GhGASA14 regulates the flowering time of upland cotton in response to GA3.

KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.

GhBRX.1, GhBRX.2, and GhBRX4.3 improve resistance to salt and cold stress in upland cotton.

Introduction: Abiotic stress during growth readily reduces cotton crop yield. The different survival tactics of plants include the activation of numerous stress response genes, such as BREVIS RADIX (BRX). Methods: In this study, the BRX gene family of upland cotton was identified and analyzed by bioinformatics method, three salt-tolerant and cold-resistant GhBRX genes were screened. The expression of GhBRX.1, GhBRX.2 and GhBRXL4.3 in upland cotton was silenced by virus-induced gene silencing (VIGS) technique. The physiological and biochemical indexes of plants and the expression of related stress-response genes were detected before and after gene silencing. The effects of GhBRX.1, GhBRX.2 and GhBRXL4.3 on salt and cold resistance of upland cotton were further verified. Results and discussion: We discovered 12, 6, and 6 BRX genes in Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. Chromosomal localization indicated that the retention and loss of GhBRX genes on homologous chromosomes did not have a clear preference for the subgenomes. Collinearity analysis suggested that segmental duplications were the main force for BRX gene amplification. The upland cotton genes GhBRX.1, GhBRX.2 and GhBRXL4.3 are highly expressed in roots, and GhBRXL4.3 is also strongly expressed in the pistil. Transcriptome data and qRT‒PCR validation showed that abiotic stress strongly induced GhBRX.1, GhBRX.2 and GhBRXL4.3. Under salt stress and low-temperature stress conditions, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and the content of soluble sugar and chlorophyll decreased in GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants compared with those in the control (TRV: 00). Moreover, GhBRX.1-, GhBRX.2- and GhBRXL4.3-silenced cotton plants exhibited greater malondialdehyde (MDA) levels than did the control plants. Moreover, the expression of stress marker genes (GhSOS1, GhSOS2, GhNHX1, GhCIPK6, GhBIN2, GhSnRK2.6, GhHDT4D, GhCBF1 and GhPP2C) decreased significantly in the three target genes of silenced plants following exposure to stress. These results imply that the GhBRX.1, GhBRX.2 and GhBRXL4.3 genes may be regulators of salt stress and low-temperature stress responses in upland cotton.

The Silencing of GhPIP5K2 and GhPIP5K22 Weakens Abiotic Stress Tolerance in Upland Cotton (Gossypium hirsutum).

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), essential enzymes in the phosphatidylinositol signaling pathway, are crucial for the abiotic stress responses and the overall growth and development of plants. However, the GhPIP5Ks had not been systematically studied, and their function in upland cotton was unknown. This study identified a total of 28 GhPIP5Ks, and determined their chromosomal locations, gene structures, protein motifs and cis-acting elements via bioinformatics analysis. A quantitative real-time PCR (qRT‒PCR) analysis showed that most GhPIP5Ks were upregulated under different stresses. A virus-induced gene silencing (VIGS) assay indicated that the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were significantly decreased, while malondialdehyde (MDA) content were significantly increased in GhPIP5K2- and GhPIP5K22-silenced upland cotton plants under abiotic stress. Furthermore, the expression of the stress marker genes GhHSFB2A, GhHSFB2B, GhDREB2A, GhDREB2C, GhRD20-1, GhRD29A, GhBIN2, GhCBL3, GhNHX1, GhPP2C, GhCBF1, GhSnRK2.6 and GhCIPK6 was significantly decreased in the silenced plants after exposure to stress. These results revealed that the silencing of GhPIP5K2 and GhPIP5K22 weakened the tolerance to abiotic stresses. These discoveries provide a foundation for further inquiry into the actions of the GhPIP5K gene family in regulating the response and resistance mechanisms of cotton to abiotic stresses.

Genome-Wide Identification of the GhANN Gene Family and Functional Validation of GhANN11 and GhANN4 under Abiotic Stress.

