Genome-wide association study for growth and fatness traits in Chinese Sujiang pigs

Growth and fatness traits are complex and economically important traits in the pig industry. The molecular basis underlying porcine growth and fatness traits remains largely unknown. To uncover genetic loci and candidate genes for these traits, we explored the GeneSeek GGP Porcine 80K SNP chip to perform a GWAS for seven growth and fatness traits in 365 individuals from the Sujiang pig, a recently developed breed in China. We identified two, 17, one and 11 SNPs surpassing the suggestively significant threshold (P < 1.86 × 10⁻⁵) for body weight, chest circumference, chest width and backfat thickness respectively. Of these SNPs, 20 represent novel genetic loci, and five and four SNPs were respectively associated with chest circumference and backfat thickness at a genome-wide significant threshold (P < 9.31 × 10⁻⁷). Eight SNPs had a pleiotropic effect on both chest circumference and backfat thickness. The most remarkable locus resided in a region between 72.95 and 76.27 Mb on pig chromosome 4, harboring a number of previously reported quantitative trait loci related to backfat deposition. In addition to two reported genes (PLAG1 and TAS2R38), we identified four genes including GABRB3, ZNF106, XKR4 and MGAM as novel candidates for body weight and backfat thickness at the mapped loci. Our findings provide insights into the genetic architecture of porcine growth and fatness traits and potential markers for selective breeding of Chinese Sujiang pigs.     

An imputation-based genome-wide association study for growth and fatness traits in Sujiang pigs

Sujiang pigs are a synthetic breed derived from Jiangquhai, Fengjing, and Duroc pigs. In this study, we sequenced the genome of 62 pigs with a coverage depth of 10× to 20×, including 27 Sujiang and 35 founder breed pigs, and we collected 360 global pigs’ genome sequence data from public databases including 39 Duroc pigs. We obtained a high-quality variant dataset of 365 Sujiang pigs by imputing the porcine 80 K single nucleotide polymorphism (SNP) Beadchip to the whole-genome scale with a total of 422 pigs as a reference panel. A dataset of 365 imputated Sujiang pigs was used to perform single-trait genome-wide association study (GWAS) and meta-analyses for growth and fatness traits. Single-trait GWAS identified 1 907, 18, and 14 SNPs surpassing the suggestively significant threshold for backfat thickness, chest circumference, and chest width, respectively. Meta-analyses identified 2 400 genome-wide significant SNPs and 520 suggestively significant SNPs for backfat thickness and chest circumference, and 719 genome-wide significant SNPs and 1 225 suggestively significant SNPs for all seven traits. According to the meta-analysis of backfat thickness and chest circumference, a remarkable region of 2.69 Mb on Sus scrofa chromosome 4 containing FAM110B, IMPAD1, LYN, MOS, PENK, PLAG1, SDR16C5 and XKR4 was identified as a candidate region. The haplotype heat map of the 2.69 Mb region verified that Sujiang pigs were derived from Duroc and Chinese indigenous pigs, especially Jiangquhai pigs. The Kruskal-Wallis test showed that haplotypes of the 2.69 Mb region significantly affected backfat thickness and chest circumference traits. We then focused on PLAG1, an important growth-related gene, and identified two synonymous SNPs with obvious differences among different breeds in the PLAG1 gene. We then performed genotyping of 365 Sujiang, 150 Duroc, 95 Jiangquhai, and 100 Fengjing pigs to confirm the above result and verified that the two variants significantly affected phenotypes of growth and fatness traits. Our findings not only provide insights into the genetic architecture of porcine growth and fatness traits but also provide potential markers for selective breeding of these traits in Sujiang pigs.     

猪esr基因反转录转座子插入多态性及其与大白猪生产性能的关联性分析

雌激素受体(Estrogen receptor,esr)介导雌激素影响相关基因表达,从而调控哺乳动物的生长和繁殖机能.为了探讨esr基因的反转录转座子多态性对猪生长性能的影响,文中应用比较基因组学和生物信息学方法,预测猪esr基因的反转录转座子插入位点,采用PCR方法验证不同品种猪中插入多态性,并将该基因型与大白猪性能...     

