Molecular Cytogenetic Markers Research Articles

Agro-Morphological and Cytogenetic Characterization of Colchicine-Induced Tetraploid Plants of Polemonium caeruleum L. (Polemoniaceae).

Polemonium caeruleum L. (Polemoniaceae) is a valuable medicinal herb with a wide spectrum of biological activities. Under natural conditions, the productivity of this species is rather low. In this study, colchicine-induced tetraploid plants (2n = 4x = 36) of P. caeruleum were obtained, and for the first time, their morphological and cytogenetic characterization was performed. In the tetraploid plants, raw material productivity and also the content of triterpene saponins were significantly higher than in the control diploids. The analysis of chromosome behavior at meiosis and FISH chromosome mapping of 45S and 5S rDNA generally demonstrated stability of both genomes in the tetraploid plants. Based on chromosome morphology and distribution patterns of the studied molecular cytogenetic markers, all chromosome pairs in karyotypes were identified, and chromosome karyograms and idiograms of P. caeruleum were constructed. The revealed specific microdiagnostic characteristics of P. caeruleum (strongly sinuous cells and anomocytic stomata of the leaf epidermis, and also glandular hairs along the veins) could be useful for raw material identification. In the obtained tetraploids, the predominance of large stomata on the lower leaf epidermis was determined. The studied tetraploids can be used in various breeding programs to obtain high-quality pharmaceutical raw materials of P. caeruleum.

Comparative Analyses of Complete Chloroplast Genomes and Karyotypes of Allotetraploid Iris koreana and Its Putative Diploid Parental Species (Iris Series Chinenses, Iridaceae).

The Iris series Chinenses in Korea comprises four species (I. minutoaurea, I. odaesanensis, I. koreana, and I. rossii), and the group includes some endangered species, owing to their high ornamental, economic, and conservation values. Among them, the putative allotetraploid, Iris koreana (2n = 4x = 50), is hypothesized to have originated from the hybridization of the diploids I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28) based on morphological characters, chromosome numbers, and genome size additivity. Despite extensive morphological and molecular phylogenetical studies on the genus Iris, little is known about Korean irises in terms of their complete chloroplast (cp) genomes and molecular cytogenetics that involve rDNA loci evolution based on fluorescence in situ hybridization (FISH). This study reports comparative analyses of the karyotypes of the three Iris species (I. koreana, I. odaesanensis, and I. minutoaurea), with an emphasis on the 5S and 35S rDNA loci number and localization using FISH together with the genome size and chromosome number. Moreover, the cp genomes of the same individuals were sequenced and assembled for comparative analysis. The rDNA loci numbers, which were localized consistently at the same position in all species, and the chromosome numbers and genome size values of tetraploid Iris koreana (four 5S and 35S loci; 2n = 50; 1C = 7.35 pg) were additively compared to its putative diploid progenitors, I. minutoaurea (two 5S and 35S loci; 2n = 22; 1C = 3.71 pg) and I. odaesanensis (two 5S and 35S loci; 2n = 28; 1C = 3.68 pg). The chloroplast genomes were 152,259–155,145 bp in length, and exhibited a conserved quadripartite structure. The Iris cp genomes were highly conserved and similar to other Iridaceae cp genomes. Nucleotide diversity analysis indicated that all three species had similar levels of genetic variation, but the cp genomes of I. koreana and I. minutoaurea were more similar to each other than to I. odaesanensis. Positive selection was inferred for psbK and ycf2 genes of the three Iris species. Phylogenetic analyses consistently recovered I. odaesanensis as a sister to a clade containing I. koreana and I. minutoaurea. Although the phylogenetic relationship, rDNA loci number, and localization, together with the genome size and chromosome number of the three species, allowed for the inference of I. minutoaurea as a putative maternal taxon and I. odaesanensis as a paternal taxon, further analyses involving species-specific molecular cytogenetic markers and genomic in situ hybridization are required to interpret the mechanisms involved in the origin of the chromosomal variation in Iris series Chinenses. This study contributes towards the genomic and chromosomal evolution of the genus Iris.