Annexins (ANNs) are a structurally conserved protein family present in almost all plants. In the present study, 27 GhANNs were identified in cotton and were unevenly distributed across 14 chromosomes. Transcriptome data and RT-qPCR results revealed that multiple GhANNs respond to at least two abiotic stresses. Similarly, the expression levels of GhANN4 and GhANN11 were significantly upregulated under heat, cold, and drought stress. Using virus-induced gene silencing (VIGS), functional characterization of GhANN4 and GhANN11 revealed that, compared with those of the controls, the leaf wilting of GhANN4-silenced plants was more obvious, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were lower under NaCl and PEG stress. Moreover, the expression of stress marker genes (GhCBL3, GhDREB2A, GhDREB2C, GhPP2C, GhRD20-2, GhCIPK6, GhNHX1, GhRD20-1, GhSOS1, GhSOS2 and GhSnRK2.6) was significantly downregulated in GhANN4-silenced plants after stress. Under cold stress, the growth of the GHANN11-silenced plants was significantly weaker than that of the control plants, and the activities of POD, SOD, and CAT were also lower. However, compared with those of the control, the elasticity and orthostatic activity of the GhANN11-silenced plants were greater; the POD, SOD, and CAT activities were higher; and the GhDREB2C, GhHSP, and GhSOS2 expression levels were greater under heat stress. These results suggest that different GhANN family members respond differently to different types of abiotic stress.

Cotton RSG2 Mediates Plant Resistance against Verticillium dahliae by miR482b Regulation.

Cotton Verticillium wilt, mainly caused by Verticillium dahliae, has a serious impact on the yield and quality of cotton fiber. Many microRNAs (miRNAs) have been identified to participate in plant resistance to V. dahliae infection, but the exploration of miRNA's function mechanism in plant defense is needed. Here, we demonstrate that the ghr-miR482b-GhRSG2 module mediates cotton plant resistance to V. dahliae infection. Based on the mRNA degradation data and GUS fusion experiments, ghr-miR482b directedly bonds to GhRSG2 mRNA to lead to its degradation. The knockdown and overexpression of ghr-miR482b through virus-induced gene silencing strategies enhanced (decreased by 0.39-fold in disease index compared with the control) and weakened (increased by 0.46-fold) the plant resistance to V. dahliae, respectively. In addition, silencing GhRSG2 significantly increased (increased by 0.93-fold in disease index) the plant sensitivity to V. dahliae compared with the control plants treated with empty vector. The expression levels of two SA-related disease genes, GhPR1 and GhPR2, significantly decreased in GhRSG2-silenced plants by 0.71 and 0.67 times, respectively, and in ghr-miR482b-overexpressed (OX) plants by 0.59 and 0.75 times, respectively, compared with the control, whereas the expression levels of GhPR1 and GhPR2 were significantly increased by 1.21 and 2.59 times, respectively, in ghr-miR482b knockdown (KD) plants. In sum, the ghr-miR482b-GhRSG2 module participates in the regulation of plant defense against V. dahliae by inducing the expression of PR1 and PR2 genes.

Genome-wide investigation and expression profiles of the NPF gene family provide insight into the abiotic stress resistance of Gossypium hirsutum.

Membrane transporters encoded by NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NPF) genes, which play crucial roles in plant growth, development and resistance to various stresses, are involved in the transport of nitrate (NO3 -) and peptides. In several plant species, NPF genes are involved in the resistance to abiotic stresses; however, whether the whole NPF gene family in cotton contributes to this resistance has not been systematically investigated. Here, 201 genes encoding NPF proteins with a peptide transporter (PTR) domain were confirmed in three different Gossypium species, namely, Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii. The NPF proteins in these three Gossypium species and Arabidopsis thaliana were classified into three different subfamilies via phylogenetic analysis. Among the genes that encode these proteins, most GhNPF genes in the same subfamily contained similar gene structures and conserved domains. Predictions of the promoters of these genes revealed that the cis-acting elements included phytohormone- and light-responsive elements, indicating that some of these genes might be expressed in response to abiotic stress. Furthermore, 52 common potential candidate genes in 98 GhNPFs were predicted to exhibit specific spatiotemporal expression patterns in different tissues based on two RNA sequencing (RNA-seq) datasets. Finally, the gene expression profiles of abiotic stress indicated that 31 GhNPF genes were upregulated in at least one treatment period. Under abiotic stress for 12 and 24 h, the expression of GhNPF8 was upregulated upon cold treatment but downregulated with heat treatment, salt treatment and drought treatment. Furthermore, the expression of genes GhNPF8, GhNPF54 and GhNPF43 peaked at 6 h after heat and salt treatment. These results indicated that these genes exhibit underlying characteristics related to responses to abiotic stress. The verification of NPFs and analysis of their expression profiles in different tissues and in response to different abiotic stresses of cotton provide a basis for further studying the relationship between abiotic stress resistance and nitrogen (N) transport in cotton, as well as identifying candidate genes to facilitate their functional identification.

