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Molecular cytogenetic characterisation of Elytrigia ×mucronata, a natural hybrid of E. intermedia and E. repens (Triticeae, Poaceae)
Ladislava Paštová ORCID: orcid.org/0000-0003-4394-51971,2,
Alexander Belyayev1 &
Václav Mahelka1
Interspecific hybridisation resulting in polyploidy is one of the major driving forces in plant evolution. Here, we present data from the molecular cytogenetic analysis of three cytotypes of Elytrigia ×mucronata using sequential fluorescence (5S rDNA, 18S rDNA and pSc119.2 probes) and genomic in situ hybridisation (four genomic probes of diploid taxa, i.e., Aegilops, Dasypyrum, Hordeum and Pseudoroegneria).
The concurrent presence of Hordeum (descended from E. repens) and Dasypyrum + Aegilops (descended from E. intermedia) chromosome sets in all cytotypes of E. ×mucronata confirmed the assumed hybrid origin of the analysed plants. The following different genomic constitutions were observed for E. ×mucronata. Hexaploid plants exhibited three chromosome sets from Pseudoroegneria and one chromosome set each from Aegilops, Hordeum and Dasypyrum. Heptaploid plants harboured the six chromosome sets of the hexaploid plants and an additional Pseudoroegneria chromosome set. Nonaploid cytotypes differed in their genomic constitutions, reflecting different origins through the fusion of reduced and unreduced gametes. The hybridisation patterns of repetitive sequences (5S rDNA, 18S rDNA, and pSc119.2) in E. ×mucronata varied between and within cytotypes. Chromosome alterations that were not identified in the parental species were found in both heptaploid and some nonaploid plants.
The results confirmed that both homoploid hybridisation and heteroploid hybridisation that lead to the coexistence of four different haplomes within single hybrid genomes occur in Elytrigia allopolyploids. The chromosomal alterations observed in both heptaploid and some nonaploid plants indicated that genome restructuring occurs during and/or after the hybrids arose. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile. Hybridisation in Triticeae allopolyploids clearly and significantly contributes to genomic diversity. Different combinations of parental haplomes coupled with chromosomal alterations may result in the establishment of unique lineages, thus providing raw material for selection.
Hybridisation and polyploidisation are the major driving forces underlying plant evolution [1,2,3,4]. While hybridisation through genome merging may lead to the formation of new hybrid species, polyploidisation can stabilise hybrid breeding behaviour [5].
Hybridisation can occur between species of the same ploidy level (homoploid hybridisation) and between species of different ploidy levels (heteroploid hybridisation). In either case, the formation of new hybridogenous species in sympatry requires the presence of reproductive barriers between the hybrid and its parents. In the absence of reproductive barriers, newly formed hybrids can backcross with one or both parental species and form hybrid swarms [6].
The newly formed hybrid possesses a novel combination of genomes, which can manifest improved or enhanced qualities for certain characteristics (hybrid vigour or heterosis). However, hybrid vigour decreases in every subsequent generation of a hybrid's progeny because of the decreasing proportion of heterozygotes.
Allopolyploidisation, i.e., the multiplication of chromosome sets in hybrids, is a mechanism by which the decay of hybrid vigour can be avoided. In allopolyploids, the advantage of heterosis is largely retained due to the enforced pairing of homologous chromosomes and limited intergenomic recombination [1].
The involvement of unreduced gametes in mating is considered to be the prevailing mechanism for the origin of polyploids [7]. Alternative polyploid formation mechanisms include polyspermy (fertilisation by more male gametes; [8]) and (somatic) genome doubling [9]. Thus, the established allopolyploids can generate high ploidy level cytotypes in polyploid complexes.
The changes that occur after polyploid hybrid formation include diverse processes at the molecular, chromosomal, and organismal levels [10]. Coexistence of formerly isolated genomes is often followed by chromosomal restructuring, which contributes to genome variation beyond the merging of genomes [11].
The tribe Triticeae is an extensively studied group of grasses in which hybridisation and polyploidisation have significantly contributed to present-day diversity. Depending on the classification, Triticeae comprises approximately 350–500 taxa in 27–37 genera [12,13,14]. In total, 23 basic genomes were distinguished by Löve, who referred to them to as haplomes [13].
The basic chromosome number in Triticeae is x = 7. The majority of species are allopolyploids, with ploidy levels ranging from tetraploid (2n = 4x) to dodecaploid (2n = 12x). The structure of the tribe is reticulate when certain haplomes are involved in the formation of more than one allopolyploid species [13].
Elytrigia ×mucronata (Opiz) Prokudin [syn. Agropyron mucronatum Opiz, E. apiculata (Tscherning) Jirásek], the subject of this study, is a natural hybrid between E. intermedia (Host) Nevski [syn. Elymus hispidus (Opiz) Melderis, Thinopyrum intermedium (Host) Barkworth & D.R. Dewey] and E. repens (L.) Nevski [syn. Elymus repens (L.) Gould]. Both parental species occur predominantly as hexaploids (2n = 6x = 42) in the study area (Czech Republic), but a minority nonaploid cytotype (2n = 9x = 63) was recorded for E. repens [15, 16]. Natural hybridisation between the hexaploids E. intermedia and E. repens appears to be common and is bi-directional, and evidence of backcrosses has been found [16]. Therefore, no obvious reproductive barriers exist in this species complex. In addition to the prevailing hexaploid E. ×mucronata, hybrids with higher ploidy occur rarely, including heptaploids (2n = 7x = 49) in one population and nonaploids in three populations. The origin of the nonaploid hybrids has been assumed to have involved the fusion of unreduced and reduced gametes, where the unreduced gamete was donated by either of the parental species or their hybrid [16]. The origin of the heptaploids is unknown.
Both parental species are perennial allopolyploids with complex evolutionary histories [17,18,19,20]. The composition of the hexaploid Elytrigia repens genome is assumed to include two subgenomes originating from Pseudoroegneria (haplome symbol St) and one subgenome originating from Hordeum (H) (genomic formula StStStStHH). In addition to the major genome constituents, the E. repens genome contains hints of other lineages from within and outside of Triticeae, demonstrating that the species must have acquired additional genetic material from distant sources [17, 18, 21]. A portion of the foreign genetic material found in E. repens has been inherited from its parental species [22].
The genomic constitution of the allohexaploid intermediate wheatgrass E. intermedia has not yet been satisfactorily resolved (for discussion, see [20, 23]). A consensus has been reached that the species is composed of three distinct subgenomes, one of which originated from Pseudoroegneria (St). The identity of the other two subgenomes remains controversial, particularly due to assumed contributions from Thinopyrum bessarabicum (Eb), Th. elongatum (Ee), Dasypyrum (V), Secale (R), and Aegilops (D) [19, 20, 23, 24]. In this respect, the presence of potential local varieties cannot be entirely excluded. Nevertheless, data from the analysis of native Central European specimens from the Czech Republic (from the same area from which the studied hybrids originated) clearly suggest contributions from Dasypyrum (V) and Aegilops (D) [19, 23]. Therefore, we concluded that these genera represent the donors of the two other subgenomes in this allopolyploid, and we therefore assume the genomic formula of E. intermedia to be StStDDVV.
Molecular cytogenetics techniques have given rise to new possibilities for studying the genomic constitution of hybrid plants. In particular, genomic in situ hybridisation (GISH) is suitable for studying the origins of allopolyploid species (e.g., [18, 25]). Moreover, both numerical and structural chromosomal alterations (especially intergenomic translocations) can be detected in allopolyploids [26, 27]. Furthermore, fluorescence in situ hybridisation (FISH) employing known repetitive sequences as chromosomal markers provides information on their physical localisation within the genome and allows us to study genome restructuring after polyploidisation events [28, 29].
In this study, we performed molecular cytogenetic analyses of three cytotypes of the allopolyploid hybrid E. ×mucronata to characterise their genomic constitutions and elucidate their genome dynamics following hybrid formation. In particular, sequential GISH and FISH were employed to reveal (1) the genomic constitution of the hexaploid, heptaploid, and nonaploid cytotypes of E. ×mucronata; (2) chromosomal alterations in the studied genotypes; and (3) the patterns of the ribosomal 5S and 18S rDNA units with the Triticeae-specific repeat pSc119.2, primarily with respect to their assignment to particular haplomes.
Plant material for this study is available in previous studies [15, 16]. The experiments were performed on two hexaploid (2n = 6x = 42), two heptaploid (2n = 7x = 49), and three nonaploid (2n = 9x = 63) Elytrigia ×mucronata plants from five Central European localities (Table 1, Fig. 1). While the hexaploid and nonaploid plants were used in previous studies focused on genome size variation and natural hybridisation ([15, 16]; for details see Methods Plant material), the heptaploids were studied for the first time in this paper. The plants were identified based on morphological, flow cytometric, and ITS diagnostic markers [15, 16].
Table 1 List of the localities of the analysed plants
Map showing the location of the studied plants of E. ×mucronata. As complementary information, the locations of the parental species analysed in other studies (see text) is given. Blue circle (int) – Elytrigia intermedia; red circle (hyb) – E. ×mucronata; empty circle (rep) – E. repens; combined circle (int + hyb + rep) – all three taxa originated from the locality
Hexaploid Elytrigia ×mucronata
Genome composition
We analysed two hexaploid (2n = 6x = 42) E. ×mucronata plants. The plants 10–1 and 17–4 originated from localities 'Paví vrch' and 'Vrbčany', respectively, (Table 1). After GISH, we observed identical hybridisation patterns in both analysed plants, which consisted of 21 St (Pseudoroegneria) + 7 H (Hordeum) + 7 D (Aegilops) + 7 V (Dasypyrum) chromosomes (Fig. 2a and c; Table 2). A signal was missing in the centromeric regions of Dasypyrum-labelled chromosomes. We are currently not convinced that this pattern is an indication of chromosomal translocations. In contrast, only one of the Hordeum-labelled chromosomes exhibited a Pseudoroegneria probe signal in the centromeric region, suggesting the presence of an intergenomic translocation (Figs. 2a, c and 3).
Mitotic metaphase chromosomes of hexaploid E. ×mucronata after sequential FISH and GISH experiments. a and c GISH in plants 10–1 and 17–4: Aegilops tauschii (digoxigenin, anti-DIG-FITC, yellow pseudocolour), Dasypyrum villosum (biotin, streptavidin-Cy3, blue pseudocolour), Hordeum bogdanii (biotin, streptavidin-Cy3, green pseudocolour), and Pseudoroegneria spicata (Cy5, red). b and d FISH in plants 10–1 and 17–4: 5S rDNA (Cy5, red), 18S rDNA (digoxigenin, anti-DIG-FITC, green), and pSc119.2 (Cy3, white pseudocolour; applied only in 10–1 – b). After FISH, chromosomes were counterstained with DAPI. Structural chromosomal alterations are indicated by numerals (1). Scale bars = 10 μm
Table 2 Number of rDNA and pSc119.2 sites in E. ×mucronata
Schematic patterns of rDNA and pSc119.2 sites revealed in three cytotypes of E. ×mucronata. Only chromosomes exhibiting a signal after FISH are shown. Chromosomes with identical/similar patterns within each haplome are arranged into groups indicated with roman numerals. St haplome – dark red, D haplome – yellow, H haplome – green, V haplome – blue, 5S rDNA – red, 18S rDNA – light green, and pSc119.2 – white. Different types of structural chromosomal alterations are indicated by numerals above the chromosomes (1–7). Total numbers of chromosomes of a particular haplome are given (bottom right side). One Pseudoroegneria chromosome was missing in the analysed metaphase of 41–5
Chromosomal structural variation
