Cytogenetic characterization and description of an XX/XY1Y2 sex chromosome system in catfish Harttia carvalhoi (Siluriformes, Loricariidae)

Karyotypic and cytogenetic characteristics of catfish Harttia carvalhoi (Paraíba do Sul River basin, S?o Paulo State, Brazil) were investigated using differential staining techniques (C-banding, Ag-staining) and fluorescent in situ hybridization (FISH) with 18S and 5S rDNA probes. The diploid chromosome number of females was 2n = 52 and their karyotype was composed of nine pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric and four pairs of acrocentric chromosomes. The diploid chromosome number of males was invariably 2n = 53 and their karyotype consisted of one large unpaired metacentric, eight pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric, four pairs of acrocentric plus two middle-sized acrocentric chromosomes. The differences between female and male karyotypes indicated the presence of a sex chromosome system of XX/XY1Y2 type, where the X is the largest metacentric and Y1 and Y2 are the two additional middle-sized acrocentric chromosomes of the male karyotype. The major rDNA sites as revealed by FISH with an 18S rDNA probe were located in the pericentromeric region of the largest pair of acrocentric chromosomes. FISH with a 5S rDNA probe revealed two sites: an interstitial site located in the largest pair of acrocentric chromosomes, and a pericentromeric site in a smaller metacentric pair of chromosomes. Translocations or centric fusions in the ancestral 2n = 54 karyotype is hypothesized for the origin of such multiple sex chromosome systems where females are fixed translocation homozygotes whereas males are fixed translocation heterozygotes. The available cytogenetic data for representatives of the genus Harttia examined so far indicate large kayotype diversity.     

Major cytogenetic landmarks and karyotype analysis inDaucus carota and other Apiaceae

Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution. Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives. Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci. All had single loci for both 5S and 18S-25S (nucleolar organizing region) rDNA, located on two different chromosome pairs. The 5S rDNA was on the short arm of a metacentric chromosome pair in D. crinitus (2n = 22) and D. glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome. For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S-25S rDNA sites. Variability for number and position of the 5S rDNA was also observed. FISH signals enabled us to identify 20-40% of the chromosome complement among species examined. Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus.     

Use of the chromosomal co-location of the minor 5S and the major 28S rDNA as a cytogenetic marker within the genus Leuciscus (Pisces, Cyprinidae)

The chromosomes of three species from the genus Leuciscus (the ide L. idus, the European chub L. cephalus and the common dace L. leuciscus) were examined with the FISH technique for 5S and 28S rDNA probes. The analysis showed that among the three examined species, 5S rDNA signals were located on two large and four small subtelocentric chromosomes in L. leuciscus, on one large and five small subtelocentric chromosomes in L. idus, while in L. cephalus the probe signals were found on two metacentric chromosomes and one large and one small subtelocentric chromosome pairs. In all analysed species, the 28S rDNA probe signals were placed on only one chromosome pair, subtelocentric in the common dace and the European chub, and submetacentric in the ide. The three species differed in the number of sites in which both probe signals were present. In conclusion, the co-location of the 5S and 28S rDNA proved to be a useful cytogenetic marker among the studied fishes. Moreover, this marker could be adapted to other cyprinids.     

Comparative cytogenetics of giant trahiras Hoplias aimara and H. intermedius (Characiformes, Erythrinidae): chromosomal characteristics of minor and major ribosomal DNA and cross-species repetitive centromeric sequences mapping differ among morphologically identical karyotypes

Karyotype and cytogenetic characteristics of 2 species of giant trahiras, Hopliasintermedius, S?o Francisco river basin, and Hopliasaimara, Arinos river (Amazon basin), were examined by conventional (C-banding, Ag-NOR, DAPI/CMA(3) double-staining) and fluorescent in situ hybridization (FISH) with 5S, 18S rDNA probes and cross-species Cot-1 DNA probing. Both species invariably had diploid chromosome number 2n = 50 and identical karyotypes composed of 10 pairs of metacentric and 15 pairs of submetacentric chromosomes. On the other hand, staining with base-specific fluorochromes (CMA(3), DAPI) and FISH mapping of repetitive DNA sequences showed extensive interspecific differences: while the genome of H. aimara had one submetacentric pair bearing CMA(3)-positive (DAPI-negative) sites, that of H. intermedius had 4 such pairs; while FISH with a 5S rDNA probe showed one (likely homologous) signal-bearing pair, that with 18S rDNA displayed one signal-bearing pair in H. intermedius and 2 such pairs in H. aimara. Cross-species FISH probing with Cot-1 DNA prepared from total DNA of both species showed no signals of Cot-1 DNA from H. aimara on chromosomes of H. intermedius but reciprocally (Cot-1 DNA from H. intermedius on chromosomes of H. aimara) displayed signals on at least 4 chromosome pairs. Present findings indicate (i) different composition of repetitive sequences around centromeres, (ii) different NOR phenotypes and (iii) distinct taxonomic status of both giant trahira species.     

