Antibacterial activity of Arthrobacter strains isolated from Great Gobi A Strictly Protected Area, Mongolia.

Desert soil hosts many microorganisms, whose activities are essential from an ecological viewpoint. Moreover, they are of great anthropic interest. The knowledge of extreme environments microbiomes may be beneficial for agriculture, technology, and human health. In this study, 11 Arthrobacter strains from topsoil samples collected from the Great Gobi A Strictly Protected Area in the Gobi Desert, were characterized by a combination of different techniques. The phylogenetic analysis, performed using their 16S rDNA sequences and the most similar Arthrobacter sequences found in databases, revealed that most of them were close to A. crystallopoietes, while others joined a sister group to the clade formed by A. humicola, A. pascens, and A. oryzae. The resistance of each strain to different antibiotics, heavy-metals, and NaCl was also tested as well as the inhibitory potential against human pathogens (i.e., Burkholderia ssp., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus ssp.) via cross-streaking, to check the production of metabolites with antimicrobial activity. Data obtained revealed that all strains were resistant to heavy metals and were able to strongly interfere with the growth of many of the human pathogens tested. The volatile organic compounds (VOCs) profile of the 11 Arthrobacter strains was also analyzed. A total of 16 different metabolites were found, some of which were already known for having an inhibitory action against different Gram-positive and Gram-negative bacteria. Isolate MS-3A13, producing the highest quantity of VOCs, is the most efficient against Burkholderia cepacia complex (Bcc), K. pneumoniae, and coagulase-negative Staphylococci (CoNS) strains. This work highlights the importance of understanding microbial populations' phenotypical characteristics and dynamics in extreme environments to uncover the antimicrobial potential of new species and strains.

The Great Gobi A Strictly Protected Area: Characterization of Soil Bacterial Communities from Four Oases.

Understanding how microbial communities survive in extreme environmental pressure is critical for interpreting ecological patterns and microbial diversity. Great Gobi A Strictly Protected Area represents an intriguing model for studying the bacterial community since it is a protected and intact wild area of the Mongolian desert. In this work, the composition of a bacterial community of the soil from four oases was characterized by extracting total DNA and sequencing through the Illumina NovaSeq platform. In addition, the soil's chemical and physical properties were determined, and their influence on shaping the microbial communities was evaluated. The results showed a high variability of bacterial composition among oases. Moreover, combining specific chemical and physical parameters significantly shapes the bacterial community among oases. Data obtained suggested that the oases were highly variable in physiochemical parameters and bacterial communities despite the similar extreme climate conditions. Moreover, core functional microbiome were constituted by aerobic chemoheterotrophy and chemoheterotrophy, mainly contributed by the most abundant bacteria, such as Actinobacteriota, Pseudomonadota, and Firmicutes. This result supposes a metabolic flexibility for sustaining life in deserts. Furthermore, as the inhabitants of the extreme regions are likely to produce new chemical compounds, isolation of key taxa is thus encouraged.

Fine-scale spatial genetic structure and dispersal among Italian smooth newt populations in a rural landscape.

Amphibians are particularly sensitive to habitat loss and fragmentation caused by the intensification and modernization of farming occurring in the second half of the twentieth century in the Mediterranean basin. However, artificial water bodies, associated with traditional husbandry, proved to be important surrogate for amphibian feeding and reproduction. Here, multilocus genotypes were used to investigate the spatial population structure of Lissotriton vulgaris meridionalis and the role of drinking troughs in supporting viable breeding populations within a rural landscape interested by traditional husbandry and agriculture. Our genetic analysis highlighted the conservation value and the potential stepping-stone function of artificial aquatic sites in the dispersal of the species and for the gene flow maintenance. Indeed, populations of drinking troughs show allelic richness and heterozygosity levels comparable to those from natural ponds and there is no great evidence of genetic bottlenecks. A complex system of artificial aquatic sites and few natural wetlands was identified sustaining a well-structured network of demes highly interconnected with themselves and natural aquatic sites. The conservation of the identified genetic clusters may be useful to prevent further population declines and future loss of genetic diversity within the study area characterized by scarce natural wetlands that frequently dried because of agricultural practices and strong seasonality. Site-specific protection measures are needed to contrast the progressive disappearance of drinking troughs observed in the last years in Italy because of the abandonment of traditional farming practices in favour of modern agriculture and intensive farming.

