Nengshui Ding

Genome-wide identification of quantitative trait loci for carcass composition and meat quality in a large-scale White Duroc × Chinese Erhualian resource population.

Carcass and meat quality traits are economically important in pigs. In this study, 17 carcass composition traits and 23 meat quality traits were recorded in 1028 F(2) animals from a White Duroc x Erhualian resource population. All pigs in this experimental population were genotyped for 194 informative markers covering the entire porcine genome. Seventy-seven genome-wide significant quantitative trait loci (QTL) for carcass traits and 68 for meat quality were mapped to 34 genomic regions. These results not only confirmed many previously reported QTL but also revealed novel regions associated with the measured traits. For carcass traits, the most prominent QTL was identified for carcass length and head weight at 57 cM on SSC7, which explained up to 50% of the phenotypic variance and had a 95% confidence interval of only 3 cM. Moreover, QTL for kidney and spleen weight and lengths of cervical vertebrae were reported for the first time in pigs. For meat quality traits, two significant QTL on SSC5 and X were identified for both intramuscular fat content and marbling score in the longissimus muscle, while three significant QTL on SSC1 and SSC9 were found exclusively for IMF. Both LM and the semimembranous muscle showed common QTL for colour score on SSC4, 5, 7, 8, 13 and X and discordant QTL on other chromosomes. White Duroc alleles at a majority of QTL detected were favourable for carcass composition, while favourable QTL alleles for meat quality originated from both White Duroc and Erhualian.

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.

Susceptibility towards Enterotoxigenic Escherichia coli F4ac Diarrhea Is Governed by the MUC13 Gene in Pigs

Enterotoxigenic Escherichia coli (ETEC) F4ac is a major determinant of diarrhea and mortality in neonatal and young pigs. Susceptibility to ETEC F4ac is governed by the intestinal receptor specific for the bacterium and is inherited as a monogenic dominant trait. To identify the receptor gene (F4acR), we first mapped the locus to a 7.8-cM region on pig chromosome 13 using a genome scan with 194 microsatellite markers. A further scan with high density markers on chromosome 13 refined the locus to a 5.7-cM interval. Recombination breakpoint analysis defined the locus within a 2.3-Mb region. Further genome-wide mapping using 39,720 informative SNPs revealed that the most significant markers were proximal to the MUC13 gene in the 2.3-Mb region. Association studies in a collection of diverse outbred populations strongly supported that MUC13 is the most likely responsible gene. We characterized the porcine MUC13 gene that encodes two transcripts: MUC13A and MUC13B. Both transcripts have the characteristic PTS regions of mucins that are enriched in distinct tandem repeats. MUC13B is predicated to be heavily O-glycosylated, forming the binding site of the bacterium; while MUC13A does not have the O-glycosylation binding site. Concordantly, 127 independent pigs homozygous for MUC13A across diverse breeds are all resistant to ETEC F4ac, and all 718 susceptible animals from the broad breed panel carry at least one MUC13B allele. Altogether, we conclude that susceptibility towards ETEC F4ac is governed by the MUC13 gene in pigs. The finding has an immediate translation into breeding practice, as it allows us to establish an efficient and accurate diagnostic test for selecting against susceptible animals. Moreover, the finding improves our understanding of mucins that play crucial roles in defense against enteric pathogens. It revealed, for the first time, the direct interaction between MUC13 and enteric bacteria, which is poorly understood in mammals.

Correlations between fat depot traits and fatty acid composition in abdominal subcutaneous adipose tissue and longissimus muscle: Results from a White Duroc × Erhualian intercross F2 population

The aim of this study was to quantify the partial correlation coefficients (r(p)) between fat depot traits (FDT) and the fatty acid composition of abdominal subcutaneous adipose tissue and LM intramuscular fat in 639 F(2) pigs derived from a White Duroc × Chinese Erhualian cross. Fat depot traits are classified into 2 groups: 1 is adipose tissues (abdominal subcutaneous adipose tissue weight, mesenteric adipose tissue weight, perirenal adipose tissue weight, and backfat thickness at 4 locations); the other is LM [intramuscular fat content (IMF) and marbling score]. Correlations of FDT within classification groups were markedly greater (P < 0.001) than those observed between the 2 groups (r(p) = 0.62 vs. 0.26), indicating variability in fat content of muscle is relatively independent of amount of carcass fat. In general, fatter pigs had greater (P < 0.05) proportions of SFA and MUFA, and less PUFA, than leaner pigs. However, the relationships of individual fatty acids with FDT varied. We found that the amounts of some fatty acids regarded as neutral (e.g., stearic acid) or beneficial (e.g., palmitoleic acid and linolenic acid) for human health were associated with smaller amount of adipose tissues, or merely with greater IMF (P < 0.05). Therefore, we conclude that increasing the proportions of these neutral or healthy fatty acids can be achieved without reducing the IMF of LM, which is positively related to eating quality.

