© 2008 The Japan Mendel Society Cytologia 73(3): 221–228, 2008 The First Cytogenetic Study of the Malayan porcupine, Hystrix brachyuran (Rodentia, Hystricidae) by Conventional Staining and G-banding Technique Alongkoad Tanomtong1,*, Praween Supanuam1, Wiwat Sangpakdee1, Pornnarong Siripiyasing2, Pawarisa Boonhan1 and Sarawut Kaewsri3 1 Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Major of Biology, Faculty of Science and Technology, Mahasarakham Rajabhat University, Muang, Mahasarakham 44000, Thailand 3 Program in Applied Biology, Department of Science, Faculty of Science, Buriram Rajabhat University, Muang, Buriram 31000, Thailand Received May 24, 2008; accepted July 12, 2008 Summary This research was the first cytogenetic study of the Malayan porcupine (Hystrix brachyura) from Songkla Zoo, Thailand. Blood samples were taken from two males and two females and then subjected to standard whole blood T-lymphocyte culture. The samples were harvested by colchicine-hypotonic-fixation-air-drying technique and followed by conventional staining and G- banding with Giemsa’s. The results showed that the diploid number was 2nϭ66, and the fundamental number (NF) was 127 and 128 in male and female, respectively. The autosomes consist of 6 large submetacentric, 20 large acrocentric, 6 medium submetacentric, 18 medium acrocentric, 2 medium telocentric, 4 small submetacentric, 6 small acrocentric and 2 small telocentric chromosomes. We found the nucleolar organizer regions (NORs), the representative chromosome marker, which are lo- cated on the long arms of the pair submetacentric autosomes 9 and 13. The X chromosome was a large metacentric chromosome, while the Y chromosome was the smallest telocentric chromosome. G-banding technique indicated that the number of bands was 236. All of these results can be use as the useful basic genetic information for H. brachyuran cytogenetic study. The karyotype formula can be shown as following: ϭ smϩ a ϩ smϩ a ϩ t ϩ smϩ aϩ t ϩ 2n(66) L6 L 20 M6 M 18 M 2 S4 S6 S 2 sex-chromosomes Key words Cytogenetics, Chromosome, Malayan porcupine (Hystrix brachyuran) The family Hystricidae (old world porcupine) is the only Thai Hystricomorpha. The Malayan porcupine belongs to the kingdom Animalia, phylum Chordata, class Mammalia, order Rodentia, family Hystricidae, subfamily Hystricinae, genus Hystrix and species Hystrix brachyuran. The gen- eral characteristics of the H. brachyuran are: the front half of the body is covered with short, dark brown spines (about 30–50 mm. long), while long, pointed quills (many of which are 200–300 mm. long) protrude from the hindquarters. The long, posterior quills are whitish, usually with a distinct blackish rings which are located about a half to two-thirds of the distance from the base. There is a whitish crest (about 150 mm. long) on the neck and upper back and there is a distinct whitish cres- cent on the neck and shoulders. The tail is short, with two types of quills, long, black and white, pointed quills and rattling quills. The rattling quills are hollow, open-ended, flattened