Fig. 2: Evolutionary trajectory of Rf4 haplotypes in Oryza species.

Current Rf4 haplotypes may have originated from an ancestral type of Rf4 (Anc-Rf4, H69), which first emerged at Copy-a in the oldest wild rice O. meyeriana (GG-genome). Sequence variation, gene duplication, and recombination events resulted in new one-copy (H12-H27) or two-copy (H28-H68) haplotypes of R/rf4a-likes (a group of Rf4a-like or rf4a-like variants with SNPs) and/or R/rf4b-likes (a group of Rf4b-like or rf4b-like variants with SNPs) in wild rice. During evolution, along with natural and human selections, the nascent one-copy and two-copy Rf4 and/or rf4 haplotypes gradually migrated into the lineages of O. rufipogon, O. nivara, and O. sativa. Rf4 haplotypes were not detected in tested accessions of O. longistaminata, O. barthii, and O. glaberrima. Among the eight Rf4 haplotypes (H1–H8) in modern cultivars, three two-copy haplotypes (indicated by asterisks “*”, the percentages on the left showed frequency of specific haplotype in the tested accessions) are predominant in restorer lines. H1 (Rf4a^M-Rf4b^M) and H7 (Rf4a^I-rf4b) haplotypes first appeared in O. nivara and O. australiensis (EE-genome), respectively, while H8 (rf4a-Rf4b^M) is present only in landraces and modern cultivars of O. sativa. The one-copy variant Rf4a^I in H2 of modern cultivars was only found in H7, suggesting that H2 may be derived from loss of rf4b in H7 from O. rufipogon. Other one-copy haplotypes such as H3 (rf4aus), H4 (rf4i), H5 (rf4j) and H6 (Rf4b^M) first emerged in O. sativa, O. australiensis, O. officinalis (CC-genome), and O. glumaepatula (AA-genome), respectively. The number in the brackets next to each species represents frequency of the haplotypes detected in the species. MYA: million years ago (divergence time).