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

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

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。     

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.     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.     

Molecular cytogenetics studies in Reichardia tingetana: Physical mapping of heterochromatin,telomere repeats,and 5S and 45S rDNA by 4′, 6‐diamidino‐2‐phenylindole and fluorescence in situ hybridization

Abstract Molecular cytogenetics studies of A‐T‐rich regions, telomeres, and 5S and 45S rDNA sites on the chromosomes of Reichardia tingetana Roth (2n= 16; diploid) were done using 4′, 6‐diamidino‐2‐phenylindole (DAPI) and fluorescence in situ hybridization (FISH). The species were collected from three geographically isolated populations at Borg El Arab (salt marsh habitat), and Rashed and Shosha (sandy clay habitats) in Egypt. The three populations showed the chromosome number of all plants are diploid except for two tetraploid samples from Shosha. Plants from both Rashed and Shosha showed similarity in the distribution of six DAPI bands on six chromosomes, whereas those of Borg El Arab showed a distribution of 16 bands on 14 chromosomes. The FISH signals of the telomeres, and 5S and 45S rDNA, were at the telomeres of all chromosomes, two interstitial, and four terminal, respectively. The combination of DAPI and FISH showed colocalization of the DAPI bands with two 5S and two 45S rDNA loci. The increased number of DAPI bands in the cytotypes from the salt marsh habitat could indicate natural genetic adaptation through increasing the heterochromatin of A‐T‐rich regions.     

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.     

Reduction of chromosome number in Eleocharis subarticulata (Cyperaceae) by multiple translocations

Eleocharis subarticulata is recorded as the third species of Cyperaceae with a reduced chromosome number ( n  = 3), following reports on Rhynchospora tenuis ( n  = 2) and Fimbristylis umbellaris ( n  = 3). For Eleocharis, the numbers recorded to date vary from 2 n  = 10 to 2 n  = c. 196, with x  = 5 as the possible basic number. The karyotype of E. subarticulata was studied using conventional staining (mitosis and meiosis), C-CMA₃/DAPI banding, and FISH with 45S rDNA and telomere probes. The chromosomes showed no primary constrictions, as expected in the holocentric chromosomes of Cyperaceae. The meiotic behaviour was abnormal, with a single multivalent ring of six chromosomes at metaphase I, resulting from multiple translocations. At anaphase I six chromatids migrated to each pole, evidencing the inverted meiosis, and these groups were also visible at metaphase II. The C-CMA₃/DAPI banding technique showed only four terminal GC-rich blocks. FISH with 45S rDNA probes revealed four terminal signals, probably associated with GC-rich blocks. The telomeric probe located terminal signals in all the chromosomes, besides a hybridization site in the middle of the large pair. The occurrence of ectopic telomeric sites has not been described previously for plants with holokinetic karyotypes and with reduced chromosome numbers. These data reinforce the hypothesis of the reduction in chromosome number by multiple translocations.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 457–464.     

Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected complexity in a rye translocation used in chromosome mapping

Using fluorescence in situ hybridization (FISH) with probe pScT7, three different 5S rDNA loci were detected in the satellite of rye chromosome 1R (5SDna-R1) and in the short arms of chromosomes 3R (5SDna-R3) and 5R (5SDna-R2) respectively. All three loci showed polymorphism for the hybridization signal intensity. In order to determine the localization of these rye 5S rDNA multigene loci with higher precision within the corresponding chromosome arms, the probe pScT7 was physically mapped by FISH in relation to the following five translocations (Wageningen Tester Set): T850W (1RS/4RL), T248W (1RS/6RS), T273W (1RS/5RL), T305W (2RS/5RS) and T240W (3RS/5RL). Accurate physical maps of the translocation breakpoints had previously been made using electron microscope analysis of spread pachytene synaptonemal complexes of heterozygotes for the different translocations. The results indicate that locus 5SDna-R3 is located between the breakpoint of translocation T240W and the telomere, whereas locus 5SDna-R2 is located between the breakpoint of translocation T305W and the centromere, the hybridization of probe pScT7 on T305W translocated chromosomes demonstrating the complex nature of this translocation. On the other hand, the simultaneous detection of probes pScT7 and pTA71 (18S-5.8S-26S rDNA) with two different fluorochromes, indicated that the breakpoints of translocations T850W and T248W are located between loci Nor-R1 and 5SDna-R1.     

