ANL5 is Identified as a Major Gene Responsible for Long Anther in SSSLs of O. rufipogon

Anther length is an important outcrossing-related trait that influences the hybrid seed production in rice mainly by determining the pollen grain numbers of the restorer lines. However, the molecular genetic mechanism underlying anther length is still poorly understood. Wild rice species generally exhibit outcrossing characteristics with long anthers and thus is the valuable germplasm resources for mining the beneficial genes for long anthers. In this study, we investigated the anther lengths of the single-segment substitution lines (SSSLs) of O. rufipogon in the background of Huajingxian74 (HJX74), and identified a SSSL, SR51, with the significant long anthers. Using an F 2 population derived from HJX74 and SR51, a major QTL, qANLr5-1 , was mapped between markers RM6339 and RM6313 within the substituted segment interval on chromosome 5. By further substitution mapping among the secondary SSSLs (sSSSLs) of SR51, qANLr5-1 was delimited to a 13.6 kb region containing five ORFs. Through sequence alignment and relative expression analyses, four ORFs, ORF1 , ORF2 , ORF3 , and ORF5 , were selected as the possible candidate genes of qANLr5-1 . Investigation of the anther length in the CRISPR/Cas9 knockout lines of these four genes in SR51 revealed that only the homozygous knockout lines of ORF2 ( LOC_Os05g49320 ) exhibited significantly reduced anther length (reduced by 0.22–0.31 mm), as well as significantly reduced pollen grain numbers per anther (reduced by 29.11–30.28%) compared with SR51, yet retained normal pollen fertility and anther dehiscence. ORF2 was thus confirmed as the causal gene, ANTHER LENGTH 5 ( ANL5 ), of qANLr5-1 . ANL5 encodes the ribosomal protein L7/L12 C-terminal domain containing protein, and it was clustered with the known RPL12 family proteins among the monocot and dicot plant species. ANL5 locus was primarily divided into five haplotypes in Oryza sativa and wild rice ( Oryza rufipogon ). ANL5 regulated the anther length by controlling the longitudinal elongation of the anther epidermal cells. These results laid a foundation for exploring the genetic mechanism of anther length as a mating system related trait during domestication of rice, and provided the beneficial gene for improvement of anther length and pollen grain numbers in restorer lines of hybrid rice.