Analysis of non‐synonymous SNPs of the porcine SERPINA6 gene as potential causal variants for a QTL affecting plasma cortisol levels on SSC7

Recently, the SERPINA6 gene encoding corticosteroid‐binding globulin (CBG) has been proposed as a candidate gene for a quantitative trait locus (QTL) affecting cortisol level on pig chromosome 7. The QTL was repeatedly detected in different lines, including a Piétrain × (German Landrace × German Large White) cross (PiF1) and purebred German Landrace (LR). In this study, we investigated whether the known non‐synonymous polymorphisms c.44G>T, c.622C>T, c.770C>T, c.793G>A, c.832G>A and c.919G>A of SERPINA6 are sufficient to explain the QTL in these two populations. Our investigations revealed that SNPs c.44G>T, c.622C>T, c.793G>A and c.919G>A are associated with cortisol level in PiF1 (P < 0.01). Haplotype analysis showed that these associations are largely attributable to differences between a major haplotype carrying SNPs c.793G>A and c.919G>A and a haplotype carrying SNPs c.44G>T and c.622C>T. Furthermore, some SNPs, particularly c.44G>T and c.622C>T and the carrier haplotype, showed association with meat quality traits including pH and conductivity (P < 0.05). In LR, the non‐synonymous SNPs segregate at very low frequency (<5%) and/or show only weak association with cortisol level (SNPs c.832G>A and c.919G>A; P < 0.05). These findings suggest that the non‐synonymous SNPs are not sufficient to explain the QTL across different breeds. Therefore, we examined whether the expression of SERPINA6 is affected by cis‐regulatory polymorphisms in liver, the major organ for CBG production. We found allelic expression imbalance of SERPINA6, which suggests that its expression is indeed affected by genetic variation in cis‐acting elements. This represents candidate causal variation for future studies of the molecular background of the QTL.     

Effect of FASN gene on milk yield and milk composition in the Chinese Holstein dairy population

Fatty acid synthase (FASN) is a multifunctional protein that catalyzes de novo synthesis of fatty acids in cells. It plays a key role in the lipid biosynthesis as well as in the general metabolism of all living animals. We herein investigated polymorphisms of FASN. As a result, six single nucleotide polymorphisms (SNPs) were found and then genotyped in 752 Chinese Holstein cows. It was found that g.17924A>G was non‐synonymous, g.13965 C>T, g.16907 T>C and g.18663T>C were synonymous mutations and two other two SNPs, g.8948 C>T (ss491228481) and g.14439T>C (rs133498277), were in intronic sequences of the gene. All such identified SNPs were found to be associated with milk yield and composition traits (P = 0.0441 to <0.0001). Significant additive and allele substitution effects were observed for three yield traits at all six loci as well (P < 0.05 to <0.01). Complete linkage disequilibrium among the five SNPs, with the exception of g.8948 C>T, was observed.     

A post‐GWAS confirming the SCD gene associated with milk medium‐ and long‐chain unsaturated fatty acids in Chinese Holstein population

The stearoyl‐CoA desaturase (delta‐9‐desaturase) gene encodes a key enzyme in the cellular biosynthesis of monounsaturated fatty acids. In our initial genome‐wide association study (GWAS) of Chinese Holstein cows, 19 SNPs fell in a 1.8‐Mb region (20.3–22.1 Mb) on chromosome 26 underlying the SCD gene and were highly significantly associated with C14:1 or C14 index. The aims of this study were to verify whether the SCD gene has significant genetic effects on milk fatty acid composition in dairy cattle. By resequencing the entire coding region of the bovine SCD gene, a total of six variations were identified, including three coding variations (g.10153G>A, g.10213T>C and g.10329C>T) and three intronic variations (g.6926A>G, g.8646G>A and g.16158G>C). The SNP in exon 3, g.10329C>T, was predicted to result in an amino acid replacement from alanine (GCG) to valine (GTG) in the SCD protein. An association study for 16 milk fatty acids using 346 Chinese Holstein cows with accurate phenotypes and genotypes was performed using the mixed animal model with the proc mixed procedure in sas 9.2. All six detected SNPs were revealed to be associated with six medium‐ and long‐chain unsaturated fatty acids (P = 0.0457 to P < 0.0001), specifically for C14:1 and C14 index (P = 0.0005 to P < 0.0001). Subsequently, strong linkage disequilibrium (D′ = 0.88–1.00) was observed among all six SNPs in SCD and the five SNPs (rs41623887, rs109923480, rs42090224, rs42092174 and rs42091426) within the 1.8‐Mb region identified in our previous GWAS, indicating that the significant association of the SCD gene with milk fatty acid content traits reduced the observed significant 1.8‐Mb chromosome region in GWAS. Haplotype‐based analysis revealed significant associations of the haplotypes encompassing the six SCD SNPs and one SNP (rs109923480) in a GWAS with C14:1, C14 index, C16:1 and C16 index (P = 0.0011 to P < 0.0001). In summary, our findings provide replicate evidence for our previous GWAS and demonstrate that variants in the SCD gene are significantly associated with milk fatty acid composition in dairy cattle, which provides clear evidence for an increased understanding of milk fatty acid synthesis and enhances opportunities to improve milk‐fat composition in dairy cattle.     

