 22 of which were distributed over 17 chromosomes. Bioinformatic analysis revealed that 20 GhZFP genes originated from whole genome duplications and two originated from dispersed duplication events, indicating that whole genome duplication is the main force in the expansion of the GhZFP gene family. Most GhZFP8 subfamily genes, except for GhZFP8-3, were highly expressed during fiber cell growth, and were induced by brassinosteroids in vitro. Furthermore, we found that a large number of GhZFP genes contained gibberellic acid responsive elements, auxin responsive elements, and E-box elements in their promoter regions. Exogenous application of these hormones significantly stimulated the expression of these genes. CONCLUSIONS: Our findings reveal that GhZFP8 genes are involved in cotton fiber development and widely induced by auxin, gibberellin and BR, which provides a foundation for the identification of more downstream genes with potential roles in phytohormone stimuli, and a basis for breeding better cotton varieties in the future.

Transcriptome analysis and identification of genes associated with fruiting branch internode elongation in upland cotton.

BACKGROUND: Appropriate plant architecture can improve the amount of cotton boll opening and allow increased planting density, thus increasing the level of cotton mechanical harvesting and cotton yields. The internodes of cotton fruiting branches are an important part of cotton plant architecture. Thus, studying the molecular mechanism of internode elongation in cotton fruiting branches is highly important. RESULTS: In this study, we selected internodes of cotton fruiting branches at three different stages from two cultivars whose internode lengths differed significantly. A total of 76,331 genes were detected by transcriptome sequencing. By KEGG pathway analysis, we found that DEGs were significantly enriched in the plant hormone signal transduction pathway. The transcriptional data and qRT-PCR results showed that members of the GH3 gene family, which are involved in auxin signal transduction, and CKX enzymes, which can reduce the level of CKs, were highly expressed in the cultivar XLZ77, which has relatively short internodes. Genes related to ethylene synthase (ACS), EIN2/3 and ERF in the ethylene signal transduction pathway and genes related to JAR1, COI1 and MYC2 in the JA signal transduction pathway were also highly expressed in XLZ77. Plant hormone determination results showed that the IAA and CK contents significantly decreased in cultivar XLZ77 compared with those in cultivar L28, while the ACC (the precursor of ethylene) and JA contents significantly increased. GO enrichment analysis revealed that the GO categories associated with promoting cell elongation, such as cell division, the cell cycle process and cell wall organization, were significantly enriched, and related genes were highly expressed in L28. However, genes related to the sphingolipid metabolic process and lignin biosynthetic process, whose expression can affect cell elongation, were highly expressed in XLZ77. In addition, 2067 TFs were differentially expressed. The WRKY, ERF and bHLH TF families were the top three largest families whose members were active in the two varieties, and the expression levels of most of the genes encoding these TFs were upregulated in XLZ77. CONCLUSIONS: Auxin and CK are positive regulators of internode elongation in cotton branches. In contrast, ethylene and JA may act as negative regulators of internode elongation in cotton branches. Furthermore, the WRKY, ERF and bHLH TFs were identified as important inhibitors of internode elongation in cotton. In XLZ77(a short-internode variety), the mass synthesis of ethylene and amino acid conjugation of auxin led to the inhibition of plant cell elongation, while an increase in JA content and degradation of CKs led to a slow rate of cell division, which eventually resulted in a phenotype that presented relatively short internodes on the fruiting branches. The results of this study not only provide gene resources for the genetic improvement of cotton plant architecture but also lay a foundation for improved understanding of the molecular mechanism of the internode elongation of cotton branches.

Auxin-mediated statolith production for root gravitropism.

Root gravitropism is one of the most important processes allowing plant adaptation to the land environment. Auxin plays a central role in mediating root gravitropism, but how auxin contributes to gravitational perception and the subsequent response are still unclear. Here, we showed that the local auxin maximum/gradient within the root apex, which is generated by the PIN directional auxin transporters, regulates the expression of three key starch granule synthesis genes, SS4, PGM and ADG1, which in turn influence the accumulation of starch granules that serve as a statolith perceiving gravity. Moreover, using the cvxIAA-ccvTIR1 system, we also showed that TIR1-mediated auxin signaling is required for starch granule formation and gravitropic response within root tips. In addition, axr3 mutants showed reduced auxin-mediated starch granule accumulation and disruption of gravitropism within the root apex. Our results indicate that auxin-mediated statolith production relies on the TIR1/AFB-AXR3-mediated auxin signaling pathway. In summary, we propose a dual role for auxin in gravitropism: the regulation of both gravity perception and response.

