Plant height is a key agronomic trait that influences plant architecture and mechanical harvesting suitability in cotton; however, the molecular mechanisms underlying its dynamic development remain unclear. In this study, two recombinant inbred line (RIL) populations sharing CCRI127 as a common paternal parent (RIL-GH07, n = 150; RIL-2358B, n = 276) were developed. Based on stable plant-height performance across multiple environments, tall and short extreme lines were selected from the two RIL populations for transcriptome sequencing. By integrating differential expression analysis with weighted gene co-expression network analysis (WGCNA), we identified hub genes associated with cotton plant height development, characterized the molecular features and core pathways governing dynamic stem elongation at different growth stages, thereby providing insights into the transcriptional regulation of plant height development in cotton. The two RIL populations showed broadly similar plant-height growth patterns, with slow elongation at 15 DOS, rapid elongation during 30-60 DOS, and reduced growth after 70 DOS. Transcriptome differential expression analysis identified 15,052 non-redundant DEGs, which exhibited clear population- and stage-specific expression patterns. In the GH07 population, the largest number of DEGs was detected at 15 DOS (7193), whereas in the 2358B population relatively large numbers of DEGs were maintained at both 30 DOS (3839) and 70 DOS (3118). Analysis of DEGs shared by the two populations across four developmental stages showed that, in addition to genes with consistent expression trends, each stage also contained a substantial number of DEGs with opposite expression directions. WGCNA identified 25 gene expression modules, among which the green and yellow modules were significantly positively correlated with plant height. Functional enrichment analysis indicated that genes in these two modules were mainly enriched in hormone regulation and signal transduction, protein modification and degradation, and intracellular transport. Seven hub genes were identified by integrating intramodular connectivity and kME values. Functional prediction suggested that these genes may play important roles in cotton plant height development. This study provides genetic resources and a theoretical basis for subsequent functional validation of cotton plant height-related genes and the improvement of plant architecture in cotton.

Climate change has increased the sensitivity of cropping systems to environmental variability, making the genetic dissection of G × E and phenotypic plasticity essential for adaptive breeding. In this study, a recombinant inbred line population of upland cotton was evaluated across 16 environments from multi-year and multi-location field trials to assess the plasticity of yield- and fiber-quality traits. By integrating the Finlay–Wilkinson regression model with CERIS sliding-window analyses, 13 key meteorological factors and 4 principal component variables were identified, together with their effective time windows. QTL mapping based on trait plasticity slopes and environmental response slopes detected 172 environment-associated loci. A major locus at 188 Mb on chr10 influenced boll weight and seed index in response to multiple environmental variables, including photoperiod, cumulative radiation, growing degree days, and the product of radiation and photoperiod, suggesting a key regulatory site for environmental signal perception. Haplotype analysis further supported functional divergence of alleles within this region in relation to environmental adaptation. Overall, this study identifies critical environmental factors shaping cotton yield and fiber quality and reveals multiple G × E–related loci underlying environmental responsiveness and phenotypic plasticity, providing a theoretical foundation for environmentally informed precision breeding.

Ammonium transporters (AMTs) represent a class of proteins within the ammonium transporter domain, which play an important role in mediating the transmembrane transport of NH4+ in plants. However, research on AMT genes in foxtail millet remains limited. In this study, members of the AMT gene family in foxtail millet were identified at the whole genome level through bioinformatic analysis. The gene structure, evolutionary relationships, chromosomal localization, interspecies collinearity, cis-acting elements, and expression patterns of SiAMT members were systematically analyzed. The results revealed that there were nine SiAMT family members in foxtail millet, with molecular weights ranging from 49.5 to 53.8 kDa. Phylogenetic analysis classified them into three groups, which were unevenly distributed across chromosomes. The analysis of promoter cis-acting elements identified multiple regulatory elements, including light-, anaerobic-, and hormone-responsive elements. Collinearity analysis showed that the divergence time of AMT family members in foxtail millet and rice was more recent compared to Arabidopsis thaliana. The expression levels of SiAMT members varied across different tissues of foxtail millet, with most SiAMT family members showing high expression in roots, while SiAMT7 was significantly expressed in leaves. qRT-PCR analysis showed that SiAMT1 was significantly down-regulated in roots, stems, and leaves under salt stress. This study provides a theoretical foundation for further investigation into the functions of the AMT gene family.