Weather, sex and body condition affect post-fledging migration behaviour of the greater flamingo Phoenicopterus roseus.

BACKGROUND: Understanding which intrinsic and extrinsic factors dictate decision-making processes such as leaving the natal area or not (migratory vs resident strategy), departure time, and non-breeding destination are key-issues in movement ecology. This is particularly relevant for a partially migratory meta-population in which only some individuals migrate. METHODS: We investigated these decision making-processes for 40 juvenile greater flamingos Phoenicopterus roseus fledged in three Mediterranean colonies and equipped with GPS-GSM devices. RESULTS: Contrary to the body size and the dominance hypotheses, juveniles in better body condition were more likely to migrate than those in worse conditions, which opted for a residence strategy. Flamingo probability of departure was not associated with an increase in local wind intensity, but rather with the presence of tailwinds with departure limited to night-time mostly when the wind direction aligned with the migratory destination. Moreover, a positive interaction between tailwind speed and migration distance suggested that juveniles opted for stronger winds when initiating long-distance journeys. In contrast to previous studies, the prevailing seasonal winds were only partially aligned with the migratory destination, suggesting that other factors (e.g., adults experience in mix-aged flocks, availability of suitable foraging areas en route, density-dependence processes) may be responsible for the distribution observed at the end of the first migratory movement. We found potential evidence of sex-biased timing of migration with females departing on average 10 days later and flying ca. 10 km/h faster than males. Female flight speed, but not male one, was positively influenced by tailwinds, a pattern most likely explained by sexual differences in mechanical power requirements for flight (males being ca. 20% larger than females). Furthermore, juveniles considerably reduced their flight speeds after 400 km from departure, highlighting a physiological threshold, potentially linked to mortality risks when performing long-distance non-stop movements. CONCLUSION: These results suggest that not only intrinsic factors such as individual conditions and sex, but also extrinsic factors like weather, play critical roles in triggering migratory behaviour in a partially migratory metapopulation. Furthermore, social factors, including conspecific experience, should be taken into consideration when evaluating the adaptive processes underlying migration phenology, flight performance, and final destination selection.

Anthropogenic hybridization and its influence on the adaptive potential of the Sardinian wild boar (Sus scrofa meridionalis).

The wild boar (Sus scrofa meridionalis) arrived in Sardinia with the first human settlers in the early Neolithic with the potential to hybridize with the domestic pig (S. s. domesticus) throughout its evolution on the island. In this paper, we investigated the possible microevolutionary effects of such introgressive hybridization on the present wild boar population, comparing Sardinian wild specimens with several commercial pig breeds and Sardinian local pigs, along with a putatively unadmixed wild boar population from Central Italy, all genotyped with a medium density SNP chip. We first aimed at identifying hybrids in the population using different approaches, then examined genomic regions enriched for domestic alleles in the hybrid group, and finally we applied two methods to find regions under positive selection to possibly highlight instances of domestic adaptive introgression into a wild population. We found three hybrids within the Sardinian sample (3.1% out of the whole dataset). We reported 11 significant windows under positive selection with a method that looks for overly differentiated loci in the target population, compared with other two populations. We also identified 82 genomic regions with signs of selection in the domestic pig but not in the wild boar, two of which overlapped with genomic regions enriched for domestic alleles in the hybrid pool. Genes in these regions can be linked with reproductive success. Given our results, domestic introgression does not seem to be pervasive in the Sardinian wild boar. Nevertheless, we suggest monitoring the possible spread of advantageous domestic alleles in the coming years.

Possible origins and implications of atypical morphologies and domestication-like traits in wild golden jackals (Canis aureus).

