Salt tolerance during germination is a complex trait involving coordinated regulation of multiple developmental processes, yet its shared genetic architecture remains largely unknown. Here, we integrated genome-wide association study (GWAS) and multi-trait GWAS (MTAG) to dissect the pleiotropic basis of salt tolerance in 355 upland cotton accessions by using 2.52 million high-quality SNPs. A total of 517 and 534 significantly associated SNPs were identified by GWAS and MTAG, respectively, revealing 79 salt-tolerance-related QTLs. A major locus, qRST-D08-8, was refined to a 73.8 kb interval containing 12 candidate genes, among which GhWRKY21-D08 (Ghir_D08G020650) was supported by expression profiling and functional assays. Virus-induced gene silencing (VIGS) indicated that GhWRKY21-D08 enhances salt tolerance in cotton by increasing antioxidant enzyme activities to scavenge reactive oxygen species (ROS). Notably, a cis-regulatory SNP (-718 C/A) in the promoter region significantly altered transcriptional activity, linking regulatory variation to phenotypic divergence. Population genetic analyses suggest that the favorable haplotype (Hap1) has undergone recent positive selection during breeding without compromising yield-related traits. These findings highlight the role of cis-regulatory variation in shaping adaptive traits and provide a valuable target for molecular breeding of salt-tolerant cotton.

Improving lint percentage (LP) through genetic breeding is crucial for stabilizing cotton fiber yield. To dissect the genetic basis of LP, a recombinant inbred line (RIL) population consisting of 300 lines were derived from a cross between the high-LP cultivar ‘CRI50’ and the low-LP standard line ‘TM-1’. Multi-environment phenotypic investigations revealed that LP showed a continuous distribution with high heritability (H²=81.65%), indicating complex genetic control with strong genetic determination. Bulked segregant RNA-seq (BSR-seq) analysis of extreme LP pools identified a major quantitative trait locus (QTL), qLP-D03-1, on chromosome D03. Further GWAS and QTL hotspot analysis narrowed the candidate region to approximately 250 kb on chromosome D03. Within this region, GhLPA1 (Ghicr24_D03G124100), encoding a ribosomal protein with extraribosomal functions, was identified as the key candidate gene, and its function was confirmed by virus-induced gene silencing (VIGS) analysis. Haplotype analysis showed that the ‘CRI50’-type allele (Hap1) conferred significantly higher LP than the ‘TM-1’-type allele (Hap2). Furthermore, a kompetitive allele-specific PCR (KASP) marker developed from a functional SNP in the 3′ untranslated region (UTR) of GhLPA1 effectively differentiated genotypes with contrasting LP, which providing a valuable marker for marker-assisted selection (MAS) in cotton breeding.

Cotton (Gossypium spp.), an economically and strategically significant crop in China, faces challenges such as rising cultivation costs and conflicts between grain and cotton cultivation. These challenges underscore the need for enhancing yields per unit area. In response, this study employs deep learning techniques, combined with high-throughput angle detection, to conduct genome-wide association studies (GWAS) on 355 upland cotton accessions, identify key SNPs and candidate genes for plant architecture by fruit branch angle (FBA) influences planting density, yield, and mechanized harvesting. A convolutional neural network (CNN)-based software was developed for rapid and accurate branch angle detection, showing high correlation with both AutoCAD and manual measurements. Significant phenotypic variation in FBA was observed across various cotton planting regions, with the Northwest Inland Region (NIR) exhibiting notably smaller angles. In total, 107 significant Single Nucleotide Polymorphisms (SNPs) were detected across 45 quantitative trait loci (QTL), and three potential candidate genes (Ghir_A11G034910, Ghir_D05G007790, and Ghir_D05G031350) were identified, providing insights into the genetic basis of FBA and presenting valuable genetic resources for cotton breeding programs.

