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.

Identification of chromosomes by fluorescence in situ hybridization in Gossypium hirsutum via developing oligonucleotide probes.

Discrimination of chromosome is essential for chromosome manipulation or visual chromosome characterization. Oligonucleotide probes can be employed to simplify the procedures of chromosome identification in molecular cytogenetics due to its simplicity, fastness, cost-effectiveness, and high efficiency. So far, however, visual identification of cotton chromosomes remains unsolved. Here, we developed 16 oligonucleotide probes for rapid and accurate identification of chromosomes in Gossypium hirsutum: 9 probes, of which each is able to distinguish individually one pair of chromosomes, and seven probes, of which each distinguishes multiple pairs of chromosomes. Besides the identification of Chrs. A09 and D09, we first find Chr. D08, which carries both 45S and 5S rDNA sequences. Interestingly, we also find Chr. A07 has a small 45S rDNA size, suggesting that the size of this site on Chr. A07 may have reduced during evolution. By the combination of 45S and 5S rDNA sequences and oligonucleotide probes developed, 10 chromosomes (Chrs. 3-7, and 9-13) in A subgenome and 7 (Chrs. 1-2, 4-5, and 7-9) in D subgenome of cotton are able to be recognized. This study establishes cotton oligonucleotide fluorescence in situ hybridization technology for discrimination of chromosomes, which supports and guides for sequence assembling, particularly, for tandem repeat sequences in cotton.

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.

RVE2, a new regulatory factor in jasmonic acid pathway, orchestrates resistance to Verticillium wilt.

Verticillium dahliae, one of the most destructive fungal pathogens of several crops, challenges the sustainability of cotton productivity worldwide because very few widely-cultivated Upland cotton varieties are resistant to Verticillium wilt (VW). Here, we report that REVEILLE2 (RVE2), the Myb-like transcription factor, confers the novel function in resistance to VW by regulating the jasmonic acid (JA) pathway in cotton. RVE2 expression was essentially required for the activation of JA-mediated disease-resistance response. RVE2 physically interacted with TPL/TPRs and disturbed JAZ proteins to recruit TPL and TPR1 in NINJA-dependent manner, which regulated JA response by relieving inhibited-MYC2 activity. The MYC2 then bound to RVE2 promoter for the activation of its transcription, forming feedback loop. Interestingly, a unique truncated RVE2 widely existing in D-subgenome (GhRVE2D) of natural Upland cotton represses the ability of the MYC2 to activate GhRVE2A promoter but not GausRVE2 or GbRVE2. The result could partially explain why Gossypium barbadense popularly shows higher resistance than Gossypium hirsutum. Furthermore, disturbing the JA-signalling pathway resulted into the loss of RVE2-mediated disease-resistance in various plants (Arabidopsis, tobacco and cotton). RVE2 overexpression significantly enhanced the resistance to VW. Collectively, we conclude that RVE2, a new regulatory factor, plays a pivotal role in fine-tuning JA-signalling, which would improve our understanding the mechanisms underlying the resistance to VW.

Transcriptome Analysis Revealed that GhPP2C43-A Negatively Regulates Salinity Tolerance in an Introgression Line from a Semi-Wild Upland Cotton.

Salt damage is a major threat to sustainable cotton production owing to the limited arable land in China, which is mainly occupied by the production of staple food crops. Salt-stress-tolerant cotton varieties are lacking in production, and the mechanisms underpinning salt stress tolerance in cotton remain enigmatic. Here, DM37, an intraspecific introgression line from Gossypium hirsutum race yucatanense acc TX-1046 into the G. hirsutum acc TM-1 background, was found to be highly tolerant to salt stress. Its seed germination rate and germination potential were significantly higher than those of the recipient TM-1 under salt stress. Physiological analysis showed that DM37 had a higher proline content and peroxidase activity and lower Na+/K+ ratios at the seedling stage, which is consistent with a higher seedling survival rate after durable salt stress. Furthermore, comparative transcriptome analysis revealed that responsive patterns to salt stress in DM37 were different from those in TM-1. Weighted correlation network analysis demonstrated that co-expression modules associated with salt stress in DM37 also differed from those in TM-1. From this analysis, GhPP2C43-A, a phosphatase gene, was found to exhibit negative regulation of salt stress tolerance verified by virus-induced gene silencing and the genration of transgenic Arabidopsis. Gene expression showed that GhPP2C43-A in TM-1 was induced by durable salt stress but not in DM37, probably attributable to a variation in the cis-element in its promoter, thereby conferring different salt stress tolerance. These results provide new genes/germplasms from semi-wild cotton in salt-stress-tolerant cotton breeding, as well as new insight into the mechanisms underpinning salt stress tolerance in cotton.