Deciphering the origins of phenotypic variations in natural animal populations is a challenging topic for evolutionary and conservation biologists. Atypical morphologies in mammals are usually attributed to interspecific hybridisation or de-novo mutations. Here we report the case of four golden jackals (Canis aureus), that were observed during a camera-trapping wildlife survey in Northern Israel, displaying anomalous morphological traits, such as white patches, an upturned tail, and long thick fur which resemble features of domesticated mammals. Another individual was culled under permit and was genetically and morphologically examined. Paternal and nuclear genetic profiles, as well as geometric morphometric data, identified this individual as a golden jackal rather than a recent dog/wolf-jackal hybrid. Its maternal haplotype suggested past introgression of African wolf (Canis lupaster) mitochondrial DNA, as previously documented in other jackals from Israel. When viewed in the context of the jackal as an overabundant species in Israel, the rural nature of the surveyed area, the abundance of anthropogenic waste, and molecular and morphological findings, the possibility of an individual presenting incipient stages of domestication should also be considered.

Microsatellite Characterization and Panel Selection for Brown Bear (Ursus arctos) Population Assessment.

An assessment of the genetic diversity and structure of a population is essential for designing recovery plans for threatened species. Italy hosts two brown bear populations, Ursus arctos marsicanus (Uam), endemic to the Apennines of central Italy, and Ursus arctos arctos (Uaa), in the Italian Alps. Both populations are endangered and occasionally involved in human-wildlife conflict; thus, detailed management plans have been in place for several decades, including genetic monitoring. Here, we propose a simple cost-effective microsatellite-based protocol for the management of populations with low genetic variation. We sampled 22 Uam and 22 Uaa individuals and analyzed a total of 32 microsatellite loci in order to evaluate their applicability in individual identification. Based on genetic variability estimates, we compared data from four different STR marker sets, to evaluate the optimal settings in long-term monitoring projects. Allelic richness and gene diversity were the highest for the Uaa population, whereas depleted genetic variability was noted for the Uam population, which should be regarded as a conservation priority. Our results identified the most effective STR sets for the estimation of genetic diversity and individual discrimination in Uam (9 loci, PIC 0.45; PID 2.0 × 10-5), and Uaa (12 loci, PIC 0.64; PID 6.9 × 10-11) populations, which can easily be utilized by smaller laboratories to support local governments in regular population monitoring. The method we proposed to select the most variable markers could be adopted for the genetic characterization of other small and isolated populations.

Endophytic Bacteria Associated with Origanum heracleoticum L. (Lamiaceae) Seeds

Seed-associated microbiota are believed to play a crucial role in seed germination, seedling establishment, and plant growth and fitness stimulation, due to the vertical transmission of a core microbiota from seeds to the next generations. It might be hypothesized that medicinal and aromatic plants could use the seeds as vectors to vertically transfer beneficial endophytes, providing plants with metabolic pathways that could influence phytochemicals production. Here, we investigated the localization, the structure and the composition of the bacterial endophytic population that resides in Origanum heracleoticum L. seeds. Endocellular bacteria, surrounded by a wall, were localized close to the aleurone layer when using light and transmission electron microscopy. From surface-sterilized seeds, cultivable endophytes were isolated and characterized through RAPD analysis and 16S RNA gene sequencing, which revealed the existence of a high degree of biodiversity at the strain level and the predominance of the genus Pseudomonas. Most of the isolates grew in the presence of six selected antibiotics and were able to inhibit the growth of clinical and environmental strains that belong to the Burkholderia cepacia complex. The endophytes production of antimicrobial compounds could suggest their involvement in plant secondary metabolites production and might pave the way to endophytes exploitation in the pharmaceutical field.

"Guess Who's Coming to Dinner": Molecular Tools to Reconstruct multilocus Genetic Profiles from Wild Canid Consumption Remains.

Non-invasive genetic sampling is a practical tool to monitor pivotal ecological parameters and population dynamic patterns of endangered species. It can be particularly suitable when applied to elusive carnivores such as the Apennine wolf (Canis lupus italicus) and the European wildcat (Felis silvestris silvestris), which can live in overlapping ecological contexts and sometimes share their habitats with their domestic free-ranging relatives, increasing the risk of anthropogenic hybridisation. In this case study, we exploited all the ecological and genetic information contained in a single biological canid faecal sample, collected in a forested area of central Italy, to detect any sign of trophic interactions between wolves and European wildcats or their domestic counterparts. Firstly, the faecal finding was morphologically examined, showing the presence of felid hair and claw fragment remains. Subsequently, total genomic DNA contained in the hair and claw samples was extracted and genotyped, through a multiple-tube approach, at canid and felid diagnostic panels of microsatellite loci. Finally, the obtained individual multilocus genotypes were analysed with reference wild and domestic canid and felid populations to assess their correct taxonomic status using Bayesian clustering procedures. Assignment analyses classified the genotype obtained from the endothelial cells present on the hair sample as a wolf with slight signals of dog ancestry, showing a qi = 0.954 (C.I. 0.780-1.000) to the wolf cluster, and the genotype obtained from the claw as a domestic cat, showing a qi = 0.996 (95% C.I. = 0.982-1.000) to the domestic cat cluster. Our results clearly show how a non-invasive multidisciplinary approach allows the cost-effective identification of both prey and predator genetic profiles and their taxonomic status, contributing to the improvement of our knowledge about feeding habits, predatory dynamics, and anthropogenic hybridisation risk in threatened species.

