Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(2): 81-88, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1544 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Weera Thongnetr, Surachest Aiumsumang, Alongklod Tanomtong, Sumalee Phimphan (2022) Classical chro- mosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand. Caryolo- gia 75(2): 81-88. doi: 10.36253/caryolo- gia-1544 Received: January 23, 2022 Accepted: July 07, 2022 Published: September 21, 2022 Copyright: © 2022 Weera Thongnetr, Surachest Aiumsumang, Alongklod Tanomtong, Sumalee Phimphan. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand Weera Thongnetr1, Surachest Aiumsumang2, Alongklod Tanomtong3, Sumalee Phimphan2,* 1 Walai Rukhavej Botanical Research institute, Mahasarakham University, Kantharawi- chai, Maha Sarakham 44150, Thailand 2 Biology program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 3 Program of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand *Corresponding author. E-mail: sumalee.phi@pcru.ac.th Abstract. The objectives of this study were to examine size, shape, diploid number (2n), fundamental number (NF), NORs region, and distribution of microsatellite by using Fluorescence in situ hybridization technique (FISH) and to establish the karyo- type and standard idiogram of sandstone geckos (Gekko petricolus Taylor, 1962). Sand- stone gecko distributed in the sandstone mountains in Laos, Cambodia, and Thailand. Five male and five female specimens were collected from Ubon Ratchathani and Muk- dahan provinces, Thailand. The metaphase cells were directly prepared from the bone marrow cells. Chromosomes were stained by conventional staining, NORs-banding and FISH techniques. The results found that the diploid number was 38 chromosomes. The fundamental number was 54. The karyotype composed of 4 large metacentric, 4 large acrocentric, 2 large telocentric, 4 medium acrocentric, 2 medium telocentric, 2 small submetacentric, 2 small acrocentric and 18 small telocentric chromosomes. No morphological difference was identified between sex chromosomes of male and female specimens. The NORs appeared to telomere of the long arm of chromosome pair 17. The study displayed that the distribution of microsatellite using (CA)15 and (GAA)10 probes distributed throughout the genome. However, (CA)15 sequences concentrated in the telomere. The karyotype formula G. petricolus is as follow: 2n (38) = Lm4+La4+Lt2+ Ma4+Mt2+Ssm2+Sa2+St18. Keywords: Karyotype, Gekko petricolus, Ag-NOR, FISH microsatellite. INTRODUCTION The sandstone gecko (Gekko petricolus) belongs to family Gekkoni- dae, a genus mainly found in subtropical limestone areas, near the Tropic of Cancer, such as southern China, India, Myanmar, Thailand, Vietnam, Malaysia, Indonesia, and other countries in Southeast Asia (Li et al. 1996). 82 Weera Thongnetr et al. The genus Gekko comprises over 30 species and a few undescribed species distributed in East and Southeast Asia, and western Oceania (Kluge 2001; Toda 2008). The Gekko petricolus group currently contains 10 spe- cies, namely G. boehmei, G. badenii, G. canaensis, G. flavimaritus, G. grossmanni, G. lauhachindai, G. petrico- lus, G. russelltraini, G. takouensis, and G. vietnamensis. (Ngo et al. 2009; Panitvong et al. 2010; Ngo and Gamble 2011; Rösler et al. 2011; Luu et al. 2015; Rujirawan et al. 2019). Karyological analyses in Gekko have differenti- ated species based on mitotic metaphase chromosomal morphology while sporadic reports have based the spe- cies differentiation on meiotic metaphase chromosomal morphology. The chromosome study of Gekkonid that have been reported such as; Hemidactylus: diploid num- ber (2n) ranging from 40-56 and mostly 40 or 46 (De Smet 1981; Patawang and Tanomtong 2015b), Gehyra: mostly 44 (King, 1984), Ptychozoon: 2n = 34 and 42 (Ota and Hikida, 1988), Paroedura: diploid number ranging from 31-38 and mostly 36 (Aprea et al. 2013; Koubova et al. 2014), Phelsuma: 2n = 36 (Aprea et al., 1996), Dixonius: 2n = 42 (Ota et al., 2001), and genus Gekko, there are several reports on cytogenetic studies of G. gecko, namely, G. gecko: 2n=38 (Singh 1974; Wu and Zhao 1984; Solleder and Schmid, 1984; Trifonov et al. 2011; Qin et al. 2012; Patawang et al. 2014), G. hok- ouensis: 2n=38 (Chen et al. 1986; Shibaike et al. 2009; Kawai et al. 2009), G. japonicus: 2n=38 (Yoshida and Itoh, 1974; Shibaike et al., 2009; Trifonov et al., 2011), G. shibatai and G. vertebralis: 2n=38 (Shibaike et al. 2009), G. Vittatus and G. ulikovskii: 2n=38 (Trifonov et al. 2011), G. tawaensis: 2n=38 (Ota, 1989a; Shibaike et al. 2009), G. taylori: 2n=42 (Ota and Nabhitabhata 1991), G. monarchus: 2n=44 (Ota et al. 1990), G. yakue- nsis, G. petricolus and G. smithii: 2n=38-42 ( Ota, 1989), G. kikuchii: 2n=44 ( Ota, 1989a), G. chinensis: 2n=40 (Lau et al. 1997), G. subpalmatus: 2n=38 (Wu and Zhao 1984), G. swinhonis: 2n=38 (Chen et al. 1986) (Table 1). Most gekkonid chromosome complements consist of acrocentric or telocentric chromosomes which gradually decreases in size whereas the karyotype evolution with- in the group is accompanied by fusions, Robertsonian fissions and pericentric inversions (Gorman 1973). From the previous reports, there are no studies of G. petri- colus chromosome or karyotypic analyses. The present study of the cytogenetics of G. gecko provides the first report on the conventional staining, Ag-NOR banding and fluorescence in situ hybridization techniques. Data provided here can gain us the knowledge of cytogenetic information which can be used as a basis to comprehen- sively examine the taxonomy and evolutionary relation- ship of Gekko species. MATHERIAL AND METHODS Sample collection We obtained five male and five female specimens of G. petricolus (Fig. 1) that were collected from Ubon Rat- chathani and Mukdahan provinces, Northeastern Thai- land. Chromosome preparation Chromosomes were directly prepared in vivo (Ota 1989a; Qin et al. 2012) using the following methods. The gecko intramuscular was inoculated Colchicine solution then left for 12 h. After that cut testis samples and bone marrow into small pieces. Then squashed and mixed with 0.075 M KCl. After discarding all large piece tissues, 15 mL of cell sediments were transferred to a centrifuge tube and incubated for 25–35 min. The KCl was discarded from the supernatant after centrifu- gation again at 3,000 rpm for 8 min. In fresh cool fixa- tive, cells were fixed (3 methanol : 1 glacial acetic acid), gradually added to make a volume of 8 mL, before being centrifuged again at 3,000 rpm for 8 min. Afterward the supernatant was expelled. Fixation was repeated until the supernatant was clear whereas the pellet was mixed with 1 mL fixative. The mixture was dropped onto a clean and cold slide by micropipette followed by air-dry- ing technique. Chromosome staining With 20% of Giemsa solution, the slides were con- ventionally stained for 30 minutes (Patawang et al. 2014). After that, to remove excess stain, the slides were rinsed thoroughly with running tap water and were placed in air-dry at room temperature. Ag-NOR banding was analysed according to the method of Howell and Black (1980). Two drops each of 50% silver nitrate and 2% gel- atine solutions were added to slides, respectively. Then, they were sealed with cover glasses and incubated at 60°C for 5-10 minutes. They were also soaked in distilled water until the cover glasses were separated. Finally, the slides were placed in air-dry at room temperature. They were observed under microscope. Chromosome checks Metaphase figures were analysed following the