Polymorphisms of Mitochondrial ATPASE 8/6 Genes and Association with Milk Production Traits in Holstein Cows

The maternal effect has been widely proposed to affect the production traits in domestic animals. However, the sequence polymorphisms of mitochondrial DNA (mtDNA) and association with milk production traits in Holstein cows have remained unclear. In this study, we investigated the single nucleotide polymorphisms (SNPs) of mtDNA ATPase 8/6 genes and association with four milk production traits of interest in 303 Holstein cows. A total of 18 SNPs were detected among the 842 bp fragment of ATPase 8/6 genes, which determined six haplotypes of B. taurus (H1-H4) and B. indicus (H5-H6). The mixed model analysis revealed that there was significant association between haplotype and 305-day milk yield (MY). The highest MY was observed in haplotype H4. However, we did not detect statistically significant differences among haplotypes for the traits of milk fat (MF), milk protein (MP), and somatic cell count (SC). The overall haplotype diversity and nucleotide diversity of ATPase 8/6 genes were 0.563 ± 0.030 and 0.00609 ± 0.00043, respectively. The results suggested that mitochondrial ATPase 8/6 genes could be potentially used as molecular marker to genetically improve milk production in Holstein cows.     

A candidate gene association study for nine economically important traits in Italian Holstein cattle

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty‐six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity–functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty‐two SNPs were significantly associated (proportion of false positives <0.05) with different traits: 16 with MSCC, 15 with PY, 14 with MY, 12 with PFT, eight with longevity, eight with FY, eight with PP, five with FP and two with fertility. In particular, a SNP in the promoter region of the PRLR gene was associated with eight of nine traits. DGAT1 polymorphisms were highly associated with FP and FY. Casein gene markers were associated with several traits, confirming the role of the casein gene cluster in affecting milk yield, milk quality and health traits. Other SNPs in genes located on chromosome 6 were associated with PY, PP, PFT, MY (PPARGC1A) and MSCC (KIT). This latter association may suggest a biological link between the degree of piebaldism in Holstein and immunological functions affecting somatic cell count and mastitis resistance. Other significant SNPs were in the ACACA, CRH, CXCR1, FASN, GH1, LEP, LGB (also known as PAEP), MFGE8, SRC, TG, THRSP and TPH1 genes. These results provide information that can complement QTL mapping and genome‐wide association studies in Holstein.     

Correlation analysis between three novel SNPs of the Src gene in bovine and milk production traits

As an essential signaling modulator, Src gene appears to be necessary for increased expression of the prolactin receptor, normal downstream signaling, and alveolar cell organization. In this study, we detected the polymorphism of Src gene by polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) and DNA sequencing methods in 985 individuals from three Chinese cattle breeds. Three novel single nucleotide polymorphisms (SNPs) (g.14062C>T ss161151834, g.17302G>A ss161151835, g.18107T>C ss161151836) were detected. Least squares analysis showed that cows with g.14062C>T-CC genotypes and g.18107T>C-TT genotypes had the highest protein rate, while the cows with g.17302G>A-GG genotype had higher 305 d milk yield (p < 0.05), fat yield (p < 0.01) and protein yield (p < 0.01) than the ones with genotypes g.17302G>A-GA. These results revealed the statistical significant effects of three SNPs of the Src gene on the milk production traits in Chinese Holstein. In addition, based on the nine genotypes constructed from 27 combined haplotypes, the association analysis between combined haplotypes and milk production traits was carried out. Statistic results showed that the cows with combined haplotypes H2H4(CCGATT) had the highest fat rate and the highest protein rate and the cows with combined haplotypes H1H8(TCGATC) and H3H7(TCGGCC) had greater 305 d mild yield than H1H2(CCAATC)(P < 0.05). Our finding demonstrated that the Src gene possibly contributed to conducting association analysis and can be recognized as genetic marker in milk production traits and other performance for animal breeding and genetics.     

Genome‐wide association analysis and gene set enrichment analysis with SNP data identify genes associated with 305‐day milk yield in Holstein dairy cows

Milk production traits, such as 305‐day milk yield (305MY), have been under direct selection to improve production in dairy cows. Over the past 50 years, the average milk yield has nearly doubled, and over 56% of the increase is attributable to genetic improvement. As such, additional improvements in milk yield are still possible as new loci are identified. The objectives of this study were to detect SNPs and gene sets associated with 305MY in order to identify new candidate genes contributing to variation in milk production. A population of 781 primiparous Holstein cows from six central Washington dairies with records of 305MY and energy corrected milk were used to perform a genome‐wide association analysis (GWAA) using the Illumina BovineHD BeadChip (777 962 SNPs) to identify QTL associated with 305MY (P < 1.0 × 10^?5). A gene set enrichment analysis with SNP data (GSEA‐SNP) was performed to identify gene sets (normalized enrichment score > 3.0) and leading edge genes (LEGs) influencing 305MY. The GWAA identified three QTL comprising 34 SNPs and 30 positional candidate genes. In the GSEA‐SNP, five gene sets with 58 unique and 24 shared LEGs contributed to 305MY. Identification of QTL and LEGs associated with 305MY can provide additional targets for genomic selection to continue to improve 305MY in dairy cattle.     