Analysis of Drought Tolerance and Associated Traits in Upland Cotton at the Seedling Stage.

(1) Background: Upland cotton (Gossypium hirsutum L.) is the most important natural fiber worldwide, and it is extensively planted and plentifully used in the textile industry. Major cotton planting regions are frequently affected by abiotic stress, especially drought stress. Drought resistance is a complex, quantitative trait. A genome-wide association study (GWAS) constitutes an efficient method for dissecting the genetic architecture of complex traits. In this study, the drought resistance of a population of 316 upland cotton accessions was studied via GWAS. (2) Methods: GWAS methodology was employed to identify relationships between molecular markers or candidate genes and phenotypes of interest. (3) Results: A total of 8, 3, and 6 SNPs were associated with the euphylla wilting score (EWS), cotyledon wilting score (CWS), and leaf temperature (LT), respectively, based on a general linear model and a factored spectrally transformed linear mixed model. For these traits, 7 QTLs were found, of which 2 each were located on chromosomes A05, A11, and D03, and of which 1 was located on chromosome A01. Importantly, in the candidate regions WRKY70, GhCIPK6, SnRK2.6, and NET1A, which are involved in the response to abscisic acid (ABA), the mitogen-activated protein kinase (MAPK) signaling pathway and the calcium transduction pathway were identified in upland cotton at the seedling stage under drought stress according to annotation information and linkage disequilibrium (LD) block analysis. Moreover, RNA sequencing analysis showed that WRKY70, GhCIPK6, SnRK2.6, and NET1A were induced by drought stress, and the expression of these genes was significantly different between normal and drought stress conditions. (4) Conclusions: The present study should provide some genomic resources for drought resistance in upland cotton. Moreover, the germplasm of the different phenotypes, the detected SNPs and, the potential candidate genes will be helpful for molecular marker-assisted breeding studies about increased drought resistance in upland cotton.

QTL delineation for five fiber quality traits based on an intra-specific Gossypium hirsutum L. recombinant inbred line population.

Gossypium hirsutum L. is the most important fiber crop worldwide and contributes to more than 95% of global cotton production. Marker-assisted selection (MAS) is an effective approach for improving fiber quality, and quantitative trait loci (QTL) mapping of fiber quality traits is important for cotton breeding. In this study, a permanent intra-specific recombinant inbred line (RIL) population containing 137 families was used for fiber quality testing. Based on a previously reported high-density genetic map with an average marker distance of 0.63 cM, 186 additive QTLs were obtained for five fiber quality traits over five consecutive years, including 39 for fiber length (FL), 36 for fiber strength (FS), 50 for fiber uniformity (FU), 33 for micronaire (MC) and 28 for fiber elongation (FE). Three stable QTLs, qMC-A4-1, qMC-D2-3 and qFS-D9-1, were detected in four datasets, and another eight stable QTLs, qMC-A4-2, qMC-D11-2, qFU-A9-1, qFU-A10-4, qFS-D11-1, qFL-D9-2, qFL-D11-1 and qFE-A3-2, were detected in three datasets. The annotated genes in these 11 stable QTLs were collected, and these genes included many transcription factors with functions during fiber development. 33 QTL coincidence regions were found, and these involved nearly half of the total QTLs. Four chromosome regions containing at least 6 QTLs were promising for fine mapping. In addition, 41 pairs of epistatic QTLs (e-QTLs) were screened, including 6 for FL, 30 for FS, 2 for FU and 3 for MC. The identification of stable QTLs adds valuable information for further QTL fine mapping and gene positional cloning for fiber quality genetic detection and provides useful markers for further molecular breeding in enhancing fiber quality.

Genome-wide identification of the GhARF gene family reveals that GhARF2 and GhARF18 are involved in cotton fibre cell initiation.

