Functional implication of an Arg307Gly substitution in corticosteroid-binding globulin, a candidate gene for a quantitative trait locus associated with cortisol variability and obesity in pig

We previously reported that corticosteroid-binding globulin gene (Cbg) may be the causal gene of a quantitative trait locus associated with cortisol levels, fat deposition, and muscle content in a pig intercross. Sequence analysis of parental animals allowed us to identify four amino-acid substitutions. Here we have examined if any of these single amino acid substitutions could be responsible for the difference in CBG binding and affinity for cortisol between the parental breeds, using in vitro assays of Cbg variants after transfection of mammalian cells. Additionally, the Cbg coding region was analyzed in samples from a synthetic pig line to study association between polymorphism and CBG biochemical properties, carcass composition, and meat quality. Both in vitro transfection assays and the association studies suggest a role of the Arg307Gly mutation in increasing CBG capacity (by >70%) and decreasing CBG affinity for cortisol (by 30%). The Ile265Val substitution may also have an effect on decreasing CBG affinity for cortisol by 25%. The mutations Ser15Ile and Thr257Met do not seem to have an effect on CBG parameters. The Arg307Gly substitution was the only mutation associated with a parameter of meat quality and no mutation was linked to carcass composition.     

Evidence for new alleles in the protein kinase adenosine monophosphate-activated gamma(3)-subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality.

Several quantitative trait loci (QTL) affecting muscle glycogen content and related traits were mapped to pig chromosome 15 using a three-generation intercross between Berkshire x Yorkshire pigs. On the basis of the QTL location the PRKAG3 (protein kinase, AMP-activated, gamma(3)-subunit) gene was considered to be a good candidate for the observed effects. Differences in the PRKAG3 gene sequences of the founder animals of the intercross were analyzed. The RN(-) mutation previously reported was not present in the cross but three missense substitutions and a polymorphic short interspersed element (SINE) were identified. To confirm the hypothesis that at least one of these mutations was associated with differences in meat quality, >1800 animals from several unrelated commercial lines were genotyped for the candidate substitutions and an association study was performed. The results demonstrate the presence of new economically important alleles of the PRKAG3 gene affecting the glycogen content in the muscle and the resulting meat quality. Haplotype analysis was shown to resolve the effects of PRKAG3 more clearly than analysis of individual polymorphisms. Because of their prevalence in the more common commercial breeds, the potential implications for the pig industry and consumers are considerably greater than the original discovery of the RN(-) mutation. Furthermore, these results illustrate that additional alleles of genes involved in major mutations may play a significant role in quantitative trait variation.     

Quantitative trait loci for fatty acid composition in longissimus dorsi and abdominal fat: results from a White Duroc × Erhualian intercross F2 population

A whole-genome scan was performed on 660 F₂ animals including 250 barrows and 410 gilts in a White Duroc × Erhualian intercross population to detect quantitative trait loci (QTL) for fatty acid composition in the longissimus dorsi muscle and abdominal fat. A total of 153 QTL including 63 genome-wide significant QTL and 90 suggestive effects were identified for the traits measured. Significant effects were mainly evident on pig chromosomes (SSC) 4, 7, 8 and X. No association was detected on SSC3 and 11. In general, the QTL detected in this study showed distinct effects on fatty acid composition in the longissimus muscle and abdominal fat. The QTL for fatty acid composition in abdominal fat did not correspond to those identified previously in backfat and the majority of QTL for the muscle fatty acid composition were mapped to chromosomal regions different from previous studies. Two regions on SSC4 and SSC7 showed significant pleiotropic effects on monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA) in both longissimus muscle and abdominal fat. Another two QTL with significant multi-faceted effects on MUFA and PUFA in the longissimus muscle were found each on SSC8 and SSCX. Chinese Erhualian alleles were associated with increased ratios of MUFA to saturated fatty acid at most of the QTL detected, showing beneficial effect in terms of human health.     