Genomic Analysis of Endophytic Bacillus-Related Strains Isolated from the Medicinal Plant Origanum vulgare L. Revealed the Presence of Metabolic Pathways Involved in the Biosynthesis of Bioactive Compounds.

Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant Origanum vulgare L. and able to antagonize the growth of opportunistic pathogens of cystic fibrosis patients. The in silico analysis revealed the presence of gene clusters involved in the production of antimicrobial compounds, such as paeninodin, paenilarvins, polymyxin, and paenicidin A. Endophytes' adaptation to the plant microenvironment was evaluated through the analysis of the presence of antibiotic resistance genes in the four genomes. The diesel fuel degrading potential was also tested. Strains grew in minimum media supplemented with diesel fuel, but no n-alkanes degradation genes were found in their genomes, suggesting that diesel fuel degradation might occur through other steps involving enzymes catalyzing the oxidation of aromatic compounds.

A reduced SNP panel to trace gene flow across southern European wolf populations and detect hybridization with other Canis taxa.

Intra- and inter-specific gene flow are natural evolutionary processes. However, human-induced hybridization is a global conservation concern across taxa, and the development of discriminant genetic markers to differentiate among gene flow processes is essential. Wolves (Canis lupus) are affected by hybridization, particularly in southern Europe, where ongoing recolonization of historic ranges is augmenting gene flow among divergent populations. Our aim was to provide diagnostic canid markers focused on the long-divergent Iberian, Italian and Dinaric wolf populations, based on existing genomic resources. We used 158 canid samples to select a panel of highly informative single nucleotide polymorphisms (SNPs) to (i) distinguish wolves in the three regions from domestic dogs (C. l. familiaris) and golden jackals (C. aureus), and (ii) identify their first two hybrid generations. The resulting 192 SNPs correctly identified the five canid groups, all simulated first-generation (F1) hybrids (0.482 ≤ Qi ≤ 0.512 between their respective parental groups) and all first backcross (BC1) individuals (0.723 ≤ Qi ≤ 0.827 to parental groups). An assay design and test with invasive and non-invasive canid samples performed successfully for 178 SNPs. By separating natural population admixture from inter-specific hybridization, our reduced panel can help advance evolutionary research, monitoring, and timely conservation management.

Phylogeography and population genetic structure of the European roe deer in Switzerland following recent recolonization.

In the early 1800s, the European roe deer (Capreolus capreolus) was probably extirpated from Switzerland, due to overhunting and deforestation. After a federal law was enacted in 1875 to protect lactating females and young, and limiting the hunting season, the roe deer successfully recovered and recolonized Switzerland. In this study, we use mitochondrial DNA and nuclear DNA markers to investigate the recolonization and assess contemporary genetic structure in relation to broad topographic features, in order to understand underlying ecological processes, inform future roe deer management strategies, and explore the opportunity for development of forensic traceability tools. The results concerning the recolonization origin support natural, multidirectional immigration from neighboring countries. We further demonstrate that there is evidence of weak genetic differentiation within Switzerland among topographic regions. Finally, we conclude that the genetic data support the recognition of a single roe deer management unit within Switzerland, within which there is a potential for broad-scale geographic origin assignment using nuclear markers to support law enforcement.

Cross-Amplification in Strigiformes: A New STR Panel for Forensic Purposes.

