Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

Steinbock und Gemse im Alpenraum – genetische Variabilität und biochemische Differenzierung zwischen den Arten

Ibex and chamois of the Alps – genetic variability and biochemical differentiation between species Genetic variability of 46 specimens of ibex (Capra ibex ibexL.) from Graubünden/Switzerland was investigated by means of horizontal starch gel electrophoresis. Twenty four isoenzyme systems were examined in kidney and liver tissues. From 38 loci scored 5.3 % were polymorphic (PGM-2, ADA-1) and average heterozygosity was 0.0235. The comparatively low proportion of polymorphic loci can be interpreted as the result of a bottleneck in alpine ibex during the last centuries. Based on a ¯-value of 0,2746 a time of divergence between ibex and chamois of about 1370000 years was calculated.     

Band karyotypes and specific types of heterochromatins in several species of European percid fishes (Percidea,Pisces)

The chromosomes of the European Percidae (Lucioperca lucioperca L., Gymnocephalus cernuus L., Gymnocephalus schraetser L. and Perca fluviatilis L.) were analyzed by means of silver staining chromomycin A₃/distamycin A/DAPI and DAPI/actinomycin D fluorescence banding techniques. The nucleolus organizer regions (NORs) were localized at the satellite stalks of chromosome no. 16 in Lucioperca lucioperca and Perca fluviatilis, and of chromosome no. 18 in both Gymnocephalus species. Bright chromomycin A₃ fluorescence clusters were associated with them.Bright distamycin A-DAPI and DAPI/actinomycin D heterochromatic blocks were detected in Lucioperca lucioperca and the Gymnocephalus species.     

Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.     

Comparative evaluation of effort, capture and handling effects of drive nets to capture roe deer (Capreolus capreolus), Southern chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica)

The objective of this study is to assess the usefulness of drive nets to capture roe deer (Capreolus capreolus), Southern chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica), comparing the results obtained with other capture methods and amongst the three species. Sixty-five drive net capture operations using beaters were conducted from January 1998 to September 2004. A total of 161 wild ungulates (31 roe deer, 95 Southern chamois and 35 Spanish ibexes) were captured. The average number of animals captured per operation was 1.07 for roe deer, 3.96 for Southern chamois and 2.92 for Spanish ibex. The average number of person–days per captured animal was 21.5, 7.1 and 10.6 for roe deer, Southern chamois and Spanish ibex, respectively. Specificity was 100% for Southern chamois and Spanish ibex (only the target species captured) and 77.5% for roe deer. Risk for the animals (mortality plus injuries) was 3.23% for roe deer, 5.27% for Southern chamois and 0% for Spanish ibex, whereas injuries to the operators occurred with 3.1% of the handled animals. Sex ratio was skewed towards females in roe deer, towards males in Southern chamois and balanced in Southern chamois. Drive nets showed good performance, although many operators were required. Safety for the animals and specificity were higher than traditionally attributed to this capture method. It is concluded that drive nets are an efficient and safe method to capture many ungulate species.     

Analysis of heterochromatin by combination of C-banding and CMA3 and DAPI staining in two fish species (Pimelodidae,Siluriformes)

The chromosomes of Steindachneridion sp. (2n = 56) and Rhamdia quelen (2n = 58) were analyzed by C-banding (CB) and Chromomycin A₃ (CMA₃) and 4,6-diamidino-2-phenylindole (DAPI) staining, separately and consecutively, in order to understand the role of base-specific fluorochrome treatment after CB. Both species' chromosomes shared common staining profiles as follows. CB with Giemsa (CBG) revealed weak heterochromatic blocks in the telomeric regions of some chromosomes and conspicuous bands on the short arms of one chromosome pair, where nucleolar organizer regions (NORs) were evidenced by silver-staining. Without CB pretreatment, the NORs were stained conspicuously with CMA₃, but not with DAPI. The latter uniformly stained all chromosomes, but leaving the NORs pale. Combination of CMA₃ or DAPI staining with CB showed distinctive fluorescent blocks in the NOR-bearing short arms of the single chromosome pair along with several bright fluorescent signals on other chromosomes, which were not evidenced by single CMA₃ or DAPI staining. These results suggest a modification of chromatin structure by CB treatment, which may increase the stainability of CMA₃ and DAPI.     

