Comparative genomic and phylogenetic analysis
A comparative genomic analysis was performed among 16 plant genomes, including 12 Gramineae species, 1 Bromeliaceae species, 1 Musaceae species, 1 Orchidaceae species and 1 Brassicaceae species. For the three allotetraploid species (O. kokonorica , C. songorica andE. tef ), their subgenomes were used. In total, 34,019 O. kokonorica genes (70.00%) were clustered into 25,903 gene families, of which 8,961 families were shared with the other four Gramineae species (O. sativa, C. songorica, Z. mays, O. thomaeum ) and 1,387 were unique to the O. kokonorica genome (Figure 2B; Table S11). These unique gene families were predicted to have functions involved in ‘regulation of salicylic acid metabolic process’ (GO:0010337), ‘amino acid transport’ (GO:0006865), and ‘male-female gamete recognition during double fertilization forming a zygote and endosperm’ (GO:0080173)( Figure S6).
The same 16 plant species were used to infer the phylogeny of O. kokonorica based on 381 single-copy orthologous genes. Subgenomes of the three allotetraploid species were used for the phylogenetic reconstruction and estimation of divergence times. As expected, O. kokonorica displayed the closest relationship with C. songorica . The diploid progenitors of subgenomes A and B (now likely extinct) ofO. kokonorica and C. songorica diverged from their common ancestor ~18.19 Ma (Figure 2A). The ancestor of O. kokonorica and C. songorica diverged from O. thomaeum~20.86 Ma, which together diverged from E. tef~26.19 Ma (Figure 2A). The phylogenetic relationships among these 16 species were the same as those recovered from previous studies (Wang et al., 2022).
The divergence times estimated by phylogenetic and K s analyses between subgenomes of O. kokonorica and C. songoricasuggested that the two species (or the two genera) may have shared one paleo-allotetraploidy event. The A and B progenitors were estimated to diverge from each other ~18.19 Ma, corresponding to the overlapped K s peaks between A-B subgenomes of the two species (OkoA–CsoB, OkoB–CsoA; Figure 2C). The K s analysis also showed that K s peaks of OkoA–CsoA, OkoB–CsoB, and Oko–Cso overlapped, which indicated that the OrinusCleistogenes divergence, A subgenomes divergence and B subgenomes divergence occurred at the same time, i.e., ~10 Ma (Figure 2A). In other words, the two genera diverged from the same allotetraploidy ancestor, and the allotetraploidy event occurred between 18.19–10 Ma.
We identified 495 expanded and 6,400 contracted gene families in the A subgenome of O. kokonorica compared to the other plant species, and the corresponding numbers in the B subgenome was 549 and 6,556, respectively. However, compared with O. kokonorica , the number of expanded gene families in C. songorica was larger (1,234 in A and 1,350 in B subgenomes), whereas the number of contracted families was much lower (1,645 in A and 1,491 in B subgenomes; Figure 2A; Table S12). GO analysis revealed that the expanded orthogroups of O. kokonorica were significantly enriched in the functional terms ‘programmed cell death involved in cell development’, ‘jasmonic acid biosynthetic process’ and ‘regulation of double-strand break repair’ (Figure S7; Table S13). KEGG analysis of these expanded gene families revealed significant enrichment in the “DNA repair and recombination proteins pathway”, “flavonoid biosynthesis pathway” and “environmental adaptation pathway” (Figure S8; Table S14). Several markedly expanded gene families were functionally associated with the maintenance of homologous recombination and DNA repair during adaptation to the high plateau.