Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Summary Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.Contribution from the Department of Agronomy and published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2730. This research was supported in part by the USDA/SEA Competitive Research Grant 5901-0410-9-0334-0, USDA/ SEA-CSU Cooperative Research Grant 12-14-5001-265 and Colorado State University Hatch Project. This paper was presented partly at the Fourth International Barley Genetics Symposium, Edinburgh, Scotland, July 22–29, 1981     

Time-dependent AluI action on human chromosomes

We have analyzed the pattern of AluI digestion over time on human chromosomes in order to monitor the evolution of the in situ enzyme action. Short treatments followed by Giemsa staining produce a G-like banding effect, whereas longer treatments produce a C-like banding pattern. However, when Propidium iodide staining is used, it reveals a uniform bright fluorescence after short AluI digestions and C bands when longer treatments are developed. We propose that C banding is the result of a uniform DNA removal in non centromeric regions taking place after a critical time point, the initial G like banding being produced by changes in the DNA-proteins interactions.     

Heterochromatic banding pattern in two Brazilian populations of Aedes aegypti

We analysed samples of Aedes aegypti from São José do Rio Preto and Franca (Brazil) by C‐banding and Ag‐banding staining techniques. C‐banding pattern of Ae.aegypti from São José do Rio Preto examined in metaphase cells differed from Franca. The chromosomes 2, 3 and X showed centromeric C‐bands in both populations, but a slightly stained centromeric band in the Y chromosome was observed only in São José do Rio Preto. In addition, the X chromosome in both populations and the Y chromosome of all individuals from São José do Rio Preto showed an intercalary band on one of the arms that was absent in Franca. An intercalary, new band, lying on the secondary constriction of chromosome 3 was also present in mosquitoes of both populations. The comparison of the present data with data in the literature for Ae.aegypti from other regions of the world showed that they differ as to the banding pattern of sex chromosomes and the now described intercalary band in chromosome 3. The observations suggested that the heterochromatic regions of all chromosomes are associated to constitute a single C‐banded body in interphase cells. Ag‐banding technique stained the centromeric regions of all chromosomes (including the Y) and the intercalary C‐band region of the X chromosome in both populations. As Ae.aegypti populations are widespread in a great part of the world, the banding pattern variations indicate environmental interactions and may reveal both the chromosome evolutionary patterns in this species and the variations that may interfere with its vector activity.     

C-banding patterns in the AustralianBulbine (Liliaceae): The perennial group,B. bulbosa s. lato

C-banding studies support earlier evidence thatB. bulbosa, as a previously circumscribed, is heterogeneous, consisting of three distinct entities: (1) theB. bulbosa complex (B. bulbosa s. str.) at 4x (2n = 24), 8x (2n = 48) and 12x (2n = 72) ploidy levels, (2) the rock lily and (3) the Kroombit population (both 2n = 46). Each of these three main groups has a distinctive banding profile, though centromeric and telomeric dot bands, variably expressed, are common to all. In theB. bulbosa complex, substantial heterochromatin development, apart from bands associated with the NORs on chromosomes 1 L, 2 S and 3 L, occurs only at the terminal regions of the short arms of the large and middlesized acrocentric chromosomes, with considerable polymorphic and polytypic variation in the number and size of the heterochromatic blocks, especially at the 4x level. Queensland 8xB. bulbosa populations differ in having terminal heterochromatin, probably associated with NORs, on 11 S and 12 S, and in having some strong interstitial bands. The differences appear to correlate with attributes relating to flower morphology, and may have systematic significance. The karyotypes of rock lily and Kroombit are somewhat similar but the former has a characteristic C-band profile with multiple interstitial bands on chromosomes 1–5 and 7–9, whereas the latter has only one interstitial band on chromosome 9.First contribution of a series on cytoevolution in the AustralianBulbine. Two introductory papers in Austral. J. Bot.34 (2)     

Chromosome banding and essential oils composition of Brazilian accessions of <Emphasis Type="Italic">Lippia alba</Emphasis> (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.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

