Chromosomal structural variations, particularly translocation and inversion, play important roles in karyotype evolution, adaptation divergence, and speciation (Dobzhansky and Sturtevant, 1938; Rieseberg, 2001; Todesco et al., 2020). In heterozygotes with rearranged chromosomes, recombination between rearranged regions produces chromatin with duplicated and deficient genes. During the first meiotic division, disjunction patterns can cause varying degrees of sterility in the gametophytic and sporophytic phases. Because viable recombinants from inversion heterozygotes are extremely rare, inversions have been classically defined as recombination suppressors. Gene flow between rearranged regions is greatly restricted by recombination suppression (Livingstone and Rieseberg, 2004). Adaptive genes can be held together in chromosomal regions with structural variations via recombination suppression (Rieseberg, 2001; Livingstone and Rieseberg, 2004). Recombination suppression is typically involved in both inversion and translocation (Davisson and Akeson, 1993; Jiang et al., 2007; Dumas et al., 2015; Todesco et al., 2020). In addition, novel variations can be induced by altering the gene expression pattern and producing new chimeric genes near chromosome breakpoints (Lister et al., 1993; Hewitt et al., 2014).

Elymus nutans Griseb. is a well-known perennial and caespitose grass belonging to the genus Elymus L. in the Triticeae tribe of the family Poaceae, which comprises approximately 150 perennial and exclusively polyploid species (Löve, 1984). E. nutans is widely distributed in Central and Eastern Asia and the Himalayas (Bor, 1960; Lu, 1993; Chen and Zhu, 2006). E. nutans is an allopolyploid with a genome constitution of StStHHYY (2n = 6x = 42). The St genome originates from the genus Pseudoroegneria, the H genome from Hordeum, and the Y genome’s origin is unknown and still on debating (Wang et al., 1994; Lei et al., 2022; Liu et al., 2022). E. nutans distributed on the Qinghai-Tibet Plateau show highly divergent adaptation, growing on grassland, bushland, wetland, mountain slopes, and swales, at altitudes from 1,000 to 5,000 m, and is tolerant to various environmental stresses associated with high altitudes, such as cold and drought. E. nutans shows great variability in the Qinghai-Tibet Plateau, in morphological characteristics (Zhang et al., 2009), inter-simple sequence repeat analysis (Chen S. Y. et al., 2009), sequence-related amplified polymorphism (Chen Z. H. et al., 2009), amplified fragment length polymorphism (Yan et al., 2010), gliadin variation (Miao et al., 2011), and simple sequence repeats analysis (Chen et al., 2013). In addition, E. nutans in this region showed high chromosomal variation. Chromosomal rearrangements (translocations or inversions) were first suggested from regular analysis of chromosome pairing in a hybrid between parents from two different populations (Lu, 1993). Later, 42 chromosomes of E. nutans were well recognized and allocated three different genomes (St, H, and Y) by using fluorescence in situ hybridization (FISH) with tandem repetitive sequences and total genomic DNA as FISH probes (Dou et al., 2009, 2017). High molecular karyotype variations were identified in intra- and inter-populations of E. nutans, and highly frequent intergenomic translocations were identified, and chromosomal variations with repeat amplification and deletions were exclusively identified in the aforementioned studies (Dou et al., 2009, 2017). However, chromosomal inversions are still difficult to determine because of technical limitations. Moreover, the rise in chromosomal variation in E. nutans is still unknown.

Meiosis is a unique process in which four haploid daughter cells are generated from a diploid parent cell after a single round of DNA replication and two consecutive nuclear divisions. Accurate segregation requires coordinated execution of conserved processes occurring throughout the two meiotic cell divisions. Meiotic recombination is closely connected to homologous chromosome pairing, crossovers, and reciprocal chromosome segment exchange forms. Recombination during meiosis is initiated by forming double-strand breaks (DSBs) in chromosomal DNA (Zamariola et al., 2014). Misrepair of DNA DSBs, particularly by non-homologous end-joining DSBs, may lead to primary chromosomal structural variations (Schubert, 2007; Gaeta and Chris Pires, 2010). In heterozygotes with rearranged chromosomes, chromosomal abnormalities can occur in different meiosis stages, which can cause variations in the second chromosome structure (Livingstone and Rieseberg, 2004; Schubert, 2007). Low to high frequencies of heterozygotes have been revealed in domesticated and wild populations of E. nutans by molecular karyotyping (Dou et al., 2009, 2017). The association between karyotype heterozygosity and low seed fertility has also been demonstrated, reflecting the involvement of chromosome rearrangements in the heterozygotes of E. nutans (Liu et al., 2021). In this study, an individual of E. nutans with a heterozygous karyotype and low fertility was used, and detailed cytological observations were carried out at different meiosis stages with each chromosome clearly identified by FISH. The results are expected to reveal the particular inversions previously undiscovered and help to elucidate the rise of chromosome variation in E. nutans.

