Karyotype and Idiogram of the Axis Deer (Axia axis

Cytologia 82(1) Special Issue: 91–98

© 2017 The Japan Mendel Society

Karyotype and Idiogram of the Axis Deer (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 Animals 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-resolution 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 differentiated. 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: t t t 2n (66) = Lm2 + Lsm 2 + L 2 + M 6 + S52 + Sex-chromosomes

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

The family Cervidae (infraorder Pecora, suborder Ruminantia, order Artiodactyla) has at least 90 species, 20 genera, four tribes (Muntiacini, Cervini, Capreolini and Rangiferini) and two subfamilies (Cervinae and Capreolinae) (Janis and Scott 1987, Nowak 1999, Groves 2006, Wilson and Reeder 2006). The classification of deer in the family Cervidae use external morphology, biogeography, physiology, cytogenetics and especially deciduous cranial appendages. According to the presence or absence of antlers, all deer species were classified into antlered and antlerless deer. The extant deer species have diverse karyotypes; their diploid chromosome numbers range from 2n= 6 in the female Indian muntjac (Muntiacus muntjak vaginalis) (Wurster and Benirschke 1970) to 2n=80 in the Capreolus capreolus pygargus, * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.82.91

which make them an ideal group for chromosomal evolution studies. It is proposed that the ancestral cervid karyotype is 2n=70 based on comparative karyotype analysis of many deer species (Neitzel 1987, Tanomtong et al. 2005, 2010, Huang et al. 2005). The axis deer (Axis axis), also known as chital deer or spotted deer, is a deer which commonly inhabits wooded regions of South Asia including India, Sri Lanka, Nepal, Bangladesh, Bhutan and Pakistan. The axis deer skin is pinkish fawn, marked with white spots, and its underparts are also white (Fig. 1). Its antlers, which it sheds annually, are usually three-pronged and curve in a lyre shape and may extend to 75 cm. Compared to the hog deer (Hyelaphus porcinus), its close relative, the axis deer has a more cursorial build. It also has a more advanced morphology with antler pedicles being proportionally short and its auditory bullae being smaller. It also has large nares. Their lifespans are around eight

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Fig. 1.

Cytologia 82(1) Special Issue

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 two subspecies: common axis deer (A. a. axis) and Sri Lankan axis deer (A. a. ceylonensis). These animals are widespread in many countries. This species is listed on CITES appendix III and is an IUCN least concern species (Grzimek 2004, Groves 2006). To date, there are few cytogenetic reports of the Axis axis, such as those of Hsu and Benirschke (1974), Asher et al. (1999), Bonnet-Garnier et al. (2003) and Shanthi et al. (2008), which report karyotypes of the conventional, RBG-, QFH-banding and chromosome painting of this animal. Our present study shows a confirmation and

comparison finding with previous reports. Moreover, the finding obtained here is the first report on standardized karyotype and idiogram measurements by GTG-, highresolution GTG- and Ag-NOR-banding techniques. This cytogenetic information will represent basic knowledge and can be applicable to genetic diversity, taxonomy, conservation and evolution. Materials and methods Blood samples of the Axis axis (two males and two females) were collected from Khon Kaen Zoo, Thailand

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Karyotype and Idiogram of the Axis Deer (Axia axis, Cervidae) by Conventional Staining

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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

1 2* 3 4* 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 X Y

2.519 0.000 3.303 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

5.749 7.556 4.707 4.779 4.773 4.655 4.110 3.904 3.884 3.804 3.748 3.664 3.562 3.524 3.440 3.355 3.278 3.204 3.157 3.087 3.024 2.988 2.941 2.891 2.866 2.810 2.804 2.751 2.624 2.545 2.441 2.285 6.393 2.345

8.269 8.156 8.010 4.779 4.773 4.655 4.110 3.904 3.884 3.804 3.748 3.664 3.562 3.524 3.440 3.355 3.278 3.204 3.157 3.087 3.024 2.988 2.941 2.891 2.866 2.810 2.804 2.751 2.624 2.545 2.441 2.285 6.393 2.345

