Molecular Identification of Indian Crocodile Species: PCR‐RFLP Method for Forensic Authentication*

Abstract: South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile‐based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA‐based identification of species using PCR‐RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species‐specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

Forensic Identification of Indian Snakeroot (Rauvolfia serpentina Benth. ex Kurz) Using DNA Barcoding

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species‐specific DNA polymorphisms. We found two species‐specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species‐specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot.     

A Three‐Locus,PCR‐based Method for Forensic Identification of Plant Material

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant‐derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA‐based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad‐spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL‐F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species‐targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.     

Forensic Identification of CITES Protected Slimming Cactus (Hoodia) Using DNA Barcoding

Slimming cactus (Hoodia), found only in southwestern Africa, is a well‐known herbal product for losing weight. Consequently, Hoodia extracts are sought‐after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA‐trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI‐MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

Single Nucleotide Polymorphism Assay for Genetic Identification of Lophophora williamsii*

Lophophora is a member of the Cactaceae family, which contains two species: Lophophora williamsii and L. diffusa. Lophophora williamsii is an illegal plant containing mescaline, a hallucinogenic alkaloid. In this study, a novel method based on a single nucleotide polymorphism (SNP) assay was developed for identifying L. williamsii; this assay reliably detects SNPs within chloroplast DNA (rbcL, matK, and trnL-trnF IGS) and was validated for identifying Lophophora and L. williamsii simultaneously. The chloroplast DNA sequences from four L. williamsii and three L. diffusa plants were obtained and compared using DNA sequence data from approximately 300 other Cactaceae species available in GenBank. From this sequence data, a total of seven SNPs were determined to be suitable for identifying L. williamsii. A multiplex assay was constructed using the ABI PRISM^® SNaPshot™ Multiplex Kit (Applied Biosystems, Forster City, CA) to analyze species-specific SNPs. Using this multiplex assay, we clearly distinguished the Lophophora among 19 species in the Cactaceae family. Additionally, L. williamsii was distinguished from L. diffusa. These results suggest that the newly developed assay may help resolve crimes related to illegal distribution and use. This multiplex assay will be useful for the genetic identification of L. williamsii and can complement conventional methods of detecting mescaline.     

Cytochrome b based genetic differentiation of Indian wild pig (Sus scrofa cristatus) and domestic pig (Sus scrofa domestica) and its use in wildlife forensics

The Indian wild pig (Sus scrofa cristatus) is a protected species and listed in the Indian Wildlife (Protection) Act, 1972. The wild pig is often hunted illegally and sold in market as meat warranting punishment under law. To avoid confusion in identification of these two subspecies during wildlife forensic examinations, we describe genetic differentiation of Indian wild and domestic pigs using a molecular technique. Analysis of sequence generated from the partial fragment (421 bp) of mitochondrial DNA (mtDNA) cytochrome b (Cyt b) gene exhibited unambiguous (> 3%) genetic variation between Indian wild and domestic pigs. We observed nine forensically informative nucleotide sequence (FINS) variations between Indian wild and domestic pigs. The overall genetic variation described in this study is helpful in forensic identification of the biological samples of wild and domestic pigs. It also helped in differentiating the Indian wild pig from other wild pig races. This study indicates that domestic pigs in India are not descendent of the Indian wild pig, however; they are closer to the other wild pig races found in Asia and Europe.     

DNA条形码技术在法庭科学种属鉴定中的应用

DNA条形码(DNA Barcoding)技术提出10余年,目前已成为国内外食品安全和动物保护等相关领域种属鉴定的新热点,该技术可实现生物样本的快速、准确识别和种属鉴定,为司法实践提供所需的科学证据。本文综述了DNA条形码技术的原理、发展及应用前景。     

基于12s rRNA的犀牛角制品DNA条形码鉴定分析

目的通过对几种涉案犀牛角制品的12s rRNA条形码序列比对分析,探析12s rRNA在犀牛角制品种属鉴定中的应用可行性。方法以3个案件中的涉案犀牛角制品为材料,采用改良的基因组DNA提取方法,PCR扩增DNA条形码片段12s rRNA。结果通过序列比对与分析,表明12s rRNA可将涉案犀牛角制品鉴定到种的水平。结论 DNA条形码12s rRNA可以作为一个新手段对形态上无法鉴别的犀牛角制品进行准确的种属鉴定,为案件的定性与量刑提供可靠的依据。     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

Identification of a forensic case using microscopy and forensically informative nucleotide sequencing (FINS): A case study of small Indian civet (Viverricula indica)