Molecular cytogenetics of Hedysarum gmelinii Ledeb. and H. setigerum Turcz. (Fabaceae) from populations of Southern Siberia

For the first time, a comparative karyotype analysis of closely related species Hedysarum gmelinii andH. setigerum (Hedysarum section Multicaulia) grown in Southern Siberia, has been performed by molecular cytogeneticmarkers. Chromosome numbers in karyotypes of these species were specified – 2n = 4х = 32. In some accessions, additionalB chromosomes were revealed. FISH analyses indicated high similarities in chromosome morphology and also patternsof chromosomal distributions of 45S and 5S rDNA clusters in karyotypes of H. gmelinii and H. setigerum, which confirmsthe close relationship between their genomes.

Characterization, Identification and Evaluation of Wheat-Aegilops sharonensis Chromosome Derivatives.

Aegilops sharonensis, a wild relative of wheat, harbors diverse disease and insect resistance genes, making it a potentially excellent gene source for wheat improvement. In this study, we characterized and evaluated six wheat-A. sharonensis derivatives, which included three disomic additions, one disomic substitution + monotelosomic addition and two disomic substitution + disomic additions. A total of 51 PLUG markers were developed and used to allocate the A. sharonensis chromosomes in each of the six derivatives to Triticeae homoeologous groups. A set of cytogenetic markers specific for A. sharonensis chromosomes was established based on FISH using oligonucleotides as probes. Molecular cytogenetic marker analysis confirmed that these lines were a CS-A. sharonensis 2Ssh disomic addition, a 4Ssh disomic addition, a 4Ssh (4D) substitution + 5SshL monotelosomic addition, a 6Ssh disomic addition, a 4Ssh (4D) substitution + 6Ssh disomic addition and a 4Ssh (4D) substitution + 7Ssh disomic addition line, respectively. Disease resistance investigations showed that chromosome 7Ssh of A. sharonensis might harbor a new powdery mildew resistance gene, and therefore it has potential for use as resistance source for wheat breeding.

Cytogenetic and genetic data support Crossodactylus aeneus Müller, 1924 as a new junior synonym of C. gaudichaudii Duméril and Bibron, 1841 (Amphibia, Anura).

The nominal anuran species Crossodactylus gaudichaudii Duméril and Bibron, 1841 and Crossodactylus aeneus Müller, 1924 are indistinguishable based on adult and larval morphology, being subject of taxonomic doubts. Here, we describe the karyotypes of C. gaudichaudii and C. aeneus, using classical and molecular cytogenetic markers. In addition, we used sequences of the H1 mitochondrial DNA to infer their phylogenetic relationships by Maximum Likelihood (ML) and Maximum Parsimony (MP) approaches and species delimitation test (by bPTP approach). The karyotypic data do not differentiate C. gaudichaudii and C. aeneus in any of the chromosome markers assessed. In both phylogenetic analyses, C. gaudichaudii and C. aeneus were recovered into a strongly supported clade. The species delimitation analysis recovered the specimens assigned to C. gaudichaudii and C. aeneus as a single taxonomic unit. Taken the cytogenetic and genetic results together with previous studies of internal and external morphology of tadpoles and biacoustic pattern, C. gaudichaudii and C. aeneus could not be differentiated, which supports the hypothesis that they correspond to the same taxonomic unit, with C. aeneus being a junior synonym of C. gaudichaudii.

Importance of molecular cytogenetic markers in plant breeding

Importance of molecular cytogenetic markers in plant breeding

Karyotypic mosaicism and molecular cytogenetic markers in the scleractinian coral Acropora pruinosa Brook, 1982 (Hexacorallia, Anthozoa, Cnidaria)

Molecular cytogenetic investigation was carried out on the scleractinian coral, Acropora pruinosa (A. pruinosa). Conventional Giemsa staining techniques for karyotyping, such as G- and C-banding, were conducted. Karyotype analysis showed mosaicism of cells with two different chromosome numbers, 28 and 29, in the same embryo. The C-band positive portions appeared on the centromeres of most chromosomes and along the entire length of the unpaired chromosome 15 in cells with 29 chromosomes. Fluorescence in situ hybridization (FISH) revealed that the loci of rRNA genes (rDNA), 5S and 18S/28S, were located on chromosomes 4 and 2, respectively. Whole genome hybridization (WGH) with sperm DNA showed distinct signals not only on all centromeric regions but also on a whole unpaired chromosome 15. Comparative genomic hybridization (CGH) using DNAs from sperm and unfertilized eggs revealed that the unpaired chromosome 15 has sperm-specific DNA sequences. We therefore hypothesized the existence of a sex-related chromosome in A. pruinosa. In this study, we have presented a tentative karyotype of this coral, based on banding results, and described FISH, WGH, and CGH results. Moreover, we successfully cloned and sequenced three clones; AP-5S for 5S rDNA, AP-18S for 18S/28S rDNA, and AP-unpaired chromosome 15q for the unpaired chromosome 15-specific DNA. These molecular cytogenetic approaches will help establish a more exact karyotype of corals and promote understanding of coral genetics, including chromosome evolution. These findings will help to verify the method of classification of complex scleractinian corals.

