Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach

Combining different approaches (resequencing of portions of 54 obesity candidate genes, literature mining for pig markers associated with fat deposition or related traits in 77 genes, and in silico mining of porcine expressed sequence tags and other sequences available in databases), we identified and analyzed 736 SNP within candidate genes to identify markers associated with back fat thickness (BFT) in Italian Large White sows. Animals were chosen using a selective genotyping approach according to their EBV for BFT (276 with most negative and 279 with most positive EBV) within a population of ≈ 12,000 pigs. Association analysis between the SNP and BFT has been carried out using the MAX test proposed for case-control studies. The designed assays were successful for 656 SNP: 370 were excluded (low call rate or minor allele frequency <5%), whereas the remaining 286 in 212 genes were taken for subsequent analyses, among which 64 showed a P(nominal) value <0.1. To deal with the multiple testing problem in a candidate gene approach, we applied the proportion of false positives (PFP) method. Thirty-eight SNP were significant (P(PFP) < 0.20). The most significant SNP was the IGF2 intron3-g.3072G>A polymorphism (P(nominal) < 1.0E-50). The second most significant SNP was the MC4R c.1426A>G polymorphism (P(nominal) = 8.0E-05). The third top SNP (P(nominal) = 6.2E-04) was the intronic TBC1D1 g.219G>A polymorphic site, in agreement with our previous results obtained in an independent study. The list of significant markers also included SNP in additional genes (ABHD16A, ABHD5, ACP2, ALMS1, APOA2, ATP1A2, CALR, COL14A1, CTSF, DARS, DECR1, ENPP1, ESR1, GH1, GHRL, GNMT, IKBKB, JAK3, MTTP, NFKBIA, NT5E, PLAT, PPARG, PPP2R5D, PRLR, RRAGD, RFC2, SDHD, SERPINF1, UBE2H, VCAM1, and WAT). Functional relationships between genes were obtained using the Ingenuity Pathway Analysis (IPA) Knowledge Base. The top scoring pathway included 19 genes with a P(nominal) < 0.1, 2 of which (IKBKB and NFKBIA) are involved in the hypothalamic IKKβ/NFκB program that could represent a key axis to affect fat deposition traits in pigs. These results represent a starting point to plan marker-assisted selection in Italian Large White nuclei for BFT. Because of similarities between humans and pigs, this study might also provide useful clues to investigate genetic factors affecting human obesity.     

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.     

A genomewide association study in divergently selected lines in rabbits reveals novel genomic regions associated with litter size traits

Uterine capacity (UC), defined as the total number of kits from unilaterally ovariectomized does at birth, has a high genetic correlation with litter size. The aim of our research was to identify genomic regions associated with litter size traits through a genomewide association study using rabbits from a divergent selection experiment for UC. A high-density SNP array (200K) was used to genotype 181 does from a control population, high and low UC lines. Traits included total number born (TNB), number born alive (NBA), number born dead, ovulation rate (OR), implanted embryos (IE) and embryo, foetal and prenatal survivals at second parity. We implemented the Bayes B method and the associations were tested by Bayes factors and the percentage of genomic variance (GV) explained by windows. Different genomic regions associated with TNB, NBA, IE and OR were found. These regions explained 7.36%, 1.27%, 15.87% and 3.95% of GV, respectively. Two consecutive windows on chromosome 17 were associated with TNB, NBA and IE. This genomic region accounted for 6.32% of GV of TNB. In this region, we found the BMP4, PTDGR, PTGER2, STYX and CDKN3 candidate genes which presented functional annotations linked to some reproductive processes. Our findings suggest that a genomic region on chromosome 17 has an important effect on litter size traits. However, further analyses are needed to validate this region in other maternal rabbit lines.     

A retrospective analysis of allele frequency changes of major genes during 20 years of selection in the Italian Large White pig breed

In this study, we investigated whether a selection programme based on boar genetic evaluation obtained with a classical BLUP animal model can change allele frequencies in a pig population. All Italian Large White boars born from 1992 to 2012 with estimated breeding value reliability >0.85 (n = 200) were selected among all boars of this breed. Boars were genotyped with markers in major genes (IGF2 intron3‐g.3072G>A, MC4R p.D298N, VRTN PRE1 insertion, PRKAG3 p.I199V and FTO g.276T>G). Genotyping data were analysed grouping boars in eight classes according to their year of birth. To evaluate the influence of time on allele frequencies of the genotyped markers, multinomial logistic regression models were computed. Four of five polymorphic sites (IGF2, MC4R, VRTN and FTO) showed significant (p < 0.01) changes in allele frequencies over time due to a progressive and continuous increase of one allele (associated with higher lean meat content, higher average daily gain and favourable feed: gain ratio) and, consequently, decrease of the other one, following the directional selection of the selection programme of this pig breed. The retrospective analysis that was carried out in Italian Large White boars suggests that selection based on methodologies assuming the infinitesimal model is able to modify in a quite short period of time allele frequencies in major genes, increasing the frequency of alleles explaining a relevant (non‐infinitesimal) fraction of the overall genetic variability for production traits.     

