Non‐functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.)

SUMMARY Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin‐like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin‐like domain and the arabinogalactan protein domain were predicted to cause the non‐functionalization of GbFLA19‐D and GhFLA19‐A. CRISPR/Cas9‐mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19‐D and GhFLA19‐A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA‐sequencing and quantitative reverse transcriptase–polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding. Significance Statement In this study, the cotton photosensitive genetic male sterile line, CCRI9106, was discovered to be a hybrid progeny of Gossypium hirsutum and Gossypium barbadense, and probably a product of hybrid breakdown. GoFLA19s (encoding fasciclin‐like arabinogalactan family protein) located on chromosomes A12 and D12 were identified to be the male sterility gene pair. Crucial variations were predicted to cause the defunctionalization of GbFLA19‐D and GhFLA19‐A in different cotton species. This study improves the understanding to the molecular mechanisms and evolutionary significance of hybrid breakdown in cotton.