Chromosome polymorphism in species of the genus Hedysarum L. (Fabaceae) originated from Southern Sibеria

For the first time, chromosomal polymorphism in karyotypes of three species from the section Hedysarum (= syn. Gamotion) of the genus Hedysarum L. (Fabaceae) grown in Southern Siberia has been studied with the useof molecular cytogenetic markers. This comparative molecular cytogenetic analysis revealed high similarity in morphology of chromosomes in H. alpinum L., H. austrosibiricum B. Fedtsch. and H. theinum Krasnob. as well as in patterns ofdistribution of 45S and 5S rDNA loci in their karyotypes confirming their close relationships. Considerable intra-specificpolymorphism on 45S rDNA chromosome localization was detected in H. theinum. In karyotype of H. alpinum, unlikethe other two species, two chromosome pairs bearing 5S rDNA locus were observed which could be used as additionalspecies-specific markers.

Cytogenetic markers applied to cytotaxonomy in two soybean pests: Anticarsia gemmatalis (Hübner, 1818) and Chrysodeixis includens (Walker, 1858).

Anticarsia gemmatalis (Hübner, 1818) and Chrysodeixis includens (Walker, 1858) are species of Lepidoptera that cause great damages in the soybean plantations of Brazil. Despite the importance they have in this regard, there are no studies on the chromosomal organization of these species and recently, A. gemmatalis, which belonged to the Noctuidae family, was allocated to the Erebidae family. Therefore, the objective of this paper was to analyze, through conventional and molecular cytogenetic markers, both species of Lepidoptera. A 2n = 62 was observed, with ZZ/ZW sex chromosome system and holokinetic chromosomes for both species. There was homogeneity in the number of 18S rDNA sites for both species. However, variations in heterochromatin distribution were observed between both species. The cytogenetic analyses enabled separation of the species, corroborating the transference of A. gemmatalis, from the family Noctuidae to the family Erebidae, suggesting new cytotaxonomic characteristics.

Molecular cytogenetic analyses reveal extensive chromosomal rearrangements and novel B chromosomes in Moenkhausia (Teleostei, Characidae).

The cytogenetic characteristics of five fish species of the Moenkhausia are described, based on the analysis of specimens collected in different headwater. All the species analyzed presented 2n=50 chromosomes. The C-banding revealed a similar distribution pattern of heterochromatic blocks in all the species, except Moenkhausia nigromarginata. The 5S rDNA sites were distributed on multiple chromosome pairs in all five species. Single and multiple histone H1 sites were observed in all the species, and histone H1 was shown to be co-located with the 18S rRNA gene in a single chromosome pair. The U2 snDNA gene was distributed at multiple sites in all the Moenkhausia species. The presence of B microchromosomes was confirmed in Moenkhausia forestii, while individuals of the three study populations of Moenkhausia oligolepis presented three morphologically distinct types of B chromosome. The chromosomal mapping of the 18S rDNA sites using the FISH technique revealed signals in the B chromosomes of M. forestii, while clusters of the H1 histone and U2 snDNA genes were found in the B chromosomes of M. forestii and M. oligolepis. The classical and molecular cytogenetic markers used in this study revealed ample variation in the Moenkhausia karyotypes, reflecting the dynamic nature of the chromosomal evolution.