RL SD 0.064 0.058 0.062 0.037 0.037 0.036 0.032 0.030 0.030 0.029 0.029 0.028 0.028 0.027 0.027 0.026 0.025 0.025 0.024 0.024 0.023 0.023 0.023 0.022 0.022 0.022 0.022 0.021 0.020 0.020 0.019 0.018 0.049 0.018

0.0059 0.0089 0.0076 0.0047 0.0033 0.0027 0.0028 0.0026 0.0019 0.0021 0.0019 0.0021 0.0018 0.0012 0.0012 0.0013 0.0013 0.0013 0.0012 0.0012 0.0012 0.0013 0.0012 0.0013 0.0012 0.0012 0.0014 0.0014 0.0011 0.0011 0.0013 0.0014 0.0043 0.0017

CI SD 0.438 0.000 0.702 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.017 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Size

Types

Large Large Large Medium Medium Medium Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small

Submetacentric Telocentric Metacentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric 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

1 2* 3 4* 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 X

3.011 0.000 3.267 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

5.917 8.226 4.297 5.040 4.843 4.567 4.395 4.278 3.951 3.902 3.842 3.742 3.629 3.539 3.449 3.359 3.262 3.255 3.201 3.086 3.047 2.963 2.934 2.906 2.853 2.785 2.673 2.607 2.525 2.470 2.351 2.169 6.430

8.928 8.226 7.564 5.040 4.843 4.567 4.395 4.278 3.951 3.902 3.842 3.742 3.629 3.539 3.449 3.359 3.262 3.255 3.201 3.086 3.047 2.963 2.934 2.906 2.853 2.785 2.673 2.607 2.525 2.470 2.351 2.169 6.430

Remark: *=Nucleolar organizer region/NOR.

RL SD 0.069 0.064 0.059 0.039 0.038 0.035 0.034 0.033 0.031 0.030 0.030 0.029 0.028 0.027 0.027 0.026 0.025 0.025 0.025 0.024 0.024 0.023 0.023 0.023 0.022 0.022 0.021 0.020 0.020 0.019 0.018 0.017 0.050

0.0043 0.0044 0.0058 0.0035 0.0036 0.0026 0.0016 0.0016 0.0010 0.0009 0.0009 0.0010 0.0008 0.0008 0.0008 0.0006 0.0007 0.0008 0.0008 0.0008 0.0008 0.0010 0.0010 0.0008 0.0008 0.0008 0.0008 0.0009 0.0008 0.0011 0.0011 0.0024 0.0041

CI SD 0.663 1.000 0.568 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

0.015 0.000 0.013 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Size

Types

Large Large Large Medium Medium Medium Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small Small

Submetacentric Telocentric Metacentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric Telocentric

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and then applied to cytogenetic experimentations by lymphocyte culture of whole blood samples. The culture cells were treated with a colchicine-hypotonic-fixationair-drying technique followed by conventional staining, GTG-, high-resolution GTG- and Ag-NOR banding techniques (Rooney 2001, Campiranon 2003, Sangpakdee et al. 2017). For 20 cells of each individual chromosome,

checks, length measurements, karyotype and idiogram analysis were accomplished by using a light microscope as previously described (Chaiyasut 1989, Chooseangjaew et al. 2017).

Fig. 3. Standardized idiogram of axis deer (Axis axis) 2n= 66 by conventional staining technique; the arrow indicates the satellite chromosome on the long arm of the second pair chromosome.

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

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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.