Associations of ATGL gene polymorphisms with chicken growth and fat traits

Adipose triglyceride lipase (ATGL) catalyses the initial step in triglyceride hydrolysis, so theATGL gene is a candidate for growth and fat traits in chickens. Nine reported single-nucleotide polymorphisms (SNPs) located in 3 exons of the chickenATGL gene were chosen for genotyping an F₂ population. Only 5 SNPs were confirmed for polymorphisms and used for association analyses. The results show that c.531G>A (p.E177Syn) was not associated with any growth and fat traits (P > 0.05), but c.782G>A (p.S261N) was associated with body weight (BW) on days 14, 21, 35, 63, 70, 77, cingulated fat width and abdominal fat pad weight (P< 0.05), and significantly associated with BW on days 42, 49, and 56 (P < 0.01). Significant associations of c.903C>T (p.F301Syn) with BW on days 49 and 77 days and crude protein content of breast muscle (P < 0.05), and c. 1164G>A (p.K388Syn) with BW on day 7 (P< 0.05) were also detected. Additionally, c. 1069T>C (p.L357Syn) was associated with breast muscle colour (P < 0.05), and significantly associated with crude fat (ether extract) content of breast muscle (P< 0.01). Thus the missense SNP of c.782G>A (p.S261N) was significantly associated with the largest number of chicken growth and fat traits in this study.     

Identification of genetic associations of ECHS1 gene with milk fatty acid traits in dairy cattle

Our previous genome‐wide association study identified 83 genome‐wide significant SNPs and 20 novel promising candidate genes for milk fatty acids in Chinese Holstein. Among them, the enoyl‐CoA hydratase, short chain 1 (ECHS1) and enoyl‐CoA hydratase and 3‐hydroxyacyl CoA dehydrogenase (EHHADH) genes were located near two SNPs and one SNP respectively, and they play important roles in fatty acid metabolism pathways. We herein validated whether the two genes have genetic effects on milk fatty acid traits in dairy cattle. By re‐sequencing the full‐length coding region, partially adjacent introns and 3000 bp up/downstream flanking sequences, we identified 12 SNPs in ECHS1: two in exons, four in the 3′ flanking region and six in introns. The g.25858322C>T SNP results in an amino acid replacement from leucine to phenylalanine and changes the secondary structure of the ECHS1 protein, and single‐locus association analysis showed that it was significantly associated with three milk fatty acids (P = 0.0002–0.0013). The remaining 11 SNPs were found to be significantly associated with at least one milk fatty acid (P = <0.0001–0.0040). Also, we found that two haplotype blocks, consisting of nine and two SNPs respectively, were significantly associated with eight milk fatty acids (P = <0.0001–0.0125). However, none of polymorphisms was observed in the EHHADH gene. In conclusion, our findings are the first to indicate that the ECHS1 gene has a significant genetic impact on long‐chain unsaturated and medium‐chain saturated fatty acid traits in dairy cattle, although the biological mechanism is still undetermined and requires further in‐depth validation.     

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.     

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.     

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.     

Single nucleotide polymorphisms in bovine <Emphasis Type="Italic">PRL</Emphasis> gene and their associations with milk production traits in Chinese Holsteins

Prolactin (PRL) plays a crucial role in the initiation and maintenance of lactation in mammals. In this study, seven PCR fragments representing most important functional domains of PRL gene were screened for single nucleotide polymorphisms (SNPs) in Chinese Holsteins by single-strand conformation polymorphisms and amplicons sequencing, and their genetic effects on milk production traits were evaluated. A total of four SNPs, including two in the promoter (−1043A>G and −402A>G), one in intron 1 (+2723C>T) and one in exon 4 (+8398G>A) were identified in PRL gene. Statistical results showed significant associations between the promoter genotypes and the milk performance traits in Chinese Holsteins. Cows with genotype P1-GG showed higher milk yields (P < 0.01), while cows with genotype P1-AA showed higher fat contents (P < 0.01). Haplotype analysis of two SNPs in promoter region revealed that the Hap(AG) was significantly associated with increased milk yields and Hap(AA) was associated with increased fat contents (P < 0.01). This is the second study reporting SNPs in the 5′-regulatory region of PRL gene, which interfere with milk production traits.     

