Within the Aveneae/Poeae tribe complex, most grass species were shown to have a basic chromosome number x = 7, though other basic chromosome numbers have also been found (http://www.tropicos.org/project/ipcn). Besides, a natural polyploid series as well as variation in ploidy level were revealed [4, 15, 27, 39]. Polyploidy is widespread in grass species, and it considered to play an important role in the evolution of vascular plants [53, 54]. The speciation within the Aveneae/Poeae tribe complex has been accompanied with episodes of polyploidy and intergeneric hybridization between the representatives of this tribe (especially, having hybrid or/and rearranged genomes) resulted in appearance of allopolyploid species [55-58]. Polyploidization events often seem to be associated with increases in vigor followed by adaptation of newly formed polyploids to novel conditions [55-57], so polyploids are able to colonize larger geographic ranges and/or occur in more habitats than related diploids [54]. The genera Alopecurus, Arctagrostis, Beckmania, Deschampsia and Holcus comprise widespread polyploid species and/or polymorphic forms which are highly tolerant to stressful and/or variable environmental conditions [6, 8-10, 55, 59]. The species A. aequalis, A. arundinaceus, A. latifolia, B. syzigachne, D. cespitosa, D. flexuosa, D. sukatschewii and H. lanatus are predominant grasses within the pastures of the Arctic and sub-Arctic regions [6, 10, 11]. However, only scattered data on their distribution in the vast territory of Eurasia is currently available. In the present study, we constructed the integrated schematic maps of their occurrence in the northern, central and eastern parts of Eurasia based on the currently available data [2, 6, 45-52]. These maps indicate the vast areas with multi-species occurrence and also the regions (predominantly in the Far North and the Far East) where only certain grass species are now distributed. In these regions, shortage in forage resources could be recovered through the introduction of the other Arctic grassland species with high levels of adaptation and seed productivity as well as developing new valuable cultivars with the use of promising wild morphotypes [11-13]. The grassland accessions examined in the present study were sampled in different parts of the sub-Arctic tundra (North West and Far East regions and also the highlands with sub-Arctic mountain climate) characterized by harsh climate, shallow soils, permafrost, high levels of UV radiation, low participations, strong winds, etc. In plants grown under various abiotic environmental stresses, different cytogenetic abnormalities (mixo-, aneu- and polyploidy, chromosome rearrangements, variability in chromosome number, distribution of rDNA loci and other DNA repeats, appearance of B chromosomes, etc,) are especially common [27, 37], and this karyotype diversity is considered to be related to the plasticity of plant genomes [60, 61]. Despite the stressful natural environmental conditions, the cytogenetic analysis showed that the studied accessions presented normal diploid (2n = 2x = 14, A. aequalis, A. longiglumis, B. syzigachne and H. lanatus) and tetraploid (2n = 4x = 28, A. arundinaceus, A. latifolia and D. flexuosa) karyotypes with the typical for cereals basic chromosome number x = 7, except for the paleopolyploid D. cespitosa having 2n = 26 chromosomes. Our findings agreed with the cytological data reported earlier [36, 37, 62]. Particular, for D. cespitosa and some other Deschampsia species with 2n = 26, the analysis of meiotic chromosome behavior confirmed the basic number of chromosomes (n = 13) and also their diploid status [63]. This unusual for cereals chromosome number could be related to descending dysploidy, one of the most crucial routes of post-polyploid genome diploidization, which is described for several taxa of the Aveneae/Poeae tribe complex [64].
In some cells of the studied D. cespitosa accession and also in one metaphase plate of the H. lanatus accession, we detected supernumerary small chromosomes with uncertain morphology and distinct DAPI bands. The frequency of those supernumerary chromosomes varied between individual plants as well as within root meristem of each plant. They could be referred to B chromosomes because Bs are known to be extra karyotype components which are generally smaller than the normal chromosomes (A chromosomes), often possess heterochrornatic segments and exhibit non-Mendelian inheritance [65]. B chromosomes have previously been revealed in various grass species including D. cespitosa [37, 62, 65]. Their appearance in a karyotype is associated with genome instability though functional role of Bs is still not fully understood [65-66]. In this study, the appearance of Bs in karyotypes of D. cespitosa and H. lanatus accessions could be related to environmental stress factors as a correlation between the presence of Bs in a karyotype and environmental conditions was described earlier [67-68]. At the same time, the B chromosome detected in H. lanatus could be a fragment resulted from a break involving nearby chromosome 7 (which was smaller than its homolog). Considering that Bs have not previously been described in H. lanatus, further molecular cytogenetic studies of different accessions of this species are required.
