The double-recessive genic male-sterile (ms) line ms5 ms6 has been used to develop cotton (Gos-sypium 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-A312aa), 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-A312aa) and ms6 (GhCYP703A2-DD98E,E168K,G198R) mutant pro-teins have decreased enzyme activities. Bio-chemical 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 steri-lity. Overall, our characterization uncovered func-tions 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.

Huanhuan Ma;Yuanlong Wu;Ruiling Lv;Huabin Chi;Yunlong Zhao;Yanlong Li;Hongbo Liu;Yizan Ma;Longfu Zhu;Xiaoping Guo;Jie Kong;Jianyong Wu;Chaozhu Xing;Xianlong Zhang;Ling Min

National Key Laboratory of Crop Genetic Improvement,Huazhong Agricultural University,Wuhan 430070,China;College of Biology and Agricultural Resources,Huanggang Normal University,Huanggang 438000,China;College of Plant Science and Technology,Huazhong Agricultural University,Wuhan 430070,China;Institute of Economic Crops,Xinjiang Academy of Agricultural Sciences,Xinjiang 830091,China;State Key Laboratory of Cotton Biology,Institute of Cotton Research of Chinese Academy of Agricultural Sciences,Anyang 455000,China

植物学报（英文版）

[1]Huanhuan Ma;Yuanlong Wu;Ruiling Lv;Huabin Chi;Yunlong Zhao;Yanlong Li;Hongbo Liu;Yizan Ma;Longfu Zhu;Xiaoping Guo;Jie Kong;Jianyong Wu;Chaozhu Xing;Xianlong Zhang;Ling Min-.Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton)[J].植物学报（英文版）,2022(10):2009-2025

CYP703A2,sporopollenin,ms5ms6,ms5,ms6,Gos,sypium,anthers,oxygenases,GhCYP703A2,A312aa,D98E,E168K,G198R,L11L,hydroxylate,DD98E

Cytochrome,P450,mono,plays,central,role,formation,fertility,cotton,double,recessive,genic,male,sterile,has,been,used,develop,hirsutum,hybrids,many,years,but,its,molecular,genetic,basis,remained,unclear,Here,identified,Ms5,Ms6,loci,through,map,cloning,confirmed,their,function,sterility,CRISPR,Cas9,editing,are,highly,expressed,stages,encode,cytochrome,mutant,carries,single,nucleotide,nonsense,mutation,leading,premature,chain,termination,amino,three,nonsynonymous,substitutions,Enzyme,assays,showed,that,proteins,fatty,acids,have,decreased,enzyme,activities,Bio,chemical,lipidomic,analyses,plants,C12,C18,free,phospholipid,levels,significantly,elevated,while,lack,fluorescence,around,causing,microspore,degradation,Overall,our,characterization,uncovered,providing,guidance,creating,lines,facilitate,production,therefore,exploit,heterosis,improvement

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