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.     

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae)

Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA⁺⁺/DAPI⁻ bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.     

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera,Meliponini)

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.     

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.     

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.     

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.     

Cytogenetic characterization of the invasive mussel species Xenostrobus securis Lmk. (Bivalvia: Mytilidae)

The chromosomes of the invasive black-pigmy mussel (Xenostrobus securis (Lmk. 1819)) were analyzed by means of 4',6-diamidino-2-phenylindole (DAPI) / propidium iodide (PI) and chromomycin A3 (CMA) / DAPI fluorescence staining and fluorescent in situ hybridization using major rDNA, 5S rDNA, core histone genes, linker histone genes, and telomeric sequences as probes. The diploid chromosome number in this species is 2n = 30. The karyotype is composed of seven metacentric, one meta/submetacentric, and seven submetacentric chromosome pairs. Telomeric sequences appear at both ends of every single chromosome. Major rDNA clusters appear near the centromeres on chromosome pairs 1 and 3 and are associated with bright CMA fluorescence and dull DAPI fluorescence. This species shows five 5S rDNA clusters close to the centromeres on four chromosome pairs (2, 5, 6, and 8). Three of the four core histone gene clusters map to centromeric positions on chromosome pairs 7, 10, and 13. The fourth core histone gene cluster occupies a terminal position on chromosome pair 8, also bearing a 5S rDNA cluster. The two linker histone gene clusters are close to the centromeres on chromosome pairs 12 and 14. Therefore, the use of these probes allows the unequivocal identification of 11 of the 15 chromosome pairs that compose the karyotype of X. securis.     

Comparative cytogenetic analysis of two grasshopper species of the tribe Abracrini (Ommatolampinae, Acrididae)

The grasshopper species Orthoscapheus rufipes and Eujivarus fusiformis were analyzed using several cytogenetic techniques. The karyotype of O. rufipes, described here for the first time, had a diploid number of 2n = 23, whereas E. fusiformis had a karyotype with 2n = 21. The two species showed the same mechanism of sex determination (XO type) but differed in chromosome morphology. Pericentromeric blocks of constitutive heterochromatin (CH) were detected in the chromosome complement of both species. CMA(3)/DA/DAPI staining revealed CMA(3)-positive blocks in CH regions in four autosomal bivalents of O. rufipes and in two of E. fusiformis. The location of active NORs differed between the two species, occurring in bivalents M(6) and S(9) of O. rufipes and M(6) and M(7) of E. fusiformsi. The rDNA sites revealed by FISH coincided with the number and position of the active NORs detected by AgNO(3) staining. The variability in chromosomal markers accounted for the karyotype differentiation observed in the tribe Abracrini.     

Nuclear DNA content,base composition,heterochromatin and rDNA in Picea omorika and Picea abies

Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species. Received: 18 October 2000 / 14 June 2001     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

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.     

Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.     

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.     

Cytogenetic studies of four South American species of Paullinia L. (Sapindaceae)

Four South American species of Paullinia ( P. elegans , P. meliaefolia , P. pinnata , and P. rhomboidea ) were compared using conventional chromosome staining, C-Giemsa and C-chromomycin A₃/4',6-diamidino-2-phenylindole (C-CMA₃/DAPI) banding, and fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe. All species showed a somatic complement of 2 n  = 24 chromosomes, agreeing with earlier records in some cases, and showing a tendency for the chromosome number to be conserved in this genus. The chromosome number of P. rhomboidea is a new report. The karyotypes differed in chromosome size and degree of karyotype asymmetry. The chromosomal band patterns and location of the 45S rDNA sites are reported for the first time in the genus. Terminal C-CMA₃ bands were associated with the 45S rDNA sites, but varied in number and size between the species. The occurrence of other C-Giemsa bands that were not revealed by CMA₃ suggests that more than one family of repetitive DNA may be involved in karyotype differentiation. The systematic implications of these results on the infrageneric relationships are discussed.   © 2007 The Linnean Society of London . Botanical Journal of the Linnean Society , 2007, 154 , 313–320.     

Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck]

Citrus sinensis chromosomes, although small in size, present a remarkable differentiation of bands with the fluorochromes CMA and DAPI. These bands suggest that some heteromorphisms are fixed in this species. To investigate the extension of these heteromorphisms, ten cultivars of C. sinenesis were analysed with CMA/DAPI staining and, in some of them, the 18S–5.8S–25S rRNA and 5S rRNA genes were located by in situ hybridization. CMA/DAPI staining showed exactly the same CMA⁺/DAPI⁻ banding pattern for all cultivars. In situ hybridization revealed three 18S–5.8S–25S rRNA gene sites, two proximally located on two similar chromosomes and one terminally located on a third non-related chromosome. Two 5S rRNA gene sites were observed in this species, with one located proximal to the telomeric 18S–5.8S–25S rDNA site. Both cytological approaches revealed an invariable, heterozygotic karyotype among sweet orange cultivars. Based on these data, the putative hybrid origin of the species is discussed. Received: 9 April 1999 / Accepted: 22 June 1999     

Karyotypic characterization and NOR analysis by different banding techniques of Rhamdia quelen (Pisces, Pimelodidae) from the first plateau of the Iguaçu River (Brazil)

A population of R. quelen from the first plateau of the Igua?u River (Paraná State, Brazil) was analyzed cytogenetically. A diploid set of 58 chromosomes was constituted by 32 M, 16 SM, 6 ST and 4 A (FN=116). In one individual a 2n=59 karyotype was determined due to the presence of one additional metacentric heterochromatic chromosome. NORs, detected by AgNO3 and CMA3 staining as well as fluorescence in situ hybridization with an 18S rDNA probe, are located at a terminal position on the short arms of a ST chromosome pair. C-banding marks the NORs and other weak bands distributed on telomeric regions of some chromosomes. These results give evidence that Rhamdia constitute, in terms of karyotypic macrostructure, a conserved group and support the idea that several synonymous species may be included in this genus.