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The heavy metal-binding domain is involved in heavy metal transporting and plays a significant role in plant detoxification. However, the functions of HMAs are less well known in cotton. In this study, a total of 143 GhHMAs (heavy metal-binding domain) were detected by genome-wide identification in G. hirsutum L. All the GhHMAs were classified into four groups via phylogenetic analysis. The exon/intron structure and protein motifs indicated that each branch of the GhHMA genes was highly conserved. 212 paralogous GhHMA gene pairs were identified, and the segmental duplications were the main role to the expansion of GhHMAs. The Ka/Ks values suggested that the GhHMA gene family has undergone purifying selection during the long-term evolutionary process. GhHMA3 and GhHMA75 were located in the plasma membrane, while GhHMA26, GhHMA117 and GhHMA121 were located in the nucleus, respectively. Transcriptomic data and qRT-PCR showed that GhHMA26 exhibited different expression patterns in each tissue and during fiber development or under different abiotic stresses. Overexpressing GhHMA26 significantly promoted the elongation of leaf trichomes and also improved the tolerance to salt stress. Therefore, GhHMA26 may positively regulate fiber elongation and abiotic stress. Yeast two-hybrid assays indicated that GhHMA26 and GhHMA75 participated in multiple biological functions. Our results suggest some genes in the GhHMAs might be associated with fiber development and the abiotic stress response, which could promote further research involving functional analysis of GhHMA genes in cotton.

Tetraploid cultivated cotton (Gossypium spp.) produces cottonseeds rich in protein and oil. Gossypol and related terpenoids, stored in the pigment glands of cottonseeds, are toxic to human beings and monogastric animals. However, a comprehensive understanding of the genetic basis of gossypol and gland formation is still lacking. We performed a comprehensive transcriptome analysis of four glanded versus two glandless tetraploid cultivars distributed in Gossypium hirsutum and Gossypium barbadense. A weighted gene co-expression network analysis (WGCNA) based on 431 common differentially expressed genes (DEGs) uncovered a candidate module that was strongly associated with the reduction in or disappearance of gossypol and pigment glands. Further, the co-expression network helped us to focus on 29 hub genes, which played key roles in the regulation of related genes in the candidate module. The present study contributes to our understanding of the genetic basis of gossypol and gland formation and serves as a rich potential source for breeding cotton cultivars with gossypol-rich plants and gossypol-free cottonseed, which is beneficial for improving food safety, environmental protection, and economic gains of tetraploid cultivated cotton.

Phytocyanins (PCs) are a class of plant-specific blue copper proteins that have been demonstrated to play a role in electron transport and plant development. Through analysis of the copper ligand residues, spectroscopic properties, and domain architecture of the protein, PCs have been grouped into four subfamilies: uclacyanins (UCs), stellacyanins (SCs), plantacyanins (PLCs), and early nodulin-like proteins (ENODLs). The present study aimed to identify and characterise the PCs present in three distinct cotton species (Gossypium hirsutum, Gossyium arboreum, and Gossypium raimondii) through the identification of 98, 63, and 69 genes respectively. We grouped PCs into four clades by using bioinformatics analysis and sequence alignment, which exhibit variations in gene structure and motif distribution. PCs are distributed across all chromosomes in each of the three species, with varying numbers of exons per gene and multiple conserved motifs, and with a minimum of 1 and maximum of 11 exons found on one gene. Transcriptomic data and qRT-PCR analysis revealed that two highly differentiated PC genes were expressed at the fibre initiation stage, while three highly differentiated PCs were expressed at the fibre elongation stage. These findings serve as a foundation for further investigations aimed at understanding the contribution of this gene family in cotton fibre production.

