E. Scotti

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 approach1

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.

Study of candidate genes for glycolytic potential of porcine skeletal muscle: identification and analysis of mutations, linkage and physical mapping and association with meat quality traits in pigs

Several genes (PRKAA2, PRKAB1, PRKAB2, PRKAG3, GAA, GYS1, PYGM, ALDOA, GPI, LDHA, PGAM2 and PKM2), chosen according to their role in the regulation of the energy balance and in the glycogen metabolism and glycolysis of the skeletal muscle, were studied. Eleven single nucleotide polymorphisms (SNPs) were identified in six of these genes (PRKAB1, GAA, PYGM, LDHA, PGAM2 and PKM2). Allele frequencies were analyzed in seven different pig breeds for these loci and for a polymorphism already described for GPI and for three polymorphic sites already reported at the PRKAG3 locus (T30N, G52S and I199V). Linkage mapping assigned PYGM and LDHA to porcine chromosome (SSC) 2, PKM2 to SSC7, GAA to SSC12, PRKAB1 to SSC14 and PGAM2 to SSC18. Physical mapping, obtained by somatic cell hybrid panel analysis, confirmed the linkage assignments of PRKAB1 and GAA and localized ALDOA, PRKAB2 and GYS1 to SSC3, SSC4 and SSC6, respectively. Pigs selected for the association study, for which several meat quality traits were measured, were first genotyped at the PRKAG3 R200Q polymorphic site (RN locus), in order to exclude carriers of the 200Q allele, and then were genotyped for all the mutations considered in this work. Significant associations (P ≤ 0.001) were observed for the PRKAG3 T30N and G52S polymorphic sites with meat colour (L* at 24 h post mortem). PGAM2 and PKM2 were significantly associated (P = 0.01) with drip loss percentage and glycogen content at one hour post mortem, respectively.

Investigation of candidate genes for glycolytic potential of porcine skeletal muscle: Association with meat quality and production traits in Italian Large White pigs

The objective of this study was to investigate the association of DNA markers in candidate genes for glycolytic potential on meat quality parameters (pH(1), pH(u), glycogen and lactate content and glycolytic potential of semimembranosus muscle) and estimated breeding values (EBVs) for average daily gain, lean cuts, back fat thickness, ham weight, and feed:gain ratio in 272 Italian Large White pigs. Three mutations in the PRKAG3 gene (T30N, G52S and I199V) were investigated as well as single nucleotide polymorphisms in two other skeletal muscle genes (PGAM2 and PKM2) involved in the glycolytic pathway. Association analysis with the PRKAG3 markers showed significant results (P<0.05) only for pH(1) (I199V, with significant additive effect) and lactate content (T30N), confirming, at least in part, the effects of this gene on meat quality traits. Significant association (P<0.05) was also observed for PGAM2 and ham weight EBV with significant additive and dominance effects. PKM2 was associated with average daily gain, lean cuts (P<0.001), back fat thickness and feed:gain ratio (P<0.05), with significant additive and/or dominance effects on these traits. PKM2 encodes for a key enzyme of the muscle glycolytic pathway and maps on porcine chromosome 7 where other studies have reported important QTL for the same traits. These data might suggest an important function of this gene in the mechanisms that produce the observed effects. The results will be important to evaluate the inclusion of some of these DNA polymorphisms in marker assisted selection programs.

A genome wide association study for backfat thickness in Italian Large White pigs highlights new regions affecting fat deposition including neuronal genes

