Molecular characterization, expression pattern, polymorphism and association analysis of bovine ADAMTSL3 gene

A disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like 3 (ADAMTSL3) is an important candidate gene for body measurement traits through marker-assisted selection (MAS). The objective of this study was to analyze SNP of bovine ADAMTSL3 gene and its specific expression in tissues to explore its possible correlation with body measurement traits in Bos taurus. Our genomic structural analysis showed that bovine ADAMTSL3 shares much similarity with human ADAMTSL3. Gene expression analysis indicated that the order of specific tissue expression patterns of bovine ADAMTSL3 was the testis, heart, fat, stomach, small intestine, liver, lung, trachea, kidney, spleen, large intestine and muscle. Allele frequencies demonstrated significant variance in different local cattle breeds. Also, the T1532C SNP in exon 13 and C1899T SNP in exon 15 were significantly correlated with Body Length (BL), Chest Depth (CD), Heart Girth (HG) and Pin Bone Width (PBW). Furthermore, C1899T SNP marker had significant correlation with the PBW (P = 0. 0235) based on 867 individual analysis. In the total population, combination of T1532C and C1899T SNPs significantly affected on the BL (P = 0.0028), CD (P < 0.0001), HG (P = 0.0002) and PBW (P = 0.0049). These results suggest that the ADAMTSL3 gene, as one of target genes for body measurement traits in cattle breeds, could be used as a genetic marker to select excellent body type of cattle population in the animal breeding program.     

Molecular characterization, polymorphism of bovine ZBTB38 gene and association with body measurement traits in native Chinese cattle breeds

Zinc finger and BTB domain containing 38 (ZBTB38), binding to and repressing methylated DNA, is an important candidate gene for selection of body measurement traits through marker-assisted selection (MAS). The expression of ZBTB38 is regulated in human and animal height as well as other stature indexes. Genomic structural analysis shows that bovine ZBTB38 shares much similarity with human ZBTB38. We discovered and evaluated the potential association of the single nucleotide polymorphism (SNP) of the bovine ZBTB38 gene with body measurement traits in 722 individuals. The latest findings demonstrate that the A841G SNP in exon 1 is significantly associated with Body Length (BL), Hip Height (HH) and Heart Girth (HG). Furthermore, the analysis of A841G SNP marker shows that there are significant effects on the BL (P = 0.0389) in 722 individuals, significant effects on the HH (P = 0.0173) and HG (P = 0.0147) in Qinchuan improvement steers (QI) population, as well as significant effects on the WH (P = 0.0094) in Xuelong (XL) population. These results clearly suggest that the ZBTB38 gene is among of target genes for body measurement traits in bovine reproduction and breeding, and thus provide data for establishment of an animal model using cattle to study big animal body type.     

Polymorphisms of the bovine DKK2 and their associations with body measurement traits and meat quality traits in Qinchuan cattle

The objective of this research were to detect bovine Dickkopf 2 (DKK2) gene polymorphism and analyze their associations with body measurement traits (BMT) and meat quality traits (MQT) of animals. Blood samples were taken from a total of 541 Qinchuan cattle aged from 18 to 24 months. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was employed to find out DKK2 single-polymorphism nucleotide (SNPs) and to explore their possible association with BMT and MQT. Sequence analysis of DKK2 gene revealed 2 SNPs (C29 T and A169C) in 5′ untranslated region (5′UTR) of exon 1.C29T and A164T SNPs are both synonymous mutation, which showed 2 genotypes namely (CC, CT) and (AA and AC), respectively. Association analysis of polymorphism with body measurement and meat quality traits at the two locus showed that there were significant effects on CT, BL, RL, PBW, BFT, LMA, and IFC. These results suggest that the DKK2 gene might have potential effects on BMT and MQT in Qinchuan cattle population and could be used for marker-assisted selection.     

A Common Variant Of Ubiquinol-Cytochrome c Reductase Complex Is Associated with DDH

Purpose

Genetic basis of Developmental dysplasia of the hip (DDH) remains largely unknown. To find new susceptibility genes for DDH, we carried out a genome-wide association study (GWAS) for DDH.

Methods

We enrolled 386 radiology confirmed DDH patients and 558 healthy controls (Set A) to conduct a genome-wide association study (GWAS). Quality-control was conducted at both the sample and single nucleotide polymorphism (SNP) levels. We then conducted a subsequent case-control study to replicate the association between a promising loci, rs6060373 in UQCC gene and DDH in an independent set of 755 cases and 944 controls (set B).

