SR45a plays a key role in enhancing cotton resistance to Verticillium dahliae by alternative splicing of immunity genes.

Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.

General Regulatory Factor7 regulates innate immune signalling to enhance Verticillium wilt resistance in cotton.

Sessile growing plants are always vulnerable to microbial pathogen attacks throughout their lives. To fend off pathogen invasion, plants have evolved a sophisticated innate immune system that consists of cell surface receptors and intracellular receptors. Somatic embryogenesis receptor kinases (SERKs) belong to a small group of leucine-rich repeat receptor-like kinases (LRR-RLKs) that function as co-receptors regulating diverse physiological processes. GENRAL REGULATORY FACTOR (GRF) proteins play an important role in physiological signalling transduction. However, the function of GRF proteins in plant innate immune signalling remains elusive. Here, we identified a GRF gene, GauGRF7, that is expressed both constitutively and in response to fungal pathogen infection. Intriguingly, silencing of GRF7 compromised plant innate immunity, resulting in susceptibility to Verticillium dahliae infection. Both transgenic GauGRF7 cotton and transgenic GauGRF7 Arabidopsis lines enhanced the innate immune response to V. dahliae infection, leading to high expression of two helper NLRs (hNLR) genes (ADR1 and NRG1) and pathogenesis-related genes, and increased ROS production and salicylic acid level. Moreover, GauGRF7 interacted with GhSERK1, which positively regulated GRF7-mediated innate immune response in cotton and Arabidopsis. Our findings revealed the molecular mechanism of the GRF protein in plant immune signaling and offer potential opportunities for improving plant resistance to V. dahliae infection.

Long noncoding RNA TRABA suppresses ß-glucosidase-encoding BGLU24 to promote salt tolerance in cotton.

Salt stress severely damages the growth and yield of crops. Recently, long noncoding RNAs (lncRNAs) were demonstrated to regulate various biological processes and responses to environmental stresses. However, the regulatory mechanisms of lncRNAs in cotton (Gossypium hirsutum) response to salt stress are still poorly understood. Here, we observed that a lncRNA, trans acting of BGLU24 by lncRNA (TRABA), was highly expressed while GhBGLU24-A was weakly expressed in a salt-tolerant cotton accession (DM37) compared to a salt-sensitive accession (TM-1). Using TRABA as an effector and proGhBGLU24-A-driven GUS as a reporter, we showed that TRABA suppressed GhBGLU24-A promoter activity in double transgenic Arabidopsis (Arabidopsis thaliana), which explained why GhBGLU24-A was weakly expressed in the salt-tolerant accession compared to the salt-sensitive accession. GhBGLU24-A encodes an endoplasmic reticulum (ER)-localized ß-glucosidase that responds to salt stress. Further investigation revealed that GhBGLU24-A interacted with RING-type E3 ubiquitin ligase (GhRUBL). Virus-induced gene silencing (VIGS) and transgenic Arabidopsis studies revealed that both GhBGLU24-A and GhRUBL diminish plant tolerance to salt stress and ER stress. Based on its substantial effect on ER-related degradation (ERAD)-associated gene expression, GhBGLU24-A mediates ER stress likely through the ERAD pathway. These findings provide insights into the regulatory role of the lncRNA TRABA in modulating salt and ER stresses in cotton and have potential implications for developing more resilient crops.

Genetic dissection of lint percentage in short-season cotton using combined QTL mapping and RNA-seq.

KEY MESSAGE: In total, 17 QTLs for lint percentage in short-season cotton, including three stable QTLs, were detected. Twenty-eight differentially expressed genes located within the stable QTLs were identified, and two genes were validated by qRT-PCR. The breeding and use of short-season cotton have significant values in addressing the question of occupying farmlands with either cotton or cereals. However, the fiber yields of short-season cotton varieties are significantly lower than those of middle- and late-maturing varieties. How to effectively improve the fiber yield of short-season cotton has become a focus of cotton research. Here, a high-density genetic map was constructed using genome resequencing and an RIL population generated from the hybridization of two short-season cotton accessions, Dong3 and Dong4. The map contained 4960 bin markers across the 26 cotton chromosomes and spanned 3971.08 cM, with an average distance of 0.80 cM between adjacent markers. Based on the genetic map, quantitative trait locus (QTL) mapping for lint percentage (LP, %), an important yield component trait, was performed. In total, 17 QTLs for LP, including three stable QTLs, qLP-A02, qLP-D04, and qLP-D12, were detected. Three out of 11 non-redundant QTLs overlapped with previously reported QTLs, whereas the other eight were novel QTLs. A total of 28 differentially expressed genes associated with the three stable QTLs were identified using RNA-seq of ovules and fibers at different seed developmental stages from the parental materials. The two genes, Ghir_A02G017640 and Ghir_A02G018500, may be related to LP as determined by further qRT-PCR validation. This study provides useful information for the genetic dissection of LP and promotes the molecular breeding of short-season cotton.

GbCYP72A1 Improves Resistance to Verticillium Wilt via Multiple Signaling Pathways.

