Variation and differentiation in Hordeum marinum (Poaceae)

In Hordeum marinum two subspecies are recognized, viz. ssp. marinum , 2x and ssp. gussoneanum , 2x and 4x. The tetraploid cytotype has a more eastern distribution in the Mediterranean area than the two diploids. Crosses and meiotic data in hybrids revealed that the three cytotypes are closely related. The tetraploid ssp. gussoneanum behaves like a true alloploid but evidently has a very strong diploidizing mechanism, which prevents recombination between the genomes. The isoenzyme pattern shows differences between the cytotypes and a clinal differentiation in ssp. marinum.     

Nucleolar organizing chromosomes ofRicinus

Summary Pachytene chromosome morphology was compared in nine races ofRicinus communis L. (2n = 20), using pollen mother cells (PMCs) and light microscopy. Of the ten bivalents, only the two possessing nucleolar organizing regions (NORs), chromosomes 2 and 7, exhibit structural variations among the races. The NORs are located in the short arms of these two chromosomes. Most of the observed structural variations affect these short arms, which are similar morphologically and consist largely of heterochromatic segments. The PMCs contain a single nucleolus and this is associated with the NOR of each of the two chromosomes at a particular frequency in each race. In eight races, a nucleolar constriction (NC) is present in either chromosome 2 or chromosome 7. In these races, the nucleolus is associated with the chromosome possessing an NC at a frequency of 100% and with the chromosome lacking an NC at a frequency ranging between 5.6 and 100%, depending upon the race. No microscopically visible NC is present in the ninth race. In this race, the nucleolus is associated with both chromosomes 2 and 7 at a frequency of 100%. The association of the nucleolus with a chromosome possessing an NC is at the NC and with a chromosome lacking an NC is at the terminal heterochromatic segment of the short arm. Several interpretations are offered to account for the variations in frequency of association between the nucleolus and each of the nucleolar organizing chromosomes. It is suggested that the two non-linked NORs have evolved through some intragenomic changes rather than polyploidy, that this species is highly intolerant to structural variations other than those occurring in or near the NORs, and that structural variations in the nucleolar organizing chromosomes are not associated with racial variations in plant phenotype.Paper of the Journal Series, New Jersey Agricultural Experiment Station     

Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, <Emphasis Type="Italic">Pomphorhynchus laevis</Emphasis> and <Emphasis Type="Italic">Pomphorhynchus tereticollis</Emphasis> (Acanthocephala)

We studied distribution of ribosomal DNA (rDNA) sequences along with chromosomal location of the nucleolar organizer regions (NORs) in males of two fish parasites, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Fluorescence in situ hybridization with 18S rDNA probe identified two clusters of rDNA in each species, but revealed a remarkable difference in their location on chromosomes. In P. laevis, the rDNA-FISH signals were found in long arms of the first chromosome pair and in short arms of the second pair. Whereas in P. tereticollis, rDNA clusters were located in long arms of both the first and second chromosome pairs. The divergent location of rDNA clusters in the chromosome No. 2 supports current classification of P. tereticollis, previously considered a synonym of P. laevis, as a separate species. A possible scenario of the second chromosome rearrangement during karyotype evolution of the two species involves two successive pericentric inversions. In both species, one or two prominent nucleoli were apparent within interphase nuclei stained with either silver nitrate or a fluorescent dye YOYO-1. However, a single large nucleolus was observed in early stages of mitosis and meiosis I regardless the number of rDNA clusters. Nevertheless, two bivalents with silver-stained NORs in diakinesis and two silver-stained sites in early prophase II nuclei indicated that all NORs are active. This means that each Pomphorhynchus NOR generates a nucleolus, but the resulting nucleoli have a strong tendency to associate in a large body.     

Metaphase I bound arms frequency and genome analysis in wheat-Aegilops hybrids

Summary Meiotic associations of different wheat-Aegilops variabilis and wheat-Ae. kotschyi hybrid combinations with low and high homoeologous pairing were analyzed at metaphase I. Five types of pairing involving wheat and Aegilops genomes were identified by using C-banding. A genotype that seems to promote homoeologous pairing has been found in Ae. variabilis var. cylindrostachys. Its effect is detectable in the low pairing hybrids but not in the high ones. Pairing affinity has been analyzed on the basis of metaphase I associations in the low and high homoeologous pairing hybrids, and in bivalents and multivalents in the high pairing hybrids. The results indicate that the amount of bound arms of each type of identifiable association relative to the total associations formed (relative contribution) was not maintained, either between the different levels of pairing (low and high) or between different meiotic configurations (bivalents and multivalents). These findings seem to indicate that quantifications of genomic relationships based on the amount of chromosome pairing at metaphase I must be carefully done in this type of hybrid combinations.     

