Pool‐based genome‐wide association study identified novel candidate regions on BTA9 and 14 for oleic acid percentage in Japanese Black cattle

Fatty acid composition is an important indicator of beef quality. The objective of this study was to search the potential candidate region for fatty acid composition. We performed pool‐based genome‐wide association studies (GWAS) for oleic acid percentage (C18:1) in a Japanese Black cattle population from the Hyogo prefecture. GWAS analysis revealed two novel candidate regions on BTA9 and BTA14. The most significant single nucleotide polymorphisms (SNPs) in each region were genotyped in a population (n = 899) to verify their effect on C18:1. Statistical analysis revealed that both SNPs were significantly associated with C18:1 (p = .0080 and .0003), validating the quantitative trait loci (QTLs) detected in GWAS. We subsequently selected VNN1 and LYPLA1 genes as candidate genes from each region on BTA9 and BTA14, respectively. We sequenced full‐length coding sequence (CDS) of these genes in eight individuals and identified a nonsynonymous SNP T66M on VNN1 gene as a putative candidate polymorphism. The polymorphism was also significantly associated with C18:1, but the p value (p = .0162) was higher than the most significant SNP on BTA9, suggesting that it would not be responsible for the QTL. Although further investigation will be needed to determine the responsible gene and polymorphism, our findings would contribute to development of selective markers for fatty acid composition in the Japanese Black cattle of Hyogo.     

Effect of DNA polymorphisms related to fatty acid composition in adipose tissue of Holstein cattle

Fatty acid composition of adipose tissue has been recognized as an important carcass trait because of its relationship with eating quality such as favorable beef flavor and tenderness. Therefore, we investigated the effects of genetic polymorphisms of liver X receptor, alpha (LXR), stearoyl‐CoA desaturase (SCD), Fatty acid synthase (FASN), and Fatty acid binding protein 4 (FABP4) on fatty acid composition in intramuscular fat tissue of Holstein steers. The major allele frequencies were 0.705 in SCD, 0.518 in FABP4, 0.888 in FASN, and 0.984 in LXR. Genotyping of SCD showed significant effect on C14:0, C14:1, C18:0 and saturated fatty acid (P < 0.05). In addition, the result suggested that SCD genotype possibly had effect on composition of C18:1 and monounsaturated fatty acid. Genotype of FABP4 had significant effect on composition of C16:0. Effect of LXR genotypes could not be analyze because of extremely biased genotype frequencies. Our results suggest that genotypes of SCD and FABP4 may in part affect meat quality in Holstein.     

The g.841G>C SNP of FASN gene is associated with fatty acid composition in beef cattle

The objective of the current study is to evaluate the association between fatty acid composition and fatty acid synthase gene polymorphisms as responsible mutations. For this purpose, we selected seven previously reported single nucleotide polymorphisms (SNPs) in FASN gene, including one within promoter region (g.841G>C) and six non‐synonymous SNPs (g.8805C>T, g.13126C>T, g.15532A>C, g.16024A>G, g.16039C>T, g.17924A>G), and genotyped them in Japanese Black cattle. Genotyping results revealed that g.8805 C>T and g.17924 A>G were monomorphic loci. Genome‐wide association analysis including the other five SNPs revealed that only g.841G>C showed significant associations with the percentages of C14:0, C14:1, C16:1 and C18:1 at 5% genome‐wide significance level. In order to further evaluate the effect, we genotyped g.841G>C using additional three populations, including two Japanese Black populations and a Holstein cattle population. g.16024A>G was also genotyped and included in the analysis because it has been reported to be associated with fatty acid composition in Japanese Black cattle. In the result of analysis of variance, g.841G>C showed stronger effects on fatty acid percentage than those of g.16024A>G in all populations. These results suggested that g.841G>C would be a responsible mutation for fatty acid composition and contribute to production of high‐grade beef as a selection marker in beef cattle.     