Novel SNPs of the Bovine PRLR Gene Associated with Milk Production Traits

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.     

Study on genetic variations of PPARα gene and its effects on thermal tolerance in Chinese Holstein

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR–restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.     

Genetic effects of stearoyl-coenzyme A desaturase (SCD) polymorphism on milk production traits in the Chinese dairy population

Stearoyl-CoA desaturase (SCD) is a multifunctional complex enzyme important in the cellular biosynthesis of fatty acids. The present study was to investigate the association of the SCD gene with milk production traits in dairy cattle. Two single nucleotide polymorphisms (SNPs) (g.6926A>G and g.8646A>G) in introns 3 and 4, and three SNPs (g.10153A>G, g.10213T>C and g.10329C>T) in exon 5 were identified with pooled DNA sequencing and genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry assay in 752 Chinese Holstein cows. Polymorphism g.10329C>T was predicted to result in an amino acid replacement from alanine to valine in the SCD protein. With a mixed animal model, the significant associations of the five SNPs with 305-day milk, fat and protein yields and protein percentage were determined. We further demonstrated cows with heterozygous genotypes (A/G or C/T) had highest 305 day milk yield, fat yield, protein yield and lowest protein percentage. Heterozygous cows with genotype AG at the g.6926A>G locus showed the greatest milk yield (P < 0.0001), fat yield (P < 0.0001) and protein yield (P < 0.0001) among other heterozygous genotypes at any of the loci. Dominance effects of all identified SNPs on milk, fat and protein yields and protein percentage were significant. Moreover, significant allele substitution effects at g.6926A>G locus on milk yield and at g.10213T>C on protein yield were observed. Five-locus haplotypes and strong linkage disequilibrium (D' > 0.9) between the five SNPs were also observed. The results suggest that identified polymorphisms could be potential genetic markers to improve the production performance of Chinese Holstein.     

Genetic variations of HSBP1 gene and its effect on thermal performance traits in Chinese Holstein cattle

Molecular chaperones have been understood to be preferentially transcribed to prevent perturbations in response to various stresses. In this study, three single nucleotide polymorphisms (SNPs), g.324G>C, g.589C>T and g.651C>G in Heat shock factor binding protein 1 (HSBP1) gene were found and genotyped in 930 Chinese Holstein cattle. The results indicated that only g.589C>T polymorphism locus met Hardy–Weinberg equilibrium (P > 0.05). Pair linkage disequilibrium analysis and haplotype construction of HSBP1 gene were performed using SHEsis software. Seven haplotypes were constructed and fourteen haplotype combinations were found. Association analysis showed that H2H2 haplotype combination was advantageous for thermo tolerance breeding in Chinese Holstein. The cows with H2H2 haplotype combination have lower decrease rate of milk yield than those with H2H3 haplotype combination (P < 0.05) and lower potassium content in erythrocytes (PCE) than those with H2H5 (P < 0.05), H4H4 (P < 0.05) and H4H5 (P < 0.01) haplotype combination. The association between SNP and thermo tolerance traits showed that PCE of cows with GG genotype was lower than those with CG genotype at g.651C>G locus (P < 0.01). Pair linkage disequilibrium analysis revealed that the three loci were at a strong disequilibrium state. So we presumed that the effect of H2H2 haplotype combination on thermo tolerance traits major due to the SNP of g.651C>G.     

Single nucleotide polymorphisms, haplotypes and combined genotypes of LAP3 gene in bovine and their association with milk production traits