The correct completion of meiotic cell division involves a sequence of coordinated steps during the two phases of meiosis. In this study, chromosomal abnormalities were detected at nearly every division step. Homologous chromosome recognition is a key step during meiosis. The efficacy of meiosis relies heavily on the accuracy of the chromosome pairing. Chromosome pairing is more challenging in polyploid plants because of the presence of duplicated homologous or homoeologous chromosomes. In our study, a few chromosome pairing aberrations were observed; however, no homoeologous chromosome pairing between different genomes was detected. This suggests that homologous pairing in E. nutans is well regulated by genetic elements such as the Ph1 locus in allopolyploid wheat (Riley and Chapman, 1958; Sears, 1976). The pairing of homologous chromosomes during meiosis is determined prior to and concomitant with meiotic recombination, referred to as recombination-independent and recombination-dependent chromosome pairing, respectively (Grusz et al., 2017). Ph1 has been implicated in many recombination-independent events, such as centromere pairing preprophase (Martinez-Perez et al., 2001), presynaptic alignment of chromosomes (Feldman, 1993), and changes in chromosome condensation that prevent DSB repair by homologous chromosomes (Greer et al., 2012). In this study, chromosome pairing aberrations were detected in diakinesis and metaphase I on a few chromosomes, most of which also showed FISH pattern polymorphisms between homologs in mitotic cell chromosomes. This indicates that chromosome pairing aberration is leaded by the diverse sequences or structure of the homologous chromosomes, since the parents of the investigated hybrid may be differentiated in these chromosomes or chromosomal regions. The frequency of chromosome pairing varies between different chromosomes and genomes. The St and H genomes presented distinctly higher pairing aberrations than the Y genome. These results are in accordance with the cytological investigation of E. nutans with varying fertility, suggesting that the St and H genomes may contain more chromosomal structural variations than the Y genome (Liu et al., 2021).

Chromosomal fragments were observed in both diakinesis and anaphase I. The fragments in diakinesis were mostly identified as terminal fission, which was further confirmed by identifying deletions in the corresponding chromosomes during metaphase I. The pairing and subsequent segregation of homologous chromosomes are achieved by chromosomal recombination during meiosis I through the formation of DSBs (Zickler and Kleckner, 2015). DSBs are repaired by DSB repair or by non-homologous end-joining of DSBs (Szostak et al., 1983; Gaeta and Chris Pires, 2010). Errors during non-homologous end-joining of DSBs can result in insertions and deletions at the ligation site (Pipiras et al., 1998). Chromosome pairing in E. nutans is strictly regulated by the Ph1 system, which prevents the repair of DSBs by homoeologous chromosomes (Greer et al., 2012). Therefore, the breaks in diakinesis in a few chromosomes that were not well paired were probably initiated by failing to repair the DSBs by non-homologous end-joining of DSBs. Two types of fragments were observed in anaphase I. The fragments in the equatorial plate were mostly derived from the crossover of homologous chromosomes involving paracentric inversions, which were confirmed by fragments along with bridges in a few cells. However, fragments were also detected in a few of poled chromosomes. A few of these were identified as chromatid breaks using distinct chromosomal markers. Sister chromatid cohesion is essential for faithful chromosome segregation, and the cohesion complex is highly conserved in eukaryotes (Zamariola et al., 2014). DSBs occurring in sister chromatids are repaired using the intact sisters as the repair template by homologous recombination through generating junction molecules. Defective resolution of junction molecules not only leaves DSBs unrepaired but also has the broken sisters remain entangled with the intact sisters, leading to the formation of isochromatid breaks, where both chromatids of a single chromosome are broken at the same sites, in mitotic chromosome spreads (Shimizu et al., 2020). The staggered breaks, either chromatid or isochromatid breaks, rather than ectopic exchange between inverted repetitive sequences, was suggested to be the prevalent mechanism for the generation of inversions in the melanogaster species group evidenced by genome sequences (Ranz et al., 2007). In this study, the chromosome fragments in diakinesis strongly suggested isochromatid breaks involvement. Especially, the identified de novo inversions in 3H was distinctly featured with isochromatid breaks. Whether the breaks formation in meiosis of E. nutans is the same as the chromatid breaks described in mitotic cells (Shimizu et al., 2020) needs further investigation. The sister chromatids with breaks were frequently heterozygous in anaphase I (Figures 4N,O), as are chromosomes with the isochromatid breaks in diakinesis (Figure 2). Most of the inversions identified in different ecotypes of sunflower are introgression chromosomal regions from related species (Todesco et al., 2020). If the inversions in sunflower are caused by isochromatids breaks, the sequences divergence of the introduced blocks should be considered to play a role to initiate the chromatid or isochromatid breaks. Given the high chromosome heterozygosity of the chromatid and isochromatid breaks in this study, the chromatids junction may be affected by structural variation or sequence divergence between them.

