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Metaphase chromosome plates and karyotypes of painted chorus frog (Microhyla pulchra), male (A) and female (B), by conventional staining technique, and male (C) and female (D) by Ag-NOR banding technique. The karyotype was composed of 2n=24 chromosomes. Arrows indicate nucleolar organizer regions (NORs) (Scale bars=5 µm).
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The cytogenetics of the ornamented pygmy frog (Microhyla fissipes), painted chorus frog (M. pulchra) and narrow-mouthed frog (M. heymonsi) were studied in the aspect of chromosome numbers, morphology and nucleolus organizer region (NOR) locations. For this present study, we provided the karyotype and idiogram of these three species by conventional...
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... karyotype of M. fissipes showed metacentric chromosomes in pairs 1, 2, 4-5, 8-11, and submetacen- tric chromosomes in pairs 3, 6-7 and X, Y metacentric chromosomes (Fig. 2a, b). The karyotype of M. pul- chra was composed of metacentric chromosomes in pairs 1, 2, 5-7, 12 and submetacentric chromosomes in pairs 3, 4, 8-11 (Fig. 3a, b). Meanwhile, M. heymonsi showed metacentric chromosomes in pairs 1-2, 5-11 and submetacentric chromosomes in pairs 3-4, 12 and no obvious difference in size of sex chromosomes were found (Fig. 4a, b). The type and number of chromo- somes of M. pulchra herein coincide with those previ- ously reported (Supaprom and Baimai 2002 Fig. 5a-c. For the sex chromosome, this is the first report for M. fissipes. Cytological evidence that was supportive of male heterogamety (XX/XY sex chromosome) has been published by Iturra and Veloso (1981) Table 3. Mean length of short arm chromosomes (Ls), long arm chromosomes (Ll), total arm chromosomes (LT), relative length (RL), centro- meric index (CI), and standard deviation (SD) of RL, CI from 20 metaphase cells of painted chorus frog (Microhyla pulchra), ...
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Pissaparn M, Phimphan S, Chaiyasan P, Tanoamtong A, Liehr T, Suwannapoom C, Reungsing M, Supiwong W. 2020. First chromosome analysis of Thai pufferfish Pao cochinchinensis (Steindachner, 1866). Biodiversitas 21: 4309-4316. Here first analysis of chromosomes and nucleolar organizer region (NOR) pattern in pufferfish Pao cochinchinensis (Steindachner...
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... Conventional staining and Ag-NOR banding were done using 20% Giemsa's solution and 50% silver nitrate solution adapted from Sangpakdee et al. (2015), Sangpakdee et al. (2017), Jantarat et al. )2017(, Sreeputhorn et al. (2017, Supiwong et al. (2017), Getlekha and Tanomtong (2020(. With a Cannon D-150 camera, 20 metaphase spreads were recorded for each sample. ...
This study investigated the chromosomes of three Trichopsis species (namely Trichopsis pumila, Trichopsis vittata, and Trichopsis schalleri) using conventional (Giemsa stain, Ag-NOR) and molecular cytogenetic techniques. Fluorescence in situ hybridization (FISH) with repetitive DNAs, including 5S and 18S rDNAs, and microsatellites as probes were also performed. Our results indicated a conserved diploid number of 46 (2n) for all analyzed species of both sexes, although varying in their karyotype structure. Furthermore, T. schalleri and T. vittata karyotypes were presented with only acrocentric chromosomes (46a), while T. pumila had a submetacentric pair in addition to acrocentric ones (2sm+44a). Positive Ag-NOR sites were differentially found in each species adjacent to the acrocentric chromosome's centromeric and/or interstitial sub-centromeric regions. Both 5S and 18S rDNAs vary between the analyzed species, but all were localized in multiple sites. However, the distribution pattern of microsatellites differed in each species, mostly scattering at peri-centromeric and telomeric regions. These findings show that Trichopsis species, while having a conserved diploid number, differ significantly in the distribution of repetitive sequences in their karyotypes, particularly for rDNAs. These species-specific patterns can be useful for characterizing and identifying further different species and examining the evolutionary mechanisms that drove the evolution of these fishes' karyotypes.
... Conventional staining was done using 20% Giemsa's stock solution for 30 min (Patawang et al. 2014). 50% AgNO3 and 2% gelatin were used for Ag-NOR banding technique (Patawanget al. 2017;Sangpakdee et al. 2017;Phimphan et al. 2020;Thongnetr et al. 2021). ...
