CMA Bands Research Articles

The karyotype of Clivia mirabilis analyzed by differential banding and fluorescence in-situ hybridization

The karyotype of the recently described species Clivia mirabilis was analyzed by differential chromosome staining with Giemsa, chromomycin, and DAPI and by fluorescence in-situ hybridization with 5S and 45S rDNA probes. Like the other five Clivia species it was shown to have a unique karyotype, although its karyotype was similar in several respects to that of C. gardenii, differing in having only one pair of chromosomes with CMA bands compared with two pairs in C. gardenii and lacking any DAPI-positive bands. The evolutionary relationships of the species and their karyotypes are discussed.

Differential Chromosome Banding and Isozyme Assay of Three Corchorus spp.

Karyotypes of 3 wild Corchorus spp. of Tiliaceae, namely C. tridens, C. fascicularis and C. pseudo-capsularis, were investigated after differential staining with orcein, CMA and DAPI. In addition, the activities of 3 isozymes, esterase, acid phosphatase and peroxidase in these 3 species were compared. These 3 spp. were found to possess 2n=14 metacentric chromosomes. The CMA and DAPI bands were found in either the terminal or interstitial region of the respective chromosomes of 3 species. This indicated a tendency for the accumulation of GC- and AT-rich repeats at terminal or interstitial region of the chromosomes. The number and distribution of CMA- and DAPI-bands were unique for each species. A comparison of CMA- and DAPI-banding pattern indicated the presence of paracentric inversion in the concerned areas of the respective chromosomes. Different types of heteromorphicity regarding CMA- and DAPI-banding revealed the probable occurrence of deletion and tandem duplication. In acid phosphatase, 2 common bands were found in C. fascicularis and C. pseudo-capsularis. A dark, thick peroxidase band was found in C. fascicularis whereas a light, thick band occurred in C. pseudo-capsularis. These light and dark thick bands could be used as markers for these 2 spp. Moreover, a big upper esterase band makes C. tridens unique.

Karyotype Characterization with Fluorescent Banding in One Released and Two Wild Germplasms of Hibiscus cannabinus L.

One released (HC-95) and 2 wild (CPI-72126 and KE-1) germplasms of Kenaf (Hibiscus cannabinus L.) were studied to characterize their karyotypes. These germplasms were found to possess 2n=36 chromosomes. Except 2 sub-metacentric chromosomes in CPI-72126 and 1 in HC-95, all the chromosomes of these germplasms were metacentric. No significant difference in the range of chromosomal length and total length of 2n chromosome complements was found among these germplasms. These features revealed that the germplasms have symmetric karyotypes. Four orcein stained satellites were observed in KE-1 and CPI-72126. No satellite was found in CMA and only 2 in DAPI (KE-1). The differential staining property indicated that some satellites were AT-rich and other stain specific. No DAPI band was found in 3 germplasms. However, the number and distribution of CMA bands indicated that each germplasm has distinct CMA-karyotype and thus could easily be characterized.

Giemsa staining and fluorescent chromosome banding in someVitisL. species

Giemsa staining technique and fl uorescent chromosome banding with CMA 3 and DAPI were applied to the mitotic chromosomes of Vitis champinii, V. cinerea, V. girdiana, V. labrusca, V. rotundifolia, V. rupestris and V. vinifera for the purpose of chromosome measurement and constitutive heterochromatin characterization at the cytochemical level, respectively. Both fl uorescent CMA and conventional Giemsa staining constituted a valuable tool for chromosome characterization. Karyomorphometric data obtained after Giemsa staining allowed for an average ideogram and karyotype formulae based on chromosome length for the species. V. champinii and V. girdiana distinguished from the other fi ve species by means of the total haploid chromosome length, by the longest chromosome length and by the average chromosome length. The seven grape species have moderate chromosome asymmetry values and were classifi ed on 2A Stebbins' category. Positive CMA bands were seen in all species. V. girdiana distinguished from the other species solely by the presence of two CMA + bands, while V. champinii, V. cinerea, V. labrusca, V. rotundifolia and V. rupestris had four bands. V. vinifera showed chromosome heteromorphism for CMA bands. No clearly visible DAPI + band was seen in the species. According to the present observations, it seems that the evolutionary process of speciation involving North American and European Euvitis species studied, resulted in some discrete changes in chromosome measurements and also in heterochromatin base composition of at least one species. These data enlarge the chromosomal information of the genus Vitis and make possible further comparative studies into the Vitaceae family.

