BackgroundMillennia of directional human selection has reshaped the genomic architecture of cultivated cotton relative to wild counterparts, but we have limited understanding of the selective retention and fractionation of genomic components.ResultsWe construct a comprehensive genomic variome based on 1961 cottons and identify 456 Mb and 357 Mb of sequence with domestication and improvement selection signals and 162 loci, 84 of which are novel, including 47 loci associated with 16 agronomic traits. Using pan-genome analyses, we identify 32,569 and 8851 non-reference genes lost fromGossypium hirsutumandGossypium barbadensereference genomes respectively, of which 38.2% (39,278) and 14.2% (11,359) of genes exhibit presence/absence variation (PAV). We document the landscape of PAV selection accompanied by asymmetric gene gain and loss and identify 124 PAVs linked to favorable fiber quality and yield loci.ConclusionsThis variation repertoire points to genomic divergence during cotton domestication and improvement, which informs the characterization of favorable gene alleles for improved breeding practice using a pan-genome-based approach.

Background Base editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome. Results We established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21–23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein (GhPEBP), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production. Conclusions Our data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding.

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Summary Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease‐resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock‐down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome‐wide chromatin immunoprecipitation and high‐throughput sequencing (ChIP‐seq), in combination with RNA sequencing (RNA‐seq) analyses, revealed that 43.1% of GhWRKY41‐binding genes were up‐regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance‐related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY‐WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.