The glutathione S-transferases (GSTs) are important enzymes of secondary metabolism in plants. In this study, two putative GSTs, GhGSTF1 and GhGSTF2, were identified as anthocyanin-related GSTs by the transcriptome data of the leaves of Gossypium hirsutum L. TM-1 and T586. The quantitative real-time PCR showed that GhGSTF1 and GhGSTF2 were highly expressed in red leaves and stems of Gossypium hirsutum L. T586. Orthologous genes of GhGSTF2 in two Gossypium barbadense L. 3-79 and Xinhai21 contain bases deletion in N-terminal (GbGSTF2a) and C-terminal (GbGSTF2b) respectively. Among which, GhGSTF1 and GhGSTF2 can restore pigmentation in hypocotyls of Arabidopsis thaliana mutant tt19-7 while GbGSTF2a and GbGSTF2b cannot. Furthermore, in vitro assays showed the recombinant GhGSTF1 and GhGSTF2 had Glutathione S-transferase activities. Fluorescence quenching assays showed that Cya could obviously quench the fluorescence of GhGSTF1, GhGSTF2, GbGSTF2a and GbGSTF2b to lower levels as compared to C3G. Moreover, the transient dual-luciferase assays showed that the promoters of GhGSTF1 and GhGSTF2 could be activated by GhPAP1D at different levels. GUS staining assays showed that their promoters have different activities to light. This study indicated that GhGSTF1 and GhGSTF2 play important roles in anthocyanin accumulation and the regulatory mechanism of anthocyanin accumulation in allotetraploid Gossypium are complicated.

Background WRKY transcription factors have various functions in leaf senescence, plant maturation, adaptation to stress and regulation of secondary metabolism. However, the relationship between WRKY family and cotton pigment gland development remains unclear. Result In this study, 36 WRKY group I genes were identified in G. hirsutum L. Expression pattern analysis showed that GhWRKY138 of WRKY group I had a large difference in expression between glanded and glandless plants. Virus-induced gene silencing experiments showed that the number of glands decreased significantly with gene silencing. At the same time, we found that the expression of GhMYC2-like, a key gene regulating pigment gland development, also decreased with the decrease of GhWRKY138. Conclusion GhWRKY138 may involved in the formation and development of cotton glands. This study explored the possible relationship between the WRKY transcription factor family and gland development, which further enriched the connotation of the biological function of WRKY genes. These studies provide new insights into the mechanism of gland development.

The cellulose synthase genes control the biosynthesis of cellulose in plants. Nonetheless, the gene family members of CesA have not been identified in the newly assembled genome of Gossypiumhirsutum (AD1, HEBAU_NDM8). We identified 38 CesA genes in G. hirsutum (NDM8) and found that the protein sequence of GhMCesA35 is 100% identical to CelA1 in a previous study. It is already known that CelA1 is involved in cellulose biosynthesis in vitro. However, the function of this gene in vivo has not been validated. In this study, we verified the function of GhMCesA35 in vivo based on overexpressed Arabidopsis thaliana. In addition, we found that it interacted with GhCesA7 through the yeast two-hybrid assay. This study provides new insights for studying the biological functions of CesA genes in G. hirsutum, thereby improving cotton fiber quality and yield.

Background Expansins (EXPs), a group of proteins that loosen plant cell walls and cellulosic materials, are involved in regulating cell growth and diverse developmental processes in plants. However, the biological functions of this gene family in cotton are still unknown. Results In this paper, we identified a total of 93 expansin genes in Gossypium hirsutum. These genes were classified into four subfamilies, including 67 GhEXPAs, 8 GhEXPBs, 6 GhEXLAs, and 12 GhEXLBs, and divided into 15 subgroups. The 93 expansin genes are distributed over 24 chromosomes, excluding Ghir_A02 and Ghir_D06. All GhEXP genes contain multiple exons, and each GhEXP protein has multiple conserved motifs. Transcript profiling and qPCR analysis revealed that the expansin genes have distinct expression patterns among different stages of cotton fibre development. Among them, 3 genes (GhEXPA4o, GhEXPA1A, and GhEXPA8h) were highly expressed in the initiation stage, 9 genes (GhEXPA4a, GhEXPA13a, GhEXPA4f, GhEXPA4q, GhEXPA8f, GhEXPA2, GhEXPA8g, GhEXPA8a, and GhEXPA4n) had high expression during the fast elongation stage, and GhEXLA1c and GhEXLA1f were preferentially expressed in the transition stage of fibre development. Conclusions Our results provide a solid basis for further elucidation of the biological functions of expansin genes in relation to cotton fibre development and valuable genetic resources for future crop improvement.

