© 2017 The Japan Mendel Society Cytologia 82(1) Special Issue: 91–98

Karyotype and Idiogram of the Axis (Axia axis, Cervidae) by Conventional Staining, GTG-, High-Resolution GTG-, and Ag-NOR-Banding Techniques

Hathaipat Khongcharoensuk1, Alongklod Tanomtong1*, Isara Patawang2, Praween Supanuam3, Somnuek Sornnok4 and Krit Pinthong5

1 Toxic Substances in Livestock and Aquatic Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand 2 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 3 Program in Biology, Faculty of Science, Ubon Ratchathani Rajabhat University, Ubon Ratchathani 34000, Thailand 4 Agricultural Technology Division, Department Technology and Industries, Faculty of Science and Technology, Prince of Songkla University (Pattani Campus), Muang, Pattani 94000, Thailand 5 Program in Biology, Department of Fundamental Science, Faculty of Science and Technology, Surindra Rajabhat University, Surin 32000, Thailand

Received December 22, 2014; accepted April 8, 2015

Summary The standardized karyotype and idiogram of the axis deer (Axis axis, Cervidae) at Khon Kaen Zoo, Thailand were established. Blood samples were taken from two male and two female axis deer. After standard whole blood T-lymphocytes were cultured at 37°C for 72 h in the presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, GTG-, Ag-NOR-banding and high-resolu- tion techniques were applied to stain the chromosome. The results showed that the diploid chromosome number of A. axis was 2n=66 and the fundamental number (NF) was 70 in both male and female. The types of autosomes observed were 2 large metacentric, 2 large submetacentric, 2 large telocentric, 6 medium telocentric and 52 small telocentric chromosomes. The X chromosome was a large telocentric chromosome, and the Y chromosome was a small telocentric chromosome. The GTG-banding and high-resolution techniques provided that the respective numbers of bands and locations of A. axis were 246 and 294, and each chromosome pair could be clearly dif- ferentiated. In addition, the subtelomeric q-arm of chromosome pair 2 and the telomeric q-arm of chromosome pair 4 showed clearly observable nucleolar organizer regions (NORs) and secondary constrictions. Our results are the first reports of GTG-, high-resolution GTG- and Ag-NOR-banding techniques on this species. The karyotype formula of A. axis is as follows: m sm t t t 2n (66) = L2 + L 2 + L 2 + M 6 + S 52 + Sex-chromosomes

Key words Axis deer, Axis axis, Karyotype, GTG-banding, Ag-NOR-banding.

The family Cervidae (infraorder Pecora, suborder Ru- which make them an ideal group for chromosomal evo- minantia, order Artiodactyla) has at least 90 species, 20 lution studies. It is proposed that the ancestral cervid genera, four tribes (, Cervini, Capreolini and karyotype is 2n=70 based on comparative karyotype Rangiferini) and two subfamilies ( and Capreo- analysis of many deer species (Neitzel 1987, Tanomtong linae) (Janis and Scott 1987, Nowak 1999, Groves 2006, et al. 2005, 2010, Huang et al. 2005). Wilson and Reeder 2006). The classification of deer in The axis deer (Axis axis), also known as deer or the family Cervidae use external morphology, biogeog- spotted deer, is a deer which commonly inhabits wooded raphy, physiology, cytogenetics and especially decidu- regions of South Asia including India, Sri Lanka, Ne- ous cranial appendages. According to the presence or pal, Bangladesh, Bhutan and Pakistan. The axis deer absence of antlers, all deer species were classified into skin is pinkish fawn, marked with white spots, and its antlered and antlerless deer. The extant deer species underparts are also white (Fig. 1). Its antlers, which it have diverse karyotypes; their diploid chromosome sheds annually, are usually three-pronged and curve in numbers range from 2n=6 in the female Indian a lyre shape and may extend to 75 cm. Compared to the (Muntiacus muntjak vaginalis) (Wurster and Benirschke hog deer ( porcinus), its close relative, the 1970) to 2n=80 in the capreolus pygargus, axis deer has a more cursorial build. It also has a more advanced morphology with antler pedicles being pro- * Corresponding author, e-mail: [email protected] portionally short and its auditory bullae being smaller. DOI: 10.1508/cytologia.82.91 It also has large nares. Their lifespans are around eight 92 H. Khongcharoensuk et al. Cytologia 82(1) Special Issue

Fig. 1. General characteristics of male (A) and female (B) axis deer (Axis axis).

