Associations of polymorphisms in the promoter I of bovine acetyl‐CoA carboxylase‐α gene with beef fatty acid composition

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) in the promoter I (PI) region of the bovine acetyl‐CoA carboxylase‐α (ACACA) gene and to evaluate the extent to which they were associated with lipid‐related traits. Eight novel SNPs were identified, which were AJ276223:g.2064T>A (SNP1), g.2155C>T (SNP2), g.2203G>T (SNP3), g.2268T>C (SNP4), g.2274G>A (SNP5), g.2340A>G (SNP6), g.2350T>C (SNP7) and g.2370A>G (SNP8). Complete linkage disequilibrium was observed among SNP1, 2, 4, 5, 6 and 8. Phenotypic data were collected from 573 cross‐bred steers with six sire breeds, including Hereford, Angus, Brangus, Beefmaster, Bonsmara and Romosinuano. The genotypes of SNP1/2/4/5/6/8 were significantly associated with adjusted backfat thickness. The genotypes of SNP3 were significantly associated with triacylglycerol (TAG) content and fatty acid composition of longissimus dorsi muscle (LM) in Brangus‐, Romosinuano‐ and Bonsmara‐sired cattle. Cattle with g.2203GG genotype had greater concentrations of TAG, total lipid, total saturated fatty acid and total monounsaturated fatty acid than did cattle with g.2203GT genotype. The genotypes of SNP7 were significantly associated with fatty acid composition of LM. Cattle with genotype g.2350TC had greater amounts of several fatty acids in LM than did cattle with genotype g.2350CC. Our results suggested that the SNPs in the PI region of ACACA gene are associated with variations in the fatty acid contents in LM.     

Detection of exonic variants within the melanocortin 1 receptor (MC1R) gene in Black Silky,White Leghorn and Golden duckwing Araucana chicken

The melanocortin 1 receptor (MC1R) gene can be considered a candidate functional gene for the pigmentation of plumage color. The aim of this study was to investigate the association between the genotype frequencies of g.69 T>C, g.376 G>A and g.427 A>G SNPs within the MC1R gene in Black silky (O), Golden duckwing Araucana (GA) and White Leghorn (W). The CC and AA genotype frequencies of g.69 T>C and g.427 A>G SNPs in White Leghorn (W) were both 1.000, and the TT genotype frequency of the g.69 T>C SNP in Golden duckwing Araucana (GA) was also 1.000. The GG and AA genotype frequencies of g.376 G>A and g.427 A>G SNPs in Black silky (O) were both 0.100. When a haplotype is observed using a combination of markers, a Golden duckwing Araucana (GA) can especially be distinguished when it is a TAG, TGG and TAA type in the SNP combination of the MC1R gene. In case of the CAA types, only White Leghorn (W) could specifically be distinguished. Therefore, three SNPs in MC1R may provide identification in chicken breeds.     

Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses

The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26 T > C, g.156 T > C, g.587A > G, g.598C > T, g.1485C > T, g.2115A > G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A > G and g.598C > T residing in the 5′UTR region were novel SNPs identified by this study. The g.2115A > G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.     

Association study of single nucleotide polymorphisms in JAK2 and STAT5B genes and their differential mRNA expression with mastitis susceptibility in Chinese Holstein cattle

