Adrian Linacre

Cytochrome b gene for species identification of the conservation animals

A partial DNA sequence of cytochrome b gene was used to identify the remains of endangered animals and species endemic to Taiwan. The conservation of animals species included in this study were: the formosan gem-faced civets, leopard cats, tigers, clouded leopards, lion, formosan muntjacs, formosan sika deers, formosan sambars, formosan serows, water buffalo, formosan pangolins and formosan macaques. The control species used included domestic cats, domestic dogs, domestic sheeps, domestic cattles, domestic pigs and humans. Heteroplasmy was detected in the formosan macaque, domestic pig and domestic cats. The frequencies of heteroplasmy in these animals were about 0.25% (1 in 402bp). Sequences were aligned by Pileup program of GCG computer package, and the phylogenetic tree was constructed by the neighbor-joining method. The results of sequence comparison showed that the percentage range of sequence diversity in the same species was from 0.25 to 2.74%, and that between the different species was from 5.97 to 34.83%. The results of phylogenetic analysis showed that the genetic distance between the different species was from 6.33 to 40.59. Animals of the same species, both the endangered animal species and domestic animals, were clustered together in the neighbor-joining tree. Three unknown samples of animal remains were identified by this system. The partial sequence of cytochrome b gene adopted in this study proved to be usable for animal identification.

New guidelines for the publication of genetic population data.

In 2000 a new policy concerning the publication of population genetic data was set up in Forensic Science International [1] with the introduction of a new section entitled ‘‘Announcement of population data’’. Subsequently in 2010 [2] a new section on ‘‘Forensic Population Genetics’’ was introduced, and recommendations were redefined. FSI: Genetics is one of the few journals still considering population genetic data for publication and we strongly believe that this policy has contributed to the dissemination of common standards in the field all over the world and also to motivate labs and people to embark in research in the area of forensic genetics. For this reason it is our intention to continue with this policy, and recently an associate editor exclusively devoted to this topic was appointed to the journal. Despite having defined a more detailed procedure for acceptance, our journal is still receiving a massive number of submissions of varying quality in this area. Therefore it has become necessary to raise the threshold regarding the acceptance of this type of publication to ensure a high standard of published data. In addition we want to improve the submission, reviewing and publication procedures, and to correct some aspects that we have detected such as the obligation to meet ethical standards in the collection of samples including informed consent and approval by ethical committees. For this reason, we have decided to publish new guidelines for the publication of population genetic data in the journal.

Update of the guidelines for the publication of genetic population data

Due to the massive number of submissions of varying quality with population genetic data we decided one year ago to raise the threshold regarding the acceptance of this type of publications to ensure a high standard of published data and, therefore, we updated the FSI: Genetics 2010 guidelines [1] to a new set of recommendations [2]. In the 2013 guidelines in addition to some new requirements in the procedure and regarding ethical standards, we significantly increased the number of markers and samples required for submission to the journal of papers presenting population data alone, with no additional information on new methods or other forensically relevant findings. We have been working during last year with these new recommendations. During this period, we have received a number of critical comments from authors that we have considered and therefore we have decided to make some amendments in the requirements. The first change refers to the minimal number of autosomal STRs. The 2013 guidelines indicate that for data comprising autosomal STR genotypes only, 17 different autosomal STR loci are required as a minimum. This was based on the average number of STRs that most laboratories are routinely using – in most cases forensic laboratories are using at least two PCR multiplexes (commercial kits or homemade) on their routine work for a minimal number of 17 autosomal STR markers. However, this number would exclude laboratories and national and international compilation efforts that are working with just one kit, for some of the most commonly used kits. Whilst we certainly recognize the need to restrict the number of small data sets submitted we think that the forensic community would benefit from the publication of a large and nationally important data set such as the ones that are being generated in some countries. For this reason we have decided to reinstate the 2010 recommendations requiring 15 STRs only. Concerning X chromosome a minimum number of 12 STRs will be required and for the Y chromosome a minimum of 17 STRs will be required as well, taking into account that the core minimum haplotype (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385) [3,4] must be provided if it has not been previously analyzed in the same population sample. Collaborative efforts to produce large datasets are strongly encouraged and, therefore, the minimal number of markers should not be a limitation to the publication of large National or International collaborative databasing efforts when a significant number of laboratories and samples are involved. There are also changes in the requirements for the minimum number of samples. We maintain the threshold of 500 samples for

ISFG: Recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations

The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only.