Fig. 2. Metaphase chromosome plate and karyotype of male (A) and female (B) axis deer (Axis axis) 2n=66 by conventional staining technique; scales indicate 10 µm. to 14 years. Within the axis deer were recognized comparison finding with previous reports. Moreover, the two subspecies: common axis deer (A. a. axis) and Sri finding obtained here is the first report on standardized Lankan axis deer (A. a. ceylonensis). These animals are karyotype and idiogram measurements by GTG-, high- widespread in many countries. This species is listed on resolution GTG- and Ag-NOR-banding techniques. This CITES appendix III and is an IUCN least concern spe- cytogenetic information will represent basic knowledge cies (Grzimek 2004, Groves 2006). and can be applicable to genetic diversity, , To date, there are few cytogenetic reports of the Axis conservation and evolution. axis, such as those of Hsu and Benirschke (1974), Asher et al. (1999), Bonnet-Garnier et al. (2003) and Shanthi Materials and methods et al. (2008), which report karyotypes of the convention- al, RBG-, QFH-banding and chromosome painting of Blood samples of the Axis axis (two males and two this . Our present study shows a confirmation and females) were collected from Khon Kaen Zoo, Thailand 2017 Karyotype and Idiogram of the Axis Deer (Axia axis, Cervidae) by Conventional Staining 93

Table 1. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 10 metaphases of male axis deer (Axis axis) in Thailand, 2n=66.

Chromosome pair Ls Ll LT RL±SD CI±SD Size Types 1 2.519 5.749 8.269 0.064±0.0059 0.438±0.017 Large Submetacentric 2* 0.000 7.556 8.156 0.058±0.0089 0.000±0.000 Large Telocentric 3 3.303 4.707 8.010 0.062±0.0076 0.702±0.007 Large Metacentric 4* 0.000 4.779 4.779 0.037±0.0047 0.000±0.000 Medium Telocentric 5 0.000 4.773 4.773 0.037±0.0033 0.000±0.000 Medium Telocentric 6 0.000 4.655 4.655 0.036±0.0027 0.000±0.000 Medium Telocentric 7 0.000 4.110 4.110 0.032±0.0028 0.000±0.000 Small Telocentric 8 0.000 3.904 3.904 0.030±0.0026 0.000±0.000 Small Telocentric 9 0.000 3.884 3.884 0.030±0.0019 0.000±0.000 Small Telocentric 10 0.000 3.804 3.804 0.029±0.0021 0.000±0.000 Small Telocentric 11 0.000 3.748 3.748 0.029±0.0019 0.000±0.000 Small Telocentric 12 0.000 3.664 3.664 0.028±0.0021 0.000±0.000 Small Telocentric 13 0.000 3.562 3.562 0.028±0.0018 0.000±0.000 Small Telocentric 14 0.000 3.524 3.524 0.027±0.0012 0.000±0.000 Small Telocentric 15 0.000 3.440 3.440 0.027±0.0012 0.000±0.000 Small Telocentric 16 0.000 3.355 3.355 0.026±0.0013 0.000±0.000 Small Telocentric 17 0.000 3.278 3.278 0.025±0.0013 0.000±0.000 Small Telocentric 18 0.000 3.204 3.204 0.025±0.0013 0.000±0.000 Small Telocentric 19 0.000 3.157 3.157 0.024±0.0012 0.000±0.000 Small Telocentric 20 0.000 3.087 3.087 0.024±0.0012 0.000±0.000 Small Telocentric 21 0.000 3.024 3.024 0.023±0.0012 0.000±0.000 Small Telocentric 22 0.000 2.988 2.988 0.023±0.0013 0.000±0.000 Small Telocentric 23 0.000 2.941 2.941 0.023±0.0012 0.000±0.000 Small Telocentric 24 0.000 2.891 2.891 0.022±0.0013 0.000±0.000 Small Telocentric 25 0.000 2.866 2.866 0.022±0.0012 0.000±0.000 Small Telocentric 26 0.000 2.810 2.810 0.022±0.0012 0.000±0.000 Small Telocentric 27 0.000 2.804 2.804 0.022±0.0014 0.000±0.000 Small Telocentric 28 0.000 2.751 2.751 0.021±0.0014 0.000±0.000 Small Telocentric 29 0.000 2.624 2.624 0.020±0.0011 0.000±0.000 Small Telocentric 30 0.000 2.545 2.545 0.020±0.0011 0.000±0.000 Small Telocentric 31 0.000 2.441 2.441 0.019±0.0013 0.000±0.000 Small Telocentric 32 0.000 2.285 2.285 0.018±0.0014 0.000±0.000 Small Telocentric X 0.000 6.393 6.393 0.049±0.0043 0.000±0.000 Small Telocentric Y 0.000 2.345 2.345 0.018±0.0017 0.000±0.000 Small Telocentric Remark: *=Nucleolar organizer region/NOR.

