An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

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

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

Genetic variation in the β, β‐carotene‐9′, 10′‐dioxygenase gene and association with fat colour in bovine adipose tissue and milk

β, β‐carotene‐9′, 10′‐dioxygenase (BCO2) plays a role in cleaving β‐carotene eccentrically, and may be involved in the control of adipose and milk colour in cattle. The bovine BCO2 gene was sequenced as a potential candidate gene for a beef fat colour QTL on chromosome (BTA) 15. A single nucleotide base change located in exon 3 causes the substitution of a stop codon (encoded by the A allele) for tryptophan⁸⁰ (encoded by the G allele) (c. 240G>A, p.Trp80stop, referred to herein as SNP W80X). Association analysis showed significant differences in subcutaneous fat colour and beta‐carotene concentration amongst cattle with different BCO2 genotypes. Animals with the BCO2 AA genotype had more yellow beef fat and a higher beta‐carotene concentration in adipose tissues than those with the GA or GG genotype. QTL mapping analysis with the BCO2 SNP W80X fitted as a fixed effect confirmed that this SNP is likely to represent the quantitative trait nucleotide (QTN) for the fat colour‐related traits on BTA 15. Moreover, animals with the AA genotype had yellower milk colour and a higher concentration of beta‐carotene in the milk.     

A Single Nucleotide Polymorphism and Sequence Analysis of CSN1S1 Gene Promoter Region in Chinese BOS Grunniens (YAK)

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G₃₈₆ and A₃₈₆, based on the nucleotide at position 386. The allele G₃₈₆ was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G₃₈₆ in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A_386.     

Genetic variants of the <Emphasis Type="Italic">FABP4</Emphasis> gene are associated with marbling scores and meat quality grades in Hanwoo (Korean cattle)

This study was aimed to search new genetic variants in the bovine FABP4 gene as molecular markers for meat quality and carcass traits. PCR–RFLP analysis revealed that three SNPs located at nucleotide positions g.2834C>G, g.3533T>A, and g.3691G>A were identified based on a GenBank accession number (NC_007312.4). Sequence analysis revealed that SNPs were located in intron 1 (g.2834C>G) and 2 (g.3533T>A), and an exon 3 (g.3691G>A), showing allele frequencies as 0.592, 0.579, and 0.789, respectively. Genetic variabilities of heterozygosity (He) and polymorphic information contents (PIC) were estimated for g.2834C>G (0.608 and 0.531), g.3533T>A (0.615 and 0.539), and g.3691G>A (0.498 and 0.401) loci, respectively. A SNP located in the exon 3 of FABP4 was characterized and associated with desirable increases of MS (marbling scores) and MG (meat quality grades) in Hanwoo. The statistical analysis revealed that additive effects by GG genotypes in g.3691G>A SNP were significantly greater than AA genotypes in MS and MG traits. These findings suggest that the FABP4g.3691G>A SNP will be a useful candidate locus to maximize economic benefits for cattle populations.     

Goat casein genotypes are associated with milk production traits in the Sarda breed

The aim of the current work was to analyze, in the Sarda breed goat, genetic polymorphism within the casein genes and to assess their influence on milk traits. Genetic variants at the CSN1S1, CSN2, CSN1S2 and CSN3 gene loci were investigated using PCR‐based methods, cloning and sequencing. Strong alleles prevailed at the CSN1S1 gene locus and defective alleles also were revealed. Null alleles were evidenced at each calcium‐sensitive gene locus. At the CSN3 gene locus, we observed a prevalence of the CSN3 A and B alleles; the occurrence of rare alleles such as CSN3 B'', C, C', D, E and M; and the CSN3 S allele (GenBank KF644565 ) described here for the first time in Capra hircus. Statistical analysis showed that all genes, except CSN3, significantly influenced milk traits. The CSN1S1 BB and AB genotypes were associated with the highest percentages of protein (4.41 and 4.40 respectively) and fat (5.26 and 5.34 respectively) (P < 0.001). A relevant finding was that CSN2 and CSN1S2 genotypes affected milk protein content and yield. The polymorphism of the CSN2 gene affected milk protein percentage with the highest values recorded in the CSN2 AA goats (4.35, at P < 0.001). The CSN1S2 AC goats provided the highest fat (51.02 g/day) and protein (41.42 g/day) (P < 0.01) production. This information can be incorporated into selection schemes for the Sarda breed goat.     

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.     

Effects of genetic variability of the dairy goat growth hormone releasing hormone receptor (<Emphasis Type="Italic">GHRHR</Emphasis>) gene on growth traits

Growth hormone-releasing hormone receptor (GHRHR) plays a critical role in growth hormone (GH) synthesis, release and regulation of pituitary somatotroph expansion in vertebrates. The objective of this study was to investigate variations in goat GHRHR gene and their associations with growth traits in 668 dairy goats. The results showed four novel single nucleotide polymorphisms (SNPs): NC_007302:g.5203C>T, 7307C>G, 9583G>A and 9668A>C. In detail, the novel SNP C>T in the 5203rd nucleotide identified a missense mutation: CCC (Pro)>TCC (Phe) at position 116aa of the goat GHRHR (423aa). Besides, 9583G>A and 9668A>C polymorphism were in complete linkage disequilibrium. The genetic diversity analysis revealed that the Guanzhong dairy goat possessed intermediate genetic diversity in P3 and P7 loci, and the Xinong Sannen dairy goat belonged to poor genetic diversity in P4 locus. Significant associations between the genotypes of P3 locus and body length, body height and chest circumference was observed in Guanzhong goat (P < 0.05). However, in Xinong saanen population, significant statistical difference was only found in body height and body length (P < 0.05). In P4 and P7 loci, no significant associations were detected between any variant sites and body length, body height and chest circumference, as well as for the milk traits (P > 0.05). These results strongly suggested that the goat GHRHR gene is a candidate gene that influences growth traits in dairy goat.     

