A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Cytogenetic characterization of <Emphasis Type="Italic">Lippia alba</Emphasis> and <Emphasis Type="Italic">Lantana camara</Emphasis> (Verbenaceae) from Brazil

The aim of this work was to determine the cytogenetic characteristics of Brazilian Lippia alba (Mill) N. E. Brown and Lantana camara Plum. that could be useful for future characterization of these genera. Our analyses revealed that Li. alba has 2n=30 chromosomes consisting of ten metacentric and five submetacentric pairs, while La. camara has 44 metacentric chromosomes. The large blocks of heterochromatin seen in both species suggest an apomorphic condition. Six 45S rDNA sites were detected in both species by fluorescence in situ hybridization (FISH). Two and four 5S rDNA sites were observed in Li. alba and La. camara, respectively. Meiotic analysis revealed a normal chromosomal behaviour. The number of chromosomes and the presence of 45S rDNA and 5S rDNA sites do not exclude a possible polyploid origin. The cytogenetic differences between La. camara and Li. alba may be useful markers for differentiating these species.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

Comparison of genomes of eight species of sections <Emphasis Type="Italic">Linum</Emphasis> and <Emphasis Type="Italic">Adenolinum</Emphasis> from the genus<Emphasis Type="Italic"> Linum</Emphasis> based on chromosome banding,molecular markers and RAPD analysis

Karyotypes of species sects. Linum and Adenolinum have been studied using C/DAPI-banding, Ag-NOR staining, FISH with 5S and 26S rDNA and RAPD analysis. C/DAPI-banding patterns enabled identification of all homologous chromosome pairs in the studied karyotypes. The revealed high similarity between species L. grandiflorum (2n = 16) and L. decumbens by chromosome and molecular markers proved their close genome relationship and identified the chromosome number in L. decumbens as 2n = 16. The similarity found for C/DAPI-banding patterns between species with the same chromosome numbers corresponds with the results obtained by RAPD-analysis, showing clusterization of 16-, 18- and 30-chromosome species into three separate groups. 5S rDNA and 26S rDNA were co-localized in NOR-chromosome 1 in the genomes of all species investigated. In 30-chromosome species, there were three separate 5S rDNA sites in chromosomes 3, 8 and 13. In 16-chromosome species, a separate 5S rDNA site was also located in chromosome 3, whereas in 18-chromosome species it was found in the long arm of NOR-chromosome 1. Thus, the difference in localization of rDNA sites in species with 2n = 16, 2n = 30 and 2n = 18 confirms taxonomists opinion, who attributed these species to different sects. Linum and Adenolinum, respectively. The obtained results suggest that species with 2n = 16, 2n = 18 and 2n = 30 originated from a 16-chromosome ancestor.     

Chromosomal Characteristics of rDNA in European Grayling <Emphasis Type="Italic">Thymallus thymallus</Emphasis> (Salmonidae)

The chromosomal characteristics, locations and variations of two classes of ribosomal DNA (5S and 18S) were studied in European grayling karyotype (Thymallus thymallus, Salmonidae). Major rDNA sites as revealed by sequential CMA₃/Ag staining and confirmed by in situ hybridization with a 18S rDNA probe were situated in two loci and were found to be polymorphic in size and displaying several distinct forms. The 5S rDNA was located by PRINS on three pairs of subtelocentric chromosomes, additional minor signal was present at the centromere of one metacentric element. 5S sites were not associated with NORs. The dosage compensation mechanism was proposed as an explanation of high frequency of lethal rDNA-deleted forms of the NOR-bearing chromosomes. Double variable pattern in the number and location of NORs supported the bi-directional evolution of salmonid rDNA loci.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, <Emphasis Type="Italic">Taeniura lymma</Emphasis> and <Emphasis Type="Italic">Raja montagui</Emphasis> (Chondrichthyes,Batoidea)

We used silver nitrate staining, CMA₃ and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC)_n trinucleotides, (CA)_n dinucleotides and (GTGA)_n tetranucleotides, which probably influence gene regulation.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