Table 2. Mean length of short arm chromosome (Ls), long arm chromosome (Ll), total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 10 metaphases of female axis deer (Axis axis) in Thailand, 2n=66.

Chromosome pair Ls Ll LT RL±SD CI±SD Size Types 1 3.011 5.917 8.928 0.069±0.0043 0.663±0.015 Large Submetacentric 2* 0.000 8.226 8.226 0.064±0.0044 1.000±0.000 Large Telocentric 3 3.267 4.297 7.564 0.059±0.0058 0.568±0.013 Large Metacentric 4* 0.000 5.040 5.040 0.039±0.0035 1.000±0.000 Medium Telocentric 5 0.000 4.843 4.843 0.038±0.0036 1.000±0.000 Medium Telocentric 6 0.000 4.567 4.567 0.035±0.0026 1.000±0.000 Medium Telocentric 7 0.000 4.395 4.395 0.034±0.0016 1.000±0.000 Small Telocentric 8 0.000 4.278 4.278 0.033±0.0016 1.000±0.000 Small Telocentric 9 0.000 3.951 3.951 0.031±0.0010 1.000±0.000 Small Telocentric 10 0.000 3.902 3.902 0.030±0.0009 1.000±0.000 Small Telocentric 11 0.000 3.842 3.842 0.030±0.0009 1.000±0.000 Small Telocentric 12 0.000 3.742 3.742 0.029±0.0010 1.000±0.000 Small Telocentric 13 0.000 3.629 3.629 0.028±0.0008 1.000±0.000 Small Telocentric 14 0.000 3.539 3.539 0.027±0.0008 1.000±0.000 Small Telocentric 15 0.000 3.449 3.449 0.027±0.0008 1.000±0.000 Small Telocentric 16 0.000 3.359 3.359 0.026±0.0006 1.000±0.000 Small Telocentric 17 0.000 3.262 3.262 0.025±0.0007 1.000±0.000 Small Telocentric 18 0.000 3.255 3.255 0.025±0.0008 1.000±0.000 Small Telocentric 19 0.000 3.201 3.201 0.025±0.0008 1.000±0.000 Small Telocentric 20 0.000 3.086 3.086 0.024±0.0008 1.000±0.000 Small Telocentric 21 0.000 3.047 3.047 0.024±0.0008 1.000±0.000 Small Telocentric 22 0.000 2.963 2.963 0.023±0.0010 1.000±0.000 Small Telocentric 23 0.000 2.934 2.934 0.023±0.0010 1.000±0.000 Small Telocentric 24 0.000 2.906 2.906 0.023±0.0008 1.000±0.000 Small Telocentric 25 0.000 2.853 2.853 0.022±0.0008 1.000±0.000 Small Telocentric 26 0.000 2.785 2.785 0.022±0.0008 1.000±0.000 Small Telocentric 27 0.000 2.673 2.673 0.021±0.0008 1.000±0.000 Small Telocentric 28 0.000 2.607 2.607 0.020±0.0009 1.000±0.000 Small Telocentric 29 0.000 2.525 2.525 0.020±0.0008 1.000±0.000 Small Telocentric 30 0.000 2.470 2.470 0.019±0.0011 1.000±0.000 Small Telocentric 31 0.000 2.351 2.351 0.018±0.0011 1.000±0.000 Small Telocentric 32 0.000 2.169 2.169 0.017±0.0024 1.000±0.000 Small Telocentric X 0.000 6.430 6.430 0.050±0.0041 1.000±0.000 Small Telocentric Remark: *=Nucleolar organizer region/NOR. 94 H. Khongcharoensuk et al. Cytologia 82(1) Special Issue and then applied to cytogenetic experimentations by checks, length measurements, karyotype and idiogram lymphocyte culture of whole blood samples. The culture analysis were accomplished by using a light microscope cells were treated with a colchicine-hypotonic-fixation- as previously described (Chaiyasut 1989, Chooseang- air-drying technique followed by conventional staining, jaew et al. 2017). GTG-, high-resolution GTG- and Ag-NOR banding tech- niques (Rooney 2001, Campiranon 2003, Sangpakdee et al. 2017). For 20 cells of each individual chromosome,