Genome‐wide association studies reveal additional related loci for fatty acid composition in a Duroc pig multigenerational population

The aim of the present study was to detect quantitative trait loci affecting fatty acid composition in back fat and intramuscular fat in a Duroc pig population comprising seventh‐generation pedigrees using genome‐wide association studies (GWAS). In total, 305 animals were genotyped using single nucleotide polymorphisms (SNPs) array and five selected SNPs from regions containing known candidate genes related to fatty acid synthesis or metabolism. In total, 24 genome‐wide significant SNP regions were detected in 12 traits, and 76 genome‐wide suggestive SNP regions were detected in 33 traits. The Sus scrofa chromosome (SSC) 7 at 10.3 Mb was significantly associated with C17:0 in intramuscular fat, while the SSC9 at 13.6 Mb was significantly associated with C14:0 in intramuscular fat. The SSC12 at 1.0 Mb was significantly associated with C14:0 in back fat and the SSC14 at 121.0 Mb was significantly associated with C18:0 in intramuscular fat. These regions not only replicated previously reported loci containing some candidate genes involved in fatty acid composition (fatty acid synthase and stearoyl‐CoA desaturase) but also included several additional related loci.     

Genetic mapping using 1.4M SNP array refined loci for fatty acid composition traits in Chinese Erhualian and Bamaxiang pigs

Chinese indigenous pigs display marked genetic and phenotypic differences compared with western commercial pigs. In this study, we tested the association between 660K SNPs and longissimus muscle fatty acid composition traits in Chinese Erhualian (n = 331) and Bamaxiang (n = 315) pigs based on a customized 1.4 million SNP array. We identified a total of 64 significant associations for 20 fatty acid composition traits at the p‐value threshold of 1 × 10^?6 among which 42 associations in low linkage disequilibrium (r² < .2) with previously reported loci were considered novel. We substantially improved the strength and precision of the associations at four previously detected loci near FADS2, ELOVL7, ELOVL6 and FASN genes, facilitating follow‐up candidate gene studies. Moreover, we also identified loci near ABCD2, ACSBG1, ELOVL5, HPGDS, DAGT2, ACAD10 and ACSL1 genes with function relevant to metabolism of fatty acids. In this study, valuable genetic variants and candidate genes associated with fatty acid composition traits were identified in Erhualian and Bamaxiang pigs. Some identified loci could be used to improve pork nutrition in pig breeding practice. Using the SNP array with higher marker density and less ascertainment bias improved QTL detection power and precision in Chinese indigenous pigs.     

Identification and utilization of genes associated with beef qualities

The fatty acid composition of adipose tissue in beef has been recognized as an important trait because of its relationship with beef quality, including favorable beef flavor and tenderness. Over the last decade, we have tried to identify the genes responsible for the fatty acid composition in cattle, and have found the following. (i) Genetic polymorphism of stearoyl‐CoA desaturase (SCD) is one of the responsible genes associated with fatty acid composition. The average effects of gene substitution of the SCD type A gene on the monounsaturated fatty acid (MUFA) percentage and the melting point of intramuscular fat were approximately +1.0% and ?1.0°C, respectively. (ii) Intron polymorphism of sterol regulatory element binding protein–1 (SREBP‐1) also affected MUFA. (iii) No effect of SCD or SREBP‐1 genotypes on any representative carcass traits of Japanese Black in the field population was observed. (iv) Additional genetic markers adipocytes fatty acid binding protein 4 (FABP4) and liver X receptor α also affected the fatty acid composition. (v) SCD and FABP4 significantly affected fatty acid composition in Holstein steers. These findings will bring new insight into the fat‐related carcass traits of beef cattle and will thus contribute to the beef industry.     