In the plant 10–1, in which the complete set of FISH probes was applied, 5S rDNA, 18S rDNA and pSc119.2 signals were located on all of the chromosome sets (haplomes; Figs. 2b and 3; Table 2), with 9, 10 and 18 probe hybridisation sites being observed in this plant, respectively. Pseudoroegneria-labelled chromosomes (St haplome) carried five 5S and four 18S rDNA sites. The 5S rDNA co-localised with 18S rDNA on three chromosomes; on two of these chromosomes, the 5S rDNA loci were situated proximal to 18S rDNA, and on one of them, the 5S rDNA locus was located distal to the 18S rDNA. Additionally, there were two chromosomes carrying solitary subtelomeric 5S rDNA loci and a single chromosome carrying a subtelomeric 18S rDNA locus. Within the Aegilops-labelled chromosomes (D haplome), we detected two chromosomes with co-localised 5S and 18S rDNA loci, where the latter was positioned distal to the former. The Hordeum-labelled chromosomes (H haplome) carried one 5S rDNA site and three 18S rDNA sites. On one of the chromosomes, there was a co-localised rDNA locus containing a double 18S rDNA site with an intervening 5S rDNA site (18S–5S–18S). The V haplome, corresponding to Dasypyrum, carried single 5S and 18S rDNA sites located on separate chromosomes.
FISH with pSc119.2 revealed a disproportion compared to the number of detected sites (Fig. 2b; Table 2). The Pseudoroegneria-labelled chromosomes carried three pSc119.2 sites, one of which was located on the chromosome carrying the single 18S rDNA locus on the opposite arm. A single pSc119.2 hybridisation site in the terminal region was detected within the Aegilops-labelled chromosomes. The H haplome from Hordeum carried three pSc119.2 loci. Two of these loci resided on chromosomes lacking rDNA sites, while the third was located within the co-localised rDNA locus. The seven Dasypyrum-labelled chromosomes carried eleven pSc119.2 hybridisation sites in total. Three of these chromosomes harboured pSc119.2 in both terminal chromosome regions, one chromosome carried two loci on the same chromosome arm; the other chromosomes carried only one site at one chromosome end.
A slightly different pattern was observed in the second hexaploid plant (17–4) upon analyses with FISH with rDNA probes. There were three additional 18S rDNA sites on the Pseudoroegneria-labelled chromosomes and an additional 5S rDNA site on the Aegilops-labelled chromosomes (Fig. 2c and d; Table 2). The obtained results convincingly showed that the hexaploid plants are hybrids between hexaploid E. intermedia and E. repens.
Heptaploid Elytrigia ×mucronata
The two heptaploid (2n = 7x = 49) E. ×mucronata plants C9 and C25B, both from the locality 'Čertoryje' (Table 1), were analysed. The probe hybridisation patterns after GISH slightly differed between the two examined heptaploids. In addition, 28 St + 7 H + 7 D + 7 V chromosomes were found in plant C25B (Fig. 4a), whereas plant C9 exhibited 27 St + 8 H + 7 D + 7 V chromosomes (Fig. 4c). Similar to the hexaploids, five of the Dasypyrum-labelled chromosomes lacked a probe signal in centromeric regions. Several translocations involving all four haplomes were detected in both examined plants. The translocations are described in a separate chapter (see below).
Mitotic metaphase chromosomes of heptaploid E. ×mucronata after sequential FISH and GISH experiments. a and c GISH in plants C25B and C9: Aegilops tauschii (digoxigenin, anti-DIG-FITC, yellow pseudocolour), Dasypyrum villosum (biotin, streptavidin-Cy3, blue pseudocolour), Hordeum bogdanii (biotin, streptavidin-Cy3, green pseudocolour), and Pseudoroegneria spicata (Cy5, red). b and d FISH in plants C25B and C9: 5S rDNA (Cy5, red), 18S rDNA (digoxigenin, anti-DIG-FITC, green), and pSc119.2 (Cy3, white pseudocolour – applied only in C9 – d). After FISH, chromosomes were counterstained with DAPI. Different types of structural chromosomal alterations are indicated with numerals (1, 3, 4, 6). The chromosomal segment broken from the Aegilops chromosome is connected by a dotted line (c and d). Scale bars = 10 μm
The contribution of additional chromosomes compared to the hexaploids is reflected by the increased numbers and altered patterns of the chromosomes marked by repetitive probes. There were differences between the two examined heptaploids regarding the Pseudoroegneria and Hordeum chromosome sets. The patterns within the other two haplomes were identical in both plants (although pSc119.2 was not applied in C25B).
The heptaploid C25B, which carries euploid chromosome sets for each haplome, exhibited twelve and thirteen 5S and 18S rDNA sites, respectively (Fig. 4b; Table 2). The heptaploid C9 exhibited eleven 5S and twelve 18S rDNA sites (Fig. 4d; Table 2). The differences between the two examined heptaploids are due to the acquisition of one extra Hordeum-like chromosome and the loss of one Pseudoroegneria-like chromosome in C9. Thus, in the C25B plant, the Pseudoroegneria chromosomes harboured eight 5S sites and nine 18S rDNA sites, while seven and eight corresponding sites were found in the C9 plant. The number of pSc119.2 sites on the Pseudoroegneria chromosomes in C9 remained the same as in the hexaploid (3 sites on 3 chromosomes). Two chromosomes exhibited the same pSc119.2 pattern observed in the hexaploid. The third chromosome, which displayed a pSc119.2 site on one chromosome arm, also exhibited a co-localised rDNA locus adjacent to the translocated terminal segment from a Hordeum chromosome on the opposite chromosome arm (Figs. 3 and 4c and d; Table 2).
The D haplome from Aegilops exhibited two chromosomes with co-localised 5S and 18S rDNA sites (5S situated proximally); moreover, there was an additional 5S site in the terminal region of one chromosome. No pSc119.2 sites were detected in the D haplome (in plant C9). The co-localised 18S–5S-18S locus, which was observed within the Hordeum haplome in hexaploids, was translocated to a Pseudoroegneria-like chromosome. Thus, the Hordeum haplome of the heptaploids carried only a single 18S rDNA site. However, the pSc119.2 locus residing within the original 18S–5S–18S rDNA locus in Hordeum was no longer detected within the translocated locus in Pseudoroegneria. The Dasypyrum-like chromosome set (in plant C9) harboured the same number of rDNA loci found in the hexaploids (one 5S and one 18S rDNA locus on separate chromosomes) but exhibited only nine pSc119.2 sites on six chromosomes (Figs. 3 and 4d, Table 2).
Nonaploid Elytrigia ×mucronata
Three nonaploid (2n = 9x = 63) E. ×mucronata plants from two localities were analysed (Table 1). We chose plants with two different assumed origin scenarios for the analyses. While plants 50–1 and 50–7 from locality 'Dolní Dunajovice' may have arisen from a 2n (E. repens) + n (E. intermedia) or 2n (6x E. ×mucronata) + n (E. repens) combination, the nonaploid 41–5 from locality 'Hovorany' may be characterised by either a 2n (E. intermedia) + n (E. repens) or 2n (6x E. ×mucronata) + n (E. intermedia) gamete composition [16].
There were two clear-cut GISH patterns among the analysed nonaploids, which likely reflected their distinct origins (see also Discussion). Two plants from one population (50–1 and 50–7) exhibited a 35 St + 14 H + 7 D + 7 V constitution (Fig. 5a and c). We again encountered a lack of signal from the Dasypyrum probe in the centromeric regions of Dasypyrum-labelled chromosomes. Chromosomal translocations were detected in both plants (for details, see below).
Mitotic metaphase chromosomes of nonaploid E. ×mucronata after sequential FISH and GISH experiments. a, c. and e GISH in plants 50–1, 50–7 and 41–5 (metaphase only with 62 chromosomes): Aegilops tauschii (digoxigenin, anti-DIG-FITC, yellow pseudocolour), Dasypyrum villosum (biotin, streptavidin-Cy3, blue pseudocolour), Hordeum bogdanii (biotin-streptavidin-Cy3, green pseudocolour), and Pseudoroegneria spicata (Cy5, red). b, d and f FISH in plants 50–1, 50–7 and 41–5 (metaphase only with 62 chromosomes): 5S rDNA (Cy5, red), 18S rDNA (digoxigenin, anti-DIG-FITC, green), and pSc119.2 (Cy3, white pseudocolour). After FISH, chromosomes are counterstained with DAPI. Different types of structural chromosomal alterations were indicated with numerals (1, 2, 5, 7). Scale bars = 10 μm
The genomic constitution of the 41–5 plant was different from that of the above-described plants (Fig. 5e). Based on the GISH analysis, 41–5 exhibited 28 St + 7 H + 14 D + 14 V chromosomes. Similar to the other analysed plants, the Dasypyrum-labelled chromosomes lacked a signal in centromeric regions.
The examined nonaploid plants showed variable patterns in the FISH analysis, reflecting their distinct origins and genome additivity (50–1 and 50–7 vs. 41–5). Furthermore, differences in the FISH patterns were observed between two plants with an identical genomic constitution (50–1 and 50–7). In this case, the differences were mainly due to polymorphisms within the chromosome sets originating from Pseudoroegneria. Compared with the hexaploids, there was an increase in the total number of all FISH probes in all examined nonaploids (Table 2).
Plant 50–1 harboured a total of fourteen 5S and eighteen 18S rDNA sites. The pSc119.2 probe hybridised to 21 sites on 16 chromosomes. Five chromosome sets from the Pseudoroegneria St haplome exhibited nine 5S and nine 18S rDNA sites. These rDNA sites co-localised on five chromosomes, while four chromosomes harboured solitary 5S rDNA sites, and four other chromosomes harboured solitary 18S rDNA sites (two interstitial and two subtelomeric sites). The St haplome carried four pSc119.2 loci in the terminal chromosome region, two of which resided on 18S rDNA-carrying chromosomes on opposite chromosome arms. The Aegilops-like D haplome carried two 5S rDNA sites, three 18S rDNA sites and one pSc119.2 site. Two 5S and five 18S rDNA sites were detected within the two Hordeum chromosome sets. The 5S and 18S rDNA loci co-localised on two chromosomes. In addition to the 18S–5S–18S rDNA locus observed in both the hexaploids and heptaploids, there was an additional co-localised 5S–18S rDNA locus residing on the same chromosome arm as the pSc119.2 site. In total, six pSc119.2 sites were located on six Hordeum chromosomes. One of the sites resided on the terminal chromosome segment originating from an Aegilops-like chromosome. Similar to the hexaploids, the V haplome from Dasypyrum harboured solitary 5S and 18S rDNA sites located on separate chromosomes. Five out of seven Dasypyrum-like chromosomes carried ten pSc119.2 loci (Figs. 3 and 5b; Table 2).
Plant 50–7 harboured eleven 5S and sixteen 18S rDNA sites. The pSc119.2 probe hybridised at 20 sites on 15 chromosomes. The FISH pattern was similar to that of plant 50–1, with major differences present within the Pseudoroegneria-like chromosomes. These chromosome sets carried five 5S sites and eight 18S rDNA sites. Furthermore, the Aegilops-like chromosomes bore three 5S sites instead of two; two 18S rDNA loci instead of three; and two pSc119.2 hybridisation sites. The rDNA-FISH patterns within the haplomes from Hordeum and Dasypyrum were otherwise identical in the two plants. Five pSc119.2 loci were found on five Hordeum-labelled chromosomes. The number and pattern of pSc119.2 loci were the same in the Dasypyrum chromosome set (Fig. 5d; Table 2).
For plant 41–5, 28 Pseudoroegneria-labelled chromosomes carried four 5S and seven 18S rDNA sites. All of the 5S rDNA sites co-localised with 18S rDNA sites, and three of them were located proximal to 18S rDNA. The other 18S rDNA sites resided on three chromosomes in terminal regions. There were seven pSc119.2 loci located in terminal regions, four of which were located on rDNA-carrying chromosomes.
The Hordeum haplome of the nonaploid 41–5 carried one co-localised locus with a proximal 18S site and a distal 5S rDNA site, residing on the opposite chromosome arm to the pSc119.2 site. Moreover, there was a single 18S locus on a separate chromosome. The rDNA loci in both the D and V haplomes exhibited twice the number of sites found within these haplomes in the hexaploids. The D haplome carried two pairs of chromosomes with co-localised 5S and 18S rDNA sites, and the V haplome from Dasypyrum carried two 5S and two 18S sites, all of which resided on separate chromosomes. While the V haplome harboured seventeen pSc119.2 sites on 11 chromosomes, no pSc119.2 sites were detected within the D haplome (Figs. 3 and 5f; Table 2).
Chromosomal alterations – overview
