O. sativa and O. glaberrima were independently domesticated in Asia and Africa, respectively, from different wild ancestors 1. In general, wild species obtain various alleles for resistance or tolerance to environmental stresses as adaption to a broad biogeographical diversity 13,14. This rich diversity of wild relatives allows for introgression breeding to be a prominent approach in rice to enhance agricultural traits such as resistance to biotic and abiotic stresses 19–21. Several previous studies have reported that introgression of alleles from wild germplasms enhanced the productivity and grain quality in soybeans and tomatoes 22,23. However, in rice, most introgression breeding strategies have focused on conferring pest and disease resistance to O. sativa species 19–21. In the present study, the evolutionary history of the focal genes related to grain quality and yield potential were investigated among 10 Oryza species.
The evolution and speciation of Asian rice species have remained unclear due to their high ortholog sequence similarity 1,24. To date, there are two hypotheses concerning the origin and domestication of the O. sativa species 24. The single-domestication hypothesis posits that the O. sativa species originated from a wild ancestor and the differentiation between O. sativa ssp. indica and O. sativa ssp. japonica occurred after domestication of the cultivated species 25–28. This single-domestication hypothesis is mainly supported by the molecular evidence of the identical sequences of the key domestication genes between the O. sativa subspecies, including sh4 that reduces shattering and prog1 which is associated with erect growth 25–27. In contrast, the multiple-domestication hypothesis postulates that O. sativa ssp. indica and O. sativa ssp. japonica were domesticated separately from different wild ancestors 29,30. This multiple-domestication hypothesis has gained support through the phylogenetic analyses which shows that the O. sativa subspecies are separated into distinct clades and are closer to the different wild accessions than each other 29,30. The phylogenetic tree that was constructed in this study using the true orthologs based on syntenic relationship was consistent with the single-domestication hypothesis as the O. sativa subspecies were grouped into the same clade (Figure 1). The synteny analysis revealed that the genomic structure of the O. sativa species was more conserved with O. rufipogon than those of O. nivara (Figure 2B). This result is in agreement with a previous study which reported that the O. sativa species may originate from O. rufipogon and that O. nivara is one of the ecological varieties of O. rufipogon 25–27.
Genomic similarity is a key factor that determines genetic compatibility which enables the transfer of desired traits between two species through interspecific crossing 31. In rice, several reproductive isolations had been observed in interspecific hybrids, which resulted in inviability, weakness, and sterility 32. Several genetic models have been suggested to explain the mechanisms of reproductive isolations in plants and structural variations were identified as one of leading factors of reproductive isolation 32. In riceand Arabidopsis, pollen incompatibility and inviability had been reported in their hybrids, respectively, due to a change of the gene locus caused by reciprocal gene loss of duplicated genes 15,33. Other studies have reported that chromosomal rearrangements enhance the reproductive isolation by suppressing recombination 31,34,35, which results in unbalanced gametes that may be inviable 31,34.
Suppressed recombination also increased the extent of linkage disequilibrium (LD) block, thereby restricting gene flow in potentially larger genomic regions 34,36,37. When introgression of a favorable gene from an external germplasm into a cultivar occurs, other genes that confer undesirable traits can also be transferred if the genes are located in the same LD block 38,39. This phenomenon is also known as the linkage drag problem and it is a major concern in introgression breeding which prevents breeders from introducing desirable traits into elite cultivars 40–42. Linkage drag that leads to the negative relationships among the yield potential, grain quality, and environmental resistance have been reported in O. sativa species 40,41. The synteny analysis in this study identified that O. rufipogon would be a more genetically compatible germplasm for O. sativa breeding with reduced reproductive isolation and linkage drag problems (Figure 2).