Results and discussion The cytogenetic study of Axis axis using lymphocyte culture exhibited that the chromosome number is 2n (diploid)= 66 and the fundamental number (NF, num-

ber of chromosome arms) for both sexes is 70 (Fig. 2). These results are in agreement with Hsu and Benirschke (1974), Asher et al. (1999), Bonnet-Garnier et al. (2003) and Shanthi et al. (2008). The diploid number of Cervini species (genera Axis, Cervus, Dama, Elaphurus,

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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, Rucervus, Rusa) ranges from 56 to 68 with a constant fundamental number of 70. The decrease in chromosome number is due to an increase in the number of biarmed chromosomes. Robertsonian

fusions or translocations are described as the main rearrangement involved in this variation (Bonnet-Garnier et al. 2003, O Brien et al. 2006). The autosomes of axis deer consisted of 2 large meta-

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centric, 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 (Tables 1 and 2). The conventional, GTG-, high-resolution GTG- and Ag-NOR karyotypes of Axis axis are demonstrated in Figs. 2–5, respectively. In addition, the idiograms that they illustrate are shown in Figs. 6–9, respectively. This result differs from the reports of Hsu and Benirschke (1974), Asher et al. (1999), Bonnet-Garnier et al. (2003) and Shanthi et al. (2008), which showed that the autosomes include one pair each of metacentric and submetacentric and 30 pairs of acrocentric, and the X and Y were acrocentric chromosomes too. The difference between studies may be due to the different criteria used for the classification of chromosome types. Because previous studies used the criteria of Levan et al. (1964) while the present study used the criteria of Mitelman (1995) and Turpin and Lujeune (1965), different chromosome types were found. The standardized idiogram of Axis axis by the GTGbanding technique showed a pattern of transverse light and heavy bands. The amount of banding pattern on a set of haploid number (n) including autosomes and sexchromosomes is 246 and 294 bands on metaphase and prometaphase chromosomes, respectively (Figs. 7, 8). Comparison to the reports of Herzog (1987) and Bonnet et al. (2001), which revealed the respective 353 and 409 bands of banding pattern on metaphase chromosomes for red deer (Cervus elaphus) and Vietnamese sika deer (C. nippon psuedaxis), this study showed a lower number of bands because only the bands which were clearly observed were counted. The Ag-NOR-banding clearly showed nucleolar organizer regions of the axis deer located on the subtelomeric q-arm of chromosome pair 2 and the telomeric q-arm of chromosome pair 4. There was no detection of heteromorphic NOR. This finding is consistent with Asher et al. (1999) and Bonnet-Garnier et al. (2003), who reported that chital deer had two pairs of NORs. Numerous repetitions of 28S and 18S ribosomal genes are found in NORs. Multiplication of ribosomal DNA is able to increase or decrease the size of NORs (Campiranon 2003). In summary, it can be concluded that an asymertical karyotype is an important characteristic of Axis axis. We found three types of chromosomes: metacentric, submetacentric and telocentric chromosomes. The karyotype formula for axis deer is as follows: sm t t t 2n (66)=Lm 2 +L2 +L 2 +M6 +S 52 +Sex-chromosomes Acknowledgements This work was supported by the research capability enhancement program through graduate student scholarship, Faculty of Science, Khon Kaen University and the