Seven types of structural alterations and one numerical alteration occurred in the three E. ×mucronata cytotypes. The following structural alterations were found (Table 3). (1) H/St translocation of a Pseudoroegneria-derived centromeric chromosomal segment to a Hordeum chromosome, which occurred in all the analysed plants (twice in C9, 50–1 and 50–7) (Figs. 2a, c, 4a, c and 5a, c and e) and was the only type out of the seven that has also been detected in either parental species of E. ×mucronata (the translocation was detected on a pair of chromosomes in E. repens [18]). Thus, this translocation is supposed to be inherited from a parent (see Discussion). The translocated chromosome carried a pSc119.2 site in the terminal region of one chromosome arm (when this probe was used). (2) H/St translocation, in which a Pseudoroegneria-labelled chromosome carried a Hordeum probe signal in its centromeric region, and this translocation only occurred in the nonaploid 41–5 (Fig. 5e). (3) H/St translocation occurred between terminal parts of the Hordeum and Pseudoroegneria chromosomes. Since the translocation caused acquisition of the co-localised 18S–5S–18S rDNA site by a Pseudoroegneria chromosome (Figs. 3 and 4) and disappearance of this site in Hordeum, we classified this translocation as reciprocal, and it occurred in both heptaploids (Fig. 4a and c). (4) H/St terminal translocation occurred when a Pseudoroegneria-like chromosome carried a terminal segment translocated from a Hordeum-like chromosome. This type of translocation occurred only in the heptaploids on two chromosomes (Fig. 4a and c). One of the chromosomes carried a co-localised 5S–18S rDNA site adjacent to the breakpoint and a pSc119.2 site in the terminal region of the opposite chromosome arm (pSc119.2 was applied only in C9). (5) D/H reciprocal translocation of a chromosome segment occurred between Hordeum- and Aegilops-like chromosomes. The breakpoints on both involved chromosomes were close to an 18S rDNA site, and the translocated segment of the Aegilops chromosome included a pSc119.2 site, and it was specific to the nonaploid 50–1 (Figs. 3 and 5a). (6) D/V interstitial translocation is a non-reciprocal translocation of a chromosomal segment from an Aegilops-like chromosome to a Dasypyrum-like chromosome. The translocated chromosome carried two pSc119.2 sites (in interstitial and subtelomeric positions) and was found in both heptaploid plants (Fig. 4a and c). (7) Inversion within a Hordeum-labelled chromosome, where the co-localised 5S–18S rDNA locus was in a mutually inverted position in nonaploid plants 50–1, 50–7 in comparison to 41–5 (the rDNA locus occurred on the same and opposite chromosome arms, respectively; Figs. 3 and 5b, d and f).
Table 3 Characterisation and occurrence of structural chromosomal alterations in the analysed plants and parental species
A numerical chromosomal alteration was observed in one heptaploid plant (C9). Although the total number of chromosomes in this plant was euploid, not all chromosome sets exhibited multiples of seven chromosomes. One missing chromosome from the Pseudoroegneria chromosome set was compensated by an extra chromosome from Hordeum. The identity of the extra and missing chromosomes was traceable based on comparison with the second heptaploid; the extra Hordeum chromosome was one of the two chromosomes with the translocated centromeric region from Pseudoroegneria, although one Pseudoroegneria-like chromosome carrying a co-localised locus for 5S and 18S rDNA was missing (Fig. 4c and d; Table 3).
The occurrence of natural hybrids with high ploidy levels has rarely been documented for hybrid complexes from the Triticeae tribe. For example, there are reports of a heptaploid hybrid between Thinopyrum junceum and Elytrigia repens from Sweden [30] and a nonaploid hybrid of Elytrigia pycnantha and E. repens from France [31]. GISH analysis of the latter hybrid revealed the presence of four haplomes within this nonaploid, which consisted of four chromosome sets from Pseudoroegneria, two chromosome sets from Agropyron, two chromosome sets from Thinopyrum and one chromosome set from Hordeum. These results demonstrate that regular meiosis in higher polyploids may occur in hybrid complexes within Triticeae. However, to the best of our knowledge, the presence of four haplomes has not been shown in hexaploid and heptaploid natural hybrids.
In this study, we show that Elytrigia ×mucronata is an allopolyploid of high complexity, in which all three studied hybrid cytotypes comprised all four haplomes (D, H, St, V) present in the parental species E. repens and E. intermedia. In all but one case (see below), all of the chromosome sets in the hybrids were euploid and consisted of seven chromosomes. While the genomic constitution (i.e., the type and number of specific haplomes/basic genomes) of the E. ×mucronata hybrids reflects the ploidy level of particular cytotypes, it also depends on the type of gametes involved in the origination of particular plants.
If we assume that regular meiosis occurs in the parental species, then the E. ×mucronata hexaploid would harbour three chromosome sets from Pseudoroegneria and one chromosome set each from Hordeum, Aegilops and Dasypyrum (genomic formula StStStHDV). In addition to these chromosome sets, the heptaploid cytotype harboured an additional chromosome set from Pseudoroegneria (StStStStHDV). The genomic constitution of the nonaploid cytotypes differed between plants. Two plants exhibited 35 St + 14 H + 7 D + 7 V chromosomes, while the other nonaploid harboured 28 St + 7 H + 14 D + 14 V chromosomes. We assume that this difference reflects the distinct origins of the nonaploid cytotypes [16] (see below).
Origin of different cytotypes
The genomic constitutions of the analysed plants allowed us to partly infer the types of gametes that gave rise to their origination (Fig. 6). The hexaploid cytotype of E. ×mucronata most likely originated through the merging of two reduced gametes from both parental species.
Proposed scenarios of the origination of E. ×mucronata cytotypes. The analysed cytotypes (6x, 7x, 9x) are presented in coloured boxes; for each cytotype, the genomic formula is given. For parental species, the contributions of unreduced (2n, solid boxes) and reduced (n, dashed boxes) gametes are considered. The contributions of unreduced and reduced gametes are indicated with solid and dashed arrows, respectively. Arrows of the same colour represent one potential scenario
As mentioned above, nonaploid plants most likely originated through the fusion of reduced and unreduced gametes [15, 16]. Mahelka et al. [16] suggested different scenarios for the origin of the hybrid nonaploids 50–1, 50–7 and 41–5 (plants N7, N6 and N8 in the original article). While plants 50–1 and 50–7 may have arisen from 2n (E. repens) + n (E. intermedia) or 2n (6x E. ×mucronata) + n (E. repens) combinations, nonaploid 41–5 may represent either 2n (E. intermedia) + n (E. repens) or 2n (6x E. ×mucronata) + n (E. intermedia) gamete compositions. Since the alternative gamete combinations result in the same genomic compositions, we are not able to discern which scenario truly led to the formation of the analysed nonaploids by using GISH. The involvement of hexaploid E. ×mucronata hybrids in the formation of the nonaploids seems to be more likely because hybrids might more easily produce unreduced gametes than pure species due to disturbed meiosis [9]. The heptaploid cytotype likely resulted from heteroploid hybridisation; however, the exact mode of its origination is difficult to determine. One possibility is that the heptaploid originated after a cross between a hexaploid and an octoploid (2n = 8x = 56). If the hexaploid parent was either E. repens or E. intermedia (gamete n = 3x = 21 = StStH or StDV, respectively), then the gamete from the octoploid would have to have been n = 4x = 28 = StStDV or StStStH, respectively (Fig. 6). However, no octoploid plants have been recorded from this locality. Alternatively, the heptaploids could have originated after a cross between a hexaploid and a nonaploid, in which the latter was the donor of the gamete comprising four chromosome sets. Such a scenario has been observed in Elytrigia wheatgrasses, where a heptaploid plant was found among progeny of the nonaploid hybrid 50–1 collected in the field [16]. It is likely that the pollen donor was either E. repens or E. intermedia. Unfortunately, the genomic constitution of this particular heptaploid was not analysed.
Chromosomal alterations in E. ×mucronata
Chromosomal alterations occurred in all three cytotypes and involved all four haplomes. Most of the translocations involved St and H chromosomes, while the V chromosome from Dasypyrum was involved in only one translocation. The question is whether this difference simply occurred because St and H chromosomes outnumber chromosomes from the other haplomes, or if it stems from different levels of karyotype stability, which may have a strong effect on chromosome restructuring and aneuploidy in Triticeae [32].
Structural chromosomal alterations have been reported in other perennial species harbouring an H and/or St haplome. Dou et al. [33] found two types of non-reciprocal translocations between H and St haplomes and two types of reciprocal translocations between H and Y haplomes in Elymus nutans (2n = 6x = 42, StStHHYY). Different frequencies of chromosomal alterations between particular haplomes were observed in Kengyilia thoroldiana (2n = 6x = 42, StStPPYY). The frequency of P/Y translocations was higher than that of P/St translocations, while no translocations were observed between the chromosomes of the St and Y haplomes [34].
The question arises of how frequently and at which stage of hybrid formation do translocations occur? Without knowledge on the parental species, it is problematic to infer whether particular structural rearrangements have been inherited from the parental species, or whether they have originated de novo in hybrids. Cytogenetic analyses of local accessions of parental species E. intermedia and E. repens showed that in E. repens, one pair of Hordeum chromosomes carried a centromeric H/St 'translocation' [18], corresponding to what we called Type 1 translocation here. In contrast, no translocation that would resemble those observed in the E. ×mucronata plants analysed here were observed in the other parental species E. intermedia [19, 23]. Therefore, we concluded that all but one (type 1) chromosomal alterations in the three cytotypes of E. ×mucronata appeared during hybrids' formation.
In any case, the presence of only one such translocation in the hexaploid hybrid indicates the occurrence of regular meiosis in the E. repens parent. Thus, it appears that no de novo translocations appeared in the E. ×mucronata hexaploid. The presence of two such translocations in two nonaploids (50–1 and 50–7) and one translocation in the other nonaploid (41–5) is consistent with both alternative origins of these nonaploids.
Furthermore, the presence of the D/H reciprocal translocation (type 5) in the nonaploid 50–1 indicates that this plant is not a primary nonaploid hybrid between E. intermedia and E. repens, since recombination between H and D haplomes (which do not co-exist in any of the parents) must have occurred in the hybrid plant. Therefore, an origin involving a 2n (6x E. ×mucronata) + n (E. repens) combination seems more likely in this plant.
The notably higher frequency of chromosomal alterations observed in heptaploid plants contrasted with the results for the other analysed cytotypes. Three of the four types of translocations (types 3, 4, and 6) were not found in other cytotypes, suggesting that several multivalents between homoeologous chromosomes must have occurred during the formation of the gametes, giving rise to the heptaploid plants.
The concurrent presence of both chromosomes with reciprocally translocated segments indicates alternate segregation (i.e., the translocated chromosomes do not segregate and are present in a single gamete). It is unlikely that this state originated from the fusion of two unbalanced gametes. Non-reciprocal translocations can be non-reciprocal per se or may result from adjacent segregation, where normal and translocated chromosomes segregate into one gamete [35].
Moreover, the numerical chromosomal alteration observed in one heptaploid plant was an example of hidden aneuploidy [27, 32], i.e., the absence of one chromosome is compensated by the acquisition of an extra chromosome from another chromosome set.
Chromosomes were also observed carrying differentiating signals in the centromeric regions in Dasypyrum-like chromosomes. This feature has previously been reported for E. intermedia species [19, 23]. Further research is required to reveal the true nature of these GISH patterns and determine whether they resulted from chromosome restructuring or sequence homology.
Mapping of repetitive DNA in E. ×mucronata hybrids
The evolution of ribosomal DNA genes in relation to allopolyploidy is an intensively studied issue (e.g., [36, 37]). rDNA loci are valuable chromosome markers, and the mapping of rDNA loci using in situ hybridisation allows for the evaluation of the progenitor-derivative patterns and positional dynamics of ribosomal loci within allopolyploid genomes. rDNA genes in allopolyploid species may experience contrasting and barely predictable patterns of evolution, ranging from loss of some loci with respect to their progenitors (Zingeria—[38]) over nearly complete additivity (e.g., Thinopyrum ponticum—[39]; Nicotiana—[40]) to increasing the number of rDNA loci (Triticum—[41]). Triticeae grasses are characterised by the ability to change the positions of rDNA loci [42,43,44], which may occur via the dispersion of minor loci, followed by rDNA array magnification and deletion of the original loci thereafter. Loss of rDNA loci is one potential mechanism accelerating the process of concerted evolution [45].