The cellulose synthase genes control the biosynthesis of cellulose in plants. Nonetheless, the gene family members of CesA have not been identified in the newly assembled genome of

Amino acid transporters (AATs) are a kind of membrane proteins that mediate the transport of amino acids across cell membranes in higher plants. The AAT proteins are involved in regulating plant cell growth and various developmental processes. However, the biological function of this gene family in cotton fiber development is not clear. In this study, 190, 190, 101, and 94 full-length AAT genes were identified from Gossypiumhirsutum, G. barbadense, G. arboreum, and G. raimondii. A total of 575 AAT genes from the four cotton species were divided into two subfamilies and 12 clades based on phylogenetic analysis. The AAT genes in the four cotton species were distributed on all the chromosomes. All GhAAT genes contain multiple exons, and each GhAAT protein has multiple conserved motifs. Transcriptional profiling and RT qPCR analysis showed that four GhATT genes tend to express specifically at the fiber initiation stage. Eight genes tend to express specifically at the fiber elongation and maturity stage, and four genes tend to express specifically at the fiber initiation and elongation stages. Our results provide a solid basis for further elucidating the biological function of AAT genes related to cotton fiber development and offer valuable genetic resources for crop improvement in the future.

WRKY transcription factors had multiple functions in plant secondary metabolism, leaf senescence, fruit ripening, adaptation to biotic and abiotic stress, and plant growth and development. However, knowledge of the group III WRKY subfamily in fiber development in upland cotton (Gossypium hirsutum L.) is largely absent. Previous studies have shown that there were 21 putative group III WRKY members in G. hirsutum L. These putative amino acid sequences from the III WRKY group were phylogenetically clustered into three clades. Multiple alignment, conservative motif analysis, and gene structure analysis showed that the members clustered together in the phylogenetic tree had similar motifs and gene structures. Expression pattern analysis revealed that variation in the expression levels of these genes in different tissues and fiber development stages. To better understand the functions of putative group III WRKY genes in G. hirsutum L., we selected the cotton fiber initiation-related gene GhWRKY53 for cloning and functional identification. The subcellular localization experiment of GhWRKY53 in Nicotiana tabacum leaves showed that it was located in the nucleus. The heterologous expression of GhWRKY53 in Arabidopsis thaliana could significantly increase the density of trichomes. Twelve proteins that interacted with GhWRKY53 were screened from the cotton fiber cDNA library by yeast two-hybrid experiment. This study findings lay a foundation for further research on the role of the GhWRKY53 during cotton fiber development and provide a new insight for further studying putative group III WRKY genes in G. hirsutum L. Our research results also provide vital information for the genetic mechanism of high-quality cotton fiber formation and essential genetic resources for cotton fiber quality improvement.

Fuzzless mutants are ideal materials to decipher the regulatory network and mechanism underlying fuzz initiation and formation. In this study, we utilized two Gossypium arboreum accessions differing in fuzz characteristics to explore expression pattern differences and discriminate genes involved in fuzz development using RNA sequencing. Gene ontology (GO) analysis was conducted and found that DEGs were mainly enriched in the regulation of transcription, metabolic processes and oxidation–reduction-related processes. Weighted gene co-expression network analysis discerned the MEmagenta module highly associated with a fuzz/fuzzless trait, which included a total of 50 hub genes differentially expressed between two materials. GaFZ, which negatively regulates trichome and fuzz formation, was found involved in MEmagenta cluster1. In addition, twenty-eight hub genes in MEmagenta cluster1 were significantly up-regulated and expressed in fuzzless mutant DPL972. It is noteworthy that Ga04G1219 and Ga04G1240, which, respectively, encode Fasciclin-like arabinogalactan protein 18(FLA18) and transport protein, showed remarkable differences of expression level and implied that they may be involved in protein glycosylation to regulate fuzz formation and development. This module and hub genes identified in this study will provide new insights on fiber and fuzz formation and be useful for the molecular design breeding of cotton genetic improvement.