Cohabitation of a <Emphasis Type="Italic">Brucella melitensis</Emphasis> infected Alpine ibex (<Emphasis Type="Italic">Capra ibex</Emphasis>) with domestic small ruminants in an enclosure in Gran Paradiso National Park,in Western Italian Alps

After the first report of Brucella melitensis infection from a 7-year-old alpine ibex (Capra ibex) buck living in Gran Paradiso National Park (GPNP), further studies demonstrated the presence of the infection in ibex and chamois. Considering that livestock herds keep on sharing pastures with more than 3,500 ibex and 9,000 chamois in the park, our aim was to demonstrate under controlled conditions the possibility of Brucella infection passing from wild ruminants to livestock. A 7-year-old male alpine ibex with clinical signs of brucellosis and serologically positive was released in a 5,000 m² enclosure together with five goats and two sheep rams. Due to poor condition, ibex was suppressed at day 40, domestic ruminants stayed into the enclosure potentially contaminated by ibex for further 38 days. During this period, we had monitored our animals taking blood from domestic ruminants every 15 days and tested the serum to Rose Bengal agglutination test and Complement Fixation test. Domestic animals tested negative at serology at all sampling time and at isolation, while B. melitensis biovar 3 was isolated from ibex tissues. Our data show that transmission of infection from ibex to livestock is not easy. After 40 days of strict cohabitation and 38 days of permanence in an area where an infected ibex lives, no one of the domestic animals contracted infection. In spite of the limitation of our field trial, we have demonstrated that long direct and indirect contact between alpine ibex and domestic animals will not easily lead to an infection of the latter. Further investigations are needed to confirm our results and evaluate the effective risk of B. melitensis transmission from alpine ibex to livestock.     

Heterochromatins and band karyotypes in three species of salmonids

Summary The heterochromatins of rainbow trout (Salmo gairdneri R.), brown trout (Salmo trutta fario L.) and brook trout (Salvelinus fontinalis M.) were characterized by sequential chromomycin A₃/distamycin A/DAPI (CDD) and DAPI/actinomycin D (DAPI/AmD) fluorescence. On most biarmed chromosomes, an equilocal localization of prominent DAPI/AmD positive, chromomycin A₃ negative, AT-rich blocks at the centromeres were observed in all three species. Band karyotypes of the three species were established. In rainbow trout, several DAPI/AmD positive heterochromatin blocks behaved positive in a silver-staining method. Mitotic and interphase studies proved the presence of inter-individual NOR variation in brown trout. The NORs of brook trout were localized on chromosomes 5, 10, 14, 15 and 29.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural chromosome differentiation between Triticum timopheevii and T. turgidum and T. aestivum

 Chromosome pairing at metaphase-I was analyzed in F₁ hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (A^tA^tGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AA^tBG, AA^tBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AA^tBGD hybrids. T. timopheevii chromosomes 1A^t, 2A^t, 5A^t, 7A^t, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6A^tS/1GS, 1GS/4GS, 4GS/4A^tL, and 4A^tL/3A^tL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats. Received: 15 June 1998 / Accepted: 19 August 1998     

Chromosomal differentiation and genome size in three European mountain Lilium species

 Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A₃ (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species. Received June 21, 2002; accepted October 4, 2002 Published online: Febraury 7, 2003     