Differentiated ZZ/ZW sex chromosomes in Apareiodon ibitiensis (Teleostei,Parodontidae): cytotaxonomy and biogeography

Conventional and molecular chromosomal analyses were carried out on three populations of Apareiodon ibitiensis sampled from the hydrographic basins of the São Francisco River and Upper Paraná River (Brazil). The results reveal a conserved diploid number (2n = 54 chromosomes), a karyotype formula consisting of 50 m‐sm + 4st and a ZZ/ZW sex chromosome system that has not been previously identified for the species. C‐banding analysis with propidium iodide staining revealed centromeric and terminal bands located in the chromosomes of the specimens from the three populations and allowed the identification of heteromorphism of heterochromatin regions in the Z and W chromosomes. The number of 18S sites located through fluorescent in situ hybridization (FISH) varied between the populations of the São Francisco and Upper Paraná Rivers. The location of 5S rDNA sites proved comparable in one pair of metacentric chromosomes. Thus, the present study proposes a ZZ/ZW sex chromosome system for A. ibitiensis among the Parodontidae, and a hypothesis is presented regarding possible W chromosome differentiation stages in this species through DNA accumulation, showing geographical variations for this characteristic, possibly as a consequence of geographical reproductive isolation.     

Karyotype and chromosomal characteristics of Ag–NOR sites and 5S rDNA in European smelt, Osmerus eperlanus

Karyotype and cytogenetic characteristics of European smelt Osmerus eperlanus were investigated using different staining techniques (sequential Ag-, CMA3 and DAPI banding) and PRINS to detect 5S rDNA and telomeric sites. The diploid chromosome number was invariably 2n = 56 and karyotype composed of 5 pairs of metacentrics, 9 pairs of subtelocentrics and 14 pairs of subtelo- to acrocentrics. The DAPI-positive heterochromatic regions were found in centromeric positions on bi-armed chromosomes and few acrocentrics. Additionally, some interstitial DAPI-positive bands were identified on three pairs of submetacentric chromosomes. The nucleolar organizer regions (NORs) were detected in the short (p) arms of the largest metacentric pair of chromosomes No. 1. Sequential banding (Giemsa-, AgNO₃ and CMA₃ stainings) revealed NOR sites corresponding to achromatic regions but not associated with CMA₃-positive blocks of heterochromatin located on either side of NORs. Individuals from the analyzed population had this conspicuous pair of chromosomes always in heterozygous combination. A complex inversion system was hypothesized to be involved in the origin of the observed variation but analysis with telomeric PRINS and PNA-FISH did not reveal any Interstitial Telomeric Sites (ITS). Hybridization signals were confined exclusively to terminal chromosomal regions. The 5S ribosomal sites as revealed by PRINS were found to be invariably located in the short (p) arms of four pairs of subtelocentric chromosomes. Cytotaxonomic comparisons of the present results with the voluminous available cytogenetic data-set from salmoniform and esociformes fishes appear to support the recent view, based on robust molecular-based phylogeny, that salmoniform and osmeriform fishes are not as closely related as previously assumed.     

Cytological characterization of heterochromatin and rDNA inPinus radiata andP. taeda

Fluorochrome C-banding ofPinus radiata andP. taeda metaphase chromosomes showed many pericentromeric DAPI bands and interstitial CMA bands inP. radiata, and centromeric and interstitial CMA bands inP. taeda. Giemsa C-band patterns differed between the species with centromeric bands inP. radiata but no consistent bands inP. taeda. A karyotype ofP. radiata was developed based on banding patterns that distinguished all but two of the 12 pairs of chromosomes. In situ hybridization (ISH) using probes for high-copy ribosomal DNA (rDNA) showed 10 pairs of 18S–25S sites and two pairs of 5S sites in both species. Most of the sites were interstitial or centromeric.     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.     