Evidence of hexaploid karyotype in shortnose sturgeon

A karyotype analysis by several staining techniques was carried out on triplicate samples of the shortnose sturgeon, Acipenser brevirostrum. The chromosome number was found to be 2n = 372 +/- 6. A representative karyotype of 374 chromosomes was composed of 178 metacentrics/submetacentrics and 196 telocentrics/acrocentrics and microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible on 14 chromosomes. The signals obtained with a PstI satellite DNA probe appeared on 12 chromosomes. The FISH with a 5S rDNA probe revealed fluorescent signals on 6 chromosomes. These last results, compared with 2 signals in species with about 120 chromosomes and 4 in species with 240, support the hypothesis that A. brevirostrum is a hexaploid species, probably of hybrid origin. Based on these results, we propose a model explaining speciation events occurring in sturgeons by hybridization, genome duplication, and diploidization.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Conservation of the 5S-bearing chromosome pair and co-localization with major rDNA clusters in five species of Astyanax (Pisces,Characidae)

Major and 5S ribosomal genes have been localized in chromosomes from five fish species, genus Astyanax, using in situ hybridization (FISH) with 28S and 5S rDNA probes. In situ signals for the major rDNA co-localized with the 5S rDNA clusters in the pericentromeric region of one marker chromosome in all five species analyzed. The conserved localization of these two rDNA clusters in the five related Astyanax species was considered as indicative of a close relationship among them. The use of these molecular markers for elucidating evolutionary relationships among closely related taxa is discussed.     

Chromosomal divergence and maintenance of sympatric Characidium fish species (Crenuchidae, Characidiinae)

Cytogenetic studies were performed in two syntopic species of Characidium, C. lauroi and Characidium sp. cf. C. alipioi, from Ribeir?o Grande, Paraíba do Sul river basin. Both species have diploid number 2n=50 chromosomes, but differ in chromosome shape, C-banding pattern and location of nucleolar organizing regions. In Characidium sp. cf. C. alipioi a new type of ZW sex chromosome system composed of equal sized metacentric chromosomes is reported for the first time in the genus Characidium. Species of Characidium with a sex chromosome system form a monophyletic group. Variations in this system are interpreted as resulting from geographic isolation among allopatric species.     

Chromosomal localization of 18S and 5S rDNA using FISH in the genus <Emphasis Type="Italic">Tor</Emphasis> (Pisces,Cyprinidae)

Dual color fluorescence in situ hybridization (FISH) was performed to study the simultaneous chromosomal localization of 18S and 5S ribosomal genes in the genus Tor for the first time. The 18S and 5S rDNAs in four Tor species were amplified, sequenced and mapped on the metaphase chromosomes. The number and distribution of 18S and 5S rDNA clusters were examined on metaphase chromosome spreads using FISH. The specimens of T. chelynoides, T. putitora and T. progeneius showed six bright fluorescent signals of 18S rDNA and T. tor exhibited ten such signals. The 5S rDNA signals were present only on one pair of chromosomes in all the four Tor species. Ag-NORs were observed on two pairs of chromosomes in T. chelynoides, T. putitora, T. progeneius and four pairs in T. tor. Comparison of the observed 18S rDNA FISH signals and Ag-NORs strongly suggested a possible inactivation of NORs localized at the telomeres of a subtelocentric and telocentric chromosome pairs in all four species. The 5S rDNA contained an identical 120 bp long coding region and 81 bp long highly divergent non-transcribed spacers in all species examined. 18S and 5S rDNA sequencing and chromosomal localization can be a useful genetic marker in species identification as well as phylogenetic and evolutionary studies.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