Combined ecological niche modelling and molecular phylogeography revealed the evolutionary history of Hordeum marinum (Poaceae)--niche differentiation, loss of genetic diversity, and speciation in Mediterranean Quaternary refugia

The Hordeum marinum species group consists of two annual grasses of western Eurasian saline meadows or marshes. The two grasses split in the Quaternary about two million years ago. Hordeum marinum and the diploid of Hordeum gussoneanum (2x) co-occur throughout the Mediterranean basin, while the autotetraploid cytotype of H. gussoneanum (4x) overlaps with its diploid progenitor geographically only in the utmost Eastern Mediterranean, extending from there eastwards into Asia. Using chloroplast sequences of the trnL-F region, six newly developed chloroplast microsatellite loci, ecological predictive models based on climate data, and the present geographical distribution of the two species we analysed differentiation processes in the H. marinum group. The chloroplast data indicated clear differences in the history of both species. For H. marinum we found a subdivision between genetically variable populations from the Iberian Peninsula and the more uniform populations from the remaining Mediterranean. As an explanation, we assume Pleistocene fragmentation of an earlier widespread population and survival in an Iberian and a Central Mediterranean glacial refuge. Chloroplast variation was completely absent within the cytotypes of H. gussoneanum, indicating a severe and recent genetic bottleneck. Due to this lack of chloroplast variation only the combination of ecological habitat modelling with molecular data analyses allowed conclusions about the history of this taxon. The distribution areas of the two cytotypes of H. gussoneanum overlap today in parts of Turkey, indicating an area with similar climate conditions during polyploid formation. However, after its origin the polyploid cytotype underwent a pronounced ecological shift, compared to its diploid progenitor, allowing it to colonize mountainous inland habitats between the Mediterranean basin and Afghanistan. The extant sympatric occurrence of H. marinum and H. gussoneanum 2x in the Mediterranean region is interpreted as a result of secondary contact after fast Holocene range expansion out of different ice age refugia.     

Heterochromatin, synaptonemal complex, and NOR activity in the somatic and germ cells of a male domestic dog, Canis familiaris (Mammalia, Canidae)

C-banding and silver staining of the somatic and germ cells of the male domestic dog. Canis familiaris, have shown that: (1) the amount of C-banding is small compared to most other mammalian species, (2) three pairs of autosomes have nucleolus organizer regions (NORs) at the terminal ends of their long arms, whereas the Y chromosome has an NOR on the terminal end of the short arm, (3) the organization of the synaptonemal complex (SC) is similar to that of other mammalian species, (4) a distinct SC is formed between the long arm of the Y chromosome and probably the short arm of the X chromosome, and (5) the differential axes of both sex chromosomes do not demonstrate fusiform thickenings nor do they stain darkly with silver as do the XY bivalents in many other mammalian species.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.     

Cytogenetic analyses of a Salvelinus hybrid reveal an evolutionary relationship between the parental species

Mitotic analyses of the brook trout (Salvelinus fontinalis) x arctic char (S. alpinus) hybrids (sparctic trout) revealed a mode of 2n = 82 with 18 metacentric and 64 acrocentric chromosomes. The brook trout had 2n = 84 with 16 metacentric chromosomes and the arctic char had 2n = 80 with 20 metacentric chromosomes; both species are derivatives of a single tetraploid event. Variable multivalent-like configurations that may be centromeric associations of bivalents were observed in C-banded pachytene figures of female sparctic trout. Metaphase I analyses of sparctic trout males indicated that two fusions of nonhomologous acrocentric chromosomes representing two duplicated chromosome sets must have occurred in the arctic char after its evolutionary divergence from the brook trout. A mode of seven tetravalent rods per cell suggests that preferential multivalent pairing occurs in the sparctic hybrid; metaphase I analyses of S. alpinus males revealed a mode of only five tetravalent rods per cell. The presence of multivalents implies that the arctic char, like the brook trout, is still undergoing diploidization. Cytochemical detection of the nucleolar organizer region (NOR) revealed intra- and interspecific as well as intraindividual variability in the numbers and types of chromosomes (metacentric or acrocentric) on which NORs appeared in arctic char and sparctic trout. Brook trout only had NORs on acrocentric chromosomes. This may indicate that different chromosomal fusions occurred in the evolution of brook trout from arctic char.     

