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.