Genome‐wide association study for feedlot average daily gain in Nellore cattle (Bos indicus)

The genome‐wide association study (GWAS) results are presented for average daily gain (ADG) in Nellore cattle. Phenotype of 720 male Bos indicus animals with information of ADG in feedlots and 354 147 single‐nucleotide polymorphisms (SNPs) obtained from a database added by information from Illumina Bovine HD (777 962 SNPs) and Illumina BovineSNP50 (54 609) by imputation were used. After quality control and imputation, 290 620 SNPs remained in the association analysis, using R package Genome‐wide Rapid Association using Mixed Model and Regression method GRAMMAR‐Gamma. A genomic region with six significant SNPs, at Bonferroni‐corrected significance, was found on chromosome 3. The most significant SNP (rs42518459, BTA3: 85849977, p = 9.49 × 10^?8) explained 5.62% of the phenotypic variance and had the allele substitution effect of ?0.269 kg/day. Important genes such as PDE4B, LEPR, CYP2J2 and FGGY are located near this region, which is overlapped by 12 quantitative trait locus (QTLs) described for several production traits. Other regions with markers with suggestive effects were identified in BTA6 and BTA10. This study showed regions with major effects on ADG in Bos indicus in feedlots. This information may be useful to increase the efficiency of selecting this trait and to understand the physiological processes involved in its regulation.     

UTS2R gene polymorphisms are associated with fatty acid composition in Japanese beef cattle

Fatty acid composition of beef adipose tissue is one of its important traits because a high proportion of monounsaturated fatty acid is related to favorable beef flavor and tenderness. In this study, we searched polymorphisms in full length coding DNA sequence of urotensin 2 recepter and investigated the effects on fatty acid composition (C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, monounsaturated fatty acid, saturated fatty acid). Eight single nucleotide polymorphisms (SNP) were identified by sequence comparison among eight animals, including five Japanese Black and three Holstein cattle. One of these SNP (c.866C>T) was predicted to cause amino acid substitutions (P289L) and the other seven synonymous SNP, including c.267C>T, were presumed to be in linkage disequilibrium. Therefore we selected two SNP (c.267C>T and c.866C>T) for further analysis. We investigated associations between these genotypes and fatty acid composition in three Japanese Black populations (n = 560, 245 and 287) and a Holstein population (n = 202). Tukey‐Kramer's honestly significant difference test revealed that CC genotype in c.267C>T indicated lower C14:0 and higher C18:1 than the other genotypes in Japanese Black cattle and CC genotype in c.866C>T showed lower C16:1 than CT genotype in Holstein cattle (P < 0.05). These results suggested that these genotypes would contribute to production of high‐grade meat as selection markers in beef cattle.     

Allelic frequencies and association with carcass traits of six genes in local subpopulations of Japanese Black cattle

Marker‐assisted selection (MAS) is expected to accelerate the genetic improvement of Japanese Black cattle. However, verification of the effects of the genes for MAS in different subpopulations is required prior to the application of MAS. In this study, we investigated the allelic frequencies and genotypic effects for carcass traits of six genes, which can be used in MAS, in eight local subpopulations. These genes are SCD, FASN and SREBP1, which are associated with the fatty acid composition of meat, and NCAPG, MC1R and F11, which are associated with carcass weight, coat color and blood coagulation abnormality, respectively. The frequencies of desirable alleles of SCD and FASN were relatively high and that of NCAPG was relatively low, and NCAPG was significantly associated with several carcass traits, including carcass weight. The proportions of genotypic variance explained by NCAPG to phenotypic variance were 4.83 for carcass weight. We thus confirmed that NCAPG is a useful marker for selection of carcass traits in these subpopulations. In addition, we found that the desirable alleles of six genes showed no negative effects on carcass traits. Therefore, selection using these genes to improve target traits should not have negative impacts on carcass traits.     

