Whole-genome sequence analysis reveals differences in population management and selection of European low-input pig breeds

The use of a relatively small number of international high-output or commercial breeds largely explains the increase in livestock productivity over the past decades. In parallel, the number of commercial populations is even decreasing due to consolidation of breeding stock and breeding companies [1]. While high productive breeds may not compete with low-input breeds in marginal regions or extensive production, FAO has expressed concern due to the shift from local breeds to high-output animals [2]. Local breeds may be more resistant than high-performance breeds to local diseases, may be better adapted to local climate, and may be adapted to poorer food quality [2, 3]. These characteristics of local breeds are very relevant for humans living in developing countries where local domestic animals are an important source of protein. Local breeds are also appreciated in developed countries for their cultural heritage value, and as producers of traditional and high quality meat products [4, 5]. Increasingly, local heritage breeds are recognized for their potential in sustainable or organic food production systems. Moreover, they represent a yardstick against which to compare highly selected breeds and allowing the detection of genes under selection [6]. Lastly, local breeds are claimed to harbour a large amount of the variation within livestock species [7, 8], and as such are recognized as important genetic reservoirs that need to be protected for future food security [9].

Despite all those inherent properties of local breeds, the long term survival of many of them is not assured [9]. Inbreeding is particularly relevant in local breeds that have low population numbers [5, 8]. The loss of genetic diversity within a breed due to drift and inbreeding can have direct consequences for reduction of survival, reproduction efficiency and capacity of adaptation to environmental changes [10]. The reduction in reproduction and growth rates is particularly relevant for local livestock breeds as it can directly lead to economic loss. Minimising inbreeding is, therefore, a major goal to guarantee the sustainability and maintenance of domestic populations of livestock species.

Genetic characterization of livestock breeds by applying genetic marker technology is needed to enhance breeding and to better direct biodiversity conservation strategies. In pigs, the Porcine SNP60 Bead-array [11] is a commercially available marker system extensively used in genetic studies (e.g. [12, 13]). More recently, whole-genome re-sequencing has emerged as an economically feasible tool for assessing genomic variation among populations [14]. In contrast to the commercially available SNP chip, the study of the whole genome sequence provides the opportunity of performing unbiased and comprehensive studies to characterize genetic diversity [15], regions of homozygosity [16], and scanning the pig genome to detect signatures of selection [17, 18]. The study of entire genomes increases the availability of information on neutral loci, and thereby the accuracy of estimates of demographically important parameters, such as the inbreeding coefficient (F) [19, 20]. Next generation sequencing (NGS) also allows for direct assessment of polymorphisms in coding regions that could have consequences in selective processes. For instance, genes involved in local adaptation, or alleles responsible for inbreeding depression can be analysed [19].

In this study, we first assess and compare genetic diversity of low-input breeds from Europe by integrating high-density SNP and re-sequencing data. Secondly, we explore the role of local breeds as reservoirs for genetic variation in a domesticated species. Finally, we assessed differences between local and commercial populations in terms of functional variation and explore evidences for inbreeding in local breeds that could lead to inbreeding depression.

The advances in sequencing technologies now allows sequencing whole genomes in multiple individuals [19, 28]. However, the cost of this technology is still high, and budgets for conservation genetics research are limited. While high-density SNP panels allow the study of a representative sample size of a population at a much lower cost, there is a concern regarding the ascertainment bias implicit in the use of SNP chips [29]. This concern is even higher for local pig populations since they were not considered in the design the Porcine SNP60 Beadchip [11]. In this study, we found a high correlation between diversity estimates derived from the Illumina porcine 60SNP Beadchip and NGS data when local European breeds were analyzed. These results indicate that the Illumina porcine 60SNP Beadchip provides reliable estimates of genomic diversity for comparative studies between European populations, despite the expected bias. Nevertheless, English breeds showed greater diversity with NGS compared to 60K data than expected compared to expected values derived from all populations combined. These results may highlight the influence of historical breeding practices, whereby Asian pigs were used to improve local English pigs during the late 18^th and 19^th century [14, 30]. Despite the additional diversity found in English pigs owing to Asian introgression, some English pigs display high levels of ROH and potentially damaging mutations as a the result of recent inbreeding and could indicate that these breeds are prone to inbreeding depression.

SNP variants were annotated and potential deleterious effects were predicted with Polyphen2. Recessive deleterious alleles can be a major cause of inbreeding depression in populations with low genetic diversity [31]. In our study we find the largest number of putative deleterious mutations in those animals that also have the highest percentage of the genome covered by ROH and the lowest genetic diversity, i.e. Mangalica and Cinta Senese breeds, and in the breeds Tamworth and Gloucester Old Spots. Genomic diversity in these breeds was lower than almost all domestic and wild populations from Europe and Asia [16] corroborating the hypothesis that damaging mutations can accumulate, due to drift, in populations with high levels of inbreeding. A similar relation between genetic diversity and proportion of deleterious alleles has been described in human populations [32] and is thought to be caused by a less effective purifying selection as effective size decreases. This finding points out the need to develop conservation programs for endangered livestock populations that are very prone to high levels of inbreeding.

We found non-synonymous, high allele frequency differences (fixed for different alleles) at non-synonymous sites to be overrepresented in genes involved in immune response, anatomical development, behaviour, and sensory perception between commercial and local populations. Local breeds tend to be reared in traditional systems without being subjected to intense artificial selection (e.g. BLUP, GBLUP selection) as applied to commercial pig populations. As a result of years of different selection pressures and environments, genomic variations underlying phenotypic differences can be expected. We have specifically focussed on non-synonymous variants because they will alter the amino acid sequence of gene products, which may result in different phenotypes [33]. Although phenotypic change is expected to a large extent to result from regulation of genes, rather than differences in amino acid sequences, regulatory important variations are currently difficult to predict reliably and were therefore not considered in this study.

The gene AZGP1 stimulates lipid degradation in adipocytes and subsequently is considered a lipid-mobilizing factor [34]. This gene is linked with obesity in humans and its expression is inversely associated with body weight and percentage of body fat in mice and humans [35, 36]. In pigs, a 20 Mb QTL in chromosome 3 [37] for abdominal fat weight overlaps this gene. Mangalica, Cinta Senese and one European wild boar are homozygous for a derived allele annotated as probably damaging. This allele is absent in commercial pigs and also in some local pigs. The inferred status of the allele as ‘probably damaging’ may, for pig, rather result in having a large effect on the phenotype. Whereas pigs used to be bred for high fat deposition, in modern pig production systems lean meat is desired. AZGP1 also overlaps with a 16 Mb QTL for ear size, area and weight [38] and a 8.5 Mb QTL for vertebra number [39]. Ear morphology traits have been traditionally used to define breed standards. We observed a non-random overrepresentation of non-synonymous SNPs overlapping with QTLs related with ear morphology. This is in agreement with Wilkinson et al. [6] who found signatures of diversifying selection between pig breeds from Europe in genomic regions associated with ear morphology. Related to vertebra number, we found a fixed non-synonymous mutation in the Mangalica and heterozygous genotype in Iberian and Casertana breeds within the gene PLAG1 that has been related with stature in humans and cattle [40, 41]. Rubin et al. [18] concluded a strong signature of selection in the domestic pig genome at PLAG1. These data suggest that the mutations found in the genes AZGP1 and PLAG1 may represent signatures of different selection pressures between local breeds as Mangalica and commercial pigs. Another compelling example of potential differential selection between commercial and local populations is represented by the two mutations found in the bitter taste receptor TAS2R40. The high variability within the family of taste receptor genes has been suggested a consequence of adaptation of populations to specific dietary repertoires and environment [42], such as prevention of consumption of plant toxins [43].

It has been observed that selection for economically important traits tends to increase the susceptibility to environmental factors [44, 45]. In our study, ancestral mutations classified as benign in genes involved in immune related genes such as IL12RB2 and STAB1, were observed in several local pigs. The IL12RB2 subunit plays an important role in Th1 cell differentiation that is critical for an effective immune response against different types of pathogens [46]. The three mutations observed in this gene overlap with important QTLs in pig production such as back fat thickness and intramuscular fat content [47, 48]. The fact that mutations in IL12RB2 can lead to a defective IFN-gamma response to microorganisms [49, 50], suggests that disadvantageous genotypes could have been maintained in commercial populations.

The EIF2AK3 gene overlaps with QTLs for osteochondrosis score [51] and feet and leg conformation [52]. Moreover, the permutation test using all the non-synonymous SNPs showed non-random overrepresentation of SNPs overlapping with QTLs for leg conformation. Interestingly, this gene encompasses functions of bone mineralization, chondrocyte development insulin secretion and fat cell differentiation and has being related with the Wolcott-Rallison syndrome in humans [53]. Leg weakness is a major concern in growing pigs raised under modern production systems and osteochondrosis is considered to be the primary cause of this syndrome. Indeed, forced selection for high growth capacity predisposes to these disorders due to an imbalance between the development of the skeletal system and muscle [54]. The allelic differences between local and commercial pigs within the EIF2AK3 gene could underlie strong directional selection in commercial breeds. The fact that the same alleles are segregating in both wild boar and low-input breeds supports this hypothesis.

The genes discussed above had different fixed alleles for non-synonymous SNPs between commercial and local pigs. The presence of both alleles, the ancestral and the derived, in wild boars indicates that the variation was present before domestication. While differences in allele frequencies of SNPs in genes such as AZGP1 and TAS2R40 may underlie a rapid adaptation to different environments, it can also occur due to drift effects in small populations in the absence of selection, or even if the allele is in fact disadvantageous. The fixed alleles in EIF2AK3 and IL12RB2 could potentially result in disadvantageous phenotypes in high-output breeds owing to the strong artificial selection for production traits. We demonstrated that genetic variability found in wild populations is also being preserved in local breeds at genomic sites with potential phenotypic effect. This further highlights the importance of preserving local breeds as a source of genomic diversity that could be used in future selection programs of commercial pigs. However, the results presented also highlight high levels of ROHs, inbreeding and potentially damaging mutations that threat the future of local pig breeds, emphasizing the need of implementing conservation programmes to preserve the genomic variability of low-input breeds.

In this study, we assessed genetic diversity of low-input breeds from different European regions by integrating high-density SNP and re-sequencing data. The comparison of the two marker system estimations provided insights for strategies to the genetic characterization of local breeds. Furthermore, the re-sequenced local pigs were compared with re-sequenced commercial pigs to report candidate mutations responsible for phenotypic divergence among those groups of breeds. We observed that local pig breeds are an important source of genomic variation within-species, and thereby, they represent a genomic stock that could be important for future adaptation to long-term changes in the environment or consumers preferences. However, high levels of inbreeding threaten the long term survival of some of the local breeds studied.