Characterization and validation of bovine Gonadotripin releasing hormone receptor (GNRHR) polymorphisms

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4 bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.     

High incidence of single nucleotide polymorphisms in the prion protein gene of native Brazilian Caracu cattle

Different alleles of the human and ovine prion protein gene correlate with a varying susceptibility to transmissible spongiform encephalopathies. However, the pathogenic implications of specific polymorphisms in the bovine prion protein gene (PRNP) are only poorly understood. Previous studies on the bovine PRNP gene investigated common European and North American cattle breeds. As a consequence of decades of intensive breeding for specific traits, these modern breeds represent only a small fraction of the bovine gene pool. In this study, we analysed PRNP polymorphisms in the native Brazilian Caracu breed, which developed in geographical isolation since the 16th century. A total of 10 single nucleotide polymorphisms (SNPs) were discovered in the coding region of the Caracu PRNP gene. Eight of the SNPs occurred at high frequencies in Caracu cattle (variant allele frequencies = 0.10–0.76), but were absent or only rarely observed in European and North American breeds. One of the Caracu SNPs was associated with an amino acid exchange from serine to asparagine (f = 0.17). This SNP was not detected in Holstein–Friesian, Simmental and German Gelbvieh and was only rarely detected in beef cattle (f = 0.01). We found 17 haplotypes for PRNP in the Caracu breed.     

Characterization and validation of bovine gonadotripin releasing hormone receptor (GNRHR) polymorphisms

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4 bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.     

The polymorphisms of bovine melanocortin-3 receptor pseudogene

Mammalian melanocortin-3 receptor (MC3R) plays an important role in the central control of energy homeostasis, and several functional polymorphisms of mc3r have been detected. Interestingly, the bovine mc3r was a pseudogene, and its polymorphisms and function remain to be investigated. Single-strand conformation polymorphism (SSCP) showed 5, 2 and 3 genotypes in fragment F1, F2 and F3 of mc3r in seven cattle breeds, respectively. All genotypes revealed novel sequences. Three SNPs 657G > T, 756C > T, 822T > C were detected in fragment F1, five SNPs 1091T > C, 1133T > C, 1144C > T, 1259T > C and 1319G > A were detected in fragment F2, and two SNPs 1687G > A, 1860C > T were detected in F3. The SNPs in fragment F1 and F2 were located at exon 2. The five SNPs in fragment F2 demonstrated a tight linkage disequilibrium status. Variation detected here might have an impact on the function of bovine mc3r pseudogene.     

Association of bovine KISS1 single nucleotide polymorphisms with reproductive traits in Indian Cattle

Kisspeptins, a family of neuropeptide encoded by the Kiss1 gene, have emerged as crucial regulator of fertility and reproduction by regulating the hypothalamic–pituitary–gonadal axis. The present study was aimed to identify and associate SNPs in the KISS1 gene with reproductive traits in cattle of Indian origin. DNA samples collected from 300 individual cows of three Indian dairy breeds (Gir, Kankrej and Frieswal) of cattle were used in the study. The SNPs of KISS1 gene were identified with PCR-RFLP and sequence analysis using two sets of primer pairs. A total of 5 SNPs were identified in the targeted region of which, two were selected for screening the population and association studies. The analysis revealed that genotypes of rs442633552G>A and rs42022871C>T had a significant association with dry period. The SNP rs42022871C>T also established significant role in milk production traits, and selection of TT-genotyped animals will improve the reproduction and production potential of the animals.     

深县猪mtDNA CytB基因的遗传多样性与母系起源研究

 研究过氧化物酶体增殖激活受体α (PPARα) 基因第7外显子SNP位点与牛的部分胴体、肉质性状的相关性。以6个牛品种(秦川牛、南阳牛、郏县红牛、鲁西牛、安格斯、夏南牛) 共计717个个体为研究对象,采用PCR SSCP结合DNA测序方法对PPARα基因第7外显子进行SNP检测,并该SNP位点与108头秦川牛的部分胴体、肉质性状的相关性进行分析。结果发现在PPARα基因第7外显子的184位检测到C→T突变,在南阳牛、秦川牛、郏县红牛、安格斯、鲁西牛、夏南牛这6个群体中等位基因E/F的频率分别是0.225/0.775,0.151/0.849,0.125/0.875,0.123/0.877,0.070/0.930, 0.157/0.783;遗传学指标结果显示：秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛这5个群体属于低度多态(PIC<0.25),南阳牛为中度多态（PIC=0.288）;相关分析结果显示：该位点与秦川牛的背膘厚和胴体长两个性状显著相关,表现为FF基因型个体在背膘厚性状方面显著高于EE和EF基因型个体(P<0.05),FF基因型个体的胴体长显著高于EE基因型个体(P<0.05)。这一位点可能是影响牛背膘厚和胴体长的主效QTN或与之紧密连锁,可做为肉牛选育的候选分子标记。     