Whole‐genome resequencing to identify candidate genes for the QTL for oleic acid percentage in Japanese Black cattle

In our previous study, we detected a QTL for the oleic acid percentage (C18:1) on BTA9 in Japanese Black cattle through a genome‐wide association study (GWAS). In this study, we performed whole‐genome resequencing on eight animals with higher and lower C18:1 to identify candidate polymorphisms for the QTL. A total of 39,658 polymorphisms were detected in the candidate region, which were narrowed to 1993 polymorphisms within 23 genes based on allele differences between the high and low C18:1 groups. We subsequently selected three candidate genes, that is, CYB5R4, MED23, and VNN1, among the 23 genes based on their function in fatty acid metabolism. In each candidate gene, three SNPs, that is, CYB5R4 c.*349G > T, MED23 c.3700G > A, and VNN1 c.197C > T, were selected as candidate SNPs to verify their effect on C18:1 in a Japanese Black cattle population (n = 889). The statistical analysis showed that these SNPs were significantly associated with C18:1 (p < 0.05), suggesting that they were candidates for the QTL. In conclusion, we successfully narrowed the candidates for the QTL by detecting possible polymorphisms located within the candidate region. It is expected that the responsible polymorphism can be identified by demonstrating their effect on the gene's function.     

Genome‐wide association study for carcass traits,fatty acid composition,chemical composition,sugar, and the effects of related candidate genes in Japanese Black cattle

We performed a genome‐wide association study (GWAS) and candidate gene analysis to: (i) evaluate the effectiveness of the GWAS in our small population by performing GWAS for carcass weight (CW) and fatty acid composition; (ii) detect novel candidate regions affecting non‐CW carcass traits, chemical composition and sugar; and (iii) evaluate the association of the candidate genes previously detected in CW and fatty acid composition with other economically important traits. A total of 574 Japanese Black cattle and 40 657 Single nucleotide polymorphisms were used. In addition, candidate gene analyses were performed to evaluate the association of three CW‐related genes and two fatty acid‐related genes with carcass traits, fatty acid composition, chemical composition and sugar. The significant regions with the candidate genes were detected for CW and fatty acid composition, and these results showed that a significant region would be detectable despite the small sample size. The novel candidate regions were detected on BTA23 for crude protein and on BTA19 for fructose. CW‐related genes associated with the rib‐eye area and fatty acid composition were identified, and fatty acid‐related genes had no relationship with other traits. Moreover, the favorable allele of CW‐related genes had an unfavorable effect on fatty acid composition.     

Variations in genes involved in fat metabolism and their association with ultrasonic and carcass traits in Japanese Black steers

Polymorphisms in genes involved in lipid metabolism have been reported to be associated with fatty acid composition of adipose tissue. However, the relationship of these polymorphisms with premortem ultrasonic traits is unknown. The objective of this study, therefore, was to assess the association between polymorphisms in fatty acid synthase, stearoyl‐coenzyme A desaturase (SCD), sterol regulatory element‐binding protein 1 (SREBP1), diacylglycerol acyltransferase 1, and nuclear receptor subfamily 1, group H, number 3 genes with ultrasonic and carcass traits in Japanese Black steers (n = 300) under progeny testing at the Livestock Improvement Association of Miyazaki. To have a comprehensive analysis of the association between the aforementioned genetic polymorphisms and ultrasonic traits, longitudinal measurements of ultrasonic traits were taken. Furthermore, the association of these genetic polymorphisms and carcass traits was evaluated. The polymorphisms in the SCD gene and SREBP1 were associated (P < 0.05) with some ultrasonic traits at multiple stages. To add to that, the polymorphisms were associated (P < 0.05) with some carcass traits. These findings suggest that the polymorphisms in SCD and SREBP1 are functional mutations or could be related to mutations that can aid in selection to improve some ultrasonic and carcass traits.     