Key message The fuzzless gene GaFzl was fine mapped to a 70-kb region containing a GIR1 gene, Cotton_A_11941, responsible for the fuzzless trait in Gossypium arboreum DPL972. Abstract Cotton fiber is the most important natural textile resource. The fuzzless mutant DPL972 (Gossypium arboreum) provides a useful germplasm resource to explore the molecular mechanism underlying fiber and fuzz initiation and development. In our previous research, the fuzzless gene in DPL972 was identified as a single dominant gene and named GaFzl. In the present study, we fine mapped this gene using F2 and BC1 populations. By combining traditional map-based cloning and next-generation sequencing, we mapped GaFzl to a 70-kb region containing seven annotated genes. RNA-Sequencing and re-sequencing analysis narrowed these candidates to two differentially expressed genes, Cotton_A_11941 and Cotton_A_11942. Sequence alignment uncovered no variation in coding or promoter regions of Cotton_A_11942 between DPL971 and DPL972, whereas two single-base mutations in the promoter region and a TTG insertion in the coding region were detected in Cotton_A_11941 in DPL972. Cotton_A_11941 encoding a homologous gene of GIR1 (GLABRA2-interacting repressor) in Arabidopsis thaliana is thus the candidate gene most likely responsible for the fuzzless trait in DPL972. Our findings should lead to a better understanding of cotton fuzz formation, thereby accelerating marker-assisted selection during cotton breeding. Electronic supplementary material The online version of this article (10.1007/s00122-019-03330-3) contains supplementary material, which is available to authorized users.

GL2-interacting-repressor (GIR) family members may contribute to fiber/fuzz formation via a newly discovered unique pathway in Gossypium arboreum. There are similarities between cotton fiber development and the formation of trichomes and root hairs. The GL2-interacting-repressors (GIRs) are crucial regulators of root hair and trichome formation. The GaFzl gene, annotated as GaGIR1, is negatively associated with trichome development and fuzz initiation. However, there is relatively little available information regarding the other GIR genes in cotton, especially regarding their effects on cotton fiber development. In this study, 21 GIR family genes were identified in the diploid cotton species Gossypium arboreum; these genes were divided into three groups. The GIR genes were characterized in terms of their phylogenetic relationships, structures, chromosomal distribution and evolutionary dynamics. These GIR genes were revealed to be unequally distributed on 12 chromosomes in the diploid cotton genome, with no GIR gene detected on Ga06. The cis-acting elements in the promoter regions were predicted to be responsive to light, phytohormones, defense activities and stress. The transcriptomic data and qRT-PCR results revealed that most GIR genes were not differentially expressed between the wild-type control and the fuzzless mutant line. Moreover, 14 of 21 family genes were expressed at high levels, indicating these genes may play important roles during fiber development and fuzz formation. Furthermore, Ga01G0231 was predominantly expressed in root samples, suggestive of a role in root hair formation rather than in fuzz initiation and development. The results of this study have enhanced our understanding of the GIR genes and their potential utility for improving cotton fiber through breeding.

Background Upland cotton provides the most natural fiber in the world. During fiber development, the quality and yield of fiber were influenced by gene transcription. Revealing sequence features related to transcription has a profound impact on cotton molecular breeding. We applied convolutional neural networks to predict gene expression status based on the sequences of gene transcription start regions. After that, a gradient-based interpretation and an N-adjusted kernel transformation were implemented to extract sequence features contributing to transcription. Results Our models had approximate 80% accuracies, and the area under the receiver operating characteristic curve reached over 0.85. Gradient-based interpretation revealed 5' untranslated region contributed to gene transcription. Furthermore, 6 DOF binding motifs and 4 transcription activator binding motifs were obtained by N-adjusted kernel-motif transformation from models in three developmental stages. Apart from 10 general motifs, 3 DOF5.1 genes were also detected. In silico analysis about these motifs’ binding proteins implied their potential functions in fiber formation. Besides, we also found some novel motifs in plants as important sequence features for transcription. Conclusions In conclusion, the N-adjusted kernel transformation method could interpret convolutional neural networks and reveal important sequence features related to transcription during fiber development. Potential functions of motifs interpreted from convolutional neural networks could be validated by further wet-lab experiments and applied in cotton molecular breeding.

