Meiosis is the primary process in the sexual life cycle of most eukaryotes, during which haploid gametes are generated from diploid germ cells. It involves one round of DNA replication, followed by two rounds of chromosome segregation. Cytological homologous chromosome pairing is a crucial step in meiosis I, involves key events, such as chromosomal recombination and crossover interference. At least one chiasma (obligatory chiasma) per bivalent is required to ensure proper chromosomal segregation, while multiple chiasmata are associated with a high frequency of genetic recombination at this stage (Jones and Franklin, 2006; Zickler and Kleckner, 2023). During meiosis, crossover rates (recombination) are unevenly distributed along chromosomes, thereby influencing the generation of novel genotypes and the efficacy of selection (Brazier et al., 2022). Owing to the positive association between recombination and gene density, crossover patterns are considered critical for efficient chromosomal shuffling, however, the evolutionary significance of diverse recombination patterns remains unclear (Brazier and Glémin, 2022).

Polyploidy, or whole-genome duplication (WGD), comprises two primary forms, allopolyploidy, which arises from interspecific hybridization and genome doubling, and autopolyploidy, which results from genome doubling within a species. Both mechanisms play a significant role in plant evolution (Soltis et al., 2015; Heslop-Harrison et al., 2023). Meiosis in polyploids is challenging owing to the presence of more than two homologs. Cytological diploidization of autopolyploids can be accomplished by crossover frequency reduction and redistribution (Grandont et al., 2013; Yant et al., 2013; Bomblies et al., 2015). In most established allopolyploids, bivalents form predominantly within each subgenome, and disomic inheritance is maintained due to persistent heterozygosity between subgenomes (Pikaard, 2001; Bomblies and Madlung, 2014). However, neo-allopolyploids exhibit extensive homeologous recombination (Zhang et al., 2013). Additionally, newly formed autopolyploids and allopolyploids exhibit an increased number of crossover events (Pecinka Alex et al., 2011). Polyploidy may promote meiotic recombination, and the consequent rapid generation of genetic diversity likely contributes to its prevalence (Pecinka Alex et al., 2011). High recombination rates are associated with recombination hotspots and gene-rich and diverse regions (Lawrence et al., 2017; Brazier and Glémin, 2022). Variations in the crossover rate and redistribution may significantly affect the shifting of genetic diversity, such as recombination hot spot, although they may enhance the adaptive potential of closely related species in challenging natural environments (Smukowski and Noor, 2011). Evaluating meiotic recombination in species with varying ploidy levels from a stable and long-term-adapted taxon could help clarify how recombination variation during polyploidization contributed to the enhanced adaptability of polyploids.

Triticeae (Poaceae) contains approximately 100 annual and 400 perennial taxa (Dewey, 1983). This economically crucial tribe includes genera, such as Triticum, Hordeum, and Secale, which include widely domesticated crops like wheat, barley, and rye. This tribe includes species with varying ploidy levels, serving as both a vital genetic resource for food security and a prominent model system for investigating the interactions among hybridization, chromosomal evolution, and biological diversification (Parisod and Badaeva, 2020; Kroupin et al., 2023). Hordeum bogdanii is a diploid species (2n = 2x = 14) with an H genome, whereas H. brevisubulatum is an autopolyploid (2n = 28) (Landström et al., 1984; Al-Saghir, 2016). The similar cytological patterns between the two species indicate that H. brevisubulatum originated as an autopolyploid from a Hordeum species carrying the common H genome, such as H. bogdanii (Dou et al., 2016). Elymus sibiricus L. is an allopolyploid with a genome constitution of StStHH (2n = 28), where St and H are derived from Pseudoroegneria (Neveski) Löve and Hordeum L., respectively (Dewey, 1983). H. bogdanii is a widely distributed Asiatic species, ranging from western Iran to eastern China (Yang et al., 1987), whereas H. brevisubulatum is distributed from Western Turkey to eastern China (Bothmer, 1979). Both species show superior adaptability to saline-alkaline soils (Garthwaite et al., 2005; Zhang et al., 2020). E. sibiricus is widely distributed across the Northern Hemisphere, with particular preponderance in Sweden, northern Asia, Japan, and North America (Baum et al., 2012). It usually grows on moist meadows, riparian sands, among open woodland, on sunny or semi-shade mountains or valley slopes at elevations ranging from 1,000 to 4,000 meters (Lu, 1993). The superior adaptability of the H genome to saline-alkaline soils is not distinctly manifested in E. sibiricus. Investigation on the crossover variation among three closely related Triticeae species, which share a common progenitor H genome, may provide valuable insights into their diverse adaptive strategies. Due to the large chromosome size in the Triticeae species, chiasmata resulting from crossing over are clearly visible during diakinesis, with each chiasma representing a single crossover (Fu and Sears, 1973), chromosome synteny across species can be clearly determined cytologically (Danilova et al., 2012; Liu et al., 2023). In this study, the variation in chiasmata of the H genome was analyzed cytologically during diakinesis and metaphase I across species of different ploidy levels. Chromosomal synteny of the H genome in was ambiguously determined in H. hordeum (2H), H. brevisubulatum, and E, sibiricus. The results of the variations in the crossover rate and redistribution at the chromosomal level provide important insights into the evolution and adaptation of the H genome during polyploidization, facilitating further genomic and molecular investigations.