猪TLR4基因外显子1新等位基因的分离及遗传变异分析

文章采用PCR-SSCP方法对亚洲野猪、3个引进的商业化品种和10个中国地方猪品种共893个个体TLR4基因外显子1的遗传变异进行了检测,旨在系统分析国内外猪种TLR4基因的多态性,为探讨该基因在免疫和防御系统中发挥的作用提供依据。结果,在猪TLR4基因外显子1中分离到新的等位基因,共检测到3个等位基因,6种基因型。其中杜洛克检测到AA、BB、CC、AB、AC、BC基因型,有杜洛克血统的苏太猪中检测到BB、CC、BC基因型,长白猪、约克夏中检测到CC、BC基因型,野猪及所有10个中国地方猪品种TLR4基因外显子1高度保守,只检测到CC基因型,中国地方猪品种和引进品种TLR4基因外显子1多态性存在极显著的差异。3种基因型中CC型与GenBank中的序列一致,BB和AA基因型分别存在G93C同义突变位点和G194A无义突变位点,这2个变异位点与抗逆性和一般抗病力的关系值得进一步深入研究。     

Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits

Three non-synonymous single nucleotide polymorphisms (T221C, T232A and C233T) were detected in exon 4 of the porcine leptin receptor (LEPR) gene. The T232A substitution could be identified as a (Tsp509I) restriction fragment length polymorphism. The frequency of genotype TT varied in six genetic groups from 0.62 (Duroc) to 0.99 (Polish Large White). Sequencing of exon 4, performed for 30 animals, revealed that only two intragenic haplotypes (TC and AT at nucleotide position 232-233) were present. The phenotypic effect of the Tsp509I polymorphism was tested for the Polish Landrace (n = 241) and a synthetic line 990 (n = 243). There was no statistical evidence for the direct effect of the LEPR polymorphisms on fatness traits. However, in Polish Landrace allele A at position 232 was associated with thicker backfat over shoulder.     

Detection of quantitative trait loci for growth and fatness in pigs

A quantitative trait locus (QTL) analysis of growth and fatness data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six boars and 23 F1 sows, the progeny of six LW boars and six MS sows, produced 530 F2 males and 573 F2 females. Nine growth traits, i.e. body weight at birth and at 3, 10, 13, 17 and 22 weeks of age, average daily gain from birth to 3 weeks, from 3 to 10 weeks and from 10 to 22 weeks of age, as well as backfat thickness at 13, 17 and 22 weeks of age and at 40 and 60 kg live weight were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: 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. Both methods revealed highly significant gene effects for growth on chromosomes 1, 4 and 7 and for backfat thickness on chromosomes 1, 4, 5, 7 and X, and significant gene effects on chromosome 6 for growth and backfat thickness. Suggestive QTLs were also revealed by both methods on chromosomes 2 and 3 for growth and 2 for backfat thickness. Significant gene effects were detected for growth on chromosomes 11, 13, 14, 16 and 18 and for backfat thickness on chromosome 8, 10, 13 and 14. LW alleles were associated with high growth rate and low backfat thickness, except for those of chromosome 7 and to a lesser extent early-growth alleles on chromosomes 1 and 2 and backfat thickness alleles on chromosome 6.     

