Elerrrophoresis zyxwvutsrqponm 1997, zyxwvutsrqponml 18, 1693-1700 zyxwvutsrqponml Genetic variation in lions and tigers zyx 1693 Pattabhiraman Shankaranarayanan' Moinak Baneiee'' Raj Kumar Kacke? Ramesh Kumar Aggarwal' Lalji Singh'. 'Center for Cellular and Molecular Biology, Hyderabad, India 'Zoological Survey of India, Calcutta, India 'Center for DNA Fingerprinting and Diagnostics, Housed on the campus of the Center for Cellular and Molecular Biology, Hyderabad, India Genetic variation in Asiatic lions and Indian tigers Previous reports have suggested that Asiatic lions and tigers are highly inbred and exhibit very low levels of genetic variation. Our analyses on these species have shown much higher degrees of polymorphism than reported. Randomly amplified polymorphic DNA (RAPD) analysis of 38 Asiatic lions, which exist as a single population in the Gir Forest Sanctuary in India, shows an average heterozygosity of 25.82 zyxwv O/o with four primers. Sperm motility studies by our col- leagues corroborate this data. In Indian tigers, microsatellite analysis of five CA repeat loci and multilocus fingerprinting using Bkm 2(8) probe on a popu- lation of 22 individuals revealed a heterozygosity of 22.65 %. Microsatellite analysis at loci Fca 77 and Fca 126 revealed polymorphism amongst the Asiatic X African lion hybrids, which has enabled us to use these as markers to discri- minate the pure Asiatic lions from the hybrids. A similar analysis was used to identify hybrids of Indian and Siberian tigers through polymerase chain reac- tion (PCR) amplification of hair samples. To ascertain the variation which existed before the population bottleneck at the turn of the present century, microsatellite analysis was performed on 50- to 125-year-old skin samples from museum specimens. Our results show similar levels of genetic variability as in the present population (21.01 Yo). This suggests that low genetic variability may be the characteristic feature of these species and not the result of intensive inbreeding. DNA fingerprinting studies of Asiatic lions and tigers have helped in identifying individuals with high genetic variability which can be used for conservation breeding programs. 1 Introduction The habitat of the Asiatic lion (Puntheru leo persica) ranged across southwestern Asia, extending from Syria to Northern India, as recently as 200 years ago [l]. The subspecies became extinct in Syria, Iraq, Iran, Afgha- nistan and Pakistan in the latter part of the 19'h century. At present, Asiatic lions exist as a single relict popula- tion isolated since the 1880s [l] in the Gir Forest Sanc- tuary and the surrounding forest in the Gujarat state in western India. Large-scale hunting had reduced the population to less than twenty at the turn of the present century. Conservation efforts have brought this number up to zyxwvutsr 350. O'Brien et zyxwvutsrqp al. [2-41 reported a total absence of variation at each of 46 allozyme loci in 28 individuals of Asiatic lions of Gir Forest in contrast to several African lion (Punthera leo leo) populations which showed moder- ate levels of allozyme variation at the same loci. Lack of genetic variation in the Asiatic lion was correlated with a high incidence of morphologically abnormal sperma- tozoa and low levels of circulating testosterone, a critical hormone for spermatogenesis [4]. Based on these obser- vations, the prediction was made [2-41 that the Asiatic lion is a severely endangered species that has suffered a population bottleneck or a series of bottlenecks followed by inbreeding in their recent history. The Indian tiger (Panthera tigris tigris), is another critically endangered felid. The population of this animal has dwindled from Correspondence: Dr. Lalji Singh, Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India. (Tel: +0091-40- 7171252; Fax: +0091-40-7150008; E-mail: lalji@ccmb.globemail.com) Nonstandard abbreviations: Bkm, banded krait minor satellite DNA; OTU, operational taxonomic unit; RAPD, randomly amplified poly- morphic DNA Keywords: Genetic variation zyxwvutsrqp I Randomly amplified polymorphic DNA / Microsatellites / Lions / Tigers / Conservation 40 000 individuals at the beginning of this century to the present figure of approximately 3500. Poaching for bones and hair, which have purported medicinal properties, and habitat shrinkage have been the major causes for the drastic reduction in number. Furthermore, the lions and tigers presently kept in various Indian zoos are sus- pected to be hybrids between Asiatic and African lions, and Indian and Siberian tigers. The present study was, therefore, undertaken (i) to examine the extent of inbreeding in the Asiatic lion and Indian tiger population, (ii) to identify pure Asiatic lion from the hybrid between Asiatic and African lion, and pure Indian tiger from the hybrid between Indian tiger and Siberian tiger, and (iii) to identify the lions and tigers showing extensive genetic variation for selective breeding for their proper genetic management. To answer the above questions we have used random ampli- fied polymorphic DNA (RAPD) and microsatellite anal- yses. For RAPD, short arbitrary oligonucleotides were used as primers in a PCR reaction to generate a DNA pattern. The advantages of this technique are that no prior sequence information of the genomes under inves- tigations is required, it covers the genome more extensi- vely than other techniques, and minute amounts of DNA are sufficient to carry out the analysis. RAPD, therefore, has been used to study many animal and plant genomes [5-71. Microsatellites, due to their abundance in the genome and high mutation rates, prove useful in addres- sing questions in dynamics of populations. They have been used in conservation biology to assess the levels of genetic diversity and plan future strategies as in the case of the bottlenecked wombat and endangered Ethiopian wolf [8, 91. We have also used banded krait minor satel- lite DNA (Bkm)-derived 2(8) multilocus DNA finger- * Presently at the Rajiv Gandhi Center of Biotechnology, Thiruvanan- thapuram, India 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1997 0173-0835/97/0909-1693 $17.50+.50/0