In all investigated hybrid cytotypes, the 5S and 18S rDNA loci were located on all chromosome sets representing the different haplomes. Therefore, the rDNAs of the E. ×mucronata hybrid reflect the principle of genome additivity to a certain degree. However, because E. ×mucronata is a hybrid between two allopolyploid species, the dynamics of the rDNA loci of this hybrid are traceable, provided that the pattern in both parental species is understood. Thus, we characterised specimens of both E. repens and E. intermedia from the same distribution area from which the hybrids originated ([18, 23], Mahelka, Kopecký, unpubl. data). In both parental species, we encountered some reorganisation of rDNA loci with respect to their diploid progenitors, which likely occurred following the origination of the allopolyploids (for discussion, see [18, 23]). The patterns of the rDNA loci of both E. repens and E. intermedia are shown in Additional file 1: Table S1. Notably, severe elimination of all but one minor 45S rDNA locus likely occurred within the Hordeum subgenome in E. repens (genomic formula StStStStHH). Similarly, the elimination of some 45S rDNA loci occurred within the Pseudoroegneria- and Dasypyrum-like subgenomes in E. intermedia (genomic formula StStDDVV). In both species, 5S rDNA was less heavily affected by the loss of the loci than 45S rDNA.
In this study, we predicted the theoretical numbers of rDNA loci in hybrid E. ×mucronata cytotypes (Additional file 1: Table S1) by assuming complete additivity of the parental rDNA loci. From comparison of the observed data, we can infer the positional dynamics of rDNA loci in the hybrids. For this purpose, we consider the 18S and 45S probes to be equally informative.
The total numbers of 5S rDNA loci fell well into the expected numbers in all hybrid plants examined. In the nonaploid 41–5, the number of 5S rDNA loci only fell within the expected range if the [2n (6x E. ×mucronata) + n (E. intermedia)] scenario of its origin was considered. Under the opposite scenario [2n (E. intermedia) + n (E. repens)], a lower number was observed (11 vs. 13–15 expected). This depletion was mainly caused by a smaller number of 5S rDNA sites on Pseudoroegneria chromosomes than expected (4 sites observed vs. 6–7 expected). However, we must note that the real number of loci in this plant may be higher than was observed because one chromosome was missing in the analysed metaphases. Such an observation is otherwise in agreement with the pattern found for 5S rDNA loci in the parental species, in which no major changes in the 5S rDNA loci were recorded ([23], unpubl. data).
In contrast, the total numbers of 18S rDNA sites were always higher than expected in the hybrids of all analysed cytotypes. This observation is especially interesting if we consider that severe losses of 45S rDNA loci had already occurred in both parental species [18, 23]. Restoration of some loci clearly occurred within the Hordeum and, to a lesser degree, Pseudoroegneria haplomes (see hexaploid 10–1) after the hybridisation events. In particular, while examining the Hordeum haplome, we observed co-localised 5S–18S and/or 18S–5S–18S rDNA loci in all the cytotypes (although in both heptaploids, the co-localised locus had been translocated to a Pseudoroegneria chromosome); however, this locus was not observed in E. repens [18]. This pattern was consistent in all three cytotypes, but neither the mechanism of the re-appearance of the loci nor its cause was studied.
We did not probe either of the parental species with the pSc119.2 probe. In other studies, up to 5 chromosomes with one or two pSc119.2 loci (located in a terminal or interstitial position) have been found in E. intermedia [46]. In E. repens, the total number of pSc119.2 sites ranges between 5 and 10 (one interstitial site and others in the terminal region) [47]. Although these plants come from different geographic regions, it appears that similar to 18S rDNA, the total number of pSc119.2 sites detected in E. ×mucronata was higher than that in the parental species.
Hybridisation and polyploidisation are prominent speciation mechanisms in the grass tribe Triticeae. These mechanisms have not only produced new allopolyploid lineages, but the ongoing transfer of genetic material via extensive hybridisation and introgression may also have significantly enriched the gene pools of the newly established lineages, thus providing raw material for selection. Many Triticeae wheatgrasses, including E. intermedia, represent invaluable in situ sources of genetic material that may be useful for wheat improvement. Therefore, the newly established hybrid lineages are both of interest to general science and of particular concern to wheat breeders due to their potential practical impact. We performed molecular cytogenetic analyses using genomic and fluorescence in situ hybridisation on three cytotypes of the allopolyploid E. ×mucronata, a hybrid between two allopolyploid wheatgrasses, E. intermedia and E. repens. In all three investigated cytotypes (i.e., hexaploid, heptaploid, and nonaploid), we observed coexistence of four different haplomes that occurred in the parental species, thus confirming the assumed hybrid origin of the plants. The genomic constitutions of the analysed plants allowed us to partially infer the types of gametes that gave rise to their origins. While the hexaploid cytotype of E. ×mucronata originated through the merging of two reduced gametes from both parental species, the heptaploid cytotype likely resulted from heteroploid hybridisation. The nonaploid plants most likely originated through the fusion of reduced (n) and unreduced (2n) gametes. The different genomic constitutions of the nonaploids showed that along with both parental species, the hexaploid E. ×mucronata should be considered as a donor of unreduced gametes. Several chromosomal alterations observed in both heptaploid and some nonaploid plants occurred during and/or after the formation of the hybrids. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile and produce viable offspring.
Plant material
The plant material used in this study is available as living material from previous studies [15, 16]. All investigated plants were collected by the author (VM) at localities where no permissions were necessary to collect the samples. Details on the sample locations are given in Table 1 and Fig. 1. The experiments were performed on selected hybrid plants and involved two hexaploid (2n = 6x = 42), two heptaploid (2n = 7x = 49), and three nonaploid (2n = 9x = 63) E. ×mucronata plants (Table 1). Hexaploid and nonaploid plants were used in previous studies focused on genome size variation and natural hybridisation [15, 16]. Thus, while hexaploid plants 10–1 and 17–4 correspond to accessions H-30 and H-2 from Mahelka et al. [16], nonaploid plants 41–5, 50–1 and 50–7 correspond to nonaploids N8, N7 and N6 from the same study, respectively. The heptaploids are studied for the first time in this study. The seeds of diploid Triticeae species used for probe preparation were provided by the US Department of Agriculture (USDA) National Small Grains Collection.
Collecting materials and slide preparation
Plants were cultivated in plastic pots filled with perlite in the greenhouse. Root tips were pre-treated in ice cold water for 24–33 h and fixed in fresh ethanol-acetic acid fixative (3:1, v/v).
The fixed root tips were washed in distilled water (twice) and citrate buffer (0.01 M citric acid and 0.01 M sodium citrate buffer, pH 4.8) for 5 min each. Thereafter, the root tips were treated in an enzyme mixture [1% (w/v) pectinase, 1% (w/v) pectolyase and 20% (v/v) pectinase (Sigma, St. Louis, MO, USA) in citrate buffer] for 3–4 h at 37 °C. After treatment, the digested tissue was washed in distilled water.
The slides were prepared using the smear method [48] according to a previous report [49] with several modifications. The digested tissue was carefully transferred to a microscope slide, and a suspension was produced with needles. Cold 75% acetic acid was then immediately added to the suspension, after which the slide was placed on a hot plate and stirred with a needle to spread the cells. Finally, 150 μl of cold ethanol-acetic acid fixative was added, and the slides were washed with ethanol and air-dried.
Probe preparation and in situ hybridisation
The following three probes representing repetitive chromosomal markers were used for the FISH analyses: (1) a 5S rDNA probe, (2) an 18S rDNA probe, and (3) a pSc119.2 probe. Ribosomal 5S and 18S DNAs were amplified using the primers 5SprobeF (5′-GATCCCATCAGAACTCCGAAG-3′), 5SprobeR (5′-CGGTGCTTTAGTGCTGGTATG-3′) [50], 18S-F (5′-CGAACTGTGAAACTGCGAATGGC-3′) and 18S-R (5′-TAGGAGCGACGGGCGGTGTGTG-3′) [51]. The pSc119.2 probe, a 120-bp fragment originally isolated from rye [52], was amplified from Triticum aestivum cv. Chinese Spring DNA according to the reported protocol.
The selection of the diploid species used to obtain the genomic probes was based on analyses of the genomic constitutions of the parental species [18, 19, 23]. Genomic DNAs from the diploid species Aegilops tauschii Coss. (USDA accession identifier PI542278; representing D haplome), Dasypyrum villosum (L.) P. Candargy (PI639751; V haplome), Hordeum bogdanii (PI269406; H haplome), and Pseudoroegneria spicata (Pursh) Á. Löve (PI563869; St haplome) were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The probes were labelled either via nick translation using the Biotin-Nick Translation Kit and the DIG-Nick Translation Kit (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol, or via random primer labelling [53] with direct fluorochromes (Cy3, Cy5; Amersham, Piscataway, NJ, USA).
The following three sequential in situ experiments were performed on mitotic metaphase chromosomes of E. ×mucronata: (i) FISH with the probes for 5S rDNA (Cy5), 18S rDNA (digoxigenin, anti-DIG-FITC conjugate), and pSc119.2 (Cy3, this probe was not applied in plants 17–4 and C25B); (ii) GISH with three genomic probes, from Aegilops tauschii (digoxigenin, anti-DIG-FITC conjugate), Hordeum bogdanii (biotin, streptavidin-Cy3), and Pseudoroegneria spicata (Cy5); and (iii) GISH with a genomic probe from Dasypyrum villosum (biotin, streptavidin-Cy3). The hybridisation pattern was always confirmed in 3–6 metaphases per plant.
Chromosomes and probes were simultaneously denatured at 93 °C for 3 min on a hot plate (ThermoBrite™ Slide Processing System, StatSpin, Norwood, MA, USA). Hybridisation was then performed at 63 °C for 3 h according to the relevant literature [54, 55]. Detection was completed using an anti-DIG-FITC conjugate (Roche, Indianapolis, IN, USA) or a streptavidin-Cy3 conjugate (Sigma, St. Louis, MO, USA) (1 h in 37 °C). Slides were mounted with Vectashield antifade mountant (Vector Laboratories, Burlingame, CA, USA), examined and photographed on a Zeiss Axio Imager.Z2 microscope system equipped with an ApoTome.2. Zen (Zeiss, Jena, Germany) and Adobe Photoshop software (Adobe Systems, San Jose, CA, USA) were used for merging and processing the images.
18S rDNA:
18S ribosomal DNA
5S rDNA:
5S ribosomal DNA
anti-DIG-FITC:
Anti-digoxigenin-fluorescein isothiocyanate
Cy3:
Cy3-dUTP, Cyanine 3-dUTP
4′,6-Diamidino-2-phenylindole
DNA:
Deoxyribonucleic acid
Fluorescence in situ hybridisation
GISH:
Genomic in situ hybridisation
ITS:
Internal transcribed spacer
rDNA:
Ribosomal DNA
streptavidin-Cy3:
Streptavidin-cyanine 3
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We are grateful to František Krahulec for helpful discussions and comments on the manuscript. We thank Andreas Houben for providing pSc119.2 and Michaela Jandová for help with the preparation of the map. Seeds from the plant accessions used for probe preparation were generously provided by the USDA National Small Grains Collection.
The study was financially supported by long-term research development project RVO 67985939 from the Czech Academy of Sciences, project 17-06548S from the Czech Science Foundation (to VM) and project SVV 260439 from Charles University.
The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
All data generated during this study are included in this article (and its supplementary information file).
Institute of Botany, Czech Academy of Sciences, Zámek 1, 252 43, Průhonice, Czech Republic
Ladislava Paštová, Alexander Belyayev & Václav Mahelka
Department of Botany, Charles University, Benátská 2, 128 01, Prague, Czech Republic
Ladislava Paštová
Alexander Belyayev
Václav Mahelka
AB and VM designed the study. LP performed the experiments and analysed the data. LP, AB and VM wrote the manuscript. All authors read and approved the final manuscript.
Correspondence to Ladislava Paštová.
Table S1. Theoretical patterns of rDNA loci in hexaploid (2n = 6x), heptaploid (2n = 7x), and nonaploid (2n = 9x) cytotypes of E. ×mucronata hybrids. Observed values of rDNA loci for the parental species and expected values for the investigated E. ×mucronata cytotypes. (XLSX 12 kb)
Paštová, L., Belyayev, A. & Mahelka, V. Molecular cytogenetic characterisation of Elytrigia ×mucronata, a natural hybrid of E. intermedia and E. repens (Triticeae, Poaceae). BMC Plant Biol 19, 230 (2019). https://doi.org/10.1186/s12870-019-1806-y
DOI: https://doi.org/10.1186/s12870-019-1806-y
Allopolyploidy
Chromosomal alterations
Higher polyploids
Hybridisation
GISH
Thinopyrum intermedium


