Discussion
As an endemic genus to the QTP and its adjacent regions, Orinusis mainly distributed in extreme arid regions at elevations between 2500 and 5200 m (Su et al., 2015). This genus not only serves as an important natural forage source but also has ecological significance in alpine arid regions for soil conservation due to its strong underground root system, as well as its resistance to cold, drought, soil salinity and disease (Chen et al., 2006). In this study, we report the first chromosome-scale genome assembly of O. kokonorica , and the first for this genus. Our karyotyping and comparative genomic analyses confirmed that O. kokonorica is an allotetraploid species, with all chromosomes confidently assigned to two subgenomes. Unlike most polyploid genomes in the grass family, the allotetraploid O. kokonorica has a relatively compact genome (~556 Mb), similar to other tetraploid species from the subfamily Chloridoideae, such as C. songorica (540 Mb; Zhang et al., 2021a), E. tef(576 Mb; VanBruen et al., 2020), and Leptochloa chinensis (416 Mb; Wang et al., 2022). The small genome size in these tetraploids is probably inherited from their diploid ancestors, such as that ofO. thomaeum , a diploid species closely related to these four tetraploids, whose genome is only 245 Mb in size (VanBuren et al., 2015). However, we found significantly higher proportions of repetitive elements and a fewer number of genes in O. kokonorica than those in the other three species (Table S20). LTR-RTs in O. kokonoricaexperienced rapid expansion in the very recent past of approximately 0.8 Ma compared with C. songorica (Figure S3). This time frame is congruent with the largest Naynayxungla glaciation in the QTP, which reached its maximum between 0.8 and 0.6 Ma with an ice sheet covering an area five to seven times larger than its current range (Zheng et al., 2002). The severe environmental conditions during this period may have induced the bursts of TEs (Lisch, 2013; Schrader et al., 2014), which may have contributed to the increase in the genome size of this species. Nonetheless, the enormous contraction in gene families in O. kokonorica (Figure 2A) may have counteracted this contribution to genome size. Therefore, the O. kokonorica genome might have been maintained by a reciprocal offset between recent expansion of TEs and contraction in gene families.
Our phylogenetic and K s analyses indicated that the two closely related genera, Orinus and Cleistogenes , may share one paleo-allotetraploidy event before 10 Ma. This polyploidization event is older than the ones detected in their relatives E. tef (1.1 Ma; VanBruen et al., 2020) and L. chinensis (<10.9 Ma; Wang et al., 2022). Following polyploidization, duplicated genes can be retained through neofunctionalization/subfunctionalization or reverted to a single copy through genome fractionation. Biased genome fractionation may lead to a dominant subgenome, which has often been reported in the genomes of allopolyploids especially in allopalaeopolyploids (Cheng et al., 2018), such as in Arabidopsis, bread wheat, maize, and Brassica rapa (Li et al., 2014; Schnable et al., 2011; Thomas et al., 2006; Wang et al., 2011). However, we found that the two subgenomes of O. kokonorica have retained similar numbers of genes across the chromosomes and display no significant global gene expression dominance, which is consistent with that found inC. songorica (Zhang et al., 2021a). Such non-biased homoelog expression between subgenomes in allopolyploids seems not as rare as previously thought, as this pattern is also observed in other allopolyploid species, including E. tef (VanBuren et al., 2020), pumpkin (Sun et al., 2017), and E. crus-galli (Ye et al., 2020).