The karyotype ofFestucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

The karyotypes of two populations ofFestucopsis serpentini (2n = 2x = 14) endemic to Albania were investigated in detail by Giemsa C- and N-banding, AgNO₃ staining, and in situ hybridization with an rDNA probe. The complements consisted of 14 large chromosomes, 10 metacentric and 4 SAT-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming activity was established through AgNO₃ staining; however, the rDNA probe additionally hybridized to intercalary positions in the short arms of two metacentric chromosomes revealing two inactive rDNA sites. C-banding patterns comprised from zero and up to four very small to larger, generally telomeric bands per chromosome giving low levels of constitutive heterochromatin. Similarities in chromosome morphology and C-banding patterns identified the homologous relationships of all chromosomes in one population, but of three pairs only in the other. Reliable identification of homologous chromosomes between plants was only possible for the SAT-chromosomes. A comparison between the C-banded karyotypes ofF. serpentini andPeridictyon sanctum supports their position in two genera.     

Chromosome heteromorphisms in the Japanese

Summary The nucleolus organizer regions (NORs) of variant D- and G-group chromosomes characterized by enlargements of the short arms including secondary constrictions and satellites, were examined using the silver-staining method. Of a total of nine variants examined, four were found to have double Ag-stained NORs in the enlarged short arm, two were found to involve chromosome 22, one was a 13, and one, a 14. Four of the other variants had only one Ag-stained NOR. From the positions of the NORs, three of them were judged to have enlarged satellites (two chromosomes 15 and one 22) and the other an enlarged short arm (a 15). In the remaining variant (a 14), no Agstained material was noted in the short arm, so it could not be determined whether this variant chromosome was derived from the enlargement of the short arm or from satellites. Based on the position of the Ag-stained NORs and staining intensity of the Q and C methods in the short arms, mechanisms of producing the enlarged short arms of D- and G-group chromosomes are discussed.     

Production and meiotic pairing behaviour of new hybrids of winter wheat (Triticum aestivum) x winter barley (Hordeum vulgare).

New winter wheat (Triticum aestivum L.) x winter barley (Hordeum vulgare L.) hybrids produced using cultivated varieties (wheat 'Martonvásári 9 krl'(Mv9 krl) x barley 'Igri', Mv9 krl x 'Osnova', 'Asakazekomugi' x 'Manas') were multiplied in tissue culture because of the high degree of sterility and then pollinated with wheat to obtain backcross progenies. Meiotic analysis of the hybrids Mv9 krl x 'Igri' and 'Asakazekomugi' x 'Manas' and their in vitro regenerated progenies with the Feulgen method revealed 1.59 chromosome arm associations per cell in both initial hybrids. The number of chromosome arm associations increased after in vitro culture to 4.72 and 2.67, respectively, in the two combinations. According to the genomic in situ hybridization (GISH) analysis, wheat-barley chromosome arm associations made up 3.6% of the total in the initial Mv9 krl x 'Igri' hybrid and 6.6% and 16.5% of the total in in vitro regenerated progenies of the 'Asakazekomugi' x 'Manas' and Mv9 krl x 'Igri' hybrids, respectively. The demonstration by GISH of wheat-barley chromosome pairing in the hybrids and especially in their in vitro regenerated progenies proves the possibility of producing recombinants between these two genera, and thus of transferring useful characters from barley into wheat. In vitro conditions caused an increase in chromosome arm association frequency in both combinations and in fertility in some regenerants.     

Meiotic abnormalities of the fifth pair of chromosomes in Paeonia decomposita and P. intermedia

This study revealed that there were common meiotic abnormalities on the fifth pair of chromosomes in Paeonia decomposita and P. intermedia. Short arms had a tremendous deviation between genetic distance and physical distance, when compared with the long arms. The genetic distance of the short arm was about a twenty-eighth (P. intermedia) and fiftieth (P. decomposita) of the long arm, whereas the physical distance was about one third of the long arm and the physical distance was over nine times longer than the genetic distance. In P. decomposita, the ratio between ring bivalents (both arms formed chiasma) and rod bivalents (only one arm formed chiasma) was 1.94 : 98.06, whereas in P. intermedia the ratio was 3.42 : 96.58. In both of the species, rod bivalents were much more than ring bivalents. Occurrence frequency of anaphase I bridges was low in P. decom-posita and P. intermedia, and the length of fragments varied in a range.     