Leucine aminopeptidase 3 (LAP3) is an aminopeptidase which catalyses the removal of N-terminal amino acids and is involved in protein maturation and degradation. In this study, we detected the polymorphisms of LAP3 gene by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in 916 individuals from three Chinese cattle breeds including Chinese Holstein, Luxi Yellow and Bohai Black. One novel single nucleotide polymorphism (SNP) (g.24564G>A ss196003366) and four previously deposited SNPs in the GenBank database (g.24794T>G, g.24803T>C, g.24846T>C, g.25415T>C) were detected. Three of the SNPs (g.24794T>G, g.24803T>C, g.24846T>C) were firstly found to be linked completely and regarded as a SNP g.24794M>N by PCR-SSCP and DNA sequencing in the tested breeds. The allelic frequencies and genetic indices of the SNPs were different in three Chinese cattle populations. The SNPs and their genetic effects on milk production traits in Chinese Holsteins were evaluated. Least squares analysis showed that cows with genotype MM had higher fat percentage and protein percentage than genotype NN (P<0.05); and the cows with g.25415T>C-CC genotype had higher protein rate than ones with TT genotype (P<0.05). In addition, eight haplotypes and 23 combined genotypes were identified based on the nine genotypes and the association between combined genotypes and milk production traits were analyzed. Statistic results showed that the cows with genotype combination MAT/MGC have higher protein and fat rate and lower SCS. Our finding demonstrated that the LAP3 gene possibly contributed to conducting association analysis and can be used as molecular marker in milk production traits and other performance for animal breeding.     

贵州瑶族3支系Y-DNA及线粒体DNA序列多态性分析

采用PCR－RFLP技术,通过观察由12个单核苷酸多态位点(SNPs)组成的Y染色体单倍型及由9个多态位点组成的线粒体DNA单倍型在贵州瑶族中的分布,分析贵州瑶族父系及母系遗传结构,探讨其起源及迁徙。结果显示,97份男性样本分别属于H7、H8、H9、H11 4种Y-DNA单倍型,苗瑶语系特异Y-DNA单倍型H7的平均频率为92.4%;通过对线粒体DNA基因分型,得到8种单倍型,可归入B4、B5、D4、D5和N*单倍型类群中,CoⅡ/tRNA^Lys区域间的9bp缺失平均频率为58.2%。结果提示贵州瑶族父系遗传结构单一,具有典型的苗瑶族群特征,又存在与其他族群的融合。母系遗传结构相对复杂,9 bp缺失是贵州瑶族的母系遗传结构特征。      

Three novel SNPs of the bovine Tf gene in Chinese native cattle and their associations with milk production traits

Transferrin (Tf) is a β-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.     

Six novel single-nucleotide polymorphisms in SPAG11 gene and their association with sperm quality traits in Chinese Holstein bulls

Sperm-associated antigen 11 (SPAG11) is predominant in the male reproductive tract. Similar to β-defensin, aside from its antibacterial activity, SPAG11 also has an important role in male reproductive function. In the present study, the association of bovine SPAG11 gene polymorphism with sperm quality traits was examined, including ejaculate volume, sperm concentration, fresh sperm motility, post-thaw cryopreserved sperm motility, and deformity rate of bull semen. Six novel single nucleotide polymorphisms (SNPs) of the SPGA11 gene were investigated in 426 normal mature Chinese Holstein bulls using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), PCR-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR), and DNA sequencing methods. Linkage disequilibrium analysis showed that g.1306G>A and g.1454G>A (SNP-1), and g.16904G>T, g.16974C>T, and g.17000A>G (SNP-2) are completely linked, respectively. Correlation analysis showed the SNP-2 marker had a marked effect on fresh sperm motility and sperm concentration (P<0.05). SNP-3 g.22696T>C had a marked effect on post-thaw cryopreserved sperm motility (P<0.05) and deformity rate (P<0.01). However, the presence of SNP-1 was not correlated with the sperm production traits (P>0.05). Furthermore, association analyses of the 8 haplotypes constructed from the 17 combined haplotypes and reproductive traits showed that the bulls with the combined haplotype H5H6 (GGT/TTC) have the highest ejaculate volumes and the bulls with combined haplotypes H1H1 (AAT/TTT) and H1H6 (AGT/TTC) had the highest fresh and post-thaw sperm motilities, respectively. These results indicate that new molecular markers associated with sperm quality traits can be used in marker-assisted selection in bull breeding programs.     

Effects on production traits of haplotypes among casein genes in Norwegian goats and evidence for a site of preferential recombination

In goat milk the most abundant proteins are the casein genes, CSN1S1, CSN2, CSN1S2, and CSN3. Mutations have been identified within these genes affecting the level of gene expression, and effects on milk production traits have been reported. The aim of this study was to detect polymorphisms (SNPs) in the casein genes of Norwegian goats, resolve haplotype structures within the loci, and assess the effect of these haplotypes on milk production traits. Four hundred thirty-six Norwegian bucks were genotyped for 39 polymorphic sites across the four loci. The numbers of unique haplotypes present in each locus were 10, 6, 4, and 8 for CSN1S1, CSN2, CSN1S2, and CSN3, respectively. The effects of the CSN1S1 haplotypes on protein percentage and fat kilograms were significant, as were the effects of CSN3 haplotypes on fat percentage and protein percentage. A deletion in exon 12 of CSN1S1, unique to the Norwegian goat population, explained the effects of CSN1S1 haplotypes on fat kilograms, but not protein percentage. Investigation of linkage disequilibrium between all possible pairs of SNPs revealed higher levels of linkage disequilbrium for SNP pairs within casein loci than for SNP pairs between casein loci, likely reflecting low levels of intragenic recombination. Further, there was evidence for a site of preferential recombination between CSN2 and CSN1S2. The value of the haplotypes for haplotype-assisted selection (HAS) is discussed.     