Background Gossypium hirsutum L. (cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear. Results In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing (VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Additional file 2 Fig. S1 Amplification of the full-length cDNA of GhERF105. 1: DNA marker; 2–3: the full-length cDNA of GhERF105. Fig. S2 Nucleotide and amino acid sequences of GhERF105. Symbol ‘*’ indicates the amino acid encoded by TGA, The conserved ERF domain is in black bold. Fig. S3 The phenotypes of stem inoculated with pTRV::GhERF105 and empty vector (pTRV::00). Fig. S4 The double digestion by XbaI and SmaI of result of pBI121-GhERF105-GFP construction. 1: DNA marker (DM15000), 2 double digestion by endonuclease of recombinant vector. 3 DNA marker (DM2000).

Background Gossypium hirsutum L. (cotton) is one of the most economically important crops globally. Cottonseed is the significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited by the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and other non-ruminant animals. To date, there has been some progress in the pigment gland formation, but the underlying molecular mechanism of pigment gland formation was still complicated and unclear. Results In this study, we identified an AP2/ERF transcription factor named GhERF105 (Gh_A12G1784), which was involved in the regulation of gland pigmentation, from comparative transcriptome analysis of the leaf of two pairs of glanded and glandless accessions, which are CCRI12 and CCRI12XW, L7 and L7XW. It encoded an ERF protein localized in the nucleus with transcriptional activation activity containing a conserved AP2 domain, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions These results suggest that GhERF105 participates in the pigment gland formation and gossypol biosynthesis in partial tissue of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Background: Genome sequence plays an important role in both the basic and applied studies. Gossypium raimondii, the putative contributor of the D subgenome of Upland cotton (Gossypium. hirsutum), highlights the need to improve the genome quality in a rapid and efficient way. Methods: we performed Hi-C sequencing of Gossypium raimondii and reassembled its genome based on a set of new Hi-C data and previously published scaffolds. We identified and corrected errors of initial scaffolds before reassembled into chromosomes. Result: A total of 98.42% of sequence was clustered successfully, among which 99.72% of the clustered sequence was ordered and 99.92% of the ordered sequence was oriented with high-quality. Further evaluation of results by heat-map and collinearity analysis revealed that the current reassembled genome is significantly improved than previous one. Conclusion: This improvement in Gossypium raimondii genome not only provides a better reference genome to increase study efficiency, but also offers a new way to assemble cotton genomes. Furthermore, Hi-C data of Gossypium raimondii may be used for 3D structure research or regulating analysis.

Background: Catharanthus roseus receptor-like kinase 1-like (CrRLK1Ls) proteins play important roles in cell growth, plant morphogenesis, reproduction, hormone signaling, plant immunity and stress responses in Arabidopsis. However, not much information is available about their functions during cotton fiber development.Results: We identified a total of 125, 73 and 71 full-length putative CrRLK1L genes in G. hirsutum, G. arboreum and G. raimondii, which are much greater than that of the other plants. The phylogenetic and gene structure analysis divided the cotton CrRLK1L genes into six major groups, among which only group I and II contained AtCrRLK1Ls of Arabidopsis, suggesting that other groups (group III-VI) were expanded by gene duplication during cotton evolution. Genome collinearity analysis revealed that half of the At02 genes in G. hirsutum derived from A02 of G. arboreum, while the other half (GhCrRLK1L6 and GhCrRLK1L7) originated from Dt03 and Dt02 of G. raimondii, indicating segmental duplication between noncorresponding chromosomes during polyploidization of G. hirsutum. In addition, expression and cis-element analysis revealed that only 22 GhCrRLK1Ls showed specific expression pattern during fiber development which are mainly due to the presence of binding sites for NAC, MYB and WRKY transcription factors.Conclusions: This study provides a strong foundation to further explore the molecular mechanism of CrRLK1L genes during fiber development in upland cotton.

