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.     

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.     

Exploring polymorphisms and their effects on reproductive traits of the INHA and INHβA genes in three goat breeds

In this study, we report the analysis of INHA and INHβA gene polymorphisms in 786 goats of three breeds: Xinong Saanen (SN), Guanzhong (GZ) and Boer (BG). We identified three new allelic variants: P1–C80G and/126G (GenBank accession no. HQ202573) in the three goat breeds and P2–C936T (GenBank accession no. HQ202572) in SN and GZ goat breeds. At P1 locus, AA, AB and BB genotypes were found in the three goat breeds. At P2 locus, CC and CT genotypes were found in SN and GZ goat breeds. After comparing genotype distribution within the three goat breeds, BG had conspicuous differences from SN and GZ (P < 0.001) at P2 locus. The SNP locus was in Hardy–Weinberg disequilibrium at P1 locus in the three goat breeds (P < 0.05). At P2 locus, the SNP locus was in Hardy–Weinberg disequilibrium in SN and GZ goat breeds (P < 0.05). Association of polymorphisms with litter size was done at P1 locus in the three goat breeds. The result showed that AA genotype had remarkable litter size at P1 locus in the three goat breeds (P < 0.05). Therefore, these results suggest that INHA gene is a strong candidate gene that affects litter size in goats.     

A substitution within erythropoietin receptor gene D1 domain associated with litter size in Beijing Black pig,Sus scrofa

Studies of uterine capacity and litter size in swine have suggested that erythropoietin receptor (EPOR) plays an important role in fetal survival through maturation of red blood cells. In this study, we screened the porcine EPOR gene for mutations and identified three single nucleotide polymorphisms (SNPs): two missense mutations and one synonymous mutation. We then genotyped 272 Beijing Black sows, Sus scrofa, and compared this data with litter sizes from a total of 1523 parities among the sows. The G allele of the nonsynonymous SNP, EPOR c.434A>G, was associated with greater litter size at both first parity (P < 0.05) and at later parities (P < 0.01). This SNP causes His92Arg adjacent to the fourth conserved cysteine residue in the mature protein and is in the D1 domain of the protein. Additionally, we determined the allele frequencies for this SNP among six Chinese indigenous pig breeds (Bamei, Erhualian, Laiwu Black, Mashen, Meishan and Min) and three Western commercial pig breeds (Duroc, Landrace and Large White). The c.434G allele was significantly more common among the more prolific Chinese breeds than the Western breeds, implying that EPOR c.434A>G could be a useful genetic marker to improve litter size in swine.     

Two novel intronic polymorphisms of bovine FGF21 gene are associated with body weight at 18 months in Chinese cattle

Fibroblast growth factor 21 (FGF21) is a hepatic hormone that regulates peripheral glucose tolerance, energy balance and lipid metabolism. Prior evidence suggests that FGF21 may have the potential to favorably reduce obesity. The objective of the present study was to identify single nucleotide polymorphisms (SNPs) of bovine FGF21 using 1255 animals representing the five main Chinese breeds and to investigate the effect of these SNPs on economic traits in Nanyang cattle. Four significant SNPs were identified, one was a synonymous mutation and the other three were in intronic regions. The polymorphism information content (PIC) analysis showed that four beef cattle populations (NY, JX, LX and QC) had a moderate genetic diversity at the four loci while the beef and dairy population (CRS) had a low level. Additionally, allele and genotype frequencies for the beef breeds were significantly different from CRS, implying that these mutations are possibly associated with some quantitative traits. Moreover, linkage disequilibrium analysis and haplotype frequencies were also reported. Seven different haplotypes were identified and haplotype TCCC was predominant in all five cattle breeds. Association analysis suggested that SNPs g.297C>G and g.940C>T of bovine FGF21 were associated with higher body weight at 18 months within NY cattle, which would contribute to cattle breeding and genetics through marker-assisted selection (MAS).     