Initial polyploids pose significant challenges that often form aberrant associations among the additional homologs (called multivalents) in meiosis I that can cause mis-segregation and reduced fertility (Bomblies et al., 2015). Auto- and allopolyploids may overcome this issue using distinct evolutionary solutions that suppress multivalent associations. In autopolyploids, meiotic stability can be achieved by reducing crossover (Le Comber et al., 2010), primarily by strengthening crossover interference (Morgan et al., 2021). In this study, the global chiasma (cytological crossover) number was significantly reduced from diploid H. bogdanii to autopolyploid H. brevisubulatum during diakinesis. Specifically, the absence of interstitial chiasmata on the short arms of H. brevisubulatum indicated significant crossover interference. However, H. brevisubulatum is notably characterized by the prevalence of quadrivalent configurations (44.86%), in contrast to the autopolyploid Arabidopsis arenosa, where cytological diploidization predominates and bivalents form the primary pairing configuration (Yant et al., 2013). This indicates that the quadrivalents of large chromosomes in Triticeae species are more challenging to resolve than those from small chromosomes in Arabidopsis. In most established allopolyploids, bivalents are formed within each subgenome owing to persistent heterozygosity between distinct subgenomes (Pikaard, 2001; Bomblies and Madlung, 2014). In wheat, genes such as pairing homoeologous 1 (Ph1) and Ph2, which suppress homoeologous chromosome pairing, play crucial roles in genome stabilization in allopolyploids (Ralph Riley, 1958; Luo et al., 1996; Romero et al., 2001; Serra et al., 2021). Allotetraploid E. sibiricus contains distinct H and St subgenomes and exhibits low frequencies of non-homoeologous chromosome exchange in specific cases (Liu et al., 2023). In this study, E. sibiricus exhibited disomic inheritance dominated by bivalent formation, suggesting that homeologous chromosome pairing is strictly regulated by genes such as Ph1 and Ph2. Additionally, the number of distinct chiasmata reduced from H. bogdanii to E. sibiricus across the H genome, indicating that crossover reduction may play a role in the meiotic stability of allopolyploids.