Hereditary haemochromatosis gene (HFE) H63D mutation shows an association with abnormal sperm motility

The aim of this study was to screen infertile men for HFE H63D mutation in correlation with clinical characteristics of infertile men (sperm concentration, sperm motility, morphology, testicular volume, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and total Testosterone levels) and find out if the HFE H63D mutation has an effect on male infertility. After excluding hormonal treatment, any scrotal pathology, having any systemic diseases such as diabetes mellitus, sickle cell anemia and microdeletions of the Y chromosome, a total of 148 infertile men with age range between 17 and 52-years-old (average age 29.6 ± 7.2) were enrolled into the study. Our analysis indicates that the mean FSH levels are significantly higher (6.3 ± 4.6 mIU/ml, P = 0.03), whereas sperm motility is significantly lower (36.6 ± 28.1%, P = 0.01) in the infertile men with the HFE H63D mutation compared with subjects lacking this mutation. Comparison of allele frequencies of the infertile men with Ts < 50% versus the infertile men with Ts > 50% revealed a significant difference as expected (P = 0.001, OR = 0.14, %95 CI = 0.04–0.44). Comparison of allele frequencies of infertile men with abnormal sperm motility versus infertile men with normal sperm motility revealed a highly significant difference (P = 0.005, OR = 3.11, %95 CI = 1.41–6.86). Thus, the HFE H63D mutation seems to be an important risk factor for impaired sperm motility and is clinically associated with male infertility.     

Health and immune traits of Basque and Large White pigs housed in a conventional or enriched environment

Since decades, production traits such as growth rate, feed efficiency or body composition have been drastically increased in pigs by genetic selection. Whether this selection impacted animal robustness is still unclear. In this study, we compared Large White (LW) pigs, a breed submitted to intense genetic selection for production traits, and Basque (B) pigs, a local rustic breed, reared in two different housing environments (conventional v. enriched). Adaptation to housing conditions among each breed was evaluated at the level of endocrine and immune traits. These are known to be impacted by housing conditions and breed; however, the interaction effects between genotype and environment are less described. Animals (20 per breed and housing environment) entered the experiment at 35 kg of live weight. Levels of cortisol, acute-phase inflammatory proteins, immunoglobulins and hydrogen peroxide, blood formula, lymphocyte proliferation and in-vitro cytokine expression were measured at ∼115 kg of live weight. Animals were checked for skin injuries during the growing period. At slaughter, at the average live weight of 145 kg, carcasses were examined for pathological conditions of the respiratory tract. The major result was that the two breeds exhibited differences in response to the housing environment. Among the 24 sanitary, endocrine or immune traits investigated, the housing conditions affected eight variables in both breeds (salivary cortisol at 0700 and 1900 h, severity of pneumonia at slaughter) or only in B pigs (severe skin lesions) or LW pigs (salivary cortisol at 1500 h, granulocyte numbers and lymphocyte/granulocyte ratio and lymphocyte proliferation). These observations strengthen the hypothesis that selection for high meat production level might be associated with an increased susceptibility of animals to environmental stressors.     

Mapping of the porcine oestrogen receptor 2 gene and association study with litter size in Iberian pigs

Genes encoding the oestrogen receptors (ESR) are considered candidate genes for prolificacy traits due to the key role these molecules play in the regulation of reproductive physiology. In this paper, we report the assignment of the pig ESR2 gene to porcine chromosome 1 by radiation hybrid mapping. Most of the ESR2 cDNA was sequenced from Iberian pig ovarian RNA samples and one A/G single nucleotide polymorphism (SNP) was found at exon 5, being associated with a Met/Val substitution at position 949. This SNP was genotyped using a PCR-RFLP (Hsp92II) protocol and its potential effect on litter size was evaluated in two Iberian pig populations. However, no statistically significant association between the ESR2 polymorphism and litter size was found.     

Polar overdominant inheritance of a DLK1 polymorphism is associated with growth and fatness in pigs

The polar overdominance model of inheritance was proposed to explain the non-Mendelian expression of callipyge muscular hypertrophy in sheep. The callipyge locus (CLPG) maps to the distal portion of ovine Chromosome 18 within the DLK1– GTL2 region and corresponds to human Chromosome 14q32, where uniparental disomy (UPD) of the region is associated with multiple congenital anomalies, including growth retardation and obesity. We investigated the porcine DLK1– GTL2 region in a cross of two pig breeds to determine if the callipyge polar overdominance is present in another species. Analyses of the parental origin of DLK1 polymorphism in the F2 offspring found that paternal inheritance of DLK1 allele 2 and maternal inheritance of the allele 1 was significantly associated with decreased fat deposition and increased lean muscle mass, while the opposite parental inheritance of these alleles was associated with slower prenatal and postnatal growth. These results suggest that the polar overdominance mode of inheritance is present in the pig chromosomal region that is homologous to the CLPG locus in sheep. Further study in pigs can provide important insights into understanding the molecular regulation of imprinted genes that are associated with human UPD14 and sheep callipyge phenotypes.     