Detection of novel single-nucleotide polymorphisms (SNPs) in the CYP21 gene and association analysis of two SNPs for CYP21 and ESR2 with litter size in a commercial sow population

Altogether 129 F₁ sows from a commercial sow farm with at least four litters were genotyped for the oestrogen receptor 2 gene (ESR2) and cytochrome P450 hydroxylase 21 gene (CYP21) and investigated for associations on the litter‐size parameters: total number born and number born alive. Five novel polymorphisms were found in the 3′‐untranslated region for the CYP21 gene. Genotype and allele frequencies for the CYP21 (position 3462G > A) single‐nucleotide polymorphism (SNP) were 0.434 (GG), 0.504 (AG), 0.062 (AA) and 0.69 (G):0.31 (A), respectively. No association was found between this polymorphism and litter‐size parameters. For the ESR2 gene, the SNP in exon 5 associated with an amino acid substitution MET (allele A) > VAL (allele G) was investigated. Only two genotypes were found leading to allele frequencies of 0.34 (A):0.66 (G). Only number born alive piglets were significantly increased for the AG genotype (p = 0.034) with 11.64 piglets per sow and litter in comparison with the GG genotype, leading to only 10.96 piglets per sow and litter. From these data, it can be concluded that the investigated SNP of the ESR2 gene is associated with the number of liveborn piglets in the commercial population considered, and hence could be useful in selection for litter size. Therefore, this gene should be investigated in additional populations.     

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.     

Breeding German sheep for resistance to scrapie

Susceptibility to clinical scrapie is associated with polymorphisms in the prion protein (PrP) gene. The ARR allele reduces susceptibility to clinical disease caused by all known strains of the transmissible spongiform encephalopathy (TSE) agents. For the economically important German breeds of sheep the PrP allele frequencies are well known, but this paper presents representative genotyping results for 1526 sheep from two smaller milk sheep breeds and 2446 sheep from 14 mostly indigenous land sheep breeds. The ARR allele was detected in each breed but the breed-specific ARR frequencies varied between 1 and 63 per cent. In small populations with a very low ARR frequency the ARR allele could be lost by genetic drift. A simulation study was therefore made to examine the effects of different breeding schemes in populations of different sizes on attempts to select for the ARR allele in an endangered population. In breeds in which no homozygous rams are available the breeding strategy would depend on the number of heterozygous rams, and the genotyping and selection of suitable breeding ewes would reduce the time required to achieve a highly resistant population. In general, in all the breeds a selection programme to achieve 99 per cent ARR homozygous genotypes would be feasible in six to nine generations, depending on the initial allele frequencies. In small populations the inbreeding rate may increase if no specific mating plans are developed by the breeding organisations.     

Novel polymorphism of the canine dopamine receptor D4 gene intron II region

Various dog breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of these characteristics are under genetic control. However, little is known about genes related to behavioral traits in canine species. In humans, it has been reported that the dopamine receptor D4 gene (DRD4) includes polymorphism at several regions that relate to personality or psychiatric disorders. In an earlier study by the authors of the present study, the polymorphisms in canine DRD4 exon III and exon I regions were reported. In the present study, a novel polymorphism in canine DRD4 intron II was found based on a 17 base pair insertion/deletion, and the two alleles detected were named P (shorter allele) and Q (longer allele). The allelic distribution in 28 breeds of dog, including a total of 1114 unrelated individuals, were then investigated. Both P and Q alleles were detected in most of the breeds investigated; however, the frequencies of P and Q differed greatly between breeds. With respect to classification based on breed origin, P and Q alleles were frequent in Occidental and Oriental breeds, respectively. Furthermore, two subspecies of wolves, the ancestors of dogs, were analyzed for the comparison of allele frequencies with dogs, and the P allele was predominant in both European and Chinese wolves.     

Canine orosomucoid (alpha‐1 acid glycoprotein) variants and their influence on drug plasma protein binding

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed‐breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n = 12). Free and total drug plasma concentrations were quantified by liquid chromatography–mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n = 1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n = 21). Free drug fractions did not differ significantly (p > .05; Mann‐Whitney U) between plasma collected from dogs with c.70AA (n = 4) and those with c.70GG (n = 8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.     

Mutations in bone morphogenetic protein 15 and growth differentiation factor 9 genes are associated with increased litter size in fat-tailed sheep breeds

The objective of this study was to investigate association between GDF9 and BMP15 gene polymorphism and litter size in fat-tailed sheep, a total of 97 mature ewes from four breeds (Afshari=19; Baluchi=18; Makui=30 and Mehraban=30) were genotyped for the BMP15 HinfI and GDF9 HhaI polymorphisms by PCR-RFLP technique. The highest and lowest mutant allele frequencies were found in Makui (0.27) and Afshari (0.10) sheep for the BMP15 gene and in Afshari (0.24) and Mehraban (0.18) sheep for the GDF9 gene, respectively. Litter size was significantly influenced by genotype of the ewe for two genes (P < 0.01). Heterozygous genotypes for both loci showed higher litter size than homozygous genotypes (P < 0.01). None of the individuals carried homozygous genotype for both of the GDF9 and BMP15 variants in these breeds. The individuals carrying the mutant allele for one of the investigated candidate gene still showed fertile phenotype. Thus, existence of homozygosity at one of the BMP15 and GDF9 variant is not probably able to block normal hormonal pathway of reproduction in fat-tailed sheep.     

