Black rat (Rattus rattus) genomic variability characterized by chromosome painting

Black rats are of outstanding interest in parasitology and infective disease analysis. We used chromosome paints from both the mouse(Mus musculus) and the Norway rat(Rattus norvegicus) to characterize the genome of two Black rat subspecies from Italy. Both subspecies have two large metacentrics (n. 1, 4) not present in the Norway rat (2n = 42).Rattus rattus rattus has a diploid number of 2n = 38, whileRattus rattus frugivorous has two small metacentric “supernumerary” or B chromosomes for a diploid number of 2n = 38 + 2B. The 21 mouse paints gave 38 signals on theR. r. rattus karyotype and 39 signals in theR. r. frugivorous karyotype. The two metacentrics, not present inR. norvegicus, were hybridized by mouse 16/1/17 and mouse 4/10/15. These chromosomes are homologous to: RRA1 = RNO 5/7, and RRA4 = RNO 9/11 and not “4/7” and “11/12” as previously reported. Furthermore, the synteny of Chr 13 of theR. r. frugivorous withR. norvegicus Chr 16 and mouse Chrs 8/14 is not complete, because there is a small pericentromeric insertion of RNO Chr 18 (mouse Chr 18). If we consider only the two metacentrics, RRA1 and RRA4, the principal differences betweenR. norvegicus andR. rattus, then we can propose the derived synteny of 124 genes in the black rat. A comparison of the Z index between rats and mice shows an acceleration of genomic evolution among genus, species, and subspecies. The chromosomal differences betweenR. r. rattus xR. r. frugivorous suggest that they may be classified as different species because hybrids would produce 50% unbalanced gametes.     

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.     

Evolutionary conservatism in arrangement of genetic material

The diploid number of the Rhesus macaque, Macaca mulatta, is 42. All chromosomes are biarmed and all constitutive heterochromatins are centromeric. The diploid number of the African Green monkey, Cercopithecus aethiops, is 60. Again all chromosomes are biarmed, but seven pairs possess very short second arms which are heterochromatic. The heterochromatins of remaining chromosomes are centromeric. Using G-banding and deleting the heterochromatic short arms, the chromosomes of the African Green monkey can be artificially fused to reconstruct a karyotype of the Rhesus with only one pair of unmatched small metacentrics. In addition to the Robertsonian type of translocations, several sets of centromere-telomere translocations were found. The latter type of translocation reduced three arms into two. Thus the fundamental number can be changed by two mechanisms: growing extra heterochromatic arms and the centromere-telomere fusions.     

Evolution of karyotypes and differentiation in 13 Rattus Species

Karyotypes of 13 Rattus species collected in Asia and Oceania were analysed with special interest to karyotype evolution and species differentiation. They were classified into three groups according to their karyotype similarity. Four species (R. annandalei, R. exulans, R. muelleri and R. norvegicus) with 2n=42 and a karyotype similar to some of the polymorphic karyotypes in the Asian black rats (R. rattus) are classified into the first group. Pericentric inversion of some acrocentrics seemed to have caused the differentiation of these species. The other four species (R. bowersii, R. fuscipes, R. leucopus and R. conatus) with similar karyotypes as the above group, but lower chromosome numbers than 2n=42 are classified into the second group. Robertsonian fusion in some acrocentrics observed in the first group are suggested to have caused the development of the species in this group. The remaining four species (R. sabanus, R. canus, R. huang and R. niviventer) with karyotypes markedly different from the above two in having a fewer number of small metacentrics are classified into the third group. They seemed to be more primitive karyotypes than the other Rattus species. By the comparison between the polymorphic karyotypes in the black rat, and karyotypes in its related species it was suggested that the former had occurred as primary events to the differentiation of the latter. Parallelism between the karyotype evolution and the species differentiation was discussed.Contribution No. 874 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (No. 92159 and 92332).     