Cotton fiber quality-defined by length, strength, and fineness-directly influences the commercial value of textile products, with fiber length being one of the most critical parameters in industrial procurement. In upland cotton (Gossypium hirsutum), fiber development occurs through four overlapping stages, of which the elongation phase (2-20 d post-anthesis, DPA) is pivotal for determining final fiber length. Recent studies have identified a diverse set of genes regulating fiber elongation via distinct molecular mechanisms, categorized into six functional classes: Phytohormone-associated genes; transcription factor-associated genes; cellulose-, lignin-, and sucrose-associated genes; lipid-associated genes; cytoskeleton-associated genes; and other functionally diverse genes. By synthesizing their roles and hierarchical interactions, this review constructs comprehensive genetic networks governing fiber elongation. This work provides a molecular blueprint for precision breeding strategies to enhance cotton fiber length, offering actionable insights for breeding programs aimed at improving fiber quality.

Soil salinization significantly limis crop yield and lowers produce quality. Wild plant species have developed various strategies to cope with soil salinity. However, the molecular responding mechanism to salt stress of domesticated crops is still open to discussion. Combining analysis of quantitative trait locus (QTL) mapping and transcriptome sequencing is an effective approach to identify candidate genes and study their regulation mechanisms of plant salt tolerance. In this study, 294 BC5F3:5 chromosome segment substitution lines (CSSLs), which were constructed by introgressing chromosome segments of Gossypium barbadense Hai1 into G. hirsutum CCRI36 background, were utilized to evaluate relative germination rate (RGR) of seeds and relative survival rate (RSR) of seedlings under NaCl stress. Two salt-tolerant (ST) and salt-sensitive (SS) lines were screened from the CSSLs. With the basis of the previous SSR-based genotyping data, a total of 14 QTLs of RGR and RSR relating to salt tolerance were detected. Meanwhile, RNA-seq and physiological and biochemical indexes of ST and SS were detected. With the combination of weighted gene co-expression network analysis (WGCNA) and QTL intervals, we identified nine hub genes, four of which have nonsynonymous mutations in the protein sequences between G. hirsutum and G. barbadense. The unqiue common candidate gene located between qRGR-12–1 and qRSR-12–1, namely GH_A12G0809, were chosen to conduct functional validation vis VIGS, which confirmed its negative regulatory contribution to salt tolerance in cotton. Our results establish a foundation for elucidating the molecular mechanisms governing cotton's defense against salt stress.

The DUF538 gene family, harboring unknown functional proteins, has been reported to take active roles in plant development and response to adversities, while few studies of genome-wide identification and functional verification have been performed in cotton. Hence, two ancestral diploid species, G. arboretum and G. raimondii, and two cultivated tetraploid ones, G. hirsutum and G. barbadense, were chosen in this study to investigate the cotton DUF538 gene family, resulting in 37, 37, 70, and 70 members identified, respectively. A phylogenetic tree was constructed on these cotton DUF538 genes, together with 22 A. thaliana ones, which were divided into seven groups unevenly distributed across nearly all chromosomes. High-degree conservatism, while rich in diversity, was separately observed in gene structure and conserved motif analyses between the same groups and different groups, and a great number of gene-replication events were detected from intraspecific and interspecific collinearity analyses, implying this was the driving force for DUF538 family expansion. Multiple cis-acting elements relevant to adversity-stress responses were found in the promoter region, which were consistent with the transcriptome expression analyses in response to low-temperature and drought stress and Verticillium wilt infection. Coincidentally, GhDUF538-7 showed the core position in the protein-protein interaction network and was identified in the overlapping region of the interval of four reported VW resistance-related QTLs. The gene function of GhDUF538-7 was verified via gene cloning, relative expression-pattern detection, and virus-induced gene silencing (VIGS) experiment. The TRV:DUF538-7 plants showed more serious VW symptoms, significantly severe disease indices, relatively higher fungal biomass, and increased brown vascular bundles compared with TRV:00 plants. Significantly lower expression levels of marker genes PR4 and MYC2 in jasmonate signaling pathways indicated GhDUF538-7 as a potentially positive regulatory factor in plant defense via hormone signal transduction. This study not only broadened the research perspective of evolution and functional differentiation of the cotton DUF538 gene family, but it also revealed the cooperative relationship between DUF538-7 and the JA pathway for further molecular mechanisms of cotton resistance to VW infection.