Karyological, Molecular and Cytogenetical Studies on Tree Plants in V.N. Sukachev Institute of Forest

Aim. The aim of the article is to find out the karyological features and conduct molecular and cytogenetic analysis on tree plants for biological diversity studies, solve problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and new method of fluorescent in situ hybridization (FISH) had been used. Results . More than 250 populations and provenances of representatives of different genera of gymnosperms from the families Pinaceae, Cupressaceae, Ephedraceae and tree angiosperms from the families Rosaceae, Aceraceae, Polygonaceae, Nitrariaceae, Rhamnaceae, Fabaceae, Ericaceae were studied. Investigations had been conducted in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms and unique trees had been studied. The variability of chromosome numbers and a wide range of chromosomal mutations had been revealed. Use of fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allowed to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of those species. Analysis of the chromosome numbers of woody angiosperms showed a significant role of polyploidy in their evolution. Conclusions. The studies on karyotypes in tree species showed their diversity, variability of chromosome numbers, higher level of chromosomal anomalies and their wide spectrum in extreme conditions, under introduction and also in cultivars and abnormal form of trees. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes. The role of polyploidy in the evolution of woody angiosperms was shown.

Karyological and cytogenetic research of the conifers of boreal zone by classic and new methods

Aim. Establishing of karyological features and conducting of cytogenetic analysis on conifer plants for biological diversity studies, solving of problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and fluorescent in situ hybridization (FISH). Results. More than 150 populations and provenances of representatives of different conifer genera from the Pinaceae and Cupressaceae families were studied. The studies were carried out in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms have been studied. The variability of chromosome numbers and a wide range of chromosomal mutations have been revealed. Fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allows to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of these species. Conclusions. The studies of chromosomes in species of the Pinaceae and Cupressaceae families showed a karyotypic diversity and chromosomal anomalies in extreme conditions and under introduction. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes.
 Keywords: chromosomes, nucleolar loci, chromosome mutations, Pinaceae, Cupressaceae.

Molecular cytogenetic characterization of repetitive sequences comprising centromeric heterochromatin in three Anseriformes species.

The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.

Pipeline for the Rapid Development of Cytogenetic Markers Using Genomic Data of Related Species.

Repetitive DNA including tandem repeats (TRs) is a significant part of most eukaryotic genomes. TRs include rapidly evolving satellite DNA (satDNA) that can be shared by closely related species, their abundance may be associated with evolutionary divergence, and they have been widely used for chromosome karyotyping using fluorescence in situ hybridization (FISH). The recent progress in the development of whole-genome sequencing and bioinformatics tools enables rapid and cost-effective searches for TRs including satDNA that can be converted into molecular cytogenetic markers. In the case of closely related taxa, the genome sequence of one species (donor) can be used as a base for the development of chromosome markers for related species or genomes (target). Here, we present a pipeline for rapid and high-throughput screening for new satDNA TRs in whole-genome sequencing of the donor genome and the development of chromosome markers based on them that can be applied in the target genome. One of the main peculiarities of the developed pipeline is that preliminary estimation of TR abundance using qPCR and ranking found TRs according to their copy number in the target genome; it facilitates the selection of the most prospective (most abundant) TRs that can be converted into cytogenetic markers. Another feature of our pipeline is the probe preparation for FISH using PCR with primers designed on the aligned TR unit sequences and the genomic DNA of a target species as a template that enables amplification of a whole pool of monomers inherent in the chromosomes of the target species. We demonstrate the efficiency of the developed pipeline by the example of FISH probes developed for A, B, and R subgenome chromosomes of hexaploid triticale (BBAARR) based on a bioinformatics analysis of the D genome of Aegilops tauschii (DD) whole-genome sequence. Our pipeline can be used to develop chromosome markers in closely related species for comparative cytogenetics in evolutionary and breeding studies.

Stability of DNA content and molecular cytogenetic markers in the genus Stylosanthes (Fabaceae)