Fig. 3. Standardized idiogram of axis deer (Axis axis) 2n=66 by Fig. 5. Standardized idiogram of axis deer (Axis axis) 2n=66 by conventional staining technique; the arrow indicates the Ag-NOR banding technique. The arrow indicates satellite satellite chromosome on the long arm of the second pair chromosome on the subtelomeric long arms of second pair chromosome. and telomeric NOR on the fourth pair.

Fig. 4. Metaphase chromosome plate and karyotype of male (A) and female (B) axis deer (Axis axis) 2n=66 by Ag-NOR banding technique, scales indicate 10 µm. 2017 Karyotype and Idiogram of the Axis Deer (Axia axis, Cervidae) by Conventional Staining 95

Fig. 6. Metaphase chromosome plate and karyotype of male (A) and female (B) axis deer (Axis axis) 2n=66 by GTG-banding technique; scales indicate 10 µm.

Fig. 7. Standardized idiogram of axis deer (Axis axis) 2n=66 by GTG-banding technique.

ber of chromosome arms) for both sexes is 70 (Fig. 2). Results and discussion These results are in agreement with Hsu and Benirschke The cytogenetic study of Axis axis using lymphocyte (1974), Asher et al. (1999), Bonnet-Garnier et al. (2003) culture exhibited that the chromosome number is 2n and Shanthi et al. (2008). The diploid number of Cer- (diploid)=66 and the fundamental number (NF, num- vini species (genera Axis, , , Elaphurus, 96 H. Khongcharoensuk et al. Cytologia 82(1) Special Issue

Fig. 8. Prometaphase chromosome plate and karyotype of male (A) and female (B) axis deer (Axis axis) 2n=66 by GTG-banding high-resolution technique; scales indicate 10 µm.

Fig. 9. Standardized idiogram of axis deer (Axis axis) 2n=66 by GTG-banding high-resolution technique.

Hyelaphus, Panolia, , ) ranges from 56 fusions or translocations are described as the main rear- to 68 with a constant fundamental number of 70. The rangement involved in this variation (Bonnet-Garnier decrease in chromosome number is due to an increase et al. 2003, O’Brien et al. 2006). in the number of biarmed chromosomes. Robertsonian The autosomes of axis deer consisted of 2 large meta- 2017 Karyotype and Idiogram of the Axis Deer (Axia axis, Cervidae) by Conventional Staining 97 centric, 2 large submetacentric, 2 large telocentric, 6 Toxic Substances in Livestock and Aquatic Animals Re- medium telocentric and 52 small telocentric chromo- search Group, KhonKaen University. We also thank the somes. The X chromosome was a large telocentric chro- director of Khon Kaen Zoo for valuable help. Thanks are mosome, and the Y chromosome was a small telocentric also expressed intended to the staff of this zoo for their chromosome (Tables 1 and 2). The conventional, GTG-, good cooperation. high-resolution GTG- and Ag-NOR karyotypes of Axis axis are demonstrated in Figs. 2–5, respectively. In ad- References dition, the idiograms that they illustrate are shown in Figs. 6–9, respectively. This result differs from the re- Asher, G. W., Gallagher, D. S., Tate, M. L. and Tedford, C. 1999. ports of Hsu and Benirschke (1974), Asher et al. (1999), Hybridization between (Cervus nippon) and axis deer (Axis axis). J. Hered. 90: 236–240. Bonnet-Garnier et al. (2003) and Shanthi et al. (2008), Bonnet, A., Thevenon, S., Claro, F., Gantier, M. and Hayes, H. 2001. which showed that the autosomes include one pair each Cytogenetic comparison between and of metacentric and submetacentric and 30 pairs of acro- : R-banded karyotypes and FISH mapping. Chromosome centric, and the X and Y were acrocentric chromosomes Res. 9: 673–687. too. 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