Verticillium wilt, primarily caused by Verticillium dahliae, represents a major constraint on both quality and yield in upland cotton (Gossypium hirsutum). Calcium (Ca2+) functions as a pivotal second messenger in plant signal transduction, regulating the expression of stress-induced genes. The Soybean gene Regulated by Cold-2 (SRC2), which encodes a protein containing C2 domains, is known to play important roles in plant development and environmental adaptation. In this study, a total of 31 SRC2 members were identified in five cotton species and classified into five distinct groups. Analyses of gene structure and conserved protein motifs revealed that SRC2 genes are evolutionarily conserved. GhSRC2 genes were widely expressed in various cotton tissues and showed responsiveness to cold, heat, drought and salt stresses. Notably, GhSRC2-3D expression was significantly induced upon V. dahliae infection. Subcellular localization assays indicated that GhSRC2-3D localizes to the cell membrane. Complementation of Arabidopsis src2 mutant with GhSRC2-3D restored resistance to V. dahliae, while a C2 domain deletion variant (ΔGhSRC2-3D) failed to confer resistance. Furthermore, down-regulation of GhSRC2-3D mediated by virus-induced gene silencing (VIGS) compromised V. dahliae resistance in upland cotton. Collectively, our findings demonstrate the conserved role of GhSRC2-3D in plant defense against V. dahliae infection and underscore the essential contribution of its C2 domain to protein function.

Leucine-rich repeat extensins (LRXs) are essential regulators of plant development, cell wall integrity, and stress responses. However, genome-wide LRX studies in cotton are limited. Analysis of four Gossypium species identified 29, 28, 16, and 16 LRX genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis resolved these 89 genes into four subfamilies (I-IV). Structural annotation revealed that cotton LRX family members exhibit conserved domain architectures. This finding was corroborated by motif analysis, which revealed notable conservation in the motif compositions of most cotton LRX proteins, suggesting functional conservation across evolutionary lineages. Distinct spatiotemporal expression patterns were uncovered between G. hirsutum and G. barbadense. Prolonged exposure to extreme temperatures induced widespread down-regulation of most GhLRX genes, whereas genes in subgroup IV were significantly up-regulated under salt and drought stress conditions, respectively. Notably, GhLRX7 showed a more proactive responding profile to Verticillium wilt (VW) infection, which was therefore selected for functional validation employing virus-induced gene silencing in the cotton cultivars MBI9626 and CCRI36. Phenotypic analysis of silenced plants revealed exacerbated disease symptoms compared to wild-type controls, providing direct evidence implicating GhLRX7 as a key contributor to defense against VW.

Gibberellins (GAs) play a crucial regulatory role in the growth and development of cotton (Gossypium hirsutum L.). Through bioinformatics analyses, we identified a total of 39 GA2ox genes (encoding gibberellin 2-oxidases) in the cotton genome, designated GhGA2ox1 to GhGA2ox39. Based on phylogenetic analysis, these genes were classified into five groups. We further examined their gene structures, conserved motifs, and chromosomal distributions, revealing that members within the same group shared similar structural and motif organizations. Collinearity and cis-element analyses provided important insights into the evolutionary history and regulatory potential of the GA2ox gene family in cotton. Notably, using nucleotide diversity (π) and population differentiation (FST) analyses across the entire family, we screened and identified nine candidate genes that underwent strong artificial selection during cotton domestication and improvement. Further haplotype-phenotype association analysis identified GH_D09G0919 (GhGA2ox31) as a key regulator of Plant Height (PH). To validate their regulatory roles, we analyzed the genotype distribution in accessions with extreme phenotypes. The results revealed divergent selection histories for these two loci: the favorable allele of GH_D01G0720 (GhGA2ox23) was already fixed in the tested population, whereas GH_D09G0919 maintained significant natural variation. Specifically, the Hap2 allele of GH_D09G0919 was significantly enriched in the shortest accessions compared to the tallest ones. Importantly, quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed that the Hap2 allele drives significantly higher gene expression in leaves, suggesting that enhanced GA catabolism underlies the compact phenotype. Additionally, transcriptomic profiling revealed the tissue-specific expression patterns of candidate genes, implying their functional roles in development. Furthermore, functional validation using the Arabidopsis mutant of the homologous gene (AtGA2ox8) confirmed its conserved role in regulating plant height, as the mutant exhibited a distinct short-stature phenotype. These results uncover valuable genetic resources for molecular breeding to shape compact cotton architecture. Collectively, this study aims to analyze the evolutionary patterns of the cotton GA2ox gene family and to identify key genes that regulate plant height under artificial selection, providing theoretical support for molecular breeding of compact plant types.