Cotton is one of the major industrial crops that supply natural fibers and oil for industries. This study was conducted to understand the mechanism of delayed gland morphogenesis in seeds of Gossypium bickii. In this study, we compared glandless seeds of G. bickii with glanded seeds of Gossypium arboreum. High-throughput sequencing technology was used to explore and classify the expression patterns of gland-related genes in seeds and seedlings of cotton plants. Approximately 131.33 Gigabases of raw data from 12 RNA sequencing samples with three biological replicates were generated. A total of 7196 differentially-expressed genes (DEGs) were identified in all transcriptome data. Among them, 3396 genes were found up-regulated and 3480 genes were down-regulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were performed to identify different functions between genes unique to glandless imbibed seeds and glanded seedlings. Co-expression network analysis revealed four modules that were identified as highly associated with the development of glandless seeds. Here the hub genes in each module were identified by weighted gene co-expression network analysis (WGCNA). In total, we have selected 13 genes involved in transcription factors, protein and MYB-related functions, that were differentially expressed in transcriptomic data and validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). These selected genes may play an important role for delayed gland morphogenesis. Our study provides comprehensive insight into the key genes related to glandless traits of seeds and plants, and can be further exploited by functional and molecular studies.

Additional file 1: Fig. S1 Genetic map of G. arboreum genome. Fig. S2 Arrangement of G. arboreum scaffolds within reassembled G. arboreum chromosome 12 (A_A12). Fig. S3 Collinearity among homologous chromosomes 12 of three cotton species. Fig. S4 Alignments of reassembled G. arboreum chromosome A_A12 with the whole genome of G. hirsutum.Fig. S5 Dotplot representation with the previously assembled G. arboreum chromosome.

BackgroundGenome sequencing technologies have been improved at an exponential pace but precise chromosome-scale genome assembly still remains a great challenge. The draft genome of cultivatedG. arboreumwas sequenced and assembled with shotgun sequencing approach, however, it contains several misassemblies. To address this issue, we generated an improved reassembly ofG. arboreumchromosome 12 using genetic mapping and reference-assisted approaches and evaluated this reconstruction by comparing with homologous chromosomes ofG. raimondiiandG. hirsutum.ResultsIn this study, we generated a high quality assembly of the 94.64 Mb length ofG. arboreumchromosome 12 (A_A12) which comprised of 144 scaffolds and contained 3361 protein coding genes. Evaluation of results using syntenic and collinear analysis of reconstructedG. arboreumchromosome A_A12 with its homologous chromosomes ofG. raimondii(D_D08) andG. hirsutum(AD_A12 and AD_D12) confirmed the significant improved quality of current reassembly as compared to previous one. We found major misassemblies in previously assembled chromosome 12 (A_Ca9) ofG. arboreumparticularly in anchoring and orienting of scaffolds into a pseudo-chromosome. Further, homologous chromosomes 12 ofG. raimondii(D_D08) andG. arboreum(A_A12) contained almost equal number of transcription factor (TF) related genes, and showed good collinear relationship with each other. As well, a higher rate of gene loss was found in corresponding homologous chromosomes of tetraploid (AD_A12 and AD_D12) than diploid (A_A12 and D_D08) cotton, signifying that gene loss is likely a continuing process in chromosomal evolution of tetraploid cotton.ConclusionThis study offers a more accurate strategy to correct misassemblies in sequenced draft genomes of cotton which will provide further insights towards its genome organization.

Long noncoding RNAs (lncRNAs) play important roles in various biological regulatory processes in yeast, mammals, and plants. However, no systematic identification of lncRNAs has been reported in Gossypium arboreum. In this study, the strand-specific RNA sequencing (ssRNA-seq) of samples from cotton fibers and leaves was performed, and lncRNAs involved in fiber initiation and elongation processes were systematically identified and analyzed. We identified 5,996 lncRNAs, of which 3,510 and 2,486 can be classified as long intergenic noncoding RNAs (lincRNAs) and natural antisense transcripts (lncNAT), respectively. LincRNAs and lncNATs are similar in many aspects, but have some differences in exon number, exon length, and transcript length. Expression analysis revealed that 51.9% of lincRNAs and 54.5% of lncNATs transcripts were preferentially expressed at one stage of fiber development, and were significantly highly expressed than protein-coding transcripts (21.7%). During the fiber and rapid elongation stages, rapid and dynamic changes in lncRNAs may contribute to fiber development in cotton. This work describes a set of lncRNAs that are involved in fiber development. The characterization and expression analysis of lncRNAs will facilitate future studies on their roles in fiber development in cotton.