The T allele at the g.1471620G>T in the EDG1 gene associated with high marbling in Japanese Black cattle is at a low frequency in breeds not selected for marbling

Our previous study detected a single nucleotide polymorphism (SNP), g.1471620G > T , in the 5' flanking region of the endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, which has been considered as a positional functional candidate for the gene responsible for marbling, and showed association of the g.1471620G > T SNP with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the g.1471620G > T SNP among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Short Horn, Holstein, and Brown Swiss breeds. The T allele at the g.1471620G > T SNP associated with high marbling was found at high frequency in Japanese Black breed that has been subjected to a strong selection for high marbling, while the allele was absent or at very low frequencies in the other breeds that have not been strongly selected for high marbling. Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequency of the T allele at the g.1471620G > T SNP in the EDG1 .     

Polymorphisms in growth hormone gene and their associations with calf weight in Japanese Black cattle

The objectives of this study were to detect effective genetic polymorphisms of bovine growth hormone (bGH) gene associated with calf weight in Japanese Black cattle. Fifty‐eight sires and 47 breeding cows were used to detect the polymorphisms in exons by single‐strand conformation polymorphism (SSCP). Four homozygous and six heterozygous SSCP genotypes were identified in exon 5. Although each single nucleotide polymorphism (SNP) had been reported, these genotypes were caused by three SNPs at the nucleotide positions 2141, 2277 and 2291. Four haplotypes C‐C‐A, G‐C‐A, C‐C‐C and G‐T‐A were newly identified. It was suggested that other haplotypes not detected in this study may not exist, considering the allele frequencies reported in Bos taurus and Bos indicus, and the migrating process of native Japanese cattle. Thereafter, we examined associations between the detected polymorphic sites in exon 5 by PCR – restriction fragment length polymorphism and calf weight using 53 breeding dams and 135 calves. The birth weights of calves with haplotype G‐C‐A are significantly lighter and calves' weights produced by cows with such haplotype are also lighter at 30 days old, using regression analysis. Although further research is necessary, these results may serve as a useful criterion to select breeding stocks, especially in maternal abilities.     

Polymorphism of <Emphasis Type="Italic">GDF</Emphasis>9 gene and its association with litter size in goats

Growth differentiation factor 9 (GDF9) was studied as a candidate gene for high prolificacy in goats. The polymorphism of exon 1 and flanking of GDF9 gene was detected by PCR-SSCP in five goat breeds with different prolificacy. Three genotypes (AA, AB and BB) were detected in goat breeds joined and two silent mutations (c.183A>C and c.336C>T) were identified in comparison genotype AA with genotype BB. Heterozygous genotype AB and wild type BB were detected in all five goat breeds and homozygous genotype AA was only detected in Jining Grey goats. The frequencies of genotypes AA, AB and BB were 0.18, 0.42 and 0.40 in Jining Grey goats, respectively. The genotype distribution was different (P < 0.01) between high prolificacy breed (Jining Grey goat) and low prolificacy breeds (Boer, Wendeng Dairy, Liaoning Cashmere and Beijing native goats). The Jining Grey goat does with genotype AA and AB had 0.72 (P < 0.01) and 0.56 (P < 0.01) kids more than those with genotype BB, respectively. The does with genotype AA had 0.16 (P > 0.05) kids more than those with genotype AB. These indicated that the allele A may have certain correlation with prolificacy in Jining Grey goats.     

Polymorphism of the Melatonin Receptor Genes and its Relationship with Seasonal Reproduction in the Gulin Ma Goat Breed