A genome-wide scan reveals candidate susceptibility loci for pig hernias in an intercross between White Duroc and Erhualian.

Pig scrotal/inguinal and umbilical hernias are the most prevalent congenital disorders in pigs and often cause animal welfare problems and economic loss. To identify susceptibility loci for these traits, a genome-wide scan with 194 microsatellite markers covering the pig genome was performed in a White Duroc x Erhualian resource population with 23 scrotal/inguinal F(2) animals, 50 umbilical F(2) animals, and their unaffected siblings. A sex-average linkage map with a total length of 2,350.3 cM and an average marker interval of 12.84 cM was constructed. Both nonparametric genome-wide linkage (NPL) analysis and transmission disequilibrium test (TDT) were implemented to detect closely linked markers. The NPL analysis revealed 11 chromosomal regions on SSC1, 2, 3, 6, 7, 8, 10, and 11 for umbilical hernia and 5 regions on SSC2, 4, 8, 13, and 16 for scrotal/inguinal hernia, whereas the TDT test identified susceptibility loci for umbilical hernia on SSC1, 2, 4, 7, 10, 13, 14, and 15 and for scrotal/inguinal hernias on SSC2, 8, 10, and 18. The most promising loci were SWR1928 on SSC7 and SW830 on SSC10 for umbilical hernia, and SW933 on SSC8 for scrotal hernia, which were consistently detected by both NPL and TDT. Several previously reported chromosomal regions for scrotal/inguinal hernia were confirmed, and new evidence for linkage with this pig defect was found. Moreover, susceptibility loci for pig umbilical hernia were detected for the first time.

Genetic variation within coat color genes of MC1R and ASIP in Chinese brownish red Tibetan pigs

Melanocortin receptor 1 (MC1R) and agouti signaling protein (ASIP) are two major genes affecting coat color phenotypes in mammals, and inactivation mutations in the MC1R gene are responsible for red coat color in European pig breeds. Conversely, the gain-of-function ASIP mutations block MC1R signaling and lead to the production of red pheomelanin. Chinese Tibetan pigs have three types of coat color phenotypes, including brownish red, solid black and black with patches of brownish red and white. Herein, we investigated variations of the MC1R and ASIP genes in Tibetan pigs. The results showed that the brownish red Tibet pig had the dominant black MC1R allele (E(D1)). No loss-of-function mutation in MC1R responsible for red coat color in European breeds was observed in this breed. No causal mutation for the red coat color phenotype was found in the coding sequence of the ASIP gene. A novel missense mutation c.157G > A was firstly identified in exon 2 of ASIP, which was further genotyped in 285 pigs from five Chinese breeds and three Western breeds having different coat color phenotypes. Nearly all pigs were GG homozygotes. In conclusion, no functional variant responsible for brownish red coloration was found in the coding region of MC1R and ASIP in Tibetan pigs.

Genetic Variation of Porcine Prostaglandin-endoperoxide Synthase 2 (PTGS2) Gene and Its Association with Reproductive Traits in an Erhualian × Duroc F2 Population

Identification of major genes that genetically impact female fertility is important for successful selection of high prolificacy pig lines. Because it is the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins (PGs), which are important for ovulation, fertilization, implantation, decidualization and parturition, prostaglandin-endoperoxide synthase 2 gene (PTGS2) is a potential candidate gene affecting porcine reproductive traits. In this study, a PCR-RFLP was used to genotype a total of 1 031 animals, including 661 from twelve Chinese local pig breeds, 190 from three Western pig breeds and 180 F2 sows from Nanchang pig resource family. Differences in frequency distributions of PTGS2 among twelve Chinese and three Western pig breeds and populations generally agree with their prolificacy. The allele frequencies in Lower Changjiang River Basin Type pig breeds, North China Type and Central China Type breeds are significantly different from those in South China Type, Plateau Type and Western pig breeds (P<0.001). And no significant differences were observed among Lower Changjiang River Basin Type, North China Type, Central China Type pig breeds, between South China Type and Western pig breeds, in part because of similar fertility patterns. And notable associations as well as reliable additive and dominant effects were not detected in an Erhualian x Duroc F2 population (P>0.05). Whereas, there is a trend for animals with one copy of the favourable A allele to have an increased TNB (total number of piglet born) and TBA (the number of piglets born alive) and a decreased SB (stillborn pigs) trait. Considering its crucial role in reproductive pathways, the PTGS2 gene deserves further study.