Auxin signalling plays an essential role in regulating plant development. Auxin response factors (ARFs), which are critical components of auxin signalling, modulate the expression of early auxin-responsive genes by binding to auxin response factor elements (AuxREs). However, there has been no comprehensive characterization of this gene family in cotton. Here, we identified 56 GhARF genes in the assembled Gossypium hirsutum genome. This gene family was divided into 17 subfamilies, and 44 members of them were distributed across 21 chromosomes. GhARF6 and GhARF11 subfamily genes were predominantly expressed in vegetative tissues, whereas GhARF2 and GhARF18 subfamily genes were highly expressed during seed fibre cell initiation. GhARF2-1 and GhARF18-1 were exclusively expressed in trichomes, organs similar to cotton seed fibre cells, and overexpression of these genes in Arabidopsis enhances trichome initiation. Comparative transcriptome analysis combined with AuxRE prediction revealed 11 transcription factors as potential target genes of GhARF2 and GhARF18. Six of these genes were significantly expressed during seed fibre cell initiation and were bound by GhARF2-1 and GhARF18-1 in yeast one-hybrid assays. Our results suggest that GhARF2 and GhARF18 genes may be key regulators of cotton seed fibre initiation by regulating the expression of several transcription factor genes. This study deepens our understanding of auxin-mediated initiation of cotton seed fibre cells and helps us in breeding better cotton varieties in the future.

Functional analysis of nine cotton genes related to leaf senescence in Gossypium hirsutum L.

Leaf senescence is defined as a deterioration process that continues to the final developmental stage of leaf. This process is usually regulated by both external and internal factors. There are about 5356 senescence associated genes belonging to 44 plant species. A great number of these genes were identified in Arabidopsis. Leaf senescence can be regulated by many transcription factors. In this study, nine gene families were selected according to their expression levels during leaf senescence from our laboratory database. Phylogenetic tree was constructed by MEGA6. Cultivated cotton CCRI-10 seeds were sown in the experimental field of Institute of Cotton Research of CAAS for profiling and leaf development stages analysis. For abiotic (drought and salt) stress and phytohormone (ABA, SA, ET and JA) treatments, CCRI-10 seeds were sown in potting soil at 25 °C in a chamber room. Total RNA was isolated from various samples and the cDNA prepared for qRT-PCR. The comparative CT method was applied to calculate the relative expression levels of genes. For phylogenetic tree, nine cotton genes were divided into two groups, most of homologous genes in previous studies showed roles in phytohormones and abiotic stress. Expression profiling of the nine genes showed different patterns of tissue specific expression. In leaf development stages, majority of cotton genes showed high expression in early and complete senescence stage. Furthermore, most of cotton genes have positive or negative response to phytohormones and abiotic stress. Based on the results of this study, we found four cotton genes CotAD_07559, CotAD_37422, CotAD_21204 and CotAD_54353 as candidate genes for leaves senescence and abiotic stress.

iTRAQ-Based Quantitative Proteomic Analysis Reveals Cold Responsive Proteins Involved in Leaf Senescence in Upland Cotton (Gossypium hirsutum L.).

Premature leaf senescence occurs in the ultimate phase of the plant, and it occurs through a complex series of actions regulated by stress, hormones and genes. In this study, a proteomic analysis was performed to analyze the factors that could induce premature leaf senescence in two cotton cultivars. We successfully identified 443 differential abundant proteins (DAPs) from 7388 high-confidence proteins at four stages between non-premature senescence (NS) and premature senescence (PS), among which 158 proteins were over-accumulated, 238 proteins were down-accumulated at four stages, and 47 proteins displayed overlapped accumulation. All the DAPs were mapped onto 21 different categories on the basis of a Clusters of Orthologous Groups (COG) analysis, and 9 clusters were based on accumulation. Gene Ontology (GO) enrichment results show that processes related to stress responses, including responses to cold temperatures and responses to hormones, are significantly differentially accumulated. More importantly, the enriched proteins were mapped in The Arabidopsis Information Resource (TAIR), showing that 58 proteins play an active role in abiotic stress, hormone signaling and leaf senescence. Among these proteins, 26 cold-responsive proteins (CRPs) are significantly differentially accumulated. The meteorological data showed that the median temperatures declined at approximately 15 days before the onset of aging, suggesting that a decrease in temperature is tightly linked to an onset of cotton leaf senescence. Because accumulations of H2O2 and increased jasmonic acid (JA) were detected during PS, we speculate that two pathways associated with JA and H2O2 are closely related to premature leaf senescence in cotton.