chro- mosome classification of Turpin and Lejeune (1965). Chromosomes were categorized as submetacentric (sm), 83Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand metacentric (m), telocentric (t), and acrocentric (a). The Fundamental Number (NF: number of chromosome arms) was gained by assigning a value of two to meta- centric, submetacentric and acrocentric chromosomes and one to acrocentric chromosomes. Fluorescence in situ hybridization technique The use of microsatellite investigations which were described by Kubat et al. (2008) was followed here with slight modifications. These sequences were directly labeled with Cy3 at the 5´-terminal during synthesis by Sigma (St. Louis, MO, USA). Fluorescence in situ Table 1 The karyotype reviews among the genes Gekko (Gekkonidae, Squamata). Species 2n NF Karyotype formulas NORs FISH Locations References Gekko gekko 38 50 6m+4sm+2a+26t P4 - Thailand Patawang (2014) 38 44 6bi-arms+32uni-arms - - - Singh (1974) 38 - - - - - Wu and Zhao (1984) 38 46 8bi-arms+30uni-arms - - - Solleder and Schmid (1984) 38 - - - - - Trifonov et al. (2011) 38 50 8 m+2sm+2st(a)+26t Laos Qin et al. (2012) 38 48 8 m+2sm+28t - - China Qin et al. (2012) G. hokouensis 38 56 4 m+6sm+20t+8bi-arms* P19 - China, Chen et al. (1986) 38 58/59 2 m+8sm+10a+16t+Z(t)+W(a) - - Japan Kawai et al. (2009) 38 58 4 m+8sm+18t+8bi-arms* P19 - Taiwan Shibaike et al. (2009) 38 58/59 4 m+8sm+16t+8bi-arms*+Z(t)W(sm) P19 - Japan Shibaike et al. (2009) 38 42 2m+18sm+16a+ZW P19 FISH mapping Thailand Srikulnath (2015) G. japonicus 38 - - - - - Yoshida and Itoh (1974) 38 58 4m+8sm+8st+18t P17 - Japan Shibaike et al. (2010) 38 - - - - - Trifonov et al. (2011) G. shibatai 38 58 4m+8sm+18t+8bi-armed P19 - Japan Shibaike et al. (2009) G. vertebralis 38 62 4m+14sm+14t+6bi-arme* P19 - Japan Shibaike et al. (2009) G. vittatus 38 - - - Trifonov et al. (2011) G. ulikovskii 38 - - - Trifonov et al. (2011) G. tawaensis 38 58 4m+8sm+18t+8bi-armed P19 - Japan Shibaike et al. (2009) 38 56 - P19 - Japan Ota (1989a) G. taylori 42 - - - - Thailand Ota and Nabhitabhata (1991) G. monarchus 44 46 - - - Malaysia Ota et al. (1990) G. yakuensis 38 56 - P19 - Japan Ota (1989a) G. petricolus 38 54 - - - Thailand Ota (1989a) G. kikuchii 44 50 - - - Taiwan Ota (1989a) G. chinensis 40 46 6bi-armed+34uni-armed - - China Lau et al. (1997) G. smithii 38 48 - - - - Ota (1989a) G. subpalmatus 38 58 - - - - Wu and Zhao (1984) G. swinhonis 38 66 - - - Chen et al. (1986) G. petricolus 38 54 4m+2sm+10a+22t P17 (CA)15, (GAA)10 Thailand Present study Remarks: 2n = diploid chromosome number, NORs = nucleolar organizer regions, NF = fundamental number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t = telocentric chromosome, *=small bi- arms chromosome, and - = not available. Figure 1. General characteristic of G. petricolus (scale bar = 5 cm). 84 Weera Thongnetr et al. hybridization (FISH) was performed under highly rig- orous conditions on mitotic chromosome spreads (Pin- kel et al. 1986). After denaturation of chromosomal DNA in 70% formamide/ 2×SSC at 70 °C, spreads were incubated in 2×SSC for 4 min at 70 °C. The hybridiza- tion mixture (2.5 ng/μL probes, 2 μg/μL salmon sperm DNA, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridization was performed overnight at 37 °C in a moist chamber con- taining 2×SSC. The post hybridization wash was fulfilled with 1×SSC for 5 min at 65 °c. A final wash was oper- ated at room temperature in 4×SSCT for 5 min. Finally, the chromosomes were counterstained with DAPI (1.2 μg/mL), mounted in antifading solution (Vector, Burl- ingame, CA, USA), and analyzed in fluorescence micro- scope Nikon ECLIPSE. RESULTS AND DISCUSSION Mitotic chromosome features from Giemsa staining Karyomorphology of the G. petricolus revealed that the number of diploid chromosome (2n) was 38 and the fundamental number was 54. The karyotype composed of 4 large metacentric, 4 large acrocentric, 2 large telo- centric, 4 medium acrocentric, 2 medium telocentric, 2 small submetacentric, 2 small acrocentric and 18 small telocentric chromosomes. (Table 2 and Figs. 1A-B). The karyotype formula of G. petricolus 2n (38) = Lm4+La4+Lt 2+Ma4+Mt2+Ssm2+Sa2+St18. The diploid chromosome num- ber is following previous studies of Gekkos. However, overall karyotypes of G. petricolus was similar to other Gekko, diploid number ranging from 38-42 and mostly 38 (Singh 1974; Wu and Zhao 1984; Solleder and Schmid 1984; Yoshida and Itoh 1974; Ota 1989a; Ota et al. 1990; Ota and Nabhitabhata 1991; Lau et al. 1997; Chen et al. 1986; Kawai et al. 2009; Shibaike et al. 2009; Trifonov et al. 2011; Qin et al. 2012; Patawang et al. 2014). Proximity of chromosome number and karyotype feature within genus Gecko represent a close evolutionary line in the group. This species exhibit no sex differences in karyo- types between males and females (Figs. 2A-B). No cyto- logically distinguishable sex chromosome was observed to be similar to G. shibatai, G. tawaensis, G. yakuensis, G. vertebralis, G. japonicas, G. vittatus, G. ulikovskii, G. chinensis, G. kikuchii, G. monarchus, G. petricola, G. smithii, G. subpalmatus, G. swinhonis and G. Tay- lori (Shibaike et al. 2009) and other lizards in Thailand (Satrawaha and Ponkanid, 1988, Wongwattana et al., 2001). This study is different from the study of Kawai Table 2. Karyomorphological details of the G. petricolus, 2n = 38. Chro. Pair Ls (µm) Ll (µm) LT (µm) RL±SD CI±SD Sizes Types 1 2.350 3.284 5.563 0.116±0.006 0.589±0.017 Large metacentric 2 2.237 2.906 5.143 0.107±0.005 0.556±0.024 Large metacentric 3 0.967 3.130 4.047 0.085±0.003 0.767±0.033 Large acrocentric 4 0.833 2.986 3.790 0.079±0.003 0.781±0.029 Large acrocentric 5 0.000 3.294 3.229 0.067±0.004 1.000±0.000 Large telocentric 6 0.000 3.085 3.050 0.063±0.002 1.000±0.000 Medium telocentric 7 0.656 2.354 2.953 0.062±0.002 0.792±0.025 Medium acrocentric 8 0.536 2.269 2.787 0.058±0.003 0.813±0.034 Medium acrocentric 9 0.000 2.442 2.420 0.050±0.002 1.000±0.000 Small telocentric 10 0.000 2.272 2.236 0.047±0.002 1.000±0.000 Small telocentric 11 0.000 1.972 1.941 0.041±0.003 1.000±0.000 Small telocentric 12 0.000 1.817 1.774 0.037±0.002 1.000±0.000 Small telocentric 13 0.000 1.696 1.653 0.034±0.002 1.000±0.000 Small telocentric 14 0.000 1.544 1.515 0.031±0.002 1.000±0.000 Small telocentric 15 0.000 1.455 1.415 0.029±0.002 1.000±0.000 Small telocentric 16 0.374 1.049 1.388 0.029±0.002 0.748±0.039 Small acrocentric 17* 0.466 0.831 1.266 0.027±0.002 0.656±0.059 Small submetacentric 18 0.000 0.971 0.969 0.020±0.002 1.000±0.000 Small telocentric 19 0.000 0.838 0.857 0.018±0.002 1.000±0.000 Small telocentric Remarks: Ls=Short arm, Ll=Long arm, LT=Total chromosome length, CI=Centromeric Index, RL=Relative length, *=NORs bearing chro- mosomes (satellite chromosome) 85Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand Figure 2. Metaphase chromosome plate and karyotypes of G. petricolus (A, B) by conventional technique, (C, D) by Ag-NOR banding tech- nique (A, B) by FISH technique of microsatellite probe (CA)15, (G, H) by microsatellite probe (GAA)10. Scale Bars = 10 µm. 86 Weera Thongnetr et al. et al. (2009) and Shibaike et al. (2009) which revealed a ZW system (sex-chromosomes) of G. hokouensis in Japan. Geckos represent an interesting group regard- ing the evolution of sex determination mechanisms and include species possessing either environmental or genetic sex determination systems. Gecko populations without males were first discovered followed by seven parthenogenetic species (Smith 1935; Ota 1989b; Volo- bouev et al. 1993), including triploid (3n) forms in some populations (Moritz 1984). A ZW system was recently revealed in G. hokouensis, and the genetic content of its sex chromosomes was similar to that of the avian Z chromosome (Kawai et al. 2009; Shibaike et al. 2009). Nucleolar organizer region The first cytogenetic study of G. petricolus car- ried out by Ag-NOR banding technique was obtained from this research. We found the observable NORs on the region adjacent to telomere of long arm of the sub- metacentric chromosome pair 17th (Figs 2C-D). The objective of the Ag-NOR banding technique is to detect NORs which represent the location of genes that have a function in ribosome synthesis (18S and 28S ribosomal RNA). NORs produce numerous gene expressions and comprise non-histone proteins more than other chromo- some regions. Accordingly, the specific dark band (NOR- positive) is induced by the reduction of organic silver by these proteins that change silver to be dark (Sharma et al., 2002). This study according to G. japonicas had two NORs on the long arm near telomere of small bi-arms chromosome pair 17 (Shibaike et al. 2009). The NOR regions compared with other geckos, most showed two NORs appearing near telomeric region of small bi- armed or small mono-armed chromosome. An exam- ple of the previous reports of the genus Gekko had two NORs on one pair of small bi-arms chromosomes. G. gecko had two NORs on the near telomere of mono-arms chromosome pair 4 (Patawang 2014), while there were G. shibatai, G. Yakuensis, G. hokouensis, G. tawaensis, G. vertebralis and G. yakuensis which had two NORs on the long arm near telomere of small bi-arms chromo- some pair 19 (Ota 1989b; Chen et al. 1986; Shibaike et al. 2009). The use of NORs in explaining kinships depends a large extent on the uniformity of this characteristic and the degree of variety within a taxon (Yüksel and Gaffaroğlu 2008). Microsatellite pattern Microsatellites or simple sequence repeats (SSRs) are oligonucleotides of 1–6 base pairs in length, forming excessive tandem repeats of usually 4 to 40 units (Tautz and Renz 1984; Ellegren 2004; Chistiakov et al. 2006). They show ample distribution throughout eukaryotic genomes, being scattered or clustered both in euchroma- tin and heterochromatin. They are highly polymorphic regarding copy number deviation (Ellegren 2004). Fluo- rescence hybridization indicate the (CA)15 repeat showing abundance at the telomeric ends of most chromosomes (Figs. 2E-F), verifying the findings from other gekko groups investigated to date (Srikulnath 2015). Hybridiza- tion signals of (GAA)10 repeats were studied at all chro- mosomes (Figs. 2G-H), while the results clearly showed that the microsatellite repeats are in high copy num- ber on some chromosome pairs, according to previous reports on reptile groups (Pokorná et al. 2011; Matsubara et al., 2013). In this study, a comparison of the cytogenet- ic maps of G. hokouensis, enabled us to describe the pro- cesses of chromosomal reorganization in Gekkota. These cytogenetic data could also be a substantial prerequisite the reptiles’ genome projects in the future. CONCLUSIONS This study, The first cytogenetic study of G. petrico- lus. Karyotyping from metaphase spreads of G. petrico- lus showed a chromosome number of 2n = 38, NF=54. The karyotype formula is 2n (38) = Lm4+La4+Lt2+Ma4+Mt 2+Ssm2+Sa2+St18. Data obtained in this study can increase the knowledge of cytogenetic which can be used as a basis to comprehensively examine the taxonomy and evolutionary relationship of gekko species and other gek- konid. ACKNOWLEDGMENTS This research project was financially supported by Mahasarakham University, Phetchabun Rajabhat Uni- versity, Khon Kaen University and Phetchabun Rajabhat University. Figure 3. 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