|Year : 2018 | Volume
| Issue : 3 | Page : 156-160
Application of molecular markers in wildlife DNA forensic investigations
Ishani Mitra1, Soma Roy1, Ikramul Haque2
1 DNA Unit, Biology Division, Central Forensic Science Laboratory, Kolkata, West Bengal, India
2 Directorate of Forensic Science Services, Ministry of Home Affairs, CGO Complex, New Delhi, India
|Date of Web Publication||28-Sep-2018|
DNA Unit, Biology Division, Central Forensic Science Laboratory, 30, Gorachand Road, Kolkata - 700 014, West Bengal
Source of Support: None, Conflict of Interest: None
Wildlife DNA Forensic is the application of regular DNA forensic methods for proper identification of wildlife parts and their products. Recent advances in molecular genetic studies have generated a new and exciting range of possible applications of genetic methods to wildlife research, conservation, and management. These advances have led to an explosion in genetic research on wildlife for their identification at molecular level and have increased interest among researchers working in other scientific disciplines for application of genetic technology in wildlife DNA forensic field. Different molecular markers have been developed and being routinely used for analysis, such as nuclear markers (variable number of tandem repeats, single-nucleotide polymorphisms), mitochondrial markers (cytochrome b, cytochrome c oxidase subunit I, 16S rRNA, 12S rRNA, and D-Loop) and microsatellites. As soon as, a case is reported under Wildlife Protection Act (1972) the case exhibits are sent to forensic laboratories for proper analysis of species for appropriate application of law.
Keywords: Genetic techniques, Microsatellites, Mitochondrial markers, Nuclear markers, Wildlife DNA Forensic, Wildlife Protection Act 1972
|How to cite this article:|
Mitra I, Roy S, Haque I. Application of molecular markers in wildlife DNA forensic investigations. J Forensic Sci Med 2018;4:156-60
| Introduction|| |
With increasing adverse effect of natural resource depletion worldwide, the conservationists and environmentalists have awakened and wants to apply strict Wildlife Act all over the continents. The main focus of that act is to stop the illegal poaching, smuggling, and hunting of endangered and threatened wildlife creatures, apart from their protection in their particular niche. To apply the law, it becomes necessary to properly identify each crime exhibits up to species level. It is a serious worldwide concern for wildlife management to stop the illegal smuggling, hunting, and poaching of wildlife, be it for their medicinal value or ornamental body parts.
Wildlife Forensic Science is nothing but the application of established and accepted forensic techniques to identify the wildlife species and helping to answer the legal issues related to them. It has the same task as of human forensic analysis, i.e., to relate suspect, victim, and crime scene with the minute and degraded physical evidence recovered from the scene of the crime and fixing the accurate wildlife offense as well as to study the phylogenetic relationship between wild animals. Hence, Wildlife Forensics is a vital branch of Forensics, which deals with the identification of the species from the biological remnants. It is a wide range of discipline compared to human identification and takes many guises depending on the nature of the allegation. A key difference is that in alleged crimes against wildlife there can often be no “victim” to provide information regarding the investigation. In addition, the list of species encountered in Wildlife Forensic Science is extensive in contrast to the single species analyzed in human identification [Figure 1].
|Figure 1: Illegal use of Animal and their body products, (a) Group of elephants. (b) Ornaments made from illegal ivory. (c) Ornamented elephant tusk. (d) A spitting snake. (e) Collection of venom from snake. (f) Shoes made from snake skins. Sources of images are listed in references|
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From a quality control and quality assurance perspective, a complicating factor is that most accredited forensic laboratories and associated scientists do not handle non-human samples. This is due to the particular complexities of wildlife crime analysis techniques requiring a completely different expertise and skill set to those possessed by scientists in traditional forensic laboratories.
| Wildlife DNA Forensic|| |
The field of conservation genetics has developed over the past 20 years to support the application of molecular genetic analysis to problems and questions encountered in species conservation. Wildlife DNA Forensic is nothing but the application of established DNA forensic methods for the identification of wildlife specimens. It is essentially concerned with the identification of evidence items to determine the species, population, relationship, or individual identity of a sample  and technological advancement in human forensic provides a backbone for wildlife investigation. The researchers develop new approaches for the collection, analysis, and interpretation of wild confiscated biological samples in addition to generating information relevant to the management of target populations. However, the progress rate of advancements in human forensic has been more gradual than wildlife forensic because of lack of proper attention for many years, but wildlife crime investigation is often lots more complicated as compared to others investigative Sciences. There are a number of reasons or circumstances under which animal can be killed (legally or illegally), but lack of proper species-specific identification procedure and lots more complication in identification techniques, hindered the fight against wildlife crime.
Thus, DNA typing of non-human DNA is a fast developing area of research and professional practice [Figure 2]. The application of DNA typing in Wildlife Forensic Science is one of these prime uses of DNA typing and is gaining increasing profile. The use of DNA profiling in wildlife forensic science falls into the following areas:
|Figure 2: A diagrammatic process of non-human or Wildlife DNA Forensic analysis, (a-e) Wildlife specimen collection and tissue sampling. (f) Steps in genomic DNA extraction. (g) 1% Agarose gel electrophoresis to check the DNA quantity. (h) 2% Agarose gel electrophoresis to check the PCR amplified DNA products. (i) DNA sequencing. (j) Sequence alignment. Sources of images are listed in references|
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- Identification of unknown species
- Identification of gender from questioned animal
- Identification of individual animal
- Population identification
- Parentage analysis
- Study of phylogenetic history
- Expert testimony and consultation.