Identification of new single nucleotide polymorphisms affecting total number born and candidate genes related to ovulation rate in Chinese Erhualian pigs

The Chinese Erhualian pig has the highest record for litter size in the world. However, the genetic mechanism of its high prolificacy remains poorly understood. In our study, large phenotypic variations in litter size were found among Erhualian sows. Significant differences in total number born (TNB) and corpora lutea numbers were observed between sows with high and low estimated breeding values (EBVs) for TNB. To identify single nucleotide polymorphisms (SNPs) associated with TNB, a selective genomic scan was conducted on 18 sows representing the top 10% and 18 sows representing the bottom 10% of EBVs of 177 sows using Illumina Porcine SNP60 genotype data. Genome-wide fixation coefficient (FST ) values were calculated for each SNP between the high- and low-EBV groups. A total of 154 SNPs were significantly differentiated loci between the two groups. Of the top 10 highest FST SNPs, rs81399474, rs81400131 and rs81405013 on SSC8 and rs81434499 and rs81434489 on SSC 12 corresponded to previously reported QTL for litter size. The other five SNPs, rs81367039 on SSC2, rs80891106 on SSC7, rs81477883 on SSC12 and rs80938898 and rs80971725 on SSC14, appeared to be novel QTL for TNB. Significant associations between rs81399474 on SSC8 and TNB were confirmed in 313 Erhualian sows. Forty genes were identified around the top 10 highest FST SNPs, of which UCHL1, adjacent to rs81399474, and RPS6KB1 and CLTC, adjacent to rs81434499, have been reported to affect the ovulation rate in pig. The findings can advance understanding of the genetic variations in litter size of pigs.

Porcine SOX9 Gene Expression Is Influenced by an 18bp Indel in the 5’-Untranslated Region

Sex determining region Y-box 9 (SOX9) is an important regulator of sex and skeletal development and is expressed in a variety of embryonal and adult tissues. Loss or gain of function resulting from mutations within the coding region or chromosomal aberrations of the SOX9 locus lead to a plethora of detrimental phenotypes in humans and animals. One of these phenotypes is the so-called male-to-female or female-to-male sex-reversal which has been observed in several mammals including pig, dog, cat, goat, horse, and deer. In 38,XX sex-reversal French Large White pigs, a genome-wide association study suggested SOX9 as the causal gene, although no functional mutations were identified in affected animals. However, besides others an 18bp indel had been detected in the 5′-untranslated region of the SOX9 gene by comparing affected animals and controls. We have identified the same indel (Δ18) between position +247bp and +266bp downstream the transcription start site of the porcine SOX9 gene in four other pig breeds; i.e., German Large White, Laiwu Black, Bamei, and Erhualian. These animals have been genotyped in an attempt to identify candidate genes for porcine inguinal and/or scrotal hernia. Because the 18bp segment in the wild type 5′-UTR harbours a highly conserved cAMP-response element (CRE) half-site, we analysed its role in SOX9 expression in vitro. Competition and immunodepletion electromobility shift assays demonstrate that the CRE half-site is specifically recognized by CREB. Both binding of CREB to the wild type as well as the absence of the CRE half-site in Δ18 reduced expression efficiency in HEK293T, PK–15, and ATDC5 cells significantly. Transfection experiments of wild type and Δ18 SOX9 promoter luciferase constructs show a significant reduction of RNA and protein levels depending on the presence or absence of the 18bp segment. Hence, the data presented here demonstrate that the 18bp indel in the porcine SOX9 5′-UTR is of functional importance and may therefore indeed be a causative variation in SOX9 associated traits.

Mapping QTL for porcine muscle fibre traits in a White Duroc × Erhualian F2 resource population.

Muscle fibre traits are related with meat quality in meat animals. In this study, a whole-genome scan with 183 microsatellite markers covering the pig genome was performed to identify quantitative trait loci (QTL) for cross-sectional area, numerical percentage and relative area of type I, IIA and IIB myofibres, fibre number per square centimetre and total fibre number in the longissimus muscle by using 120 F(2) animals in a White Duroc x Erhualian intercross. In total, 20 QTL were mapped on pig chromosomes (SSC) 1, 2, 7, 8, 9, 11, 15, 16 and X, of which eight reached genome-wide significance levels and explained large proportions (6.53-34.63%) of phenotypic variance. Five QTL detected in this study confirmed the previous QTL reports and the others were detected for the first time. Chinese Erhualian alleles are generally associated with muscle fibre traits favourable for meat quality.