CPD staining: an effective technique for detection of NORs and other GC-rich chromosomal regions in plants.

Mitotic chromosome spreads of 16 plant species belonging to six families were analyzed using an improved combined PI and DAPI (CPD) staining procedure. Fluorescence in situ hybridization (FISH) with 45S rDNA probe was conducted sequentially on the same spreads to evaluate the efficiency and sensitivity of the technique. Fluorochrome staining with chromomycin A3 (CMA)-DAPI also was conducted to clarify the properties of the sequences involved in the CPD banded regions. Our results revealed that all of the NORs (rDNA sites) in the species tested were efficiently shown as red bands by CPD staining, and the number and position of the bands corresponded precisely to those of the 45S rDNA FISH signals, indicating that the detection sensitivity of CPD staining is similar to that of FISH. In 10 of the species tested including Aegilops squarrosa, Allium sativum, Oryza sativum ssp. indica, Oryza officinalis, Pisum sativum, Secale cereale, Setaria italica, Sorghum vulgare, Vicia faba and Zea mays, CPD bands were exhibited exclusively in their NORs, while in other six species including Hordeum vulgare, Allium cepa, Psophocarpus tetragonolobus, Arabidopsis thaliana, Brassica oleracea var. capitata and Lycopersicon esculentum, CPD bands appeared in chromosomal regions other than their NORs. The CPD bands were in accordance with the CMA bands in all species tested, indicating GC-rich sequences in the CPD bands and that the improved CPD staining procedure is specific for GC-rich regions in plant genomes. Our investigation not only elucidated the banding mechanisms of CPD, but also demonstrated that the CPD staining technique, which may be preferable to CMA staining, is an effective tool for detecting NORs and other GC-rich chromosomal regions in plants.     

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae)

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.     

Study of the Nucleolar Organizer Regions in Palinurus elephas (Crustacea: Decapoda)

Using fluorescence in situ hybridization (FISH), chromomycin (CMA₃) staining and silver staining, we studied the nucleolar organizer regions in the spiny lobster Palinurus elephas in order to extend our knowledge on the karyology of this commercially important species. Multiple NORs have been detected by FISH, and CMA₃ showed a good correspondence between the localization of GC-rich heterochromatin and the ribosomal genes mapped by FISH. In contrast, the number of Ag-positive regions was higher than the number of FISH and CMA₃ signals, which may be explained by silver staining of the kinetochores. A variability in the number of FISH and CMA₃ signals has been detected in metaphases I and II which is probably due to the occurrence of rDNA cistrons on B chromosomes.     

Karyotype analysis, banding, and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea : Scarabaeidae)

Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic chromosomes of Gymnopleurus sturmi (Scarabaeoidea : Scarabaeidae) are described. The karyotype is distinguished by a pericentric inversion polymorphism in chromosome 3, which is either acrocentric or subtelocentric. Silver staining (Ag-NOR) and chromomycin A3 (CMA3), failed to study the detection of nucleolar organizer regions (NORs), due to the extensive silver and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped the ribosomal RNA genes (rDNA). FISH with the all-human telomeric sequences (TTAGGG)n revealed a lack of homology between the telomeric probe and the telomeres of G. sturmi. This suggests that the telomeric hesanucleotide (TTAGGG)n is not so conserved within eukaryotes as it has been hypothesized.     

Chromosomal Mapping and Molecular Characterization of Ribosomal RNA Genes in Lebias fasciata (Teleostei, Cyprinodontidae)

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.     

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.     

NOR regions of polychaete worms of the genus Ophryotrocha studied by chromosome banding techniques and FISH

This article reports the results of cytogenetic analyses carried out on 10 species of polychaete worms belonging to the genus Ophryotrocha (Dorvilleidae). Nucleolar organizer regions (NORs) were characterized by Ag staining, C-banding, CMA3 staining, and ribosomal fluorescent in situ hybridization (rDNA FISH). Extensive intraspecific variation in NOR number and distribution were observed in O. costlowi, O. sp. macrovifera, O. notoglandulata, O.l. labronica, O. l. pacifica (2n = 6), O. p. puerilis, O. diadema (2n = 8), O. hartmanni, O. gracilis (2n = 10). In O. sp. robusta (2n = 10), Ag-NORs were always located on a single chromosome pair. CMA3 staining suggests a possible trend toward a GC-rich rDNA compartmentalization. In O.l. labronica, O. p. puerilis, O. diadema, and O. sp. robusta rDNA FISH shows that Ag and FISH signals coincide. Results from C-banding seem to indicate that the increased genome size (GS) observed in O. sp. macrovifera (0.8 pg) and O. hartmanni (1.16 pg) compared to the base GS value of the genus (0.4 pg) cannot be attributed to variation in the heterochromatin content.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

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     

Fragile Sites of ‘Valencia’ Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in ‘Valencia’ C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of ‘Valencia’ C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid ‘Valencia’ C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in ‘Valencia’ sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in ‘Valencia’ sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.     