Integrating genome-wide association study with RNA-seq revealed DBI as a good candidate gene for intramuscular fat content in Beijing black pigs

Increasing intramuscular fat (IMF) content can enhance the sensory quality of meat, including tenderness, juiciness, flavor, and color. Genome-wide association study and RNA-sequencing (RNA-seq) analysis were used to identify candidate IMF genes in Beijing Black pigs, a popular species among consumers in northern China. Two and three single nucleotide polymorphisms were significantly associated with IMF in SSC13 and SSC15 respectively. Solute carrier family 4 member 7 (SLC4A7) on SSC13 and insulin induced gene 2 (INSIG2), coiled-coil domain containing 93 (CCDC93), and diazepam binding inhibitor acyl-CoA binding protein (DBI) on SSC15 are good candidate genes in this population. Furthermore, RNA-seq analysis was performed between high and low IMF groups, and 534 differentially expressed genes were identified. In addition, based on differentially expressed genes, Kyoto Encyclopedia of Genes and Genomes analysis revealed that peroxisome proliferator-activated receptors and FoxO signaling pathway pathways might contribute to IMF. Moreover, the DBI gene was identified as a candidate for IMF both by genome-wide association study and RNA-seq analysis, suggesting that it might be a crucial candidate gene for influencing IMF in Beijing Black pigs.     

Identification and characterization of a new allele for the beta subunit of follicle-stimulating hormone in Chinese pig breeds

During evaluation of follicle-stimulating hormone-beta (FSHB) expression in anterior pituitary glands by an RNase protection assay (RPA), the expected fragment of 205 nucleotides at positions 759-963 was not detected in one boar that had moderate plasma and pituitary FSH concentrations. After subcloning and sequencing, mRNA from this boar lacked an 11-bp fragment (5'-CATTTGGAAAC-3') at nucleotide positions 807-817 of the 3'-untranslated region (3'-UTR, D allele). Wild-type FSHB (WT allele) was present in pituitary RNA and genomic DNA in both Meishan (MS) and White Composite (WC) pigs; whereas the D allele was present only in MS pigs (P < 0.01; 5/6 MS vs. 0/6 WC). Also, we found the D allele in five other Chinese breeds but absent in ten American Landrace, 11 Yorkshire and 17 Berkshire pigs. Additionally, the D allele had one silent nucleotide change in the coding region plus six, single nucleotide changes in the 3'-UTR.     

Genome-wide association analysis reveals genomic regions on Chromosome 13 affecting litter size and candidate genes for uterine horn length in Erhualian pigs

Litter size has a great impact on the profit of swine producers. Uterine development is an important determinant of reproduction efficiency and could hence affect litter size. Chinese Erhualian pig is one of the most prolific breeds in the world, even though large phenotypic variation in litter size was observed within Erhualian sows. To dissect the genetic basis of the phenotypic variation, we herein conducted genome-wide association studies for total number born and number born alive (NBA) of Erhualian sows. In total, one significant single nucleotide polymorphism (SNP) (P<1.78e−06) and 11 suggestive SNPs (P<3.57e−05) were identified on 10 chromosomes, confirming seven previously reported quantitative trait loci (QTL) and uncovering six QTL for litter size or uterus length. One locus on Sus scrofa chromosome (SSC) 13 (79.28 to 90.43 Mb) harbored a cluster of suggestive SNPs associated with multiparous NBA. The SNP (rs81447100) within this region was confirmed to be significantly (P<0.05) associated with litter size in Erhualian (n=313), Sutai (n=173) and Yorkshire (n=488) populations. Retinol binding protein 2 and retinol binding protein 1 functionally related to the development of uterus were located in a region of 2 Mb around rs81447100. Moreover, four genes related to embryo implantation and development were also detected around other significant SNPs. Taken together, our findings provide a potential marker (rs81447100) for the genetic improvement of litter size not only in Chinese Erhualian pigs but also in European commercial pig breeds like Yorkshire, and would facilitate the final identification of causative variant(s) underlying the effect of SSC13 QTL on litter size.     

