1 Introduction
The domestic pig is an important livestock animal that is widely used for red meat, lard, and cured goods. It is a key player in the meat industry, particularly in Europe (OECD, 2022). Previous studies have suggested that the European domestic pig (Sus scrofa domesticus) is primarily descended from European wild boars (Giuffra et al., 2000). However, recent research has challenged this notion by identifying Asian mitochondrial DNA (mtDNA) haplotypes in European Yorkshire, Duroc, and Landrace pigs. This finding suggests that there may have been some interbreeding or genetic exchange between the two populations in the past (Giuffra et al., 2000; Larson et al., 2005). Throughout history, Italy has developed various breeds of pigs, each with unique characteristics and uses, such as Cinta Senese (Tuscany region), Nero Siciliano (Sicily region), and Mora Romagnola (Emilia-Romagna region) (Franci and Pugliese, 2007). The Yorkshire breed is one of the most commonly used commercial pig breeds and was introduced to Italy in the early 20th century due to its fast growth rate and high efficiency in converting feed into meat. The Landrace breed was introduced to Italy in the mid-20th century and has since been utilized in industrial pork production. The Duroc breed originated in the United States in the 19th century and has been exported to many countries, including Italy. This breed is often used in crossbreeding programs to produce hybrid pigs with desirable traits such as meat quality and growth rate (https://www.thepigsite.com/).
Both genetic and environmental factors have an impact on the phenotypic characteristics of commercial pig breeds, such as meat quality and disease resistance (Rosenvold and Andersen, 2003). Therefore, understanding the genetic diversity of these breeds is crucial for enhancing animal production, conserving animal genetic resources, and evaluating breed performance (Bovo et al., 2020; Dadousis et al., 2022). This research can help find breeds with better phenotypic traits and the ability to adapt to difficult conditions (Bovo et al., 2020). It can also support the sustainable growth of animal production in different settings and make it easier to reach evolutionary breeding goals rapidly (Notter, 1999).
The use of genome-wide panels of single nucleotide polymorphisms (SNPs) has transformed the study of pig breeds by allowing for the examination of complex relationships among them (Muñoz et al., 2019). However, processing such vast amounts of data can be challenging, leading to the need for a more efficient approach. One potential solution is to create less dense panels using a smaller set of markers specific to each breed based on a reduced number of SNPs. This approach would require less time and effort for analysis, thus making it more feasible. Breed-specific SNPs are frequently used in conservation biology to manage and protect livestock resources (Ozerov et al., 2013; Huisman, 2017), as well as for breed identification and authentication of meat and dairy products (Russo et al., 2007; Fontanesi et al., 2010).
The use of breed-informative SNPs has shown promising results in improving desired traits in pig breeding programs. A recent study on Italian Yorkshire pigs found that selecting SNPs associated with production traits, such as lean meat content, daily gain, and feed/gain ratio, can increase the frequency of desirable alleles over time, leading to faster improvement of these traits (Fontanesi et al., 2015). Genome-wide association studies (GWAS) have also become a popular way to find genetic variants linked to important production traits like meat and carcass quality, growth, and teat number in European pig breeds (Tang et al., 2019; Fabbri et al., 2020; Bovo et al., 2021). To identify breed-informative SNPs, various analytical tools, such as Random Forests, Principal Component Analysis, Regression, allele frequency differences, and Discriminant Analysis of Principal components, have been developed (Wilkinson et al., 2011; Schiavo et al., 2020; Hayah et al., 2021; Dadousis et al., 2022). These tools can help researchers identify key genetic markers and gain a deeper understanding of the genetic basis of production traits in pig breeds.