Banding pattern analysis of polymorphic karyotypes in the black rat by a new differential staining technique

Polymorphic karyotypes of black rats (Rattus rattus) collected in Japan, Australia and India were analysed by a new differential staining technique by which banding patterns in the metaphase chromosomes are revealed. The technique consists in two steps: immersion of slides in a mixture of 2 x SSC and 0.1% (w/v) SDS (sodium dodecyl sulfate) for a few seconds at room temperature, and staining in Giemsa. By this treatment characteristic banding patterns were obtained in each chromosome pair. From the banding pattern analysis, subtelocentric pairs No. 1 and 9, which are polymorphic in respect to the acrocentrics and the subtelocentrics, were proven to have originated by pericentric inversion in the acrocentrics. The origin of two large metacentrics observed in Australian and Indian black rats was confirmed to have been developed by Robertsonian fusion of the acrocentrics No. 4 and 7 and No. 11 and 12 present in the Asian type black rat.Contribution No. 873 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (Nos. 92159 and 92332).     

Chromosomal evolution in the Brazilian lizards of genus Leposoma (Squamata, Gymnophthalmidae) from Amazon and Atlantic rain forests: banding patterns and FISH of telomeric sequences

An extensive karyotype differentiation was found among three species of gymnophthalmid lizard genus Leposoma which occur in the tropical forest areas of Brazil. We examined the chromosomes of the Amazonic species L. guianense (LOU) and L. oswaldoi (LOS) and the Atlantic forest species L. scincoides (LSC) after conventional and differential staining, and FISH of telomeric sequences. Both Amazonic species shared very similar 2n = 44 karyotypes, including 20 biarmed macrochromosomes and 24 microchromosomes (20 M + 24 m). However, the location of Ag-NORs and the amount of constitutive heterochromatin differed in these karyotypes. The Atlantic forest species L. scincoides has a very distinct karyotype with 52 acrocentric and subtelocentric chromosomes of decreasing size. Comparative R-banding analysis revealed complete homeology of the macrochromosomes of LGU and LOS and correspondence of banding patterns between LSC acrocentrics and subtelocentrics and some arms of biarmed LGU and LOS chromosomes. Pair 1 had similar banding patterns in the three species, implying the occurrence of a pericentric inversion. Interstitial telomeric bands (ITBs) detected by FISH at the pericentromeric region of some biarmed LGU and LOS chromosomes could be remnants of chromosomal rearrangements occurred during the differentiation of the karyotypes. Robertsonian rearrangements as well as pericentric inversions events probable were involved in the karyotype evolution of these Amazon and Atlantic forests species of Leposoma.     

Cytogenetic comparison of saola (Pseudoryx nghetinhensis) and cattle (Bos taurus) using G- and Q-banding and FISH

In order to cytogenetically describe the new bovid species saola (Pseudoryx nghetinhensis), comparative G- and Q-banding of saola and cattle (Bos taurus) chromosomes as well as FISH-mapping of 32 type-I markers (29 Texas markers and three additional markers) on saola chromosomes were performed. Saola was shown to have a diploid number of 2n = 50 chromosomes possessing five biarmed autosomal pairs and an acrocentric X chromosome. Homology of saola and cattle chromosomes was indicated by banding patterns and by marker hybridization suggesting that all five biarmed pairs in saola originate from centric fusions involving ten cattle autosomes. However, small intrachromosomal rearrangements cannot be excluded. In this study the first preliminary homology map of these two species is presented.     

Interrelationships among three derivative forms of the immigrans-Hirtodrosophila radiation: evidence from heterochromatin and polytene chromosomes

The phylogenetic relationships among three derivative forms of the immigrans-Hirtodrosophila radiation viz. Chaetodrosophilella, Zaprionus and the immigrans species group are examined, by comparing the banding patterns of their polytene chromosomes and by analysing the nature of their heterochromatin. Based on the results of these studies it is concluded that D. quadrilineata has a very strong affinity with the members of the immigrans species group, while the genus Zaprionus represents a very distinct evolutionary lineage. This study further indicates that D. quadrilineata is a karyotypically primitive species having five pairs of rods and one pair of dots, while the karyotype of other members of the immigrans species group appears to have undergone modifications through fusions, fissions, inversions and the addition or deletion of heterochromatin to dots and other chromosomes.     