Two missense mutations in exon 9 of caprine PRLR gene were associated with litter size

Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR‐RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G‐G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.     

Single nucleotide polymorphisms in the insulin‐like growth factor 1 (IGF1) gene are associated with growth‐related traits in farmed Atlantic salmon

Understanding the genetic basis of variation in traits related to growth and fillet quality in Atlantic salmon is of importance to the aquaculture industry. Several growth‐related QTL have been identified via the application of genetic markers. The IGF1 gene is considered a highly conserved and crucial growth‐regulating gene in salmonid species. However, the association between polymorphisms in the IGF1 gene and growth‐related traits in Atlantic salmon is unknown. Therefore, in this study, regions of the Atlantic salmon IGF1 gene were sequenced, aligned and compared across individuals. Three SNPs were identified in the putative promoter (SNP1, g.5763G>T; GenBank no. AGKD01012745 ), intron 1 (SNP2, g.7292C>T; GenBank no. AGKD01012745 ) and intron 3 (SNP3, g.4671A>C; GenBank no. AGKD01133398 ) regions respectively. These SNPs were genotyped in a population of 4800 commercial Atlantic salmon with data on several weight and fillet traits measured at harvest (at approximately 3 years of age). In a mixed model, association analysis of individual SNPs, SNP1 and SNP3 were both significantly associated with several weight traits (P < 0.05). The estimated additive effect on overall harvest weight was approximately 35 and 110 g for SNPs 1 and 3 respectively. A haplotype analysis confirmed the association between genetic variation in the IGF1 gene with overall body weight (P < 0.05) and fillet component traits (P < 0.05). Our findings suggest the identified nucleotide polymorphisms of the IGF1 gene may either affect farmed Atlantic salmon growth directly or be in population‐wide linkage disequilibrium with causal variation, highlighting their possible utility as candidates for marker‐assisted selection in the aquaculture industry.     

Identification of novel SNPs in the Sarda breed goats POU1F1 gene and their association with milk productive performance

The aim of the study was to detect polymorphism in the POU1F1 gene in Sarda breed goat, as well as to establish if SNPs could be associated with milk productive traits. The research was conducted on 129 Sarda breed goats from 4 to 5 years old, multiparous, lactating and in their third to fifth lactation. We report nine exonic and seven non-coding regions SNPs within the Sarda goat POU1F1 gene, namely, Ex 1 61 G>C; Ex 1 108 G>A; Ex 3 C>T; Ex 3 92 C>T; Ex 4 110 A>G; Ex 5 34 G>A resulting in Arg213Lys change; IVS4 641 G>A, IVS4 643 A>C, IVS4 659 G>A, IVS4 677 A>C, IVS4 G699Del, IVS4 709 C>G, Ex 6 17 G>A resulting in Arg228Ser change, Ex 6 58 G>T, Ex 6 172 T>C, 3′UTR 110 T>C. A statistically significant association was found between genotype TT, in position 17 of the exon 6 (3.1 % of frequency), and increased milk yield (P < 0.01) while genotype GT (25.6 % of frequency) was associated with a higher fat content. Genotype TT in position 58 of the exon 6 (3.9 % of frequency) was found to be associated with a higher fat (P < 0.01) and protein content (P < 0.05). Twenty-eight haplotypes were detected, but no significant association between the haplotypes and the milk production traits have been found. Our data, as well as providing new SNPs extending the POU1F1 gene characterization, evidence a relationship between polymorphism and milk production traits in Sarda goat breed.     

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.     