Molecular markers in wildlife
A molecular marker or a genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. In general, two types of markers are used for Wildlife DNA Forensic analysis.
- Mitochondrial markers
- Nuclear markers.
Markers derived from nuclear genes are not available for a majority of wildlife, but that might change. Currently, mtDNA markers dominate the wildlife area for species identification.
| Identification Using Mitochondrial Marker|| |
Mitochondrial DNA has some unique features that make it a useful tool for species identification. It is only 16.5 kb in size and encodes for only 37 genes, i.e., 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. Each cell has 100s of copies of mtDNA. Mitochondrial DNA is better protected from degradation due to its own rigid membrane which is high in protein content. It can even be found in highly degraded samples that do not contain much nuclear DNA (e.g., hair and bone). There is no proofreading activity during mtDNA replication so there is a greater chance of mutation or change in DNA sequence than in the nuclear DNA., Mitochondrial DNA is maternally inherited so all the maternal lineages will have the same mtDNA sequence., Mitochondrial markers that are used for species identification are as follows: cytochrome b (Cyt b) gene, cytochrome c oxidase subunit I (COI) gene, 12S and 16S rRNA segment and control region (D Loop) in animals; rbcL and matK (plastid genes) in plants.
Advantages of mitochondrial markers for wildlife analysis
- For the purpose of species identification, apart from the application of morphological examination, DNA-based analysis is the only option when trying to establish the presence/absence of biological material from wildlife products
- In plants, mtDNA is not used for species identification because of the low rate of sequence mutation which cannot provide species-level resolution. In fungi, internal transcribed spacer regions of 12S rRNA is used for the purpose of species identification
- Helps in the study of phylogenetic relationship between species. A phylogenetic understanding of mtDNA variation has a number of positive implications in the forensic arena. It yields a wealth of information, including:
- The identification of the major haplogroups present.
- The frequency of these haplogroups, which can be compared with other databases.
- The most informative single-nucleotide polymorphisms that differentiate the major clades, and the sites that show a tendency to reverse often on the phylogeny.
- Quality assurance by identifying potential sequencing errors in population database(s).
Disadvantages of mitochondrial markers for wildlife analysis
- If mixed or contaminated DNA samples received, the result may turn unreliable as universal primers will bind with all the samples.
- Another problem associated with the current generation sequencing of mtDNA genes is that if a hybrid species is protected by law and it is produced using a nonprotected maternal animal species, then the mtDNA analysis will result in the profile of the maternal nonprotected species.
- Heteroplasmy is yet another issue that can create problems when dealing with identification.,
| Identification Using Nuclear Marker|| |
Nuclear DNA markers are usually used for individual identification of an animal. This is mostly achieved using STR profiling of nuclear DNA based on the unique genetic profile of the animal, instead of mitochondrial DNA. While performing individual identification with STRs, generating a Random Match Probability value is also necessary if there is a match between the evidence and reference sample.
The most commonly used nuclear markers are the following: Amplified Fragment Length Polymorphism, random amplified polymorphic DNA, and short sequence repeats or Microsatellites.
Advantages of nuclear markers for wildlife analysis
- STR DNA databases are required because wild endangered species are very scarce. Work is still progressing on this field as a number of threatened or endangered animals is plenty, and advanced research on them will take more time.
- However, the work of Singh et al. discovered a DNA marker called Ple46 in four species of big cats (Panthera species) in India where different animals had different lengths of the microsatellite marker. The repeat sequence was CA bases; the domestic cat had 10 (CA) repeats, the lion had 22 repeats, the leopard had 14–15 repeats and the tiger had 7–8 repeats while the heterozygosity level for Ple46 marker was high (>75%).
- Some existing relevant databases are that of Indian Gharials  where 18 STR markers were used in different combinations for individual identification and of Tigers.
Disadvantages of nuclear markers for wildlife analysis
- The current STR profiling being used for wildlife species individualization does not have much value in forensic cases even when much sequence data are available, because most of the literature is flooded with dinucleotide STR markers which are not ideal in forensic science because of the generation of stutter products.
- At least tetra-nucleotide or larger STR markers need to be identified to be of any use in forensic identification. Therefore, much of the quality check, quality analysis, and method validation must be conducted before using STR markers for a species identification in forensic casework [Table 1].
|Table 1: Name of some Asian endangered species and wildlife DNA forensic markers|
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| Conclusion|| |
The introduction of new generation DNA-based technologies has revolutionized the modern forensic investigation. With the advent of polymerase chain reaction-based techniques, it has become a routine for forensic analysis of very little amounts of a wide range of biological samples, including badly degraded biological material. For establishing the relatedness between the species and individuals, molecular markers play an important role by comparing the genotypes at a number of polymorphic loci. With an ever-increasing wildlife crime, there is an urgent need to develop forensic processes for wildlife identification that meet international standards to make it possible to identify each and every wildlife species. In this respect, DNA-based technologies are now a realistic prospect for many independent laboratories and research centers that are involved in the study of endangered mammals, reptiles and amphibians. Therefore, additional markers need to be used for more accurate interpretation of population genetics, biodiversity, phylogeny, and forensic. It is advisable to the government organizations, zoological gardens, museums, and private keepers to become a part of a global drive, aimed at obtaining sufficient nucleotide data to aid in the identification of each individual wildlife species for Forensic and Conservation studies by providing necessary biological samples for further studies. This paper provides an overview of genetic techniques applied in our forensic laboratory for identification of endangered wildlife species for proper implication of law to stop the illegal poaching and trafficking of endangered wildlife flora and fauna.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]