Identification of new cotton fiber-quality QTL by multiple genomic analyses and development of markers for genomic breeding

Cotton fiber is one of the main raw materials for the textile industry.In recent years,many cotton fiber quality QTL have been identified,but few were applied in breeding.In this study,a genome wide association study(GWAS)of fiber-quality traits in 265 upland cotton breeding intermediate lines(GhBreeding),combined with genome-wide selective sweep analysis(GSSA)and genomic selection(GS),revealed 25 QTL.Most of these QTL were ignored by only using GWAS.The CRISPR/Cas9 mutants of GhMYB_D13 had shorter fiber,which indicates the credibility of QTL to a certain extent.Then these QTL were verified in other cotton natural populations,5 stable QTL were found having broad potential for application in breeding.Additionally,among these 5 stable QTL,superior genotypes of 4 showed an enrichment in most improved new varieties widely cultivated currently.These findings provide insights for how to identify more QTL through combined multiple genomic analysis to apply in breeding.

High-temperature stress suppresses allene oxide cyclase 2 and causes male sterility in cotton by disrupting jasmonic acid signaling

Cotton(Gossypium spp.) yield is reduced by stress. In this study, high temperature(HT) suppressed the expression of the jasmonic acid(JA) biosynthesis gene allene oxide cyclase 2(GhAOC2), reducing JA content and causing male sterility in the cotton HT-sensitive line H05. Anther sterility was reversed by exogenous application of methyl jasmonate(MeJA) to early buds. To elucidate the role of GhAOC2 in JA biosynthesis and identify its putative contribution to the anther response to HT, we created gene knockout cotton plants using the CRISPR/Cas9 system. Ghaoc2 mutant lines showed male-sterile flowers with reduced JA content in the anthers at the tetrad stage(TS), tapetum degradation stage(TDS), and anther dehiscence stage(ADS). Exogenous application of MeJA to early mutant buds(containing TS or TDS anthers) rescued the sterile pollen and indehiscent anther phenotypes, while ROS signals were reduced in ADS anthers. We propose that HT downregulates the expression of GhAOC2 in anthers, reducing JA biosynthesis and causing excessive ROS accumulation in anthers, leading to male sterility. These findings suggest exogenous JA application as a strategy for increasing male fertility in cotton under HT.

Histone H3 lysine 27 trimethylation suppresses jasmonate biosynthesis and signaling to affect male fertility under high temperature in cotton

High-temperature(HT)stress causes male sterility in crops,thus decreasing yields.To explore the possible contribution of histone modifications to male fertility under HT conditions,we defined the histone methylation landscape for the marks histone H3 lysine 27 trimethylation(H3K27me3)and histone H3 lysine 4 trimethylation(H3K4me3)by chromatin immunoprecipitation sequencing(ChIP-seq)in two differing upland cotton(Gossypium hirsutum)varieties.We observed a global disruption in H3K4me3 and H3K27me3 modifications,especially H3K27me3,in cotton anthers subjected to HT.HT affected the bivalent H3K4me3–H3K27me3 modification more than either monovalent modification.We determined that removal of H3K27me3 at the promoters of jasmonate-related genes increased their expression,maintaining male fertility under HT in the HT-tolerant variety at the anther dehiscence stage.Modulating jasmonate homeostasis or signaling resulted in an anther indehiscence phenotype under HT.Chemical suppression of H3K27me3 deposition increased jasmonic acid contents and maintained male fertility under HT.In summary,our study provides new insights into the regulation of male fertility by histone modifications under HT and suggests a potential strategy for improving cotton HT tolerance.

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton

The double-recessive genic male-sterile(ms)line ms5 ms6 has been used to develop cotton(Gossypium hirsutum)hybrids for many years,but its molecular-genetic basis has remained unclear.Here,we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing.Ms5 and Ms6 are highly expressed in stages 7–9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D.The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312(GhCYP703A2-A^(312aa)),and ms6 carries three nonsynonymous substitutions(D98E,E168K,and G198R)and a synonymous mutation(L11L).Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids,and the ms5(GhCYP703A2-A^(312aa))and ms6(GhCYP703A2-D^(D98E,E168K,G198R))mutant proteins have decreased enzyme activities.Biochemical and lipidomic analyses showed that in ms5 ms6 plants,C12–C18 free fatty acid and phospholipid levels are significantly elevated in stages 7–9 anthers,while stages 8–10 anthers lack sporopollenin fluorescence around the pollen,causing microspore degradation and male sterility.Overall,our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility,providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.