CompCytogen 15(1): 41-52 (2021) COMPARATIVE A reerrerewet open-access oven

doi: 10.3897/compcytogen.v | 5.i1.58208 Kan Cytogenetics

https://compcytogen.pensoft.net

rational Journal of Plant & Animal Cytogenetics,
Karyosystematics, and Molecular Systematics

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

Weera Thongnetr', Surachest Aiumsumang’, Rodjarin Kongkaew’,
Alongklod Tanomtong?, Chatmongkon Suwannapoom*, Sumalee Phimphan?

| Walai Rukhavej Botanical Research institute, Mahasarakham University, Kantharawichai, Maha Sarakham,
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 4 Department of Fishery, School of Agriculture and Natural Resources, University of Phayao, Muang,
Phayao, 56000, Thailand

Corresponding author: Sumalee Phimphan (joodoof@gmail.com)

Academic editor: N. Golub | Received 1 September 2020 | Accepted 16 December 2020 | Published 2 February 2021
http://zoobank.org/FC36370E-12AB-4DED-960D-0B72CCBC8008

Citation: Thongnetr W, Aiumsumang S, Kongkaew R, Tanomtong A, Suwannapoom C, Phimphan S (2021)
Cytogenetic characterisation and chromosomal mapping of microsatellite and telomeric repeats in two gecko species

(Reptilia, Gekkonidae) from Thailand. CompCytogen 15(1): 41-52. https://doi-org/10.3897/compcytogen.v15.i1.58208

Abstract

Studies of chromosomes of Cyrtodactylus jarujini Ulber, 1993 and C. doisuthep Kunya et al., 2014 to com-
pare microsatellite and TTAGGG sequences by classical and molecular techniques were conducted in Thai-
land. 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) =
Le oe Lee EN S71, +85 Ss while that of C. doisuthep was 2n (34) = Laat DE eS
+ M‘, + S™, + S*, +S. 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
spr tl AD p(CGG) SS (CGG) SAGAA), oC TA): sand,
TTAGGG repeats. The signals showed that the different patterns in each chromosome of the Gekkonids

technique, as well as microsatellite probes including (A)

depended on probe types. TTAGGG repeats showed high distribution on centromere and telomere regions,
while (A),,, (TA),,, (CGG),,, (CGG),,,

including chromosomes and some had strong signals on only a pair of homologous chromosomes. ‘These

(GAA),, and (TA),, bearing dispersed over the whole genomes

results suggest that the genetic linkages have been highly differentiated between the two species.

Copyright Weera Thongnetr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

42 Weera Thongnetr et al. / CompCytogen 15(1): 41-52 (2021)

Keywords
Ag-NOR, Cyrtodactylus doisuthep, Cyrtodactylus jarujini, FISH microsatellite, karyotype

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 Cyrtodac-
tylus 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 (Thong-
netr 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, ap-
pearance 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 rela-
tionship 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.

A comparative FISH mapping of two gecko species 43

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 Dis-
trict, 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 cav-
ity 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 (KCI). 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 (Patawang et al. 2014). Then, the slides were rinsed thoroughly with
running tap water to remove excess stain. Afterwards, the slides were allowed to air-
dry 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), sub-
metacentric (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/ 2xSSC at 70 °C, spreads were incubated in 2xSSC for 4 min at
70 °C. The hybridization mixture (2.5 ng/uL probes, 2 ug/uL 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
2xSSC. The post hybridization wash was carried out with 1xSSC for 5 min at 65 °C.
A final wash was performed at room temperature in 4xSSCT for 5 min. Finally, the
chromosomes were counterstained with DAPI (1.2 ug/mL), mounted in antifading
solution (Vector, Burlingame, CA, USA), and analyzed in fluorescence microscope

Nikon ECLIPSE.

44 Weera Thongnetr et al. / CompCytogen 15(1): 41-52 (2021)

Results

The diploid chromosome number and fundamental number

The diploid numbers in C. jarujini and C. doisuthep, were 40 and 34, respectively
(Fig. 1C, E), whereas NF was 56 in both species (Fig. 1G, I). The type chromo-
somes of metacentric, submetacentric, acrocentric and telocentric were 8-4-4-24
and 14-6-2-12. There are no sex-related chromosomal heteromorphisms in the two
species here studied.

The karyological characteristics

The karyotype of C. jarujini consists of two large metacentric, four large submetacen-
tric, six large telocentric, two medium metacentric, eight medium telocentric, four
small metacentric, four small acrocentric and ten small telocentric chromosomes. The

° oe ee 7 _ m sm t m t
karyotype formula for C. jarujini is as follows: 2n (40) = L™, + L™, +L‘, + M™,+M‘,

3cm

Cyrtodactylus jarujini

3cm

Cyrtodactylus doisuthep

Figure |. The C. jarujini specimen (A), metaphase chromosome plate and karyotypes (A=G) by con-
ventional technique, (D-H) by Ag-NOR banding technique. The C. doisuthep specimen (B), metaphase
chromosome plate and karyotypes (E=-I) by conventional technique, (F-J) by Ag-NOR banding tech-
nique. Arrows indicated Ag-NORs regions. Scale Bar: 5 um.

A comparative FISH mapping of two gecko species 45

+ S™, + S*, +S‘, or 2n (40) = 8m + 4sm + 4a + 24t. The karyotype of C. doisuthep
comprises four large metacentric, six large submetacentric, six large telocentric, two
medium metacentric, four medium telocentric, eight small metacentric, two small ac-
rocentric and two small telocentric chromosomes. The karyotype formula for C. do-
isuthep is as follows: 2n (34) = L™, + L™, + L¥. + M™, +Mt, + S™, + St, + St, or 2n (34)
= 14m + 6sm + 2a + 12t.

Ag-NOR banding

This technique highlighted active NORs on pairs 13 and 14 of C. jarujini (Fig. 1D, H)
and pairs 9 and 13 of C. doisuthep (Fig. 1E J).

Microsatellite pattern

Microsatellites (A),,, (TA),,, (CAG),,, (CGG),,, (GAA),, and (TA), abundantly dis-
tributed in some chromosomes, usually in telomeric regions of both species studied.
FISH with the telomeric probe TTAGGG revealed hybridization signals on each tel-

omere of all chromosomes (Fig. 2).

Discussion

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 resem-
ble 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 telo-
centrics, 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).

46 Weera Thongnetr et al. / CompCytogen 15(1): 41-52 (2021)

(Ano A (Ayo G
de 6)
6 7

54

(CAG)io H

6 ¢?
6 7

(CGG)io C (CGG)i0 I
86 ea ad ee
6 7 : 5 6 7

(GAA)io J
ae ae
6 7

| ee

13

(TTAGGG), L
a4 “ae

Figure 2. Karyotypes of two geckos presenting the patterns of microsatellite (A)
(GAA)

(GAG)
(TA),, and TTAGGG; C. jarujini (A-F), C. doisuthep (G=L). Scale Bars: 10 ym.

(CGG)

10? 10?

10?

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

A comparative FISH mapping of two gecko species 47

Table |. Karyotype reviews in the genera Cyrtodactylus, Gekko Laurenti, 1768 and Hemidactylus Gold-
fuss, 1820 (Gekkonidae, Squamata).

Species 2n NF Karyotype formula NORs Location Reference
Cyrtodactylus consobrinus 48 50 2bi-arm+46t - Malaysia Ota et al. (1992)
(Peters, 1871)

C. doisuthep Kunya et al., 2014 34 56 14m+6sm+2a+12t P9,13 Thailand Present study
C. interdigitalis Ulber, 1993 42 52 4m+2sm+4a+32t P12. ~— Thailand ~—s Thongnetr et al. (2019a)
C. jarujini Ulber, 1993 40 56 8m+4sm+4a+24t P13, 14 Thailand Present study
C. kunyai Pauwels et al., 2014 40 52 8m+4sm+6a+22t P12. ~— Thailand ~=—s Thongnetr et al. (2019a)
C. pubisulcus Inger, 1958 42 44 2bi-arm+40t - Malaysia Ota et al. (1992)
C. saiyok Panitvong, 2014 42 42 42t P15 Thailand = Thongnetr et al. (2019b)
Gekko chinensis Gray 1842 40 46 6bi-armed+34uni-armed = China Lau et al. (1997)
G. gecko (Linnaeus, 1758) 38 50 12bi-armed+26uni-armed < = Cohen et al. (1967)
38 50 Lm2+Lsm4+Lt4+Mt6+Sm4+Sa2+St16 P4 Thailand Patawang et al. (2014)
G. hokouensis Pope, 1928 38 56 4m+6sm+20t+8bi-armed P(L)19 ~=-China Chen et al. (1986)
G. monarchus (Schlegel, 1836) 38 46 = = Malaysia Ota et al. (1990)
G. petricolus Taylor, 1962 38 54 = es Z Ota (1989)
G. shibatai Toda et al., 2008 38 58 4m+8sm+18t+8bi-armed P(L)19 =‘ Japan Shibaike et al. (2009)
G. tawaensis Okada, 1956 38 58 4m+8sm+18t+8bi-armed P(L)19 ~—- Japan Shibaike et al. (2009)
G. taylori 42 — - - Thailand Ota and Nabhitabhata
Grossmann et Ulber, 1990 (1991)
G. vertebralis Toda et al., 2008 38 62 4m+14sm+14t+6bi-armed P(L)19 ~—- Japan Shibaike et al. (2009)
Hemidactylus brookii 40 44 4bi-armed+36t = Bhatnagar (1962)
Gray, 1854
H. flaviviridis Riippell, 1835 40 60 20bi-armed+20t - - Asana and Mahabale (1941)
46 46 46t = = Makino and Momma (1949)
40 52 12bi-armed+28t = = Branch (1980)
H. frenatus Schlegel, 1836 46 46 46t 7 ~ Makino and Momma (1949)
40 54 14bi-armed+26t P3 - King (1978)
40 46 6bi-armed+34t 7 — Darevsky et al. (1984)
H.. mabouia 42 56 14bi-armed+28t - — Becak et al. (1972)
(Moreau de Jonnés, 1818) 42 54 12bi-armed+30t = — McBee et al. (1987)

Remarks: 2n = diploid chromosome number, NORs = nucleolus organiser regions, SCR = subcentromeric regions, NF = fundamental
number (number of chromosome arms), bi-arm = bi-armed chromosome, m = metacentric, sm = submetacentric, a = acrocentric, t =
telocentric chromosome, L = large, S = small, P = chromosome pair and — = not available.

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 NOR-
bearing 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 cen-
tromere (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).

The NORs in both species of genus Cyrtodactylus exhibited at the telomeric region
on the long arm and short arm and are similar to the previous reports of the gekkonids
for the Gekkonidae family by King (1978) and Moritz and King (1985). The NORs of
Dixonius siamensis (Boulenger, 1898), G. gecko, G. hokouensis, G. shibatai, G. tawaensis,
G. vertebralis, H. frenatus and H. platyurus were found at all regions on the short arm

48 Weera Thongnetr et al. / CompCytogen 15(1): 41-52 (2021)

* JULUUONBCO08 aga wan

1 22-33 13° 19-20

: (Cioasenossone

14 15

MA. [](CAG)i0 J(CGG)o [JGAA). (cts); J mssccso),

Figure 3. Idiograms represent the (A),,, (CAG),,, (CGG),,, (GAA),,; (TA),, and TTAGGG mapping on
the chromosomes of C. jarujini (A) and C. doisuthep (B).

and that agrees with those previous reported (Asana and Mahabale 1941; Makino and
Momma 1949; Bhatnagar 1962; Cohen et al. 1967; Becak et al. 1972; King 1978;
Branch 1980; Darevsky et al. 1984; Chen et al. 1986; McBee et al. 1987; Ota 1989;
Ota et al. 1990; Ota and Nabhitabhata 1991; Lau et al. 1997; Ota et al. 2001; Shibaike
et al. 2009; Patawang et al. 2014; Trifonov et al. 2011; Trifonov et al. 2015).

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 hybridi-
sation pattern for some applied probes with the motif TAAGGG repeat showing abun-
dance at the telomeric ends of all chromosomes (Fig. 3), corroborating findings from
other gekko groups studied to date (Srikulnath 2015). Otherwise, the dinucleotides
(A),.> (CAG),,, (CGG),,, (GAA),, and (TA),, 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 (Pokorna et al. 2011; Matsubara et al. 2013).

A comparative FISH mapping of two gecko species 49

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 re-
organisation in this group. This is the first report in Thailand for the study of cytoge-
netics 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 pre-
requisite for future genome projects of gecko groups.

Acknowledgements

This research project was financially supported by Mahasarakham University, Phet-
chabun Rajabhat University and The Unit of Excellence 2020 on Biodiversity and
Natural Resources Management, University of Phayao (UoEG63005).

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