Rearranged gene order between pig and human in a QTL region on SSC 7

On porcine Chromosome 7, the region surrounding the MHC region contains QTL influencing many traits including growth, back fat thickness, and carcass composition. Towards the identification of the responsible gene(s), this article describes an increase of density of the radiated hybrid map of SSC 7 in the q11-q14 region and the comparative analysis of gene order on the porcine RH map and human genome assembly. Adding 24 new genes in this region, we were able to build a framework map that fills in gaps on the previous maps. The new software Carthagene was used to build a robust framework in this region. Comparative analysis of human and porcine maps revealed a global conservation of gene order and of distances between genes. A rearranged fragment of around 3.7 Mb was, however, found in the pig approximately 20 Mb upstream from the expected location on the basis of the human map. This rearrangement, found by RH mapping on the IMpRH 7.000 rads panel, has been confirmed by two-color FISH and by mapping on the high resolution IMNpRH2 12.000 rads panel. The rearranged fragment contains two microsatellites found at the most likely QTL location in the INRA QTL experiment. It also contains the BMP5 gene, which, together with CLPS, could be considered as a possible candidate.     

Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc × Chinese Erhualian intercross resource population

The number of vertebrae is associated with body size and meat production in pigs. To identify quantitative trait loci (QTL) for the number of vertebrae, phenotypic values were measured in 1029 individuals from a White Duroc × Chinese Erhualian intercross F₂ population. A whole genome scan was performed with 194 microsatellite markers in the F₂ population. Four genome‐wide significant QTL and eight chromosome‐wide significant QTL for the number of vertebrae were identified on pig chromosomes (SSC) 1, 2, 6, 7, 10 and 12. The most significant QTL was detected on SSC7 with a confidence interval of 1 cM, explaining 42.32% of the phenotypic variance in the thoracic vertebral number. The significant QTL on SSC1, 2 and 7 confirmed previous reports. A panel of 276 animals representing seven Western and Chinese breeds was genotyped with 34 microsatellite markers in the SSC7 QTL region. No obvious selective sweep effect was observed in the tested breeds, indicating that intensive selection for enlarged body size in Western commercial breeds did not wipe out the genetic variability in the QTL region. The Q alleles for increased vertebral number originated from both Chinese Erhualian and White Duroc founder animals. A haplotype block of approximately 900 kb was found to be shared by all Q‐bearing chromosomes of F₁ sires except for one distinct Q chromosome. The critical region harbours the newly reported VRTN gene associated with vertebral number. Further investigations are required to confirm whether VRTN or two other positional candidate genes, PROX2 and FOS, cause the QTL effect.     

Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross

The leptin receptor gene (LEPR) is a candidate for traits related to growth and body composition, and is located on SSC6 in a region where fatness and meat composition quantitative trait loci (QTL) have previously been detected in several F2 experimental designs. The aims of this work were: (i) to fine map these QTL on a larger sample of animals and generations (F3 and backcross) of an Iberian x Landrace intercross and (ii) to examine the effects of LEPR alleles on body composition traits. Eleven single nucleotide polymorphisms (SNPs) were detected by sequencing LEPR coding regions in Iberian and Landrace pig samples. Three missense polymorphisms were genotyped by pyrosequencing in 33 F0, 70 F1, 418 F2, 86 F3 and 128 individuals coming from the backcross of four F2 males with 24 Landrace females. Thirteen microsatellites and one SNP were also genotyped. Traits analysed were: backfat thickness at different locations (BF(T)), intramuscular fat percentage (IMF(P)), eye muscle area (EM(A)), loin depth (LO(D)), weight of shoulder (SH(W)), weight of ribs (RIB(W)) and weight of belly bacon (BB(W)). Different statistical models were applied in order to evaluate the number and effects of QTL on chromosome 6 and the possible causality of the LEPR gene variants with respect to the QTL. The results support the presence of two QTL on SSC6. One, at position 60-100 cM, affects BF(T) and RIB(W). The other and more significant maps in a narrow region (130-132 cM) and affects BF(T), IMF(P), EM(A), LO(D), SH(W), RIB(W) and BB(W). Results also support the association between LEPR alleles and BF(T) traits. The possible functional implications of the analysed polymorphisms are considered.     