Our analyses of comparative genomics and transcriptomics provide some new insights into the genetic basis of divergence between the two genera after paleo-allotetraploidization. First, we found two pairs of large interchomosomal rearrangements occurred specifically in B subgenome ofC. songorica . Although no substantial changes were found at the breakpoints and these translocation regions have high collinearity between the O. kokonorica and C. songorica genomes, these large genomic rearrangements may affect chromatin organization as revealed in two ecotypes of Medicago truncatula (Li et al., 2022), which may result in different epigenetic modifications and gene transcriptional activity in the two species (Zhang et al., 2021b). Nevertheless, whether these genomic rearrangements are present in all Cleistogenes species and whether these translocations affect chromatin organization needs further investigation. Second, SVs constituted a large proportion of the genomic variation that results in phenotypic variation in organisms (Ho et al., 2020). Using two methods, we built an overview of the genomic landscape of SVs between O. kokonorica and C. songorica , many of which may have contributed to the phenotypic variation and diversification of the two genera. For example, some highly-impacted-by-SV genes were enriched in “flower development.” We also found extensive SVs in the flower development and rhizome growth related genes and their vicinity (< 2 kb), which may play important roles in determining the morphological differentiation of flowers and rhizomes between the two genera (see more detailed discussion below). Finally, while both genera have strong resistance to drought, Orinus occurs in high altitude regions and has adapted to cold environments; Cleistogenes is tolerant to heat in low altitude regions (Chen et al., 2006). Consistent with this, we found most orthologous DEGs (98% for cold treatment and 89% for heat treatment) in the two species under cold and heat treatments exhibited functional divergence by differential expression in different tissues or in different directions. By comparison, more than half of the orthologous DEGs under drought treatment have conserved functions in the two species (Figure 3B). In addition, tandem copy increases in gene family members has contributed to the adaptation of O. kokonoricato the plateau environment, as also indicated in many other organisms (Ma et al., 2013; Zhu et al., 2007). Therefore, genomic rearrangements, SVs and functional innovations of orthologous genes have together played an important role in promoting divergence and speciation between the two genera after polyploidization.
Despite being closely related, Orinus and Cleistogeneshave two distinct morphological differences. Cleistogenes has a dimorphic flowering mechanism, while Orinus has elongated rhizomes, both of which ensure reproductive success under harsh environmental conditions (Guo et al., 2021; Schnee & Waller, 1986; Waller, 1980). We investigated the genetic basis of differentiation in floral development and rhizome growth between the two genera. Using AMGs in O. sativa as references, we found that many duplicated AMG copies have been lost in O. kokonorica , while C. songoricahas gained more copies. The increase in copy number of this gene family followed by functional innovations might have played an important role in the dimorphic flower development in Cleistogenes (Zhang et al., 2021a; Zhu et al., 2022). We further focused on the genes with significantly higher expression in CLs than in CHs in C. songorica . We found one copy of MADS13 and one copy ofMASD7 were lost in O. kokonorica (Figure 4; Table S18).MADS13 and MADS7 are involved in determining floral organ identities in O. sativa (Cui et al., 2010; Li et al., 2011). The higher expression in CLs compared with that in CHs of the MADS13and MADS7 copies in C. songorica may contribute to the development of CLs. Furthermore, we detected extensive SVs between the orthologous pairs of these genes, especially in the regulatory regions (Figure 4C). SVs in regulatory regions or introns may result in dosage variations in gene expression, which has been shown to play a crucial role in the variation of plant traits, especially for floral organ identity (Alonge et al., 2020; Liu et al., 2022; Qing et al., 2021). Many studies have suggested that changes in expression of a single ABCDE-class gene can easily shift the boundaries between different types of floral organs (Wuest et al., 2012; Wang et al., 2016). Therefore, extensive SVs in the AMGs may have played an important role in the differentiation of flower development between the two species. Similarly, we detected more copies of BOP genes that are all highly or moderately expressed in rhizomes of O. kokonorica and extensive SVs between BOP orthologous genes in O. kokonorica and C. songorica . BOP genes play an important role in rhizome tip stiffness, which ensures rhizome growth through the soil (Toriba et al., 2020). The copy number variation and SVs ofBOP genes may have contributed to the differentiation of rhizome morphology in the two species. However, our present data are not appropriate to gain detailed mechanistic explanations of the two morphological differentiations. Further studies involving more comparative genomic analyses associated with experimental validation would greatly enhance our understanding of the molecular mechanisms of flower and rhizome development in the two genera.