BACKGROUND:Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol.RESULTS:In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect.CONCLUSIONS:In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

Soil salinization is one of the abiotic stresses that inhibit plant growth and development, which seriously restricts global crop production. WRKY transcription factors play an important role in regulating plant responses to stress such as salt stress. In our previous study, two WRKY family genes from okra, AeWRKY32 and AeWRKY70, were significantly up-regulated and down-regulated, respectively, in response to salt stress. In this study, subcellular localization showed that they were localized to the nucleus. The down-regulation of AeWRKY32 and AeWRKY70 via whole plant virus-induced gene silencing (VIGS) increased and decreased plant sensitivity to salt stress, respectively. Ectopic expression of AeWRKY32 and AeWRKY70 led to promoted and reduced salt tolerance in transgenic Arabidopsis, respectively. There was no significant difference between transgenic plants and wild type (WT) without salt treatment. Salt stress significantly inhibited plant growth. The decrease of chlorophyll content and the increase of anthocyanin content in AeWRKY32-overexpressed transgenic plants were lower than those in the WT, while AeWRKY70-overexpressed plants had the opposite effect. Under salt stress, the AeWRKY70-overexpressed plants had the highest malondialdehyde (MDA) content, followed by the WT, and the lowest in AeWRKY32-overexpressed plants. The hydrogen peroxide (H2O2) content and superoxide anion (O2•−) generation rate were only slightly increased. Moreover, salt stress significantly increased plant proline content and antioxidant enzyme activities, which was highest in AeWRKY70-overexpressed plants except superoxide dismutase (SOD). Taken together, these results suggest that AeWRKY32 and AeWRKY70 play positive and negative roles in plant in response to salt stress, respectively.

Transcription Factors (TFs) are key regulators of how plants grow and develop. Among the diverse TF families, the Glabrous-enhancer binding protein (GeBP) family plays a key role in trichome initiation and leaf development. The specific roles of GeBP TFs in plants remain largely unexplored, although GeBP transcription factors play important roles in plants. This study identified 16 GhGeBP genes in Gossypium hirsutum, ranging from 534 bp (GhGeBP14) to 1560 bp (GhGeBP2). Phylogenetic analysis grouped 16 GhGeBP genes clustered into three subgroups, unevenly distributed across 14 chromosomes. Analysis of the cis-acting elements revealed 408 motifs in the 2 kb upstream regions of the promoters, including stress-responsive, phytohormone-responsive, and light-responsive elements. Tissue-specific expression analysis showed 8 GhGeBP genes were highly expressed across all tissues, while GhGeBP4 and GhGeBP12 were down-regulated under conditions of drought, salt, cold, and heat stress. A genome-wide association study (GWAS) identified GhGeBP4 was associated with fiber micronaire (FM) and fiber strength (FS), while GhGeBP9 was linked to the node of the first fruiting branch (NFFB) and flowering time (FT). Haplotype analysis revealed that GhGeBP4-HAP2 exhibited higher fiber quality traits, while GhGeBP9-HAP2 was associated with early maturity. The results of this study offer significant insights that are worthy of further investigation into the role of the GhGeBP gene family in G. hirsutum and promising targets for marker-assisted selection strategies in cotton breeding programs, particularly for improving fiber quality and early maturity traits.

MYB is one of the largest transcription factor families in plants. Among them, the R3-MYB transcription factor RADIALIS (RAD) plays a very important role in the flowers development in Antirrhinum majus. In this study, a R3-MYB gene similar to RAD was found by analyzing the genome of A. majus, which was named AmRADIALIS-like 1 (AmRADL1). The gene function was predicted through bioinformatics. The relative expression levels in different tissues and organs of wild-type A. majus were analyzed by qRT-PCR. AmRADL1 was overexpressed in A. majus, and the transgenic plants were analyzed by morphological observation and histological staining. The results showed that the open reading frame (ORF) of AmRADL1 gene was 306 bp in length, encoding 101 amino acids. It has typical SANT domain, and the C-terminal contains a CREB motif, which was highly homologous to tomato SlFSM1. The results of qRT-PCR showed that AmRADL1 was expressed in roots, stems, leaves and flowers, and the expression level was higher in flowers. Further analysis of its expression in different floral organs showed that AmRADL1 had the highest expression in carpel. The results of histological staining analysis of the transgenic plants showed that compared with the wild type, although the size of the carpel cells of the transgenic plants did not change significantly, the placenta area in the carpel became smaller and the number of cell decreased. In summary, AmRADL1 may be involved in the regulation of carpel development, but the specific mechanism of action in carpel remains to be further studied.