A genome‐wide association study for fat‐related traits computed by image analysis in Japanese Black cattle

The objective of this study was to identify genomic regions associated with fat‐related traits using a Japanese Black cattle population in Hyogo. From 1836 animals, those with high or low values were selected on the basis of corrected phenotype and then pooled into high and low groups (n = 100 each), respectively. DNA pool‐based genome‐wide association study (GWAS) was performed using Illumina BovineSNP50 BeadChip v2 with three replicate assays for each pooled sample. GWAS detected that two single nucleotide polymorphisms (SNPs) on BTA7 (ARS‐BFGL‐NGS‐35463 and Hapmap23838‐BTA‐163815) and one SNP on BTA12 (ARS‐BFGL‐NGS‐2915) significantly affected fat percentage (FAR). The significance of ARS‐BFGL‐NGS‐35463 on BTA7 was confirmed by individual genotyping in all pooled samples. Moreover, association analysis between SNP and FAR in 803 Japanese Black cattle revealed a significant effect of SNP on FAR. Thus, further investigation of these regions is required to identify FAR‐associated genes and mutations, which can lead to the development of DNA markers for marker‐assisted selection for the genetic improvement of beef quality.     

Effect of short‐chain fatty acids on triacylglycerol accumulation,lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells

Short‐chain fatty acids (SCFAs) are the major energy sources for ruminants and are known to regulate various physiological functions in other species. However, their roles in ruminant milk fat metabolism are still unclear. In this study, goat mammary gland epithelial cells (GMECs) were treated with 3 mmol/L acetate, propionate or butyrate for 24 h to assess their effects on lipogenesis. Data revealed that the content of triacylglycerol (TAG) and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate and butyrate treatment. In contrast, the messenger RNA (mRNA) expression of FASN and LXRα was not affected by propionate, but reduced by butyrate. Acetate had no obvious effect on the content of TAG and lipid droplets but increased the mRNA expression of SCD1 and FABP3 in GMECs. Additionally, it was observed that propionate significantly increased the relative content of mono‐unsaturated fatty acids (C18:1 and C16:1) at the expense of decreased saturated fatty acids (C16:0 and C18:0). Butyrate and acetate had no significant effect on fatty acid composition. Overall, the results from this work help enhance our understanding of the regulatory role of SCFAs on goat mammary cell lipid metabolism.     

Homozygosity of single nucleotide polymorphisms in the 3′ region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40‐kb region at the 3′ end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3′ end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14–17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15–17). A common haplotype block was identified in an approximately 20‐kb region encompassing four SNPs (SNPs # 14–17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3′ end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.     

Fine mapping of a calving QTL on Bos taurus autosome 18 in Holstein cattle

Decreased calving performance not only directly impacts the economic efficiency of dairy cattle farming but also influences public concern for animal welfare. Previous studies have revealed a QTL on Bos taurus autosome (BTA) 18 that has a large effect on calving traits in Holstein cattle. In this study, fine mapping of this QTL was performed using imputed high‐density SNP chip (HD) genotypes followed by imputed next‐generation sequencing (NGS) variants. BTA18 was scanned for seven direct calving traits in 6113 bulls with imputed HD genotypes. SNP rs136283363 (BTA18: 57 548 213) was consistently the most significantly associated SNP across all seven traits [e.g. p‐value = 2.04 × 10^?59 for birth index (BI)]. To finely map the QTL region and to explore pleiotropic effects, we studied NGS variants within the targeted region (BTA18: 57 321 450–57 625 355) for associations with direct calving traits and with three conformation traits. Significant variants were prioritized, and their biological relevance to the traits was interpreted. Considering their functional relationships with direct calving traits, SIGLEC12, CD33 and CEACAM18 were proposed as candidate genes. In addition, pleiotropic effects of this QTL region on direct calving traits and conformation traits were observed. However, the extent of linkage disequilibrium combined with the lack of complete annotation and potential errors in the Bos taurus genome assembly hampered our efforts to pinpoint the causal mutation.     