Chromosomal polymorphism caused by supernumerary chromosomes in the field mouse,Apodemus giliacus

Chromosome studies were made on 74 animals of the field mouse, Apodemus giliacus, a new form of the genus Apodemus described by Kobayashi and Hayata (1970). Extreme variations in number and morphology of chromosomes was observed. While the diploid numbers varied from 48 to 61, the number of acrocentric elements was consistently 48, except for one specimen which had 40 such elements. In contrast, the number and constitution of several biarmed elements and microchromosomes were highly variable in the complement, and, hence, responsible for the polymorphism. Karyotype analysis of meiotic chromosomes, on the basis of Giemsaand quinacrine-stained preparations, some familial studies and breeding experiments revealed that variable elements were supernumeraries of a hitherto undescribed type, whereas the 48 acrocentrics seemed to represent regular autosomes and sex elements. Most of the supernumeraries did not show pairing at metaphase I, but some did form bivalents. Usually, the supernumerary biarmed chromosomes were of regular size and fluoresced rather brightly over their entire length, suggesting that they were heterochromatic in nature. Considering the above findings and other relevant data of some allied species, the differentiation between A. giliacus and A. speciosus might have occurred rather recently, when the former species might have been involved in rapid and divergent chromosomal evolution.Contributions from the Chromosome Research Unit, Sapporo, Japan.     

Chromosome banding in the genusPinus

The chromosomes (2n=24) ofPinus densiflora Sieb. et Zucc. andP. thunbergii Parl. collected from several localities were analyzed on their fluorescent banding patterns by sequential staining with the base specifically binding fluorochromes, CMA and DAPI. In both species, the CMA-bands were localized at the proximal and/or interstitial regions of most of the chromosomes. The CMA-banding pattern was constant among the cells in a plant and was specific to respective species with a few variations. After the CMA and DAPI stainings each chromosome was identified individually. The fluorescent banding patterns of the two species were somewhat similar, but were diferent with respect to in some characters.Pinus thunbergii had two pairs of metacentric chromosomes without CMA-band and two pairs of metacentric chromosomes with an additional thin CMA-band at the interstitial region. The 10th and 11th pairs of chromosomes of both species, which showed similarity in interstitial CMA and DAPI banding and chromosome shape, had the proximal CMA-bands inP. densiflora and DAPI-band inP. thunbergii. The interspecific F₁ hybrid between the two species could easily be identified by the fluorescent banding method.     

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

The karyotypes ofP. juncea (Elymus junceus) andP. huashanica (both outbreeders) were investigated by Feulgen-staining and by C-, N-, and Agbanding, based on a single plant in cach case. Both species have 2n=2x=14 and large chromosomes, possibly a generic character. The karyotype ofP. juncea has 8 metacentrics and 6 SAT-chromosomes with minute, heterochromatic satellites while that ofP. huashanica has 9 metacentrics and 5 SAT-chromosomes only, 2 of which with small, heterochromatic satellites. The C-banding patterns ofP. juncea chromosomes comprise from one to five, mostly small, bands at distal, and terminal positions, while those ofP. huashanica chromosomes are characterized by large telomeric bands in most arms. Banding patterns and chromosome morphology allow identification of the homologues of the seven chromosome pairs inP. juncea, but of two pairs inP. huashanica only. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest thatP. juncea is more closely related toP. fragilis than either is toP. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heteromorphic sex chromosomes of lizardfish (Synodontidae): focus on the ZZ-ZW1W2 system in Trachinocephalus myops