High-density linkage map construction and QTL analysis for earliness-related traits in Gossypium hirsutum L.

BACKGROUND: Gossypium hirsutum L., or upland cotton, is an important renewable resource for textile fiber. To enhance understanding of the genetic basis of cotton earliness, we constructed an intra-specific recombinant inbred line population (RIL) containing 137 lines, and performed linkage map construction and quantitative trait locus (QTL) mapping. RESULTS: Using restriction-site associated DNA sequencing, a genetic map composed of 6,434 loci, including 6,295 single nucleotide polymorphisms and 139 simple sequence repeat loci, was developed from RIL population. This map spanned 4,071.98 cM, with an average distance of 0.63 cM between adjacent markers. A total of 247 QTLs for six earliness-related traits were detected in 6 consecutive years. In addition, 55 QTL coincidence regions representing more than 60 % of total QTLs were found on 22 chromosomes, which indicated that several earliness-related traits might be simultaneously improved. Fine-mapping of a 2-Mb region on chromosome D3 associated with five stable QTLs between Marker25958 and Marker25963 revealed that lines containing alleles derived from CCRI36 in this region exhibited smaller phenotypes and earlier maturity. One candidate gene (EMF2) was predicted and validated by quantitative real-time PCR in early-, medium- and late-maturing cultivars from 3- to 6-leaf stages, with highest expression level in early-maturing cultivar, CCRI74, lowest expression level in late-maturing cultivar, Bomian1. CONCLUSIONS: We developed an SNP-based genetic map, and this map is the first high-density genetic map for short-season cotton and has the potential to provide deeper insights into earliness. Cotton earliness-related QTLs and QTL coincidence regions will provide useful materials for QTL fine mapping, gene positional cloning and MAS. And the gene, EMF2, is promising for further study.

Identification of favorable SNP alleles and candidate genes for traits related to early maturity via GWAS in upland cotton.

BACKGROUND: Early maturity is one of the most important and complex agronomic traits in upland cotton (Gossypium hirsutum L). To dissect the genetic architecture of this agronomically important trait, a population consisting of 355 upland cotton germplasm accessions was genotyped using the specific-locus amplified fragment sequencing (SLAF-seq) approach, of which a subset of 185 lines representative of the diversity among the accessions was phenotypically characterized for six early maturity traits in four environments. A genome-wide association study (GWAS) was conducted using the generalized linear model (GLM) and mixed linear model (MLM). RESULTS: A total of 81,675 SNPs in 355 upland cotton accessions were discovered using SLAF-seq and were subsequently used in GWAS. Thirteen significant associations between eight SNP loci and five early maturity traits were successfully identified using the GLM and MLM; two of the 13 associations were common between the models. By computing phenotypic effect values for the associations detected at each locus, 11 highly favorable SNP alleles were identified for five early maturity traits. Moreover, dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between the numbers of highly favorable alleles and the phenotypic values of the target traits were identified. Most importantly, a major locus (rs13562854) on chromosome Dt3 and a potential candidate gene (CotAD_01947) for early maturity were detected. CONCLUSIONS: This study identified highly favorable SNP alleles and candidate genes associated with early maturity traits in upland cotton. The results demonstrate that GWAS is a powerful tool for dissecting complex traits and identifying candidate genes. The highly favorable SNP alleles and candidate genes for early maturity traits identified in this study should be show high potential for improvement of early maturity in future cotton breeding programs.

Global analysis of the Gossypium hirsutum L. Transcriptome during leaf senescence by RNA-Seq.