Gene evolution analysis has been widely used to investigate gene expansion, domestication process, genetic background, etc. Copy number variation (CNV) is a structural variation that alters the dosage of genes, which could result in phenotypic changes 43,44. In plants, most resistance traits are polygenic and highly affected by CNV 43,44. A previous study on durum wheat reported that frost resistance levels were determined by the CNV of the CBF-A14 gene family 45. In rice, the CNV of 28 functional genes was identified to be involved in insect resistance and response to salt stress 43. In Brassica napus, 563 resistance genes experienced 1137 CNV events including 704 deletions and 433 duplications 46. Based on the phylogenetic clustering, a total of 43 HIS1 genes were further clustered into five subclasses including HIS1 (9), HSL1 (4), HSL2 (5), HSL3 (9), and HLS4 (16) (Figure 3). We identified that the HIS1 and HSL families experienced multiple duplication events (Figure 4). In O. punctata, a tandemly duplicated HSL4 gene was identified on chromosome 6 and an additional pair of tandemly duplicated HSL4 genes were detected on chromosome 3 (Figure 4). Because these HSL4 genes of O. punctata were not found in other Oryza species, they might be duplicated after the O. punctata speciation event (Figure 4). The HSL1 genes were only identified in O. glaberrima, O. barthii, and O. sativa ssp. japonica (Figure 4). Considering that O. glaberrima was domesticated from O. barthii, the HSL1 gene probably originated from O. barthii, and then moved to O. satvia ssp. japonica via O. glaberrima or directly from O. barthii (Figure 4). In O. sativa ssp. japonica, duplication events of HSL1 and HSL4 were identified on chromosome 6 (Figure 4). Because four of the five duplicated HSL1 and HSL4 genes are located close to each other (less than 50 kb interval), we propose that the HIS1 and HSL families were mainly expanded through tandem duplication events in the Oryza species (Figure 4). These results are in agreement with a previous study where tandem duplication events were frequently identified in CNVs 47. Our gene evolution analysis can facilitate the improvement in herbicide resistance of rice cultivars through gene transferring from wild germplasm that has a high copy number of HIS1 and HSL genes such as O. punctata (Figure 4).
While HIS1 has diverse CNVs across 10 Oryza species, GBSS1, GBSS2, and BADH2 genes have at most one copy in the 10 Oryza species and the syntenic relationship of their orthologs was deeply conserved in pair-wise comparison among Oryza species (Table 1 and Figure 5A). These results suggest that these three genes descended from a common Oryza ancestor to present-day cultivars (Figure 5). The BADH2 and GBSS2 loss events were identified in O. brachyantha, O. punctata, and O. meridionalis, which suggests that the functional role of the BADH2 and GBSS2 genes were developed after speciation from O. meridionalis (Table 1). Meanwhile, no loss event of GBSS1 was identified in the Oryza species (Table 1). Plants have some critical genes that play essential roles in their survival such as photosynthesis, cell division, and reproduction 48–50. In general, the genetic diversity of essential genes is highly conserved among related species, because the malfunction of these genes directly affects their fitness in the population 48. In rice, a previous study reported that GBSS1 is expressed in the endosperm and pollen grains, while the expression of GBSS2 is limited to the vegetative tissues and pericarp 7. The starch content of the endosperm serves as the primary source for seed germination and seedling growth 7. Therefore, the prevalence of GBSS1 may be the product of selection pressure for its critical role in seed germination vigor, as wild relatives and landraces with lower germination rates were extinguished in nature or removed from the breeding pool during rice evolution and domestication. This result is consistent with our selection pressure analysis which showed that GBSS1 had the lowest Ka/Ks ratio indicating that the sequence diversity of GBSS1 is highly conserved during evolution (Figure 6).
Gene exchange is a key evolutionary mechanism that enhances the adaptability of the population against environment stresses 51. Natural introgression between the Asian cultivated and wild species is common because they are often sympatric 24,51,52. The African cultivated species have a relatively limited gene pool in the wild species compared to Asian rice and several historic introgression events from the Asian species are reported 24. GBSS1, GBSS2, and BADH2 genes are located in highly conserved synteny blocks as single-copy genes over the 10 Orzya species, which indicates they are true orthologs in the genus Orzya. Using the true orthologs of GBSS1, GBSS2, and BADH2, reconciled gene trees were constructed and estimated divergence time was calculated between orthologous gene pairs, to investigate gene transfer events of the target genes across the Oryza species (Figure S1 and Table 2). Our results proposed that three transfer events had occurred in GBSS1 from the Asian groups into the African group (O. glaberrima and O. barthii) and other wild species (O. glumaepatula and O. meridionalis) (Figure 7). In contrast, the GBSS2 was transferred from O. barthii into the Asian species including O. sativa ssp. japonica and O. nivara (Figure 7). This gene flow in the opposite direction between Asian and African groups indicates that GBSS1 and GBSS2 gained their subfunctions independently in the Asian and African rice populations, respectively, and they were spread to other regions during the domestication process. For BADH2, a gene from either O. barthii or O. glaberrima was moved into O. rufipogon, which suggests that most BADH2 alleles in japonica rice had been developed after divergence between the Asian and African groups (Figure 7) 6,53.
Overall, this study enhances our knowledge of the gene family evolution in rice and offers practical implications for rice breeding efforts, which ultimately supports the development of improved rice varieties with enhanced adaptability and productivity.