Genome-wide chromatin accessibility analysis unveils open chromatin convergent evolution during polyploidization in cotton.

Allopolyploidization, resulting in divergent genomes in the same cell, is believed to trigger a "genome shock", leading to broad genetic and epigenetic changes. However, little is understood about chromatin and gene-expression dynamics as underlying driving forces during allopolyploidization. Here, we examined the genome-wide DNase I-hypersensitive site (DHS) and its variations in domesticated allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense, AADD) and its extant AA (Gossypium arboreum) and DD (Gossypium raimondii) progenitors. We observed distinct DHS distributions between G. arboreum and G. raimondii. In contrast, the DHSs of the two subgenomes of G. hirsutum and G. barbadense showed a convergent distribution. This convergent distribution of DHS was also present in the wild allotetraploids Gossypium darwinii and G. hirsutum var. yucatanense, but absent from a resynthesized hybrid of G. arboreum and G. raimondii, suggesting that it may be a common feature in polyploids, and not a consequence of domestication after polyploidization. We revealed that putative cis-regulatory elements (CREs) derived from polyploidization-related DHSs were dominated by several families, including Dof, ERF48, and BPC1. Strikingly, 56.6% of polyploidization-related DHSs were derived from transposable elements (TEs). Moreover, we observed positive correlations between DHS accessibility and the histone marks H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H3K9ac, indicating that coordinated interplay among histone modifications, TEs, and CREs drives the DHS landscape dynamics under polyploidization. Collectively, these findings advance our understanding of the regulatory architecture in plants and underscore the complexity of regulome evolution during polyploidization.

GhALKBH10 negatively regulates salt tolerance in cotton.

The alpha-ketoglutarate-dependent dioxygenase (AlkB) gene family plays an essential role in regulating plant development and stress response. However, the AlkB gene family is still not well understood in cotton. In this study, 40 AlkB genes in cotton and Arabidopsis are identified and classified into three classes based on phylogenetic analysis. Their protein motifs and exon/intron structures are highly conserved. Chromosomal localization and synteny analysis suggested that segmental or whole-genome duplication and polyploidization events contributed to the expansion of the cotton AlkB gene family. Furthermore, the AlkB genes showed dynamic spatiotemporal expression patterns and diverse responses to abiotic stresses. Among them, GhALKBH10 was down-regulated under various abiotic stresses and its subcellular expression was localized in cytoplasm and nucleus. Silencing GhALKBH10 in cotton increased antioxidant capacity and reduced cytoplasmic Na+ concentration, thereby improved the plant tolerance to salinity. Conversely, overexpression (OE) of GhALKBH10 in Arabidopsis markedly weakened the plant tolerance to salinity. The global m6A levels measured in VIGS and OE transgenic lines showed that they were significantly higher in TRV: GhALKBH10 plants (VIGS) than in TRV: 00 plants but significantly lower in OE plants than wild-type plants under salt stress, which could be considered as a potential m6A demethylase in cotton. Our results suggest that the GhALKBH10 gene negatively regulates salt tolerance in plants, which provides information of the cotton AlkB family and an understanding of GhALKBH10 function under salt condition as well as a new gene for salt-tolerant cotton breeding.

ATP-citrate lyase B (ACLB) negatively affects cell death and resistance to Verticillium wilt.

BACKGROUND: ATP-citrate lyase (ACL) plays a pivotal role in histone acetylation and aerobic glycolysis. In plant, ACL is a heteromeric enzyme composed of ACLA (45 kD) and ACLB (65 kD). So far, the function of ACL genes in cotton still remains unknown. RESULTS: Here, we identified three ACLA homologous sequences and two ACLB homologous in each genome/sub-genome of cotton species. Silencing ACLB in cotton led to cell death at newly-grown leaves and stem apexes. Simultaneously, in ACLB-silenced plants, transcription factors related to senescence including SGR, WRKY23 and Osl57 were observed to be activated. Further investigation showed that excessive H2O2 was accumulated, salicylic acid-dependent defense response and pathogenesis-related gene expressions were evidently enhanced in ACLB-silenced plants, implying that knockdown of ACLB genes leads to hypersensitive response-like cell death in cotton seedlings. However, as noted, serious cell death happened in newly-grown leaves and stem apexes in ACLB-silenced plants, which led to the failure of subsequent fungal pathogenicity assays. To confirm the role of ACLB gene in regulating plant immune response, the dicotyledonous model plant Arabidopsis was selected for functional verification of ACLB gene. Our results indicate the resistance to Verticillium dahliae infection in the Arabidopsis mutant aclb-2 were enhanced without causing strong cell death. Ectopic expression of GausACLB-2 in Arabidopsis weakened its resistance to V. dahliae either in Col-0 or in aclb-2 background, in which the expression level of ACLB is negatively correlated with the resistance to V. dahliae. CONCLUSIONS: These results indicate that ACLB has a new function in negatively affecting the induction of plant defense response and cell death in cotton, which provides theoretical guidance for developing cotton varieties with resistance against Verticillium wilt.