眼斑拟石首鱼重复DNA序列的染色体定位

针对眼斑拟石首鱼Sciaenops ocellatus染色体标记匮乏的问题, 利用荧光原位杂交(FISH)定位了眼斑拟石首鱼的18S rDNA、5S rDNA和端粒序列。结果显示, 眼斑拟石首鱼的核型公式为2n=48t; 仅有1对18S rDNA位点, 位于第1对染色体的次缢痕部位; 有2对5S rDNA位点, FISH信号强度不等, 强信号位于第8对染色体的近着丝粒端, 弱信号位于第3对染色体的臂间。端粒FISH信号出现于所有染色体的两端, 但表现出染色体两端信号不平衡的特点, 着丝粒端FISH信号明显强于远端信号。这一特点为判定染色体的方向提供了便利。结合其他石首鱼的核型数据可以推断, 2n=48t的核型及单对近着丝粒分布的18S rDNA位点是石首鱼的共同祖征; 在石首鱼进化过程中, 曾发生活跃但不影响宏观核型的小规模重排。研究结果丰富了眼斑拟石首鱼染色体的辨识标记, 并为研究石首鱼染色体进化提供了基础数据。     

Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids

Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed.     

Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Salix viminalis L. (2n?=?38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afa-family, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

水稻45S rDNA和5S rDNA的染色体定位研究

45SrDNA和5SrDNA是水稻中与核糖体RNA合成有关的2个功能片段,有关这2个序列在水稻染色体上的位置,不同研究者的研究结果不尽相同,在获得水稻染色体清晰制片的基础上,通过FISH确定了45SrDNA序列位于水稻的第9号和第10号染色体的短臂末端,并且第9号染色体上的拷贝数多于第10号染色体,5SrDNA序列位于第11号染色体短臂靠近着丝点处。     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

Distribution of rDNA loci in the genus Glycine Willd.

The objective of this study was to examine the distribution of rDNA loci in the genus Glycine Willd. by fluorescent in situ hybridization (FISH) using the internal transcribed spacer (ITS) region of nuclear ribosomal DNA as a probe. The hybridized rDNA probe produced two distinct yellow signals on reddish chromosomes representing two NORs in 16 diploid (2n=40) species. Aneudiploid (2n=38) and aneutetraploid (2n=78) Glycine tomentella Hayata also exhibited two rDNA sites. However, the probe hybridized with four chromosomes as evidenced by four signals in two diploid species (Glycine curvata Tind. and Glycine cyrtoloba Tind.) and tetraploid (2n=80) G. tabacina (Labill.) Benth. and G. tomentella. Synthesized amphiploids (2n=80) of Glycine canescens F. J. Herm. (2n=40) and the 40-chromosome G. tomentella also showed four signals. This study demonstrates that the distribution of the rDNA gene in the 16 Glycine species studied is highly conserved and that silence of the rDNA locus may be attributed to amphiplasty during diploidization and speciation. Received: 10 October 2000 / Accepted: 6 December 2000     

Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization

An intensive karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) was carried out with three different methods. These included Feulgen staining, Giemsa C-banding, and fluorescence in situ hybridization (FISH). The mitotic chromosomes of the cucumber (2n = 2x = 14) were characterized, based on the length and arm ratio values. A C-banding analysis showed dark stains on the centromeric, telomeric, and intercalary regions of the chromosomes, except that chromosome 2 had a heavy staining in the long arm. Bicolor FISH, using 45S and 5S rDNA probes, provided additional information to identify cucumber chromosomes. The signals for 45S rDNA were detected on the pericentromeric regions of chromosomes 1, 2, and 4. The signals for 5S rDNA were on the short arm of chromosome 5. Similar band patterns (as the C-banding) were observed when the chromosomes were counter-stained with 4',6-diamidino-2-phenyoindole (DAPI). The data implied that the karyotype of the Korean cucumber cultivar is peculiar and different from previous reports.     

ZZ/ZW sex chromosome system in an undescribed species of the genus Apareiodon (Characiformes, Parodontidae)

The chromosomes of an undescribed species of the genus Apareiodon (Characiformes, Parodontidae) from the Verde River, a headwater affluent of the Tibagi River (Paraná State, Brazil), were investigated using conventional Giemsa and Ag stainings, C-banding, CMA(3) fluorescence and fluorescent in situ hybridization (FISH) using 18S and 5S rDNA probes. The diploid chromosome number was 2n = 54, with the karyotype composed of 48 meta/submetacentric and six subtelocentric chromosomes in males, and 47 meta/submetacentric + seven subtelocentric chromosomes in females. The difference is hypothesized to be due to a ZZ/ZW heteromorphic sex chromosome system, a cytotaxonomic characteristic previously observed only in some species of the genus Parodon (family Parodontidae). The presence of similar and/or identical heteromorphic sex chromosome systems might suggest that species of the genera Parodon and Apareiodon bearing ZZ/ZW heteromorphic sex chromosomes likely constitute a monophyletic group, a hypothesis to be tested by a robust phylogeny of the family.