Melatonin is thought to be the main molecule that transmits the signal of seasonal change to the neuroendocrine system in seasonal breeding species. Melatonin exerts its effects through specific melatonin receptors, MTNR1A and MTNR1B. In the present study, six native goat breeds in China and one introduced goat breed were analysed to investigate the relationship between the genetic polymorphism of receptor genes and seasonal reproduction. Sequencing results showed that there were five polymorphic mutations in the MTNR1A gene and two in the MTNR1B gene. In the MTNR1A gene, genotypes AA, AB and BB for 424C>T and genotypes CC, CD and DD for 589C>A were observed in these goat breeds. In all six native goat breeds, only genotype AA was detected. In the MTNR1B gene, genotypes EE, EF and FF for 1179G>A and genotypes GG, GH and HH for 1529A>G were detected. However, in Gulin Ma goats, the genotypes EE and HH were not found. Moreover, the base of G at position 1179 and A at position 1529 were linked (By Arlequin ver 3.1, Zoological Institute, Berne, Switzerland, http://cmpg.unibe.ch/software/arlequin3 ,D′ = 0.7496, r² = 0.4421, χ² = 489.8679, p = 0.000). Among these mutations, no amino acid change was found in MTNR1A, while both of the mutations in MTNR1B gene caused amino acid changes of R222H and S339G, respectively. The structural analysis showed that the R222H mutation occurred in the first amino acid residue of the third cytoplasmic loop, and the S339G mutation was located in the carboxyl terminus of the protein. In terms of seasonal breeding, all the genotypes we detected showed a similar kidding frequency distribution trend with a higher frequency in May–August than in January–April and in September–December. This suggests that the relationship between the polymorphisms in the MTNR1A and MTNR1B genes and seasonal breeding could not be established.     

Allele distributions of two novel SNPs within the sheep Cyp19 gene

Oestrogen is an important regulator of reproduction and growth. The key enzyme of oestrogen biosynthesis, aromatase cytochrome P450, is encoded by the Cyp19 gene. In order to generate genetic markers for the sheep Cyp19 gene, two novel single nucleotide polymorphisms (SNPs), one located in promoter 2 (P2), the other one in intron 9 (I9), were identified by a comparative sequencing approach. The allele distributions of both SNPs were investigated by means of Polymerase chain reaction‐restriction fragment length polymorphism analysis (PCR‐RFLP) in five economically relevant sheep breeds (British Milk Sheep, Carranzana, Latxa Black Face, Latxa White Face, Merino) and three ancient Hungarian breeds kept as gene reserves (Cikta, Racka, Tsigai). In British Milk Sheep, only the intronic SNP was present whereas in Merino, Cikta, Racka, Tsigai, Carranzana, Latxa Black Face and Latxa White Face, both SNPs could be found. This indicates that the newly identified SNPs can be used as markers for the Cyp19 locus in various sheep breeds.     

Detection of selection signatures for agonistic behaviour in cattle

The identification of genomic regions including signatures of selection produced by domestication and its subsequent artificial selection processes allows the understanding of the evolution of bovine breeds. Although several studies describe the genomic variability among meat or milk production cattle breeds, there are limited studies orientated towards bovine behavioural features. This study is focused on mapping genomic signatures of selection which may provide insights of differentiation between neutral and selected polymorphisms. Their effects are studied in the Lidia cattle traditionally selected for agonistic behaviour compared with Spanish breeds showing tamed behaviour. Two different approaches, BayeScan and SelEstim, were applied using genotypic 50K SNP BeadChip data. Both procedures detected two genomic regions bearing genes previously related to behavioural traits. The frequencies of the selected allele in these two regions in Lidia breed were opposite to those found in the tamed breeds. In these genomic regions, several putative genes associated with enriched metabolic pathways related to the behavioural development were identified, as neurochondrin gene (NCDN) or glutamate ionotropic receptor kainate type subunit 3 (GRIK3) both located at BTA3 or leucine‐rich repeat and Ig domain containing 2 (LINGO2) and phospholipase A2‐activating protein (PLAA) at BTA8.     

细毛羊KRT26基因多态性及其与羊毛细度的关联性分析

本研究旨在揭示影响绵羊重要经济性状的功能基因的分子遗传特征及其与细毛羊群体的遗传关系,为高效选育绵羊品种经济性状及其种质资源的保护与利用提供分子遗传学依据。试验利用PCR-SSCP、DNA测序和生物信息学对289个细毛羊的KRT26基因进行遗传变异分析及其与细毛羊羊毛细度的关联性分析。结果表明,KRT26基因在该细毛羊群体中存在AA、AB、BB 3种基因型,其基因型频率分别为0.221、0.426和0.353,A、B等位基因频率分别为0.434、0.566,细毛羊群体的多态信息含量为0.371,呈中度多态水平,且处于Hardy-Weinberg非平衡状态(P0.05)。经过BioEdit软件比对序列和Chromas软件分析测序结果显示,KRT26基因发现5处碱基突变:83bp(G/C)、86bp(T/C)、112bp(C/T)、140bp(G/A)和247bp(C/T),并通过氨基酸序列的比对结果表明,2处发生了氨基酸的替代,即Val/Ile和Asn/Lys。KRT26基因在细毛羊群体中AA基因型个体极显著高于AB和BB基因型(P0.01)。因此,KRT26基因可能作为羊毛细度性状的一个新的分子标记。