Repetitive DNAs are major components of plant genomes which have high evolution rates and can lead to genome diversity [61, 69]. Knowledge of cytogenetic positions of specific repetitive sequences (chromosomal markers) provides information on genome structure differences which is important for analysis of the structural evolution of plant chromosomes [70]. Particularly, in karyotypes of vascular plants, DAPI staining, performed after FISH or GISH procedures, reveals AT-rich heterochromatin (which comprises highly repetitive DNA sequences) as strongly stained bands [71]. Clustered localization of highly repetitive DNA sequences (large distinct DAPI-bands) was observed on chromosomes of A. arundinaceus, A. latifolia, D. cespitosa, D. flexuosa and H. lanatus whereas in karyotypes of the other species, including A. longiglumis, we detected small DAPI-bands. The similar chromosomal distribution of constitutive heterochromatin was earlier described for different Avena species with the use of C-banding technique [30-32]. The comparison of the species distribution areas and their cytogenetic peculiarities indicated that karyotypes of two accessions from sub-Arctic mountain tundra (H. lanatus and previously described D. sukatschewii [37]) possessed larger DAPI-bands (constitutive heterochromatin,) compared to the other accessions. This could be related to different pathways of plant genome reorganization (probably, for adaptation to the extreme environments) which involved highly repeated DNAs.
Physical mapping of ribosomal 35S and 5S DNA on chromosomes of diploid and polyploid plant species provides information on the structural evolution of the chromosomes carrying these sequences [28, 29, 72], whereas nucleotide similarity among diploid and polyploid rDNA copies reveals some of their phylogenetic and genomic relationships [73]. In this study, the comparative karyotypic analysis of the diploid and tetraploid accessions of Alopecurus (A. aequalis and A. arundinaceus) revealed chromosomes with similar morphology and distribution patterns of 35S rDNA, 5S rDNA and DAPI-bands (e.g., chromosome 1 in A. aequalis and chromosome 2 in A. arundinaceus; chromosome 6 in A. aequalis and chromosome 10 in A. arundinaceus) which could be related to the allopolyploid origin of the A. arundinaceus genome. Interestingly, in both Alopecurus arundinaceus and Avena longiglumis (Al genome) karyotypes, we indicated one pair of chromosomes with the similar pattern of multiple rDNAs localization (large terminal 35S rDNA and distal 5S rDNA sites in the short arm and also interstitial 5S rDNA loci in the long arm). The similar chromosome pair was earlier described in diploid and polyploid Avena species with different types of the A genome [30-34], and it could be inherited from a common progenitor at a remote period. In karyotypes of A. latifolia, H. lanatus and D. flexuosa, we observed a large chromosome carrying a distal 35S rDNA site in the short arm and interstitial 5S rDNA loci in the long arm. The occurrence of multiple rDNA sites localized in specific chromosomes may have value in chromosome identification and elucidation of evolutionary relationships and also delineation of possible break point sites [29, 74].
Currently, microsatellite DNA sequences are widely used as FISH probes for cytogenetic studies as they are major components of many plant genomes [33, 34, 75]. Particularly, it has been recently determined that FISH with the oligo-GTT probe produces six constant signals located in the pericentromeric regions of three chromosome pairs of diploid A genome Avena species with minor interspecies differences in signal intensity [34]. These data indicate genomic variations among AA species and agree with the results of C-banding analysis [30-32] and Southern hybridization [76]. In the A. longiglumis accession studied here, FISH with the oligo-(GTT)9 probe revealed not only the six cluster signals mentioned above but also a number of minor signals which demonstrated intraspecific variability in chromosomal distribution of this microsatellite motif. Interestingly, on chromosomes of two species, Avena longiglumis and Alopecurus arundinaceus, clustered (GTT)9 signals were detected in the pericentromeric regions. In karyotypes of the other studied species, only small distal or subterminal (GTT)9 sites were observed. This agree with the molecular phylogenetic and cytogenetic data reported earlier [5, 34] and could be related to distant relationships between these species.