Verticillium wilt (VW) is a soil-borne fungal plant disease. Gossypium hirsutum varieties with the widest planting area are highly susceptible to VW pathogens, because their narrow genetic background of germplasm resources causes difficulties in cultivating VW-resistant varieties through intraspecific breeding. Therefore, G. barbadense cultivars, harboring a natural VW resistance, become ideal donor materials to cultivate high-yield and multi-resistance chromosome segment substitution lines (CSSLs) through hybridization and backcrossing with G. hirsutum receptor and recurrent parent. In order to investigate the molecular mechanism of cotton response to VW infection, a BC5F3:5 CSSL MBI9626 and its parents, CCRI36 (G. hirsutum) and Hai1 (G. barbadense), were chosen to perform transcriptome and metabolome sequencing on their root samples at 0, 7, and 15 days after inoculation (DAI) of V. dahliae V991. In total, 36,564 differentially expressed genes (DEGs) and 102 differentially accumulated metabolites (DAMs) were separately identified from 12 pairwise comparison groups among the 27 samples. Of those, 125 common DEGs were found to participate in the biological processes of oxylipin metabolism, jasmonic acid (JA) biosynthesis/metabolism, and response to wounding in Gene Ontology (GO) enrichment analyses, while most of the DAMs were significantly enriched in tyrosine, purine, and phenylalanine metabolism pathways in enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG). Having performed a conjoint KEGG analysis of all the DEGs and DAMs, we found two commonly enriched pathways, namely plant hormone signal transduction and flavonoid biosynthesis, which were consistent with the enrichment annotations of the significant model in weighted gene co-expression network analysis on the 2091 DEGs identified by an intersection of the genes in 40 previous QTLs and the total DEGs of this RNA-seq data. Among the ABA signaling pathway, the gene GH_D12G0236 (GHABF3) was selected to be used to perform virus-induced gene silencing (VIGS) verification in CCRI36 and MBI9626, and GHABF3-silenced plants showed a more serious wilting phenotype, an increased disease index (DI), and higher accumulation of fungal biomass compared to their empty-vector plants. These results provide a high-efficiency strategy for screening vital genes affecting cotton VW resistance, and lay a solid foundation for further cotton molecular breeding.

Cotton is a crucial cash crop widely valued for its fiber. It is an important source of natural fiber and has diverse applications. Improving fiber quality is of significant economic and agricultural importance. Purple acid phosphatases (PAPs) are multifunctional enzymes critical for plant cell wall biosynthesis, root architecture modulation, low-phosphorus stress adaptation, and salt/ROS stress tolerance. In this study, a comprehensive genome-wide analysis of the PAP gene family was performed for four cotton species (G. hirsutum, G. barbadense, G. raimondii, and G. arboreum) to explore its potential role in improving fiber quality. A total of 193 PAP genes were identified in these species, revealing several conserved domains that contribute to their functional diversity. Phylogenetic analysis showed that the cotton PAP2 genes exhibited high homology with NtPAP12, a cell wall synthesis-related gene. Using cotton varieties with contrasting fiber thickness (EZ60, micronaire 4.5 vs. CCRI127, micronaire 3.5), qRT-PCR analysis demonstrated significantly higher expression levels of GhPAP2.2, GhPAP2.6, GhPAP2.8, and GhPAP2.9 in EZ60 fibers during 20–25 DPA compared to CCRI127. These results highlight the potential influence of PAP genes on cotton fiber development and provide valuable insights for improving fiber quality in cotton breeding.