The sun was shining, the activities were amazing and the crowds poured in to Headingley Stadium for Children's Day 2013.
The annual community event organised by The Yorkshire Cricket Foundation in partnership with the Gary Chapelow Memorial Fund and Leeds Rugby Foundation attracted a record attendance of 6,000.
Activities and tournaments took place throughout the day including archery, pony rides, viking sword fighting, basketball, Eureka to name just a few. West Yorkshire Police also provided a number of activities as part of their open day. The old police cars, crime scene and wanted posters were as always, very popular.
Children's Day was a fantastic event this year! The weather was amazing and it was great to see so many people at the ground enjoying the day and getting involved.
This year Children's Day was building up to the hotly contested cricket match between Emmerdale and West Yorkshire Police. Eric Pollard (Chris Chittle) led the Emmerdale stars but they came second best to the police side by a narrow margin.


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Urgent support call for asylum seekers
Catholic leaders from across the country have called on the Federal Government to provide urgent support to temporary visa holders and the release of asylum seekers from detention, saying they are at unacceptable risk during the coronavirus pandemic.
On 28 April they made public the concerns they raised in a private letter to Prime Minister Scott Morrison and other relevant decision-makers on 7 April as the Government's response was "not substantial".
The 44 signatories included four bishops, leaders of religious institutes, health care providers, charities and refugee advocates.
See the full article from The Catholic Weekly below.