Expression and genome polymorphism of ACSL1 gene in different pig breeds

Acyl coenzyme A long-chain 1 synthetase (ACSL1) plays a key role in animal fat synthesis and fatty acid β-oxidation. In order to research the function of the ACSL1 gene in pig, we analyzed the mRNA expression in liver, backfat and longissimus dorsi muscle by quantitative real-time PCR in Tibet pig (TP, n = 10), Diannan small ear pig (DSP, n = 10) and large white pig (LW, n = 10). The results showed that the mRNA expressions of the ACSL1 gene in liver and longissimus dorsi muscle of DSP and TP were significant higher than that of LW (P < 0.01). However, the expression in backfat of LW was significant higher than that of TP (P < 0.01) and DSP (P < 0.05). In addition, four SNPs located in 5' flanking region (T-1191C), exon 6(G173A), exon 14(C36T) and exon 17(T46C) were identified, and the allele frequencies of the four SNPs were significant different in indigenous and introduced pig breeds. The results indicated that the ACSL1 gene might be relative to the capacity of fat deposition and meat quality in pig breeds.     

A missense mutation in PPARD causes a major QTL effect on ear size in pigs

Chinese Erhualian is the most prolific pig breed in the world. The breed exhibits exceptionally large and floppy ears. To identify genes underlying this typical feature, we previously performed a genome scan in a large scale White Duroc × Erhualian cross and mapped a major QTL for ear size to a 2-cM region on chromosome 7. We herein performed an identical-by-descent analysis that defined the QTL within a 750-kb region. Historically, the large-ear feature has been selected for the ancient sacrificial culture in Erhualian pigs. By using a selective sweep analysis, we then refined the critical region to a 630-kb interval containing 9 annotated genes. Four of the 9 genes are expressed in ear tissues of piglets. Of the 4 genes, PPARD stood out as the strongest candidate gene for its established role in skin homeostasis, cartilage development, and fat metabolism. No differential expression of PPARD was found in ear tissues at different growth stages between large-eared Erhualian and small-eared Duroc pigs. We further screened coding sequence variants in the PPARD gene and identified only one missense mutation (G32E) in a conserved functionally important domain. The protein-altering mutation showed perfect concordance (100%) with the QTL genotypes of all 19 founder animals segregating in the White Duroc × Erhualian cross and occurred at high frequencies exclusively in Chinese large-eared breeds. Moreover, the mutation is of functional significance; it mediates down-regulation of β-catenin and its target gene expression that is crucial for fat deposition in skin. Furthermore, the mutation was significantly associated with ear size across the experimental cross and diverse outbred populations. A worldwide survey of haplotype diversity revealed that the mutation event is of Chinese origin, likely after domestication. Taken together, we provide evidence that PPARD G32E is the variation underlying this major QTL.     

Porcine skeletal muscle differentially expressed gene CMYA1: isolation, characterization, mapping, expression and association analysis with carcass traits