BackgroundCarcass fatness is an important trait in most pig breeding programs. Following market requests, breeding plans for fresh pork consumption are usually designed to reduce carcass fat content and increase lean meat deposition. However, the Italian pig industry is mainly devoted to the production of Protected Designation of Origin dry cured hams: pigs are slaughtered at around 160 kg of live weight and the breeding goal aims at maintaining fat coverage, measured as backfat thickness to avoid excessive desiccation of the hams. This objective has shaped the genetic pool of Italian heavy pig breeds for a few decades. In this study we applied a selective genotyping approach within a population of ~ 12,000 performance tested Italian Large White pigs. Within this population, we selectively genotyped 304 pigs with extreme and divergent backfat thickness estimated breeding value by the Illumina PorcineSNP60 BeadChip and performed a genome wide association study to identify loci associated to this trait.ResultsWe identified 4 single nucleotide polymorphisms with P≤5.0E-07 and additional 119 ones with 5.0E-07<P≤5.0E-05. These markers were located throughout all chromosomes. The largest numbers were found on porcine chromosomes 6 and 9 (n=15), 4 (n=13), and 7 (n=12) while the most significant marker was located on chromosome 18. Twenty-two single nucleotide polymorphisms were in intronic regions of genes already recognized by the Pre-Ensembl Sscrofa10.2 assembly. Gene Ontology analysis indicated an enrichment of Gene Ontology terms associated with nervous system development and regulation in concordance with results of large genome wide association studies for human obesity.ConclusionsFurther investigations are needed to evaluate the effects of the identified single nucleotide polymorphisms associated with backfat thickness on other traits as a pre-requisite for practical applications in breeding programs. Reported results could improve our understanding of the biology of fat metabolism and deposition that could also be relevant for other mammalian species including humans, confirming the role of neuronal genes on 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.

Analysis of melanocortin 1 receptor (MC1R) gene polymorphisms in some cattle breeds: their usefulness and application for breed traceability and authentication of Parmigiano Reggiano cheese

Abstract In cattle, the MC1R gene has been the subject of several studies with the aim to elucidate the biology of coat colour. Then, polymorphisms of this gene have been proposed as tools for breed identification and animal products authentication. As a first step to identify breed specific DNA markers that can be used for the traceability of mono-breed dairy cattle products we investigated, using PCR-RFLP and PCR-APLP protocols, the presence and distribution of some alleles at the MC1R locus in 18 cattle breeds for a total of 1360 animals. For each of seven breeds (Italian Holstein, Italian Brown, Italian Simmental, Rendena, Jersey, Reggiana and Modenese) a large number of animals (>70) was genotyped so the obtained results can be considered with more confidence. Allele Ed was identified only in black pied cattle (Italian Holstein and Black Pied Valdostana). Allele E (this nomenclature includes all alleles except Ed, E1 and e) was observed in Italian Brown, Rendena, Jersey, Modenese, Italian Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White and Danish Red. Allele E1 was identified in Italian Brown, Rendena, Grigio Alpina, Piedmontese, Swedish Red and White and Danish Red. The recessive allele e, known to cause red coat colour, was fixed in Reggiana and almost fixed in Italian Simmental. This allele was observed also in Italian Holstein, Italian Brown, Rendena, Jersey and Modenese albeit with low frequency. Moreover, this allele was detected in Valdostana, Pezzata Rossa d’Oropa, Piedmontese, Romagnola, Swedish Red and White, Danish Red, Charoleis and Salers. In the case of the Reggiana breed, which is fixed for allele e, the MC1R locus is highly informative with respect to breeds that carry other alleles or in which allele e is at very low frequency. In theory, using the MC1R locus it is possible to identify the presence of milk from some other breeds in Parmigiano Reggiano cheese labelled as exclusively from the Reggiana breed. This possibility was practically tested by setting up protocols to extract and analyse polymorphisms of the MC1R locus in several dairy products, including Parmigiano Reggiano cheese cured for 30 months. The lower detection limit was estimated to be 5% of non expected DNA. This test can represent a first deterrent against fraud and an important tool for the valorisation and authentication of Parmigiano Reggiano cheese obtained from only Reggiana milk.

Genetic heterogeneity and selection signature at the KIT gene in pigs showing different coat colours and patterns

Mutations in the porcine KIT gene (Dominant white locus) have been shown to affect coat colours and colour distribution in pigs. We analysed this gene in several pig breeds and populations (Sicilian black, completely black or with white patches; Cinta Senese; grey local population; Large White; Duroc; Hampshire; Pietrain; wild boar; Meishan) with different coat colours and patterns, genotyping a few polymorphisms. The 21 exons and parts of the intronic regions were sequenced in these pigs and 69 polymorphisms were identified. The grey-roan coat colour observed in a local grey population was completely associated with a 4-bp deletion of intron 18 in a single copy KIT gene, providing evidence that this mutation characterizes the I(d) allele described in the early genetic literature. The white patches observed in black Sicilian pigs were not completely associated with the presence of a duplicated KIT allele (I(p) ), suggesting that genetic heterogeneity is a possible cause of different coat colours in this breed. Selection signature was evident at the KIT gene in two different belted pig breeds, Hampshire and Cinta Senese. The same mutation(s) may cause the belted phenotype in these breeds that originated in the 18th-19th centuries from English pigs (Hampshire) and in Tuscany (Italy) in the 14th century (Cinta Senese). Phylogenetic relationships of 28 inferred KIT haplotypes indicated two clades: one of Asian origin that included Meishan and a few Sicilian black haplotypes and another of European origin.