The JAK–STAT pathway plays a key role in mediating immune responses. The genetic effects of single nucleotide polymorphisms (SNPs) in JAK2 and STAT5B were investigated for serum cytokines, mastitis indicators and productions traits in a population of 468 Chinese Holstein cattle. Pooled DNA sequencing revealed one SNP (BTA8:g.39645396A>G) in JAK2 and two SNPs (BTA19:g.43673888A>G and BTA19:g.43660093T>C) in STAT5B. A fixed effect model considering the effects of SNPs, parity, herd, season and year of calving was used by way of the general linear model procedure of sas . Genotype frequencies of these SNPs in the population were in Hardy–Weinberg equilibrium (P > 0.05). A novel SNP (g.39645396A>G) in JAK2 was predicted to change the amino acid from lysine to asparagine and was significantly associated with the somatic cell count (SCC) and somatic cell score (SCS), whereas g.43673888A>G in STAT5B was significantly associated with SCC, SCS and interleukin‐4 (IL‐4) (P < 0.05). The dominant effect of g.39645396A>G in JAK2 was significant for SCS, and its additive effect was significant for SCC, whereas the dominant effect of g.43673888A>G in STAT5B was significant for SCS and IL‐4 (P < 0.05). The combination of g.39645396A>G in JAK2 and g.43673888A>G in STAT5B showed a significant effect on SCC, SCS, IL‐4 and TNF‐α (P < 0.05). As for mRNA expression analysis, the AA genotype g.39645396A>G and GG genotype g.43673888A>G indicated higher mRNA expression level and were significantly different from other genotypes (P < 0.05). The results imply that JAK2 and STAT5B genes could be useful candidate genes, and the identified polymorphisms might potentially be strong genetic markers for selection of dairy cattle against mastitis development.     

Nucleotide variants in prion-related protein (testis-specific) gene (PRNT) and effects on Chinese and Mongolian sheep phenotypes

ABSTRACT

Studies of the ovine prion-related protein (testis-specific) gene (PRNT), including studies of genetic diversity, have highlighted its potential relationship to scrapie infection and economically important ovine traits. PRNT was previously reported to be highly polymorphic in Portuguese sheep. To characterize genetic polymorphisms in this gene in Asian sheep, a direct sequencing method was used to detect polymorphic loci in PRNT in 285 individual sheep from four Chinese and one Mongolian breeds. Seven SNP variants in PRNT were identified, including three novel variants (g.93G>A, g.162G>T, and g.190A>G) and four previously reported variants (g.17 C>T, g.112G>C, g.129C>T, and g.144A>G). In the five breeds that we analyzed, the mutation frequencies of g.190A>G in Lanzhou Fat-tail sheep (LFTS) and g.129C>T in the other four varieties were high (F>0.5). Moreover, thirteen different haplotypes that had a comparable distribution in the tested breeds were also identified; ‘C-G-G-C-A-G-A’ occurred at the highest frequency in the five sheep breeds. Additionally, we previously explored the significance of relationships between polymorphisms in PRNP or PRND and ovine growth performance. Here, we also performed correlation analysis in all tested loci. These loci polymorphisms were significantly associated with ten different growth traits (P<0.05), except for g.93G>A. Meanwhile, in contrast to a previous study, there was no significant association between the seven SNP loci analyzed and our previously reported sheep PRND or PRNP insertion/deletion mutations. Our findings may provide new insights into polymorphic variation in ovine PRNT, which may contribute to genetic improvements in economic traits that are important for sheep breeding.     

Identification of single nucleotide polymorphisms in the bovine follicle‐stimulating hormone receptor and effects of genotypes on superovulatory response traits

In dairy cows, there is evidence that failure to respond to superovulation protocols is a heritable trait. In women, genotyping for the p.N680S single nucleotide polymorphism (SNP) in the follicle‐stimulating hormone receptor (FSHR) gene may help identify poor responders before ovarian stimulation is initiated. Our objectives were to identify SNPs in the coding region of the bovine FSHR gene and to investigate the effect of FSHR genotypes on superovulatory response in Holstein cattle. Sequencing of FSHR exons 1–10 revealed seven SNPs. Three were non‐synonymous mutations (c.337C>G, c.871A>G and c.1973C>G). SNP c.337C>G encodes for a proline‐to‐alanine (p.Pro113Ala) amino acid replacement in the extracellular ligand‐binding domain of the receptor. PCR‐RFLP analyses showed that homozygous GG Holstein cows present a higher percentage of viable embryos, whereas GG and CG animals have less unfertilised oocytes. SNP c.871A>G results in an isoleucine‐to‐valine (p.Ile291Val) modification, and homozygous AA animals present lower embryo yield after superovulatory treatments. SNP c.1973C>G corresponds to a threonine‐to‐serine (p.The658Ser) modification in the intracellular carboxyl‐terminal domain of the FSHR protein, and homozygous GG Holstein cows were associated with a lower embryo yield and a higher percentage of unfertilised oocytes. Our results suggest that specific alleles of the bovine FSHR gene are associated with variations in embryo yield and in the number of unfertilised oocytes.     