Genetic structure of candidate genes for litter size in Italian Large White pigs

The aim of this work was to verify whether polymorphisms in candidate genes for litter size segregate in Italian Large White (ITLW) pigs. We genotyped 120 sows that belonged to six different farms for 10 single nucleotide polymorphisms (SNPs) of 10 different genes. Polymorphisms in the chosen genes had already been associated with litter-size traits in other pig populations and were candidates for function and/or chromosomal location. The results indicated that the CLGN, pDAZL, and RFN4 SNPs were not segregating in the genotyped samples. The remaining seven markers were polymorphic with minor allele frequencies ranging from 0.10 (AFP) to 0.48 (RBP4). Because of the observed genetic variabilities in the investigated loci, the polymorphisms in the AFP, BMPR1B, CXCL10, ESR2, GNRHR, MAN2B2, and RBP4 genes can be considered suitable markers for association studies with litter-size traits in ITLW pigs.

     

Combined use of principal component analysis and random forests identify population‐informative single nucleotide polymorphisms: application in cattle breeds

The genetic identification of the population of origin of individuals, including animals, has several practical applications in forensics, evolution, conservation genetics, breeding and authentication of animal products. Commercial high‐density single nucleotide polymorphism (SNP) genotyping tools that have been recently developed in many species provide information from a large number of polymorphic sites that can be used to identify population‐/breed‐informative markers. In this study, starting from Illumina BovineSNP50 v1 BeadChip array genotyping data available from 3711 cattle of four breeds (2091 Italian Holstein, 738 Italian Brown, 475 Italian Simmental and 407 Marchigiana), principal component analysis (PCA) and random forests (RFs) were combined to identify informative SNP panels useful for cattle breed identification. From a PCA preselected list of 580 SNPs, RFs were computed using ranking methods (Mean Decrease in the Gini Index and Mean Accuracy Decrease) to identify the most informative 48 and 96 SNPs for breed assignment. The out‐of‐bag (OOB) error rate for both ranking methods and SNP densities ranged from 0.0 to 0.1% in the reference population. Application of this approach in a test population (10% of individuals pre‐extracted from the whole data set) achieved 100% of correct assignment with both classifiers. Linkage disequilibrium between selected SNPs was relevant (r² > 0.6) only in few pairs of markers indicating that most of the selected SNPs captured different fractions of variance. Several informative SNPs were in genes/QTL regions that affect or are associated with phenotypes or production traits that might differentiate the investigated breeds. The combination of PCA and RF to perform SNP selection and breed assignment can be easily implemented and is able to identify subsets of informative SNPs useful for population assignment starting from a large number of markers derived by high‐throughput genotyping platforms.     

Association between melanocortin 4 receptor (MC4R) gene haplotypes and carcass and production traits in Italian Large White pigs evaluated with a selective genotyping approach

Melanocortin 4 receptor (MC4R) plays a key role in controlling energy homeostasis. Several studies have already reported effects on production traits of polymorphisms identified in the porcine MC4R gene. In this study we analysed data on 6 MC4R polymorphisms (c.-780C>G; c.-135C>T; c.175C>T; c.707G>A or p.Arg236His; and c.892G>A or p.Asp298Asn; c.?430A>T) genotyped from (1) two groups of Italian Large White pigs (280+280 animals) with extreme estimated breeding values (EBVs) for back fat thickness (BFT), selected among a performance tested population of about 12,000 pigs, and from (2) 19 Italian Duroc pigs. Two haplotypes, differentiated by the c.892G>A, were identified in the Duroc populations. Four haplotypes were identified in the Italian Large White population, one of which (haplotype 4) was identified for the first time in this study. Single marker and haplotype association analyses for BFT were obtained by comparing allele and haplotype frequency differences from the two extreme tails using χ² and Cochran–Armitage trend tests. Results confirmed the effects of the c.892G>A single nucleotide polymorphism (SNP) on BFT, as also defined by different distributions in the two tails of haplotypes carrying the alternative nucleotides at this polymorphic site (P<0.01). In addition different distributions of haplotype 4 in the two extreme groups suggested that it might affect the same trait (P<0.10). Association analyses for several other traits (average daily gain, ADG; feed gain ratio, FGR; weight of lean cuts; ham weight) were carried out by using EBVs and Random Residuals: significant effects (P<0.05) were only found for the p.Asp298Asn mutation on ADG and FGR. Results did not support any relevant effect of the p.Arg236His mutation on any trait. Data reported in this study contribute to better understand the role of MC4R variants in affecting production traits in pigs, a prerequisite to consider polymorphisms in this gene for marker assisted selection.