Identification of genomic regions that exhibit sexual dimorphism for size and muscularity in cattle

Sexual dimorphism, the phenomenon whereby males and females of the same species are distinctive in some aspect of appearance or size, has previously been documented in cattle for traits such as growth rate and carcass merit using a quantitative genetics approach. No previous study in cattle has attempted to document sexual dimorphism at a genome level; therefore, the objective of the present study was to determine whether genomic regions associated with size and muscularity in cattle exhibited signs of sexual dimorphism. Analyses were undertaken on 10 linear-type traits that describe the muscular and skeletal characteristics of both males and females of five beef cattle breeds: 1,444 Angus (AA), 6,433 Charolais (CH), 1,129 Hereford, 8,745 Limousin (LM), and 1,698 Simmental. Genome-wide association analyses were undertaken using imputed whole-genome sequence data for each sex separately by breed. For each single-nucleotide polymorphism (SNP) that was segregating in both sexes, the difference between the allele substitution effect sizes for each sex, in each breed separately, was calculated. Suggestively (P ≤ 1 × 10⁻⁵) sexually dimorphic SNPs that were segregating in both males and females were detected for all traits in all breeds, although the location of these SNPs differed by both trait and breed. Significantly (P ≤ 1 × 10⁻⁸) dimorphic SNPs were detected in just three traits in the AA, seven traits in the CH, and three traits in the LM. The vast majority of all segregating autosomal SNPs (86% in AA to 94% in LM) had the same minor allele in both males and females. Differences (P ≤ 0.05) in allele frequencies between the sexes were observed for between 36% (LM) and 66% (AA) of the total autosomal SNPs that were segregating in both sexes. Dimorphic SNPs were located within a number of genes related to muscularity and/or size including the NAB1, COL5A2, and IWS1 genes on BTA2 that are located close to, and thought to be co-inherited with, the MSTN gene. Overall, sexual dimorphism exists in cattle at the genome level, but it is not consistent by either trait or breed.     

Association between melanocortin 4 receptor (MC4R) gene haplotypes and carcass and production traits in Italian Large White pigs evaluated with a selective genotyping approach

Melanocortin 4 receptor (MC4R) plays a key role in controlling energy homeostasis. Several studies have already reported effects on production traits of polymorphisms identified in the porcine MC4R gene. In this study we analysed data on 6 MC4R polymorphisms (c.-780C>G; c.-135C>T; c.175C>T; c.707G>A or p.Arg236His; and c.892G>A or p.Asp298Asn; c.?430A>T) genotyped from (1) two groups of Italian Large White pigs (280+280 animals) with extreme estimated breeding values (EBVs) for back fat thickness (BFT), selected among a performance tested population of about 12,000 pigs, and from (2) 19 Italian Duroc pigs. Two haplotypes, differentiated by the c.892G>A, were identified in the Duroc populations. Four haplotypes were identified in the Italian Large White population, one of which (haplotype 4) was identified for the first time in this study. Single marker and haplotype association analyses for BFT were obtained by comparing allele and haplotype frequency differences from the two extreme tails using χ² and Cochran–Armitage trend tests. Results confirmed the effects of the c.892G>A single nucleotide polymorphism (SNP) on BFT, as also defined by different distributions in the two tails of haplotypes carrying the alternative nucleotides at this polymorphic site (P<0.01). In addition different distributions of haplotype 4 in the two extreme groups suggested that it might affect the same trait (P<0.10). Association analyses for several other traits (average daily gain, ADG; feed gain ratio, FGR; weight of lean cuts; ham weight) were carried out by using EBVs and Random Residuals: significant effects (P<0.05) were only found for the p.Asp298Asn mutation on ADG and FGR. Results did not support any relevant effect of the p.Arg236His mutation on any trait. Data reported in this study contribute to better understand the role of MC4R variants in affecting production traits in pigs, a prerequisite to consider polymorphisms in this gene for marker assisted selection.     

Relationship of the bovine growth hormone gene to carcass traits in Japanese black cattle.