cylinders mounted on a short, narrow stem. The tail is hidden by long quills because the hind part of the body gradually tapers toward the tail. The whiskers (vibrissae) are very long and black and the ears are small (Fig. 1) (Lekagul and McNeely 1977). * Corresponding author, e-mail: [email protected] 222 A. Tanomtong et al. Cytologia 73(3) The previous cytogenetic studies of por- cupine in family Hystricidae have been report- ed (Benirschke 1968, Arslan 2006). In this study, T-lymphocyte culture, conventional staining and G-banding technique were used in the comparison study with the previous stud- ies. In the future, basic knowledge and cytoge- netics of family Hystricidae could be applied to several researches and especially for their extinction protection. Fig. 1. General characteristic of the Malayan porcupine, Hystrix brachyura Vaunhan, 1972 (Rodentia, Hys- tricidae). Table 1. Mean of length short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes of 20 cells in male Malayan porcupine (Hystrix brachyura Vaunhan, 1972) 2n (diploid)ϭ66. Chromosome Chromosome Chromosome Ls Ll LT RLϮSD CIϮSD pairs size type 1 0.21 0.90 1.11 0.046Ϯ0.002 0.810Ϯ0.023 Large Acrocentric 2 0.20 0.77 0.97 0.040Ϯ0.002 0.791Ϯ0.021 Large Acrocentric 3 0.19 0.75 0.94 0.038Ϯ0.002 0.799Ϯ0.023 Large Acrocentric 4 0.18 0.71 0.89 0.037Ϯ0.002 0.796Ϯ0.039 Large Acrocentric 5 0.18 0.69 0.87 0.036Ϯ0.001 0.799Ϯ0.023 Large Acrocentric 6 0.20 0.66 0.86 0.035Ϯ0.001 0.772Ϯ0.043 Large Acrocentric 7 0.18 0.64 0.82 0.034Ϯ0.001 0.776Ϯ0.038 Large Acrocentric 8 0.32 0.51 0.83 0.034Ϯ0.001 0.614Ϯ0.021 Large Submetacentric 9 0.30 0.51 0.81 0.033Ϯ0.001 0.628Ϯ0.024 Large Submetacentric 10 0.19 0.61 0.80 0.033Ϯ0.001 0.766Ϯ0.042 Large Acrocentric 11 0.18 0.59 0.77 0.032Ϯ0.001 0.760Ϯ0.050 Large Acrocentric 12 0.18 0.58 0.76 0.031Ϯ0.001 0.765Ϯ0.039 Large Acrocentric 13 0.28 0.47 0.75 0.031Ϯ0.001 0.625Ϯ0.016 Large Submetacentric 14 0.17 0.57 0.74 0.030Ϯ0.001 0.764Ϯ0.050 Medium Acrocentric 15 0.17 0.56 0.73 0.030Ϯ0.001 0.764Ϯ0.041 Medium Acrocentric 16 0.27 0.44 0.71 0.029Ϯ0.001 0.619Ϯ0.018 Medium Submetacentric 17 0.26 0.44 0.70 0.029Ϯ0.001 0.632Ϯ0.025 Medium Submetacentric 18 0.18 0.51 0.69 0.028Ϯ0.001 0.739Ϯ0.033 Medium Acrocentric 19 0.18 0.50 0.68 0.028Ϯ0.001 0.735Ϯ0.022 Medium Acrocentric 20 0.18 0.49 0.67 0.028Ϯ0.001 0.728Ϯ0.025 Medium Acrocentric 21 0.00 0.65 0.65 0.027Ϯ0.001 1.000Ϯ0.000 Medium Telocentric 22 0.16 0.48 0.64 0.026Ϯ0.001 0.755Ϯ0.042 Medium Acrocentric 23 0.16 0.47 0.63 0.026Ϯ0.001 0.748Ϯ0.039 Medium Acrocentric 24 0.15 0.45 0.60 0.025Ϯ0.001 0.746Ϯ0.033 Medium Acrocentric 25 0.15 0.44 0.59 0.024Ϯ0.001 0.751Ϯ0.029 Medium Acrocentric 26 0.20 0.35 0.55 0.023Ϯ0.001 0.632Ϯ0.030 Medium Submetacentric 27 0.15 0.39 0.54 0.022Ϯ0.002 0.723Ϯ0.027 Small Acrocentric 28 0.00 0.51 0.51 0.021Ϯ0.001 1.000Ϯ0.000 Small Telocentric 29 0.14 0.35 0.49 0.020Ϯ0.001 0.711Ϯ0.009 Small Acrocentric 30 0.14 0.33 0.47 0.019Ϯ0.001 0.712Ϯ0.009 Small Acrocentric 31 0.15 0.27 0.42 0.017Ϯ0.001 0.643Ϯ0.034 Small Submetacentric 32 0.14 0.24 0.38 0.016Ϯ0.001 0.629Ϯ0.028 Small Submetacentric X 0.66 0.74 