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA) n , (TTAGGG) n and ribosomal genes by one-color and two-color FISH

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA) _n , and (TTAGGG) _n , and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG) _n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA) _n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

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.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Polyploidy, B chromosomes, and heterochromatin characterization of Mimosa caesalpiniifolia Benth. (Fabaceae-Mimosoideae)

Eight populations of Mimosa caesalpiniifolia Benth. were investigated using a cytogenetic approach. Here, we describe for the first time details of the karyotype including chromosome morphology, physical mapping of chromomycin A3 (CMA) 4′,6-diamidino-2-phenylindole (DAPI) and silver staining of nucleolar organizer regions (Ag-NOR banding), as well as 45S rDNA sites. All populations studied showed karyotypes with 2n?=?2x?=?26 small metacentric and submetacentric chromosomes, although some individuals exhibited 2n?=?4x?=?52 chromosomes. Moreover, we observed putative additional B chromosomes in some populations. The CMA banding and fluorescent in situ hybridization techniques revealed NOR heteromorphism on the unique pair containing 45 rDNA site (chromosome 12) while the Ag-NOR banding indicated NORs on both cytotypes. Up to two and four nucleoli were observed, respectively, on individuals with 2n?=?2x?=?26 and 2n?=?4x?=?52 chromosomes and the differences in nucleolar size seems to be directly related to NOR heteromorphism in some individuals. The data present new and important information to understand karyotypic evolution of Mimosa and Fabaceae.     

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

Polypterids are a group of Osteichthyan fish whose evolutionary relationships with closer basal ray-finned and lobe-finned fish have been disputed since their discovery. Very little is known about the evolutive karyology in the whole Polypteriformes group. In order to fill this gap, a cytogenetic analysis of Erpetoichthys calabaricus species was performed, using both classical and molecular techniques. Karyotype structure (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG)n and ribosomal 5S and 18S rRNA genes were examined in twenty specimens of E. calabaricus by using Ag-NOR, classical C-banding, sequential CMA3/4',6-diaminidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH). CMA3 marked all centromerical and some (no. 1 and no. 15) telomeric regions. Staining with Ag-NOR and CMA3 showed the presence of two NORs on the p arm of the chromosome pair no. 1. Hybridization with telomeric probes (TTAGGG)n showed signals at the end of all chromosomes. 5S rDNA was cloned and sequenced. After the alignment, the 5S rRNA sequences revealed an organization made up of two different classes of tandem arrays (type I and type II). FISH with 5S rDNA marked the telomeric regions of the small chromosome pair no. 15, while FISH with 18S rDNA marked the telomeric region of the pair no. 1. The results obtained were compared with cariological data on closer species now available in literature.     

Occurrence of pre-nucleolar bodies and 45S rDNA location on the chromosomes of the ant Mycocepurus goeldii (Forel) (Formicidae, Myrmicinae, Attini)

The ant Mycocepurus goeldii (Forel) is known for having a relict karyotype with low chromosome number and the present study help the understanding of this ant cytogenetics by describing the occurrence of pre-nucleolar bodies in their chromosomes using impregnation with silver nitrate (Ag-NOR) and the location of 45S rDNA sites by means of the FISH (fluorescent in situ hybridization) technique. Several spots were observed surrounding all chromosomes when submitted to the Ag-NOR technique. These unusual markings were observed in both chromatids of metaphase and early anaphase chromosomes, and are associated to the presence of pre-nucleolar bodies, allowing the observation of the phenomenon of nucleologenesis. Although recent studies have shown that all chromosomes of M. goeldii exhibit centromeric or pericentromeric markings for the CMA(3) fluorochrome, the FISH technique indicated the presence of 45S rDNA in only one pair of chromosomes that differed in the number of CMA(3) markings observed for this species, pointing that the other markings observed with this fluorochrome do not match the sequences in ribosomal genes.