To investigate the differences in gene expression between some obese and lean pig breeds, differential display of mRNA was employed in our previous research. One differentially expressed EST ( BI596262 ) was further identified as the porcine cardiomyopathy associated 1 ( CMYA1 ) gene because of its homology to the human CMYA1 gene. The full-length DNA of the porcine CMYA1 gene encompasses 9379 bp, including a complete open reading frame encoding 1839 amino acid residues, a 158-bp 5'-untranslated region and a 630-bp 3'-untranslated region. The porcine CMYA1 gene was assigned to chromosome 13 by the radiation hybrid panel (IMpRH). The porcine CMYA1 gene was expressed only in the striated muscle. Single nucleotide polymorphism (SNP) scanning in the coding region identified one synonymous mutation (c.1053C>T) and three missense mutations, c.1394A>G (p.His465Arg), c.1751A>G (p.Asp582Gly) and c.3290C>A (p.Thr1097Asp). The allele frequencies were tested among about 200 unrelated pigs from several pig breeds. Linkage mapping was further conducted with the SNP c.1751A>G (p.Asp582Gly) in a Berkshire × Yorkshire resource family and this confirmed that porcine CMYA1 is closely linked with Sw344 (distance  =  2 cM, LOD score is 129.47), an interesting region harbouring a QTL for back fat thickness. Association analysis in our experimental pig population showed that different genotypes of CMYA1 gene were associated with different back fat thicknesses ( P  <   0.05). Our results suggest that the porcine CMYA1 gene has effects on porcine back fat deposition and further investigation will be necessary to illustrate the underlying mechanisms.     

Volume measures for linkage disequilibrium

Background

A major QTL for fatness and growth, denoted FAT1, has previously been detected on pig chromosome 4q (SSC4q) using a Large White – wild boar intercross. Progeny that carried the wild boar allele at this locus had higher fat deposition, shorter length of carcass, and reduced growth. The position and the estimated effects of the FAT1 QTL for growth and fatness have been confirmed in a previous study. In order to narrow down the QTL interval we have traced the inheritance of the wild boar allele associated with high fat deposition through six additional backcross generations.

Results

Progeny-testing was used to determine the QTL genotype for 10 backcross sires being heterozygous for different parts of the broad FAT1 region. The statistical analysis revealed that five of the sires were segregating at the QTL, two were negative while the data for three sires were inconclusive. We could confirm the QTL effects on fatness/meat content traits but not for the growth traits implying that growth and fatness are controlled by distinct QTLs on chromosome 4. Two of the segregating sires showed highly significant QTL effects that were as large as previously observed in the F₂ generation. The estimates for the remaining three sires, which were all heterozygous for smaller fragments of the actual region, were markedly smaller. With the sample sizes used in the present study we cannot with great confidence determine whether these smaller effects in some sires are due to chance deviations, epistatic interactions or whether FAT1 is composed of two or more QTLs, each one with a smaller phenotypic effect. Under the assumption of a single locus, the critical region for FAT1 has been reduced to a 3.3 cM interval between the RXRG and SDHC loci.

Conclusion

We have further characterized the FAT1 QTL on pig chromosome 4 and refined its map position considerably, from a QTL interval of 70 cM to a maximum region of 20 cM and a probable region as small as 3.3 cM. The flanking markers for the small region are RXRG and SDHC and the orthologous region of FAT1 in the human genome is located on HSA1q23.3 and harbors approximately 20 genes. Our strategy to further refine the map position of this major QTL will be i) to type new markers in our pigs that are recombinant in the QTL interval and ii) to perform Identity-By-Descent (IBD) mapping across breeds that have been strongly selected for lean growth.     

Detection of quantitative trait loci for carcass composition traits in pigs

A quantitative trait locus (QTL) analysis of carcass composition data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. A total of 488 F2 males issued from six F1 boars and 23 F1 sows, the progeny of six LW boars and six MS sows, were slaughtered at approximately 80 kg live weight and were submitted to a standardised cutting of the carcass. Fifteen traits, i.e. dressing percentage, loin, ham, shoulder, belly, backfat, leaf fat, feet and head weights, two backfat thickness and one muscle depth measurements, ham + loin and back + leaf fat percentages and estimated carcass lean content were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half/full sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Additional analyses were performed to search for multiple linked QTL and imprinting effects. Significant gene effects were evidenced for both leanness and fatness traits in the telomeric regions of SSC 1q and SSC 2p, on SSC 4, SSC 7 and SSC X. Additional significant QTL were identified for ham weight on SSC 5, for head weight on SSC 1 and SSC 7, for feet weight on SSC 7 and for dressing percentage on SSC X. LW alleles were associated with a higher lean content and a lower fat content of the carcass, except for the fatness trait on SSC 7. Suggestive evidence of linked QTL on SSC 7 and of imprinting effects on SSC 6, SSC 7, SSC 9 and SSC 17 were also obtained.     