The bovine growth hormone gene (bGH) possesses three haplotypes, A, B and C, that differ by amino acid mutations at positions 127 and 172 in the fifth exon: (leucine 127, threonine 172), (valine 127, threonine 172) and (valine 127, methionine 172) respectively. The correlation between meat quality or carcass weight and these haplotypes was investigated in Japanese black cattle. Altogether, 940 bGH haplotypes were compared with respect to six carcass traits: carcass weight, longissimus muscle area, rib thickness, subcutaneous fat thickness, beef marbling score and beef colour. The frequency of the B haplotype was higher (0.421) than that of A (0.269) and C (0.311). High carcass weight and low beef marbling were associated with haplotype A (p < 0.05 and p < 0.01 respectively), whereas beef marbling was increased by haplotype C (p < 0.05). Estimated regression coefficient of the A haplotype substitution effect for carcass weight and beef marbling score were 5.55 (13.1% of the phenotypic SD) and -0.31 (17.0%) respectively. That of the C haplotype for beef marbling score was 0.20 (11.0%). The other traits showed no relationship to the haplotypes examined. The results of this investigation suggest that information pertaining to bGH polymorphisms in Japanese black cattle could be used to improve the selection of meat traits.     

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.     

Evaluation in beef cattle of six deoxyribonucleic acid markers developed for dairy traits reveals an osteopontin polymorphism associated with postweaning growth

The objectives of this study were to 1) estimate the allelic frequencies in US beef cattle of 6 DNA markers reported to be associated with variation in dairy production traits; and 2) evaluate the association of these markers with beef production traits. Several genetic markers have been associated with milk yield or composition, including polymorphisms in secreted phosphoprotein 1 (SPP1; also called osteopontin), growth hormone receptor (GHR), casein S1 (CSN1S1), diacylglycerol O-acyltransferase 1 (DGAT1), peroxisome proliferator-activated receptor gamma co-activator-1alpha (PPARGC1A), and ATP-binding cassette subfamily G (white) member 2 (ABCG2). Allelic frequencies for these 6 markers, and their association with 21 phenotypes, were evaluated in 2 crossbred beef cattle populations that sample influential industry sires. Five of 6 markers were segregating in beef cattle populations; the exception was ABCG2. The SPP1 marker was associated with yearling weight (P = 0.025), live weight at slaughter (P = 0.016), postweaning ADG (P = 0.007), and HCW (P = 0.007) in a large, multisire population representing the 7 most populous beef breeds in the United States. Postweaning growth trait associations were confirmed in an independent population of similar construction, including sires from tropically adapted breeds. The SPP1 marker was associated with yearling weight (P = 0.034), live weight at slaughter (P = 0.011), and postweaning ADG (P = 0.015) and showed a trend toward association with HCW (P = 0.083) in this population. Whereas DGAT1, GHR, and CSN1S1 polymorphisms showed association with some traits in individual populations, the lack of consistent predictive merit between populations indicates they may not be suited for beef cattle selection. No significant associations were observed for the PPARGC1A marker and any of 21 recorded traits, indicating this marker had no apparent value in selection for the beef cattle traits tested in these populations. The SPP1 marker had consistent associations and effect sizes (10.5 to 11.5 kg of live weight at slaughter) in both populations, providing strong evidence for utility of the SPP1 marker for postweaning growth in beef cattle.     

Signatures of selection in Charolais beef cattle identified by genome‐wide analysis

Charolais cattle are one of the most important breeds for meat production worldwide; in México, its selection is mainly made by live weight traits. One strategy for mapping important genomic regions that might influence productive traits is the identification of signatures of selection. This type of genomic features contains loci with extended linkage disequilibrium (LD) and homozygosity patterns that are commonly associated with sites of quantitative trait locus (QTL). Therefore, the objective of this study was to identify the signatures of selection in Charolais cattle genotyped with the GeneSeek Genomic Profiler Bovine HD panel consisting of 77 K single nucleotide polymorphisms (SNPs). A total 61,311 SNPs and 819 samples were used for the analysis. Identification of signatures of selection was carried out using the integrated haplotype score (iHS) methodology implemented in the rehh R package. The top ten SNPs with the highest piHS values were located on BTA 4, 5, 6 and 14. By identifying markers in LD with top ten SNPs, the candidate regions defined were mapped to 52.8–59.3 Mb on BTA 4; 67.5–69.3 on BTA 5; 39.5–41.0 Mb on BTA 6; and 26.4–29.6 Mb on BTA 14. The comparison of these candidate regions with the bovine QTLdb effectively confirmed the association (p < 0.05) with QTL related to growth traits and other important productive traits. The genomic regions identified in this study indicated selection for growth traits on the Charolais population via the conservation of haplotypes on various chromosomes. These genomic regions and their associated genes could serve as the basis for haplotype association studies and for the identification of causal genes related to growth traits.     

