A Study on Karyotype of the Asian Leopard Cat, Prionailurus bengalensis (Carnivora, Felidae) by Conventional
Staining, G-banding and High-resolution Technique
Puntivar Keawmad, Alongkoad Tanomtong* and Sumpars Khunsook
Genetics Program, Department of Biology, Faculty of Science, Khon Kaen University,
Muang, Khon Kaen, 40002, Thailand
Received January 9, 2007; accepted January 31, 2007
Summary A karyotypic study of the Asian leopard cat (Prionailurus bengalensis) in Thailand has
been made. Blood samples were taken from 2 males and 2 females. After the standard whole blood
lymphocyte culture in the presence of colchicine, the metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, G-banding and high-resolution techniques were
applied to stain the chromosomes. The results showed that 2n of the Asian leopard cat was 38, and
the fundamental number (NF) was 74 in male and female. There are 6 autosome types: A type has 4
large and 2 medium submetacentric chromosomes, B type has 6 large and 2 medium acrocentric
chromosomes, C type has 4 large metacentric chromosomes, D type has 8 small submetacentric
chromosomes, E type has 8 small metacentric chromosomes and F type are 2 small telocentric chromosomes. A pair of the short arm of chromosome E1 (chromosome pairs 14) showed a clearly observable satellite chromosomes. The X chromosome was medium submetacentric chromosome and
the Y chromosome was the smallest metacentric chromosome. From the G-banding and high-resolution techniques, the number of bands and locations in the Asian leopard cat was 183 and 236 respectively, and each chromosome pair could be clearly differentiated. We found that chromosomes A1,
A2, A3, B3, B4, C1, C2, D3, D4, E1, E3, F1 and X chromosome patterns were according to the domestic cat (Felis catus) chromosomes. Chromosomes B1, B2, D2 and E2 are similar to those of the
domestic cat. These results show the evolutionary relationship between the Asian leopard cat and the
domestic cat. The karyotype formula for the male and female Asian leopard cat is as follows:
Key words Karyotype, Asian leopard cat (Prionailurus bengalensis), Conventional staining.
Over the world, animals in the family Felidae are separated into 18 genera and 36 species. Six
genera and 9 species are found in Thailand (Wilson and Cole 2000). These are marbled cat (Pardofelis marmorata Martin 1837), fishing cat (Prionailurus viverrinus Bennett 1833), Asian leopard
cat (Prionailurus bengalensis Kerr 1792), flat-headed cat (Prionailurus planiceps Vigors and Horsfield 1872), jungle cat (Filis chaus Guldenstaedt 1776), Asiatic golden cat (Catopuma temminckii
Vigors and Horsfield 1827), clouded leopard (Neofelis nebulosa Griffish 1821), leopard (Panthera
pardus Linnaeus 1758) and tiger (Panthera tigris Linnaeus 1758) (Lekagul and McNeely 1977,
1988).
The Asian leopard cat is a small wild cat that is found in Southeast Asia. On average it is as
large as a domestic cat, but there are considerable regional differences. The fur is also quite variable; it is yellow in the southern populations, but silver-grey in the northern ones. The chest and the
lower part of the head are white. The Asian leopard cat bears black markings, that may be dependent on the subspecies spots or rosettes. It is a nocturnal animal and usually eats rodents, birds, fish,
reptiles and small mammals. It is usually a solitary animal except for during the mating season. It
has litters of 2 to 4 kittens and the gestation period can vary from 65 to 70 d. The habitat of this cat
is forests and rainforests both in low and mountainous areas, and usually not arid areas. It lives
close to watercourses and may be found at heights of up to 3000 m. The Asian leopard cat can
climb trees skillfully. It is also able to swim, but will seldom do so. (Lekagul and McNeely 1977,
1988).
Although karyotypic studies of the family Felidae have been reported (Makino and Tateishi
1952, Thuline and Norby 1961, Hsu 1962, Hsu et al. 1963a, 1963b, Hsu and Rearden 1965, Ohno
et al. 1962, Matano 1963, Chu et al. 1964, Benirschke and Low 1966, Leyhausen and Tonkin 1968,
Sutton 1968, Hard 1968, Wurster and Benirschke1967, 1968a, 1968b, Wurster 1969, Milosevic et
al. 1972, Wurster-Hill 1973, Wurster-Hill and Meritt 1974, Yang et al. 2000, Nie et al. 2002), there
are few reports of the cytogenetics of the family Felidae in this species in Thailand. Animals in this
family are conserved animals and are at risk. In this study, lymphocyte culture, conventional staining, G-banding and high-resolution technique were used to compare the results with the previous
studies. In the future, basic knowledge about family Felidae and their cytogenetics will be applied
for research and to protect them from extinction.