Two mutations in the 5′‐flanking region of the KITLG gene are associated with litter size of dairy goats

In this study, Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the 5′‐flanking region of the KITLG gene by DNA sequencing and primer‐introduced restriction analysis–polymerase chain reaction. Two novel SNPs (g.13090G>T and g.13664C>A) were identified (GenBank Accession no. KM658964). Furthermore, g.13090G>T and g.13664C>A loci were closely linked in SN and GZ breeds (r² > 0.33). Association analysis results showed that g.13090G>T and g.13664C>A SNPs significantly affected litter size (P < 0.05). The litter size of individuals with the combined genotype GG/CC from both dairy goat breeds was greater than that of individuals with TT/AA in average parity (P < 0.05). Known biochemical and physiological functions, along with our results, indicated that GG/CC could be used in marker‐assisted selection to choose individuals with greater litter size from both breeds. These results extend the spectrum of genetic variation in the caprine KITLG gene and may contribute to genetic resources and breeding of goats.     

鸡Apo-AI基因g.-163 A>T单核苷酸多态性的功能性分析

鸡Apo-AI基因g.-163 A>T单核苷酸多态性(SNP)与鸡腹脂重和腹脂率显著相关. 生物信息分析显示,该SNP位于鸡Apo AI基因转录起始位点,提示它可能是一个功能性SNP. 为确定该SNP的功能性, 本研究分别构建了含该SNP位点A和T等位基因的启动子报告基因载体,分别在DF1细胞和HepG2细胞中比较这2个等位基因对鸡Apo AI基因启动子活性的影响. 研究发现,T等位基因的启动子报告基因活性及报告基因mRNA表达水平均显著高于A等位基因（P<0.05）,表明该SNP影响基因表达,是1个功能性SNP. 本研究结果提示,鸡Apo-AI基因g.-163 A>T有望作为优质鸡育种的功能性分子标记.     

Characterization of the porcine transferrin gene (TF ) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection

Transferrin (TF)‐mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron‐depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra‐ and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed‐specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.     

Genetic polymorphisms of the bovine Fatty acid binding protein 4 gene are significantly associated with marbling and carcass weight in Hanwoo (Korean Cattle)

The objective of this study was to investigate an association between polymorphisms in the FABP4 gene and phenotypic variation for marbling and carcass weight (CWT) in a population of Hanwoo steers. We re‐sequenced 4.3 kb of the FABP4 gene region in 24 Hanwoo bulls and identified 16 SNPs and 1 microsatellite polymorphism. Of these 16 SNPs, three SNPs [g.2774G>C (intron I), g.3473A>T (intron II) and g.3631G>A (exon III, creating a p.Met >Val amino acid substitution)] were genotyped in 583 steers to assess their association with carcass traits. The g.3473A allele showed a significant increasing effect on CWT (P = 0.01) and the g.3631G allele was associated with higher marbling score (P = 0.006). One haplotype of these three SNPs (CAG) was significantly associated with CWT (P = 0.02) and marbling score (P = 0.05) and could potentially be of value for marker assisted selection in Hanwoo cattle. The CAG haplotype effect for CWT was larger (11.14 ± 5.03 kg) than the largest single locus effect of g.3473A>T (5.01 ± 2.2 kg).     

