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Cytogenetics Study and Characterization of Sumatra Serow, Capricornis Sumatraensis (Artiodactyla, Bovidae) by Classical and FISH Techniques

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Cytogenetics Study and Characterization of Sumatra Serow, Capricornis Sumatraensis (Artiodactyla, Bovidae) by Classical and FISH Techniques © 2017 The Japan Mendel Society Cytologia 82(2): 127–135 Cytogenetics Study and Characterization of Sumatra Serow, Capricornis sumatraensis (Artiodactyla, Bovidae) by Classical and FISH Techniques Sitthisak Jantarat1, Alongklod Tanomtong2*, Isara Patawang3, Somkid Chaiphech4, Sukjai Rattanayuvakorn5 and Krit Phintong6 1 Biology Program, Department of Science, Faculty of Science and Technology, Prince of Songkla University (Pattani), Pattanee, Muang 94000, Thailand 2 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 3 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Muang 50200, Thailand 4 Department of Animal Science, Rajamangala University of Technology Srivijaya Nakhonsrithammarat Campus, Nakhonsrithammarat, Thungyai 80240, Thailand 5 Department of Science and Mathematics, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin, Muang 32000, Thailand 6 Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin, Muang 32000, Thailand Received April 19, 2016; accepted December 10, 2016 Summary Karyological analysis in the Sumatra serow (Capricornis sumatraensis) from Thailand were conduct- ed. Blood samples were taken from two male and two female serows. 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. Conventional, GTG-, high-resolution, Ag-NOR banding and fluorescence in situ hybridiza- tion (FISH) were applied to stain the chromosomes. The results showed that the diploid chromosome number of C. sumatraensis was 2n=48 and the fundamental number (NF) for both sexes were 60. The types of autosomes were 2 large metacentric, 4 large submetacentric, 2 large acrocentric, 2 medium telocentric, 4 small submetacen- tric and 32 small telocentric chromosomes. The X chromosome was a medium telocentric chromosome and the Y chromosome was a smallest telocentric chromosome. From the GTG-banding and high-resolution techniques, the numbers of bands in the C. sumatraensis were 147 and 207, respectively, and each chromosome pair could be clearly differentiated. In addition, the long arm near telomere (subtelomeric region) of chromosome pairs 2, 3 and 4 showed clearly observable heteromorphic nucleolar organization regions (NORs) (2a2b, 3a3b, 4a4b). This is the first report on natural polymorphism of NORs. The microsatellites d(AC)15 accumulated at the telomeres of several pairs and interstitial sites of some chromosomes. The microsatellites d(CGG)10 highly accumulated at the cor- respondence sites of NORs. FISH with the telomeric probe revealed hybridization signals on each telomere of all chromosomes and interstitial telomeric sites were not detected. The karyotype formula could be deduced as: m sm a t sm t 2n (diploid) 48=L2422432 +L +L +M +S +S +sex-chromosomes Key words Karyotype, Chromosome, Capricornis sumatraensis, Polymorphism. The Sumatra serow (Capricornis sumatraensis) is rather short bodied, long-legged goat antelopes, and a classified under the order Artiodactyla, family Bo- short, thick neck. The coat is coarse and rather thin, the vidae, subfamilies Caprinae. All six species of serow, upper parts are usually black or grayish, with the legs namely, Japanese serow (Capricornis crispus), Tai- below the knees widely variable in color from black to wan serow (C. swinhoei), C. sumatraensis, Chinese gray. The lips are white and there is often a rufous or serow (C. milneedwardsii), red serow (C. rubidus) and white patch under the throat. The ears are long, nar- Himalayan serow (C. thar), were until recently also row and pointed, dark behind and white on the inner classified under genus Naemorhedus, which now only surface. The horns are rather thick, slightly curved and contains the gorals. They live in central or eastern Asia short with a record length of 280 mm, and the basal por- (Wilson and Reeder 2005). tion has numerous thin rings. The horns of females are The general characteristics of the C. sumatraensi are: shorter (25–50 mm), slightly thinner and less corrugated than those of males. The hoofs are short and solid with * Corresponding author, e-mail: [email protected] pedal glands on all four feet. There is also a well-devel- DOI: 10.1508/cytologia.82.127 oped facial gland located about 40 mm below the eye; the 128 S. Jantarat et al. Cytologia 82(2) Fig. 1. General characteristics of Sumatra serow, Capricornis sumatraensis (Artiodactyla, Bovidae, Caprinae). Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Sumatra serow (Capricornis sumatraensis), 2n=48 by conventional straining technique (scale bars=10 µm). Table 1. Review of cytogenetic reports of the genus Capriconis (Artiodactyla, Bovidae). Species 2n NF m sm a t X Y NORs Reference C. crispus 50 60 10 0 38 0 a a ̶ Benirschke et al. (1972) C. sumatraensis 46 58 10 0 34 0 m m ̶ Fischer and Höhn (1972) 48 60 12 0 34 0 a a ̶ Soma et al. (1982) 48 ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ Huang et al. (2005) 48 60 2 8 2 34 t t 2, 3, 4 Present study C. swinhoei 50 60 10 0 38 0 a a ̶ Soma et al. (1981) Notes: 2n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, a=acrocentric, t=telocentric chromosome, X=X-chromosome, Y=Y-chromosome, NORs=nucleolar organizer regions and ̶=not available. 2017 Cytogenetics Study and Characterization of Sumatra Serow, Capricornis sumatraensis (Artiodactyla, Bovidae) by Classical and FISH Techniques 129 Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) Sumatra serow (Capricornis sumatraensis), 2n=48 by GTG-banding technique (scale bars=10 µm). opening is not closed by a flap of skin as in sheep and ies on taxonomy and evolutionary relationships of this gazelles but is filled with numerous short hairs (Lekagul genus. Moreover, it provides useful basic information for and McNeely 1988) (Fig. 1). conservation and breeding practices, as well as for stud- The standardized karyotypes of cattle, buffalo, sheep ies on the chromosome evolution of this serow. and goat, which are the main domestic animals in the family Bovidae have been investigated by many workers Materials and methods using G-, C-, R-, Ag-NOR banding and high-resolution techniques (Di Berardino et al. 1990, Hayes et al. 1993, Blood samples of the C. sumatraensis (two males Kaftanovskaya and Serov 1994, Iannuzzi et al. 1995, and two female) were collected from Phang Nga Wild- Jantarat et al. 2009, Supanuam et al. 2010, Kenthao life Breeding Center, Thailand and then applied to et al. 2012), reflecting their economic importance. Re- cytogenetic studies by lymphocyte culture of whole cently, karyotypes of wild bovid have been reported. blood samples. The culture cells were treated with a Their bi-armed chromosomes are the result of Robertso- colchicine-hypotonic-fixation-air-drying technique fol- nian translocation or centric fusion (Cribiu et al. 1990, lowed by conventional staining, GTG-, high-resolution Claro et al. 1996, Robinson et al. 1996, Oh et al. 2011, and Ag-NOR banding techniques (Rooney 2001) as well Gomontean et al. 2009, Tanomtong et al. 2011). In genus as dual-color fluorescence in situ hybridization (FISH) Capricornis, cytogenetic studies have been performed to detect the microsatellites d(AC)15, d(CGG)10 and telo- in three species, including C. crispus (Benirschke et al. mere sites. FISH was performed under high stringency 1972), C. sumatraensis (Fischer and Höhn 1972, Soma conditions on mitotic chromosome spreads (Pinkel et al. et al. 1982, Huang et al. 2005) and C. swinhoei (Soma 1986, Liehr 2009). For 20 cells of each individual chro- et al. 1981) (Table 1). Most of these species have 2n=48 mosome, checks, length measurements, karotyping and or 50 chromosomes consisting of bi-armed and mono- idiograming were accomplished by using a light micro- armed chromosomes. scope as previously described (Chaiyasut 1989). In the present article, we indicate the first finding of NOR polymorphism and chromosome analysis in the Results and discussion C. sumatraensis from Thailand. We provide the very first report on chromosome standardization, including Cytogenetic study of C. sumatraensis using lympho- chromosome measurements of shape and size, karyotype cyte culture demonstrated that the chromosome number formulation, and idiograming. The results obtained can is 2n (diploid)=48 (Fig. 2). This is the same chromosome provide more cytogenetic information for future stud- number for the C. sumatraensis as reported in previous 130 S. Jantarat et al. Cytologia 82(2) Fig. 4. Prometaphase chromosome plates and karyotypes of male (A) and female (B) Sumatra serow (Capricornis sumatraensis), 2n=48 by high-resolution GTG-banding technique (scale bars=10 µm). studies (Soma et al. 1982, Huang et al. 2005). These mosome was a medium telocentric chromosome and the features are differences to that reported by Fischer and Y chromosome was the smallest telocentric chromo- Höhn (1972) indicating that C. sumatraensis had 2n=46. some. These features are differences to that reported The fundamental number (NF, number of chromosome by Soma et al. (1982) indicating that a C. sumatraensis arms) was 60 in both male and female, which is the same had an acrocentric X-chromosome and an acrocentric Y- as the report of Soma et al. (1982). According to the chromosome. Furthermore, Fischer and Höhn (1972) also chromosome characteristics of others in family Bovidae, reported that the sex chromosome of the C. sumatraensis gaur (2n=58, NF=58), banteng (2n=60, NF=58), cattle, had a metacentric X chromosome and a metacentric Y B. taurus (2n=60, NF=58), swamp buffalo (2n=48, chromosome. An important karyotypic difference be- NF=56), river buffalo (2n=50, NF=58), goat (2n=60, tween bovine and ovine species is the different shape of NF=58) sheep (2n=54, NF=58) and goral (2n=56, the X chromosomes.
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