DGAT1 p.K232A polymorphism in dairy and dual purpose Italian cattle breeds

The aim of this study was to evaluate the allele frequency distribution at the DGAT1 p.K232A polymorphic site in seven Italian dairy and dual purpose cattle breeds. On the whole, 651 animals belonging to Italian Holstein (116), Italian Brown (105), Italian Simmental (95), Valdostana Red Pied (95), Rendena (62), Reggiana (128) and Modenese (50) breeds were genotyped by PCR-RFLP. Sequencing was carried out to confirm results of the genotyping protocol. The DGAT1 p.232K allele was identified in Italian Holstein (25.4%), Reggiana (17.2%), and with very low frequency in Italian Simmental, Valdostana Red Pied and Rendena (<1%). In Italian Brown and Modenese, this allele was not detected. These results indicated that this polymorphic site can be considered for association studies only in Italian Holstein and Reggiana breeds. Deviation from Hardy- Weinberg equilibrium was observed in the Reggiana breed (P<0.01) in which there was an excess of heterozygous sires and absence of animals with the p.232KK genotype. This result should be further evaluated because the analysed sires represented almost all bulls available for artificial insemination in this breed. Comparison of allele frequencies at the DGAT1 locus with several other Holstein populations showed a wide range of variability, probably due to different selection strategies adopted.

Identification of polymorphisms in the rabbit (Oryctolagus cuniculus) myostatin (MSTN) gene and association analysis with finishing weight in a commercial rabbit population

Background: Myostatin (MSTN or GDF8) is a member of the transforming growth factor-b superfamily that actively represses skeletal muscle growth. Myostatin null mice generated by gene targeting showed a 2to 3-fold increase in muscle mass compared to wild type mice, primarily because of an increased number of muscle fibres, followed by muscle cell hypertrophy and suppression of body fat accumulation. Several mutations disrupting myostatin function or expression cause doublemuscle phenotypes, muscle hypertrophy, birthing difficulties, increased growth rate and increased feed conversion efficiency in cattle and sheep. Following the initiative of the Broad Institute shotgun sequencing of the rabbit genome (Ensembl Gene ID ENSOCUG00000012663 in the OryCun2 rabbit genome assembly), the rabbit MSTN gene sequence was recently assembled. No study has been carried out so far to investigate its variability or the effects of MSTN polymorphisms on rabbit production traits.

Differences of the porcine amelogenin X and Y chromosome genes (AMELX and AMELY) and their application for sex determination in pigs.

Molecular sexing in wild and domestic animals has becoming an important issue in several fields including reproduction. X and Y chromosome‐specific sequence differences of the amelogenin genes (AMELX and AMELY) have been described in different mammalian species and used for sex determination. We studied the possibility to use sequence variability between the porcine AMELX and AMELY genes for sex determination in pigs. Sequence analysis of about 400 bp of intron 3 of the porcine amelogenin genes showed the presence of a 9–10 bp deletion in AMELY gene compared to AMELX sequences. Moreover, one single nucleotide polymorphism (SNP) was detected for the AMELY sequence. Four other SNPs and 1 bp insertion differentiated three AMELX haplotypes indicating an unexpected quite high nucleotide diversity for a chromosome X region. Two sex determination assays targeting the 9–10 bp difference between AMELX and AMELY were developed. Assessment of the accuracy of the amelogenin assays to correctly sex individuals was tested on 329 pigs belonging to different breeds/lines. All analysed animals were correctly sexed with the new designed amelogenin tests. No amplification was obtained in human, cattle, goat, sheep, and horse genomic DNA. These assays can be used for sex diagnosis of small amounts of genomic DNA (20 pg) obtained from different sources including embryo biopsies, hair, meat, and other biological specimens. Thus, apart from the application in the reproduction field, these tests can be useful in several other sectors including forensics, archaeozoology, meat production, and processing as well as for quality control in sample identification. Mol. Reprod. Dev. 75: 1662–1668, 2008.