Materials and methods
Blood samples from the jugular vein were collected from 2 males and 2 females of the Asian
leopard cat, which were kept in Nakhon Ratchasima Zoo, Nakhon Ratchasima province and
Songkhla Zoo, Songkhla province, 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
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% carbon dioxide 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 1,200 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 1,200 rpm for 10 min. Cells were fixed by fresh cool fixative (methanol : glacial acetic
acid3 : 1) gradually added up to 8 ml before centrifuging again at 1,200 rpm for 10 min and the supernatant discarded. The fixation was repeated until the supernatant was clear and the pellet was
mixed with 1 ml fixative. The mixture was dropped onto a clean and cold slide using micropipette
followed by the air-dry technique. The materials or slide was conventionally stained with 20% stock
Giemsa’s solution for 30 min.
G-banding method
The G-banding technique was adapted from Kampiranont (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 10% fetal calf serum (FCS) or phosphate buffer. FCS was washed
out by 50% methanol and finaly materials or the slide was stained with 10% Giemsa’s solution for 30 min.
High-resolution staining method
After the lymphocytes were cultured for 72 h, 0.05 ml of 105 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 centrifugution 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. The cells were harvested at the exact time and stained using the G-banding procedure.
Chromosomal checks, karyotyping and idiograming
Chromosomal checks were performed on mitotic metaphase cells under a light microscope.
Twenty cells each of male and female with clearly observable and well-spread chromosomes were
selected and photographed. The length short arm chromosome (Ls) and the length long arm chromosome (Ll) were measured to calculate the total length arm chromosome (LT, LTLsLl). The
relative length (RL), the centromeric index (CI) and standard deviation (SD) of RL, CI were also
computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were
used in karyotyping and idiograming according to Hsu and Rearden (1965).
Results
Karyotypic study of the Asian leopard cat using lymphocyte culture revealed that the chromosome number is 2n (diploid)38, and the fundamental number (NF) is 74 in male and female. The
autosomes of the Asian leopard cat composed of 6 types: A type had 4 large submetacentric, 2
medium submetacentric chromosomes, B type had 6 large acrocentric, 2 medium acrocentric chro
mosomes, C type had 4 large metacentric chromosomes, D type had 8 small submetacentric chromosomes, E type had 8 small metacentric chromosomes and F type had 2 small telocentric chromosomes. In addition, a pair of the short arm of chromosome E1 (chromosome pairs 14) showed a
clearly observable satellite chromosomes. The X-chromosome was medium submetacentric chromosome and the Y chromosome was the smallest metacentric chromosome (Figs. 1–6).
The important chromosome marker of the Asian leopard cat is the asymmetrical karyotype, in
which all 4 types of chromosomes are found (metacentric, submetacentric, telocentric and acrocentric). The largest and smallest chromosomes show large size difference (approximately 5.2 fold).
The largest chromosome is submetacentric chromosome, while the second largest chromosome is
acrocentric chromosome and the Y chromosome is the smallest metacentric chromosome (Figs.
1–6). The G-banding revealed that the number of G-bands on one set of haploid chromosomes,
which includes autosomes, X and Y chromosomes, is 183 bands for the Asian leopard cat. The
number of bands in one set of prometaphase haploid chromosomes from the high-resolution
method is 236 bands (Figs. 7, 8).
Comparison of chromosome banding pattern between the Asian leopard cat and the domestic
cat (Felis catus) revealed that 13 chromosome pairs show the same pattern (pairs A1, A2, A3, B3,
B4, C1, C2, D3, D4, E1, E3, F1 and X chromosome) and 4 chromosome pairs share similarities
(pairs B1, B2, D2 and E2) (Fig. 9). This indicates that there is an evolutionary relationship between
the Asian leopard cat and the domestic cat. The data of the chromosomal checks on mitotic
metaphase cells of the Asian leopard cat are shown in Tables 1 and 2. Fig. 7 shows the idiogram for
the Asian leopard cat from the G-band staining, while Fig. 8 shows the idiogram from the high-resolution banding with landmarks, bands and sub-bands. The karyotype formula for the Asian leopard cat are as follows:
Discussion
Karyotypic study of the Asian leopard cat using lymphocyte culture revealed that the chromosome number is 2n38. This result agrees with the previous studies by Makino and Tateishi (1952)
indicating that a member of the Asian leopard cat family has 2n38. This corresponds to 2n for the
members of the family Felidae according to reports in puma (Felis concolor), Canadian lynx (F.