The evolution of CMA bands in Citrus and related genera

Most species of Citrus and related genera display a similar karyotype with 2n = 18 and a variable number of terminal heterochromatic blocks positively stained with chromomycin A(3) (CMA(+) bands). Some of these blocks are 45S rDNA sites, whereas others may correspond to the main GC-rich satellite DNA found in several Citrus species. In the present work, the distribution of the 45S rDNA and the main satellite DNA isolated from C. sinensis (CsSat) were investigated by in situ hybridization in seven species of Citrus, two species of closely related genera (Fortunella obovata and Poncirus trifoliata) and four species of the subfamily Aurantioideae, which were less related to Citrus (Atalantia monophylla, Murraya paniculata, Severinia buxifolia, and Triphasia trifolia). In Citrus, Fortunella, and Poncirus, most CMA(+) bands colocalized only with CsSat sites, whereas others colocalized only with rDNA sites. However, some of these species displayed a few CMA(+) bands that colocalized with sites of both probes and other CMA(+) bands that did not colocalized with any of the probes. On the other hand, in the four species less related to Citrus, no CsSat signal was found on chromosomes. On Southern blot, the CsSat probe hybridized with genomic DNA from Citrus, Fortunella, and Poncirus at high stringency only, while under the less stringent conditions, it also hybridized with distantly related species. Therefore, CsSat sequences are the principal component of the heterochromatic blocks of Citrus, Poncirus, and Fortunella, whereas CsSat-like sequences seem to be widespread in the subfamily Aurantioideae. These data further suggest that the variable number of terminal CMA(+) bands observed on chromosomes of Citrus and related genera are probably the consequence of amplification or reduction in the number of CsSat-like sequences distributed on chromosome termini, paralleled by mutation and homogenization events, as proposed by the library hypothesis.

Karyotype evolution and speciation of European lilies from Lilium sect. Liriotypus

AbstractThe main purpose of this study was to clarify the speciation and taxonomic status of European representatives of Lilium sect. Liriotypus (Lilium albanicum, L. bosniacum, L. bulbiferum, L. candidum, L. carniolicum, L. chalcedonicum, L. jankae, L. pomponium, L. rhodopaeum, L. pyrenaicum) based on karyotype differentiation. Clear interspecific differentiation was revealed among karyotypes of these taxa using fluorochrome (chromomycin and DAPI) bandings, fluorescent in situ hybridization and silver staining. Differences were evident in the number and position of CMA and DAPI bands and in the number, position and activity of 18S‐26S and 5S rDNA genes. It is hypothesized that the type species of Lilium sect. Liriotypus, L. candidum, represents the ancestral species from which three evolutionary directions diverged: the first towards L. chalcedonicum, the second towards L. rhodopaeum, and the third towards L. albanicum. The radiation process expanded from L. albanicum, beyond the southeast to the central Dinaric Alps where L. bosniacum is strongly represented. Furthermore, L. carniolicum evolved from northwestern populations of L. bosniacum. According to this evolutionary hypothesis, L. pomponium and L. pyrenaicum would be the youngest species.

Comparison of CMA-Karyotypes in Three Dendrobium spp.

