Tiger Hair Analysis, by S.K. Gupta

 

Tiger (Panthera tigris corbetti) hair analysis from Uttarakhand, by S.K. Gupta, Scientist from Wildlife Institute of India, Wildlife Forensic Lab, Dehradun 2010.

A report on similarity test of two hair samples of Tiger (Panthera tigris)

 

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Abstract:

Two tiger hair samples were sent by Frederique Lacraz, Society for Mahseer Conservancy, Ramnagar, Uttarakhand to Wildlife Institute of India. Those two tiger hair samples were collected in the same area (Tera village) where there has been two attacks on ladies by tigers. The reason for a DNA analysis was to identify, whether both the sample belonged to the same individual. Such issue can be dealt based on commonly used genotyping based genetic analysis. We describe here similarity test between two hair samples of Tigers (Panthera tigris). There are genetic markers used in similarity analysis are called Microsatellites. We used 5 fluorescent labelled microsatellite loci for analysis. After the comparison of all loci, the DNA typing indicates that in both hair samples alleles tested were not the same. We conclude the both the hair samples have different genotype i.e. Hair sample marked as F-1553 (174176, 130130, 182182, 124126, 166166) and Hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162) are not from the same individual.

 

Introduction:

DNA marker technologies have revolutionized molecular genetic techniques as they can contribute to the management of threatened species. However, despite a myriad of potential application, the usefulness of these techniques for making informed management decisions is generally underappreciated. Molecular genetic technique can be used to identify same individuals, parents, offspring and close relatives in captive and wild population (Liu and Cordes, 2004). Everyone, except identical twins, have a one-of-a-kind DNA genome. The DNA molecule is made of monomers called nucleotides, and the specific order in which nucleotides occur in a DNA molecule represents each individual’s unique genetic code. The analysis tests the DNA isolated to locate certain regions of chromosomes that are known to vary in length between individuals. These sites are tested; each site is called a “locus”, (“loci” – plural). Analysis of these sites in a large population reveals many different sized versions associated with each site.

 

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

 Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

 PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

Versions of a DNA sequence or a gene are called “alleles”. At one locus, there are two alleles, one come from father and other from mother. Genetic markers used in individual identification and parentage analysis are called Microsatellites, or Simple Sequence Repeats (SSRs), there are polymorphic loci present in nuclear and organelle DNA that consist of repeating units of 1-6 base pairs in length. They are typically neutral, co-dominant and are used as molecular markers which have wide-ranging applications in the field of genetics, including kinship and population studies.

Because each individual has two of each type of chromosome, one inherited from each parent and therefore has two alleles at each locus. These two alleles are sometimes identical called homozygous, but sometimes are not the same size called heterozygous. During similarity test on the basis of DNA fingerprinting, the analysis identifies the length of the two alleles found at each locus, by comparing the DNA profiles of the two different samples. In this report we document the similarity test undertaken between two hair samples of tiger sent by a NGO from Ramnagar, Uttarakhand and marked as F-1553 and R-1944.

Methodology

DNA fingerprinting determines whether two biological samples belong to same individual or not.

Extraction of DNA

Both the hair samples marked as F-1553 and R-1944 were subjected to DNA extraction. Commercially available DNeasy Tissue Kit (QIAGEN, Germany) was used to extract DNA from these samples.

PCR Amplification and electrophoresis

Samples were analysed using 5 polymorphic mcrosatellite loci developed for tiger. These primers (markers) are C6, F1, D6, FCA304 and Ple23.

PCR amplifications were performed in ABI thermal cycler GeneAmp® PCR System 2700, Applied Biosystems, Singapore) in a final volume of 10 μl, containing 25-50 ng of genomic DNA, 1X PCR buffer (applied biosystem), 2.0 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer and 1 units of AmpliTaq Gold DNA polymerase. Amplification conditions were 94°C for 10 min followed by 35 cycles at 94°C for 45s, 55 °C for 45s and 72°C for 1 min, with a final extension of 72°C for 20 min. Distilled water was taken as negative control to ensure there was no contamination in reaction mixtures. PCR products were genotyped using ABI Prism 3130 genetic analyzer.

Results

All the 5 fluorescent microsatellite were amplified successfully. Test in Table1 and Fig. 1 indicate multilocus 5 loci genotype obtained from both the hair samples. After the comparison of all loci, the genotype of both the samples is different at 2 loci. Therefore it is concluded that the hair sample marked as F-1553 having multilocus genotype (174176, 130130, 182182, 124126, 166166) is not the exactly same as the hair sample marked as R-1944 (174176, 132144, 182182, 124126, 162162).

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Conclusion:

Five different loci were used as genetic markers in similarity test concluded to determine whether two tiger hair sample sent by Society for Mahseer Conservancy, Ramnagar, Uttarakhand are derived from the same individuals. The multilocus genotype data obtained from both the hair samples indicate that these hair are having the different allele at two loci out of five loci, therefore; both the hair samples are the derivative of two different individual. Genetic id or multilocus genotyping of both the hairs are as follows.

Hair I (F-1553): 174176, 130130, 182182, 124126, 166166

Hair II (R-1944): 174176, 132144, 182182, 124126, 162162



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