为了建立以草甘膦为选择标记的农杆菌介导的棉花胚性愈伤组织转化体系,本研究将草甘膦抗性基因(aroA)转化陆地棉WC和YZ-1的胚性愈伤组织,分别以5 mg· L-1和10 mg·L-1的草甘膦浓度作为选择压,经过2～3轮筛选,获得了抗性胚性愈伤组织,诱导出胚状体并发育成苗.对T0再生植株进行PCR及草甘膦抗性检测,以WC和YZ-1为受体的转抗草甘膦基因(aroA)分别获得了33株和10株阳性植株,转化效率分别为82.5％和62.5％.该体系为棉花遗传转化提供了实验方法,获得的抗草甘膦棉花为棉花育种提供种质资源;同时抗除草剂基因还可以作为筛选标记与其它基因串联在一起转化棉花,进行基因功能验证.

Background In plants, sucrose synthase (Sus) is widely considered as a key enzyme involved in sucrose metabolism. Several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, while limited information of Sus genes is available to date for cotton. Results Here, we report the molecular cloning, structural organization, phylogenetic evolution and expression profiles of seven Sus genes ( GaSus1 to 7) identified from diploid fiber cotton ( Gossypium arboreum ). Comparisons between cDNA and genomic sequences revealed that the cotton GaSus genes were interrupted by multiple introns. Comparative screening of introns in homologous genes demonstrated that the number and position of Sus introns are highly conserved among Sus genes in cotton and other more distantly related plant species. Phylogenetic analysis showed that GaSus1 , GaSus2 , GaSus3 , GaSus4 and GaSus5 could be clustered together into a dicot Sus group, while GaSus6 and GaSus7 were separated evenly into other two groups, with members from both dicot and monocot species. Expression profiles analyses of the seven Sus genes indicated that except GaSus2 , of which the transcripts was undetectable in all tissues examined, and GaSus7 , which was only expressed in stem and petal, the other five paralogues were differentially expressed in a wide ranges of tissues, and showed development-dependent expression profiles in cotton fiber cells. Conclusions This is a comprehensive study of the Sus gene family in cotton plant. The results presented in this work provide new insights into the evolutionary conservation and sub-functional divergence of the cotton Sus gene family in response to cotton fiber growth and development.

A silkworm gene for fibroin was introduced into the upland cotton WC line by Agrobacterium-mediated transformation. PCR detection for fibroin, nptII and gus genes, Kanamycin (Km)-resistance analysis and GUS-histochemical assay were conducted on 30 regenerated plants from 9 callus lines, and 17 positive plants were obtained by these 5 screening methods. By Km-resistance analysis and PCR for fibroin, 6 homozygous lines in T3 were obtained. Southern blot and Northern bolt demonstrated that the fibroin gene was inserted into the genome of these 6 lines, stably inherited and expressed. Compared to the control, the surface structure of mature fiber in the 6 lines was significantly distorted and an increased number of convolution was observed by scanning electron microscopy (SEM). Fiber quality traits analysis indicated that fiber elongation of the 6 homozygous lines was all increased and fiber strength of 3 lines was enhanced. These results indicated that fibroin expression influenced cotton fiber structure and quality, suggesting that fibroin has great potential for improving cotton fiber quality by genetic engineering.

Cotton fiber elongation, largely achieved by cell wall loosening, is an important stage during cotton fiber development. In this present research, a fiber preferential cDNA encoding a pectate lyase (PEL) which could exclusively degrade the de-esterified pectin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. Subsequently, the corresponding PEL genes were isolated from four different cotton species and characterized. In vitro enzyme assays indicated that GhPEL really exhibited cleavage-activity against de-esterified pectin. The temporal-spatial expression analyses revealed that the GhPEL gene was preferentially expressed in fibers at 10 days-post anthesis (DPA). Antisense GhPEL transgenic cotton plants were generated by Agrobacterium-mediated transformation. Six homozygous lines, each with one or two copies of the transgene inserted as determined by southern blot analysis of the NPTII gene, were selected for further functional analysis. The GhPEL expression during fiber elongation in these transgenic lines was significantly suppressed in various degrees. Furthermore, the reduction of GhPEL enzymatic activity by decreasing GhPEL transcripts severely affected the degradation of de-esterified pectin in primary cell walls of transgenic cotton fibers, which consequently blocked cell wall loosening in early fiber development. Ultimately, the fiber elongation of all these transgenic lines was repressed. These results suggested that GhPEL may play an important role in the process of normal fiber elongation in cotton.