GhAP1-D3 positively regulates flowering time and early maturity with no yield and fiber quality penalties in upland cotton.

Flowering time (FTi) is a major factor determining how quickly cotton plants reach maturity. Early maturity greatly affects lint yield and fiber quality and is crucial for mechanical harvesting of cotton in northwestern China. Yet, few quantitative trait loci (QTLs) or genes regulating early maturity have been reported in cotton, and the underlying regulatory mechanisms are largely unknown. In this study, we characterized 152, 68, and 101 loci that were significantly associated with the three key early maturity traits-FTi, flower and boll period (FBP) and whole growth period (WGP), respectively, via four genome-wide association study methods in upland cotton (Gossypium hirsutum). We focused on one major early maturity-related genomic region containing three single nucleotide polymorphisms on chromosome D03, and determined that GhAP1-D3, a gene homologous to Arabidopsis thaliana APETALA1 (AP1), is the causal locus in this region. Transgenic plants overexpressing GhAP1-D3 showed significantly early flowering and early maturity without penalties for yield and fiber quality compared to wild-type (WT) plants. By contrast, the mutant lines of GhAP1-D3 generated by genome editing displayed markedly later flowering than the WT. GhAP1-D3 interacted with GhSOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), a pivotal regulator of FTi, both in vitro and in vivo. Changes in GhAP1-D3 transcript levels clearly affected the expression of multiple key flowering regulatory genes. Additionally, DNA hypomethylation and high levels of H3K9ac affected strong expression of GhAP1-D3 in early-maturing cotton cultivars. We propose that epigenetic modifications modulate GhAP1-D3 expression to positively regulate FTi in cotton through interaction of the encoded GhAP1 with GhSOC1 and affecting the transcription of multiple flowering-related genes. These findings may also lay a foundation for breeding early-maturing cotton varieties in the future.

Nucleotide Evolution, Domestication Selection, and Genetic Relationships of Chloroplast Genomes in the Economically Important Crop Genus Gossypium.

Gossypium hirsutum (upland cotton) is one of the most economically important crops worldwide, which has experienced the long terms of evolution and domestication process from wild species to cultivated accessions. However, nucleotide evolution, domestication selection, and the genetic relationship of cotton species remain largely to be studied. In this study, we used chloroplast genome sequences to determine the evolutionary rate, domestication selection, and genetic relationships of 72 cotton genotypes (36 cultivated cotton accessions, seven semi-wild races of G. hirsutum, and 29 wild species). Evolutionary analysis showed that the cultivated tetraploid cotton genotypes clustered into a single clade, which also formed a larger lineage with the semi-wild races. Substitution rate analysis demonstrated that the rates of nucleotide substitution and indel variation were higher for the wild species than the semi-wild and cultivated tetraploid lineages. Selection pressure analysis showed that the wild species might have experienced greater selection pressure, whereas the cultivated cotton genotypes underwent artificial and domestication selection. Population clustering analysis indicated that the cultivated cotton accessions and semi-wild races have existed the obviously genetic differentiation. The nucleotide diversity was higher in the semi-wild races compared with the cultivated genotypes. In addition, genetic introgression and gene flow occurred between the cultivated tetraploid cotton and semi-wild genotypes, but mainly via historical rather than contemporary gene flow. These results provide novel molecular mechanisms insights into the evolution and domestication of economically important crop cotton species.

Complete Chloroplast Genome Sequence of Sonchus brachyotus Helps to Elucidate Evolutionary Relationships with Related Species of Asteraceae.