BACKGROUND: Leaf senescence is an important developmental programmed degeneration process that dramatically affects crop quality and yield. The regulation of senescence is highly complex. Although senescence regulatory genes have been well characterized in model species such as Arabidopsis and rice, there is little information on the control of this process in cotton. Here, the senescence process in cotton (Gossypium hirsutum L.) leaves was investigated over a time course including young leaf, mature leaf and leaf samples from different senescence stages using RNA-Seq. RESULTS: Of 24,846 genes detected by mapping the tags to Gossypium genomes, 3,624 genes were identified as differentially expressed during leaf senescence. There was some overlap between the genes identified here and senescence-associated genes previously identified in other species. Most of the genes related to photosynthesis, chlorophyll metabolism and carbon fixation were downregulated; whereas those for plant hormone signal transduction were upregulated. Quantitative real-time PCR was used to evaluate the results of RNA-Seq for gene expression profiles. Furthermore, 519 differentially expressed transcription factors were identified, notably WRKY, bHLH and C3H. In addition, 960 genes involved in the metabolism and regulation of eight hormones were identified, of which many genes involved in the abscisic acid, brassinosteroid, jasmonic acid, salicylic acid and ethylene pathways were upregulated, indicating that these hormone-related genes might play crucial roles in cotton leaf development and senescence. However, most auxin, cytokinin and gibberellin pathway-related genes were downregulated, suggesting that these three hormones may act as negative regulators of senescence. CONCLUSIONS: This is the first high-resolution, multiple time-course, genome-wide comprehensive analysis of gene expression in cotton. These data are the most comprehensive dataset currently available for cotton leaf senescence, and will serve as a useful resource for unraveling the functions of many specific genes involved in cotton leaf development and senescence.

Genomic organization, differential expression, and functional analysis of the SPL gene family in Gossypium hirsutum.

SQUAMOSA promoter binding protein-like (SPL) genes encode plant-specific transcription factors that are involved in many fundamental developmental processes. Certain SPL genes contain sequences complementary to miR156, a microRNA (miRNA) that plays a role in modulating plant gene expression. In this study, 30 SPL genes were identified in the reference genome of Gossypium raimondii and 24 GhSPLs were cloned from Gossypium hirsutum. G. raimondii is regarded as the putative contributor of the D-subgenome of G. hirsutum. Comparative analysis demonstrated sequence conservation between GhSPLs and other plant species. GhSPL genes could be classified into seven subclades based on phylogenetic analysis, diverse intron-exon structure, and motif prediction. Within each subclade, genes shared a similar structure. Sequence and experimental analysis predicted that 18 GhSPL genes are putative targets of GhmiR156. Additionally, tissue-specific expression analysis of GhSPL genes showed that their spatiotemporal expression patterns during development progressed differently, with most genes having high transcript levels in leaves, stems, and flowers. Finally, overexpression of GhSPL3 and GhSPL18 in Arabidopsis plants demonstrated that these two genes are involved in the development of leaves and second shoots and play an integral role in promoting flowering. The flowering integrator GhSOC1 may bind to the promoter of GhSPL3 but not GhSPL18 to regulate flowering. In conclusion, our analysis of GhSPL genes will provide some gene resources and a further understanding of GhSPL3 and GhSPL18 function in flowering promotion. Furthermore, the comparative genomics and functional analysis deepened our understanding of GhSPL genes during upland cotton vegetative and reproductive growth.

Genome-wide analysis of the family 1 glycosyltransferases in cotton.

Family 1 GT, designated as UGT, is the largest and most functionally important multigene family in the plant kingdom. In this study, we carried out a genome-wide identification, analysis, and comparison of 142, 146, and 196 putative UGTs from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively. All members present the 44 amino-acid conserved consensus sequence termed the plant secondary product glycosyltransferase motif. According to the phylogenetic relationship among the cotton UGT proteins and those from other species, GrUGTs and GaUGTs could be classified into 16 major phylogenetic groups (A-P), whereas GhUGTs are classified into 15 major phylogenetic groups with a lack of group C. All cotton UGTs are dispersed throughout the chromosomes and are displayed in clusters with the same open reading frame orientation. The expansion of them appears to result from genome duplication and rearrangement. Two conserved introns, A and B, are detected in most of the intron-containing-UGTs in G. raimondii and G. arboreum, whereas only intron A is detected in the intron-containing-UGTs in G. hirsutum. Furthermore, expression patterns of the UGT genes in G. hirsutum wild type and its near isogenic fuzzless-lintless mutant at the stage of fiber initiation were analyzed using the RNA-seq data. Overall, this study not only deepens our understanding of the structure, phylogeny, evolution, and expression of cotton UGT genes, but also provides a solid foundation for further cloning and functional studies of the UGT family genes.

Proteomic analysis of anthers from wild-type and photosensitive genetic male sterile mutant cotton (Gossypium hirsutum L.).