1.40 0.059Ϯ0.006 0.556Ϯ0.024 Large Metacentric Y 0.00 0.31 0.31 0.014Ϯ0.002 1.000Ϯ0.000 Small Telocentric 2008 The First Cytogenetic Study of the Malayan porcupine 223 Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) Malayan porcupine (Hystrix brachyura Vaunhan, 1972) 2nϭ66 by conventional staining, arrows indicate the labeled NORs (nucleolar organizer regions) and sex-chromosomes. Materials and methods Blood samples from the jugular vein were collected from two males and two females H. brachyura, which were kept in Songkla Zoo, Thailand using aseptic technique. The samples were kept in 10 ml vacuum tubes containing heparin to prevent blood clotting and they were cooled on ice until arriving at the laboratory. Cell preparation The lymphocytes were cultured using the whole blood microculture technique adapted from 224 A. Tanomtong et al. Cytologia 73(3) Fig. 3. Metaphase chromosome plates and karyotypes of male (A) and female (B) Malayan porcupine (Hystrix brachyura Vaunhan, 1972) 2nϭ66 by G-banding technique, arrows indicate the labeled NORs (nucleolar organizer regions) and sex-chromosomes. Rooney (2001) and Kampiranont (2003). Cell culture The RPMI 1640 medium was prepared with 2% PHA (Phytohemagglutinin) as a mitogen and kept in blood culture bottles of 5 ml each. A blood sample of 0.5 ml was dropped into a medium bottle and well mixed. The culture bottle was loosely capped, incubated at 37°C under 5% of car- bondioxide environment and regularly shaken in the morning and evening. When reaching harvest time at the 72nd hour of incubation, colchicine was introduced and well mixed followed by further incubation for 30 min. Cell harvest The blood sample mixture was centrifuged at 1200 rpm for 10 min and the supernatant was discarded. 10 ml of hypotonic solution (0.075 M KCl) was applied to the pellet and the mixture was incubated for 30 min. KCl was discarded with the supernatant after centrifugation again at 2008 The First Cytogenetic Study of the Malayan porcupine 225 Fig. 4. Idiogram of the Malayan porcupine (Hystrix brachyura Vaunhan, 1972) 2nϭ66 by G-banding tech- nique, arrows indicate the labeled NORs (nucleolar organizer regions). 226 A. Tanomtong et al. Cytologia 73(3) Table 2. Mean of length short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from metaphase chromosomes of 20 cells in female Malayan porcupine (Hystrix brachyura Vaunhan, 1972) 2n (diploid)ϭ66. Chromosome Chromosome Chromosome Ls Ll LT RLϮSD CIϮSD pairs size type 1 0.27 0.96 1.23 0.045Ϯ0.002 0.779Ϯ0.019 Large Acrocentric 2 0.24 0.88 1.12 0.041Ϯ0.001 0.789Ϯ0.036 Large Acrocentric 3 0.23 0.86 1.09 0.040Ϯ0.001 0.788Ϯ0.021 Large Acrocentric 4 0.22 0.83 1.05 0.039Ϯ0.001 0.792Ϯ0.027 Large Acrocentric 5 0.21 0.82 1.03 0.037Ϯ0.002 0.800Ϯ0.019 Large Acrocentric 6 0.21 0.79 1.00 0.036Ϯ0.001 0.794Ϯ0.029 Large Acrocentric 7 0.20 0.78 0.98 0.036Ϯ0.001 0.797Ϯ0.023 Large Acrocentric 8 0.36 0.62 0.98 0.036Ϯ0.002 0.627Ϯ0.024 Large Submetacentric 9 0.36 0.58 0.94 0.034Ϯ0.001 0.618Ϯ0.018 Large Submetacentric 10 0.19 0.70 0.89 0.033Ϯ0.002 0.783Ϯ0.043 Large Acrocentric