In this study, chiasmata were highly conserved in distal positions but exhibited variability at interstitial positions across the three assessed species during diakinesis. The formation of crossover/chiasmata is influenced globally by multiple factors, including chromatin modification, heterochromatin, and centromere region (Zickler and Kleckner, 2023). During polyploidization, genetic factors can regulate crossover distribution in trans (Wang et al., 2021). A comparison of the recombination patterns between wild and domesticated barley landraces revealed high conservation throughout domestication, indicating that the chromatin environment that suppresses recombination is evolutionarily conserved (Dreissig et al., 2019). The variation in chiasmata sites indicates that the chromatin structure of the H genome evolved from diploid to tetraploid species, specifically from diploid to autoploid, and trans factors emerging during polyploidization may also play an important role in the chiasmata redistribution in tetraploids. A biased distribution of chiasmata was observed in the three different ploidy species, consistent with crossover-biased localization in distal regions in Triticeae species, such as barley and wheat (Higgins et al., 2014; Osman et al., 2021). Recombination rates vary between and within species (Lawrence et al., 2017; Stapley et al., 2017). Additionally, in this study, the variation in chiasmata occurrence frequency that was used to estimate the variation in recombination was revealed, with a significant reduction from diploid to tetraploid species. Recombination rates are affected by DNA methylation (Habu et al., 2015), histone modifications (Underwood et al., 2018), and nucleosome occupancy (Choi et al., 2017). Global genomic alterations have occurred since the genome shock caused by WGD in polyploids. The genetic and epigenetic roles in the modification of recombination rates during polyploidization require further assessment. Fine-scale estimations of recombination rates in natural populations of wild and domesticated barley have demonstrated that increased recombination rates are shifted toward more distal regions on the long arm of chromosomes in domesticated barley (Dreissig et al., 2019). Differences in recombination rates may have resulted from selection for elevated recombination in genomic regions containing defense response genes during barley domestication (Dreissig et al., 2019). The H. bogdanii accession used in this study was collected from a wetland habitat, while the accessions of H. brevisubulatum and E. sibiricus were obtained from arid grassland environments. Recombination rates of H genome are more distally localized in both H. brevisubulatum and E. sibiricus, suggesting that the crossover redistribution may be under strong selection and play an important role in species adaptation. Furthermore, significantly increased chiasmata frequencies were detected on the short arms of chromosomes 1H and 4H in E. sibiricus compared to H. bogdanii. Elucidation of whether this was caused by the internal or external environment will give more clues to the biological significance of crossover redistribution.

Chiasmata terminalization refers to the movement of chiasmata toward the ends of chromatid. As anticipated, chiasmata terminalization was observed from diakinesis to metaphase I in all three species. However, a high frequency (22.61%) of quadrivalent or trivalent configurations was maintained in H. brevisubulatum at metaphase I, and more pairing configurations were detected in metaphase I than during diakinesis. Frequent chromosomal numerical variations, such as monosomy, nullisomy, and trisomy were identified in H. brevisubulatum (Dou et al., 2016). This indicates that certain special pairing configurations cannot be completely resolved by chiasmata terminalization. Irregular chiasmata terminalization may lead to homologous chromosome segregation errors and result in aneuploidy.

Polyploid plants, possessing redundant genes, exhibit significant evolutionary and adaptive advantages (Soltis et al., 2015; Heslop-Harrison et al., 2023). Novel genetic variations in polyploid plants can arise from mechanisms such as gene sub functionalization, chromosome rearrangements, gene mutations, and transcriptomic and epigenetic alterations (Liu et al., 2021; Wang et al., 2021; Banouh et al., 2022; Blasio et al., 2022). However, from a macroevolutionary perspective, polyploidy was regarded as an evolutionary dead end (Van de Peer et al., 2017). In meiosis, crossing over is indispensable for the proper segregation of homologous chromosomes and generation of genetic diversity by creating novel gene combinations. In this study, the chiasmata of the H genome were significantly reduced in both number and frequency from diploid to tetraploid. This indicates that although polyploid species can acquire novel genetic variations through WGD, the genetic diversity of their sub genomes may be significantly reduced because of variations in crossover events during polyploidization. Additionally, polyploids are more prone to recurrent deleterious mutations than that of diploids (Otto, 2007; Conover and Wendel, 2022). Deleterious alleles at neighboring loci can hitchhike and reach fixation in regions of low recombination (Hartfield and Otto, 2011), where they tend to be enriched (Zhang et al., 2013). This indicates that the reduction in crossover within sub genomes from diploid to tetraploid species may contribute to the genetic load.

In this study, molecular cytology techniques, such as FISH, were employed to identify each chromosome at different stages of meiosis and observe varied configurations and frequencies. Crossovers or chiasmata can be more accurately detected using immunolocalization of crossover-specific proteins such as MLH1 or HEI10 (Higgins et al., 2014; Osman et al., 2021). A more precise description of variations in chiasmata patterns from diploid to tetraploid can be achieved by combining FISH and immunolocalization techniques.