redpajama_set_name: RedPajamaCommonCrawl


The Portland Promise Center is a thriving faith-based community development center in the heart of Louisville's Portland neighborhood. For the past 18 years, we have provided families and individuals in this economically depressed area with the resources they need to anchor their lives to a firm foundation of faith and to build futures filled with hope. Below is a list of items needed for this ministry. Our preference is for new or slightly used items to be donated. For more information, you can contact us at (502) 776-2635 or www.portlandpromise.org.


First Salvatore, then Attilio, today Paolo and the nephews Alessandro and Mauro: the Contini winery has grown and matured like the wines and successes that made it great, through the passion and commitment of four generations.
For almost 120 years a single common thread, represented by the Vernaccia di Oristano, has characterized the work of the Contini company.
Then, over the years, Vernaccia has been joined by other important productions: the historic Nieddera grape, the traditional Vermentino and Cannonau, the successful experiments such as the Karmis, the Attilio and the organic Mamaioa.
Today the company produces a complete range of wines with which it is present in the Italian market and in the foreign market.
The company continues to evolve, both in winemaking techniques and in ideas.
And this is why Contini wines resist time because they are wines that are loved and remembered forever.


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— японська драма 2008 року режисера та сценариста Хірокадзу Корееди, що розповідає про 24 години з життя родини Йокояма, яка щороку збирається у день смерті старшого сина.

У ролях
 Хіросі Абе — Рьота Йокояма
 Юі Нацукава — Юкарі Йокояма, дружина Рьоти
 Ю — Тінами Катаока, сестра Рьоти
 Кадзуя Такахасі — Нобуо Катаока, чоловік Тінамі
 Сьохей Танака — Ацусі Йокояма, син Юкарі
 Кірін Кікі — Тосіко Йокояма, матір Рьоти
 Йосіо Харада — Кьохей Йокояма, батько Рьоти
 Сусуму Тэрадзіма — працівник доставки суші

Критика
Картина отримала виключно позитивні відгуки кіноспільноти. На Rotten Tomatoes фільм має рейтинг 100%, оснований на 64 відгуках. У консенсусі сайту йдеться: «Фільм Хірокадзу Корееди спочатку може здатися невибагливим, але ця сімейна драма накладає делікатне, захопливе заклинання». На сайті Metacritic середня оцінка стрічки, на основі 21 рецензії, становить 89 зі 100

Роджер Еберт у своїй рецензії назвав Корееду послідовником іншого видатного японського режисера Ясудзіро Одзу й поставив фільму 4/4: «Болісні сімейні проблеми часто залишаються під поверхнею, вони відомі всім, але про них не говорять. «Все ще йдемо» — неймовірний новий фільм із Японії, дуже мудрий і дуже правдивий щодо цього». Кеннет Туран із «Лос-Анджелес Таймс» називає фільм правдивим і справжнім, оскільки він «м'яко розкриває тихі моменти, які визначають наші життя». Оглядачі тижневика «Тайм-Аут», Джошуа Роткоф і Девід Дженкінс, оцінили фільм на 5/5. Найнижче (В-) оцінив стрічку Овен Ґлайберман із «Ентертейнмент Віклі», якого розчарував «майже фетишистський емоційний мінімалізм».

Нагороди

Примітки

Посилання
 Офіційний сайт

Фільми Японії 2008
Фільми-драми Японії
Фільми Хірокадзу Корееди

redpajama_set_name: RedPajamaWikipedia


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'Sherlock' Season 4, Episode 2: When and where to watch 'The Lying Detective'
ByJason Berman
Sherlock fans entered the fourth season of the BBC series expecting to see the ramifications of whatever nefarious plan James Moriarty had put in place before his death at the end of season two. As it turns out, though, Sherlock, Watson and the rest of the gang have had to deal with different threats altogether.
In the season four premiere, titled "The Six Thatchers," Sherlock embarked on a case that he suspected may have been connected to Moriarty or the missing black pearl of the Borgias. Instead, he ended up stumbling upon a secret from Mary Watson's past. The end result left Sherlock and John's relationship completely in shambles.
The boys of Baker Street will have to get their act together soon, though, as episode two, titled "The Lying Detective" is set to introduce a villain that has been described as "the purest evil."
When and where to watch "The Lying Detective"
Sherlock fans have a few options for watching the second episode of season four. Of course, if you cannot wait a second longer than you have to for each episode, you can watch "The Lying Detective" on PBS at 9 p.m. Eastern. Should you live in the United Kingdom, the episode will air on BBC One at 21:00 local time.
For those that will not be able to watch live, there are several options to stay up to date. First, the episode should be available on PBS' website shortly after it airs. For those in the United Kingdom, you can watch on BBC iPlayer following the the original broadcast.
If you love Sherlock so much that you just have to own each episode, you can purchase them on Google Play, iTunes and Amazon. Viewers in Norway and other areas of the world can reportedly watch new episodes of season four on Netflix as they are released. Netflix subscribers in the United States aren't as lucky: Season four should become available sometime in June or July for U.S. subscribers, per Netflix Life. Currently episodes of Sherlock from seasons one through three (including "The Abominable Bride") are only available for streaming on Netflix.
Mic has ongoing coverage of Sherlock. Please follow our main Sherlock hub here.


This entry was posted in Horror, Humour and tagged smartphones, zombies. Bookmark the permalink.
I must admit that every time I pass a body of water (usually the Thames), I am tempted to send my phone for a swim. Perhaps a flip phone is the answer – next time I'll downgrade rather than upgrade.