TIP41L, a putative candidate gene conferring both seed size and boll weight, was fine-mapped in an introgression line of Gossypium hirsutum-Gossypium arboreum.

QTLs for yield-related traits in tetraploid cotton have been widely mapped, but QTLs introduced from diploid species into tetraploid cotton background remain uninvolved. Here, a stable introgression line with the traits of small boll and seed on Chr. A12, IL197 derived from Gossypium hirsutum (2n = AADD = 52) × Gossypium arboreum (2n = AA = 26), was employed to construct the F2 and F3 secondary populations for fine-mapping QTLs of yield-related traits. QTL analysis showed eight QTLs were detected for three traits, boll weight (BW), seed index (SI, one-hundred-seed weight in g), and lint percentage, with 3.94-28.13 % of the phenotypic variance explained. Of them, a stable major QTL, q(BW + SI)-A12-1 controlling both BW and SI and covering the shortest region in Chr. A12, was further narrowed into a 60.09 kb-interval through substitution mapping. Finally, five candidate genes were detected in the interval. The qRT-PCR analysis revealed only TIP41-like family protein (TIP41L) kept up-regulated expression and significantly lower in TM-1 than that in IL197 from -1 DPA to 15 DPA during cotton boll rapid developmental stage. Therefore, TIP41L gene is speculated as the most likely candidate gene. Comparative analysis with the other four allotetraploid species showed TIP41L gene was probably diverged after the formation of allotetraploid cotton, which may be selected and swept during domestication of modern upland cotton because small boll and seed are detrimental to fibre yield of cotton. This research would lay a solid foundation for further elucidating the molecular mechanism of cotton boll and seed development.

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.

Research progress of plant cytogenetics in Jiangsu province.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.

Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.

Genome-Wide Identification and Expression Profile Analysis of the PHT1 Gene Family in Gossypium hirsutum and Its Two Close Relatives of Subgenome Donor Species.

Phosphate transporter (PHT) is responsible for plant phosphorus (P) absorption and transport. PHT1 is a component of the high-affinity phosphate transporter system and plays pivotal roles in P absorption under P starvation conditions. However, in cotton, the number and identity of PHT1 genes that are crucial for P absorption from soil remain unclear. Here, genome-wide identification detected twelve PHT1 genes in Gossypium hirsutum and seven and eight PHT1 genes in two close relatives of the G. hirsutum genome-G. arboreum and G. raimondii, respectively. In addition, under low-phosphate treatment, the expressions of GaPHT1;3, GaPHT1;4, and GaPHT1;5 in roots were upregulated after 3 h of induction, and GhPHT1;3-At, GhPHT1;4-At, GhPHT1;5-At, GhPHT1;3-Dt, GhPHT1;4-Dt, and GhPHT1;5-Dt in the roots began to respond after 1 h of induction. Homologous pairs-GaPHT1;4 and GhPHT1;4-At; GaPHT1;5 and GhPHT1;5-At; GrPHT1;4 and GhPHT1;4-Dt, with GhPHT1;5-Dt and GhPHT1;5-At being syntenic-were all highly expressed in the roots under normal conditions. Among the genes highly expressed in the roots, GhPHT1;4-At, GhPHT1;5-At, GhPHT1;4-Dt and GhPHT1;5-Dt were continuously upregulated by P starvation. Therefore, it is concluded that these four genes might be key genes for P uptake in cotton roots. The results of this study provide insights into the mechanisms of P absorption and transport in cotton.

Fine-mapping and candidate gene analysis of qFS-Chr. D02, a QTL for fibre strength introgressed from a semi-wild cotton into Gossypium hirsutum.

Fibre strength (FS) is an important quality attribute in the modern textile industry, which is genetically controlled by quantitative trait loci (QTLs). Fine-mapping stable QTLs for FS to identify candidate genes would be valuable for uncovering the genetic basis of fibre quality traits in cotton. Here, a single segment introgression line, IL-D2-2, from the cross of (TM-1×TX-1046) reported in our previous studies, was found to have significantly improved FS compared with the recurrent parent TM-1. To fine-map the QTLs of the FS, we further crossed IL-D2-2 with its recurrent parent TM-1 to produce F2 and F2:3 populations. QTL analysis and substitution mapping showed qFS-Chr. D02 was anchored into a 550.66 kb-interval between two markers, INTR1027 and JESPR-231. This interval contained 67 genes, among which 27 genes related to cell-wall synthesis were selected to conduct qRT-PCR. The results revealed seven genes were expressed significantly differently during the fibre secondary-wall-thickening stage (10-25 days post-anthesis), three being upregulated and four downregulated in IL-D2-2. Both GH_D02G2269 (UDP-glucosyl transferase 84B1) and GH_D02G2289 (unknown function (DUF869)) with nonsynonymous SNPs in IL-D2-2 had significantly downregulated expression, suggesting they were candidates for qFS-Chr. D02. This research provides information about marker-assisted selection for cotton fibre strength improvement.