In this study, bridges were observed in anaphase I and anaphase II, whereas their frequencies were higher in anaphase II than in anaphase I. Breakage-fusion-bridge was classified as chromosome type and chromatid type by the breakage and fusion points between chromosomes and chromatids, respectively (Lukaszewski, 1995). Two main factors may be responsible for bridge formation in anaphase I. The bridges resulted from the crossover between homologous chromosomes involved in structural variations, such as inversion, and the others were de novo chromosome type breakage-fusion-bridge, derived from the breakage and fusion between homologous chromosomes in diakinesis. The frequency of fragments that occurred from chromosome breakage was higher in diakinesis than in bridges in anaphase I on a few chromosomes suggesting that the broken points were healed by initiated telomere sequences or that the breakages were fused between chromatids. The breakage and fusion in diakinesis between chromatids may be responsible for the high frequency of bridges in anaphase II.

Inversion, a kind of special chromosomal rearrangement, plays a very important role in speciation and local adaptation (Dobzhansky and Sturtevant, 1938; Rieseberg, 2001; Todesco et al., 2020). Inversions include pericentric inversion, which involves breakpoints at different distances from either side of the centromere, and paracentric inversion, where breakpoints occur on one arm at different distances from either side (Schubert, 2007). During meiosis, paracentric inversion distinctly features bridges and fragments during anaphase I. In this study, chromosome 2St showed a frequency of 2.198% bridges accompanied with fragments in anaphase I, which strongly suggests a paracentric inversion in 2St. Since more than one chromosome showed chromosome aberration in anaphase I and other steps, other pericentric inversions or paracentric inversions involving short inversed segments may still require further confirmation. In previous studies (Dou et al., 2009, 2017), many chromosome variations, except inversions, have been identified in mitotic cell analysis. In this study, inversion was first determined using meiosis analysis. E. nutans is widely distributed and adaptable to the Qinghai-Tibet Plateau. Whether the ecological differentiation of E. nutans is strongly associated with the variety of inversions still needs further elucidation. Chromosome inversion can be finely identified using advanced molecular cytogenetic methods capable of detecting structural variations using single-copy genes (Danilova et al., 2017; Said et al., 2018) and even using a new approach of long-read sequencing and optic mapping (Staňková et al., 2016; Ho et al., 2020). Our study showed that molecular cytogenetic investigation of meiosis also provided valuable information on chromosomal structural variation in a conventional and low-cost method, especially for polyploid Triticeae species with large genomes. In the future, more major inversions in E. nutans can be detected using a combination strategy.

E. nutans demonstrates a high frequency of variation of intra- and inter-population molecular karyotypes. The reason for this high chromosomal variation is unclear. In the present study, primary chromosomal structural variations derived from the misrepair of DSBs were exclusively identified, which was confirmed by the deletion of chromosomes and de novo inversions in metaphase I. In addition, secondary chromosomal structural variations resulting from the unstable products of primary structural variations were still uncovered. If the novel chromosome variants were successfully transferred to the next offspring from the gametes, the chromosome diversity would increase in this way in the populations. Meiosis abnormalities have also been reported in E. nutans plants from West Himalaya, India, depicting the phenomenon of cytomixis and associated meiotic abnormalities (viz. non-synchronous disjunction of bivalents, chromatin bridges, laggards, micronuclei, and shriveled microspores) (Singh et al., 2020). In particular, one of the three plants exhibited fragments in metaphase I, that were suggested to be B chromosomes (Singh et al., 2020). In our study, the fragments detected during diakinesis were exclusively identified as deleted fragments. Similar results have prompted the discovery of universal mechanisms responsible for meiosis abnormalities in E. nutans populations. Meiosis behavior of individual chromosomes was observed in the present study, and meiosis abnormality in the individuals was closely related to the heterozygous FISH patterns of the homologous chromosomes detected in mitotic cell metaphase. The polymorphism between homologous chromosomes suggests variation in chromosomal structure or sequence divergence in the parts or segments between them. Introgression hybridization with related species can introduce structural variations with divergent blocks in the genome (Todesco et al., 2020). E. nutans exhibits high cross-ability with other Elymus taxa (Lu, 1993), and interspecies hybrids have been frequently observed in the field (Lu et al., 2019). This suggests that the genome of E. nutans may contain diverged chromosomal segments due to introgression hybridization with related species. Meiotic recombination occurs predominantly among allelic regions of homologous chromosomes (Naranjo and Corredor, 2008). In the heterozygous state, the introduced homeologous or non-homologous segment from the same genome of the related species may be responsible for the recombination failing between the homologous chromosomes and triggers the meiosis abnormalities. Transposable elements can cause chromosomal breakage and promote chromosomal aberrations, such as translocations, inversions, deletions, duplications, and fragment formation (McClintock, 1946; Kunze et al., 1997; Bennetzen, 2000; Kidwell and Holyoake, 2001). Bursts in transposable elements can be driven by stress, environmental change, and interspecific hybridization (Belyayev, 2014). Whether activated transposable elements in a heterozygous state induce meiosis chromosomal abnormalities requires further investigation. Chromosomal variation plays an important role in the high genetic diversity and wide adaptation of E. nutans, although the mechanism to maintain it is intriguing. Investigating diverse E. nutans plants widely in a natural state or under controlled conditions using a combination of cytological and genomic tools may elucidate this phenomenon in the future.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

QD conceived and designed the experiments. BL performed the experimental work. QD and BL analyzed the data and wrote the manuscript. XT assisted in experimental work. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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