Strigiformes are affected by a substantial decline mainly caused by habitat loss and destruction, poaching, and trapping. Moreover, the increasing trend in bird trade and the growing interest in wild-caught rather than captive-bred birds are expected to encourage illegal trade. The biomolecular investigation represents a valuable tool to track illegal trade and to explore the genetic variability to preserving biodiversity. Microsatellite loci (STRs) are the most used markers to study genetic variability. Despite the availability of species-specific microsatellite loci in Strigiformes, a unique panel permitting the description of the genetic variability across species has not been identified yet. We tested 32 highly polymorphic microsatellite markers to evaluate the reliability of a unique microsatellite panel in different species of Strigiformes and its use for conservation and forensic purposes. We included in the study 84 individuals belonging to 28 parental groups and 11 species of Strigiformes. After screening polymorphic microsatellite loci, the description of genetic variability, and the kinship assessment, we characterized a final panel of 12 microsatellite loci able to identify individuals in 9 Strigiformes species. This STR panel might support the authorities in the forensic investigation for suspected smugglers and false parental claims; moreover, it can be useful to evaluate relatedness among individuals in captive-bred populations and to implement research projects finalized to the description of the genetic variability in wild populations.

First core microsatellite panel identification in Apennine brown bears (Ursus arctos marsicanus): a collaborative approach.

BACKGROUND: The low cost and rapidity of microsatellite analysis have led to the development of several markers for many species. Because in non-invasive genetics it is recommended to genotype individuals using few loci, generally a subset of markers is selected. The choice of different marker panels by different research groups studying the same population can cause problems and bias in data analysis. A priority issue in conservation genetics is the comparability of data produced by different labs with different methods. Here, we compared data from previous and ongoing studies to identify a panel of microsatellite loci efficient for the long-term monitoring of Apennine brown bears (Ursus arctos marsicanus), aiming at reducing genotyping uncertainty and allowing reliable individual identifications overtimes. RESULTS: We examined all microsatellite markers used up to now and identified 19 candidate loci. We evaluated the efficacy of 13 of the most commonly used loci analyzing 194 DNA samples belonging to 113 distinct bears selected from the Italian national biobank. We compared data from 4 different marker subsets on the basis of genotyping errors, allelic patterns, observed and expected heterozygosity, discriminatory powers, number of mismatching pairs, and probability of identity. The optimal marker set was selected evaluating the low molecular weight, the high discriminatory power, and the low occurrence of genotyping errors of each primer. We calibrated allele calls and verified matches among genotypes obtained in previous studies using the complete set of 13 STRs (Short Tandem Repeats), analyzing six invasive DNA samples from distinct individuals. Differences in allele-sizing between labs were consistent, showing a substantial overlap of the individual genotyping. CONCLUSIONS: The proposed marker set comprises 11 Ursus specific markers with the addition of cxx20, the canid-locus less prone to genotyping errors, in order to prevent underestimation (maximizing the discriminatory power) and overestimation (minimizing the genotyping errors) of the number of Apennine brown bears. The selected markers allow saving time and costs with the amplification in multiplex of all loci thanks to the same annealing temperature. Our work optimizes the available resources by identifying a shared panel and a uniform methodology capable of improving comparisons between past and future studies.

Exploring the Gut Microbiome Alteration of the European Hare (Lepus europaeus) after Short-Term Diet Modifications.

This study aimed to characterise the gut microbiome composition of European hares (Lepus europaeus) and its potential changes after a short-term diet modification. The high sensitivity of European hare to habitat changes makes this species a good model to analyse possible alterations in gut microbiome after the introduction of additional nourishment into the diet. In total, 20 pairs were chosen for the experiments; 10 pairs formed the control group and were fed with standard fodder. The other 10 pairs represented the experimental group, whose diet was integrated with apples and carrots. The DNA from fresh faecal pellets collected after 4 days from the start of the experiment was extracted and the V3-V4 hypervariable regions were amplified and sequenced using the Illumina MiSeq® platform. The obtained amplicon sequence variants were classified into 735 bacterial genera belonging to 285 families and 36 phyla. The control and the experimental groups appeared to have a homogenous dispersion for the two taxonomic levels analysed with the most abundant phyla represented by Bacteroidetes and Firmicutes. No difference between control and experimental samples was detected, suggesting that the short-term variation in food availability did not alter the hares' gut microbiome. Further research is needed to estimate significant time threshold.