Results

In the DDH GWAS discovering stage, 51 SNPs showed significance of less than 10^-4, and another 577 SNPs showed significance of less than 10-3. In UQCC, all the 12 genotyped SNPs showed as promising risk loci. Genotyping of rs6060373 in set A showed the minor allele A as a promising risk allele (p = 4.82*10^-7). In set A, the odds ratio of allele A was 1.77. Genotyping of rs6060373 in Set B produced another significant result (p = 0.0338) with an odds ratio of 1.18 for risk allele A. Combining set A and set B, we identified a total p value of 3.63*10^-6 with the odds ratio of 1.35 (1.19–1.53) for allele A.

Conclusion

Our study demonstrates common variants of UQCC, specifically rs6060373, are associated with DDH in Han Chinese population.     

Partial molecular characterization,expression pattern,polymorphism and association analysis of porcine <Emphasis Type="Italic">SKP2</Emphasis> gene

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).     

Detection of Bovine Central Nervous System Tissue as Bovine Spongiform Encephalopathy Risk Material by Real-Time Reverse Transcriptase-PCR in Raw and Cooked Beef Products

PCR-SSCP and DNA sequencing methods were applied to reveal single nucleotide polymorphisms (SNPs) in the bovine VEGF-B gene in 675 samples belonging to three native Chinese cattle breeds. We found 3 SNPs and a duplication NC_007330.5: g. [782 A>G p. (Gly112 =) (;) 1000-1001dup CT (;) 1079 C>T (;) 2129 G>A p. (Arg184Gln)]. We also observed a statistically significant association of the polymorphism (1000-1001dup CT) in intron 3 of the VEGF-B gene with the body weight of the Nanyang cattle (p < 0.05). This polymorphisms of VEGF-B gene need to be verified among a larger cattle population before it can be identified as a marker for bovine body weight.     

Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis(1)

The objective was to determine whether single nucleotide polymorphisms (SNPs) in the ANKRA2 and CD180 genes are associated with incidence of bovine respiratory disease (BRD) and presence of Mycobacterium avium subsp. paratuberculosis (MAP) in cattle. Two independent populations were used. The first population (BRD‐affected; N = 90) was composed of 31 half‐sib progeny, from a Brahman × Angus sire, that were treated for BRD. Untreated offspring from the sire were selected to serve as controls. The second population (MAP‐infected) of 330 animals of unknown parentage was evaluated for the presence of MAP in ileocecal lymph node and classified as positive or negative. Markers in both genes were assessed for association in these two populations. In the BRD‐affected population, five SNPs in the ANKRA2 gene were significantly associated (P < 0.05), and two SNPs were highly associated (P < 0.01) with incidence of BRD. In addition, two SNPs in the CD180 gene were found to be associated with this trait. In the MAP‐infected population, one SNP in the ANKRA2 gene was significantly associated (P < 0.05) with the presence or absence of MAP, and a SNP in the CD180 gene was highly associated (P < 0.01) with the trait. Haplotypes, using significant markers, showed a positive association with both incidence of BRD (P = 0.0001) and with the presence of MAP (P = 0.0032). Markers in the ANKRA2 and CD180 genes are associated with the ability of the animal to cope with pathogens.     

Identification of a fructose-1,6-bisphosphate aldolase gene and association of the single nucleotide polymorphisms with growth traits in the clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

This study investigated whether there were single nucleotide polymorphisms (SNPs) in fructose-1,6-bisphosphate aldolase (FBA) gene associated with growth traits of the clam Meretrix meretrix. A FBA gene was identified in M. meretrix and its deduced amino acid residues shared high identity with type I aldolase. The FBA (MmeFBA) mRNA expression profile was examined by real-time PCR in different tissues and the significantly high expression level in foot and adduct muscle suggests that MmeFBA is a muscle type aldolase which functions in glycolytic pathway. In the MmeFBA gene, we identified four intron SNPs and three exon SNPs including a nonsynonymous SNP (mmfbae-2). These SNPs were genotyped in 205 clams from two clam populations with significantly different growth performance. Results showed that allele frequencies of three SNPs (mmfbai-1, mmfbai-3 and mmfbae-2) and the genotype frequency of mmfbai-1 were all significantly different between the two populations. The haplotype analysis further supported the three SNPs distributed differently between the two populations. This study successively characterized three growth-related SNPs in a gene involved in energy metabolism of M. meretrix. These findings could contribute the development of phenotype-selective breeding program in M. meretrix.     