lynx), snow leopard (Uncia uncia), jaguarundi (F. yagouaroundi), cheetah (Acinonyx jabatus
jabatus), serval (F. serval), fishing cat (F. viverrina), Bob cat (Lynx rufus), marbled cat (F. marmorata), European wild cat (F. silvestris), black footed cat (F. nigripes), leopard (Panthera pardus), tiger
(P. tigris), domestic cat (F. catus) and ocelot (F. pardalis) (Makino and Tateishi 1952, Thuline and
Norby 1961, Hsu 1962, Hsu et al. 1963a, 1963b, Hsu and Rearden 1965, Ohno et al. 1962, Matano
1963, Chu et al. 1964, Benirschke and Low 1966, Leyhausen and Tonkin 1968, Sutton 1968, Hard
1968, Wurster and Benirschke 1967, 1968a, 1968b, Wurster 1969, Milosevic et al. 1972, WursterHill 1973, Wurster-Hill and Meritt 1974). However this number differs from the chromosome number of Geoffroy’s (F. geoffroyi) and Marguay (F. wiedi) which is 2n36 (Hsu 1962, Hsu et al. 1963).
The autosomes of the Asian leopard cat can be separated to 6 types: A type had 4 large submetacentric and 2 medium submetacentric chromosomes, B type had 6 large acrocentric and 2
medium acrocentric chromosomes, C type had 4 large metacentric chromosomes, D type had 8 small submetacentric chromosomes, E type had 8 small metacentric chromosomes and F type had 2
small telocentric chromosomes. This result agrees with the previous studies by Makino and Tateishi
(1952) indicating that a member of the Asian leopard cat family had 6 types of autosome: A type
had 6 submetacentric chromosomes, B type had 8 acrocentric chromosomes, C type had 4 metacentric chromosomes, D type had 8 submetacentric chromosomes, E type had 8 metacentric chromosomes and F type had 2 telocentric chromosomes. The comparison of the Asian leopard cat chromosomes with the domestic cat revealed that there is a difference in chromosomes of E and F type.
The Asian leopard cat had 4 and 1 pairs of chromosomes whereas the domestic cat had 3 and 2
pairs of chromosomes, respectively.
The fundamental number (NF) of the Asian leopard cat is 74 in male and female. For the sex
chromosomes, the X-chromosome was medium submetacentric chromosome and Y chromosome
was the smallest metacentric chromosome. This result was different from Makino and Tateishi
(1952) who reported that the X and Y chromosomes of the Asian leopard cat were submetacentric
chromosomes. The comparison of the Asian leopard cat X and Y chromosomes to the domestic cat
indicated that X chromosome was the medium submetacentric and Y chromosome was the smallest
subtelocentric (Thuline and Norby 1961, Ohno et al. 1962, Matano 1963, Chu et al. 1964, Hsu and
Rearden 1965). The comparison of the Asian leopard cat X and Y chromosomes to animals in family Felidae in Thailand namely, leopard (Panthera pardus) and clouded leopard (Pardofelis nebulosa)
found that X and Y chromosomes were submetacentric in every species (Fig. 10).
From this study, the Asian leopard cat had the chromosome marker at the short arm of chromosomes E1 (chromosome pairs 14) that was a satellite chromosomes with nucleolar organizer regions, NOR. This result agrees with previous reports about the chromosomes E1 of animals in the
family Felidae having a satellite chromosomes (Makino and Tateishi 1952, Thuline and Norby
1961, Hsu 1962, Hsu et al. 1963a, 1963b, Hsu and Rearden 1965, Ohno et al. 1962, Matano 1963,
Chu et al. 1964, Benirschke and Low 1966, Leyhausen and Tonkin 1968, Sutton 1968, Hard 1968,
Wurster and Benirschke 1967, 1968a, 1968b, Wurster 1969, Milosevic et al. 1972, Wurster-Hill
1973, Wurster-Hill and Meritt 1974) (Fig. 11).
Acknowledgement
The financial support from The Zoological Park Organization Under the Royal Patronage of
H.M. The King is gratefully acknowledged. We also thank Mr. Sopon Dumnui, Director of the Organization and Dr. Sumat Kamolnaranath, chief of the Educational Division, for valuable help. We
would like to thank the Directors of the Nakhon Ratchasima Zoo and Songkhla Zoo for the blood
samples. Thanks to the authorities and officers of these zoos for good cooperation.