Feral pigeon populations: their gene pool and links with local domestic breeds.

Columba livia is a wild bird whose domestication has led to a large number of pigeon breeds. The occasional loss or straying of domestic birds determined the origin of feral pigeons, which are now widespread all around the world. In this study, we assumed that the main contribution to feral populations is provided by domestic breeds reared in the same areas. We tested this hypothesis by analysing the variability of 12 microsatellite loci in nine Italian feral populations sampled in areas with different intensities of breeding and selecting domestic breeds. We included in the analysis samples belonging to ten domestic lineages commonly bred in Italy. The pattern of geographic differentiation of feral populations turned out to be rather complex and only partially explained by the geographic distance between populations. This pattern can be understood only when the domestic breeds were included in the analysis. In particular, feral populations located in regions with a long-lasting tradition of pigeon breeding showed a high level of admixture with domestic breeds, in particular with Racing Homer and Piacentino. Ferals from Bolzano, Venice and Sassari were characterized by unique genetic components, mostly not shared by other feral populations and by the considered domestic breeds. Our results further emphasize the complex genetic structure of feral populations whose origin can be properly investigated by taking into account the pool of domestic pigeons bred in the considered area.

A standardized approach to empirically define reliable assignment thresholds and appropriate management categories in deeply introgressed populations.

Anthropogenic hybridization is recognized as a major threat to the long-term survival of natural populations. While identifying F1 hybrids might be simple, the detection of older admixed individuals is far from trivial and it is still debated whether they should be targets of management. Examples of anthropogenic hybridization have been described between wolves and domestic dogs, with numerous cases detected in the Italian wolf population. After selecting appropriate wild and domestic reference populations, we used empirical and simulated 39-autosomal microsatellite genotypes, Bayesian assignment and performance analyses to develop a workflow to detect different levels of wolf x dog admixture. Membership proportions to the wild cluster (qiw) and performance indexes identified two q-thresholds which allowed to efficiently classify the analysed genotypes into three assignment classes: pure (with no or negligible domestic ancestry), older admixed (with a marginal domestic ancestry) and recent admixed (with a clearly detectable domestic ancestry) animals. Based on their potential to spread domestic variants, such classes were used to define three corresponding management categories: operational pure, introgressed and operational hybrid individuals. Our multiple-criteria approach can help wildlife managers and decision makers in more efficiently targeting the available resources for the long-term conservation of species threatened by anthropogenic hybridization.

An improved microsatellite panel to assess genetic variability of the Italian smooth newt (Lissotriton vulgaris meridionalis).

Habitat loss and fragmentation are threatening amphibians by increasing population isolation. However, artificial waterbodies created for livestockmay contrast this phenomenon by providing surrogate habitats for amphibians. Here, we performed a genetic study on an amphibian species, Lissotriton vulgaris meridionalis, in a rural area in central Italy where natural wetlands are disappearing and drinking troughs for cattle watering are widespread. Specifically, we tested a panel of microsatellite markers to identify a suitable tool for addressing conservation genetic issues of this species that is undergoing severe local decline. Twelve of the 20 tested loci produced reliable amplifications and were polymorphic. Three distinct units with a low level of gene flow were distinguished and the population genetic structuring overlapped with geographic distribution. Such loci will be useful to assess the genetic diversity of the species across multiscale levels for its management and conservation.

Wildlife DNA forensics against crime: resolution of a case of tortoise theft.

A paternity test was used to investigate a robbery case involving captive individuals of Greek tortoise (Testudo graeca). Six tortoises were allegedly stolen from a private breeder and offered for sale on the web by the supposed thief. The stolen tortoises were confiscated by the rangers of the State Forestry Corps (CFS). A panel of 14 autosomal microsatellite loci was used to genotype the seized tortoises and ten individuals assumed to be legally owned by the breeder. Kinship analyses reliably reconstructed the tortoise pedigree, demonstrating parent-offspring relationships among the owned and the stolen tortoises. As correctly declared by the breeder, four of the six stolen individuals belonged to the same family group of the ten legally owned tortoises. Results indicate that genetic identification procedures can provide valuable evidence and give useful support against illegal wildlife traffic.

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis.

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.