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin , and fibroin . The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T 1 plants demonstrated that multiple transgenes were inherited and expressed in progenies.

Ten genes expressed preferentially in fiber development period reported previously and 14 cotton cultivars (strains) with distinctly different fiber quality were selected in this paper. To test the relative expression values of the genes in six different fiber developmental stages, 0 day post anthesis (DPA), 5DPA, 10DPA, 15DPA, 20DPA, 23DPA, by real-time quantitative RT-PCR (qRT-PCR), and the data of fiber qualities from 14 cotton varieties (strains). The expression profile showed that GhExp1, GhCIPK1, GhSus1, GhSusA1, and GhPL genes were expressed preferentially during fiber elongation; GhACT1, GhRacA, and GhRacB genes all had high expression level in earlier stage of fiber elongation and the thickening period of secondary cell wall. Two cellulose synthase genes (GhCelA1 and GhCelA3) were expressed predominantly during late stage of fiber elongation and the thickening period of secondary cell wall. For most genes, the expression value in low expression level period had significant correlation with fiber quality, while no significant correlation was detected in preferential expression stage of these genes with an exception of GhRacA gene. The expression level of GhExp1 in 20DPA fiber of 14 cotton varieties (strains) had a significant negative correlation with fiber strength and uniformity and a significant positive correlation with fiber elongation percentage; the expression level of GhPL gene in 23DPA had a significant negative correlation with fiber length; the expression level of GhRacA gene in 5DPA and 23DPA both had a high significant positive correlation with fiber elongation percentage; the expression level of GhRacB gene in 10DPA had a significant negative correlation with fiber length and uniformity; the expression level of GhCelA1 gene in 5DPA had a significant positive correlation with fiber length and a significant negative correlation with micronaire value; the expression level of GhCelA1 gene in 10DPA had a significant negative correlation with fiber length, a significant positive cor-relation with micronaire value, and a high significant correlation with fiber elongation percentage; the correlation between the expression levels of GhCIPK1, GhACT1, GhSus1, GhSusA1and GhCelA3 gene and fiber quality indexes had not been detected.

Agrobacterium tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. The binary vector pBI121 (containing a neomycin phosphotransferase II gene npt-II as a selection marker and a uidA gene as a reporter gene) was used to research transformation efficiency. After 48 h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse granule EC. It indicated that the efficiency of transient transformation was affected by EC morphology. Transient transformation efficiency also was improved by cocultivation on the medium by adding 50 mg/L acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density increased the production of kanamycin-resistant (Km-R) calli lines. From an original 0.3 g EC, an average of 20 Km-R calli lines were obtained from a selection dish, and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments, and the GUS-positive rate of regeneration plants was about 72.6%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and gus gene. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, was integrated into the cotton genome.

泗棉3号农艺性状优良,是20世纪90年代长江流域棉区的主栽品种,利用基因工程导入外源基因进行直接改良,可以迅速获得新的品种或育种材料。以陆地棉泗棉3号的下胚轴为外植体,建立了高效的转化体系,得到了大量的转基因植株。泗棉3号的出愈率和分化率显著高于模式品种Coker312,同时它出现分化中心的主要形态不同于Coker312,其胚性愈伤组织主要来源于两类初生愈伤组织。对泗棉3号的胚性愈伤组织进行GUS检测,以及对再生植株叶片进行PCR检测后,阳性植株嫁接于温室。在温室用2063.98μmolL-1的卡那霉素点涂叶片检测nptII基因的表达和取叶片进行gus基因的组织化学检测,同时通过Southernblot分析检测,证明目的基因成功地整合到泗棉3号基因组中。