The aim of this study is to identify a breed-informative SNPs panel with high power to facilitate breed traceability and preservation efforts while also supporting breeding programs that prioritize desirable traits in these pig breeds. We anticipate that the identified SNPs will provide a useful tool for researchers and breeders alike, enabling them to make more informed decisions in animal management and breeding programs. By focusing on coding SNPs, we hope to identify genetic markers that are potentially functional, allowing for a better understanding of the underlying genetic mechanisms governing desirable production traits in commercial pig breeds. Ultimately, our research may contribute to the long-term sustainability of pig production, ensuring that we are able to meet the growing demand for animal products while preserving animal genetic diversity.
4 Discussion
Through our study, we have uncovered the genetic diversity present in three commercially important pig breeds, namely, Landrace, Yorkshire, and Duroc. These findings hold significant implications for breeding programs and conservation initiatives focused on preserving the genetic diversity within pig populations.
During our investigation, we observed notable genetic variability in our coding variants across the three breeds. Additionally, the Hardy-Weinberg equilibrium test revealed deviations from the expected population equilibrium. We also noted variations in the diversity q-values and an overall negative FIS value. The presence of an excess of heterozygosity in our dataset likely contributed to the observed HWE imbalance at 46 loci. It is noteworthy that our population does not appear to be subjected to selective pressure, and the deviations may be attributed to random mating among pig individuals, resulting in an isolate-breaking effect (Hamilton, 2021).
The identification of informative SNPs, particularly those located in coding regions, is crucial for developing cost-effective SNP panels to facilitate efficient genotyping and breeding selection. This approach can improve the accuracy and effectiveness of pig breeding programs, leading to the development of more robust and productive pig breeds (Fontanesi et al., 2015). Investigating coding SNPs is important for preventing genetic diseases caused by mutations in specific genes. By identifying these mutations and integrating them into breeding programs, the prevalence of these diseases in pig populations can be reduced, resulting in improved animal welfare and decreased economic losses for farmers (Mellencamp et al., 2008).
Previous research has identified informative SNPs for differentiating among various species, including cattle breeds (Cheong et al., 2013; Zwane et al., 2016; Bertolini et al., 2018) as well as wild boars and domestic pigs (Lorenzini et al., 2020). While previous studies have focused on identifying informative SNPs among commercial pig breeds (YO, DU, and LA) using non-coding SNPs (Schiavo et al., 2020; Hayah et al., 2021), our study aimed to identify informative SNPs using only coding variants.
In our study, we found 28 genetic markers (SNPs) that help distinguish the three pig breeds. Of these, six specific markers did not match what we expected based on the Hardy-Weinberg test.
The presence of these deviating SNPs highlights their importance as potential markers for distinguishing between the various pig breeds. However, it is essential to underscore that further comprehensive research and studies are imperative to validate and elucidate the precise roles and contributions of these SNPs in breed differentiation.
It is important to highlight that previous studies have already provided valuable insights into the implications of specific SNPs that we have identified in our research. For instance, a previous genome-wide association study (Große-Brinkhaus et al., 2015) demonstrated a significant association between the SNP ALGA0039432 and boar taint as well as testes size parameters. This finding underscores the relevance of this particular SNP in relation to these specific traits.
Moreover, our analysis identified two SNPs, namely, ALGA0060925 and DRGA0005996, as key contributors to breed differentiation. ALGA0060925 is positioned downstream on chromosome 11 and is responsible for encoding a long non-coding RNA (lncRNA). In contrast, DRGA0005996 is located on SSC5 and corresponds to the CPNE8 gene, which is responsible for producing the copine-8 protein. Copine-8 is a calcium-dependent phospholipid-binding molecule that plays a crucial role in calcium-mediated intracellular processes. It is worth noting that dysregulation of CPNE8, a member of the Copine family, has been associated with various diseases such as prion disease and gastric cancer in previous studies (Lloyd et al., 2013; Zhang et al., 2022). These findings suggest that CPNE8 may have multifaceted roles beyond breed differentiation and warrants further investigation in relation to its potential involvement in disease pathways.