Characterization of the telomere regions of scleractinian coral, <Emphasis Type="Italic">Acropora surculosa</Emphasis>

Terminal ends of vertebrate chromosomes are protected by tandem repeats of the sequence (TTAGGG). First thought to be vertebrate specific, (TTAGGG) _n has recently been identified in several aquatic invertebrates including sea urchin (Strongylocentrotus purpuratus), bay scallop (Argopecten irradians), and wedgeshell clam (Donax trunculus). We analyzed genomic DNA from scleractinian corals, Acropora surculosa, Favia pallida, Leptoria phrygia, and Goniastrea retiformis to determine the telomere sequence. Southern blot analysis suggests the presence of the vertebrate telomere repeats in all four species. Treatment of A. surculosa sperm DNA with Bal31 exonuclease revealed progressive shortening of the DNA fragments positive for the (TTAGGG)₂₂ sequence, supporting location of the repeats at the chromosome ends. The presence of the vertebrate telomere repeats in corals is evidence that the (TTAGGG) _n sequence is highly conserved among a divergent group of vertebrate and invertebrate species. Corals are members of the Lower Metazoans, the group of organisms that span the gap between the fungi and higher metazoans. Corals are the most basal organism reported to have the (TTAGGG) _n sequence to date, which suggests that the vertebrate telomere sequence may be much older than previously thought and that corals may share a number of genes with their higher relatives.     

Chromosomal studies in <Emphasis Type="Italic">Crenicichla lepidota</Emphasis> and <Emphasis Type="Italic">Australoheros facetus</Emphasis> (Cichlidae,Perciformes) from extreme Southern Brazil

Fishes of the family Cichlidae generally show low karyotype variability. Nevertheless, karyotype variants have been identified within some genera, providing information about their evolutionary history. In the present study, karyotype characteristics of Crenicichla lepidota and Australoheros facetus, two sympatric species found in the São Gonçalo-Mangueira basin, were studied. Besides conventional procedures, double fluorochromes staining chromomycin A₃/DAPI and fluorescent in situ hybridization (FISH) with rDNA probes were also used. Both species presented 2n = 48 chromosomes, but karyotypes were differentiated by fundamental number, which was equal to 70 in A. facetus and 56 in C. lepidota. Similar heterochromatin distribution patterns were also observed on the pericentromeric region of most chromosomes, although C. lepidota presented an additional heterochromatic block in the first pair. The Ag-NORs, 18S rDNA probe and CMA₃/DAPI were coincident in location on the first and second pairs of C. lepidota and A. facetus, respectively. The minor rDNA loci (5S rDNA) were found in four sites located on two distinct chromosomal pairs in C. lepidota. Although the data obtained here to C. lepidota and to A. facetus show chromosomal characteristics considered ancestral to the family, new data are presented to both species. Additionally, this study corroborates the hypothesis in which evolutionary processes like non-Robertsonian rearrangements are involved in the diversification of the major groups of Neotropical Cichlidae. Thus, the karyotype diversification observed in A. facetus have the high fundamental number pathway while C. lepidota has others evolutionary chromosomal mechanisms.     

<Emphasis Type="Italic">n</Emphasis>-Alkane assimilation and <Emphasis Type="Italic">tert</Emphasis>-butyl alcohol (TBA) oxidation capacity in <Emphasis Type="Italic">Mycobacterium austroafricanum</Emphasis> strains

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.     

Cytogenetic characterization of <Emphasis Type="Italic">Hydrangea involucrata</Emphasis> Sieb. and <Emphasis Type="Italic">H. aspera</Emphasis> D. Don complex (Hydrangeaceae)<Emphasis Type="Italic">:</Emphasis> genetic,evolutional, and taxonomic implications