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Toxic Substances in Livestock and Aquatic Animals Research Group, KhonKaen University. We also thank the director of Khon Kaen Zoo for valuable help. Thanks are also expressed intended to the staff of this zoo for their good cooperation. References Asher, G. W., Gallagher, D. S., Tate, M. L. and Tedford, C. 1999. Hybridization between sika deer (Cervus nippon) and axis deer (Axis axis). J. Hered. 90: 236–240. Bonnet, A., Thevenon, S., Claro, F., Gantier, M. and Hayes, H. 2001. Cytogenetic comparison between Vietnamese sika deer and cattle: R-banded karyotypes and FISH mapping. Chromosome Res. 9: 673–687. Bonnet-Garnier, A., Claro, F., Thevenon, S., Gautier, M. and Hayes, H. 2003. Identification by R-banding and FISH of chromosome arms involved in Robersonian translocations in several deer species. Chromosome Res. 11: 649–663. Campiranon, A. 2003. Cytogenetics. 2nd edition. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok. Chaiyasut, K. 1989. Cytogenetic and cytotaxonomy of genus Zephyranthes. Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok. Chooseangjaew, S., Tanyaros, S., Maneechot, N., Buasriyot, P., Getlekha, N. and Tanomtong, A. 2017. Chromosomal characteristics of the tropical oyster, Crassostrea belcheri Sowerby, 1871 (Ostreoida, Ostreidae) by conventional and Ag-NOR banding techniques. Cytologia 82: 3–8. Groves, C. 2006. The genus Cervus in eastern Eurasia. Eur. J. Wildl. Res. 52: 14–22. Grzimek, B. 2004. Grzimek s Animal Life Encyclopedia. Second Edition Volume 15: Mammals IV. Thomson Gale, Detroit. Herzog, S. 1987. Mechanisms of karyotype evolution in Cervus nippon Temminck. Caryologia 40: 347–353. Hsu, T. C. and Benirschke, K. 1974. An Atlas of Mammalian Chromosomes Vol. 8. Springer-Verlag, New York. Huang, L., Nie, W., Wang, J., Su, W. and Yang, F. 2005. Phylogenomic study of the subfamily Caprinae by cross-species chromosome painting with Chinese muntjac paints. Chromosome Res. 13: 389–399. Janis, C. M. and Scott, K. M. 1987. The interrelationships of higher ruminant families with special emphasis on the members of the Cervoidea. Am. Mus. Novit. 2893: 1–85. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for centromeric position on chromosome. Hereditas 2: 201–220. Mitelman, F. (ed.) 1995. An International System for Human Cytogenetic Nomenclature (ISCN 1995). Karger, Basal. Neitzel, H. 1987. Chromosome evolution of Cervidae: karyotypic and molecular aspects. In: Obe, G. and Basler, A. (eds.). Cytogenetics̶Basic and Applied Aspects. Springer-Verlag, Berlin. pp. 90–112. Nowak, R. M. 1999. Walker s Mammals of the World, 6th edition. Vol. II. Johns Hopkins University Press, Baltimore and London. pp. 1051–1135. O Brien, S. J., Menninger, J. C. and Nash, W. G. 2006. Atlas of Mammalian Chromosomes. John Wiley and Son Inc., New York. Rooney, D. E. 2001. Human Cytogenetics Constitution Analysis. Oxford University Press, Oxford. Sangpakdee, W., Phimphan, S., Tengjaroenkul, B., Pinthong, K., Neeratanaphan, L. and Tanomtong, A. 2017. Cytogenetic study of tree microhylid species (Anura, Microhylidae) from Thailand. Cytologia 82: 67–74. Shanthi, G., Balasubramanyam, D., Thangaraju, P. and Srinivasan, R. 2008. Karyological study in spotted deer. Tamilnadu Journal of

98


Veterinary and Animal Sciences 4: 244–246. Tanomtong, A., Chaveerach, A., Phanjun, G., Kaensa, W. and Khunsook, S. 2005. New records of chromosomal features in Indian muntjacs (Muntiacus muntjak) and Fea s muntjacs (M. feae) of Thailand. Cytologia 70: 71–77. Tanomtong, A., Jearranaiprepame, P. and Supiwong, W. 2010. A new polymorphism of nucleolar organizer regions (NORs) of Indian muntjac (Muntiacus muntjak) in Laos PDR. Cytologia 75: 23–30.


Turpin, R. and Lujeune, J. 1965. Les Chromosome Human (Caryotype Normal et Variations Phatologiques). Gautier-Villars, Paris. Wilson, D. E. and Reeder, D. A. M. 2006. Mammal Species of the World: A Taxonomic and Geographic Reference. Johns Hopkins University Press, London. Wurster, D. H. and Benirschke, K. 1970. Indian muntjac, Muntiacus muntjak: A deer with a low diploid chromosome number. Science 168: 1364–1366.