According to current molecular phylogenetic studies, the studied here grass species (except A. longiglumis) are included in chloroplast group 2 (Poaeae type) which is subdivided into two clades comprising genera 1) Avenella, Deschampsia, Holcus and 2) Alopecurus, Arctagrostis, Beckmannia [1]. In the present study, we used a rapid GISH approach to reveal common homologous DNA repeats in karyotypes of the studied species groups. We have previously reported that a rapid GISH procedure with genomic DNA of D. cespitosa revealed multiple large hybridization signals on chromosomes of D. sukatschewii (confirming their close relationships). We have also found that D. sukatschewii genome was rich in AT-heterochromatin [37]. Besides, the species from both Deschampsia and Holcus contain common DNA repeats CON1, CON2, COM1 and COM2 which are widespread in Poaceae [15]. In the rapid GISH assays performed in the present study, we used labelled genomic DNAs of D. sukatschewii and H. lanatus (chloroplast group 2) to reveal common DNA repeats on most studied species except H. lanatus. For H. lanatus chromosomes, we used genomic DNAs of D. sukatschewii and D. flexuosa as D. flexuosa differed karyotypically from D. sukatschewii and D. cespitosa [37]. The performed rapid MC-GISH analysis showed that species from both chloroplast groups possessed common DNA repeated sequences as clustered hybridization signals of genomic DNAs of both H. lanatus and D. sukatschewii were revealed in different positions on chromosomes of A. aequalis, A. arundinaceus, B. syzigachne, D. cespitosa and D. flexuosa. On chromosomes of A. latifolia, visual analysis revealed more hybridization signals of genomic DNA of H. lanatus (which therefore, indicated more genome similarities between these species) compared to genomic DNA of D. sukatschewii. At the same time, on chromosomes of A. longiglumis, which belongs to chloroplast group 1 (Aveneae type) [1, 5], we mostly observed dispersed hybridization signals of genomic DNAs of both H. lanatus and D. sukatschewii. Thus, our findings generally agree with the molecular phylogenetic data reported earlier [1, 5].
The genus Beckmannia comprises two perennial species, B. eruciformis and B. syzigachne. This genus has been subjected to several taxonomic revisions. Based on morphology, Beckmannia has been assigned to tribes Phalarideae, Chloriideae, Beckmanniinae, and finally Aveneae (subtribe Alopecurinae) [77]. Until recently, molecular phylogenetic studies have not separated Beckmannia in a distinct lineage, tentatively leaving it within subtribe Alopecurinae [78-80] though support for potential separating Beckmannia and Alopecurus from each other has been provided [78]. Finally, however, Soreng et al. [1] has placed the genera Alopecurus and Beckmannia in two different subtribes (Alopecurinae and Beckmanniina). In support of these recent data, the molecular cytogenetic analysis performed in the present study did not reveal any karyotypic similarities between B. syzigachne and both diploid and tetraploid Alopecurus accessions. Nevertheless, further investigations of Beckmannia species based on different chromosomal and molecular markers are necessary to clarify the phylogenetic position of Beckmannia within the Aveneae/Poeae tribe.
Distribution patterns of the examined molecular cytogenetic markers in the studied here sub-Arctic accessions of D. cespitosa and D. flexuosa agreed with our previous results obtained for non-polar accessions [37]. Nevertheless, differences in number and size of some DAPI-bands as well as in localization of several chromosomal markers (mainly due to chromosomal rearrangements) were also observed. Molecular cytogenetic analysis of these species showed that their karyotypes differed significantly from each other. These results agreed with our previous findings indicating that D. flexuosa also had basic karyotypic differences with D. antarctica, D. danthonioides, D. elongata, D. sukatschewii and D. parvula [37]. According to the phylogenetic analyses inferred from nuclear ITS and plastid trnL sequence data, D. flexuosa is regarded a better suited to the genus Avenella [81]. However, rapid GISH assays with labelled genomic DNAs of both D. sukatschewii (in the present study) and D. cespitosa [37] detected clustered signals on chromosomes of D. flexuosa indicating the presence of common homologous highly repeated DNA sequences in their genomes.
It should be noticed that phylogenetic position of the genus Deschampsia within the family Poaceae is still controversial. According to Soreng et al. [1], Deschampsia, Holcus and Vahlodea were classified in the subtribe Holcinae which is not, however, monophyletic because Holcus and Vahlodea do not form a clade with Deschampsia in the plastid and nuclear ribosomal DNA trees [21, 22, 82]. In a parallel classification of grasses, Holcinae is treated as a synonym of Airinae [4]. Some recent authors render the genus Deschampsia paraphyletic [83] or consider that Deschampsia would be better treated in its own monotypic subtribe [23]. Our findings agreed with the last of these observations as the comparative molecular cytogenetic analysis of Deschampsia species and Holcus lanatus did not reveal similarities in distribution patterns of the studied chromosomal markers.
In karyotypes of B. syzigachne, D. cespitosa and D. flexuosa accessions sampled in different parts of the sub-Arctic tundra (Far East and North West regions), chromosomal rearrangements were revealed. The presence of numerous chromosomal rearrangements in plant karyotypes is considered to be related to the genome plasticity [60, 61] and/or to speciation events [70, 84]. Accordingly, the process of genome evolution in these taxa could include chromosomal reorganization (chromosome interchanges, inversions, translocations) of the initial parental genomes.
Thus, the results of the present study provide unique information on distribution areas and cytogenomic structures of valuable Arctic and sub-Arctic pasture grass species from related genera of the Aveneae/Poeae tribe which revealed structural differences and also similar features in their karyotypes. The obtained results can be a basis for the further genetic and biotechnological studies.