Krit Pinthong

Generation of multicolor banding probes for chromosomes of different species

BackgroundThe multicolor banding (MCB/mBAND) technique provides a unique opportunity to characterize intrachromosomal rearrangements and to determine chromosomal breakpoints. Until recently, MCB probes have only been available for human and some murine chromosomes. Generation of MCB probes for chromosomes of other species, useful and required in many cytogenetics research fields, was limited by technical difficulties. MCB probes are established by chromosome microdissection followed by whole genomic DNA amplification. However, unambiguous identification of the target chromosome is required for MCB-probe establishment. Previously proposed protocols suggested G-banding staining or preliminary FISH with whole chromosome paints (WCP) as methods to identify the chromosome of interest.ResultsHere we present a complete workflow for MCB probe generation for those cases and species where chromosome morphology is too challenging to recognize target chromosomes by conventional methods and where WCP probes are not available. The workflow was successfully applied for murine chromosomes that are difficult to identify unambiguously. Additionally, we showed that glass-needle based microdissection enables establishment of a whole set of WCP paints by microdissection of individual chromosomes of a single metaphaseConclusionsThe present method can be applied for generation of whole or region-specific DNA probes for species, where karyotyping of G-banded chromosomes is challenging due to similar chromosome morphology and/or chromosome banding patterns.

First detailed reconstruction of the karyotype of Trachypithecus cristatus (Mammalia: Cercopithecidae)

BackgroundThe chromosomal homologies of human (Homo sapiens = HSA) and silvered leaf monkey (Trachypithecus cristatus = TCR) have been previously studied by classical chromosome staining and by fluorescence in situ hybridization (FISH) applying chromosome-specific DNA probes of all human chromosomes in the 1980s and 1990s, respectively.ResultsHowever, as the resolution of these techniques is limited we used multicolor banding (MCB) at an ~250-band level, and other selected human DNA probes to establish a detailed chromosomal map of TCR. Therefore it was possible to precisely determine evolutionary conserved breakpoints, orientation of segments and distribution of specific regions in TCR compared to HSA. Overall, 69 evolutionary conserved breakpoints including chromosomal segments, which failed to be resolved in previous reports, were exactly identified and characterized.ConclusionsThis work also represents the first molecular cytogenetic one characterizing a multiple sex chromosome system with a male karyotype 44,XY1Y2. The obtained results are compared to other available data for old world monkeys and drawbacks in hominoid evolution are discussed.

Comprehensive characterization of evolutionary conserved breakpoints in four New World Monkey karyotypes compared to Chlorocebus aethiops and Homo sapiens.

Comparative cytogenetic analysis in New World Monkeys (NWMs) using human multicolor banding (MCB) probe sets were not previously done. Here we report on an MCB based FISH-banding study complemented with selected locus-specific and heterochromatin specific probes in four NWMs and one Old World Monkey (OWM) species, i.e. in Alouatta caraya (ACA), Callithrix jacchus (CJA), Cebus apella (CAP), Saimiri sciureus (SSC), and Chlorocebus aethiops (CAE), respectively. 107 individual evolutionary conserved breakpoints (ECBs) among those species were identified and compared with those of other species in previous reports. Especially for chromosomal regions being syntenic to human chromosomes 6, 8, 9, 10, 11, 12 and 16 previously cryptic rearrangements could be observed. 50.4% (54/107) NWM-ECBs were colocalized with those of OWMs, 62.6% (62/99) NWM-ECBs were related with those of Hylobates lar (HLA) and 66.3% (71/107) NWM-ECBs corresponded with those known from other mammalians. Furthermore, human fragile sites were aligned with the ECBs found in the five studied species and interestingly 66.3% ECBs colocalized with those fragile sites (FS). Overall, this study presents detailed chromosomal maps of one OWM and four NWM species. This data will be helpful to further investigation on chromosome evolution in NWM and hominoids in general and is prerequisite for correct interpretation of future sequencing based genomic studies in those species.

RAPD and barcode analyses of groupers of the genus Epinephelus.

The diversity of Epinephelus species was investigated throughout Thailand. Random amplified polymorphic DNA successfully produced 1300 bands that were phylogenetically informative and used to construct cladograms. Values of pairwise genetic similarity (S) within species ranged from 0.65 in E. erythrurus to 0.99 in E. malabaricus. The interspecific values of S ranged from 0.23 between E. malabaricus and E. bleekeri to 0.66 between E. coeruleopunctatus and E. erythrurus. The intraspecific nucleotide variation ranged from 0.037 to 0.159 in the mitochondrially encoded 16S RNA (MT-RNR2) region and from 0.003 to 0.157 for the mitochondrially encoded cytochrome c oxidase I (MT-CO1) region. All sequences were submitted individually to GenBank. The barcode sequences of Thai species of Epinephelus were aligned to the same species found in GenBank. For the MT-RNR2 gene region, intraspecific nucleotide variation ranged from 0.000 to 0.121, and interspecific nucleotide variation ranged from 0.003 to 0.146. For the MT-CO1 gene region, intraspecific nucleotide variation ranged from 0.000 to 0.140, and interspecific nucleotide variation ranged from 0.000 to 0.166. The MT-RNR2 data indicate that some species, including E. bleekeri from India and E. malabaricus from Thailand are not monophyletic. Additionally, the MT-CO1 data indicated that E. bleekeri, E. quoyanus and E. coeruleopunctatus are not monophyletic. The sequences of E. lanceolatus from each country are highly conserved, with genetic distances ranging from 0.000 to 0.003. Another important result from this study is that the barcode sequence from Thai E. erythrurus was previously not present in the GenBank.