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.     

Porcine insulin receptor substrate 4 (<Emphasis Type="Italic">IRS4</Emphasis>) gene: cloning,polymorphism and association study

Using PCR and inverse PCR techniques we obtained a 4,498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine insulin receptor substrate 4 (IRS4) gene and its proximal promoter. The 1,269 amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesses the same domains and the same number of tyrosine phosphorylation motifs as the human protein. We detected substitution FN424076:g.96C<G in the promoter region that segregates in Meishan and a synonymous substitution FN424076:g.1829T<C in the coding sequence with allele C present only in Meishan. Linkage mapping placed the IRS4 gene at position 82 cM on the current USDA–USMARC linkage map of porcine chromosome X. Association analyses were performed on 555 animals of 12th–15th generation of the Meishan × Large White cross and showed that both SNPs were highly significantly associated with backfat depth (P = 0.0005) and that the SNP FN424076:g1829T<C was also associated with loin depth (P = 0.017). The Meishan alleles increased back fat depth and decreased loin depth. IRS4 can be considered a positional candidate gene for at least some of the QTL located at the centromeric region of porcine chromosome X.     

The differential expression of alternatively spliced transcripts and imprinting status of MEG9 gene in cows

Meg9/Mirg (maternally expressed gene 9/microRNA containing gene), a non‐coding RNA (ncRNA) comprising many alternatively splicing isoforms, has been identified as maternally expressed in mouse and sheep, but its imprinting status and splicing variants are still unknown in cattle. In this study, we found three splicing variants of the cattle MEG9 gene expressed in a tissue‐specific manner. A single nucleotide polymorphism site (SNP c.1354C>G) was identified in exon 3 of cattle MEG9 and used to distinguish between monoallelic and biallelic expression. Our results showed that MEG9 exhibited monoallelic expression in all examined cattle tissues by comparing sequencing results between genomic DNA and cDNA levels at the c.1354C>G SNP site, suggesting that MEG9 is imprinted in cattle.     

The CDKN2A/p16INK4a 5′UTR sequence and translational regulation: impact of novel variants predisposing to melanoma

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5′UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.‐27del23, c.‐93‐91delAGG) were classified as causal mutations (score ≥3), along with the c.‐21C>T, c.‐34G>T, and c.‐56G>T, which had already been studied by a subset of assays. The novel c.‐42T>A as well as the previously described c.‐67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.‐14C>T, c.‐20A>G, c.‐25C>T+c.‐180G>A, c.‐30G>A, c.‐40C>T, c.‐45G>A, c.‐59C>G, c.‐87T>A, c.‐252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5′UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16^INK4a translation.     

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.     

Genetic association between bovine NOD2 polymorphisms and infection by Mycobacterium avium subsp. paratuberculosis in Holstein‐Friesian cattle

Nucleotide‐Binding Oligomerization Domain 2 (NOD2) has been reported to be a candidate gene for Mycobacterium avium subsp. paratuberculosis (MAP) infection in a Bos taurus × Bos indicus mixed breed based on a genetic association with the c.2197T>C single nucleotide polymorphism (SNP). Nevertheless, this SNP has also been reported to be monomorphic in the B. taurus species. In the present work, 18 SNPs spanning the bovine NOD2 gene have been analysed in a genetic association study of two independent populations of Holstein‐Friesian cattle. We found that the C allele of SNP c.*1908C>T, located in the 3′‐UTR region of the gene, is significantly more frequent in infected animals than in healthy ones, which supports the idea that the bovine NOD2 gene plays a role in susceptibility to MAP infection. However, in silico analyses of the NOD2 nucleotide sequence did not yield definitive data about a possible direct effect of SNP c.*1908C>T on susceptibility to infection and led us to consider its linkage disequilibrium with the causative variant. A more exhaustive genetic association study including all putative, functional SNPs from this gene and subsequent functional analyses needs to be conducted to achieve a more complete understanding of how different variants of NOD2 may affect susceptibility to MAP infection in cattle.     

A novel 62‐bp indel mutation in the promoter region of transforming growth factor‐beta 2 (TGFB2) gene is associated with body weight in chickens

Transforming growth factor‐beta 2 (encoded by TGFB2) is a growth factor that regulates a plethora of cellular functions. In this study, we sequenced the promoter and full‐length exon region of the chicken TGFB2 and found two mutations (g.‐640C>T and g.‐851_‐790del) within the promoter. The two polymorphisms were genotyped in 1030 pedigreed hens recorded for body weight at 7 (BW7), 9 (BW9), 11 (BW11), 13 (BW13), 17 (BW17) weeks old, egg weight at 36 weeks of age (EW36) and egg numbers from the age at first egg (AFE) to 40 weeks of age (EN40). Despite the fact that no mutations were found to have statistically significant genetic effects on egg production, the association results of growth traits showed that both g.‐640C>T and g.‐851_‐790del had significant effects on body weights and that both genotype g.‐640TT and g.‐851_‐790wt/wt were positive for body weight performance. Therefore, the polymorphisms of TGFB2, especially the g.‐851_‐790del mutation associated with body weight at almost all periods, could be potential useful genetic markers to improve the growth of Beijing You chickens.