Differences in hepatic gene expression as a major distinguishing factor between Korean native pig and Yorkshire

There are phenotypic differences between Korean native pig (KNP) and Yorkshire (YS) breeds due to different interests in selection. YS has been selected for industrial interests such as a growth rate and lean meat production, while KNP has been maintained as a regional breed with local interests such as disease resistance and fat content in and between muscle. A comparison of gene expression profiles from liver tissue reflected overall long-term effects of artificial selection for these two pig breeds. Based on minimum positive false discovery rate (less than 10%) and fold change (|FC|>1.5), 73 differentially expressed genes (DEGs) were identified. Functional analysis of these DEGs indicated clear distinctions in signaling capacity related to epidermal growth factor (EGF), extracellular structure, protein metabolism, and detoxification. Hepatic DEGs demonstrated the importance of hormonal and metabolic capabilities to differences between these two pig breeds.     

Molecular characterization and association analysis of porcine CA3

Carbonic anhydrase 3 (CA3) is a member of the carbonic anhydrase family, which plays an important role in various cell processes. In this paper, molecular characterization revealed that CA3 genomic DNA consists of seven exons and six introns, spans about 10.5 kb and maps to porcine chromosome 4q11-->q14. Results of expression profiles showed that the expression levels of CA3 increased in skeletal muscles from prenatal 33- to 65-day-old Chinese Tongcheng pigs. These levels subsequently decreased to a steady state in prenatal 90-day-old, postnatal 2-day-old, postnatal 28-day-old, and pregnant 65-day-old pigs. The expression patterns of Chinese Tongcheng pig embryos were different from that of Landrace pig embryos. CA3 was expressed at higher levels in skeletal muscle and liver than in kidney, lung, stomach, intestine, and brain, but was not detected in heart and spleen. Statistical analysis showed the CA3 gene polymorphism was different between Chinese indigenous and introduced commercial western pig breeds, and was associated with intramuscular fat content and percentage of ham of pigs.     

QTL analysis of back fat thickness and carcass pH in an F2 intercross between Landrace and Korean native pigs

In this study, we conducted a genome-wide linkage analysis to identify the quantitative trait loci (QTL) that influence back fat thickness and carcass pH in an F(2) intercross between Landrace and Korean native pigs. Eight phenotypes related with back fat thickness and carcass pH were measured in more than 960 F(2) progeny. All experimental animals were subjected to genotypic analysis using 173 microsatellite markers located throughout the pig genome. The GridQTL program, based on the least squares regression model, was used to perform the QTL analysis. We identified 22 genome-wide significant QTL in 9 chromosomal regions (SSC1, 2, 5, 6, 7, 8, 12, 15, and 16) and 29 suggestive QTL in 16 chromosomal regions (SSC2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, 15, 16, 17, 18, and X). On SSC5, we detected a QTL affecting back fat thickness that accounted for 4.8 % of the phenotypic variance, which was the highest test statistic (F-ratio = 50.3 under the additive model, nominal P value = 2.5 × 10(-12)) observed in this study. Additionally, we showed that there were significant QTL on SSC16 affecting carcass pH traits. In conclusion, the QTL identified in this study together with associated positional candidate genes could play an important role in determining the genetic structure underlying the variation of back fat thickness and carcass pH in pigs.     

