Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand

. 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 karyotype 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: 2 n (38) = L m4 +L a4 +L t2 + M a4 +M t2 +S sm2 +S a2 +S t18 .


INTRODUCTION
The sandstone gecko (Gekko petricolus) belongs to family Gekkonidae, 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).

Sample collection
We obtained five male and five female specimens of G. petricolus (Fig. 1) that were collected from Ubon Ratchathani and Mukdahan provinces, Northeastern Thailand.

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 centrifugation again at 3,000 rpm for 8 min.In fresh cool fixative, 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-drying technique.

Chromosome staining
With 20% of Giemsa solution, the slides were conventionally 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% gelatine 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 chromosome classification of Turpin and Lejeune (1965).Chromosomes were categorized as submetacentric (sm), metacentric (m), telocentric (t), and acrocentric (a).The Fundamental Number (NF: number of chromosome arms) was gained by assigning a value of two to metacentric, 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   hybridization (FISH) was performed under highly rigorous conditions on mitotic chromosome spreads (Pinkel 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 hybridization 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 containing 2×SSC.The post hybridization wash was fulfilled with 1×SSC for 5 min at 65 °c.A final wash was operated 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, Burlingame, CA, USA), and analyzed in fluorescence microscope Nikon ECLIPSE.

Nucleolar organizer region
The first cytogenetic study of G. petricolus carried 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 submetacentric 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 chromosome regions.Accordingly, the specific dark band (NORpositive) 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 biarmed or small mono-armed chromosome.An example 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 chromosome 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 euchromatin and heterochromatin.They are highly polymorphic regarding copy number deviation (Ellegren 2004).Fluorescence 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).Hybridization signals of (GAA) 10 repeats were studied at all chromosomes (Figs.2G-H), while the results clearly showed that the microsatellite repeats are in high copy number 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 cytogenetic maps of G. hokouensis, enabled us to describe the processes 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. petricolus.Karyotyping from metaphase spreads of G. petricolus showed a chromosome number of 2n = 38, NF=54.The karyotype formula is 2n (38) = L m 4 +L a 4 +L t 2 +M a 4 +M t 2 +S sm 2 +S a 2 +S t 18 .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 gekkonid.

ACKNOWLEDGMENTS
This research project was financially supported by Mahasarakham University, Phetchabun Rajabhat University, Khon Kaen University and Phetchabun Rajabhat University.