Sonchus brachyotus DC. possesses both edible and medicinal properties and is widely distributed throughout China. In this study, the complete cp genome of S. brachyotus was sequenced and assembled. The total length of the complete S. brachyotus cp genome was 151,977 bp, including an LSC region of 84,553 bp, SSC region of 18,138 bp, and IR region of 24,643 bp. Sequence analyses revealed that the cp genome encoded 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The GC content was 37.6%. One hundred mononucleotide microsatellites, 4 dinucleotide microsatellites, 67 trinucleotide microsatellites, 4 tetranucleotide microsatellites, and 1 long repeat were identified. The SSR frequency of the LSC region was significantly greater than that of the IR and SSC regions. In total, 175 SSRs and highly variable regions were recognized as potential cp markers. By analyzing the IR/LSC and IR/SSC boundaries, structural differences between S. brachyotus and 6 other species were detected. According to phylogenetic analyses, S. brachyotus was most closely related to S. arvensis and S. oleraceus. Overall, this study provides complete cp genome resources for S. brachyotus that will be beneficial for identifying potential molecular markers and evolutionary patterns of S. brachyotus and its closely related species.

An RTM-GWAS procedure reveals the QTL alleles and candidate genes for three yield-related traits in upland cotton.

BACKGROUND: Cotton (Gossypium spp.) fiber yield is one of the key target traits, and improved fiber yield has always been thought of as an important objective in the breeding programs and production. Although some studies had been reported for the understanding of genetic bases for cotton yield-related traits, the detected quantitative trait loci (QTL) for the traits is still very limited. To uncover the whole-genome QTL controlling three yield-related traits in upland cotton (Gossypium hirsutum L.), phenotypic traits were investigated under four planting environments and 9244 single-nucleotide polymorphism linkage disequilibrium block (SNPLDB) markers were developed in an association panel consisting of 315 accessions. RESULTS: A total of 53, 70 and 68 significant SNPLDB loci associated with boll number (BN), boll weight (BW) and lint percentage (LP), were respectively detected through a restricted two-stage multi-locus multi-allele genome-wide association study (RTM-GWAS) procedure in multiple environments. The haplotype/allele effects of the significant SNPLDB loci were estimated and the QTL-allele matrices were organized for offering the abbreviated genetic composition of the population. Among the significant SNPLDB loci, six of them were simultaneously identified in two or more single planting environments and were thought of as the stable SNPLDB loci. Additionally, a total of 115 genes were annotated in the nearby regions of the six stable SNPLDB loci, and 16 common potential candidate genes controlling target traits of them were predicted by two RNA-seq data. One of 16 genes (GH_D06G2161) was mainly expressed in the early ovule-development stages, and the stable SNPLDB locus (LDB_19_62926589) was mapped in its promoter region. CONCLUSION: This study identified the QTL alleles and candidate genes that could provide important insights into the genetic basis of yield-related traits in upland cotton and might facilitate breeding cotton varieties with high yield.

Abscisic acid associated with key enzymes and genes involving in dynamic flux of water soluble carbohydrates in wheat peduncle under terminal drought stress.

Remobilization of stem water soluble carbohydrates (WSC) can supply crucial carbon resources for grain filling under drought stress, while the regulatory metabolism associated with abscisic acid (ABA) is still limited. Two cultivars, LJ196 (drought-tolerant) and XD18 (drought-prone), were pot-grown under well-watered (WW) and drought-stressed (DS) conditions. Concentrations of WSC components and ABA, and fructan metabolizing enzymes and genes were investigated in peduncle after anthesis. When compared with those under the WW, LJ196 remained higher grain yield and grain-filling rate than XD18 under the DS. During the early period of grain filling (0-14 DAA), DS increased concentrations of total WSC and its components, but thereafter substantially reduced them. The gene expression levels and enzymatic activities of fructan 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH) showed similar trends, whereas those of fructan: fructan 1-fructosyltransferase (1-FFT), and sucrose: fructan 6-fructosyltransferase (6-SFT) were depressed and declined over the period of examination. LJ196 still showed higher levels of ABA and fructan metabolizing. The ABA concentration under the DS was positively and significantly correlated with total WSC and fructan concentration, and expression levels of these enzymes and genes as well, with more prominently with those of 6-FEH. Presumably, ABA could enhance fructan hydrolysis by strongly up-regulating the gene expression and enzymatic activity of 6-FEH to accelerate WSC remobilization. However, stem WSC induced by DS could be not fully remobilized to grains, due to its weaker correlation with grain-filling rate and finally indicating lower grain yield. The findings would provide useful information for wheat production under water-deficit environments.