Cytogenetics of the skinks (Reptilia, Scincidae) from Thailand; IV: newly investigated karyotypic features of Lygosoma quadrupes and Scincella melanosticta

Abstract This study analyzed the karyotype of Lygosoma quadrupes and Scincella melanosticta from northeastern Thailand. Mitotic chromosomes were prepared directly from intestinal epithelial cells of three L. quadrupes and nine S. melanosticta. The chromosomes were stained by conventional staining technique. The karyotype was determined for each species based on at least 20 well-spread cells. Both L. quadrupes and S. melanosticta showed the same diploid number of 2n = 30, but differed in identical chromosome morphology. No sex chromosome heteromorphisms were evident in L. quadrupes; in contrast, S. melanosticta showed the ZZ/ZW sex determination system. The respective karyotype formulae of L. quadrupes and S. melanosticta were deduced as:

Additional description of karyotype and meiotic features of Takydromus sexlineatus (Squamata, Lacertidae) from northeastern Thailand

This study determined the standardized karyotype and idiogram of Takydromus sexlineatus from Surin Province in northeastern Thailand. Mitotic metaphase chromosomes from bone marrow cells and different meiotic stage from testicular cells respectively were studied after staining with conventional Giemsa and Ag-NOR banding techniques for chromosome identification. The study was carried out with samples taken from both male and female T. sexlineatus, the sex of which was identified by external examination. The results showed that the diploid chromosome number of T. sexlineatus was 2n = 38 and the fundamental number (NF) was 38 in both male and female. Although chromosome study was done in both sexes of T. sexlineatus, this study does not identify the sex chromosomes. This is because of the limitation of the staining techniques used. Therefore, we did not conclude on the sex chromosome system in this species and all chromosomes were taken as autosomes. The types of autosomes observed were 16 large telocentric, 14 medium telocentric, and 6 small telocentric macrochromosomes and 2 microchromosomes. Our result is the new record in Thailand on this species and NOR characteristics by silver staining is the first report on NOR banding in this species. The karyotype formula of T. sexlineatus thus was found as: 2n(38) = L16t + M14t + S6t + 2 microchromosomes.

Karyotypic study of five Lutjanid species using conventional and Ag-NORs banding techniques

The first cytogenetic comparisons of five snapper species from Thailand were presented here. Renal cell samples were taken from blacktail snapper (Lutjanus fulvus), five lined snapper (L. quinquelineatus), dory snapper (L. fulviflamma), brownstripe red snapper (L. vitta), and mangrove red snapper (L. argentimaculatus). The mitotic chromosome preparation was prepared directly from kidney cells. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results exhibited that all five snapper species have the diploid chromosome numbers of 2n = 48 and the fundamental numbers (NF) of 48. The presences of large, medium, and small telocentric chromosomes were 22-24-2, 24-20-4, 36-10-2, 28-16-4 and 36-10-2, respectively. The Ag- NORs banding technique provides the pair of nucleolar organizer regions (NORs) at subcentromeric region of the long arm of the respective telocentric chromosome pairs 9, 1, 3, 4 and 9. Their karyotype formulas is as follows: L. fulvus (2n = 48): L22t + M24t + S2t, L. quinquelineatus (2n = 48): L24t + M20t + S4t, L. fulviflamma (2n = 48): Lt36 + Mt10 + St2, L. vitta (2n = 48): L28t + M16t + S4t, and L. argentimaculatus (2n = 48): L36t + M10t + S2t.

Molecular Cytogenetic Analysis of One African and Five Asian Macaque Species Reveals Identical Karyotypes as in Mandrill

Background: The question how evolution and speciation work is one of the major interests of biology. Especially, genetic including karyotypic evolution within primates is of special interest due to the close phylogenetic position of Macaca and Homo sapiens and the role as in vivo models in medical research, neuroscience, behavior, pharmacology, reproduction and Acquired Immune Deficiency Syndrome (AIDS). Materials & Methods: Karyotypes of five macaque species from South East Asia and of one macaque species as well as mandrill from Africa were analyzed by high resolution molecular cytogenetics to obtain new insights into karyotypic evolution of old world monkeys. Molecular cytogenetics applying human probes and probe sets was applied in chromosomes of Macaca arctoides, M. fascicularis, M. nemestrina, M. assamensis, M. sylvanus, M. mulatta and Mandrillus sphinx. Established two- to multicolor-fluorescence in situ hybridization (FISH) approaches were applied. Locus-specific probes, whole and partial chromosome paint probes were hybridized. Especially the FISH-banding approach multicolor-banding (MCB) as well as probes oriented towards heterochromatin turned out to be highly efficient for interspecies comparison. Conclusion: Karyotypes of all seven studied species could be characterized in detail. Surprisingly, no evolutionary conserved differences were found among macaques, including mandrill. Between the seven here studied and phenotypically so different species we expected several via FISH detectable karyoypic and submicroscopic changes and were surprised to find none of them on a molecular cytogenetic level. Spatial separation, may explain the speciation and different evolution for some of them, like African M. sylvanus, Mandrillus sphinx and the South Asian macaques. However, for the partially or completely overlapping habitats of the five studied South Asian macaques the species separation process can also not be deduced to karyotypic separation.

Karyotype analysis of two groupers, Epinephelus species (Serranidae)

This is an initial report of a cytogenetic study in two economically important species of Epinephelus, namely E. bleekeri and E. coeruleopunctatus, using the G-banding technique. Chromosome numbers of the two species are 2n = 48. The differences were found to be karyotype variations, with 48 telocentric chromosomes in E. bleekeri and two submetacentric and 46 telocentric chromosomes in E. coeruleopunctatus. Statistically, there were no karyotype variations between male and female individuals of each of the species. This basic knowledge is highly important for other technologies, such as those used in hybridization programming.