西门塔尔牛与我国地方黄牛的杂种优势预测分析

旨在利用覆盖全基因组和与性状相关的SNPs标记分析西门塔尔牛和地方黄牛两个亲本群体的遗传结构,通过亲本种群之间的遗传距离预测不同杂交组合在生长、胴体和肉质性状上的杂种优势。本研究选择来自内蒙古锡林郭勒盟乌拉盖管理区牧场的1 222头西门塔尔牛和8个地方黄牛品种190头共组成8个杂交组合,对亲本群体进行遗传结构分析。利用牛770K SNP芯片对两个亲本群体进行基因型分型,通过牛QTL数据库筛选与目的性状对应的QTLs,对全基因组SNP位点进行映射分析得到与性状相关的SNP标记。利用两种SNP标记构建状态同源矩阵,计算各杂交组合亲本间的遗传距离。结果,所有亲本群体聚成3类,西门塔尔牛聚成一类,北方黄牛（蒙古牛、西藏牛和柴达木牛）聚成一类,南方黄牛（昭通牛、平武牛、南丹牛、文山牛和凉山牛）聚成一类。西门塔尔牛与北方黄牛在PCA图上的距离较近,说明两者之间亲缘关系相对较近,遗传背景差异较小。在各性状中,遗传距离最大的亲本组合都是西门塔尔牛与南丹牛杂交组合。除了大理石花纹评分性状是西门塔尔牛与昭通牛之间的遗传距离最小,其余性状遗传距离最小的亲本组合都是西门塔尔牛与平武牛杂交组合。据此推测,各性状杂种优势较优的可能组合是西门塔尔牛与南丹牛杂交组合。     

Genetic variation in monoamine oxidase A and analysis of association with behaviour traits in beef cattle

Husbandry of beef cattle requires animals that do not behave aggressively or timidly. The enzyme monoamine oxidase A and the coding gene (MAOA) play an important role in the complex regulation of behaviour. The complete coding region and a part of the non‐coding sequence of the bovine MAOA gene have been analysed in 20 German Angus and 20 German Simmental bulls and cows with the aim of detecting genetic variability. These two cattle breeds are known to differ regarding their behaviour during handling. Five single nucleotide polymorphisms (SNPs) were identified, three of which were found in the coding region of the gene (exons III and XV). One of the SNPs located in exon XV ( NC_007331.3 :g.80340C>T) was found to be a non‐synonymous mutation. The minor allele frequency of this resulting amino acid substitution was significantly different between 543 German Angus and 417 German Simmental calves (0.39 and 0.49, respectively). The potential functional impact of this polymorphism has been tested by in silico analysis, as well as by association analysis using behaviour scores of the genotyped calves for three behaviour tests that assessed the animals’ temperament during tethering, weighing or social separation. In silico analysis did not deliver consistent results arguing for or against a functional impact of the studied amino acid substitution on the function of the biological protein. No significant association was found between this MAOA polymorphism and the behaviour‐related scores analysed in the study.     

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.     

Pleiotropic effects of the goat prolactin receptor genotype on milk fatty acid composition

In the lactating mammary gland, prolactin (PRL) stimulates the synthesis of lactose as well as fatty acid uptake, lipogenesis, and triacylglycerol synthesis. Associations between bovine PRL receptor (PRLR) genotype and fat yield have been reported, which illustrates the role of PRL in conveying lipids toward the udder as well as in stimulating their local synthesis during lactation. Conversely, and to the best of our knowledge, the effects of PRLR genotype on milk fatty acid content have not been studied so far in any mammalian species. In this study, we sequenced most of the coding region of the caprine PRLR gene in several individuals from the Malagueña and Murciano-Granadina breeds. This approach allowed us to identify 2 long and short mRNA isoforms, produced by alternative splicing, and 4 single-nucleotide polymorphisms (SNPs), namely, c.177T>C, c.1131G>A, c.1201G>A and c.1355C>T. Two of these SNPs are nonsynonymous and involve G401R (c.1201G>A) and T452I (c.1355C>T) amino acid substitutions in the cytoplasmic domain of PRLR, which plays a fundamental role in signal transduction. Performance of an association analysis with milk composition traits in a Murciano-Granadina goat population revealed highly suggestive effects on palmitoleic acid content, whereas suggestive effects were detected for other fatty acids, such as palmitic and linoleic. These results are consistent with the pleiotropic effects of PRL on mammary gland lipid metabolism and milk composition.     

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.