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Aviation Week posted an online article last week that pointed once again to the possibility of accelerated development of a Lockheed Skunk Works designed hypersonic aircraft, now widely known as the SR-72. But the report not only talked about industry buzz and hints from Lockheed that the program may be more mature than originally thought, it also conveyed a sighting account of a demonstrator aircraft.
"All things considered, we are, and have been hearing a tremendous amount of information about what is clearly a highly classified program, coming from the contractor directly no less. This is highly peculiar. And parts of this technology have been in the works for decades. The "combined cycle" propulsion system capable of taking an aircraft from sitting on the runway well through mach five has been in development since at least the mid 2000s by Lockheed and its partner on the project Aerojet Rocketdyne. Additionally, past hypersonic vehicle tests by the USAF have also garnered valuable insights into this extreme flight environment, and there are likely many other major breakthroughs gained over the years from programs we know nothing about.
So why are we getting bread crumb trails dropped to Aviation Week periodically as to the maturity of the technology and the program? Doing so seems like a very strange move considering we don't even hear a peep about far less exotic technological developments—like the existence of advanced unmanned combat air vehicles for instance.
I would posit that it is very possible the Skunk Works and its USAF partners are actually farther along in the SR-72's development than they let on. Possibly even past the point of a small demonstrator, and already fielding a full-size prototype, or even an operational capability. In other words, what we may be hearing about are post-dated echoes of the program's past. The only other reason as to why Lockheed would be so willing to blabber about their hypersonic flying unicorn is that they need money from Uncle Sam to fund it. But that seems unlikely as it is highly doubtful the company has funded the concept's development for over a decade on its own, beyond a relatively small amount of money granted by NASA to explore the technology. In fact, Weiss says that Lockheed is not alone on the project in any way in his latest statement, noting that "DARPA and the services" are directly involved."
...Considering how competent Lockheed and their Air Force colleagues are at keeping secrets, the SR-72's strange "gray world" existence makes little sense. And hey, something has to live in that huge new remote hangar at Area 51 right? Maybe that facility's tenant is the aircraft being described still as a "paper concept" here today.
In the end, I would suggest being open to taking SR-72 related news as more of an indication of what is, instead of what could be."
...According to information provided to Aviation Week, one such technology demonstrator, believed to be an unmanned subscale aircraft, was observed flying into the U.S. Air Force's Plant 42 at Palmdale, where Skunk Works is headquartered. The vehicle, which was noted landing in the early hours at an unspecified date in late July, was seen with two T-38 escorts. Lockheed Martin declined to comment directly on the sighting."
We have no clue where, or more accurately who Aviation Week got this report from but it has spread through the aviation and defense communities like wildfire. The problem with publishing something like this without the author providing further context is that it is taken by many as fact, which it is not.
The sighting itself, as described, has its own issues. First off, we have no index regarding the quality of the source. Second, if they did indeed see a clandestine aircraft, how would they even know what they were looking at and be able to decipher the aircraft's specific mission or affiliated program?
We don't know the exact design configuration of a hypersonic demonstrator even if it exists. All we have been told by Lockheed is that it would be approximately the size of an F-22. But even that could have changed. Additionally, as noted by the article, this sighting didn't occur during daylight hours, so how did the viewer even make out enough details on the aircraft to be able to make a guess as to its origins one way or another?
Then there is the question of who the aircraft even belonged to if the sighting of something previously undisclosed did indeed occur. Boeing, Northrop Grumman, and Lockheed all have massive compounds at Plant 42. The airfield is huge. Even seeing where an aircraft taxis to from an approach end of one of its runways is questionable, especially for a clandestine test article flying in low light conditions.
Even the T-38 element of the story seems a little odd. Chase is usually provided by F-16s these days, although Boeing owns T-38 chase aircraft as does NASA—both of which have a large presence at Plant 42. Edwards AFB to the north of Palmdale also has T-38s assigned, although once again, they aren't used for chase duties of high performance aircraft like the F-16s are.
Once again, the quality of the source is key here. If it was a bystander/layman the whole thing is highly speculative as normal people misidentify familiar aircraft in broad daylight, let alone in the dark. Although it would be a much more reliable source, it is doubtful that is was someone from inside Plant 42's fence line, as blabbering about this kind of thing is not conducive to employment for most jobs there. That doesn't mean it's impossible, just not probable.
If the sighting is true, and some exotic test aircraft did fly back to Plant 42 instead of being shipped there as is known to be common, it could have belonged to Boeing or Northrop Grumman, not just Lockheed Martin. And above all else, how is it certain that what the person saw was indeed a high-speed demonstrator instead of one of a whole slew of rumored black programs or ones that otherwise remain totally unknown?
Also, it is worth noting, that there have been no other major sightings of such an aircraft in recent months, at least any that are rumored to have occurred and discussed within the communities of people who have high-interest in this type of thing—something we here at The War Zone monitor very closely. So this sighting appears to be unique if it is truly accurate.
None of this is to say that a hypersonic technology demonstrator isn't flying, or hasn't been for some time. Once again, we surmised as such months ago. It's just this sighting account was conveyed with such little detail that its validity is highly questionable. As we have discussed in the past, by most indications there are a whole slew clandestine flight test programs underway at any given time, and if the account is to be believed, any one of these could have been the aircraft in question. We don't even have a description of the aircraft spotted.
But there can be no doubt that the Pentagon is becoming highly concerned with America's "hypersonic gap" as its peer state competitors forge ahead quickly with the game-changing technology. So far it seems that America's potential foes have concentrated on the development of hypersonic cruise missile technology. With the USAF feeling behind the curve, a hypersonic platform, a reusable aircraft capable of launching its own weapons and carrying reconfigurable sensors, is likely a prime developmental target that can close that gap and reach a position of superiority in the hypersonic realm in one fell swoop.
So as we said months ago, could there be a hypersonic demonstrator aircraft our there? Yes. In fact it seems more probable than possible, and this aircraft could have existed in the past, not the present. But is the supposed sighting at Plant 42 as reported by Aviation Week further evidence of its existence? Without more details, no it is not.
While the latest tests gather scientific data, the USAF is looking for companies could build an actual weapon.


For a premium selection of gently used vehicles near Bourbonnais and Kankakee at affordable prices, look no further than Pearl Chrysler Dodge Jeep RAM of Peotone.
Whether you're looking for a smaller, sportier car or something larger and more spacious, we've got you covered. We carry the best in used Chrysler vehicles like the Chrysler 300, used Jeep vehicles like the Jeep Grand Cherokee, used Dodge vehicles like the Dodge Challenger, and used RAM vehicles like the RAM 1500. We also stock up on a variety of vehicles from other popular brands like the Ford F-150 truck, the Honda CR-V SUV, the Kia Soul, and more.
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text:
www.eddieandmarthaadcock.com
History of the Lottery
Written by adminss on December 23, 2022 in Gambling News with no comments.
A lottery is a form of gambling in which a player bets on a series of numbers drawn randomly. The winner is rewarded with prizes or a lump sum. These games are popular in many parts of the world, including the United States and the Middle East. However, the laws of each jurisdiction vary.
Lotteries are used by governments to raise funds for public projects, as well as for the poor. For instance, the Continental Congress used lotteries to raise money for the Colonial Army. In addition, several colonies in the French and Indian War used them to raise funds for troops. Several religious congregations in the US also rely on lotteries for their finances.
Although lotteries were banned in France for two centuries, they were reinstated in the 17th century. During this period, the number of lotteries grew so quickly that it became a source of battle between the monarchy and the church. At the same time, the income from lotteries was so large that it helped to repair the city of Rome. Emperor Augustus was one of the first people to use lottery profits to repair the city.
Before the 18th century, lotteries were held in various towns in the Netherlands and France, including Antwerp and Ghent. They were often organized so that a percentage of the proceeds would go to charity. There were also numerous private lotteries, which were organized for religious orders.
In the early 19th century, some bishops in the United States criticized lotteries as a form of exploiting the poor. Some historians, however, argue that lotteries were a source of financial support for religious congregations, colleges, and other institutions.
Eventually, state and local lotteries were legalized in the US. Throughout the 19th century, lotteries were a common way for the government to generate revenues. However, legal debates and declining sales kept the amount of revenue down.
Fortunately, the lottery industry has grown to become a major part of the United States economy. Today, the lottery is a favorite way to gamble. It is played in most states and the District of Columbia. Players can choose from several different games, including Mega Millions and Powerball. Ticket purchases are usually made through land-based stores. But modern lottery solutions offer players convenience and security. Moreover, there are online lotteries that may not be available in the U.S.
Lotteries are popular in Canada, Mexico, Spain, the United Kingdom, the United States, and Japan. As with any other form of gambling, the rules of playing the lottery depend on each jurisdiction. The odds of winning are low, but the prizes can be very large. Depending on the rules of each lottery, a player can win a lump sum or an annuity.
Today, lottery games are played in more than 100 countries, including Australia. The Online Lotery Market report includes a detailed analysis of the market. It also includes trends, development, and consumer behavior analysis. Ultimately, it is expected that the lottery industry will grow by 9.1% between 2018 and 2026.
Nomor Togel Sydney Pools Terkuat Hari Ini
Dapatkan hasil nomor togel sydney pools hari ini dengan tabel data sdy lengkap. Result pengeluaran sdy dapat digunakan sebagai angka keluaran sdy paling akurat.
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AC-2/Yellow
The Yellow cable system (also known as Atlantic Crossing 2, AC-2) is a 6,400 km trans-Atlantic submarine cable system linking the USA and the UK.
The AC-2 was put into service in September 2000, with an initial design capacity of 320 Gbps on 4 fiber pairs.
In May 1999, Level3 decided to lay the Yellow transatlantic cable system, with 4 fiber pairs. At that time, demand was heavily outstripping supply. Other cables in place at the time included AC1, TAT 12/13. The construction and maintenance agreement for TAT14 had been signed, but the RFS date was not scheduled until late 2000. In 1999, long term indefeasible rights of use (IRU) for STM1/OC3 was priced at approximately $3 million and above. Coupled with this, the cost of ongoing operations and maintenance was in the region of $150,000 per year per STM1/OC3. The initial cost was relatively high for a single company, therefore the decision was taken to defray the large upfront project cost by entering into a strategic partnership.
Level3 signed a coconstruction agreement with Global Crossing which took two out of the four fiber pairs on the Yellow cable system. Operating in this environment meant Level 3 already recouped half the cost for Yellow before construction even began. Global Crossing marketed their two fiber pairs of the Yellow cable system as Atlantic Crossing 2, or AC-2. Global Crossing's private Atlantic Crossing 1 (AC-1) cable system was ready for service in 1999.
Level3 owned two fiber pairs and signed an IRU agreement on a fiber pair with Viatel. In this way, Level3 recovered approximately 70% of capital costs on the Yellow cable system before the system even entered service.
The AC-2/Yellow cable lands at Level3's cable landing station at Bellport, Long Island, NY, and Global Crossing's cable landing station at Bude, the UK.
After the acquisition of Global Crossing by Level3 and the followed acquistion of Level3 by Century Link, AC-1 and AC-2/Yellow are now wholly owned by Century Link.