Furthermore, several other SNPs within our dataset have been previously associated with various phenotypic traits. For example, the intergenic variant ASGA0077916 has demonstrated a significant correlation with the fatty acid composition of the Longissimus dorsi muscle (Sambache Tayupanta, 2016). Another SNP of interest, ASGA0072056, is located on SSC16 within the RETREG1 gene, responsible for encoding the reticulophagy regulator 1. Dysregulation of the RETREG1 gene has been linked to the development of numerous diseases (Islam et al., 2018). In the context of viral diseases, other studies have highlighted the relationship between the absence of the RETREG1 protein and heightened replication of Dengue and Zika viruses (Lennemann and Coyne, 2017). ASGA0008283 is an intergenic variant on SSC1. ASGA0072056 and ASGA0008283 have been shown to be determinant factors in tracing the breeding farm of domesticated pigs (Kwon et al., 2017).
Lastly, ALGA0078229 is situated on SSC14 within the RET gene, which encodes the proto-oncogene tyrosine-protein kinase receptor RET. Dysregulation of RET has been implicated in the development of various tumor types (Zhao et al., 2023). Additionally, a previous study found a significant association between ALGA0078229 and meat quality in German Landrace pigs (Ponsuksili et al., 2014).
Moreover, we conducted a comprehensive investigation to identify the biological processes associated with the SNPs that exhibited deviations from Hardy-Weinberg equilibrium. Notably, one genome-wide association study demonstrated a significant association between ALGA0077162 and immune-relevant traits in the Landrace breed (Dauben et al., 2021). Additionally, ASGA0050304 was identified as a quantitative trait locus strongly linked to intramuscular fat (IMF) in the gluteus medius (GM) and longissimus dorsi (LD) muscles of Duroc pigs (González Prendes, 2017).
Regarding the Functional Enrichment Analysis, our results have revealed three enriched functions that involve three important parts: the nucleosome, the DNA packaging complex, and the structural components of chromatin. These components play crucial roles in DNA packaging, organization, and gene expression, thereby ensuring the efficient functioning of critical nuclear processes such as transcription, replication, and DNA repair (Alberts et al., 2002). Nucleosomes were identified as the most significant function with the lowest p-value. Previous studies have demonstrated a correlation between increased circulating nucleosomes and inflammation as well as autoimmune diseases (Schwarzenbach et al., 2011; Pisetsky, 2012). Therefore, nucleosomes are believed to have the potential to initiate immune responses (Rönnefarth et al., 2006). Moreover, the activation of chromatin is vital for the immune response, with receptor engagement triggering reaction cascades that activate transcription factors and the chromatin template (Paz and Josefowicz, 2021). This synergistic activation of select genes is particularly evident in macrophages during inflammation, where they can rapidly express hundreds of genes (Paz and Josefowicz, 2021), thus highlighting the intricate relationship between chromatin dynamics and immune processes. Investigating these functions and their underlying molecular mechanisms could offer new insights into the regulation of gene expression associated with chromatin abnormalities.
In summary, our study highlights the effectiveness of DAPC in evaluating the genetic structure and admixture levels of pig breeds. The obvious breed-specific separation of individuals seen in the DAPC and PCA plots supports our findings that these three pig breeds have distinct genetic backgrounds. Despite using only coding variants, the SNPs selected by the DAPC approach were able to assign individuals to their respective breeds with a 74% probability of correct assignment. Although this may not match the assignment rate achieved with the full dataset, it is still a significant accomplishment and highlights the importance of carefully selecting impactful genetic markers for analysis. As a result, targeting coding regions associated with traits of interest provides a more straightforward analysis of genome-wide variants and yields more explicit results.
The SNPs discovered in this study have the potential to be used as markers for pig breed identification and conservation initiatives. Further research with larger sample sizes can provide a more comprehensive understanding of the genetic structure of these pig breeds and identify additional coding SNPs that contribute to breed differentiation. By conducting further investigations and experiments, we can gain a deeper understanding of the functional significance and underlying mechanisms of these identified SNPs.