The exhibits obtained in wildlife offence cases quite often present a challenging situation for the forensic expert. The selection of proper approach for analysis is vital for a successful analysis. A generalised forensic analysis approach should proceed from the use of non-destructive techniques (morphological and microscopic examination) to partially destructive and finally destructive techniques (DNA analysis). The findings of non-destructive techniques may sometime be inconclusive but they definitely help in steering further forensic analysis in a proper direction. We describe a recent case where a very small dried skin piece (< 0.05 mg) with just one small trimmed guard hair (0.4 cm) on it was received for species identification. The single guard hair was examined microscopically to get an indication of the type of species. We also describe the extraction procedure with a lower amount of sample, using an automated extraction method (Qiagen Biorobot EZ1^®) and PCR amplification of three mitochondrial genes (16s rRNA, 12s rRNA and cytochrome b) for species identification. Microscopic examination of the single hair indicated a viverrid species but the initial DNA analysis with 16s rRNA (through NCBI BLAST) showed the highest homology (93%) with a hyaenid species (Hyaena hyaena). However, further DNA analysis based on 12s rRNA and cytochrome b gene proved that the species was indeed a viverrid i.e. Viverricula indica (small Indian civet). The highest homology shown with a Hyaenid species by the 16s rRNA sequence from the case sample was due to lack of a 16s rRNA sequence for Viverricula indica in the NCBI data base. The case highlights the importance of morphological and microscopic examinations in wildlife offence cases. With respect to DNA extraction technology we found that automatic extraction method of Biorobot EZ1^® (Qiagen) is quite useful with less amount of sample (much below recommended amount).     

Rapid and non-destructive identification of claws using ATR-FTIR spectroscopy–A novel approach in wildlife forensics

Differentiation and identification of Royal Bengal Tiger (Panthera tigris tigris) and Indian Leopard (Panthera pardus fusca) claws is a challenging task in wildlife forensics, due to similarity in their morphology, anatomy and chemical compositions as both the species are closely related to each other genetically. ATR-FTIR spectroscopy, which offers a non-destructive and safe alternative technique to other conventional methods, has been employed in the present work to differentiate claws of Royal Bengal Tiger and Indian Leopard. An attempt has been made to differentiate 31 reference claw samples from 16 different Royal Bengal Tigers, 15 different Indian Leopards, and 10 fake claws using ATR-FTIR spectroscopy supplemented with PCA, PLS-DA, and LDA. PCA could not distinguish the samples of two closely related species among themselves as well as from the fake claws. On the other hand, PLS-DA and LDA models both yielded highly significant classification rate for differentiation among the samples of Royal Bengal Tiger, Indian Leopard, and their fake counterparts. Further, seven blind claw samples that were pretended to be unknown to the analyst of both the species are also examined and identified correctly to their respective groups. The R-Square value obtained for PLS-DA model to differentiate Royal Bengal Tiger, Indian Leopard, and fake claws is 0.99, which is highly significant for predictive accuracy. This study shows that ATR-FTIR spectroscopy with PLS-DA/LDA has a potential to present a rapid, non-destructive, reliable, and eco-friendly approach for the accurate identification and differentiation of Royal Bengal Tiger and Indian Leopard claws.     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.     

DNA Barcoding in Forensic Entomology – Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species,

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High‐quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1‐5P’ DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high‐quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.     

Molecular identification of forensically important fly species in Spain using COI barcodes

Species identification with DNA barcodes has been proven to be effective on different organisms and, particularly, has become a routinely used and quite accurate tool in forensic entomology to study necrophagous Diptera species. In this study, we analysed 215 specimens belonging to 42 species of 17 genera, from 9 different Diptera families. Flies were collected in 39 Spanish localities of the Iberian Peninsula sampled across three years in the four seasons. Intraspecific variation ranged from 0 to 2.46% whereas interspecific variation fluctuated from 3.07 to 14.59%, measuring 651 pb of the cytochrome oxidase subunit one (COI) gene. Neighbour-Joining analysis was carried out to investigate the molecular identification capabilities of the barcoding region, recovering almost all species as distinct monophyletic groups. The species groupings were generally consistent with morphological and molecular identifications. This work, which is the first with this intensive and extensive sampling in this area, shows that the COI barcode is an appropriate marker for unambiguous identification of forensically important Diptera in Spain.     

An Assessment of the Utility of Universal and Specific Genetic Markers for Opium Poppy Identification

Abstract: The proper identification of illicit plants such as Papaver somniferum L (opium poppy) is important for law enforcement agencies. The identification of opium poppy was presently tested using 10 genetic markers that are universal for all plants or specific to a few poppy plants. The genetic distances of universal markers such as nuclear internal transcribed spacer (ITS), 18S rRNA, plastid rbcL, and trnL‐trnF intergenic spacer (IGS) of 14 species included in the Papaveraceae and Fumariaceae family were acquired by sequence comparisons. Both the ITS region and trnL‐trnF IGS showed high levels of interspecific divergence. Six Papaver genera‐specific markers were developed from coding regions involved in morphine biosynthesis. Three markers (TYDC, NCS, and BBE) produced amplicons only in opium poppy, providing a presence/absence test for opium poppy, while three additional markers (CYP80B1, SAT, and COR) were genus specific. These 10 markers might be useful for the forensic DNA analysis of opium poppy.     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.