An intronic polymorphism of IRF4 gene influences gene transcription in vitro and shows a risk association with childhood acute lymphoblastic leukemia in males

The interferon regulatory factor (IRF) family of DNA-binding proteins regulates expression of interferon-inducible genes with roles in the immune response and carcinogenesis. IRF4 is involved in the differentiation of B and T cells and is overexpressed in B-cell malignancies as a result of c-REL (NF-κB) hyperactivation. IRF4 polymorphisms are associated with susceptibility to chronic lymphoid leukemia (CLL) and non-Hodgkin lymphoma (NHL). We examined 13 IRF4 SNPs in 114 cases of childhood acute lymphoblastic leukemia (ALL) and 388 newborn controls from Wales (U.K.) using TaqMan assays. IRF4 intron 4 SNP rs12203592 showed a male-specific risk association (OR = 4.4, 95% CI = 1.5 to 12.6, P = 0.007). Functional consequences of the C > T substitution at this SNP were assessed by cell-based reporter assays using three different cell lines. We found a repressive effect of the rs12203592 wildtype allele C on IRF4 promoter activity (P < 0.001) but no repression by the variant allele in any cell line tested. Thus, homozygosity for the rs12203592 variant allele would result in increased IRF4 expression. This increase would be compounded by high levels of NF-κB activity in males due to the absence of estrogen. IRF4 differs from other IRFs in its anti-interferon activity which interferes with immune surveillance. We propose that a detailed study of IRF4 can provide information on the mechanism of the sex effect and the role of immune surveillance in childhood ALL development.     

Development of an in silico coding gene SNP map in pigs

A total of 5450 sequences obtained from the NCBI pig SNP database were consolidated into 465 unique sequences (189 singleton sequences and 276 contigs). These 465 sequences contained 1787 putative SNPs and had strong sequence homology to 433 human protein-coding genes based on blast analyses. These genes were assigned to the pig QTL maps ( http://www.animalgenome.org/QTLdb/pig.html ) via the human and pig comparative maps established by a pig radiation hybrid (RH) map. The SNP information characterized from this study provides a useful functional gene variation resource to facilitate QTL data mining in the pig genome.     

No evidence of association of the osteocalcin gene HindIII polymorphism with bone mineral density in Chinese women

Osteoporosis is a major health problem, mainly characterized by low bone mineral density (BMD). Osteocalcin (also known as BGP, for bone Gla protein) is a significant biomarker of bone turnover and thus the BGP gene has been considered as an important candidate gene for osteoporosis. A few studies on the relationship between variants of the BGP gene and BMD variation, via traditional association and/or linkage methods, have yielded conflicting results. In the present study, we simultaneously tested linkage and/or association of the BGP HindIII polymorphism with BMD in a large cohort of pre-menopausal Chinese women. A total of 1,263 subjects from 402 Chinese nuclear families were examined. Each family consists of both parents and at least one daughter aged between 20-45 years. BMDs at the lumbar spine and hip were measured by dual-energy X-ray absorptiometry (DXA). Using the QTDT (quantitative transmission disequilibrium test) program, we did not detect significant evidence of linkage or association between the BGP HindIII polymorphisms and the BMD variation at any skeletal site. Our data do not support the BGP gene having a major effect on BMD variation in pre-menopausal Chinese women.     

PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene

The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.