Donbundit N, Bausriyod P, Tanomtong A, Srisamoot N, Sumontha M, Thongnetr W, Patawang I, Supiwong W, Ditcharoen S, Muanglen N, Kaewmad P. 2022. Karyomorphological delineation, and the NOR loci on the sex chromosome in three species of Chrysopeleinid (Chrysopeleinae: Colubridae) from Thailand. Biodiversitas 23: 3813-3819. The description of chromosomal characteristics of three snakes from Thailand namely, Ahaetulla fusca, A. prasina, and Chrysopelea ornata was by using conventional staining and Ag-NOR banding techniques. The results showed that the same diploid chromosome number (2n) = 36 (16 macrochromosomes + 20 microchromosomes) and the fundamental number (NF) was 54 while the karyotype formulae in these three species are as follows: A. fusca; 2n(36) = Lm6 + Sm2+ Ssm4 + Sa2+ ZZ/ZW + 20 microchromosomes, A. prasina; 2n(36) = Lm6+ Sm6+ Sa2+ ZZ/ZW + 20 microchromosomes and C. ornata; 2n(36) = Lm4+ Lsm2+ Sm2 + Ssm4+ Sa2+ ZZ/ZW + 20 microchromosomes. Besides, the ZZ/ZW sex chromosome was observed in all species, whereas C. ornata was discovered the heteromorphic sex chromosome for the first time. Nucleolar organizer regions (NORs) are located on telomeric region of one pair in all species, while located on microchromosomes in A. fusca and A. prasina, whereas C. ornata was detected on sex chromosomes. Therefore, the Z chromosome revealed the NOR on the telomeric region of the long arm, while NOR of the W chromosome was located on the telomeric region of the short arm.
... The chromosome preparation was followed the method of Supiwong et al. (2010), Tanomtong (2011), Sangpakdee et al. (2015 and Sangpakdee et al. (2017). Both male and female of H. temminckii were intraperitoneally injected with 1 mL/100 g of body weight of 0.05% colchicine and then sacrificed after one hour. ...
The first chromosomal study of kissing Gourami, Helostoma temminckii from Thailand was karyotypically performed. Kidney cells were obtained from ten male and ten female specimens. Chromosomes were conventionally stained, and Ag-NOR banding was carried out. Conventional staining mainly demonstrated telocentric chromosome with diploid number (2n) and fundamental number (NF) as 48. The Ag-NOR banding technique showed the position of nucleolar organizer regions (NORs) at the end of the telomeres of chromosome pair four. The karyotype consisted of 16 large, 30 medium, and two small telocentric chromosomes. Thus, the karyotype formula can be represented as follows: L t 16 +M t 30 +S t 2.
... The chromosomes were then stained by three techniques. Conventional staining technique was carried out by using 20% Giemsa's solution (Sangpakdee et al. 2015;Sangpakdee et al. 2017;Chaiyasan et al. 2018). Ag-NOR staining was conducted by using 50% silver nitrate solution (Sreeputhorn et al. 2017;Supiwong et al. 2017;Getlekha and Tanomtong 2020), and C-banding has performed the method of Supiwong et al. (2019). ...
Supiwong W, Wongchantra P, Thongnetr W, Mingkwan B, Chaiyasan P, Pinmongkhonkul S, Pinthong K, Tanomtong A. 2021. Comparative cytogenetic analysis of fishes in the genus Trichopodus (Osphronemidae) in Thailand. Biodiversitas 22: 3029-3036. Comparative cytogenetic study of four species of the genus Trichopodus including T. leerii, T. microlepis, T. pectoralis, and T. trichopterus from Thailand, was carried out. The specimens were collected from the Basins throughout Thailand. Chromosome preparation was directly performed from the kidney tissues. Conventional staining by Giemsa solution, Ag–NOR banding by silver nitrate solution and C-banding by NaOH solution were conducted to stain the chromosomes. Results showed that four species studied have the same diploid number and fundamental number as 46 composing of all telocentric chromosomes. Karyotypes consisting of large-medium-small sizes in T. leerii, T. microlepis, T. pectoralis and T. trichopterus were 16–28–2, 14–32–0, 20–26–0 and 10–30–6 chromosomes, respectively. The marker chromosomes which present the NOR positions are the single pair in all species but there are differences in the locations and pairs such as pairs no. 1, 7, 2 and 1, respectively. Most species except T. trichopterus, NOR sites locate at interstitial region adjacent to the centromere. T. trichopterus had the telomeric NORs. Constitutive heterochromatin blocks displayed at the centromeric/pericentromeric regions of all chromosomes in T. leerii and T. microlepis whereas in T. pectoralis and T. trichopterus, those presented at not only centromeres of several chromosome pairs, but they also were found at interstitial sites. The obtained finding, cytogenetic data has species-specific. Thus, it can be used for further study on taxonomy. Moreover, this data shows the close relationship of genetics among these species so that the application for breeding may be used in the future.
... testudineus (Sá-Gabriel andMolina 2005). A single NORbearing chromosome is a common characteristic found in many fish groups as well as vertebrates (Supiwong et al. 2010, 2012a,b, 2013b, Chooseangjaew et al. 2017Kasiroek et al. 2017;Phimphan et al. 2017;Sangpakdee et al. 2017;Chaiyasan et al. 2018). NOR mapping is frequently used to compare variations, as well as to identify and explain speciation. ...
Pissaparn M, Phimphan S, Chaiyasan P, Tanoamtong A, Liehr T, Suwannapoom C, Reungsing M, Supiwong W. 2020. First chromosome analysis of Thai pufferfish Pao cochinchinensis (Steindachner, 1866). Biodiversitas 21: 4309-4316. Here first analysis of chromosomes and nucleolar organizer region (NOR) pattern in pufferfish Pao cochinchinensis (Steindachner, 1866) was undertaken. Chromosomal preparations were obtained from kidney of P. cochinchinensis from Chi River basin in Thailand. Chromosomal characteristics were analyzed by Giemsa staining, Ag-NOR banding as well as fluorescence in situ hybridization (FISH) using microsatellites d(CA)15 and d(CGG)10 probes. P. cochinchinensis had 2n = 40 with the fundamental number (NF) 74, both in male and female. The karyotype exhibited 12 metacentric (m), 10 submetacentric (sm), 12 acrocentric (a) and 6 telocentric (t) chromosomes. No differentiated heteromorphic sex chromosomes were observed. NORs were located on short arms adjacent to telomere of the metacentric chromosome pair 4, which coincide with signals of d(CGG)10 probe. FISH with d(CGG)10 sequences were also displayed at the telomeres of most other chromosomes, whereas d(CA)15 repeats highly accumulated throughout almost all entire chromosomes except for centromeric regions. The results of conventional Giemsa staining presented the differentiation even the same genus. The l ocalization of NORs on one pair of chromosomes only is a common characteristic found in many fish groups as well as other vertebrates. Mapping of two distinct microsatellites demonstrated the remarkable chromosomal diversification that characterizes evolution in the genus Pao. Both, conventional and molecular cytogenetics are excellent tools to study, and better understand chromosomal evolution, as well as to uncover biodiversity among fishes.
... The slide was conventionally stained with 20% stock Giemsa's solution for 30 min. After that, the slides were soaked in distilled water (Sangpakdee et al., 2017). ...
... C-banding has been largely used in amphibians to compare karyotypes and to distinguish species with the same diploid number (Bogart 1970, Cuevas and Formas 2003, Nogueira et al. 2015, Sangpakdee et al. 2017, Targueta et al. 2018. Moreover, homogeneous C-banding patterns among related species has been associated with low genetic differentiation (Pellegrino et al. 1997, Lourenço et al. 1998, Bruschi et al. 2012) and enrichment of repetitive elements, characteristic of amphibian chromosomes (Schmid et al. 1978, Bruschi et al. 2012, Zlotina et al. 2017. ...
South American frogs of the genus Eupsophus Fitzinger, 1843 comprise 10 species. Two of them, Eupsophus vertebralis Grandison, 1961 and E. emiliopugini Formas, 1989 belong to the Eupsophus vertebralis group, exhibiting 2n = 28. Fundamental number differences between these species have been described using conventional chromosome staining of few specimens from only two localities. Here, classical techniques (Giemsa, C-banding, CMA 3 /DAPI banding, and Ag-NOR staining), and fluorescence in situ hybridization (FISH, with telomeric and 28S ribosomal probes), were applied on individuals of both species collected from 15 localities. We corroborate differences in fundamental numbers (FN) between E. vertebralis and E. emiliopugini through Giemsa staining and C-banding (FN = 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes and Ag-NOR staining confirmed the active nucleolus organizer regions signal on pair 5 for both species. Nevertheless, one E. emiliopugini individual from the Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. Chromosomal rearrangements, rRNA gene dosage control, mobile NORs elements, and/or species hybridization process could be involved in this interpopulation chromosomal variation.
... In anurans, both female and male heterogameties are present and the occurrence of heteromorphic sex chromosomes is rare. To date, among the already karyotyped anuran species, 21 species with XX/XY and 24 species with ZZ/ZW systems present heteromorphic sex chromosomes (see review by Schmid et al., 2012;Saba & Tripathi, 2014;Patawang et al., 2014;Sangpakdee et al., 2017). In addition, heteromorphic sex chromosomes were already found in multiple sex chromosome systems (derived from Yautosome fusions) in species of the genera Pristimantis and Strabomantis (family Craugastoridae) and in Eleutherodactylus cavernicola (family Eleutherodactylidae) (Schmid et al., 1992(Schmid et al., , 2002a(Schmid et al., , 2003(Schmid et al., , 2010. ...
Initially identified as Astyanax schubarti on the basis of morphological characteristics, its chromosomal analysis revealed a unique diploid number in the genus. With 42 chromosomes and the impossibility of homologous pairing, the karyotype of the individual was compared to a set of haploid complements of Astyanax schubarti (2n = 36 chromosomes) and Astyanax fasciatus (2n = 48 chromosomes). Natural hybrids are rare. The viability of a hybrid between species with such chromosomal discrepancy may offer important hypotheses to explain the morphological, molecular, and cytogenetic diversity of the genus.
... In anurans, both female and male heterogameties are present and the occurrence of heteromorphic sex chromosomes is rare. To date, among the already karyotyped anuran species, 21 species with XX/XY and 24 species with ZZ/ZW systems present heteromorphic sex chromosomes (see review by Schmid et al., 2012;Saba & Tripathi, 2014;Patawang et al., 2014;Sangpakdee et al., 2017). In addition, heteromorphic sex chromosomes were already found in multiple sex chromosome systems (derived from Yautosome fusions) in species of the genera Pristimantis and Strabomantis (family Craugastoridae) and in Eleutherodactylus cavernicola (family Eleutherodactylidae) (Schmid et al., 1992(Schmid et al., , 2002a(Schmid et al., , 2010. ...
The order Anura currently encompasses over 6,800 amphibian species distributed in 56 families and is an interesting group for cytogenetic studies. Whereas some groups of species present conservative karyotypes, others are highly variable in diploid number or number/location of diverse chromosomal markers, such as nucleolus organizer regions, heterochromatic bands and specific satellite DNA sites. In some cases, karyotypic variation overcomes morphological diversification, turning cytogenetics into a useful tool for taxonomy. Special variation is observed with respect to sex chromosomes and sex determination systems, with both female and male heterogametes observed. Although most species already karyotyped do not show sex chromosome heteromorphism, distinct levels of differentiation are observed between the sex chromosomes of several species, which makes this group particularly interesting for studies of sex chromosome evolution. In this chapter, we explore the use of cytogenetic data for studies of frogs as well as the insights that hypotheses of phylogenetic relationships have added to this issue. In addition, we provide a brief review of PcP190 satellite DNA (with new data for the genus Engystomops), sex chromosome systems and B chromosomes found in Anura.
... Blood samples of the H. porcinus (two males and two females) were collected from Khon Kaen Zoo, Thailand and then applied to cytogenetic studies by lymphocyte culture of whole blood samples. The culture cells were treated with a colchicine-hypotonic-fixationair-drying technique followed by conventional staining, GTG-, high-resolution and Ag-NOR banding techniques (Rooney 2001, Campiranon 2003, Sangpakdee et al. 2017. For 20 cells of each individual chromosome, checks, length measurements, karyotype and idiogram analysis were accomplished by using a light microscope as previously described (Chooseangjaew et al. 2017). ...
Standardized karyotype and idiogram of Indian hog deer (Hyelaphus porcinus) at Khon Kaen Zoo, Thailand was explored. Blood samples were taken from two male and two female deer. After standard whole blood T-lymphocytes were cultured at 37°C for 72 h in presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, GTG-, high-resolution and Ag-NOR banding techniques were applied to stain the chromosome. The results show that the diploid chromosome number of H. porcinus was 2n=68, the fundamental number (NF) was 70 in both males and females. The types of autosomes observed were 6 large telocentric, 18 medium telocentric and 42 small telocentric chromosomes. The X chromosome was large metacentric chromosome and Y chromosome was small submetacentric chromosome. The GTG-banding and high-resolution techniques showed that the numbers of bands and locations in H. porcinus are 239 and 301, respectively, and each chromosome pair could be clearly differentiated. In addition, the subtelomeric q-arm of chromosome pairs 1 and 2 showed clearly observable nucleolar organizer regions (NORs). Our results are the first reports of GTG-, high-resolution and Ag-NOR banding techniques on this species. The karyotype formula of H. porcinus is as follows:
