SNP discovery and association study for growth, fatness and meat quality traits in Iberian crossbred pigs.

Iberian pigs and its crosses are produced to obtain high-quality meat products. The objective of this work was to evaluate a wide panel of DNA markers, selected by biological and functional criteria, for association with traits related to muscle growth, fatness, meat quality and metabolism. We used 18 crossbred Iberian pigs with divergent postnatal growth patterns for whole genome sequencing and SNP discovery, with over 13 million variants being detected. We selected 1023 missense SNPs located on annotated genes and showing different allele frequencies between pigs with makerdly different growth patterns. We complemented this panel with 192 candidate SNPs obtained from literature mining and from muscle RNAseq data. The selected markers were genotyped in 480 Iberian × Duroc pigs from a commercial population, in which phenotypes were obtained, and an association study was performed for the 1005 successfully genotyped SNPs showing segregation. The results confirmed the effects of several known SNPs in candidate genes (such as LEPR, ACACA, FTO, LIPE or SCD on fatness, growth and fatty acid composition) and also disclosed interesting effects of new SNPs in less known genes such as LRIG3, DENND1B, SOWAHB, EPHX1 or NFE2L2 affecting body weight, average daily gain and adiposity at different ages, or KRT10, NLE1, KCNH2 or AHNAK affecting fatness and FA composition. The results provide a valuable basis for future implementation of marker-assisted selection strategies in swine and contribute to a better understanding of the genetic architecture of relevant traits.

Consequences of a low protein diet on the liver and longissimus dorsi transcriptome of Duroc × Iberian crossbred pigs.

Low protein diets supplied during the growing period of pigs can diminish their growth rate and increase the intramuscular fat (IMF) content which affects the sensorial and technological characteristics of the products. In the present study, the effects of a low protein diet supplied during the growing period of Duroc × Iberian crossbred pigs on several phenotypic traits and on liver and longissimus dorsi transcriptome were analysed at the beginning (EARLY) and at the end (LATE) of the growing period. Two experimental groups of 10 crossbred pigs each were fed two isocaloric diets with different protein content: control diet (C) with 16.5% protein and 0.8% lysine and low protein diet (LP) with 11% CP and 0.6% lysine. Animals fed LP diet have a slower growth than those fed C diet, but no effect of LP diet was observed on the IMF content. The transcriptomes of liver and longissimus dorsi were characterised and quantified through RNA-sequencing (RNA-seq). In liver, 134 and 480 differentially expressed annotated genes and new isoforms (DEGs) were detected between C and LP diets for EARLY and LATE animals, respectively. In muscle, 128 and 68 DEGs were detected at EARLY and LATE time-points. Functional interpretation revealed that LP diet may inhibit immune system molecules and processes in both tissues at EARLY stage. In liver, the DEGs mainly affect lipid and cholesterol metabolic processes, while in muscle, the expression changes would be involved in growth, development and meat quality. In conclusion, a low protein diet supplied during the growing period seems to slow down the growth of Duroc × Iberian crossbred pigs, but it also seems to affect multiple biological processes that could compromise the immune system of Duroc × Iberian crossbred pigs. Therefore, these results question the adequacy of this type of regime in Duroc × Iberian pigs that must be studied in greater depth before being implemented.

Genome-wide detection of copy number variants in European autochthonous and commercial pig breeds by whole-genome sequencing of DNA pools identified breed-characterising copy number states.

In this study, we identified copy number variants (CNVs) in 19 European autochthonous pig breeds and in two commercial breeds (Italian Large White and Italian Duroc) that represent important genetic resources for this species. The genome of 725 pigs was sequenced using a breed-specific DNA pooling approach (30-35 animals per pool) obtaining an average depth per pool of 42×. This approach maximised CNV discovery as well as the related copy number states characterising, on average, the analysed breeds. By mining more than 17.5 billion reads, we identified a total of 9592 CNVs (~683 CNVs per breed) and 3710 CNV regions (CNVRs; 1.15% of the reference pig genome), with an average of 77 CNVRs per breed that were considered as private. A few CNVRs were analysed in more detail, together with other information derived from sequencing data. For example, the CNVR encompassing the KIT gene was associated with coat colour phenotypes in the analysed breeds, confirming the role of the multiple copies in determining breed-specific coat colours. The CNVR covering the MSRB3 gene was associated with ear size in most breeds. The CNVRs affecting the ELOVL6 and ZNF622 genes were private features observed in the Lithuanian Indigenous Wattle and in the Turopolje pig breeds respectively. Overall, the genome variability unravelled here can explain part of the genetic diversity among breeds and might contribute to explain their origin, history and adaptation to a variety of production systems.

Development of a 64 SNV panel for breed authentication in Iberian pigs and their derived meat products.

Spanish legislation regulates the labelling of Iberian pig meat and dry-cured products, which are labelled as "Ibérico" or "100% Ibérico" when they come from Duroc x Iberian crossbred or Iberian purebred pigs. Although the analytical authentication of breed origin is not mandatory, a genetic diagnostic tool is demanded by producers and consumers. We have designed a 64 Single Nucleotide Variant genotyping panel displaying extreme allelic frequencies between Duroc and Iberian purebred samples. Average proportions of Iberian alleles of 0.99, 0.01, 0.77 and 0.48 were estimated by admixture clustering analysis of known origin samples, for Iberian and Duroc purebred, 75% Iberian and 50% Iberian classes, respectively. A supervised analysis with 1419 samples showed some overlapping between contiguous classes, but the calculated degrees of separability ranged from 0.800 to 0.996, exceeding the threshold value (0.70) for considering suitable for prediction. Therefore, this panel is a useful genetic tool to infer purebred or crossbred Iberian origin of live animals, meat and dry-cured products.