Banding patterns in newt chromosomes by the giemsa stain

Specific banding patterns can be produced on the mitotic chromosomes of the newt species Triturus vulgaris meridionalis and T. italicus by using the Giemsa stain technique. These bands are most useful cytogenetic markers in karyotyping, since they facilitate identification of the individual elements of the complements. Evaluation of the shape of chromosomes as well as of the banding patterns produced by the Giemsa stain indicates that the karyotypes of T. vulgaris meridionalis and T. italicus are differentiated: hence the specific distinction of the two Salamandrids, still debated by taxonomists, appears supported by chromosome evidence. — Most of the bands seem to correspond to the heterochromatic tracts observable on mitotic chromosomes from embryos and larvae either untreated or submitted to cold treatment. Besides, the comparison of mitotic karyotypes and lampbrush maps shows that the bands located near the centromeric regions of mitotic chromosomes probably correspond to the so-called bars visible on either side of centromeres of lampbrush chromosomes, while some of the subterminal bands may correspond to the sphere.This work was financially supported by C. N. R., Roma.     

Chromosome banding in the genusPinus

Somatic chromosomes (2n=24) ofPinus luchuensis Mayr at metaphase were observed by fluorescent banding methods with chromomycin A₃ (CMA) and DAPI. CMA-bands appeared at the interstitial and/or proximal regions of nearly all chromosomes. DAPI-bands appeared at the interstitial and/or centromeric regions of nearly all chromosomes, and pairs of DAPI-dots appeared at the centromeric regions. Each homologous pair of chromosomes in the chromosome complement was identified by the CMA and DAPI fluorescent banding patterns. The interstitial CMA-bands were mostly localized at the secondary constrictions of the Feulgen-stained chromosomes. The fluorescent banding pattern ofP. luchuensis was very similar to that ofP. thunbergii, but was different from that ofP. densiflora.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Karyotype evolution and phylogenetic analyses in the genus Cardiospermum L. (Paullinieae,Sapindaceae)

Cardiospermum L. belongs to the Paullinieae tribe (Sapindaceae) and comprises 16 species. Of these, 12 species are present in South America and all occur in Brazil. Cardiospermum shows the most variable chromosome number of the tribe. Phylogenetic relationships within the genus Cardiospermum, especially with other species of the tribe, are poorly understood. This research focuses on characterisation of the karyotypic features of Cardiospermum using conventional cytogenetic methods, CMA/DAPI chromosome banding and fluorescence in situ hybridisation (FISH). To elucidate the phylogeny of the genus, the nuclear markers ITS1 and ITS2 were sequenced and analysed using maximum parsimony and Bayesian inference. Cardiospermum shows important diversity in basic numbers, with x = 7, 9, 10, 11 and 12. All species studied have metacentric and submetacentric chromosomes, some species have subtelocentric chromosomes, while telocentric chromosomes are absent. The interphase nuclei differentiate the Cardiospermum species into two groups. The CMA₃/DAPI chromosome banding revealed the presence of an AT‐rich terminal region in C. corindum, C. grandiflorum and C. urvilleoides, whereas GC‐rich regions were found in C. grandiflorum, C. halicacabum var. halicacabum, C. halicacabum var. microcarpum, C. heringeri and C. integerrimum. FISH revealed syntenic and non‐syntenic distribution of the 18‐5.8‐26S and 5S rDNA. The syntenic distribution always occurred in the short arms of the same chromosome in all of the species. The phylogenetic relationships reveal, in part, the taxonomic arrangement of the genus Cardiospermum.     

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.     

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.     

Nucleolus-organizer regions and heterochromatin in three species of Bovidae

Ag-NOR staining and a counterstain-enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI staining = CDD-method) have been applied to ibex (Capra ibex L.), chamois (Rupicapra rupicapra L.) and bison (Bison bison L.) chromosomes.Chromomycin A₃ visualization led to a well defined R-banding pattern along the chromosome arms and to a clear demonstration of centric heterochromatic bands of variable size. The nucleolus organizer regions (NORs) were found in the telomeric regions of the chromosomes 2, 3, 4, 5 and 28 of the ibex, of the chromosomes 1/3 (short arm), 2, 4, 5 and 28 of the chamois and of the chromosomes 2, 3, 4, 11 and 28 of the bison.