Single nucleotide polymorphisms associated with thermoregulation in lactating dairy cows exposed to heat stress

Dairy cows with increased rectal temperature experience lower milk yield and fertility. Rectal temperature during heat stress is heritable, so genetic selection for body temperature regulation could reduce effects of heat stress on production. One aim of the study was to validate the relationship between genotype and heat tolerance for single nucleotide polymorphisms (SNPs) previously associated with resistance to heat stress. A second aim was to identify new SNPs associated with heat stress resistance. Thermotolerance was assessed in lactating Holsteins during the summer by measuring rectal temperature (a direct measurement of body temperature regulation; n = 435), respiration rate (an indirect measurement of body temperature regulation, n = 450) and sweating rate (the major evaporative cooling mechanism in cattle, n = 455). The association between genotype and thermotolerance was evaluated for 19 SNPs previously associated with rectal temperature from a genomewide analysis study (GWAS), four SNPs previously associated with change in milk yield during heat stress from GWAS, 2 candidate gene SNPs previously associated with rectal temperature and respiration rate during heat stress (ATPA1A and HSP70A) and 66 SNPs in genes previously shown to be associated with reproduction, production or health traits in Holsteins. For SNPs previously associated with heat tolerance, regions of BTA4, BTA6 and BTA24 were associated with rectal temperature; regions of BTA6 and BTA24 were associated with respiration rate; and regions of BTA5, BTA26 and BTA29 were associated with sweating rate. New SNPs were identified for rectal temperature (n = 12), respiration rate (n = 8) and sweating rate (n = 3) from among those previously associated with production, reproduction or health traits. The SNP that explained the most variation were PGR and ASL for rectal temperature, ACAT2 and HSD17B7 for respiration rate, and ARL6IP1 and SERPINE2 for sweating rate. ARL6IP1 was associated with all three thermotolerance traits. In conclusion, specific genetic markers responsible for genetic variation in thermoregulation during heat stress in Holsteins were identified. These markers may prove useful in genetic selection for heat tolerance in Holstein cattle.     

Genome‐wide association studies identified variants for taurine concentration in Japanese Black beef

We performed genome‐wide association studies (GWAS) using the BovineSNP50 array to detect significant single nucleotide polymorphisms (SNPs) that may affect the concentration of 22 free amino acids and three peptides in Japanese Black beef cattle. A total of 574 Japanese Black cattle and 40,657 SNPs from the array were used for this study. Genome‐wide significant SNPs were detected for β‐alanine (three SNPs on chromosomes 22 and 29) and taurine (26 SNPs on chromosome 22). Importantly, the top two SNPs for taurine were highly significant (p = 6.2 × 10^?21), and the frequency of the increase‐concentration allele (Q) for taurine was found to be 0.73. The Q allele frequency of this population was similar to that of the other unrelated Japanese Black cattle, but different from that of the other breeds. In addition, the significant SNPs were not associated with carcass traits or fatty acid compositions. Interestingly, the top three of the four most significant SNPs for taurine were located near solute carrier family 6, member 6 (SLC6A6), which is a membrane transporter for taurine. We also found two associated variants in the 5′‐upstream region of SLC6A6; however, they were less significantly associated than the SNPs from the BovineSNP50 array.     

Interactions of bovine oocytes with follicular elements with respect to lipid metabolism

Among many factors, lipid metabolism within the follicular environment emerges as an important indicator of oocyte quality. In the literature a crucial significance is described concerning follicular fluid (FF) composition as well as messenger RNA (mRNA) expression in follicular cells. The aim of this study was to describe the relationship between oocyte, FF and follicular cells with regard to lipid metabolism. The set of data originating from individual follicles comprised: lipid droplets (LD) number in oocytes (BODIPY staining), mRNA expression of seven genes in cumulus and granulosa cells (SCD, FADS2, ELOVL2, ELOVL5, GLUT1, GLUT3, GLUT8; real time polymerase chain reaction) and fatty acid (FA) composition in FF (gas chromatography). Obtained results demonstrate significant correlation between oocyte lipid droplets number and FA composition in FF. However, gene expression studies show significant correlation between LD number and GLUT1 gene only. Moreover, the present experiment revealed correlations between FA content in FF and expression of several genes (SCD, FADS2, ELOVL5, GLUT8) in granulosa cells, whereas only the SCD gene in cumulus cells. We suggest that the results of our experiment indicate the importance of glucose : lipid metabolism balance, which contributes to better understanding of energy metabolism conversion between oocytes and the maternal environment.