Role of phasiRNAs from two distinct phasing frames of GhMYB2 loci in cis- gene regulation in the cotton genome.

BACKGROUND: Phased small interfering RNA (phasiRNA) is primarily derived from the 22-nt miRNA targeting loci. GhMYB2, a gene with potential roles in cotton fiber cell fate determination, is a target gene of miR828 and miR858 in the generation of phasiRNAs. RESULTS: In the presented work, through the evaluation of phasing scores and phasiRNA distribution pattern, we found that phasiRNAs from GhMYB2 were derived from the 3' cleavage fragments of 22-nt miR828 and 21-nt miR858 respectively. These two miRNA targeting sites initiated two phasing frames on transcripts of one locus. By means of RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we further demonstrated that phasiRNAs derived from the two phasing frames played a role in cis-regulation of GhMYB2. The phasiRNAs derived from GhMYB2 were expressed in the somatic tissues, especially in anther and hypocotyl. We further employed our previous small RNA sequencing data as well as the degradome data of cotton fiber bearing ovules, anthers, hypocotyls and embryogenic calli tissues published in public databases, to validate the expression, phasing pattern and functions of phasiRNAs. CONCLUSIONS: The presenting research provide insights of the molecular mechanism of phasiRNAs in regulation of GhMYB2 loci.

Genome sequencing of the Australian wild diploid species Gossypium australe highlights disease resistance and delayed gland morphogenesis.

The diploid wild cotton species Gossypium australe possesses excellent traits including resistance to disease and delayed gland morphogenesis, and has been successfully used for distant breeding programmes to incorporate disease resistance traits into domesticated cotton. Here, we sequenced the G. australe genome by integrating PacBio, Illumina short read, BioNano (DLS) and Hi-C technologies, and acquired a high-quality reference genome with a contig N50 of 1.83 Mb and a scaffold N50 of 143.60 Mb. We found that 73.5% of the G. australe genome is composed of various repeat sequences, differing from those of G. arboreum (85.39%), G. hirsutum (69.86%) and G. barbadense (69.83%). The G. australe genome showed closer collinear relationships with the genome of G. arboreum than G. raimondii and has undergone less extensive genome reorganization than the G. arboreum genome. Selection signature and transcriptomics analyses implicated multiple genes in disease resistance responses, including GauCCD7 and GauCBP1, and experiments revealed induction of both genes by Verticillium dahliae and by the plant hormones strigolactone (GR24), salicylic acid (SA) and methyl jasmonate (MeJA). Experiments using a Verticillium-resistant domesticated G. barbadense cultivar confirmed that knockdown of the homologues of these genes caused a significant reduction in resistance against Verticillium dahliae. Moreover, knockdown of a newly identified gland-associated gene GauGRAS1 caused a glandless phenotype in partial tissues using G. australe. The G. australe genome represents a valuable resource for cotton research and distant relative breeding as well as for understanding the evolutionary history of crop genomes.

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

BACKGROUND: Gossypium australe F. Mueller (2n = 2x = 26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis. RESULTS: Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds. CONCLUSIONS: The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

A CC-NBS-LRR gene induces hybrid lethality in cotton.

Hybrid lethality forms a reproductive barrier that has been found in many eukaryotes. Most cases follow the Bateson-Dobzhansky-Muller genetic incompatibility model and involve two or more loci. In this study, we demonstrate that a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) gene is the causal gene underlying the Le4 locus for interspecific hybrid lethality between Gossypium barbadense and G. hirsutum (cotton). Silencing this CC-NBS-LRR gene can restore F1 plants from a lethal to a normal phenotype. A total of 11 099 genes were differentially expressed between the leaves of normal and lethal F1 plants, of which genes related to autoimmune responses were highly enriched. Genes related to ATP-binding and ATPase were up-regulated before the lethal syndrome appeared; this may result in the conversion of Le4 into an active state and hence trigger immune signals in the absence of biotic/abiotic stress. We discuss our results in relation to the evolution and domestication of Sea Island cottons and the molecular mechanisms of hybrid lethality associated with autoimmune responses. Our findings provide new insights into reproductive isolation and may benefit cotton breeding.