Verticillium dahliae is a fungal pathogen that causes Verticillium wilt (VW), which seriously reduces the yield of cotton owing to biological stress. The mechanism underlying the resistance of cotton to VW is highly complex, and the resistance breeding of cotton is consequently limited by the lack of in-depth research. Using quantitative trait loci (QTL) mapping, we previously identified a novel cytochrome P450 (CYP) gene on chromosome D4 of Gossypium barbadense that is associated with resistance to the nondefoliated strain of V. dahliae. In this study, the CYP gene on chromosome D4 was cloned together with its homologous gene on chromosome A4 and were denoted as GbCYP72A1d and GbCYP72A1a, respectively, according to their genomic location and protein subfamily classification. The two GbCYP72A1 genes were induced by V. dahliae and phytohormone treatment, and the findings revealed that the VW resistance of the lines with silenced GbCYP72A1 genes decreased significantly. Transcriptome sequencing and pathway enrichment analyses revealed that the GbCYP72A1 genes primarily affected disease resistance via the plant hormone signal transduction, plant-pathogen interaction, and mitogen-activated protein kinase (MAPK) signaling pathways. Interestingly, the findings revealed that although GbCYP72A1d and GbCYP72A1a had high sequence similarity and both genes enhanced the disease resistance of transgenic Arabidopsis, there was a difference between their disease resistance abilities. Protein structure analysis revealed that this difference was potentially attributed to the presence of a synaptic structure in the GbCYP72A1d protein. Altogether, the findings suggested that the GbCYP72A1 genes play an important role in plant response and resistance to VW.

Membrane Localized GbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt.

Verticillium wilt (VW) is a soil-borne fungal disease caused by Verticillium dahliae Kleb, which leads to serious damage to cotton production annually in the world. In our previous study, a transmembrane protein 214 protein (TMEM214) gene associated with VW resistance was map-based cloned from Gossypium barbadense (G. barbadense). TMEM214 proteins are a kind of transmembrane protein, but their function in plants is rarely studied. To reveal the function of TMEM214s in VW resistance, all six TMEM214s were cloned from G. barbadense in this study. These genes were named as GbTMEM214-1_A/D, GbTMEM214-4_A/D and GbTMEM214-7_A/D, according to their location on the chromosomes. The encoded proteins are all located on the cell membrane. TMEM214 genes were all induced with Verticillium dahliae inoculation and showed significant differences between resistant and susceptible varieties, but the expression patterns of GbTMEM214s under different hormone treatments were significantly different. Virus-induced gene silencing analysis showed the resistance to VW of GbTMEM214s-silenced lines decreased significantly, which further proves the important role of GbTMEM214s in the resistance to Verticillium dahliae. Our study provides an insight into the involvement of GbTMEM214s in VW resistance, which was helpful to better understand the disease-resistance mechanism of plants.

Identification of superior parents with high fiber quality using molecular markers and phenotypes based on a core collection of upland cotton (Gossypium hirsutum L.).

The combination of molecular markers and phenotypes to select superior parents has become the goal of modern breeders. In this study, 491 upland cotton (Gossypium hirsutum L.) accessions were genotyped using the CottonSNP80K array and then a core collection (CC) was constructed. Superior parents with high fiber quality were identified using molecular markers and phenotypes based on the CC. The Nei diversity index, Shannon's diversity index, and polymorphism information content among chromosomes for 491 accessions ranged from 0.307 to 0.402, 0.467 to 0.587, and 0.246 to 0.316, with mean values of 0.365, 0.542, and 0.291, respectively. A CC containing 122 accessions was established and was categorized into eight clusters based on the K2P genetic distances. From the CC, 36 superior parents (including duplicates) were selected, which contained the elite alleles of markers and ranked in the top 10% of phenotypic values for each fiber quality trait. Among the 36 materials, eight were for fiber length, four were for fiber strength, nine were for fiber micronaire, five were for fiber uniformity, and ten were for fiber elongation. In particular, the nine materials, 348 (Xinluzhong34), 319 (Xinluzhong3), 325 (Xinluzhong9), 397 (L1-14), 205 (XianIII9704), 258 (9D208), 464 (DP201), 467 (DP150), and 465 (DP208), possessed the elite alleles of markers for at least two traits and could be given priority in breeding applications for a more synchronous improvement of fiber quality. The work provides an efficient method for superior parent selection and will facilitate the application of molecular design breeding to cotton fiber quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01300-0.

Genome-Wide Introgression and Quantitative Trait Locus Mapping Reveals the Potential of Asian Cotton (Gossypium arboreum) in Improving Upland Cotton (Gossypium hirsutum).