BACKGROUND: Male sterility is a common phenomenon in flowering plant species, and it has been successfully developed in several crops by taking advantage of heterosis. Using space mutation breeding of upland cotton, a novel photosensitive genetic male sterile (PGMS) mutant was isolated. To take advantage of the PGMS lines in cotton hybrid breeding, it is of great importance to study the molecular mechanisms of its male sterility. RESULTS: Delayed degradation of the PGMS anther tapetum occurred at different developmental stages as shown by analysis of anther cross-sections. To gain detailed insights into the cellular defects that occurred during PGMS pollen development, we used a differential proteomic approach to investigate the protein profiles of mutant and wild-type anthers at the tetrad, uninucleate and binucleate pollen stages. This approach identified 62 differentially expressed protein spots, including 19 associated with energy and metabolic pathways, 7 involved with pollen tube growth, 5 involved with protein metabolism, and 4 involved with pollen wall development. The remaining 27 protein spots were classified into other functional processes, such as protein folding and assembly (5 spots), and stress defense (4 spots). These differentially expressed proteins strikingly affected pollen development in the PGMS mutant anther and resulted in abnormal pollen grain formation, which may be the key reason for its male sterility. CONCLUSIONS: This work represents the first study using comparative proteomics between fertile and PGMS cotton plants to identify PGMS-related proteins. The results demonstrate the presence of a complicated metabolic network in anther development and advance our understanding of the molecular mechanisms of microgamete formation, providing insights into the molecular mechanisms of male sterility.

Genome-wide analysis of the WRKY gene family in cotton.

WRKY proteins are major transcription factors involved in regulating plant growth and development. Although many studies have focused on the functional identification of WRKY genes, our knowledge concerning many areas of WRKY gene biology is limited. For example, in cotton, the phylogenetic characteristics, global expression patterns, molecular mechanisms regulating expression, and target genes/pathways of WRKY genes are poorly characterized. Therefore, in this study, we present a genome-wide analysis of the WRKY gene family in cotton (Gossypium raimondii and Gossypium hirsutum). We identified 116 WRKY genes in G. raimondii from the completed genome sequence, and we cloned 102 WRKY genes in G. hirsutum. Chromosomal location analysis indicated that WRKY genes in G. raimondii evolved mainly from segmental duplication followed by tandem amplifications. Phylogenetic analysis of alga, bryophyte, lycophyta, monocot and eudicot WRKY domains revealed family member expansion with increasing complexity of the plant body. Microarray, expression profiling and qRT-PCR data revealed that WRKY genes in G. hirsutum may regulate the development of fibers, anthers, tissues (roots, stems, leaves and embryos), and are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses. Group I members, representing the ancestral form, seem to be insensitive to abiotic stress, with low expression divergence. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication, leading to sensitivity to diverse stresses. This study provides fundamental information to inform further analysis and understanding of WRKY gene functions in cotton species.

The Contributions of Sub-Communities to the Assembly Process and Ecological Mechanisms of Bacterial Communities along the Cotton Soil-Root Continuum Niche Gradient.

Soil microbes are crucial in shaping the root-associated microbial communities. In this study, we analyzed the effect of the soil-root niche gradient on the diversity, composition, and assembly of the bacterial community and co-occurrence network of two cotton varieties. The results revealed that the bacterial communities in cotton soil-root compartment niches exhibited a skewed species abundance distribution, dominated by abundant taxa showing a strong spatial specificity. The assembly processes of the rhizosphere bacterial communities were mainly driven by stochastic processes, dominated by the enrichment pattern and supplemented by the depletion pattern to recruit bacteria from the bulk soil, resulting in a more stable bacterial community. The assembly processes of the endosphere bacterial communities were determined by processes dominated by the depletion pattern and supplemented by the enrichment pattern to recruit species from the rhizosphere, resulting in a decrease in the stability and complexity of the community co-occurrence network. The compartment niche shaped the diversity of the bacterial communities, and the cotton variety genotype was an important source of diversity in bacterial communities within the compartment niche. We suggest that the moderate taxa contribute to significantly more changes in the diversity of the bacterial community than the rare and abundant taxa during the succession of bacterial communities in the cotton root-soil continuum.