Evidence for the effect of serotonin receptor 1A gene (HTR1A) polymorphism on tractability in Thoroughbred horses

Tractability, or how easily animals can be trained and controlled, is an important behavioural trait for the management and training of domestic animals, but its genetic basis remains unclear. Polymorphisms in the serotonin receptor 1A gene (HTR1A) have been associated with individual variability in anxiety‐related traits in several species. In this study, we examined the association between HTR1A polymorphisms and tractability in Thoroughbred horses. We assessed the tractability of 167 one‐year‐old horses reared at a training centre for racehorses using a questionnaire consisting of 17 items. A principal components analysis of answers contracted the data to five principal component (PC) scores. We genotyped two non‐synonymous single nucleotide polymorphisms (SNPs) in the horse HTR1A coding region. We found that one of the two SNPs, c.709G>A, which causes an amino acid change at the intracellular region of the receptor, was significantly associated with scores of four of five PCs in fillies (all Ps < 0.05) and one PC in colts (P < 0.01). Horses carrying an A allele at c.709G>A showed lower tractability. This result provides the first evidence that a polymorphism in a serotonin‐related gene may affect tractability in horses with the effect partially different depending on sex.     

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.     

Association of a single nucleotide polymorphism in the akirin 2 gene with economically important traits in Korean native cattle

The akirin 2 gene, located on chromosome 9 in cattle, was previously reported to be associated with nuclear factor‐kappa B (NF‐κB), involved in immune reactions and marbling of meat. To determine whether a single nucleotide polymorphism (SNP) in akirin 2 is associated with economically important traits of Korean native cattle, the c.*188G>A SNP DNA marker in the 3′‐UTR region of akirin 2 was analyzed for its association with carcass weight, longissimus muscle area and marbling. The c.*188G>A SNP was genotyped by polymerase chain reaction restriction fragment length polymorphism, and the frequency of the AA, AG, and GG genotypes were 6.82%, 71.29% and 21.88% respectively. This SNP was significantly associated with longissimus muscle area (Bonferroni corrected P < 0.05), and marbling score (Bonferroni corrected P < 0.01). These results suggest that the c.*188G>A SNP of akirin 2 might be useful as a DNA marker for longissimus muscle area and marbling scores in Korean native cattle.     

Genome‐wide association studies for milk production traits in Valle del Belice sheep using repeated measures

Genome‐wide association studies (GWASes) have become a powerful tool for identifying genomic regions associated with important traits in livestock. Milk production traits in dairy sheep are measured at different time points during their life span. Using phenotypic data generated from longitudinal traits could improve the power of association studies but until now have received less attention in GWASes as a methodology and has not been implemented. The aim of this study was to carry out a GWAS for milk production traits in Valle del Belice sheep using repeated measures. After quality control, 469 ewes and 37 228 SNPs were retained for the analysis, and phenotypic data included 5586 test‐day records for five milk production traits (milk yield, MY; fat yield and percentage, FY and F%; protein yield and percentage, PY and P%). Nine SNPs located within or close to known genes were found to be associated with milk production traits. In particular, rs398340969, associated with both milk yield and protein yield, is located within the DCPS gene. In addition, rs425417915 and rs417079368, both associated with both fat percentage and protein percentage, are located within the TTC7B gene and at 0.37 Mb within the SUCNR1 gene respectively. In summary, the use of repeated records was beneficial for mapping genomic regions affecting milk production traits in the Valle del Belice sheep.     

Associations between newly discovered polymorphisms in the Bos taurusgrowth hormone receptor gene and performance traits in Holstein–Friesian dairy cattle

Variations in the growth hormone receptor (GHR) gene sequence are associated with performance traits in cattle. For example, the single nucleotide polymorphism (SNP) F279Y in transmembrane exon 8 has a strong association with milk yield. In this study, 32 previously unreported, putative novel SNPs (31 in the 5′ non‐coding region) were identified by resequencing ～19 kb of the GHR gene in genomic DNA from 22 cattle of multiple breeds. A population of 848 Holstein–Friesian AI sires was subsequently genotyped for the 32 putative novel SNPs and seven published SNPs (including F279Y, one in exon 1A promoter and five in exon 10). Associations between each segregating SNP and genetic merit for performance were quantified in the 848 Holstein–Friesians using weighted animal linear mixed models. Six of the published SNPs and seven of the novel SNPs were associated with at least one of the traits – milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score, calving interval, survival and growth and size traits. Even when the allelic substitution effect (P < 0.001) of F279Y was accounted for, the allelic substitution effect of one of the novel SNPs (GHR4.2) in the 5′ non‐coding region of GHR was associated with a lactation milk yield of 37.46 kg (P < 0.001). GHR4.2 and F279Y were not in linkage disequilibrium (r² = 0.00, D’ = 0.04) in the 848 Holstein–Friesians, indicating that their association with milk yield was independent.