Identification of big game species by a universal cytochrome B primer pair through High-Resolution Melting

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Abstract

Species identification by DNA barcodes has become in recent years a very useful tool in molecular biology. But the degradation level shown in some samples, makes regular barcodes impractical for cases in which DNA is fragmented as it happens commonly in forensic casework. Therefore, the use of mini barcodes with sizes below 200 bp is recommended to achieve optimal amplifications. The present study is focused on the identification of five Iberian Peninsula big game species, Red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), brown bear (Ursus arctos) and wolf (Canis lupus), using a pair of universal primers that amplify a 148 bp Cytochrome B mini barcode. The analysis of amplicons was made using High-Resolution Melting and the results have shown a correct identification of the five big game species analyzed even in degraded samples.

Introduction

Levels of monitoring and control of poaching have been increased during last years, reducing the number of cases [1] but it still requires new tools for genetic identification of species in forensic casework.

DNA of forensic evidences from cinegetic samples is commonly highly degraded [2]. The small size of the fragments of the degraded DNA requires amplicons less than 200 bp for optimum sample amplification [3]. The analysis of reduced amplicons can be carried out by High-Resolution Melting (HRM). This technique has advantages such as low cost and short time, bringing the same level of discrimination of species that analytical methods based on Sanger sequencing.

The Cytochrome B (CytB) is a mitochondrial gene that has been widely used in taxonomic and forensic studies [4], [5]. The CytB provides an accurate reconstruction of the phylogeny of mammals, in the levels of super-order, order and family [6]. In addition, this locus maintains a high level of phylogenetic discrimination even when short amplicons are used.

The aim of this study was to design a pair of primers able to amplify a highly variable small region of 148 bp from the CytB gene for making a panel able to identify five game species by HRM technique.

Section snippets

Material and methods

Individuals from a total of seven species belonging to four families of two different orders were analyzed (Table 1). Muscle tissues and stool samples were obtained from several hunting associations and Cabarceno Nature Park (Cantabria, Spain). For the extraction of muscle tissues, 5 mg were taken and DNA was extracted by the salting out method, following the protocol of the Gentra Puregene tissue kit (Qiagen). 200 mg of stool samples were taken for DNA extraction using the Stool QIAmp DNA Mini

Results and discussion

The selection of species was performed taking into account sensitive big game species. On the one hand the three species most hunted by number of individuals in the Iberian Peninsula were chosen, red deer, roe deer and wild boar [1]. On the other hand, protected species because of their small number of individuals were included such as the brown bear and the wolf. Samples of two domestic species, dog and pig, were included to verify that different HRM profiles were obtained in wild and domestic

Conclusion

A pair of primers of Cytb gene that discriminated by HRM five game species of high economic and ecological value has been obtained. These primers amplify a small region of 148 bp and have been shown equally useful when amplify degraded samples.

Acknowledgements

A. Lopez-Oceja and D. Gamarra thank the Department of Economic Development and Competitiveness of the Government of the Basque Country for their predoctoral fellowships and the IT-833-13 Consolidated Group and UFI11/32 UPV/EHU for funding. Special thanks to Cabarceno Nature Park (Cantabria, Spain).

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Cited by (1)

  • Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene

    2017, Food Chemistry
    Citation Excerpt :

    HRM analysis could also be an adequate screening method in single source meat samples of unknown species not included in the present study, since it will show different melting profiles due to differences in the cyt b fragment analyzed. This fact has been noticed when comparing data with those previously described in Lopez-Oceja, Gamarra, Jiménez-Moreno, and de Pancorbo (2015), where differences in melting profiles obtained with the same pair of universal primers were observed in big game species. Hence, in samples with different melting profiles to those determined in this study, further analysis with techniques such as Sanger sequencing would be necessary to establish the species to which they belong.

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