Virescence, as a recognizable phenotype in the early development stage of cotton, is not only available for research on chloroplast development and photosynthesis but also for heterosis exploitation in cotton. In current study, for fine mapping of virescent-1 (v1) in cotton, three populations with a total of 5 678 individuals were constructed using T582 which has the virescent trait. Tobacco rattle virus, TRV1 and TRV2 (pYL156), were used as vectors for the virus-induced gene silencing (VIGS) assay. The v1 gene was fine-mapped to a 20 kb interval on chromosome 20 of tetraploid cotton. We identified only one candidate gene with four single nucleotide polymorphisms between parents, among which the single nucleotide polymorphism at the position of 1 082 base pair caused the change of amino acid residue from Arg (3–79) to Lys (T582). The relative expression of the candidate gene in virescent plants was extensively lower than that in normal plants. Nullification of the gene by VIGS significantly turned the green leaf of normal cotton plants into yellow. We named this candidate gene as GhRVL. This study will facilitate the further research on virescent formation, and will be useful for breeding of hybrid cottons.

The fuzzless gene GaFzl was fine mapped to a 70-kb region containing a GIR1 gene, Cotton_A_11941, responsible for the fuzzless trait in Gossypium arboreum DPL972. Cotton fiber is the most important natural textile resource. The fuzzless mutant DPL972 (Gossypium arboreum) provides a useful germplasm resource to explore the molecular mechanism underlying fiber and fuzz initiation and development. In our previous research, the fuzzless gene in DPL972 was identified as a single dominant gene and named GaFzl. In the present study, we fine mapped this gene using F

Summary Functional genomics has transformed from futuristic concept to well‐established scientific discipline during the last decade. Cotton functional genomics promise to enhance the understanding of fundamental plant biology to systematically exploit genetic resources for the improvement of cotton fibre quality and yield, as well as utilization of genetic information for germplasm improvement. However, determining the cotton gene functions is a much more challenging task, which has not progressed at a rapid pace. This article presents a comprehensive overview of the recent tools and resources available with the major advances in cotton functional genomics to develop elite cotton genotypes. This effort ultimately helps to filter a subset of genes that can be used to assemble a final list of candidate genes that could be employed in future novel cotton breeding programme. We argue that next stage of cotton functional genomics requires the draft genomes refinement, re‐sequencing broad diversity panels with the development of high‐throughput functional genomics tools and integrating multidisciplinary approaches in upcoming cotton improvement programmes.

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.

Sucrose synthase (Sus) is a key enzyme in plant sucrose metabolism. In cotton, Sus (EC 2.4.1.13) is the main enzyme that degrades sucrose imported into cotton fibers from the phloem of the seed coat. This study demonstrated that the genomes of Gossypium arboreum L., G. raimondii Ulbr., and G. hirsutum L., contained 8, 8, and 15 Sus genes, respectively. Their structural organizations, phylogenetic relationships, and expression profiles were characterized. Comparisons of genomic and coding sequences identified multiple introns, the number and positions of which were highly conserved between diploid and allotetraploid cotton species. Most of the phylogenetic clades contained sequences from all three species, suggesting that the Sus genes of tetraploid G. hirsutum derived from those of its diploid ancestors. One Sus group (Sus I) underwent expansion during cotton evolution. Expression analyses indicated that most Sus genes were differentially expressed in various tissues and had development-dependent expression profiles in cotton fiber cells. Members of the same orthologous group had very similar expression patterns in all three species. These results provide new insights into the evolution of the cotton Sus gene family, and insight into its members' physiological functions during fiber growth and development.