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July 2022 Supply Chain News You Can Use
calendar 2 August 4, 2022
Meeting the Growing Need for Same-day Delivery
Key Point: Online shopping exploded in 2020. The pandemic kept us home, and Amazon provided two-day delivery. Suddenly, ordering anything from a new pair of shoes to a hamburger and fries via computer seemed much easier and smarter than driving to the store and risking contact with COVID.
For the most part, Amazon held up under the onslaught of orders, and other retailers jumped on the delivery-to-your-door bandwagon. Now, though, consumers are demanding same-day delivery. Is it possible for businesses to step up to the plate and get the job done?
Senpex CEO Anar Mammadov cites some eye-opening statistics about the topic:
"The number of shoppers expecting same-day delivery nearly tripled in a year. While only 24% of shoppers placed same-day delivery orders in 2020, 68% say fast shipping was a deciding factor in their 2021 orders. To put this in perspective, in 2019, same-day delivery amounted to a $5.87 billion market in the U.S. By 2024, the market is estimated to be worth $15.6 billion."
In this article, Mammadov discusses the challenges of scaling, how to integrate tools, and how to start scaling. This is a high-level view of the challenge, but certainly a thought and conversation starter.
The number of shoppers expecting same-day delivery nearly tripled in a year. Are you one of them?
Inventory Glut Leads an Amazon, Walmart Supplier to Cut Purchase Orders
Key Point: With inflation looming and monkeypox now joining COVID as a major public health concern, shoppers are cutting back on spending. And when consumers shop less often, retailers cut inventory — which means suppliers get fewer orders and workers get fewer hours. It's a logical, domino-type progression that ripples through the economy quietly, like a sneaker wave that makes you run further up the shore in a hurry.
Supply Chain Dive's concise coverage of how one company is handling the cutback in orders is an inside look at how a business can maintain health — even in the face of diminished demand.
Helen of Troy's CEO put it like this:
"Now that we ourselves are reducing [inventory] and they're reducing theirs, it'll lead [to] an opportunity to re-normalize for both and it'll be for the benefit of all over time. It's just painful on the path from here to there."
How far should a supplier cut back on inventory, though? Get the full scoop right here.
The big retailers are cutting back on orders from their suppliers. How do suppliers handle the cutbacks? Tricky question.
What Is Crowdsourced Delivery, and How Does it Work?
Key Point: We've heard of crowdsourcing money and discounts, but what is "crowdsourced delivery?" In a broad sense, the term refers to a B2B transaction in which the merchant hands the delivery function over to another business. Ordering from Taco Bell, for instance, and soon finding a Grubhub delivery driver at your door with your chili cheese burritos.
Earlier in this issue of NYCU Supply Chain, we referred to the rapid growth in demand for one-day delivery. Crowdsourcing is one of the ways a business can deliver on that demand. And Phoenix Logistics makes the case in this brief article.
Crowdsourced delivery: Are you using it? Do you like it?
E-commerce Becoming Daily Habit for Many [Infographic]
Key Point: If you like statistics, you'll love this infographic from Freight Waves. Like a match made in heaven, e-commerce is proving the shopping method of choice for more and more consumers.
Which delivery method do Millennials prefer: curbside pickup or home delivery? (The answer may surprise you.) What percentage of shoppers place an online order each month? How many Americans now own a smart device (other than their phone)?
You can get those answers and more by clicking here and diving in.
This Freight Waves infographic delivers some thought-provoking statistics about the rapid growth of e-commerce: https://www.freightwaves.com/news/daily-infographic-e-commerce-quickly-becoming-daily-habit-for-many.
10 years of Amazon robotics
Europe's Heat Wave Threatens Key Shipping Waterway
FedEx Ground scales back Sunday delivery coverage
How Machine-Learning Solutions Drive Order Management Excellence


ExploreLearning is excited about the recent launch of Interactive Case Studies. The program engages students to become active learners by presenting a scenario that puts them in the role of STEM professionals tasked with solving a real-world problem.
Creating the cases that will be most beneficial for your teachers and students is our priority. With this in mind ExploreLearning is seeking feedback from South Carolina Science Educators to determine the biggest pain points in middle and high school science.
Below you will be able to rank the science content areas that you believe your teachers and students need the most help with. The valuable feedback that you provide will inform directly into the development schedule for Interactive Case Studies.


does not count as an "access" and is therefore compliant.
What about address of submembers of a volatile aggregate?
Are either of the members of this initializer list complaint?
All your examples are compliant with rule 13.1. Taking the address of a volatile variable is not a "persistent side effect".
In the &REG1 and &REG2.bar examples, neither the & or * operator are evaluated - see C99 6.5.3.2(3).
misra-c wrote: All your examples are compliant with rule 13.1. Taking the address of a volatile variable is not a "persistent side effect".
I don't see how the exception in C99 6.5.3.2(3) applies in the case of &REG2.bar since the operand of the & operator in this case is the result of the member access operator, not the * or  operators. Can you please clarify your position and/or rationale for this part of the example?
Yes, you are correct. We had misparsed the macro expansion for &REG2.bar when writing the response.
Our position is that taking the address of REG2.bar is not a volatile access and there is no violation of rule 13.1.
The reason for this is as follows. REG2 expands to an lvalue. According to C99 6.3.2.1(2) an lvalue, appearing as the left operand of the "." operator, is not converted to a value (i.e. there is no memory access). According to C99 6.5.2.3(2), the result of the "." operator is an lvalue. Then according to C99 6.5.3.2(3) the "address of"(&) operator converts its operand to its address and again, no memory access is involved.


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Are Rihanna and Drake Dating Again? This Might Convince You That Something Is Up
Chelsea Stewart
It's been more than a year and a half since Rihanna gave the heartbreaking news that she and Drake were no longer friends. The singers had known each other since the mid-2000s and had a close relationship, so it was crushing to hear that they'd had a falling out. But things are looking up.
During the week of Oct. 20, Rihanna and Drake reportedly reunited not once, but twice. And if that's not a sign that they're back on good terms, we don't know what is.
Drake and Rihanna performing at an event | Photo by Kevork Djansezian/Getty Images
Why Rihanna and Drake fell out
Drake and Rihanna have had an on-off relationship since they met back in 2005. But things have really dried up in recent years.
In case you need a refresher, Rihanna suggested to Vogue in May of 2018 that things became awkward after Drake put her "on blast" at the 2016 MTV Video Music Awards. During the event, Drake presented her with the Vanguard Award, giving a three-minute speech where he professed his love for her.
Rihanna winced at the moment in the interview with Vogue.
"The VMAs is such a fan-focused awards show, so having that energy around me, and knowing the people who had received the award in the past, made it feel like a big deal. Waiting through that speech was probably the most uncomfortable part. I don't like too many compliments; I don't like to be put on blast," she told the publication.
When asked about her current relationship with Drake, Rihanna said, "We don't have a friendship now, but we're not enemies either. It is what it is."
Reports say that Rihanna and Drake just reconnected
The blog, The Shade Room, reports that Rihanna attended a private party that Drake threw on Oct. 21, where the Care Package rapper "pulled a cute move." He reportedly had McDonald's delivered to the event for the Barbadian beauty and the rest of her crew.
Two days later, Drake threw another party in celebration of his 33rd birthday, which Rihanna reportedly also attended. A source told Hollywood Life that they talked about their shared interests, including basketball.
Neither of them has commented on their apparent reconnection. However, Drake later posted a collection of Instagram photos from the party and opened up about the "unbreakable bonds" he shares with many of the guests.
"I've spent many nights over the last 4 or 5 years feeling like people did not like me or resented me for things beyond my control," Drake wrote. "I would sometimes settle into this mindset for months at a time. Today I woke up and looked at my phone and realized how much positive energy and how many unbreakable bonds I have in my life from incredible people."
I've spent many nights over the last 4 or 5 years feeling like people did not like me or resented me for things beyond my control. I would sometimes settle into this mindset for months at a time. Today I woke up and looked at my phone and realized how much positive energy and how many unbreakable bonds I have in my life from incredible people. All the messages and posts just reminded me that I am covered in a blanket of love and support and I should never second guess that. Thank you for a truly uplifting day. Love you all forever.
A post shared by champagnepapi (@champagnepapi) on Oct 24, 2019 at 8:08pm PDT
Are Rihanna and Drake back together?
We highly doubt that anything romantic is happening here. As many of us know, Rihanna is in a relationship with Saudi businessman, Hassan Jameel. They've been together for more than two years, and Rihanna is admittedly in love with him.
"Rihanna is in such a great place with Hassan right now and she's at a point in her life where she doesn't want to hold on to any past grudges or negative feelings she may have had about any of her exes," a source close to the singer told Hollywood Life on Oct. 25.
Drake, on the other hand, is reportedly single — for now, at least. He recently sparked dating rumors with Kylie Jenner after they reportedly got close at his birthday party.
It seems that he and RiRi are friends, and nothing more than that.


On 16 October 2014 the Digital Baltic Public Conference is beeing held in Kraków, Poland. The theme of the conference is Open Data, PSI and Digital Innovations.
The final agenda for the conference is going to be published online in August. For more information about the conference or to register your interest to participate, please contact Ida Kullgren, Värmland County Administrative Board.


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