The subsection Asperae of genus Hydrangea L. (Hydrangeaceae) has been investigated for three reasons: several ambiguous classifications concerning Hydrangea aspera have been published, unexpected differences in genome size among seven accessions have been reported Cerbah et al. (Theor Appl Genet 103:45–51, 2001), and two atypical chromosome numbers (2n = 30 for Hydrangea involucrata and 2n = 34 for H. aspera) have been found when all other species of the genus present 2n = 36. Therefore, these two species and four subspecies of Hydrangea in all 29 accessions were analyzed for their genome size, chromosome number, and karyotype features. This investigation includes flow cytometric measurements of nuclear DNA content and bases composition (GC%), fluorochrome banding for detection of GC- and AT-rich DNA regions, and fluorescent in situ hybridisation (FISH) for chromosome mapping of 5 S and 18 S-5.8 S-26 S rDNA genes. In the H. aspera complex, the genome size ranged from 2.98 (subsp. sargentiana) to 4.67 pg/2C (subsp. aspera), an exceptional intraspecific variation of 1.57-fold. The mean base composition was 40.5% GC. Our report establishes the first karyotype for the species H. involucrata, and for the subspecies of H. aspera which indeed present different formulae, offering an element of discrimination. FISH and fluorochrome banding revealed the important differentiation between these two species (H. involucrata and H. aspera) and among four subspecies of the H. aspera complex. Our results are in agreement with the Chinese classification that places the groups Kawakami and Villosa as two different species: Hydrangea villosa Rehder and Hydrangea kawakami Hayata. This knowledge can contribute to effective germplasm management and horticultural use.     

Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

Conserved 5S and variable 45S rDNA chromosomal localisation revealed by FISH in <Emphasis Type="Italic">Astyanax scabripinnis</Emphasis> (Pisces,Characidae)

Fluorescence in situhybridisation (FISH) and double FISH experiments were carried out to ascertain the chromosomal distribution pattern of the 45S and 5S ribosomal (r) DNAs in four populations of the characid fish Astyanax scabripinnis – a group considered to be a species complex for its wide karyotypical and morphological diversity. The results regarding the 45S rDNA agreed with this hypothesis, since these sites showed intra- and inter-populational, numerical and positional variations. However, the data obtained with the 5S rDNA probe revealed a highly conserved chromosomal distribution pattern of these sequences among individuals of each population, as well as among the populations analysed. We consider this contrasting situation as a functional divergence between 45S and 5S ribosomal DNAs, which may reflect the localisation of these sequences in distinct nuclear compartments, leading them to undergo differentiated evolutionary processes.     

Karyotypic characterization of <Emphasis Type="Italic">Trachemys dorbigni</Emphasis> (Testudines: Emydidae) and <Emphasis Type="Italic">Chelonoidis (Geochelone) donosobarrosi</Emphasis> (Testudines: Testudinidae), two species of Cryptodiran turtles from Argentina

We describe for the first time the karyotypes of two species of Cryptodiran turtles from Argentina, namely, Trachemys dorbigni (Emydidae) and Chelonoidis (Geochelone) donosobarrosi (Testudinidae). The karyotype of T. dorbigni (2n = 50) consists of 13 pairs of macrochromosomes and 12 pairs of microchromosomes, whereas the karyotype of C. donosobarrosi (2n = 52) consists of 11 pairs of macrochromosomes and 15 pairs of microchromosomes. Fluorescence in situ hybridization (FISH) with a (TTAGGG)n telomeric probe showed that the chromosomes of these species have four telomeric signals, two at each end, indicating that none of the chromosomes of T. dorbigni and C. donosobarrosi are telocentric. The fact that no interstitial telomeric signals were observed after FISH, suggests that interstitial telomeric sequences did not have a major role in the chromosomal evolution of these species. Additional data will be needed to elucidate if interstitial telomeric sequences have a major role in the karyotypic evolution of Testudines.     

Comparative cytogenetic study of the forms of <Emphasis Type="Italic">Macleaya cordata</Emphasis> (Willd.) R. Br. From different localities

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of 26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.     

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.     

Localization of 5S and 25S rRNA genes on Somatic and meiotic chromosomes in <Emphasis Type="Italic">Capsicum</Emphasis> species of chili pepper

The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, an-nuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense, frutescens, and chinense, and four in baccatum, with the exceptions that ‘CM334’ of annuum had three loci and ‘tabasco’ of frutescens had one locus. ‘CM334’-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from ‘CM334’ plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili.