The Standard Karyotype of the Asiatic Jackal, Canis Aureus (Carnivora, Canidae) from Thailand
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© 2015 The Japan Mendel Society Cytologia 80(1): 3–13 The Standard Karyotype of the Asiatic Jackal, Canis aureus (Carnivora, Canidae) from Thailand Alongklod Tanomtong1*, Prapakorn Chaiyaphan1, Praween Supanuam2, Therdsak Puramongkol3, Nuntiya Maneechot4 and Nantana Jangsuwan5 1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Biology Program, Faculty of Science, Ubonratchatani Rajabhat University, Ubon Ratchatani, Muang 34000, Thailand 3 Faculty of Agro-Industrial Technology, Rajamangala University of Technology Tawan-ok, Chanthaburi Campus, Chanthaburi, Khao Kitchagoot 22210, Thailand 4 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin, Muang 32000, Thailand 5 Applied Biology Program, Faculty of Science and Technology, Thepsatri Rajabhat University, Lopburi, Muang 15000, Thailand Received November 28, 2013; accepted September 18, 2014 Summary This study conducted karyological analysis and detection of nucleolar organizer regions (NORs) in the Asiatic jackal (Canis aureus Linnaeus, 1758) from Khao Suan Kwang Zoo, Khon Kaen Province, Thailand. Blood samples were taken from two male and two female jackals. After standard whole blood lymphocytes had been cultured at 37°C for 72 h in the presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Giemsa’s staining, GTG- banding, high-resolution banding and Ag-NOR banding techniques were used to stain chromosomes. The results showed that the diploid chromosome number of C. aureus was 2n=78 and the fundamental numbers (NF) of both sexes were 80. The types of autosomes were 18 large telocentric, 18 medium telocentric and 40 small telocentric chromosomes. The X chromosome was the largest metacentric chromosome and the Y chromosome was the smallest acrocentric chromosome. In addition, the long arm near the centromere of chromosome pair 7 and the long arm near the telomere of chromosome pairs 10 and 28 contained NORs. From the GTG-banding and high-resolution banding techniques, the numbers of bands were 205 and 269, respectively, and each chromosome pair could be clearly differentiated. The karyotype formula for C. aureus is: 2n (diploid) t t t 78=L18+M18+S40+sex chromosomes. Key words Asiatic jackal, Canis aureus, Karyotype, Chromosome. The family Canidae is composed of 16 genera and 36 species of wolves, jackals, foxes and dogs (Nowak 1999). In Thailand, there are three species; domestic dog (Canis familiaris), Asiatic jackal (Ca. aureus) and Asian wild dog (Cuon alpinus). In Thailand, Ca. aureus may be confused with Cu. alpinus, but they differ morphologically in being generally greyish brown, not reddish, and by having the shoulder hairs tipped black, forming a saddle-like pattern. Also, the muzzle is not blackish as it is in Cu. alpinus, and the tail is short and blackish in only its distal third (Lekagul and McNeely 1988). Comparative analysis of chromosomes has also proved very useful because canids have a rich diversity of chromosome morphology ranging from species such as the red fox (Vulpes vulpes, * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.3 4 A. Tanomtong et al. Cytologia 80(1) Fig. 1. General characteristics of the Asiatic jackal (Canis aureus Linnaeus, 1758) from Thailand. 2n=34) and cape fox (V. corsac, 2n=36), which have a low diploid chromosome number and all metacentric autosomes, to the Ca. familiaris, Coyote (Ca. latrans), gray wolf (Ca. lupus) and Cu. alpinus, which have a high diploid chromosome number (2n=78) and all acrocentric autosomes. The primitive canid karyotype has been reshuffled in different lineages in a way that reveals the phylogenetic history of the group. The evolutionary sequence of chromosomal rearrangements is deduced by differentially staining chromosomes and matching segments of similar banding patterns in different species (Wayne et al. 1987). The karyotype of C. aureus was first reported by Matthey (1954) and shows a diploid chromosome number (2n) of 74. In the present study, conventional staining, GTG-banding, high- resolution banding and Ag-NOR banding techniques were used for a more detailed chromosome characterization of C. aureus, and aspects of chromosome evolution of the family Canidae were discussed. Materials and methods The blood samples were collected by aseptic technique from two male and two female jackals (Fig. 1), kept in Khao Suan Kwang Zoo, Khon Kaen Province, Thailand. The samples were placed in 10 ml vacuum tubes containing heparin to prevent blood clotting and cooled on ice until arriving at the laboratory. Lymphocytes were cultured using the whole blood microculture technique adapted from Rooney (2001). Cell culture Five millilitres of RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and kept in blood culture flasks. Blood samples of 0.5 ml aliquots were dropped into a medium bottle and mixed well. The culture bottles were loosely capped, incubated at 37°C under a 5% carbon dioxide environment and regularly shaken in the morning and evening. After harvesting at the 72 h of incubation, colchicine was added and mixed well, followed by further incubation for 30 min. Cell harvest The blood sample mixture was centrifuged at 3,000 rpm for 5 min and the supernatant was discarded. Ten millilitres of hypotonic solution (0.075 M KCl) was applied to the pellet and the mixture incubated for 30 min. KCl was discarded from the supernatant after centrifugation again at 3,000 rpm for 5 min. Cells were fixed in a fresh cool fixative (3 methanol : 1 glacial acetic acid) 2015 The Standard Karyotype of the Asiatic Jackal Canis aureus from Thailand 5 gradually added up to 8 ml before centrifuging again at 3,000 rpm for 5 min, and the supernatant was discarded. The fixation was repeated until the supernatant was clear, then the pellet was mixed with 1 ml fixative. The mixture was dropped onto a clean and cold slide by a micropipette followed by the air-dry technique. The slide was conventionally stained with 20% Giemsa’s solution for 30 min. G-banding method The G-banding technique was adapted from Campiranont (2003). The slide was well dried and then soaked in working trypsin (0.025% trypsin EDTA) at 37°C before the termination of trypsin activity by washing the slide with Sorensen’s buffer. The slide was stained with 20% Giemsa’s solution for 30 min. High-resolution banding method The high-resolution banding technique was adapted from Rooney (2001). After the lymphocytes were cultured for 72 h, 0.05 ml of 10-5 M methotrexate was applied into the cultured lymphocytes to induce synchronization. The mixture was incubated again for 17 h before the methotrexate was discarded with the supernatant by centrifugation at 2,800 rpm. The pellet was mixed with 5 ml of the RPMI 1640 medium and centrifuged at 2,800 rpm. The supernatant was discarded before the cultured cells were released by adding 0.2 ml thymidine and incubating for 5 h and 15 min. Ag-NOR banding method Two drops each of 50% silver nitrate and 50% gelatin were added on slides, respectively which were sealed with cover glasses and incubated at 60°C for 3 h. They were then soaked in distilled water until cover glass separation. The slide was stained with 20% Giemsa’s solution for 1 min (Howell and Black 1980). Chromosomal checks, karyotyping and idiograming Chromosome counting was performed on mitotic metaphase cells under a light microscope. Twenty clearly observable and well-spread chromosomes of each male and female were selected and photographed. The length of short arm chromosome (Ls) and the length of long arm chromosome (Ll) were measured and calculated to the length of total arm chromosome (LT, LT=Ls+Ll). The relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL and CI were estimated. CI was also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming. Results and discussion The Ca. aureus showed a diploid chromosome number of 78 chromosomes, comprising 38 pairs of autosomal telocentric chromosomes (18 large, 18 medium and 40 small chromosomes) and two sex chromosomes, the largest metacentric X-chromosome and the smallest acrocentric Y-chromosome (Fig. 2). The fundamental number (NF, number of chromosome arm) were 80 in both sexes. This is different from a previous study by Matthey (1954), who reported that Ca. aureus was 2n=74. Species described from the family Canidae have 2n between 34 and 78. Most chromosomes are mono-armed in species with higher diploid chromosome numbers, while in species with lower 2n, they are bi-armed (Hatanaka and Galetti 1999). In the family Canidae a wide range of diploid chromosome numbers is observed (Wayne et al. 1987). Moreover, an extensive chromosome and karyotype polymorphism in some species has been described (Table 1) (Rogalska-Niznik et al. 2003). Ca. aureus, Ca. familiaris, Ca. latrans, Ca. 6 A. Tanomtong et al. Cytologia 80(1) Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Asiatic jackals (Canis aureus), 2n=78 by conventional staining technique. Scale bar indicates 10 μm. Table 1. Review of canids (foxes, wolves, jackal and dogs) cytogenetic publications in the family Canidae (Carnivora). Species 2n Karyotype formulas NF Sex chro. References Canis aureus 78 76t 80 X (m) Y (a) Present study 74 ̶ 76 ̶ Matthey (1954) Ca. familiaris 78 76a 80 X (m) Y (m) Selden et al. (1975) 78 ̶ ̶ ̶ Yang et al. (1999) 78 ̶ ̶ ̶ Graphodatsky et al. (2000) Ca. latrans 78 ̶ ̶ ̶ Wurster-Hill and Centerwall (1982) Ca. lupus 78 ̶ ̶ ̶ Wurster-Hill and Centerwall (1982) Cerdocyon thous 74 ̶ ̶ ̶ Nash et al. (2001) Cuon alpinus 78 ̶ ̶ ̶ Graphodatsky et al.