Three orchid species, namely Dendrobium moschatum Sw., D. aphyllum Roxb. and D. aggregatum Roxb, were investigated by classical cytogenetics and CMA fluorescent banding methods. Dendrobium moschatum was found to possess 2n=40 chromosomes whereas, 2n=38 chromosomes found in D. aphyllum and D. aggregatum. The centromeric formula was 34 m+6 sm in D. moschatum, 38 m in D. aphyllum and 32 m+6 sm in D. aggregatum. No gradual decrease of chromosomal length was observed in these species indicated as primitive type. The total length of 2n chromosome complements in D. moschatum was 55.74 μm and 59.94 μm in D. aphyllum. Total length was remarkably less in D. aggregatum (44.15 μm). A pair of bright CMA bands was found in the interphase nuclei of these 3 species. In metaphase stage, 6 CMA bands were found in D. moschatum. The number of CMA bands was 4 and 10 in D. aphyllum and D. aggregatum, respectively. The percentage of CMA banded region in D. aphyllum was 4.76% while it was about double in D. moschatum (11.33%) and 3 times in D. aggregatum (15.38%). The CMA karyotypic features suggested that number, distribution and location of CMA bands were found to be species-specific.

Chromosome banding and essential oils composition of Brazilian accessions of Lippia alba (Verbenaceae)

The essential oil components and a karyotypic analysis of five Lippia alba (Verbenaceae) accessions from Brazil were performed with the objective of investigating the variation among different populations. The chemistry analysis allowed the grouping of the accessions in two main chemotypes: neral chemotype (LaCat, LaJF and LaRJ) and linalool chemotype (LaGua and LaVC). However, large karyotypic differences, verified by different chromosome banding techniques, were not detected. The results presented the same chromosome number for all accessions (2n = 30) with 10 metacentric chromosomes and 5 submetacentric. The chromosome banding showed great blocks of constitutive heterochromatin (C-bands) around the centromeric region, which was rich in AT bases (DAPI+), while the CMA bands were observed only in terminal regions of six chromosomes. Through Ag-NOR techniques, only two active pairs of NORs were detected on the three pairs of secondary constrictions (the NOR activity is discussed). This work relates the pattern of heterochromatin for Lippia alba for the first time.

Different chromatin fractions of tomato ( Solanum lycopersicum L.) and related species

Conventional chromosome staining has suggested that more than 75% of the tomato chromosomes are constituted by heterochromatin. In order to determine whether more deeply stained proximal regions are classic heterochromatin, the distributions of C-bands and chromomycin A 3 (CMA) bands, and the prophase condensation patterns, were analysed in tomato. In this and most other species of the tomato clade, the 5S and 45S rDNA sites were also localised. In tomato, CMA banding was similar to C-banding. After conventional staining, all species displayed large condensed heteropycnotic regions that did not correspond to C-bands or CMA bands. Analyses of the CMA banded karyotypes revealed a low heterochromatin content. Around 12–17% of the chromatin of tomato was CMA + and 1/4 to 1/5 of this heterochromatin corresponded to 45S rDNA. In other species, the CMA + heterochromatin showed extensive variation (8–35%), but was never near the values found in the literature for tomato. These data suggest the existence of three principal fractions of chromatin in tomato and related species: the late condensed euchromatin corresponding to the terminal regions of the chromosomes, the precocious condensed euchromatin that occupies the major part of the chromosomes and the constitutive heterochromatin that represents those regions revealed by C-bands.

Comparison of Orcein and CMA Stained Karyotypes in Three Morphological Forms of Typhonium trilobatum L. (Araceae)

The karyotypes in 3 morphological forms of Typhonium trilobatum (L.) Schott such as the Green form, the Dark purple form and the Light purple form were compared after staining with orcein and CMA. The Green form and the Light purple form were found to possess 2n =18 chromosomes. However, 2n =17 chromosomes were observed in the Dark purple form. One chromosome in pair IX was absent in this form. The karyotypic features revealed that the Dark purple form might be originated from the Green form as monosomic. The centromeric formula was determined 16m+2ac in the Green form, 11m+4sm+2ac in the Dark purple form and 13m+5sm in the Light purple form. The Dark purple form showed more heterogeneous karyotype and may be considered as more advanced than the other 2 forms. No CMA band was found in the Green form. The Dark purple and the Light purple forms were found to possess distinct CMA banding pattern. With the help of CMA banding, it was possible to identify some chromosomes in these 2 forms. Few chromosome pairs in both the forms showed heteromorphicity in respect of CMA banding pattern indicating the occurrence of structural aberration. Morphology, distinct karyotypic features and CMA banding pattern suggested for placing the Green form in different taxonomic rank, while the Dark purple form might be considered as a monosomic variety of Typhonium trilobatum.

