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, 35]. 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].
We found 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 = 4x = 26 chromosomes. Our findings agreed with the cytological data on these species reported earlier [35, 37, 60]. Despite the stressful natural environmental conditions (low temperatures, strong winds, low participations, high ultraviolet radiation, etc.), aneuploid karyotypes were not revealed in the accessions examined in this study. At the same time, different ploidy levels including di-, tri-, tetra- and mixoploid cytotypes had earlier been described in several taxa of the almost cosmopolitan Deschampsia genus which probably were related to the genome plasticity of this species [27, 37, 60].
In karyotypes of the accessions of the most widespread species D. cespitosa and H. lanatus, we detected supernumerary B chromosomes (Bs). In D. cespitosa, these supernumerous chromosomes were observed earlier [37], while in H. lanatus, they were revealed for the first time. Bs are dispensable components of the genome exhibiting non-Mendelian inheritance which are found in all eukaryotic phyla and are thought to stand for a specific type of selfish DNA [61]. The molecular mechanism and a functional role of Bs still remain unclear; though their appearance in a karyotype is associated with genome instability [62]. Particularly, the appearance of Bs in karyotypes of D. cespitosa and H. lanatus could be induced by environmental stress factors as a correlation between the presence of Bs in a karyotype and environmental conditions was described earlier [63-64].
After polyploidization or under the environmental stress, different genetic alterations could occur (structural chromosome rearrangements, aneuploidy, aneusomaty, direct changes in the DNA sequence (point mutations), loss of duplicated genes and gene conversion) [57]. In Poaceae, repetitive DNA sequences are a major genomic component which have high evolution rates and can play an important role in speciation processes [65]. 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 [66]. Particularly, in karyotypes of vascular plants, DAPI staining, performed after FISH or GISH procedures, reveals AT-rich heterochromatin which consists largely of highly repetitive ('satellite') DNA) as strongly stained bands [67]. In karyotypes of the studied species, different patterns of AT-heterochromatin distribution (DAPI-banding) were revealed. 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 or dispersed DAPI-bands. The similar nature of chromosomal distribution of constitutive heterochromatin was earlier described for different Avena species with the use of C-banding technique [30-32]. In the present study, the revealed patterns of DAPI-banding were specific to the examined species and were used for chromosome identification.
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, 68], whereas nucleotide similarity among diploid and polyploid rDNA copies reveals some of their phylogenetic and genomic relationships [69]. 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). It has been shown that the genus Avena comprises species with diploid (2n = 2x = 14) A and C genomes which have structural variants (for A genomes: As, Al, Ad, Ap and Ac; for C genome: Cv and Cp), tetraploid (2n = 4x = 28) AB and AC genomes and hexaploid (2n = 6x = 42) ACD genomes. Also, it was assumed that A genome could be a possible progenitor of B and D genomes [39-40]. The above-mentioned chromosome pair with multiple 35S and 5S rDNA localization 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. Such an 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, 70].
Currently, microsatellite DNA sequences are widely used as FISH probes for cytogenetic studies as they are major components of many plant genomes [33, 34, 71, 72]. 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 [73]. 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 subtelomere (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 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, more hybridization signals of genomic DNA of H. lanatus were detected (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) [74]. Until recently, molecular phylogenetic studies have not separated Beckmannia in a distinct lineage, tentatively leaving it within subtribe Alopecurinae [75-77] though support for potential separating Beckmannia and Alopecurus from each other has been provided [75]. 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 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 [78]. 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, 79]. In a parallel classification of grasses, Holcinae is treated as a synonym of Airinae [4]. Some recent authors render the genus Deschampsia paraphyletic [80] or consider that Deschampsia would be better treated in its own monotypic subtribe [23]. Our findings agree 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.
The analysis of distribution of the examined markers in karyotypes of B. syzigachne, D. cespitosa and D. flexuosa revealed different chromosomal rearrangements. The presence of numerous chromosomal rearrangements in plant genomes is considered to be related with speciation events [66, 81]. Accordingly, the process of genome evolution in these taxa could include chromosomal reorganization (chromosome interchanges, inversions, translocations) of the initial parental genomes.
Thus, the comparative molecular cytogenetic analysis of valuable Arctic and sub-Arctic grassland species from the related genera revealed structural differences and also similar features in their karyotypes. The obtained results can be a basis for the further genetic and biotechnological studies.