Genetic dissection of stem WSC accumulation and remobilization in wheat (Triticum aestivum L.) under terminal drought stress.

BACKGROUND: The accumulation and remobilization of stem water soluble carbohydrates (WSC) are determinant physiological traits highly influencing yield potential in wheat against drought stress. However, knowledge gains of the genetic control are still limited. A hexaploid wheat population of 120 recombinant inbred lines were developed to identify quantitative trait loci (QTLs) and to dissect the genetic basis underlying eight traits related to stem WSC under drought stress (DS) and well-watered (WW) conditions across three environments. RESULTS: Analysis of variance (ANOVA) revealed larger environmental and genotypic effects on stem WSC-related traits, indicating moderate heritabilities of 0.51-0.72. A total of 95 additive and 88 pairs of epistatic QTLs were identified with significant additive and epistatic effects, as well as QTL× water environmental interaction (QEI) effects. Most of additive QTLs and additive QEIs associated with drought-stressed environments functioned genetic effects promoting pre-anthesis WSC levels and stem WSC remobilization to developing grains. Compared to other genetic components, both genetic effects were performed exclusive contributions to phenotypic variations in stem WSC-related traits. Nineteen QTL clusters were identified on chromosomes 1B, 2A, 2B, 2D, 3B, 4B, 5A, 6A, 6B and 7A, suggestive of the genetic linkage or pleiotropy. Thirteen additive QTLs were detectable repeatedly across two of the three water environments, indicating features of stable expressions. Some loci were consistent with those reported early and were further discussed. CONCLUSION: Stem WSC-related traits were inherited predominantly by additive and QEI effects with a moderate heritability. QTL cluster regions were suggestive of tight linkage or pleiotropy in the inheritance of these traits. Some stable and common loci, as well as closely linked molecular markers, had great potential in marker-assisted selection to improve stem WSC-related traits in wheat, especially under drought-stressed environments.

Genetic Detection of Lint Percentage Applying Single-Locus and Multi-Locus Genome-Wide Association Studies in Chinese Early-Maturity Upland Cotton.

Upland cotton (Gossypium hirsutum L.) is the most important source of natural fiber in the world. Early-maturity upland cotton varieties are commonly planted in China. Nevertheless, lint yield of early-maturity upland cotton varieties is strikingly lower than that of middle- and late-maturity ones. How to effectively improve lint yield of early maturing cotton, becomes a focus of cotton research. Here, based on 72,792 high-quality single nucleotide polymorphisms of 160 early-maturing upland cotton accessions, we performed genome-wide association studies (GWASs) for lint percentage (LP), one of the most lint-yield component traits, applying one single-locus method and six multi-locus methods. A total of 4 and 45 significant quantitative trait nucleotides (QTNs) were respectively identified to be associated with LP. Interestingly, in two of four planting environments, two of these QTNs (A02_74713290 and A02_75551547) were simultaneously detected via both one single-locus and three or more multi-locus GWAS methods. Among the 42 genes within a genomic region (A02: 74.31-75.95 Mbp) containing the above two peak QTNs, Gh_A02G1269, Gh_A02G1280, and Gh_A02G1295 had the highest expression levels in ovules during seed development from 20 to 25 days post anthesis, whereas Gh_A02G1278 was preferentially expressed in the fibers rather than other organs. These results imply that the four potential candidate genes might be closely related to cotton LP by regulating the proportion of seed weight and fiber yield. The QTNs and potential candidate genes for LP, identified in this study, provide valuable resource for cultivating novel cotton varieties with earliness and high lint yield in the future.

Multi-Locus Genome-Wide Association Studies of Fiber-Quality Related Traits in Chinese Early-Maturity Upland Cotton.