Karyotype Analysis in Three Morphological Forms of Colocasia fallax Schott.

The 3 forms of Colocasia fallax Schott. complex, namely green petiole form, light purple petiole form and deep purple petiole form, available in Bangladesh were studied cytogenetically to confirm their taxonomic status after staining with orcein and CMA. The green petiole form and the deep purple petiole form were found to possess 2n=28 chromosomes whereas 2n=30 chromosomes were observed in the light purple petiole form. The centromeric formula was determined as 20 m+8 sm in the green petiole form, 26 m+2 sm+2 ac in the light purple petiole form and 24 m+4 sm in the deep purple petiole form. Acrocentric chromosomes were found only in the light purple petiole form. 4 satellites were found in the green petiole form after orcein staining while only 1 satellite observed in CMA staining of the same form. These satellites showed stain specific nature in this form. 8 and 11 CMA positive bands were found in the green petiole form and the deep purple petiole form, respectively. Only 2 centromeric CMA positive bands were found in the light purple petiole form. Different CMA banding pattern and percentage of GC-rich repeats were found in these 3 forms. CMA banding was able to identify some chromosomes in the green petiole form and the deep purple petiole form. The diploid chromosome numbers and overall karyotypic features indicated that the light purple petiole form possessed quite different genomes than the other 2 forms and thus may be placed in a different taxonomic rank. Except a minute difference in the karyotypic features, the green petiole form and the deep purple petiole form possessed almost entirely similar genomes and therefore could be considered as different varieties of Colocasia fallax.

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.

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.

Parental chromosome differentiation in citrangequat [ Fortunella sp. × ( Citrus sinensis × Poncirus trifoliata)] using CMA banding patterns

The efficiency of chromomycin A 3 (CMA) staining was examined for parental chromosome differentiation in citrange [ Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and citrangequat ( Fortunella sp. × citrange). All of the accessions analyzed had the same chromosome number of 2n = 18. CMA staining revealed six characteristic banding patterns on the basis of the number and position of CMA positive bands (CMA +) as follows; A: two terminal and one proximal band, B: one terminal and one proximal band, C: two terminal bands, D: one terminal band, E: no band, and F: one proximal band. Chromosome CMA banding patterns of the accessions were 1A + 1B + 2C + 13D + 1F in Fortunella margarita, 2B + 2C + 7D + 7E in ‘Fukuhara’ orange, 2B + 10D + 6E in Poncirus trifoliata, 1B + 1C + 10D + 6E in citrange and 1A + 1C + 11D + 4E + 1F in citrangequat. The results of this study confirmed the intergeneric and tri-generic hybridity of citrange and citrangequat, respectively.

Karyotype diversity and the origin of grapefruit

Grapefruit is a group of citrus of recent origin, probably resulting from a cross between pummelo and sweet-orange. Aiming to investigate this putative origin and the genetic variability among grapefruit cultivars, the karyotype of six grapefruits, two pummelos, and one tangelo cultivar (grapefruit x tangerine) were analyzed using sequential CMA/DAPI double staining and FISH with rDNA probes. The karyotypes of grapefruit 'Duncan' and 'Foster' differ from those of 'Flame', 'Henderson', 'Marsh' and 'Rio Red'. The former have two chromosomes with a single CMA(+) band in both terminal regions (C type chromosome) and six chromosomes with only one CMA(+) terminal band (D type), whereas the latter have three C and five D type chromosomes. All accessions investigated exhibited two chromosomes with 5S rDNA but a variable number of 45S rDNA. The two former grapefruits displayed four 45S rDNA sites, whereas the remaining grapefruit cultivars had five. The two pummelos showed identical karyotypes, homozygous for CMA(+) bands and their four rDNA sites. From each pummelo chromosome pair one chromosome seems to be present in grapefruit karyotypes. The different grapefruit karyotypes might result from independent crosses between pummelos of different karyotypic constitution and sweet-oranges. The chromosome markers found in the tangelo 'Orlando' and the position of their two 45S rDNA confirm the grapefruit 'Duncan' and the tangerine 'Dancy' as their parents.