The karyotype and DNA content of four lizardfish species (family Synodontidae), that is, Saurida elongata, Synodus ulae, Synodus hoshinonis and Trachinocephalus myops, were analyzed. The karyotype of T. myops significantly differed from that of the other three species having diploid chromosome number of 48 with mainly acrocentric chromosomes and the ZZ-ZW sex chromosome system. The chromosome number of male T. myops was 2n=26, while that of female T. myops was 2n=27. The karyotype consisted of 11 pairs of metacentrics, one pair of acrocentrics and, in addition, two large metacentrics in the male and a single large metacentric, a distinctly small subtelocentric and a microchromosome in the female. C-banding demonstrated that in the female the subtelocentric chromosome and the microchromosome were heterochromatic. The karyotype of T. myops was thought to be derived from a 48 chromosome type synodontid fish through the involvement of Robertsonian rearrangement; the rearrangement of the sex chromosomes proceeded during karyotype evolution. Among the chromosomes, the large metacentrics were determined to be neo-Z (a fusion of the original Z and an autosome), the microchromosomes the W₁ (originally W), and the subtelocentric chromosomes the W₂ (derived from an autosome pair). The miniaturization of W₁ and W₂ chromosomes and their heterochromatinization suggested that sex chromosomes in this species have been already highly differentiated. The findings on DNA content implied that the karyotype of T. myops evolved by centric fusion events without loss in DNA amount.     

The late replication banding patterns of chromosomes are highly conserved in the genera Rana,Hyla, and Bufo (Amphibia: Anura)

Late replication banding and C-banding analyses were performed on the metaphase chromosomes of six species and one subspecies of Palearctic water frogs, genus Rana. Although C-banding patterns showed interspecific or intersubspecific variation, late replication banding patterns of all 13 chromosome pairs of these species were homologous. Minor differences of banding patterns were observed only in chromosomes 2, 7 and 13. Close comparison of the late replication banding patterns with those of three non-water frog species of Rana, and one each of Hyla and Bufo, provided important information on interspecific and intergeneric variability. In the Rana species, the banding patterns of all 13 pairs were homologous except for those some regions of 8 pairs. In one species each of Hyla and Bufo that was examined, the six large chromosome pairs (Nos. 1-6) showed banding homologies. Furthermore, among the Rana, Hyla and Bufo species the four large chromosome pairs (Nos. 1-3, 5 of Rana and Hyla, and Nos. 1, 3–5 of Bufo) shared banding homologies. These results show that the large chromosomes have been highly conserved in the evolutionary history of the three genera.     

How many times has polyploidization occurred during acipenserid evolution? New data on the karyotypes of sturgeons (Acipenseridae,Actinopterygii) from the Russian Far East

The karyology of species of sturgeon from the Russian Far East demonstrates that the karyotype of the Sakhalin sturgeon (Acipenser mikadoi) includes 262 ± 4 chromosomes with 80 biarmed chromosomes and the number of chromosome arms (NF) 342 ± 4, the karyotype of the Amur sturgeon (A. schrenckii) includes 266 ± 4 chromosomes with 92 biarmed chromosomes and NF 358 ± 4, and the karyotype of the kaluga (A. dauricus) consists of 268 ± 4 chromosomes with 100 biarmed chromosomes and NF 368 ± 4. These results prove that all western Pacific sturgeon species are from a tetraploid origin, based on a recent ploidy scale. This suggests that at least three polyploidization events have occurred during the evolution of Acipenseridae. However, if polyploid species originated by hybridization between diploid species, there may have been more polyploidization events in this group of fishes.     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

The location of DNA homologous to human satellite III DNA in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Radioactive RNA with sequences complementary to human DNA satellite III was hybridised in situ to metaphase chromosomes of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla) and the orangutan (Pongo pygmaeus). A quantitative analysis of the radioactivity, and hence of the chromosomal distribution of human DNA satellite III equivalent sequences in the great apes, was undertaken, and the results compared with interspecies chromosome homologies based upon Giemsa banding patterns. In some instances DNA with sequence homology to human satellite III is present on the equivalent (homologous) chromosomes in identical positions in two or more species although quantitative differences are observed. In other cases there appears to be no correspondence between satellite DNA location and chromosome homology determined by banding patterns. These results differ from those found for most transcribed DNA sequences where the same sequence is located on homologous chromosomes in each species.     