References
Benirschke, K. and Low, R. J. 1966. Chromosome studies of four carnivores. Mammalian Chromosome Newsletter 21:
148–160.
Chaiyasut, K. 1989. Cytogenetics and cytotaxonomy of the family Zephyranthes. Department of Botany, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand.
Chu, E. H. Y., Thuline, H. C. and Norby, D. E. 1964. Triploid-diploid chimerism in a male tortoiseshell cat. Cytogenetics 3:
1–8.
Hard, W. L. 1968. The karyotype of a male cheetah, Acinonyx jabatus jabatus. Mamm. Chrom. Newsletter 9: 16–25.
Hsu, T. C. 1962. Two species of cats with 36 chromosomes. Mamm. Chrom. Newsletter 8: 4–10.
—, Rearden, H. H. and Luquette, G. F. 1963a. Karyological studies of nine species of Felidae. America Naturalist 97:
225–235.
—, Arrighi, F. E. and Luquette, G. F. 1963b. Karyological studies of nine species of Felidae. America Naturalist 97:
225–232.
— and Rearden, H. H. 1965. Further karyological studies on Felidae. Chromosoma (Berl.) 16: 365–371.
Kampiranont, A. 2003. Cytogenetics. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand.
Lekagul, B. and McNeely, J. A. 1977. Mammals of Thailand. 1st ed. Kurusapha Ladprao Press: Bangkok, Thailand.
— and — 1988. Mammals of Thailand. 2nd ed. Sahakarn Bhaet: Bangkok, Thailand.
Leyhausen, P. and Tonkin, B. A. 1968. Comment on the karyotypes of the leopard cat and fishing cat. Mamm. Chrom.
Newsletter 9: 78–85.
Makino, S. and Tateishi, S. 1952. A comparison of the chromosomes of the lion, Chinese leopard cat and house cat. J. Morphology 90: 93–99.
Matano, Y. 1963. A study of the chromosome in the cat. Japan J. Gene. 38: 147–152.
Milosevic, M., Zivkovic, S. and Isakovic, I. 1972. Hromozomske karakteristike nekin carnivora Iz Srbij. Genetika (Beogard) 4: 1–9.
Nie, W., Wang, J., O’Brien, P. C. M., Fu, B. and Ying, T. 2002. The genome phylogeny of domestic cat, red panda and
mustelid species revealed by comparative chromosome painting and G-banding. Chrom. Res. 10: 209–222.
Ohno, S., Stenius, C., Weiler, C. P., Trujillo, J. M., Kaplan, W. D. and Kinosita, R. 1962. Early meiosis of male germ cells in
fetal testis of Felis domestica. Exp. Cell Res. 27: 401–410.
Rooney, D. E. 2001. Human Cytogenetics: Constitutional Analysis. Oxford University Press: Oxford.
Sutton, D. A. 1968. Karyotype of a female bobcat, Lynx rufus californicus. Mamm. Chrom. Newsletter 9: 249–254.
Thuline, H. C. and Norby, D. E. 1961. Spontaneous occurrence of chromosome abnormality in cats. Science 134: 544–546.
Wilson, D. E. and Cole, F. R. 2000. Common names of mammals of the world. Smithsonian Institution: United States of
America.
Wurster, D. H. and Benirschke, K. 1967. Chromosome numbers in thirty species of Carnivora. Mamm. Chrom. Newsletter
8: 195–216.
— and — 1968a. Comparative cytogenetic studies in the order Carnivora. Chromosoma 24: 336–382.
— and — 1968b. Karyotype of four more species of cats. Mamm. Chrom. Newsletter 9: 236–245.
— 1969. Cytogenetic and phylogenetic studies in Canivora. In: Comparative Mammalian Cytogenetics Benirschke, K., ed.,
Springger-Verlag, N.Y.
Wurster-Hill, D.H. 1973. Chromosome of eight species from five families of Carnivora. J. Mammalian 54: 763–782.
— and Meritt, D. A., Jr. 1974. The G-band chromosomes of the marbled cat, Felis marmorata. Mamm. Chrom. Newsletter
15: 14–19.
Yang, F., Graphodatsky, A. S., O’Brien, P. C. M., Colabella, A., Solanky, N., Squire, M., Sargan, D. R. and Ferguson-Smith,
M. A. 2000. Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat,
dog and human. Chrom. Res. 8: 393–404.
