Development of a 64 SNV panel for breed authentication in Iberian pigs and their derived meat products
Introduction
The exceptional sensorial and nutritional characteristics of Iberian meat and dry-cured products, highly appreciated by the consumers, are strongly associated to their Iberian pig breed origin. However, Iberian pigs are usually crossed with individuals from Duroc breed in order to accelerate their lean growth and improve the percentage on carcass of premium-cuts (Serrano, Valencia, Nieto, Lázaro, & Mateos, 2008; Ventanas, Ventanas, Jurado, & Estévez, 2006). Actually, the percentage of Duroc x Iberian crossbred slaughtered pigs far exceeds that of Iberian purebred, being 83.83% vs 16.17% of the total of Iberian census in 2018 (https://infoiberico.com/datos-y-censos/censos-de-cerdos-ibericos/). According to the Spanish legislation (RD 4/2014, https://www.boe.es/diario_boe/txt.php?id=BOE-A-2014-318) that regulates the labelling of Iberian fresh meat and dry-cured products (hams, shoulders and loin) they should only proceed from the progenies of Iberian dams mated with Iberian, Duroc or Duroc x Iberian crossbred sires. Accordingly, products are labelled as “100% Ibérico” when they come from Iberian purebred pigs or generically products are labelled as “Ibérico” even when they come from Duroc x Iberian crossbred animals. In this case, the percentage of Iberian breed must be indicated specifying if they come from pigs with a 75% of Iberian breed (progeny of Duroc x Iberian sires) or 50% of Iberian breed (progeny of Duroc sires).
Taking this into account, a consumers' concern on the authenticity of these highly-prized products exists, both in Spain and other countries where these products are exported to. As a result a full traceability of their genetic origin is increasingly demanded. DNA tests based on genetic markers are a useful tool to check the breed origin both in pigs as in and their derived products.
During the last years, several approaches have been proposed to discriminate between Iberian and Duroc background using different genetic markers. Firstly, different microsatellite panels were checked (Fabuel, Barragan, Silio, Rodriguez, & Toro, 2004; García et al., 2006) but the breed-specific alleles are not very common and they display low frequencies (Barragan, Rodriganez, Garcia-Casco, Martin-Palomino, & Rodriguez, 2007) resulting in inefficient diagnostic markers. In addition, the efficiency of microsatellite panels to differentiate between purebred, 50% or 75% crossbred individuals has been questioned by genetic simulations (Rodriguez-Ramilo, Toro, & Fernandez, 2010).
Although microsatellites are more informative than SNPs, SNP panels have replaced the formers since SNPs are more abundant, and their genotyping is more reproducible and cheaper than microsatellite genotyping allowing an easy interpretation and automation. Different SNP markers have been investigated so far for their usefulness to differentiate between Duroc and Iberian genetic origin, such as private alleles of Iberian or Duroc breeds identified in several nuclear (Fernández et al., 2004) and mitochondrial (Alves et al., 2009) genes. However, these panels did not completely allow discriminating between Duroc purebred and Duroc x Iberian crossbred individuals. The genotyping of Iberian and Duroc purebred animals with the PorcineSNP60 beadchip (Ramos et al., 2009) allowed the detection of SNPs with divergent allelic frequencies between these breeds. A low density customed panel containing a subset of 96 SNPs with extreme allelic frequencies for these breeds was developed and used to successfully verify the breed origin of Duroc x Iberian crossbred individuals (Muñoz et al., 2013). However, this panel had several disadvantages, as its cost and the fact that the observed differences between frequencies were lower than expected in the analysed population. On the other hand, simulations carried out in a subsequent study showed that 64 SNPs were enough for assigning each individual to its correspondent genetic group. The type I error values (probability of rejecting true null hypothesis) were equal to 0.019 for 50% and the corresponding values for type II error (probability of accepting false null hypothesis) were 0.018 (Barragan et al., 2015). Besides, the use of a more efficient low density genotyping technology and a larger sampling improved the results obtained by Muñoz et al. (2013).
The design of the PorcineSNP60 beadchip (Ramos et al., 2009), was focused on the most variable SNPs of a wild boar population and four cosmopolitan breeds, not including the Iberian, therefore its design may not be the best for our purpose. High throughput sequencing technologies allow obtaining the whole genome sequence of an individual in a quick, reliable and relatively cheap way. The whole genome re-sequencing and posterior Single Nucleotide Variance (SNV) calling of Iberian and Duroc individuals representative of each breed would permit to discover genetic variants not contained in the PorcineSNP60 beadchip so far. Therefore, the general objective of the present study was to develop a genetic tool that improves the previous ones through: i) the selection of new SNV markers from whole genome sequencing of Iberian and Duroc samples, ii) the assessing and validation of this panel as a diagnosis tool for the authentication of the purebred and crossbred origin of Iberian pigs and their meat products.
Section snippets
DNA extraction
The DNA was extracted using the NucleoSpin blood kit (Macherey-Nagel, Germany) for blood samples, NucleoSpin Food (Macherey-Nagel, Germany) for muscle tissue and dry-cured products and Ron's Tissue DNA Mini Kit (BIORON, Germany) for ear biopsies following the manufacturer's instructions. Concentration of DNA samples was adjusted to 50 ng/μl for subsequent analysis.
Genomic sequencing
DNA samples used for sequencing were obtained from five Duroc animals of diverse lines and 15 Iberian pigs from different genetic
Genomic sequencing and SNVs detection
A total of 2725 million reads were obtained after pair-end sequencing of the 12 Iberian and five Duroc animals and 750 million reads were obtained from the additional three Iberian individuals sequenced with higher coverage. After trimming, 2941 million reads were used for further analysis. An average of 86.35% of the reads was mapped against the Sscrofa 10.2 porcine. The average coverage after mapping was 5.71×.
The pipeline GATK + samtools allowed detecting an average of 5.21 and 5.66 million
Discussion
The European Union Regulation (EU) no: 1169/2011 controls the food labelling in the European Union and establishes that the consumers should be properly informed about the food they consume. The Spanish Norm RD 4/2014 regulates the production and labelling of Iberian pig meat and dry-cured products. Regarding the genetic origin of the raw material, it only admits purebred Iberian or crossbred pigs with a proportion of Duroc alleles lower or equal to 0.50. However, it does not specify about the
Conclusions
The designed panel of 64 SNVs with divergent allelic frequencies between the Iberian and Duroc breeds is a useful genetic tool to infer the purebred or crossbred Iberian origin of live animals, meat and dry-cured products. Its low cost and easy implementation enable its use as an additional control tool for the enforcement of the Spanish regulation of the Iberian pig production.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
This work was funded by the RZ2012-00006-00-00 grant from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). We want to thanks to breeder association AECERIBER and interprofessional association ASICI for providing samples.
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