Gossypium arboreum (2n=2x=26, A2), the putative progenitor of the At-subgenome of Gossypium hirsutum (2n=4x=52, AD), is a repository of genes of interesting that have been eliminated during evolution/domestication of G. hirsutum. However, its valuable genes remain untapped so far due to species isolation. Here, using a synthetic amphiploid (AADDA2A2) previously reported, we developed a set of 289 G. arboreum chromosome segment introgression lines (ILs) in G. hirsutum by expanding the backcrossing population and through precise marker-assisted selection (MAS) although complex chromosomal structural variations existed between parents which severely hindered introgression. Our results showed the total coverage length of introgressed segments was 1,116.29 Mb, representing 78.48% of the At-subgenome in the G. hirsutum background, with an average segment-length of 8.69 Mb. A total of 81 co- quantitative trait loci (QTLs) for yield and fiber quality were identified by both the RSTEP-ADD-based QTL mapping and the genome-wide association study (GWAS) analysis, with 1.01-24.78% of the phenotypic variance explained. Most QTLs for boll traits showed negative additive effects, but G. arboreum still has the potential to improve boll-number traits in G. hirsutum. Most QTLs for fiber quality showed negative additive effects, implying these QTLs were domesticated in G. hirsutum compared with G. arboreum and, a small quantity of fiber quality QTLs showing positive additive effects, conversely; however, indicates that G. arboreum has the underlying genes of enhancing fiber quality of G. hirsutum. This study provides new insights into the breeding genetic potential of G. arboreum, lays the foundation for further mining favorable genes of interest, and provides guidance for inter-ploidy gene transference from relatives into cultivated crops.

Genome-wide quantitative trait loci reveal the genetic basis of cotton fibre quality and yield-related traits in a Gossypium hirsutum recombinant inbred line population.

Cotton is widely cultivated globally because it provides natural fibre for the textile industry and human use. To identify quantitative trait loci (QTLs)/genes associated with fibre quality and yield, a recombinant inbred line (RIL) population was developed in upland cotton. A consensus map covering the whole genome was constructed with three types of markers (8295 markers, 5197.17 centimorgans (cM)). Six fibre yield and quality traits were evaluated in 17 environments, and 983 QTLs were identified, 198 of which were stable and mainly distributed on chromosomes 4, 6, 7, 13, 21 and 25. Thirty-seven QTL clusters were identified, in which 92.8% of paired traits with significant medium or high positive correlations had the same QTL additive effect directions, and all of the paired traits with significant medium or high negative correlations had opposite additive effect directions. In total, 1297 genes were discovered in the QTL clusters, 414 of which were expressed in two RNA-Seq data sets. Many genes were discovered, 23 of which were promising candidates. Six important QTL clusters that included both fibre quality and yield traits were identified with opposite additive effect directions, and those on chromosome 13 (qClu-chr13-2) could increase fibre quality but reduce yield; this result was validated in a natural population using three markers. These data could provide information about the genetic basis of cotton fibre quality and yield and help cotton breeders to improve fibre quality and yield simultaneously.

Identification of genomewide single-nucleotide polymorphisms associated with presummer, summer and autumn bolls in upland cotton.

Presummer, summer, and autumn bolls (PSB, SB and AB, respectively) in cotton are related to both maturity and yield. Therefore, studying their genetic basis is important for breeding purposes. In this study, we developed an association analysis panel consisting of 169 upland cotton accessions. The panel was phenotyped for PSB, SB and AB across four environments and genotyped using a Cotton SNP80K array. Single-nucleotide polymorphisms (SNPs) associated with these three traits were identified by a genomewide association study. A total of 53,848 high-quality SNPs were screened, and 91 significant trait-associated SNPs were detected. Of the 91 SNPs 33 were associated with PSB, 21 with SB and 37 with AB. Three SNPs for PSB (TM10410, TM13158 and TM21762) and five for AB (TM13730, TM13733, TM13834, TM29666 and TM43214) were repeatedly detected in two environments or by two methods. These eight SNPs exhibited high phenotypic variation of more than 10%, thus allowing their use formarker-assisted selection. The candidate genes for target traits were also identified. These findings provide a theoretical basis for the improvement of early maturity and yield in cotton breeding programmes.

A genome-wide association study of early-maturation traits in upland cotton based on the CottonSNP80K array.

Genome-wide association studies (GWASs) efficiently identify genetic loci controlling traits at a relatively high resolution. In this study, variations in major early-maturation traits, including seedling period (SP), bud period (BP), flower and boll period (FBP), and growth period (GP), of 169 upland cotton accessions were investigated, and a GWAS of early maturation was performed based on a CottonSNP80K array. A total of 49,650 high-quality single-nucleotide polymorphisms (SNPs) were screened, and 29 significant SNPs located on chromosomes A6, A7, A8, D1, D2, and D9, were repeatedly identified as associated with early-maturation traits, in at least two environments or two algorithms. Of these 29 significant SNPs, 1, 12, 11, and 5 were related to SP, BP, FBP, and GP, respectively. Six peak SNPs, TM47967, TM13732, TM20937, TM28428, TM50283, and TM72552, exhibited phenotypic contributions of approximately 10%, which could allow them to be used for marker-assisted selection. One of these, TM72552, as well as four other SNPs, TM72554, TM72555, TM72558, and TM72559, corresponded to the quantitative trait loci previously reported. In total, 274 candidate genes were identified from the genome sequences of upland cotton and were categorized based on their functional annotations. Finally, our studies identified Gh_D01G0340 and Gh_D01G0341 as potential candidate genes for improving cotton early maturity.

Molecular Mapping of Restriction-Site Associated DNA Markers In Allotetraploid Upland Cotton.

Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits.