Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(2): 19-28, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2668 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Supanuam, P., Jantarat, S., Khawporntip, W., Kraiprom, T., Buatip, S., Jumrusthanasan, S., Kaewsri, S., Donbundit, N., Ditcharoen, S., Thong- netr, W., Phimphan, S., & Tanomtong, A. (2024). First Chromosome Characteri- zation and Repetitive DNA of Barred Gliding Lizard, Draco taeniopterus Günther, 1861 (Draconinae: Agamidae: Squamata). Caryologia 77(2): 19-28. doi: 10.36253/caryologia-2668 Received: April 04, 2024 Accepted: September 21, 2024 Published: November 10, 2024 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus Günther, 1861 (Draconinae: Agamidae: Squamata) Praween Supanuama, Sitthisak Jantaratb,*, Warakarn Khawporn- tipb, Thaintip Kraipromc, Somsak Buatipb, Sarun Jumrusthanasand, Sarawut Kaewsrid, Nattasuda Donbundite, Sukhonthip Ditcharoene, Weera Thongnetrf, Sumalee Phimphang, Alongklod Tanomtonge aBiology Program, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Rat- chathani, Thailand; bDepartment of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Campus, Thailand; cDepartment of Agricultural and Fishery Science, Faculty of Science and Technology, Prince of Songkla University, Pattani Cam- pus, Thailand; dBiology Program, Faculty of Science, Buriram Rajabhat University, Bur- iram, Thailand; eDepartment of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; fDivision of Biology, Department of Science, Faculty of Science and Tech- nology, Rajamangala University of Technology Krungthep, Bangkok, Thailand; gBiology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetch- abun, Thailand *Corresponding author. E-mail: sitthisak.j@psu.ac.th Abstract. This research was the first report on karyological analysis and distribution patterns of repetitive DNA using the fluorescence in situ hybridization (FISH) tech- nique on the barred gliding lizard, Draco taeniopterus Günther, 1861. The 10 male and 10 female specimens were collected from Than To district, Yala province, Thailand. Chromosome preparation was performed by direct method using bone marrow and testis. The chromosomes were stained using conventional staining, NOR-banded, and FISH technique with d(GC)15, d(TA)15, d(CAG)10, and d(CAA)10 microsatellite probes. The karyotype of the barred gliding lizard reveals a diploid chromosome number of 34 and a fundamental chromosome number of 46, comprising of 8 pairs of large metacentric chromosomes, 2 pairs of small metacentric chromosomes, 2 pairs of large submetacentric chromosomes, and 22 pairs of microchromosomes, no sex chromo- some detection between male and female karyotype. The metaphase I showed 17 biva- lents and metaphase II showed haploid, n=17. The NOR is observed on the telomeric region of the last microchromosome pair 17th. Microsatellite repeat patterns indicate the presence of d(GC)15 and d(CAG)10 show specific regions, 2qter and 3qter respec- tively. While d(TA)15 and d(CAA)10, show cumulative signals dispersed throughout the chromosomes. This research can provide additional fundamental information for future genetic studies. The barred gliding lizard has the following karyotype formula: 2n=34=Lm 8+Lsm 2+Sm 2+22mi. Keywords: Draco taeniopterus, Chromosome, Cytogenetics, Repetitive DNA. http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2668 https://doi.org/10.36253/caryologia-2668 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode mailto:sitthisak.j@psu.ac.th 20 Praween Supanuam et al. INTRODUCTION Flying lizard genus Draco are classified in family Agamidae, subfamily Draconinae which consists of 34 genera and 272 species, the important genera such as Acanthosaura, Calotes, Diploderma, Draco, Gonocepha- lus, Japalura and Sitana. In the genus Draco, 40 species are found, which is distributed from Southwest India through Southeast Asia, including the Malay Penin- sula, the Philippines, and Thailand (Honda et al. 2000; McGuire & Heang 2001; Hoser 2014; Denzer et al. 2015; Nampochai et al. 2021). Barred gliding lizard or spotted flying dragon (Draco taeniopterus Günther, 1861), It is a species that indicates the abundance of tropical rainforest ecosystems. The typical characteristics of this lizard are its small body size (66-78 mm from the tip of the mouth to the anus and the tail 136-153 mm long), flat body, small head, the extended dulap (chin flap) is yellow-orange. Patagium has 4-5 distinct dark transverse bands alternative to light transverse bands and presence of light spots in the mid- dle of dark bands. These colors are useful to help cam- ouflage there to match the bark. Tympanum uncovered with scales. Snout without a series of scales forming a Y-shaped figure. Nostril directed upward (Figure 1). The Draco taeniopterus is found in Myanmar, Thailand, Cam- bodia, and Malaysia. The conservation status of this spe- cies is least concern (Honda et al. 1999; 2000; Srichairat et al. 2014; 2015; 2017; Visoot et al. 2023) Cytogenetic review of the genus Draco has only 2 reports in 3-4 species with conventional technique. The Draco karyotype is 2n=34 with 16 macrochromosomes and 18 microchromosomes, without sex-chromosomes. The karyotype report of Draconinae also not prevailing. The diploid number of this subfamily has appeared in several genera, Gonocephalus (2n=36), Calotes (2n=32- 34), Japalura (2n=34,46), Sitana (2n=34, 36, 46) (Ota and Hikida 1989; Sharma and Nakhasi 1980; Li et al. 1981; Ota 1988; Solleder and Schmid 1988; Ota et al. 1992; Kritpetcharat et al. 1999; Diong et al. 2000; Ota et al. 2002; Singh and Banerjee 2004; Zongyun et al. 2004; Patawang et al. 2015a). This research is first report on molecular cytoge- netics of the genus Draco. Conventional, meiotic con- figuration, Ag-NOR banding and molecular cytogenetic techniques using microsatellite DNA probes including, d(TA)15, d(CG)15, d(CAA)10 and d(CAG)10 were applied to detect. This study is useful for taxonomy, conservation and basic and in-depth cytogenetic information of this species. MATERIALS AND METHODS The 10 male and 10 female specimens, barred glid- ing lizard (Draco taeniopterus) were collected from Ban Wang Sai, Mae Wat subdistrict, Than To District, in Yala Province, Thailand. The flying lizard were transferred to the laboratory and identified according to the morpho- logical criteria of KEY (Chan-Ard et al. 2015; Das 2015). Experiments were performed in accordance with ethi- cal protocols, as approved by the Ethics Committee of Prince of Songkla University (Ref No.AI003/2022). Chromosomes were directly prepared in vivo (Pata- wang et al., 2018a) as follows. The animals were inject- ed on their abdominal cavity with colchicine. Then leaved for 24 hours. Chromosome preparation con- taining bone marrow for mitosis and testis for meiosis were conducted by the colchicine-hypotonic-fixation- air drying technique. The chromosomes were stained with 20% Giemsa’s for 30 minutes and identified for NORs by Ag-NOR staining according to Howell and Black (1980) and Verma and Babu (1995). Chromosom- al checks were performed on mitotic metaphase cells under light microscope. FISH experiments were performed under high stringency conditions (Yano et al. 2017) to classify microsatellite sequences, specifically (TA)15, (GC)15, (CAA)10, and (CAG)10. These sequences were directly labeled by Cy3 at the 5’end during synthesis (Sigma, St. Louis, MO, USA) . FISH was performed under stringent conditions and hybridization occurred overnight in a moist chamber at 37 °C. Chromosomes were counter- stained with 4’,6-Diamidino-2-phenylindole dihydro- chloride (DAPI, 1.2 μg/ml) mounted in antifade solu- Figure 1. General characteristics (A.) and its patagium (B.) of barred gliding lizard, Draco taeniopterus, Draconinae, Agamidae) from Ban Wang Sai, Mae Wat subdistrict, Than To District, in Yala Province, Thailand. 21First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus tion (Vector, Burlingame, CA, USA,) (Aiumsumang et al. 2022; Patawang et al. 2022; Prasopsin et al. 2022; Thongnetr et al. 2022). At least 20 metaphase spreads per individual were analyzed to confirm the diploid number, karyotype structure, NORs and FISH data. Images were cap- tured using an Axioplan II microscope (Carl Zeiss Jena GmbH, Germany) with CoolSNAP and processed using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). Chromosomes were classified accord- ing to centromere position as metacentric (m), submeta- centric (sm) and acrocentric (a) (Tanomtong et al. 2019). For the chromosomal arm number (NF; fundamental number), m, sm, a were scored as bi-armed while t as mono-armed. The microchromosomes are chromosomes that are 5 times less long than the largest pair of chro- mosomes (Patawang et al. 2016; 2017; 2018b). RESULTS AND DISCUSSION Barred gliding lizard (Draco taeniopterus) had a diploid number of 34. The karyotype comprised eight large metacentric, two large submetacentric, two small metacentric and 22 microchromosomes. The fundamen- tal number was 46 in both sexes and no sex chromo- some heteromorphisms were evident (Table 1 and Fig- ure 2). The karyotype formula of Draco taeniopterus is 2n=34=Lm 8+Lsm 2+Sm 2+22mi. The diploid chromosome number is following previous studies of 4 species of genus Draco (Ota and Hikida 1989, Kritpetcharat et al. 1999). The diploid numbers of subfamily Draconinae are 2n=32-46 in 7 genera 21 species (Ota and Hikida 1989; Sharma and Nakhasi 1980; Li et al. 1981; Ota 1988; Sol- leder and Schmid 1988; Ota et al. 1992; Kritpetcharat et al. 1999; Diong et al. 2000; Ota et al. 2002; Singh and Banerjee 2004; Zongyun et al. 2004; Patawang et al. 2015a). Some species of Draconinae has polymorphism, the Calotes versicolor from India has 2n=32 and 34, the Japarula swinhonis swinhonis from Taiwan has 2n=36, 40, and 46. This species exhibits no sex differences in karyotypes between males and females, no cytologi- cally distinguishable sex chromosome was observed to be similar to the Draco cornutus, D. haematopogon, D. quinquefasciatus and D. belliana. The karyotypes of this genus are quite similar. All species have 12-16 macro- metacentric or submetacentric chromosomes, and 18-22 microchromosomes. The mechanism of chromosomes rearrangement maybe fission, fusion and/or pericentric inversion. Comparative chromosome studies of sub- family Draconinae is show on Table 2 (Ota and Hikida 1989; Sharma and Nakhasi 1980; Li et al. 1981; Ota 1988; Solleder and Schmid 1988; Ota et al. 1992; Krit- petcharat et al. 1999; Diong et al. 2000; Ota et al. 2002; Table 1. Mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), length of total chromosomes (LT), relative length (RL), centromeric index (CI) and standart deviation (SD) from 10 metaphases of male and female of barred gliding lizard (Draco taeniopterus), 2n (diploid)=34. Chromosome pairs Ls (µm) Ll (µm) LT (µm) CI±SD RL±SD Chromosome size Chromosome type 1 7.69 8.61 16.30 0.528±0.000 0.184±0.000 Large metacentric 2 5.97 8.45 14.41 0.586±0.000 0.163±0.000 Large metacentric 3 5.37 6.09 11.46 0.532±0.000 0.129±0.000 Large metacentric 4 5.25 5.59 10.84 0.515±0.000 0.122±0.000 Large metacentric 5 2.87 6.15 9.02 0.683±0.000 0.102±0.000 Large submetacentric 6 2.83 3.44 6.27 0.550±0.000 0.070±0.000 Small metacentric 7 0.00 2.22 2.22 1.000±0.000 0.025±0.000 microchromosome 8 0.00 2.13 2.13 1.000±0.000 0.024±0.000 microchromosome 9 0.00 2.06 2.06 1.000±0.000 0.023±0.000 microchromosome 10 0.00 2.04 2.04 1.000±0.000 0.023±0.000 microchromosome 11 0.00 1.96 1.96 1.000±0.000 0.022±0.000 microchromosome 12 0.00 1.89 1.89 1.000±0.000 0.021±0.000 microchromosome 13 0.00 1.67 1.67 1.000±0.000 0.019±0.000 microchromosome 14 0.00 1.66 1.66 1.000±0.000 0.019±0.000 microchromosome 15 0.00 1.71 1.71 1.000±0.000 0.019±0.000 microchromosome 16 0.00 1.58 1.58 1.000±0.000 0.018±0.000 microchromosome 17* 0.00 1.45 1.45 1.000±0.000 0.016±0.000 microchromosome * = NORs bearing chromosomes. 22 Praween Supanuam et al. Singh and Banerjee 2004; Zongyun et al. 2004; Pata- wang et al. 2015a). The first cytogenetic study of Draco taeniopterus car- ried out by Ag-NOR banding technique was obtained from this research. We found NORs observed in the region adjacent the last smallest microchromosomes (pair 17th) (Figure 3). The report of NOR position in Draconi- nae was located on telomeric region of q-arm of pair 2nd in 4 species of Calotes including C. cristatellus, C. emma, C. mystaceus, and C. versicolor (Solleder and Schmid Figure 2. Metaphase plates and standardized karyotypes of male (A.), female (B.) and Idiogram (C.) of barred gliding lizard, Draco taeniop- terus, 2n=34 by conventional staining (Scale bars = 10 µm). 23First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus 1988; Patawang et al. 2015a). The NOR position of Draco taeniopterus was more conserved than the genus Calotes. Chromosomes of barred gliding lizard testis for meiosis was observed. The metaphase I has 17 bivalents comprising 6 ring bivalents of macrochromosomes and 11 small rod bivalents of microchromosomes. The meta- phase II has n=17 haploid comprising 5 metacentric, 1 submetacentric macrochromosomes and 11 microchro- mosomes (Figure 4). The meiosis karyotypes of other species in Agamidae are showed in lndo-Chinese water dragon, Physignathus cocincinus which has 2n =36 with 6 ring bivalents of metacentric or submetacentric mac- rochromosomes and 12 rod bivalent of microchromo- somes (Patawang et al. 2015b). In addition, the meiotic Table 2. Comparative chromosome studies of subfamily Draconinae. Species 2n Karyotype NOR Locality References Acanthosaura armata 32 12m+20mi - Malaysia Ota et al. (2002) Bronchocela cristatella 34 14m+20mi - Singapore Ota et al. (2002) 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) Calotes cristatellus 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) C. emma 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 12m+22mi - Malaysia Ota et al. (2002) 34 - - India Singh and Banerjee (2004) C. jerdoni 34 12m/sm+22mi - India Sharma and Nakhasi (1980) 34 - - India Singh and Banerjee (2004) C. mystaceus 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 - - India Singh and Banerjee (2004) 34 10m+2m+22mi 2qter Thailand Patawang et al. (2015a) C. versicolor 34 12m/sm+22mi - India Sharma and Nakhasi (1980) 34 12m/sm+22mi 2qter Asia Solleder and Schmid (1988) 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) 34 12m+22mi Singapore Ota et al. (2002) 32, 34 - - India Singh and Banerjee (2004) 34 12m/sm+22mi 2qter Thailand Patawang et al. (2015a) Draco cornutus 34 16m+18mi - Malaysia Ota and Hikida (1989) D. haematopogon 34 16m+18mi - Malaysia Ota and Hikida (1989) D. quinquefasciatus 34 16m+18mi - Malaysia Ota and Hikida (1989) D. belliana 34 12m/sm+22mi - Thailand Kritpetcharat et al. (1999) D. taeniopterus 34 10m+2sm+22mi 17 Thailand This study Diploderma splendidum (as Japarula splendida) 34 12m+22mi - China Zongyun et al. (2004) Di. swinhonis (as Japarula swinhonis swinhonis) 36 40 46 10bi+26a 6bi+34a 46a - - - Central Taiwan Central Taiwan Northern Taiwan Ota (1988) Gonocephalus chamaeleontinus 42 22m+20mi - Malaysia Diong et al. (2000) G. liogaster 42 22m+20mi - Malaysia Diong et al. (2000) G. bellii 42 22m+20mi - Malaysia Diong et al. (2000) G. grandis 42 30m/sm+12t - Boeneo Ota et al. (1992) 42 22m+20mi - Malaysia Diong et al. (2000) G. myotympanum 42 30m/sm+12t - Boeneo Ota et al. (1992) G. robinsonii 32 12m+20mi - Malaysia Diong et al. (2000) Japalura variegata 34 - - India Singh and Banerjee (2004) J. varcoae 34 12m+22mi - China Li et al. (1981) Ptyctolaemus gularis 34 12m/sm+22mi - India Sharma and Nakhasi (1980) Remark: 2n=diploid number, m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric, mi=microchromosome, qter=terminal region of long arm. 24 Praween Supanuam et al. configurations of another lizard were revealed in butter- fly lizard, Leiolepis reevesii rubritaeniata (Agamidae) and long-tailed grass lizard, Takydromus sexlineatus (Lacerti- dae) (Phimphan et al. 2013; Patawang et al. 2018b). Microsatellite repeat patterns of Draco taeniopterus indicated the presence of d(GC)15 and d(CAG)10 showed specific regions, 2qter and 3qter respectively. While d(TA)15 and d(CAA)10, showed cumulative signals dis- Figure 3. Metaphase plates and standardized karyotypes of male (A.), female (B.) and Idiogram (C.) of barred gliding lizard, Draco taeniop- terus, 2n=34 by Ag-NOR banding, arrows indicate NORs (Scale bars = 10 µm). 25First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus persed throughout the chromosomes (Figure 5 and 6). The microsatellite loci were highly evolved loci. There- fore, in many species there may be different forms of repetitive sequences. Most of them were found distrib- uted throughout the genome. But in some species it was found on the telomere position. However, in some spe- cies it may be found in a specific location. This study, the short tandem repeats of d(GC)15 was found on q-arm telomeric of pair 2nd and d(CAG)10 was found on q-arm telomeric of pair 3rd. The molecular cytogenetics apply- ing microsatellite probe of family Agamidae in previ- ous study has Leiolepis reevesii rubritaeniata, 2n=36 (12bi+24mi) using (TTAGGG)n probes presented on tel- omeric and interstitial some chromosomes and Tympa- nocryptis lineata and Rankinia diemensis, 2n=32 (12bi+ 20mi) using (TTAGGG)7 presented on centromeric and telomeric region in some chromosomes (Jantarat et al. 2018; Srikulnath et al. 2009; Alam et al. 2021). We sug- gest employing GC and CAG probes in different Drago to have a deeper understanding of the relationship. The barred gliding lizard, Draco taeniopterus from Than To district, Yala province, Thailand has 2n=34, NF=46. The karyotype comprises four pairs of large metacentric chromosomes, one pairs of small meta- centric chromosomes, one pairs of large submetacen- tric chromosomes, and 11 pairs of microchromosomes. The metaphase I showed 17 bivalents and metaphase II showed haploid, n=17. 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The role of chromosomal rearrangements, polyploidy, and genome size variation in the diversity and ecological distribution of Asparagus L. species: a landscape cytogenetics meta-analysis approach Paris Fouroutan1, Masoud Sheidai2,*, Fahimeh Koohdar3 First chromosome characterization and repetitive DNA of Barred Gliding Lizard, Draco taeniopterus Günther, 1861 (Draconinae: Agamidae: Squamata) Praween Supanuama, Sitthisak Jantaratb,*, Warakarn Khawporntipb, Thaintip Kraipromc, Somsak Buatipb, Sarun Jumrusthanasand, Sarawut Kaewsrid, Nattasuda Donbundite, Sukhonthip Ditcharoene, Weera Thongnetrf, Sumalee Phimphang, Alongklod Tanomtonge Cytotoxic assessment of aqueous extracts of Heliotropium keralense Sivar. & Manilal on Allium cepa root tip cells Athira. R. Mohan, Nisha Joseph* Chromosomal variations and genetic diversity in subpopulations of Senna alexandrina Mill. from Western Thar, India Sunita Arora, Monika Vyas, Meena Barupal* Application of Genomic In Situ Hybridization (GISH) and tandem repeat sequence amplification for identification of Erianthus – Saccharum introgression Valiya Purakkal Sobhakumari*, Krishnasamy Mohanraj Mapping of five classes of repetitive DNAs and microsatellite repeats in the genome of the Rainbow Shark, Epalzeorhynchos frenatum (Fowler, 1934) Kamika Sribenja1, Nuntaporn Getlekha2,* Natural hybridization between Iris minutoaurea Makino and Iris odaesanensis Y. N. Lee in Korea: evidence from cytological traits Bokyung Choi, Tae-Soo Jang*