A polymorphism in the porcine miR‐208b is associated with microRNA biogenesis and expressions of SOX‐6 and MYH7 with effects on muscle fibre characteristics and meat quality

MicroRNAs (miRNAs) encoded by the myosin heavy chain (MHC) genes are muscle‐specific miRNAs (myomiRs) and regulate the expression of MHC isoforms in skeletal muscle. These miRNAs have been implicated in muscle fibre types and their characteristics by affecting the heterogeneity of myosin. In pigs, miR‐208b and miR‐499 are embedded in introns of MYH7 and MYH7b respectively. Here, we identified a novel single nucleotide polymorphism (SNP) in intron 30 of MYH7 by which porcine miR‐208b is encoded. Based on the association study using a total of 487 pigs including Berkshire (n = 164), Landrace (n = 121) and Yorkshire (n = 202), the miR‐208b SNP (g.17104G>A) had significant effects on the proportions of types I and IIb fibre numbers (P < 0.010) among muscle fibre characteristics and on drip loss (P = 0.012) in meat quality traits. Moreover, the SNP affected the processing of primary miR‐208b into precursor miR‐208b with a marginal trend towards significance (P = 0.053), thereby leading to significant changes in the levels of mature miR‐208b (P = 0.009). These SNP‐dependent changes in mature miR‐208b levels were negatively correlated with the expression levels of its target gene, SOX‐6 (P = 0.038), and positively associated with the expression levels of its host gene, MYH7 (P = 0.046). Taken together, our data suggest that the porcine miR‐208b SNP differentially represses the expression of SOX‐6 by regulating miRNA biogenesis, thereby affecting the expression of MYH7 and the traits of muscle fibre characteristics and meat quality.     

Novel Polymorphisms in Porcine 3′UTR of the Leptin Gene,Including a Rare Variant within Target Sequence for MIR-9 Gene in Duroc Breed,not Associated with Production Traits

The leptin gene (LEP) is considered as a functional candidate for production traits in livestock due to its crucial role in energy homeostasis. Because polymorphisms in regulatory sequences may affect gene expression, we searched for them in the 3′UTR of LEP and analyzed their association with production traits. Four breeds and a composite line were studied. In the Polish Landrace and Polish Large White breeds, 8 SNPs and 1 indel were observed; whereas, in the Duroc breed, 9 specific SNPs were found. Pietrain and Line 990 were monomorphic. One SNP (g.+168C>T), observed in the Duroc breed only, was located within a target site for microRNA (miR-9). Association studies showed a weak association between one SNP (c.+846C>T) and abdominal fat weight in the Polish Landrace only. Thus, we concluded that contribution of polymorphisms in the 3′UTR to phenotypic variability of pig production traits is marginal. Moreover, we presented an overview of known polymorphisms (128) in the pig leptin gene.     

Polymorphism identification in goat GNRH1 and GDF9 genes and their association analysis with litter size

This study investigated the polymorphisms of GNRH1 and GDF9 genes in 641 goats of three breeds: Xinong Saanen, Guanzhong and Boer. Two allelic variants were identified in the GNRH1 gene (JN645280:g.3548A>G and JN645281:g.3699G>A) and one allelic variant was found in the GDF9 gene (JN655693:g.4093G>A). Furthermore, g.4093G>A was a missense mutation (p.Val397Ile of GDF9). Results of an association analysis indicated that SNPs g.3548A>G and g.4093G>A had significant effects on litter size (P < 0.05). The combination genotypes of SNPs g.3548A>G, g.3699G>A and g.4093G>A also affected litter size with the C₅ genotype having the highest litter size in the first, third, fourth and average parity. Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the GNRH1 and GDF9 genes could serve as genetic markers for litter size in goat breeding.     

Identification of a functional SNP in the 3′‐UTR of caprine MTHFR gene that is associated with milk protein levels

Xinong Saanen (n = 305) and Guanzhong (n = 317) dairy goats were used to detect SNPs in the caprine MTHFR 3′‐UTR by DNA sequencing. One novel SNP (c.*2494G>A) was identified in the said region. Individuals with the AA genotype had greater milk protein levels than did those with the GG genotype at the c.*2494 G>A locus in both dairy goat breeds (P < 0.05). Functional assays indicated that the MTHFR:c.2494G>A substitution could increase the binding activity of bta‐miR‐370 with the MTHFR 3′‐UTR. In addition, we observed a significant increase in the MTHFR protein level of AA carriers relative to that of GG carriers. These altered levels of MTHFR protein may account for the association of the SNP with milk protein level.