Genetic Variability and Linkage Disequilibrium Patterns in the Bovine <Emphasis Type="Italic">DNAJA1</Emphasis> Gene

Correlation between expression level of the bovine DNAJA1 gene and meat tenderness was recently found in Charolais longissimus thoracis muscle samples, suggesting that this gene could play an important role in meat tenderness. Here, we report the validation of polymorphisms within the bovine DNAJA1 gene, and the haplotype variability and extent of linkage disequilibrium in the three main French beef breeds (Blonde d’Aquitaine, Charolais, Limousin). Genotyping 18 putative SNPs revealed that 16 SNPs were polymorphic within the breeds tested. Two SNPs were removed from further analyses as one SNP had a low genotyping call rate, while the other SNP was not in Hardy–Weinberg equilibrium. The degree of heterozygosity observed for the remaining 14 SNPs varied between breeds, with Charolais being the breed with the highest genetic variation and Blonde d’Aquitaine the lowest. Linkage disequilibrium and haplotype structure of DNAJA1 were different between breeds. Eighteen different haplotypes, including three shared by all breeds, were discovered, and two to three tag SNPs (depending on the breed) are sufficient to capture all the genetic variability seen in these haplotypes. The results of this study will facilitate the design of optimal future association studies evaluating the role of the DNAJA1 gene in meat tenderness.     

Comprehensive evaluation of the estrogen receptor α gene reveals further evidence for association with type 2 diabetes enriched for nephropathy in an African American population

We previously investigated the estrogen receptor α gene (ESR1) as a positional candidate for type 2 diabetes (T2DM), and found evidence for association between the intron 1-intron 2 region of this gene and T2DM and/or nephropathy in an African American (AA) population. Our objective was to comprehensively evaluate variants across the entire ESR1 gene for association in AA with T2DM and end stage renal disease (T2DM–ESRD). One hundred fifty SNPs in ESR1, spanning 476 kb, were genotyped in 577 AA individuals with T2DM–ESRD and 596 AA controls. Genotypic association tests for dominant, additive, and recessive models, and haplotypic association, were calculated using a χ² statistic and corresponding P value. Thirty-one SNPs showed nominal evidence for association (P < 0.05) with T2DM–ESRD in one or more genotypic model. After correcting for multiple tests, promoter SNP rs11964281 (nominal P = 0.000291, adjusted P = 0.0289), and intron 4 SNPs rs1569788 (nominal P = 0.000754, adjusted P = 0.0278) and rs9340969 (nominal P = 0.00109, adjusted P = 0.0467) remained significant at experimentwise error rate (EER) P ≤ 0.05 for the dominant class of tests. Twenty-three of the thirty-one associated SNPs cluster within the intron 4–intron 6 regions. Gender stratification revealed nominal evidence for association with 35 SNPs in females (352 cases; 306 controls) and seven SNPs in males (225 cases; 290 controls). We have identified a novel region of the ESR1 gene that may contain important functional polymorphisms in relation to susceptibility to T2DM and/or diabetic nephropathy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

<Emphasis Type="Italic">BRCA1</Emphasis>: a new candidate gene for bovine mastitis and its association analysis between single nucleotide polymorphisms and milk somatic cell score

Bovine mastitis is a very complex and common disease of dairy cattle and a major source of economic losses to the dairy industry worldwide. In this study, the bovine breast cancer 1, early onset gene (BRCA1) was taken as a candidate gene for mastitis resistance. The main object of this study was to investigate whether the BRCA1 gene was associated with mastitis in cattle. Through DNA sequencing, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Created Restriction Site PCR (CRS-PCR) methods, three SNPs (G22231T, T25025A, and C28300A) were detected and twenty-four combinations of these SNPs were observed. The single SNP and their genetic effects on somatic cell score (SCS) were evaluated and a significant association with SCS was found in C28300A. The mean of genotype EE was significantly lower than those of genotypes EF and FF. The results of combined genotypes analysis of three SNPs showed that BBDDFF genotype with the highest SCS were easily for the mastitis susceptibility, whereas AACCEE genotype with the lowest SCS were favorable for the mastitis resistance. The information provided in the present study will be very useful for improving mastitis resistance in dairy cattle by marker-assisted selection.     

The polymorphisms of bovine <Emphasis Type="Italic">VEGF</Emphasis> gene and their associations with growth traits in Chinese cattle

PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of VEGF gene in 671 individuals belonging to three Chinese indigenous cattle breeds including Nanyang, Jiaxian Red and Qinchuan. Three haplotypes (A, B and C), four observed genotypes (AA, AB, BB and AC) and three new SNPs (6765T>C ss130456744, 6860A>G ss130456745, 6893T>C ss130456746) were detected. The analysis suggested that one SNP (ss130456744) in the bovine VEGF gene had significant effects on birth weight, body weight and heart girth at 6 months old in the Nanyang breed (P < 0.05). The results showed that the SNP (ss130456744) in intron 2 of the VEGF gene is associated with early development and growth of Chinese cattle. These findings raise hope that this polymorphism can be a molecular breeding marker in breeding strategies through marker assisted selection (MAS) in Chinese domestic cattle.     