Development of Marker Chromosomes in Three Varieties of Vigna radiata L. (Fabaceae)

Karyotype analysis of 3 released varieties in Vigna radiata L. viz. Barimung-2, Barimung-3, Barimung-5 has been carried out after staining with orcein, CMA and DAPI. These varieties were found to possess 2n=22 chromosomes. Four terminal CMA-positive bands were found in Barimung-2, 3 such bands in Barimung-3 and 4 in Barimung-5. The terminal CMA positive bands showed deep DAPI-negative reversible bands in the 3 varieties of V. radiata. One chromosome of Barimung-5 was fluoresced entirely with CMA and DAPI. DAPI-positive and DAPI-negative band occurred in another chromosome of Barimung-5. A pair of interstitial CMA-negative band was observed in chromosome pair V of only Barimung-2. These portions were stained brightly with DAPI. These kinds of reversible banded chromosomes could be easily identified. Polymorphism regarding the number and location of DAPI-positive bands were observed in these varieties. That may due to either minute deletion or high condensation of heterochromatic region at the respective loci. Few chromosomes of each variety showed characteristic CMA and DAPI bands. Therefore, with the help of CMA and DAPI it was possible to develop marker chromosomes for authentic identification of three varieties in V. radiata.

CMA Banding Patterns of Chromosomes in Major Citrus Species

CMA Banding Patterns of Chromosomes in Major Citrus Species

A simple chromosomal marker can reliably distinguishes Poncirus from Citrus species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A(3 )(CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata ("Barnes", "Fawcett", "Flying Dragon", "Pomeroy", "Rubidoux", "USDA") and one P. trifoliata x C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA(+) banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus x Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm.

Chromosomal organization and phylogenetic relationships in Hypochaeris species (Asteraceae) from Brazil

The association of cytogenetic and molecular techniques has contributed to the analysis of chromosome organization and phylogeny in plants. The fluorochrome GC-specific CMA 3 , fluorescent in situ hybridization (FISH) and RAPD (Random Amplified Polymorphic DNA) markers were used to investigate chromosome structure and genetic relationships in Hypochaeris (Asteraceae). Seven species native to South America, and two species introduced from Europe (H. glabra and Hypochaeris sp) were studied. FISH with rDNA probes identified one or two loci of 18S-5.8S-25S rDNA in the South American Hypochaeris species and one locus in the European species. Only one 5S rDNA locus was seen in all species studied. Blocks of GC-rich heterochromatin (CMA-positive bands) associated to 18S-5.8S-25SrDNA loci were detected in all species investigated. Co-location of 5S rDNA and CMA bands was also observed, except for three South American species and Hypochaeris sp. In two South American species, additional CMA bands not related to rDNA were observed on the long arm of chromosome 2, near to the centromere. Hypochaeris glabra exhibited additional CMA-positive signals distributed at pericentromeric regions, on the short arms of all chromosomes. A total of 122 RAPD markers were used to determine the genetic relationships among species. The level of polymorphism was very high, revealing two genetic groups comprising the South American and the European species, thus supporting a previous hypothesis of monophyly of the South American Hypochaeris species. The coefficients of genetic similarity between European and South American species were 0.35, on average. Polymorphism was also high within the two groups. The genetic associations observed with RAPD markers were consistent with chromosome characteristics. Species carrying similar distribution of 45S rDNA loci and CMA-positive signals were included in the same group revealed by RAPDs. Cytogenetic and molecular data support the view that not only chromosome rearrangements, but also changes in DNA sequence took place during the diversification of the South American Hypochaeris species.

Variation in Chromosome Numbers, CMA Bands and 45S rDNA Sites in Species of Selaginella (Pteridophyta)

Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers. The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites. The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20). The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.