Summary 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.

In plants, long non-coding RNAs regulate disease resistance against fungi and other pathogens. However, the specific mechanism behind this regulation remains unclear. In this study, we identified disease resistance-related lncRNAs as well as their regulating genes and assessed their functions by infection of cotton (Gossypium) chromosome segment substitution lines with Verticillium dahliae. Our results demonstrated that lncRNA7 and its regulating gene Pectin methylesterase inhibitor 13 (GbPMEI13) positively regulated disease resistance via the silencing approach, while ectopic overexpression of GbPMEI13 in Arabidopsis (Arabidopsis thaliana) promoted growth and enhanced resistance to V. dahliae. In contrast, lncRNA2 and its regulating gene Polygalacturonase 12 (GbPG12) negatively regulated resistance to V. dahliae. We further found that fungal disease-related agents, including the pectin-derived oligogalacturonide (OG), could down-regulate the expression of lncRNA2 and GbPG12, leading to pectin accumulation. Conversely, OG up-regulated the expression of lncRNA7, which encodes a plant peptide phytosulfokine (PSK-α), which was confirmed by lncRNA7 overexpression and UPLC-MS experiments. We showed that PSK-α promoted IAA accumulation and activated GbPMEI13 expression through Auxin Response Factor 5 (ARF5). Since it is an inhibitor of pectin methylesterase (PME), GbPMEI13 promotes pectin methylation and therefore increases the resistance to V. dahliae. Consistently, we also demonstrated that GbPMEI13 inhibits the mycelial growth and spore germination of V. dahliae in vitro. In this study, we demonstrated that lncRNA7, lncRNA2, and their regulating genes modulate cell wall defense against V. dahliae via auxin-mediated signaling, providing a strategy for cotton breeding.

Circular RNAs (circRNAs), a class of recently discovered non-coding RNAs, play a role in biological and developmental processes. A recent study showed that circRNAs exist in plants and play a role in their environmental stress responses. However, cotton circRNAs and their role in Verticillium wilt response have not been identified up to now. In this study, two CSSLs (chromosome segment substitution lines) of G.barbadense introgressed into G. hirsutum, CSSL-1 and CSSL-4 (a resistant line and a susceptible line to Verticillium wilt, respectively), were inoculated with V. dahliae for RNA-seq library construction and circRNA analysis. A total of 686 novel circRNAs were identified. CSSL-1 and CSSL-4 had similar numbers of circRNAs and shared many circRNAs in common. However, CSSL-4 differentially expressed approximately twice as many circRNAs as CSSL-1, and the differential expression levels of the common circRNAs were generally higher in CSSL-1 than in CSSL-4. Moreover, two C-RRI comparisons, C-RRI-vs-C-RRM and C-RRI-vs-C-RSI, possessed a large proportion (approximately 50%) of the commonly and differentially expressed circRNAs. These results indicate that the differentially expressed circRNAs may play roles in the Verticillium wilt response in cotton. A total of 280 differentially expressed circRNAs were identified. A Gene Ontology analysis showed that most of the ‘stimulus response’ term source genes were NBS family genes, of which most were the source genes from the differentially expressed circRNAs, indicating that NBS genes may play a role in Verticillium wilt resistance and might be regulated by circRNAs in the disease-resistance process in cotton.

We cloned the GbABR1 gene from highly resistant Gossypium barbadense Xinhai15 based on the candidate genes screened by transcriptome sequencing that were related to resistance to Verticillium wilt. A sequence characteristic analysis showed that GbABR1 was an ERF subfamily B4 member and was a new member of the AP2 family of sea-island cotton. The GbABR1 gene was expressed highly in roots compared with the levels in leaves and stems in cotton. Expression was enhanced significantly in cotton after infection by Verticillium dahliae, indicating that GbABR1 probably plays an important role in the response to biotic stress. The results of subcellular localisation showed that GFP:GbABR1 was localised to the nucleus. GbABR1 silencing via the virus-induced gene silencing (VIGS) method indicated that the incidence of disease and the disease index in VIGS-silenced plants were much higher than in the control after infection by Verticillium dahliae. The GbABR1-overexpressing Arabidopsis plants showed similar resistance to Verticillium dahliae compared to the wild type. These results indicate that the GbABR1 gene plays a positive role in resistance to Verticillium wilt. The GbABR1-overexpressing Arabidopsis plants presented dwarfism, early maturation and early bolting compared with wild-type plants, suggesting that GbABR1 also participates in growth and development.

Verticillium wilt causes devastating loss of yield and quality in many crops, including cotton. To determine the molecular mechanism of resistance to verticillium wilt in cotton, we isolated a new cytochrome P450 gene, CYP94C1, and analysed its function. We obtained the complete open reading frame, which encodes a protein of 500 amino acids. The results of the functional analysis showed that resistance to verticillium wilt was enhanced when the gene was silenced using the virus-induced gene silencing (VIGS) method in cotton.