Early-maturity varieties of upland cotton are becoming increasingly important for farmers to improve their economic benefits through double cropping practices and mechanical harvesting production in China. However, fiber qualities of early-maturing varieties are relatively poor compared with those of middle- and late- maturing ones. Therefore, it is crucial for researchers to elucidate the genetic bases controlling fiber-quality related traits in early-maturity cultivars, and to improve synergistically cotton earliness and fiber quality. Here, multi-locus genome-wide association studies (ML-GWAS) were conducted in a panel consisting of 160 early-maturing cotton accessions. Each accession was genotyped by 72,792 high-quality single nucleotide polymorphisms (SNPs) using specific-locus amplified fragment sequencing (SLAF-seq) approach, and fiber quality-related traits under four environmental conditions were measured. Applying at least three ML-GWAS methods, a total of 70 significant quantitative trait nucleotides (QTNs) were identified to be associated with five objective traits, including fiber length (FL), fiber strength (FS), fiber micronaire (FM), fiber uniformity (FU) and fiber elongation (FE). Among these QTNs, D11_21619830, A05_28352019 and D03_34920546 were found to be significantly associated with FL, FS, and FM, respectively, across at least two environments. Among 96 genes located in the three target genomic regions (A05: 27.95 28.75, D03: 34.52 35.32, and D11: 21.22 22.02 Mbp), six genes (Gh_A05G2325, Gh_A05G2329, Gh_A05G2334, Gh_D11G1853, Gh_D11G1876, and Gh_D11G1879) were detected to be highly expressed in fibers relative to other eight tissues by transcriptome sequencing method in 12 cotton tissues. Together, multiple favorable QTN alleles and six candidate key genes were characterized to regulate fiber development in early-maturity cotton. This will lay a solid foundation for breeding novel cotton varieties with earliness and excellent fiber-quality in the future.

Genome-wide identification and expression analyses of the pectate lyase (PEL) gene family in cotton (Gossypium hirsutum L.).

BACKGROUND: Pectin is a major component and structural polysaccharide of the primary cell walls and middle lamella of higher plants. Pectate lyase (PEL, EC 4.2.2.2), a cell wall modification enzyme, degrades de-esterified pectin for cell wall loosening, remodeling and rearrangement. Nevertheless, there have been few studies on PEL genes and no comprehensive analysis of the PEL gene family in cotton. RESULTS: We identified 53, 42 and 83 putative PEL genes in Gossypium raimondii (D5), Gossypium arboreum (A2), and Gossypium hirsutum (AD1), respectively. These PEL genes were classified into five subfamilies (I-V). Members from the same subfamilies showed relatively conserved gene structures, motifs and protein domains. An analysis of gene chromosomal locations and gene duplication revealed that segmental duplication likely contributed to the expansion of the GhPELs. The 2000 bp upstream sequences of all the GhPELs contained auxin response elements. A transcriptomic data analysis showed that 62 GhPELs were expressed in various tissues. Notably, most (29/32) GhPELs of subfamily IV were preferentially expressed in the stamen, and five GhPELs of subfamily V were prominently expressed at the fiber elongation stage. In addition, qRT-PCR analysis revealed the expression characteristics of 24 GhPELs in four pollen developmental stages and significantly different expression of some GhPELs between long- and short-fiber cultivars. Moreover, some members were responsive to IAA treatment. The results indicate that GhPELs play significant and functionally diverse roles in the development of different tissues. CONCLUSIONS: In this study, we comprehensively analyzed PELs in G. hirsutum, providing a foundation to better understand the functions of GhPELs in different tissues and pathways, especially in pollen, fiber and the auxin signaling pathway.

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.

Characterization and functional analysis of GhWRKY42, a group IId WRKY gene, in upland cotton (Gossypium hirsutum L.).