Coenzyme Q (CoQ) was an electron carrier within the mitochondrial respiratory chain, serves as a cofactor for various mitochondrial dehydrogenases thereby playing a significant role in plant growth and development. In this study, GhCoQ9 was identified through transcriptomic analysis of cotton under salt stress. GhCoQ9 participates in the synthesis of coenzyme Q. Under salt stress condition, cotton plants with GhCoQ9 silenced exhibited more pronounced growth inhibition compared to the control group. Additionally, the catalase (CAT) and superoxide dismutase (SOD) levels were significantly increased, while the level of malondialdehyde (MDA) showed an upward trend. Microscopic analysis of cotton leaves revealed that under salt stress conditions GhCoQ9 -silenced plants exhibited reduced stomatal aperture, cellular destruction, cell shrinkage and deformation, and increased intercellular space compared to the control plants. The ultrastructure of GhCoQ9-silenced plants exhibited increased susceptibility to salt stress, primarily manifested as damage to the chloroplast and mitochondrial structures. We also investigated the function of CoQ9 in Arabidopsis, the results showed that coq9 mutant exhibited reduced germination rate and significant inhibited growth under salt stress. These findings suggest that GhCoQ9 plays a crucial role in the adaptation of cotton to salt stress by preserving the integrity of chloroplast and mitochondrial structures.

Cottonseed is a globally significant oilseed crop due to its high contribution to vegetable oil supply. The process of lipid accumulation is essential for seed maturation and oil buildup. In this study, we analyzed lipid metabolites and gene expression patterns related to fatty acid synthesis in two cotton genotypes with varying oil content. Our lipid analysis identified 588 kinds of lipids in developing embryos of upland cotton, with glycerophospholipids (64.29 %), glycerolipids (17.69 %), and saccharolipids (13.61 %) being the main components. Transcriptome analysis of key genes involved in fatty acid biosynthesis and lipid droplet formation revealed potential regulatory regions influencing lipid content in developing embryos of upland cotton. This research provides valuable insights into the lipidome profiles during embryo development and lays the groundwork for future investigations on lipid accumulation in economically important crops.

Understanding the influence of environmental factors on cotton performance is crucial for enhancing yield and fiber quality in the context of climate change. This study investigates genotype-by-environment (G×E) interactions in cotton, using data from 250 recombinant inbred lines (CCRI70 RILs) cultivated across 14 diverse environments in China’s major cotton cultivation areas. Our findings reveal that environmental effects predominantly influenced yield-related traits (boll weight, lint percentage, and the seed index), contributing to 34.7% to 55.7% of their variance. In contrast fiber quality traits showed lower environmental sensitivity (12.3–27.0%), with notable phenotypic plasticity observed in the boll weight, lint percentage, and fiber micronaire. Employing six machine learning models, Random Forest demonstrated superior predictive ability (R2 = 0.40–0.72; predictive Pearson correlation = 0.63–0.86). Through SHAP-based interpretation and sliding-window regression, we identified key environmental drivers primarily active during mid-to-late growth stages. This approach effectively reduced the number of influential input variables to just 0.1–2.4% of the original dataset, spanning 2–9 critical time windows per trait. Incorporating these identified drivers significantly improved cross-environment predictions, enhancing Random Forest accuracy by 0.02–0.15. These results underscore the strong potential of machine learning to uncover critical temporal environmental factors underlying G×E interactions and to substantially improve predictive modeling in cotton breeding programs, ultimately contributing to more resilient and productive cotton cultivation.