Genesis of bivalent pairing in hexaploid clump Verbena

Both 6x Verbena aubletia (n=15) and 2x V. tenuisecta (n=5) form bivalents during meiosis, however, their 4x F₁ hybrid (V. aubletia × V. tenuisecta) shows almost complete homoeologous pairing involving on average 19.74 out of its 20 chromosomes. In 10% cells there are 4IV+2II indicating that essentially there may be 4 homoeologous sets of 5 chromosomes each in the F₁ hybrid. Evidently, V. aubletia is segmental allo-hexaploid involving 3 homoeologous genomes (A₁A₁ A₂A₂ A₃A₃). Whether its cytologically diploid behaviour is the result of a multivalent suppressor system or due to an acute property of preferential pairing, cannot be answered with certainty. In either case intergenomal homoeologies are totally suppressed resulting in bivalent pairing, meiotic isolation of the 3 genomes and institution of normal fertility.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

Chromosome banding study of European catfish,Silurus glanis (Pisces,Siluridae)

The karyotype of European catfish (Silurus glanis L.) was analyzed sequentially by means of silver staining and the chromomycin A₃ (CMA₃)/distamycin A (DA)/DAPI fluorescence technique and by C-banding, respectively. The nucleolus organizer regions (NORs) were localized on the submetacentric pair No. 14. Brilliant CMA₃ fluorescent heterochromatin blocks corresponded to the NORs visualized by silver staining. No DA/DAPI-bright positive fluorescent patterns were detected while C-banding led to the detection of specific banding patterns on several chromosome pairs.—Using these banding data, the karyotype of S. glanis was redescribed.     

Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A₂ and A₃ subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).     

Cytogenetic and molecular analysis of the pufferfish Tetraodon fluviatilis (Osteichthyes)

In view of their compact genome, pufferfish (Tetraodontiformes) have been proposed as model animal for the study of the vertebrate genome. Despite such interest, cytogenetic information about puffers is still scanty. To fill this gap, a cytogenetic analysis of T. fluviatilis has been performed using both classical and molecular techniques. C-banding, followed by DAPI staining, evidenced that in T. fluviatilis, like all other puffer species so far examined, heterochromatin is essentially AT-rich and it is located at centromeres, whereas staining with CMA₃, silver staining and FISH with a 28S ribosomal RNA gene DNA probe showed 2–4 nucleolar organizing regions (NORs) located in heterochromatic regions in the considered puffer species. FISH with the 5S probe put in evidence both in T. fluviatilis and in T. nigroviridis only a 5S cluster per haploid genome that is physically unlinked with the major ribosomal RNA genes including the 28S rRNA genes. Hybridization with the (TTAGGG)_n probe showed in all the puffers brightly fluorescent signals uniform both in size and intensity at the end of all the chromosomes. Finally, mariner-like elements (MLEs) have been identified in T. fluviatilis and they have located into the NOR-associated heterochromatin.     

Interaction between rye B-chromosomes and wheat genetic systems controlling homoeologous pairing

The interactive effect on homoeologous pairing of rye B-chromosomes with the absence of both pairing suppressor (3A, 3D, 5B) and promotor (3B, 5A, 5D) chromosomes of common wheat (Triticum aestivum L.) is analyzed by comparison of pairing at Metaphase I of 27-, 27+2B, 28- and 28+2B-chromosome plants. These plants were obtained from crosses between the respective wheat monosomics (2n=41) and rye plants (Secale cereale L.) carrying or not carrying two B-chromosomes (2n=14 or 14+2Bs). —The effect of rye B-chromosomes on pairing depends on the function of the wheat chromosome which is absent in the appropriate hybrids, i.e., rye B-chromosomes have a suppressor effect on pairing when the pairing suppressing wheat chromosomes 3A, 3D or 5B are absent, while they behave as promotors when the pairing promoting chromosomes 3B, 5A or 5D are absent.     

Nucleotide Composition of the Cold-Sensitive Heterochromatic Regions in Paris hainanensis Merrill

Cold-induced decondensation of heterochromatic regions (CSR-bands) in Paris hainanensis(=Daiswa hainanensisMerrill Takht.) (2n= 10; 10 + b) was studied. The comparison of CSR-banding patterns with those obtained by nucleotide-specific staining with fluorochromes DAPI and chromomycin A₃demonstrated that low temperatures induced decondensation only of large AT-rich heterochromatic regions. It is suggested that this is characteristic of all plant species.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.