Cytogenetic characterization of <Emphasis Type="Italic">Eurysternus caribaeus</Emphasis> (Coleoptera: Scarabaeidae): evidence of sex-autosome fusion and diploid number reduction prior to species dispersion

Mitotic and meiotic chromosomes of several populations of Eurysternus caribaeus (Coleoptera: Scarabaeidae) were analysed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining and fluorescent in situ hybridization (FISH). All specimens showed 2n = 8 in their karyotypes, with a neo-XY sex system (Y is a submetacentric and X a metacentric) and three pairs of submetacentric autosomes. The analysis of constitutive heterochromatin (CH) revealed small blocks located in the centromeric region of all chromosomes which do not present positive staining under the fluorochromes CMA3 and DAPI. Silver nitrate staining revealed that the nucleolar organizer region (NORs) is associated with the sex chromosomes. The FISH technique revealed that rDNA sites in the X and Y are different in size. Data from different populations indicate that the diploid number reduction (2n = 8) observed in E. caribaeus is established and presumably has preceded the dispersion of this species. Moreover, this reduction occasioned the translocation of rDNA sites to the sex chromosomes, X and Y, an uncommon pattern in Scarabaeidae that was observed for the first time by the FISH in this work.     

Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization

A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes cma3/da and dapi/da, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects.     

Comparative karyology of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata (Phyllostomidae, Chiroptera): banding patterns, base-specific fluorochromes and FISH of ribosomal genes

This paper provides new data on chromosomes of Brazilian vampire bats Desmodus rotundus and Diphylla ecaudata. These species were analyzed by GTG, CBG- and CB-DAPI banding, AgNO3/CMA3 sequential staining, base-specific fluorochrome dyes and in situ hybridization with 18S rDNA probe. C-banding (CBG) revealed constitutive heterochromatin in the pericentromeric regions in all autosomes and the X and Y chromosomes appeared entirely heterochromatic in both species. CB-DAPI revealed a coincident banding pattern to that obtained by CBG. Triple staining CMA3/DA/DAPI revealed an R-banding and a weak G-banding pattern in the karyotypes. Sequential AgNO3/CMA3 staining showed a NOR located interstitially on the long arm of pair 8 in D. rotundus and on the short arm of pair 13 in D. ecaudata. FISH with a rDNA probe confirmed the location and number of NORs; a difference neither in intensity nor in size of hybridization signal was detected between homologues for both species.     

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.     

Lack of correspondence between CMA3-, Ag-positive signals and 28S rDNA loci in two Iberian minnows (Teleostei, Cyprinidae) evidenced by sequential banding

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations.     

Comparative molecular cytogenetic analysis of three <Emphasis Type="Italic">Leuciscus</Emphasis> species (Pisces,Cyprinidae) using chromosome banding and FISH with rDNA

A comparative molecular cytogenetic analysis was performed on three species of the genus Leuciscus viz. ide L. idus, chub L. cephalus and dace L. leuciscus distributed in Poland, using C-, Ag- and chromomycin A₃ (CMA₃)-stainings and fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Although the three species examined shared 2n = 50 chromosomes and the largest acrocentric chromosome pair in the complement, they were characterized with karyotypic differences in terms of the number of uni- and biarmed chromosomes and the localization of nucleolar organizer regions (NORs) revealed by Ag-staining and FISH. L. idus and L. cephalus showed the rDNA sites on the long arms of one submetacentric (SM) chromosome pair and on the short arms of one subtelocentric (ST) chromosome pair, respectively. These NORs were CMA₃-positive, GC-rich and C-positive heterochromatic sites in both species. Such chromosome banding features were also true for four NORs localizing on one of each SM and ST pair in L. leuciscus, but considerable numerical NOR polymorphism became apparent with Ag-staining and FISH due to a different combination of these NOR-bearing SMs and STs in this dace. The present results indicate that the molecular cytogenetic analysis applied herein may become useful to elucidate the karyotype evolution and phylogenetic relationships among the species in the genus Leuciscus and other related groups.     

Cytogenetic studies in South American species of Serjania (Sapindaceae: Paullinieae)

Abstract

Serjania Mill. (Paullinieae) is considered the most important neotropical genus of Sapindaceae due to species number and its widespread distribution. In this study, 14 species belonging to three sections were analyzed using conventional staining, C/CMA/DAPI banding, and fluorescence in situ hybridization (FISH) with a 18S-5.8S-26S rDNA probe. New chromosome counts are reported for Serjania crassifolia, Serjania platycarpa, and Serjania regnellii, all with 2n = 24, which is remarkably constant for Serjania. The karyotypes are moderately asymmetric, and variations observed in A1 and A2 indices show resemblances between S. platycarpa, Serjania hebecarpa, and S. crassifolia, and between Serjania communis, Serjania gracilis, and S. regnellii. The banding pattern was homogeneous in Serjania. C/DAPI bands (AT-rich sites) were not clearly evidenced, but changes in the number and position of GC-rich sites (CMA bands) were observed. These segments were associated with 18S-5.8S-26S rDNA sites. The significance of the results is discussed in relation to chromosomal data available for the genus and in regard to the infrageneric treatment of Serjania.     

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.