A Post-GWAS Replication Study Confirming the PTK2 Gene Associated with Milk Production Traits in Chinese Holstein

Our initial genome-wide association study (GWAS) demonstrated that two SNPs (ARS-BFGL-NGS-33248, UA-IFASA-9288) within the protein tyrosine kinase 2 (PTK2) gene were significantly associated with milk production traits in Chinese Holstein dairy cattle. To further validate if the statistical evidence provided in GWAS were true-positive findings, a replication study was performed herein through genotype-phenotype associations. The two tested SNPs were found to show significant associations with milk production traits, which confirmed the associations observed in the original study. Specifically, SNPs lying in the PTK2 gene were also detected by sequencing 14 unrelated sires in Chinese Holsteins and a total of thirty-three novel SNPs were identified. Thirteen out of these identified SNPs were genotyped and tested for association with milk production traits in an independent resource population. After Bonferroni correction for multiple testing, twelve SNPs were statistically significant for more than two milk production traits. Analyses of pairwise D’ measures of linkage disequilibrium (LD) between all SNPs were also explored. Two haplotype blocks were inferred and the association study at haplotype level revealed similar effects on milk production traits. In addition, the RNA expression analyses revealed that a non-synonymous coding SNP (g.4061098T>G) was involved in the regulation of gene expression. Thus the findings presented here provide strong evidence for associations of PTK2 variants with dairy production traits and may be applied in Chinese Holstein breeding program.     

Performance of single nucleotide polymorphisms versus haplotypes for genome-wide association analysis in barley

Genome-wide association studies (GWAS) may benefit from utilizing haplotype information for making marker-phenotype associations. Several rationales for grouping single nucleotide polymorphisms (SNPs) into haplotype blocks exist, but any advantage may depend on such factors as genetic architecture of traits, patterns of linkage disequilibrium in the study population, and marker density. The objective of this study was to explore the utility of haplotypes for GWAS in barley (Hordeum vulgare) to offer a first detailed look at this approach for identifying agronomically important genes in crops. To accomplish this, we used genotype and phenotype data from the Barley Coordinated Agricultural Project and constructed haplotypes using three different methods. Marker-trait associations were tested by the efficient mixed-model association algorithm (EMMA). When QTL were simulated using single SNPs dropped from the marker dataset, a simple sliding window performed as well or better than single SNPs or the more sophisticated methods of blocking SNPs into haplotypes. Moreover, the haplotype analyses performed better 1) when QTL were simulated as polymorphisms that arose subsequent to marker variants, and 2) in analysis of empirical heading date data. These results demonstrate that the information content of haplotypes is dependent on the particular mutational and recombinational history of the QTL and nearby markers. Analysis of the empirical data also confirmed our intuition that the distribution of QTL alleles in nature is often unlike the distribution of marker variants, and hence utilizing haplotype information could capture associations that would elude single SNPs. We recommend routine use of both single SNP and haplotype markers for GWAS to take advantage of the full information content of the genotype data.     

A comparison of sequence-based polymorphism and haplotype content in transcribed and anonymous regions of the barley genome.

Direct estimates of sequence diversity provides an abundant source of DNA polymorphisms based on single nucleotide polymorphisms (SNPs). The frequency and distribution of nucleotide diversity within 23 genes associated with grain germination in barley were determined in a sample of accessions representing European cultivars, landraces, and wild barley accessions from throughout the fertile crescent. The overall nucleotide diversity ranged from 0.0021 to 0.0189 with a single nucleotide change being detected every 78 bp and insertion-deletion events being observed every 680 bp. Within the cultivated (H. vulgare) genepool, a small number of haplotypes were detected, the total number of haplotypes observed in H. spontaneum was almost double that detected in H. vulgare (46 and 26, respectively). Distinct haplotypes were observed in the H. spontaneum and landrace genepools, which are highly divergent from H. vulgare. A comparison of SNP-based haplotype data with EST-derived SSRs and genomic SSRs revealed a similar trend of decreasing variability in the cultivated genepool. However, the number of unique alleles identified in the cultivated sample was much greater with genomic SSRs (18%) compared with only 2.1% for SNPs and 3.8% for EST-derived SSRs. The potential utility of SNPs and EST-derived SSRs for association mapping in barley is discussed.