Fine Mapping and Functional Analysis of the Multiple Sclerosis Risk Gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2^nd SRCR domain with susceptibility to MS (P _{max(T) permutation} = 1×10⁻⁴). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4⁺ naïve cells, P = 0.0001; CD8⁺ naïve cells, P<0.0001; CD4⁺ and CD8⁺ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4⁺ and CD8⁺ T cells.     

Associations of polymorphisms in TXNIP and gene–environment interactions with the risk of coronary artery disease in a Chinese Han population

Single nucleotide polymorphisms (SNPs) in thioredoxin‐interacting protein (TXNIP) gene may modulate TXNIP expression, then increase the risk of coronary artery disease (CAD). In a two‐stage case–control study with a total of 1818 CAD patients and 1963 controls, we genotyped three SNPs in TXNIP and found that the variant genotypes of SNPs rs7212 [odds ratio (OR) = 1.26, P = 0.001] and rs7211 (OR = 1.23, P = 0.005) were significantly associated with increased CAD risk under a dominant model. In haplotype analyses, compared with the reference haplotype, haplotype ‘G‐T’ had a 1.22‐fold increased risk of CAD (P = 0.003). We also observed the cumulative effects of SNPs rs7212 and rs7211 on CAD risk and the severity of coronary atherosclerosis. Moreover, the gene–environment interactions among the variant genotypes of SNP rs7212, smoking habit, alcohol drinking habit and history of type 2 diabetes were associated with a 3.70‐fold increased risk of CAD (P < 0.001). Subsequent genotype‐phenotype correlation analyses further observed the significant effects of SNP rs7212 on TXNIP mRNA expression, plasma TXNIP and malondialdehyde levels. Taken together, our data suggest that TXNIP SNPs may individually and cumulatively affect CAD risk through a possible mechanism for regulating TXNIP expression and gene–environment interactions.     

Novel SNPs in the bovine ADIPOQ and PPARGC1A genes are associated with carcass traits in Hanwoo (Korean cattle)

Adiponectin (ADIPOQ) modulates several biological processes including energy homeostasis, glucose and lipid metabolism. The bovine ADIPOQ gene was located near the QTL affecting marbling, ribeye muscle area and fat thickness on BTA1. The gene encoding peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) was located within the QTL region of the traits on BTA6. Moreover, its protein product has various biological functions such as cellular energy homeostasis, including adaptive thermogenesis, adipogenesis and gluconeogenesis. Therefore, the ADIPOQ and PPARGC1A genes are a positional and functional candidate gene for carcass traits in beef cattle. The objectives of this study were to identify polymorphisms in the bovine ADIPOQ and PPARGC1A genes, to evaluate their associations with carcass traits in Hanwoo (Korean cattle) population. We identified nine SNPs in the ADIPOQ gene. Two SNPs (DQ156119: g.1436T > C and DQ156119: g.1454A > G) in the promoter region were recognized as new SNPs identified in Hanwoo. Association analysis indicated that the g.1454A > G SNP genotype was significantly associated with effects on LMA (P = 0.004) and BF (P = 0.021). The ADIPOQ haplotype was also found to have significant effect on the LMA. In the PPARGC1A gene, we identified 11 SNPs in the two unexplored regions (intron 3 and 5). Among them, seven SNPs were located in intron 3 and four SNPs were located in intron 5. Of these 11 putative novel SNPs, two SNPs (AY839822: g.292C > T and AY839823: g.1064C > T) with minor allele frequency (MAF) > 0.20 were examined for associations with carcass traits. The association analysis revealed that both SNPs in PPARGC1A gene were significantly associated with LMA (P < 0.05). These findings suggest that the SNPs of bovine ADIPOQ and PPARGC1A genes may be a useful molecular marker for selection of carcass traits in Hanwoo.     

Effect of Single Nucleotide Polymorphisms in IGFBP2 and IGFBP3 Genes on Litter Size Traits in Berkshire Pigs

Litter size is among the most important traits in swine breeding. However, information on the genetics of litter size in pigs is lacking. In this study, we identified single nucleotide polymorphisms (SNPs) in the insulin-like growth factor binding protein 2 and 3 (IGFBP2 and IGFBP3) genes in Berkshire pigs and analyzed their association with litter size traits. The IGFBP2 SNP was located on chromosome 15 intron 2 (455, A?>?T) and the IGFBP3 SNP was on chromosome 18 intron 2 (53, A?>?G). The AT type of IGFBP2 and the GG type of IGFBP3 had the highest values for all litter size traits including total number born (TNB), number of pigs born alive, and breeding value according to TNB. Homozygous GG pigs expressed higher levels of IGFBP3 mRNA in the endometrium than pigs of other genotypes, and a positive correlation was observed between litter size traits and IGFBP3 but not IGFBP2 expression level. These results suggest that SNPs in the IGFBP2 and the IGFBP3 gene are useful biomarkers for increasing the reproductive productivity of Berkshire pigs.