Cytogenetic characterisation and chromosomal mapping of microsatellite and telomeric repeats in two gecko species (Reptilia, Gekkonidae) from Thailand

Abstract Studies of chromosomes of Cyrtodactylus jarujini Ulber, 1993 and C. doisuthep Kunya et al., 2014 to compare microsatellite and TTAGGG sequences by classical and molecular techniques were conducted in Thailand. Karyological typing from a conventional staining technique of C. jarujini and C. doisuthep showed diploid chromosome numbers of 40 and 34 while the Fundamental Numbers (NF) were 56 in both species. In addition, we created the chromosome formula of the chromosomes of C. jarujini showing that 2n (40) = Lsm1 + Lsm2 + Lt3 + Mm1 + Mt4 + Sm2 + Sa2 + St5 while that of C. doisuthep was 2n (34) = Lsm3 + Lm2 + Lt3 + Mm1 + Mt2 + Sm4 + Sa1 + St1. Ag-NOR staining revealed NOR-bearing chromosomes in chromosome pairs 13 and 14 in C. jarujini, and in chromosome pairs 9 and 13 in C. doisuthep. This molecular study used the FISH technique, as well as microsatellite probes including (A)20, (TA)15, (CGG)10, (CGG)10, (GAA)10, (TA)15 and TTAGGG repeats. The signals showed that the different patterns in each chromosome of the Gekkonids depended on probe types. TTAGGG repeats showed high distribution on centromere and telomere regions, while (A)20, (TA)15, (CGG)10, (CGG)10, (GAA)10 and (TA)15 bearing dispersed over the whole genomes including chromosomes and some had strong signals on only a pair of homologous chromosomes. These results suggest that the genetic linkages have been highly differentiated between the two species.


Abstract
Studies of chromosomes of Cyrtodactylus jarujini Ulber, 1993 andC. doisuthep Kunya et al., 2014 to compare microsatellite and TTAGGG sequences by classical and molecular techniques were conducted in Thailand. Karyological typing from a conventional staining technique of C. jarujini and C. doisuthep showed diploid chromosome numbers of 40 and 34 while the Fundamental Numbers (NF) were 56 in both species. In addition, we created the chromosome formula of the chromosomes of C. jarujini showing that 2n (40) = L sm 1 + L sm 2 + L t 3 + M m 1 + M t 4 + S m 2 + S a 2 + S t 5 while that of C. doisuthep was 2n (34) = L sm 3 + L m 2 + L t 3 + M m 1 + M t 2 + S m 4 + S a 1 + S t 1 . Ag-NOR staining revealed NOR-bearing chromosomes in chromosome pairs 13 and 14 in C. jarujini, and in chromosome pairs 9 and 13 in C. doisuthep. This molecular study used the FISH technique, as well as microsatellite probes including (A) 20 , (TA) 15 , (CGG) 10 , (CGG) 10 , (GAA) 10 , (TA) 15 and TTAGGG repeats. The signals showed that the different patterns in each chromosome of the Gekkonids depended on probe types. TTAGGG repeats showed high distribution on centromere and telomere regions, while (A) 20 , (TA) 15 , (CGG) 10 , (CGG) 10 , (GAA) 10 and (TA) 15 bearing dispersed over the whole genomes including chromosomes and some had strong signals on only a pair of homologous chromosomes. These results suggest that the genetic linkages have been highly differentiated between the two species.

Introduction
Bent-toed geckos (genus Cyrtodactylus Gray, 1827) in Thailand have been classified into approximately 24 species (Chuaynkern and Chuaynkern 2012). Cyrtodactylus jarujini ranges from Nong kai, Bueng Kan and Nakhon Phanom Provinces, Thailand. More recently, Sumontha et al. (2008), found it in two caves on two sandstone hills, Phu Sing and Phu Thok, where it remained by day on the walls and crevices and emerged from the caves at night. Both in Phu Sing and Phu Thok, syntropy was found with the cave-dwelling agamid Mantheyus phuwuanensis (Manthey and Nabhitabhata 1991). It has also been recorded from central and northern Laos (Stuart 1999), but the exact identity of the Lao populations has to be re-evaluated (Fig. 1A). In contrast C. doisuthep is known only from Doisuthep in the Doi Suthep-Pui Range, Mueang District, Chiang Mai Province, northern Thailand (Fig. 1B).
Only 13% of gekkonid species have been karyotyped (Olmo and Signorino 2005) and were studied with conventional cytogenetic methods, including routine staining, as well as R-, NOR-and C-banding (Moritz 1983;Olmo and Signorino 2005;Shibaike et al. 2009). However, a small number of species were studied by molecular cytogenetic techniques (Kawai et al. 2009). The diploid number amongst gekkonid lizards ranges from 2n = 28 to 46 with most of the karyotypes composed of 28-46 chromosomes (Gorman 1973;Olmo 1986;Schmid et al. 1994). There are five karyotyped Cyrtodactylus species: C. consobrinus 2n = 48, NF = 50, C. pubisulcus 2n = 42, NF = 44 (Ota et al. 1992); C. interdigitalis 2n = 42, NF = 52 and C. kunyai 2n = 40, NF = 52 (Thongnetr et al. 2019a); C. saiyok 2n = 42, NF = 42 (Thongnetr et al. 2019b). The typical karyotype consists of a gradual series of telocentric chromosomes (sometimes with a few metacentric) and there is no distinction between macro-and microchromosomes, the centromere often being subterminal (Gorman 1973). Karyotype evolution within the group is accompanied by fissions and fusions and pericentric inversions (Gorman 1973;Olmo and Signorino 2005). This information on chromosomes is considered important along with other information for identification of the species (Campiranont 2003), especially the identification of related species, because of similarity of shape, appearance and other phenotypic expressions that are presumed to be associated with the genotype. Information from sequences of DNA allows us to understand the creation of a phylogenetic tree (dendrogram), because these characteristics often have a particular pattern. Information on chromosomes can be used to identify the phylogenetic relationship between species and population of animals (Lauhajinda and Taksintum 2006). Therefore, it is necessary to study the karyology of this group. In addition, geckos could be affected by the actions of humans in their use of household objects and agricultural chemicals. Thus, the gecko is one of the important groups of animals that can serve as a model for studying the environmental impact from human actions in the future.

Material and methods
The samples of C. jarujini and C. doisuthep were collected from the Phu Wua, Ban Phaeng District, Nakhonphanom Province and Doi Suthep-Pui Range, Mueang District, Chiang Mai Province, Thailand, (permission from an ethical committee ID U1-04498-2559). Chromosomes were directly prepared in vivo (Ota et al. 1990) by 0.1% colchicine were injected into the animals' intramuscular and abdominal cavity and left for 8-10 hours. Bone marrow, liver and testis (male) were cut into small pieces and then mixed with 0.075 M potassium chloride (KCl). After discarding all large cell pieces, 15 ml of cell suspension was transferred to a centrifuge tube and incubated 30-40 minutes, then centrifuged at 3,000 rpm for 8 minutes. Cells were fixed in fresh cool fixative of methanol:glacial acetic acid (3:1) and gradually made up to 8 ml before centrifuging again at 3,000 rpm for 8 minutes, whereupon the supernatant was discarded. Fixation was repeated until the supernatant was clear and the pellet was mixed with 1 ml fixative. Using conventional Giemsa staining, a drop of the mixture was added to a clean and cold slide by micropipette followed by the air-dry technique. The slide was conventionally stained with 20% Giemsa solution for 30 minutes . Then, the slides were rinsed thoroughly with running tap water to remove excess stain. Afterwards, the slides were allowed to airdry at room temperature. Ag-NOR banding was analysed following 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. Afterwards, they were then soaked in distilled water until the cover glasses were separated. Finally, the slides were allowed to air-dry at room temperature and observed under microscope. Metaphase figures were analysed according to the chromosome classification of Chaiyasut (1989) and Turpin and Lejeune (1965). Chromosomes were classified as metacentric (m), submetacentric (sm), acrocentric (a) and telocentric (t). The Fundamental Number (NF: number of chromosome arms) is obtained by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to acrocentric chromosomes. The use of microsatellite probes described by Kubat et al. (2008) was followed here with slight modifications. These sequences were directly labelled with Cy3 at the 5´-terminal during synthesis by Sigma (St. Louis, MO, USA). Fluorescence In Situ Hybridization (FISH) was performed under highly stringent 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 carried out with 1×SSC for 5 min at 65 °C. A final wash was performed 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.

Karyological data of the genus Cyrtodactylus
The species in the Cyrtodactylus exhibited a variable chromosome number, ranging from 34 to 42, however, the most frequent numbers were 40 and 42. The present study showed that the chromosome numbers of C. jarujini and C. doisuthep were 40 and 34, respectively. The fundamental number was 56 in both species. These results showed difference and accordance with others Cyrtodactylus that have been reported (Table 1). The karyological characteristics of C. jarujini and C. doisuthep obtained in the present study are the first report of chromosome sizes and the chromosome types in these species. In different species of Cyrtodactylus, different karyological characteristics can be found. However, overall, of these karyotypes of C. jarujini and C. doisuthep resemble those of other Cyrtodactylus species and other gekkonids, which comprised many mono-armed (telocentric) and few bi-armed chromosomes (meta-or submetacentric). For those gekkonid chromosomes which have been reported previously, most species showed that the karyotype comprises of many mono-armed chromosomes and few bi-armed chromosomes. The present results of C. jarujini and C. doisuthep agreed with the chromosomal evolution line hypothesis within the gekkonid group (Trifonov et al. 2011). The karyotype of C. jarujini and C. doisuthep showed the gradient of most telocentrics, while comprising of a few bi-armed chromosomes. These features conform to the hypothesis of re-arrangement from ancestral karyotype by Robertsonian fissions, fusions or pericentric inversions (Gorman 1973;King 1987).

Active NOR sites
Nucleolus organiser regions (NORs) are chromosome sites which contain the 18S and 28S ribosomal RNA genes. If these regions were active during the interphase prior to mitosis, they can be detected by silver nitrate staining (Howell and Black 1980). In the present study, the chromosome markers of both Cyrtodactylus are determined by using the Ag-NOR banding technique as shown in Table 1. C. jarujini had the acrocentric chromosome pair 13 and metacentric chromosome pair 14, which were the NORbearing chromosome. Pair 13 NORs were located on the short arm near the telomere (telomeric NOR) and the pair 14 NORs were located on the short arm near the centromere (centromeric NOR). C. doisuthep had the metacentric, two chromosome pair 9 and pair 13 which were the NOR-bearing chromosomes. Pair 9 NORs were located on the arm near the telomere (telomeric NOR) on both sides and the pair 13 NORs were located on the arm near the telomere (telomeric NOR).

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 abundant distribution throughout eukaryotic genomes, being dispersed or clustered both in euchromatin or heterochromatin. They are highly polymorphic regarding copy number variations (Ellegren 2004). In our present study both species exhibited the same general hybridisation pattern for some applied probes with the motif TAAGGG repeat showing abundance at the telomeric ends of all chromosomes (Fig. 3), corroborating findings from other gekko groups studied to date (Srikulnath 2015). Otherwise, the dinucleotides (A) 20 , (CAG) 10 , (CGG) 10 , (GAA) 10 and (TA) 15 accumulated exclusively in telomeric and subtelomeric chromosomal regions. However, the results clearly indicate 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).

Conclusions
In this study, the comparison of the cytogenetic maps of two Cyrtodactylus species (C. jarujini and C. doisuthep) enabled us to delineate the process of chromosomal reorganisation in this group. This is the first report in Thailand for the study of cytogenetics of both species. Therefore, the cytogenetic data obtained can be used to benefit cytotaxonomy and the study of evolution of geckos, as well as being an essential prerequisite for future genome projects of gecko groups.