Stylosanthes (Leguminosae) comprises about 50 species being some of them with great economic importance. Some species are widely used as forages, to recover degraded areas due to its adaptability to low fertility soils and nitrogen fixing capacity as well as a green fertilizer. The genus is distributed in tropical and subtropicals areas. Brazil is considered the larger diversity center, with great inter and intraspecific variation. Breeding studies have increased, however, the taxonomic situation of the group remains unsettled and controversial. The occurrence of polyploidy, hybrids and many additional synonyms reinforce the importance to add new information about the genus. Cytogenetic data are scarce, even considering as an important tool to give relevant information about the genome. Here we describe, for the first time, karyotypic aspects and the DNA content of diploid species (S. grandifolia, S. gracilis, S. hipocampoides, S. acuminata and four varieties of S. guianensis) by using molecular cytogenetic techniques (CMA/DAPI banding and in situ hybridization) and flow cytometry. Seeds germination was induced by temperature variation (18° to 40°C) and growth hormones (CEPA 0.1mM) in a germination chamber. Meristematic roots were used to slides preparation. Chromosomes were stained with CMA/DAPI for banding patterns analysis and ribosomal 5S, 45S rDNA sites were mapped by in situ hybridization technique. To estimate the DNA content, Pisum sativum were used as a reference standard. The chromosomal morphometry showed a symmetrical karyotype with few variations among the species. The number of CG rich regions ranged from 2 to 4. Regarding 5S and 45S rDNA, two sites were observed in most species, excepting in S. acuminata, in which four 45S markers were identified. DNA content estimation was about 2.50pg in all species. These new data constitutes an important contribution in the study of evolutionary history of the group, opening new possibilities for future comparisons among genomes of different Stylosanthes species and also being of great value for breeding programs of species with economic importance.

Mapping of Hieracium (Asteraceae) chromosomes with genus-specific satDNA elements derived from next-generation sequencing data

The highly repetitive DNA fraction of the eukaryotic genome is considered a mobile, rapidly changing entity, thus reflecting trajectories of short-term evolutionary change. It consists of several large classes in which transposable elements and satellite DNA (satDNA) predominate. Despite a growing awareness of its structure and functional significance, the evolutionary dynamics of repetitive elements and, particularly, satDNA remain poorly characterized. Next-generation sequencing (NGS) has opened up new possibilities for high-throughput genome analysis. Here, we applied satDNA repeatome elements derived from NGS data as probes for fluorescence in situ hybridization to characterize the karyotypes of three diploid hawkweed species of the predominantly polyploid apomictic genus Hieracium, namely H. intybaceum, H. prenanthoides and H. alpinum. Three cluster-distributed, genus-specific satDNA elements that are not present in the sister genus Pilosella were identified; notably, one element spans the functional centromeres. Each of the investigated diploids possessed a species-specific assortment of detected repeats. Their utilization as molecular-cytogenetic markers, in combination with ribosomal DNA loci, allowed for the development of a system to identify the individual chromosomes of the Hieracium species, thus providing a basis for the future investigation of karyotype evolution in diploid hawkweeds and for exploring satDNA dynamics in hybrids and apomicts of allopolyploid origin.

Tandem repeats of Allium fistulosum associated with major chromosomal landmarks.

Tandem repeats are often associated with important chromosomal landmarks, such as centromeres, telomeres, subtelomeric, and other heterochromatic regions, and can be good candidates for molecular cytogenetic markers. Tandem repeats present in many plant species demonstrate dramatic differences in unit length, proportion in the genome, and chromosomal organization. Members of genus Allium with their large genomes represent a challenging task for current genetics. Using the next generation sequencing data, molecular, and cytogenetic methods, we discovered two tandemly organized repeats in the Allium fistulosum genome (2n = 2C = 16), HAT58 and CAT36. Together, these repeats comprise 0.25% of the bunching onion genome with 160,000 copies/1C of HAT58 and 93,000 copies/1C of CAT36. Fluorescent in situ hybridization (FISH) and C-banding showed that HAT58 and CAT36 associated with the interstitial and pericentromeric heterochromatin of the A. fistulosum chromosomes 5, 6, 7, and 8. FISH with HAT58 and CAT36 performed on A. cepa (2n = 2C = 16) and A. wakegi (2n = 2C = 16), a natural allodiploid hybrid between A. fistulosum and A. cepa, revealed that these repeats are species specific and produced specific hybridization patterns only on A. fistulosum chromosomes. Thus, the markers can be used in interspecific breeding programs for monitoring of alien genetic material. We applied Non-denaturing FISH that allowed detection of the repeat bearing chromosomes within 3h. A polymorphism of the HAT58 chromosome location was observed. This finding suggests that the rapid evolution of the HAT58 repeat is still ongoing.

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbrücker, 1983.