BACKGROUND: WRKY transcription factors (TFs) participate in various physiological processes of plants. Although WRKY genes have been well studied in model plants, knowledge of the functional roles of these genes is still extremely limited in cotton. RESULTS: In this study, a group IId WRKY gene from cotton, GhWRKY42, was isolated and characterized. Our data showed that GhWRKY42 localized to the nucleus. A transactivation assay in yeast demonstrated that GhWRKY42 was not a transcriptional activator. A ß-glucuronidase (GUS) activity assay revealed that the promoter of GhWRKY42 showed fragment deletion activity in Nicotiana tabacum and was mainly expressed in the roots, stems and leaves of ProGhWRKY42::GUS transgenic Arabidopsis plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that GhWRKY42 was up-regulated during leaf senescence and was induced after exposure to abiotic stresses. Constitutive expression of GhWRKY42 in Arabidopsis led to a premature aging phenotype, which was correlated with an increased number of senescent leaves, reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). In addition, virus-induced gene silencing (VIGS) was used to silence the endogenous GhWRKY42 gene in cotton, and this silencing reduced plant height. CONCLUSIONS: Our findings indicate that GhWRKY42 is involved in abiotic stress responses, premature leaf senescence and stem development. This work establishes a solid foundation for further functional analysis of the GhWRKY42 gene in cotton.

Genome-wide association study identified genetic variations and candidate genes for plant architecture component traits in Chinese upland cotton.

KEY MESSAGE: Thirty significant associations between 22 SNPs and five plant architecture component traits in Chinese upland cotton were identified via GWAS. Four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits. A candidate gene, Gh_D03G0922, might be responsible for plant height in upland cotton. A compact plant architecture is increasingly required for mechanized harvesting processes in China. Therefore, cotton plant architecture is an important trait, and its components, such as plant height, fruit branch length and fruit branch angle, affect the suitability of a cultivar for mechanized harvesting. To determine the genetic basis of cotton plant architecture, a genome-wide association study (GWAS) was performed using a panel composed of 355 accessions and 93,250 single nucleotide polymorphisms (SNPs) identified using the specific-locus amplified fragment sequencing method. Thirty significant associations between 22 SNPs and five plant architecture component traits were identified via GWAS. Most importantly, four peak SNP loci located on chromosome D03 were simultaneously associated with more plant architecture component traits, and these SNPs were harbored in one linkage disequilibrium block. Furthermore, 21 candidate genes for plant architecture were predicted in a 0.95-Mb region including the four peak SNPs. One of these genes (Gh_D03G0922) was near the significant SNP D03_31584163 (8.40 kb), and its Arabidopsis homologs contain MADS-box domains that might be involved in plant growth and development. qRT-PCR showed that the expression of Gh_D03G0922 was upregulated in the apical buds and young leaves of the short and compact cotton varieties, and virus-induced gene silencing (VIGS) proved that the silenced plants exhibited increased PH. These results indicate that Gh_D03G0922 is likely the candidate gene for PH in cotton. The genetic variations and candidate genes identified in this study lay a foundation for cultivating moderately short and compact varieties in future Chinese cotton-breeding programs.

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.

Identification of the group IIa WRKY subfamily and the functional analysis of GhWRKY17 in upland cotton (Gossypium hirsutum L.).

WRKY transcription factors play important roles in plant defense, stress response, leaf senescence, and plant growth and development. Previous studies have revealed the important roles of the group IIa GhWRKY genes in cotton. To comprehensively analyze the group IIa GhWRKY genes in upland cotton, we identified 15 candidate group IIa GhWRKY genes in the Gossypium hirsutum genome. The phylogenetic tree, intron-exon structure, motif prediction and Ka/Ks analyses indicated that most group IIa GhWRKY genes shared high similarity and conservation and underwent purifying selection during evolution. In addition, we detected the expression patterns of several group IIa GhWRKY genes in individual tissues as well as during leaf senescence using public RNA sequencing data and real-time quantitative PCR. To better understand the functions of group IIa GhWRKYs in cotton, GhWRKY17 (KF669857) was isolated from upland cotton, and its sequence alignment, promoter cis-acting elements and subcellular localization were characterized. Moreover, the over-expression of GhWRKY17 in Arabidopsis up-regulated the senescence-associated genes AtWRKY53, AtSAG12 and AtSAG13, enhancing the plant's susceptibility to leaf senescence. These findings lay the foundation for further analysis and study of the functions of WRKY genes in cotton.