A comparative chromosome study of twenty killifish species of the genus Fundulus (Teleostei: Cyprinodontidae)

Female karyotypes from ovarian cell cultures of 20 species of killifish (Fundulus) ranged in diploid number from 32 to 48, but in arm number (NF) from 48 to 52. The small F chromosomes, which constituted the fundamental elements in the karyotype, were evenly graded in length. The large biarmed chromosomes (L), which were about twice the length of the average Fs, characterized only those species with 2N less than 48 chromosomes. And among these species, an increase in complement by a pair of L's was always accompanied by a decrease of two pairs of A's, indicating Robertsonian changes by the centric fusion of two A's to form one L chromosome. Other diagnostic chromosome characters included: the number and structure of biarmed and satellited F chromosomes and the percentage of F's with relatively short short-arms (SSA). Besides centric fusion, mechanisms of chromosomal evolution in Fundulus probably included pericentric inversion, producing biarmed F chromosomes from acrocentric F's and partial loss of a chromosome segment producing smaller biarmed F chromosomes from larger ones. The percentage of SSA chromosomes generally decreases from relatively primitive to specialized species. The presumably most primitive species have only SSA type acrocentric F chromosomes. The 20 Fundulus species were classified into 2 major groups according to the percentage of SSA chromosomes: the SSA group, including 3 subgroups, had more than 50% SSA's; the LSA group, including 2 subgroups, had fewer than 50% SSA's. This classification based only on karyotypic characters generally agreed with others based on gross morphological characters. A possible evolutionary scheme is proposed to account for the derived killifish karyotypes.     

Chromosomal description of the rodent generaOecomys andNectomys from Brazil

We analysed karyotypes of five taxa of the rodent generaOecomys andNectomys, trapped in 14 localities in an area ranging from 8° to 29°S on Brazilian territory.Oecomys cf.concolor, collected in the Amazon and in two localities of the Cerrado biome, showed a 2n=60 karyotype constituted by a pair of large subtelocentric chromosomes, a small metacentric pair and 27 acrocentric pairs. The X chromosome was a large submetacentric and a subtelo-submetacentric, the morphology of the latter showing variable C-banding patterns. In all three localities the Y chromosome was a medium size heterochromatic acrocentric. Two individuals from the Cerrado had a heterochromatic acrocentric B-chromosome.Oecomys cf.bicolor presented two cytotypes, 2n=80 in the specimens from the Cerrado biome and 2n=82 in individuals trapped in the Amazon. The 2n=80 cytotype 1 showed a large subtelocentric, 22 biarmed pairs (medium to small) and 16 acrocentric autosomal pairs. The karyotype of the 2n=82 cytotype 2 is constituted by 15 biarmed chromosomes (median to small) and 25 acrocentric pairs with heterochromatic blocks at pericentromeric regions. The sexual pairs were the same (large submetacentric X and median acrocentric Y) in both cytotypes. InO. cf.concolor and in both cytotypes ofO. cf.bicolor the nucleolar organizer regions were observed in 1-3 pairs, located in the short arms.Nectomys genus presented two cytotypes, 2n=52–55 (N. rattus, with 0–3 biarmed heterochromatic accessory chromosomes) and 2n=56–59 (N. squamipes, bearing 0–3 biarmed, heterochromatic, B-chromosomes). These 2 cytotypes occupy disjunct regions of South America, with overlapping areas in the Brazilian states of Pernambuco, Bahia, and Mato Grosso do Sul.     

Molecular cytogenetic study of three common Mediterranean limpets, Patella caerulea, P. rustica and P. ulyssiponensis (Archaeogastropoda, Mollusca)

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.