Species identification of rhinoceros horns using the cytochrome b gene

Material suspected of originating from species of Rhinoceros is frequently seized by forensic organizations investigating trade in endangered species. At present identification of the species is possible by DNA sequencing of the material, such as powdered rhinoceros horns. The unambiguous identification of rhino products using a 402 bp fragment of cytochrome b gene was investigated. This DNA sequence may not only assist in the identification of the unknown sample, but can be used to determine the phylogenetic relationships of rhinoceros species. Sequences of suspect rhinoceros horns were compared with the sequences registered in GenBank. The maximum value of genetic distance among white rhinoceros was 0.0176, and 0.0333 among black rhinoceros. In the comparison among rhinoceros species, the greatest genetic distance was between black and Indian rhinoceros (0.1564). The rhinoceros sequences extracted from GenBank and 13 samples in this study were clustered and separated from other mammals. Holstein cow was used as an out-group and was clustered with cattle in the phylogenetic tree. The results of this phylogenetic study also showed that there were four major branches among rhinoceros species from a common origin. The amplification of the 402 bp fragment of the cytochrome b gene was found to be able to detect rhinoceros DNA even in the ratio of 1:19 with Holstein cow DNA. In the initial identification of species from unknown powdered material, all the unknown samples were found to be from rhinoceroses. In phylogenetic analysis, the results supported the morphological hypothesis. The method used in this study can be applied in the identification of processed products of rhinoceros horns, such as sculptures, daggers, powders or even mixture powdered prescriptions.

Reconstructing Mammalian Phylogenies: A Detailed Comparison of the Cytochrome b and Cytochrome Oxidase Subunit I Mitochondrial Genes

The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cyt b more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cyt b correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cyt b also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5–2.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.

Generation of DNA profiles from fabrics without DNA extraction

DNA profiles can be obtained from fabrics where a person has made direct contact with clothing. A standard approach is to cut out a section of the fabric and then use a commercially available method to extract and isolate the DNA. Alternative methods to isolate DNA include the use of adhesive tape to remove traces of cellular material from the fabric prior to extraction. We report on a process to obtain full DNA profiles using direct amplification from a range of fabrics. The absence of an extraction step both reduces the opportunity for contamination and reduces the loss of DNA during the extraction process, increasing the sensitivity of the process of generating a DNA profile. The process does not require the use of commercially available extraction kits thus reducing the cost of generating a DNA profile from trace amounts of starting material. The results are in part dependent upon the nature of the fabric used to which the DNA has been transferred.

An overview to the investigative approach to species testing in wildlife forensic science

The extent of wildlife crime is unknown but it is on the increase and has observable effects with the dramatic decline in many species of flora and fauna. The growing awareness of this area of criminal activity is reflected in the increase in research papers on animal DNA testing, either for the identification of species or for the genetic linkage of a sample to a particular organism. This review focuses on the use of species testing in wildlife crime investigations. Species identification relies primarily on genetic loci within the mitochondrial genome; focusing on the cytochrome b and cytochrome oxidase 1 genes. The use of cytochrome b gained early prominence in species identification through its use in taxonomic and phylogenetic studies, while the gene sequence for cytochrome oxidase was adopted by the Barcode for Life research group. This review compares how these two loci are used in species identification with respect to wildlife crime investigations. As more forensic science laboratories undertake work in the wildlife area, it is important that the quality of work is of the highest standard and that the conclusions reached are based on scientific principles. A key issue in reporting on the identification of a particular species is a knowledge of both the intraspecies variation and the possible overlap of sequence variation from one species to that of a closely related species. Recent data showing this degree of genetic separation in mammalian species will allow greater confidence when preparing a report on an alleged event where the identification of the species is of prime importance. The aim of this review is to illustrate aspects of species testing in wildlife forensic science and to explain how a knowledge of genetic variation at the genus and species level can aid in the reporting of results.

Comparison of Cannabis sativa by Random Amplification of Polymorphic DNA (RAPD) and HPLC of cannabinoids: a preliminary study

Methods are described for the HPLC and genetic analysis of herbal Cannabis sativa. The latter method was applied to 17 plants grown simultaneously, at the same site. Sixteen of these samples were also compared using HPLC, which resulted in differentiation of the plants into 3 groups. Within two of these groups, the members could not be distinguished. By RAPD analysis, using certain combinations of primers and cladistic analysis, differentiation was possible between all but two of the plants. The use of the RAPD technique enables differentiation between samples that cannot be differentiated by HPLC analysis alone.

Forensic Applications of Infrared Imaging for the Detection and Recording of Latent Evidence

Abstract:  We report on a simple method to record infrared (IR) reflected images in a forensic science context. Light sources using ultraviolet light have been used previously in the detection of latent prints, but the use of infrared light has been subjected to less investigation. IR light sources were used to search for latent evidence and the images were captured by either video or using a digital camera with a CCD array sensitive to IR wavelength. Bloodstains invisible to the eye, inks, tire prints, gunshot residue, and charred document on dark background are selected as typical matters that may be identified during a forensic investigation. All the evidence types could be detected and identified using a range of photographic techniques. In this study, a one in eight times dilution of blood could be detected on 10 different samples of black cloth. When using 81 black writing inks, the observation rates were 95%, 88% and 42% for permanent markers, fountain pens and ball‐point pens, respectively, on the three kinds of dark cloth. The black particles of gunshot residue scattering around the entrance hole under IR light were still observed at a distance of 60 cm from three different shooting ranges. A requirement of IR reflectivity is that there is a contrast between the latent evidence and the background. In the absence of this contrast no latent image will be detected, which is similar to all light sources. The use of a video camera allows the recording of images either at a scene or in the laboratory. This report highlights and demonstrates the robustness of IR to detect and record the presence of latent evidence.