Arabinogalactan proteins (AGPs) constitute a diverse class of hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. However, their functional characterization in cotton (Gossypium spp.) remains limited. As a globally significant economic crop, cotton serves as the primary source of natural fiber, making it essential to understand the genetic mechanisms underlying its growth and development. This study aims to perform a comprehensive genome-wide identification and characterization of the AGP gene family in Gossypium spp., with a particular focus on elucidating their structural features, evolutionary relationships, and functional roles. A genome-wide analysis was conducted to identify AGP genes in Gossypium spp., followed by classification into distinct subfamilies based on sequence characteristics. Protein motif composition, gene structure, and phylogenetic relationships were examined to infer potential functional diversification. Subcellular localization of a key candidate gene, GhAGP50, was determined using fluorescent protein tagging, while gene expression patterns were assessed through β-glucuronidase (GUS) reporter assays. Additionally, hormonal regulation of GhAGP50 was investigated via treatments with methyl jasmonate (MeJA), abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA). A total of 220 AGP genes were identified in Gossypium spp., comprising 19 classical AGPs, 28 lysine-rich AGPs, 55 AG peptides, and 118 fasciclin-like AGPs (FLAs). Structural and functional analyses revealed significant variation in gene organization and conserved motifs across subfamilies. Functional characterization of GhAGP50, an ortholog of AGP18 in Arabidopsis thaliana, demonstrated its role in promoting epidermal hair formation in leaves and stalks. Subcellular localization studies indicated that GhAGP50 is targeted to the nucleus and plasma membrane. GUS staining assays revealed broad expression across multiple tissues, including leaves, inflorescences, roots, and stems. Furthermore, hormonal treatment experiments showed that GhAGP50 expression is modulated by MeJA, ABA, IAA, and GA, suggesting its involvement in hormone-mediated developmental processes. This study presents a comprehensive genome-wide analysis of the AGP gene family in cotton, providing new insights into their structural diversity and functional significance. The identification and characterization of GhAGP50 highlight its potential role in epidermal hair formation and hormonal regulation, contributing to a deeper understanding of AGP functions in cotton development. These findings offer a valuable genetic resource for future research aimed at improving cotton growth and fiber quality through targeted genetic manipulation.

Cottonseed oil is rich in unsaturated fatty acids (UFAs), making it suitable for use as edible oil. Fatty acid desaturases (FADs) play a major role in the conversion of monounsaturated fatty acids (MUFAs) to polyunsaturated fatty acids (PUFAs). In total, 39 GhFAD genes were detected in upland cotton and divided into five groups in the present study. Gene structure and domain analysis showed that GhFAD members within each group were highly conserved. Cis-elements associated with environmental stress and hormone responses were identified in GhFAD promoters. The predicted transcription factors and miRNAs targeting these genes suggest extensive roles for GhFADs in diverse stress conditions. Analysis of expression profiles indicated that GhFAD genes participate extensively in developmental processes and stress adaptation in cotton. Among these, the concurrent high expression of GhFAD2-1 and low expression of GhFAD3 are consistent with the typical fatty acid profile of cottonseed oil. GhFAD3-2 and GhFAD3-1 exhibit a complementary expression profiles, suggesting they may operate in a relay manner during fiber development. Additionally, experimental evidence established that GhFAD2-3 is involved in the cold stress response. This research delivers a thorough characterization of the GhFAD genes in upland cotton, thereby establishing a solid groundwork for future functional genomics studies.

We identified two splicing variants of GhLSM1B (GhLSM1BS and GhLSM1BL) with distinct expression patterns and predicted 3D structures, despite sharing the same nuclear localization. Overexpression of GhLSM1BS, but not GhLSM1BL, accelerated cotton callus proliferation and altered cell morphology during somatic embryogenesis, accompanied by altered expression of CYP450 family genes and elevated brassinosteroid levels.

Verticillium wilt (VW) severely limits the cotton yield and fiber quality. Marker-assisted selection is an efficient strategy for breeding resistant varieties. In this study, a high-density genetic map was constructed by using an F8:9 recombinant inbred line (RIL) population derived from CCRI70. Phenotypic data on disease incidence (DINC) and disease index (DI) were collected across six environments. A total of 59 QTLs for DINC and 60 QTLs for DI were identified, with three and six stable across multiple environments, respectively. These QTLs formed 18 clusters across 13 chromosomes, showing consistent additive effects. Transcriptome analysis revealed eight differentially expressed candidate genes within stable QTL regions. Among them, GH_D05G1495, GH_A09G1013, and GH_D05G1683 were further validated by virus-induced gene silencing as key genes conferring V. dahliae resistance in cotton. This study provides valuable genetic resources for improving Verticillium wilt resistance in cotton breeding.