Corydoras Lacepède, 1803 is the most specious genus of Corydoradinae subfamily and many of its species are still unknown in relation to molecular cytogenetic markers. However, the diploid number and karyotypic formula were recorded for many species of this group. In current study, we provided the first cytogenetic information of Corydoras carlae Nijssen & Isbrücker, 1983, an endemic fish species from Iguassu River basin, Paraná State, Brazil. The individuals were collected in Florido River, a tributary of Iguassu River and analysed with respect to diploid number, heterochromatin distribution pattern, Ag-NORs and mapping of 5S and 18S ribosomal genes. The karyotype of this species comprises 46 chromosomes arranged in 22m+22sm+2st. The heterochromatin is distributed in centromeric and pericentromeric positions in most of the chromosomes, and also associated with NORs. The Ag-NORs were detected in the terminal position on the long arm of the metacentric pair 6. The double-FISH technique showed that 5S rDNA and 18S rDNA were co-localized in the terminal portion on the long arm of the metacentric pair 6. This condition of co-localization of ribosomal genes in Corydoras carlae seems to represent a marker for this species.

Genomic organization of repetitive DNAs highlights chromosomal evolution in the genus Clarias (Clariidae, Siluriformes).

BackgroundThe genus Clarias (Clariidae, Siluriformes) contains at least 61 species naturally spread over vast regions of Asia, India and Africa. However, Clarias species have also been introduced in many different countries and represent the most widespread catfishes in the world. These fishes are also known as “walking catfishes” due to their ability to move over land. A large degree of chromosomal variation has been previously found in this family, mainly using conventional cytogenetic investigations, with diploid chromosome numbers ranging between 48 and 100. In this study, we analyzed the karyotype structure and distribution of four repetitive DNA sequences (5S and 18S rDNAs and (CA)15 and (GA)15 microsatellites) in three Clarias species (C. batrachus, C. gariepinus, C. macrocephalus), as well as in a probable natural hybrid of the two latter species from different Thailand river basins.ResultsClarias gariepinus and C. macrocephalus had 2n = 56 and 2n = 54, respectively, as well as karyotypes composed mainly by metacentric and submetacentric chromosomes. Their karyotypes differed in the number and location of 5S and 18S rDNA sites and in the degree of microsatellite accumulation. An intermediate chromosomal pattern incorporating those of the parental species was found in the probable hybrid, confirming its interspecific origin. Clarias batrachus had 2n = 104 chromosomes and its karyotype was dominated by mainly acrocentric elements, indicating that unusual multiple centric fissions were involved in its karyotype differentiation. The karyotype of this species presented an unexpected dispersion of ribosomal DNAs, possessing 54 and 12 sites of 5S and 18S rDNAs, respectively, as well as a high accumulation and differential distribution of both microsatellite repeats, representing ‘hot spots’ for chromosomal rearrangement.ConclusionBoth conventional and molecular cytogenetic markers were useful tools for demonstrating remarkable evolutionary dynamism and highlighting multiple chromosomal rearrangements and hybridization events correlated with the notable karyotypic diversity of these walking catfishes.

Fish Karyome version 2.1: a chromosome database of fishes and other aquatic organisms.

A voluminous information is available on karyological studies of fishes; however, limited efforts were made for compilation and curation of the available karyological data in a digital form. ‘Fish Karyome’ database was the preliminary attempt to compile and digitize the available karyological information on finfishes belonging to the Indian subcontinent. But the database had limitations since it covered data only on Indian finfishes with limited search options. Perceiving the feedbacks from the users and its utility in fish cytogenetic studies, the Fish Karyome database was upgraded by applying Linux, Apache, MySQL and PHP (pre hypertext processor) (LAMP) technologies. In the present version, the scope of the system was increased by compiling and curating the available chromosomal information over the globe on fishes and other aquatic organisms, such as echinoderms, molluscs and arthropods, especially of aquaculture importance. Thus, Fish Karyome version 2.1 presently covers 866 chromosomal records for 726 species supported with 253 published articles and the information is being updated regularly. The database provides information on chromosome number and morphology, sex chromosomes, chromosome banding, molecular cytogenetic markers, etc. supported by fish and karyotype images through interactive tools. It also enables the users to browse and view chromosomal information based on habitat, family, conservation status and chromosome number. The system also displays chromosome number in model organisms, protocol for chromosome preparation and allied techniques and glossary of cytogenetic terms. A data submission facility has also been provided through data submission panel. The database can serve as a unique and useful resource for cytogenetic characterization, sex determination, chromosomal mapping, cytotaxonomy, karyo-evolution and systematics of fishes.Database URL: http://mail.nbfgr.res.in/Fish_Karyome