NORs inheritance analysis in crossings including individuals from two stocks of rainbow trout (Oncorhynchus mykiss)

Silver nitrate staining of rainbow trouts (Oncorhynchus mykiss) chromosomes, for the identification of the nucleolar organizing regions (NORs), revealed that in individuals from Núcleo Experimental de Salmonicultura de Campos do Jord?o (Brazil) NORs were located in the long arms of submetacentric pair while in specimens from Mount Shasta (USA) NORs were located in the short arms of a submetacentric pair. Cytogenetic analysis of the offspring, obtained through artificial crosses including individuals from both stocks, allowed the identification of NORs in two submetacentric chromosomes, one in the short arms and the other in the long arms, confirming the effectiveness of the hybridization process. Complementary results obtained using the FISH technique with 18S and 5S rDNA probes showed that NOR-bearing chromosomes exhibited a cluster of 5S genes located in tandem with the 18S gene cluster in both stocks. The results allow us to suggest that the difference in NOR-bearing chromosomes found between the two stocks is likely to be due to pericentric inversion involving the chromosome segment where 18S and 5S rDNA genes are located. The presence of ribosomal genes in the long arms of a submetacentric chromosome is apparently a particular characteristic of the rainbow trout stock of Campos do Jord?o and might be used as a chromosome marker in studies of controlled crosses in this species.     

Nucleolus,nucleolar chromosomes,and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

Summary The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of³H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of³H-uridine uptake showed that the oocyte was not at a resting stage. The possible cytogenetic consequences of these observations are discussed.     

Localization of rRNA genes in Phyllostomidae bats reveals silent NORs in Artibeus cinereus

We analyzed the nucleolus organizer regions (NORs) of thirteen bats from genera Phyllostomus, Phylloderma, Trachops, Tonatia, Sturnira, Platyrrhinus, Artibeus and Glossophaga. We used silver staining and FISH with rDNA probe. Nine species had only one Ag-NOR-bearing chromosome pair. Artibeus lituratus, A. jamaicensis and A. fimbriatus presented multiple Ag-NORs located in the short arms of pairs 5, 6 and 7, and an additional mark in the long arm of one chromosome 5 in A. fimbriatus. Artibeus cinereus showed Ag-NORs in the chromosome pairs 10 and 13. The chromosomal location of rRNA genes using FISH agreed with the number and position of NORs in all but one species. In A. cinereus the hybridization signals were seen in three chromosome pairs 9, 10 and 13. This suggests the occurrence of silent NORs in pair 9. Differences in the size and intensity of the hybridization signals were also observed in the pair 9 of A. cinereus.     

Dynamic of nucleolus organizer regions and karyotype evolution in Indian pygmy field mice

Ag-NOR staining and fluorescence in situ hybridization with rDNA probes showed an unusually high number of NORs in the Indian pygmy field mice, Mus booduga and the M. terricolor complex. The chromosomal location of the NORs was also altered in terricolor, they were shifted from the proximal regions of the long arms to the tips of the perceptible heterochromatic short arms of the acrocentric autosomes. The results suggested dispersion of the NORs in the booduga-terricolor lineage probably by transposition, and relocalization of the NORs in the terricolor complex by centric reorganization during the process of replacement of the Mus musculus-related AT-rich heterochromatin with the terricolor-specific heterochromatin.     

Cytological studies on F1 hybrids of Solanum integrifolium with S. melongena and S. melongena var. insanum

S. integrifolium (2n = 24) can easily be crossed as the pistillate parent with S. melongena (2n = 24) and S. melongena var. insanum (2n = 24). However, crosses in the other direction do not succeed. Both hybrids are vigorous. Chromosome association at diakinesis and metaphase I was studied. Chromosome associations higher than bivalents were observed in the hybrids indicating structural repatterning of chromosomes. The modal chromosome association in hybrids was twelve bivalents per PMC. This is suggestive of the retention of ancestral chromosome homeologies by the taxa concerned. Despite regular meiosis both hybrids were highly pollen-sterile (about 95%), which was attributed to segregational events of the recombined chromosomes.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.