Cotton is a widely grown crop to produce natural textile fiber, and improving the fiber strength (FS) is one of the main targets that cotton breeders focus on. The long-term natural selection and domestication have produced abundant germplasm resources of Gossypium hirsutum, and exploring genetic underpinnings underlying these FS innovation in elite collections is crucial. PCAMP is proposed as a most optimized NGS based bulked segregant analysis (NGS-BSA) for the high-resolution identification of markers linked to specific genomic regions through pairwise comparing multiple BSA bulks. In this study, we firstly applied PCAMP to resolved the FS genetic architecture in G. hirsutum cv. CCRI127. As an extension for PCAMP approach, graded bulks were constructed using F-2 segregants with the FS phenotype revalidated by F-2:3 lines, and then, a major QTL was eventually narrowed to 2.47 Mb from 8.14 Mb generated by traditional BSA approaches. Subsequently, through a saturated genetic map constructed in this locus, an novel FS gene, GhCKX1, predicted to produce a cytokinin (CTK) oxidase was isolated. It can negatively modulate the CTK signaling circuit via irreversible degradation of CTKs, resulting in an additional cell wall thickness to xylem tracheary elements in transgenic lines of Arabidopsis thaliana. Thus, the GhCKX1 gene will be an potential genetic target, with which, we can genetically manipulate the secondary wall synthesis in unicellular cotton fibers.

Background Epidermal patterning factor / -like (EPF/EPFL) gene family encodes a class of cysteine-rich secretory peptides, which are widelyfound in terrestrial plants.Multiple studies has indicated that EPF/EPFLs might play significant roles in coordinating plant development and growth, especially as the morphogenesis processes of stoma, awn, stamen, and fruit skin. However, few research on EPF/EPFL gene family was reported in Gossypium. Results We separately identified 20 G. raimondii, 24 G. arboreum, 44 G. hirsutum, and 44 G. barbadense EPF/EPFL genes in the 4 representative cotton species, which were divided into four clades together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 17 Selaginella moellendorffii ones based on their evolutionary relationships. The similar gene structure and common motifs indicated the high conservation among the EPF/EPFL members, while the uneven distribution in chromosomes implied the variability during the long-term evolutionary process. Hundreds of collinearity relationships were identified from the pairwise comparisons of intraspecifc and interspecific genomes, which illustrated gene duplication might contribute to the expansion of cotton EPF/EPFL gene family. A total of 15 kinds of cis-regulatory elements were predicted in the promoter regions, and divided into three major categories relevant to the biological processes of development and growth, plant hormone response, and abiotic stress response. Having performing the expression pattern analyses with the basic of the published RNA-seq data, we found most of GhEPF/EPFL and GbEPF/EPFL genes presented the relatively low expression levels among the 9 tissues or organs, while showed more dramatically different responses to high/low temperature and salt or drought stresses. Combined with transcriptome data of developing ovules and fibers and quantitative Real-time PCR results (qRT-PCR) of 15 highly expressed GhEPF/EPFL genes, it could be deduced that the cotton EPF/EPFL genes were closely related with fiber development. Additionally, the networks of protein-protein interacting among EPF/EPFLs concentrated on the cores of GhEPF1 and GhEPF7, and thosefunctional enrichment analyses indicated that most of EPF/EPFLs participate in the GO (Gene Ontology) terms of stomatal development and plant epidermis development, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DNA or base excision repair. Conclusion Totally, 132 EPF/EPFL genes were identified for the first time in cotton, whose bioinformatic analyses of cis-regulatory elements and expression patterns combined with qRT-PCR experiments to prove the potential functions in the biological processes of plant growth and responding to abiotic stresses, specifically in the fiber development. These results not only provide comprehensive and valuable information for cotton EPF/EPFL gene family, but also lay solid foundation for screening candidate EPF/EPFL genes in further cotton breeding.