Analysis of non‐synonymous SNPs of the porcine SERPINA6 gene as potential causal variants for a QTL affecting plasma cortisol levels on SSC7

Recently, the SERPINA6 gene encoding corticosteroid‐binding globulin (CBG) has been proposed as a candidate gene for a quantitative trait locus (QTL) affecting cortisol level on pig chromosome 7. The QTL was repeatedly detected in different lines, including a Piétrain × (German Landrace × German Large White) cross (PiF1) and purebred German Landrace (LR). In this study, we investigated whether the known non‐synonymous polymorphisms c.44G>T, c.622C>T, c.770C>T, c.793G>A, c.832G>A and c.919G>A of SERPINA6 are sufficient to explain the QTL in these two populations. Our investigations revealed that SNPs c.44G>T, c.622C>T, c.793G>A and c.919G>A are associated with cortisol level in PiF1 (P < 0.01). Haplotype analysis showed that these associations are largely attributable to differences between a major haplotype carrying SNPs c.793G>A and c.919G>A and a haplotype carrying SNPs c.44G>T and c.622C>T. Furthermore, some SNPs, particularly c.44G>T and c.622C>T and the carrier haplotype, showed association with meat quality traits including pH and conductivity (P < 0.05). In LR, the non‐synonymous SNPs segregate at very low frequency (<5%) and/or show only weak association with cortisol level (SNPs c.832G>A and c.919G>A; P < 0.05). These findings suggest that the non‐synonymous SNPs are not sufficient to explain the QTL across different breeds. Therefore, we examined whether the expression of SERPINA6 is affected by cis‐regulatory polymorphisms in liver, the major organ for CBG production. We found allelic expression imbalance of SERPINA6, which suggests that its expression is indeed affected by genetic variation in cis‐acting elements. This represents candidate causal variation for future studies of the molecular background of the QTL.     

Detection of novel SNPs and mapping of the fatness QTL on pig chromosome 7q1.1-1.4 region

Many QTLs for fatness traits have been mapped on pig chromosome 7q1.1-1.4 in various pig resource populations. Eight novel markers, including seven SNPs and one insertion or deletion within BTNL1, COL21A1, PPARD, GLP1R, MDFI, GNMT, ABCC10, and PLA2G7 genes, as well as two previously reported SNPs in SLC39A7 and HMGA1 genes, were genotyped in Large White and Meishan pig breeds. Except for two SNPs in HMGA1 and ABCC10 genes, allele frequencies of the other eight markers are highly significant different between Chinese indigenous Meishan breeds and Large White pig breeds. Eight polymorphic sites were then used for linkage and QTL mapping to refine the fatness QTL in a Large White × Meishan F(2) resource population. Five chromosome-wise significant QTLs were detected, of which the QTLs for leaf fat weight, backfat thickness at 6-7th rib and rump, and mean backfat thickness were narrowed to the interval between PPARD and GLP1R genes and the QTL for backfat thickness at thorax-waist between GNMT and PLA2G7 genes on SSC7p1.1-q1.4.     

Detection of quantitative trait loci for growth- and fatness-related traits in a large-scale White Duroc × Erhualian intercross pig population

Growth and fatness are economically important traits in pigs. In this study, a genome scan was performed to detect quantitative trait loci (QTL) for 14 growth and fatness traits related to body weight, backfat thickness and fat weight in a large-scale White Duroc × Erhualian F(2) intercross. A total of 76 genome-wide significant QTL were mapped to 16 chromosomes. The most significant QTL was found on pig chromosome (SSC) 7 for fatness with unexpectedly small confidence intervals of ～2 cM, providing an excellent starting point to identify causal variants. Common QTL for both fatness and growth traits were found on SSC4, 5, 7 and 8, and shared QTL for fat deposition were detected on SSC1, 2 and X. Time-series analysis of QTL for body weight at six growth stages revealed the continuously significant effects of the QTL on SSC4 at the fattening period and the temporal-specific expression of the QTL on SSC7 at the foetus and fattening stages. For fatness traits, Chinese Erhualian alleles were associated with increased fat deposition except that at the major QTL on SSC7. For growth traits, most of White Duroc alleles enhanced growth rates except for those at three significant QTL on SSC6, 7 and 9. The results confirmed many previously reported QTL and also detected novel QTL, revealing the complexity of the genetic basis of growth and fatness in pigs.