Genomic diversity, linkage disequilibrium and selection signatures in European local pig breeds assessed with a high density SNP chip.

Genetic characterization of local breeds is essential to preserve their genomic variability, to advance conservation policies and to contribute to their promotion and sustainability. Genomic diversity of twenty European local pig breeds and a small sample of Spanish wild pigs was assessed using high density SNP chips. A total of 992 DNA samples were analyzed with the GeneSeek Genomic Profiler (GGP) 70 K HD porcine genotyping chip. Genotype data was employed to compute genetic diversity, population differentiation and structure, genetic distances, linkage disequilibrium and effective population size. Our results point out several breeds, such as Turopolje, Apulo Calabrese, Casertana, Mora Romagnola and Lithuanian indigenous wattle, having the lowest genetic diversity, supported by low heterozygosity and very small effective population size, demonstrating the need of enhanced conservation strategies. Principal components analysis showed the clustering of the individuals of the same breed, with few breeds being clearly isolated from the rest. Several breeds were partially overlapped, suggesting genetic closeness, which was particularly marked in the case of Iberian and Alentejana breeds. Spanish wild boar was also narrowly related to other western populations, in agreement with recurrent admixture between wild and domestic animals. We also searched across the genome for loci under diversifying selection based on FST outlier tests. Candidate genes that may underlie differences in adaptation to specific environments and productive systems and phenotypic traits were detected in potentially selected genomic regions.

Direct and maternal additive effects are not the main determinants of Iberian piglet perinatal mortality.

Data of 127,800 Iberian piglets were used to study genetic parameters of mortality at birth at the piglet level. These records proceed from three data sets: 4,987 litter of 2,156 sows of a dam line, 2,768 litter of 817 sows of a complete diallel cross between four Iberian strains and 7,153 litter of 2,113 sows of the Torbiscal composite line. Perinatal mortality was considered as a binary trait, and Bayesian threshold animal models were fitted to separately analyse the three data sets. The posterior means of direct heritability were 0.010, 0.004 and 0.003, and those of maternal heritability were 0.034, 0.011 and 0.014 for dam line, diallel cross and Torbiscal line, respectively. Important effects of litter size and parity order were inferred in the three data sets, of within-breed cross-breeding parameters in the diallel cross and of sex and sow handling in the Torbiscal line Therefore, the inclusion of perinatal mortality in the objective of selection is questionable in this breed and strategies for reducing piglet mortality successful in other breeds should be considered.

Assessing effective population size, coancestry and inbreeding effects on litter size using the pedigree and SNP data in closed lines of the Iberian pig breed.

The complete pedigree of two closed Iberian pig lines (Gamito and Torbiscal), with 798 and 4077 reproducers, has been used to measure the evolution of coancestry (f) and inbreeding (F) for autosomal and X-linked genes along 16 and 28 respective equivalent discrete generations. At the last generation, the mean values of each line were f = 0.41 and 0.22, F = 0.35 and 0.18, fX  = 0.46 and 0.22 and FX  = 0.47 and 0.19, respectively. Other calculated parameters were the effective number of founders (final values, 6.8 and 35.2) and non-founders (1.5 and 2.4), founder genome equivalents (1.2 and 2.3) and effective population size (16.0 and 57.7). Measures of Torbiscal effective size based on rates of coancestry (66.1), inbreeding (65.0) and linkage disequilibrium (71.0) were estimated from whole-genome SNP genotyping data. Values of new and old inbreeding and their respective rates by generation were computed to detect purging effects of natural selection. The analysis of 6854 Torbiscal litters showed significant negative impacts of new and fast inbreeding on litter size, as expected from the purging hypothesis: -0.20 born piglets per litter by a 10% of new inbreeding, and -0.03 and -0.02 piglets by 1% of total and new inbreeding rates, respectively. The analysis performed on 1274 litters of the Gamito line failed to show purging effects. The only significant results were reductions in -0.91 and -0.17 piglets by a 10% of old and X-linked genes inbreeding, respectively. These results may be useful for some practical issues in conservation programs of farm or captive wild animals.

Genetic parameters for meat and fat quality and carcass composition traits in Iberian pigs.

Carcass composition and quality traits were measured in heavy Iberian pigs after extensive handling. The weights of trimmed hams, forelegs and loins were recorded on 2170-2553 pigs, intramuscular fat content in M. longissimus (IMF) on 1489 and percentages of palmitic, stearic, oleic and linoleic acids in the subcutaneous fat on 1495 pigs, slaughtered at about 160 kg live weight. For carcass traits the heritability estimates ranged from 0.28 to 0.41. Heritability estimate for IMF was 0.25 and estimates of heritability for the measured fatty acids ranged from 0.29 to 0.41. The estimated genetic correlations (rg) suggest antagonism between IMF and weights of hams (rg=-0.30) and loins (rg=-0.33) and an unfavourable positive association of premium cuts with linoleic acid content (rg: 0.31-0.57). The incorporation of meat and fat quality traits to the selection aims of the breeding scheme for Iberian pigs seems advisable.