Sequence variation of bovine prion protein gene in Japanese cattle (Holstein and Japanese Black)

To assess relationships between nucleotide polymorphisms of the prion protein (PRNP) gene and susceptibility to bovine spongiform encephalopathy (BSE), we investigated polymorphisms in the open reading frame (ORF) and 2 upper regions of the PRNP gene from 2 Japanese cattle breeds: 863 healthy Holstein cattle, 6 BSE-affected Holstein cattle, and 186 healthy Japanese Black (JB) cattle. In the ORF, we found single-nucleotide polymorphisms (SNPs) at nucleotide positions 234 and 576 and found 5 or 6 copies of the octapeptide repeat, but we did not find any amino acid substitutions. In the upper region, we examined 2 sites of insertion/deletion (indel) polymorphisms: a 23-bp indel in the upper region of exon 1, and a 12-bp indel in the putative promoter region of intron 1. A previous report suggests that the 23-bp indel polymorphism is associated with susceptibility to BSE, but we did not find a difference in allele frequency between healthy and BSE-affected Holstein cattle. There were differences in allele frequency between healthy Holstein and JB cattle at the 23- and 12-bp indels and at the SNPs at nucleotide positions 234 and 576, but there was no difference in allele frequency of the octapeptide repeat. We identified a unique PRNP gene lacking a 288-bp segment (96 amino acids) in DNA samples stocked in our laboratory, but this deletion was not found in any of the 1049 cattle examined in the present study. The present results provide data about variations and distribution of the bovine PRNP gene.     

Frequencies of bovine PrP gene polymorphisms in Holstein and Japanese Black bulls in Japan

We screened for Japanese Black and Holstein bull sire samples to detect single nucleotide polymorphisms (SNPs) involving animo‐acid substitutions in the bovine prion gene in the entire coding region of the PRNP gene. Although three silent SNPs were found, we could not detect any SNP with animo‐acid substitution. We also examined the polymorphism of the octapeptide repeat number in these samples. There was no homozygous bull with repeat number 5. The frequency of heterozygous (6/5) bulls was 8% in the Japanese Black bull and 4% in the Holstein bull, respectively. The bull samples used in this study contain popular elite sires, so it appears that the polymorphisms of prion protein (PrP) are rather difficult to find in these two breeds in Japan, except for polymorphism of the octapeptide repeat number.     

Frequencies of PRNP gene polymorphisms in Vietnamese dairy cattle for potential association with BSE

Summary Since 2004, significant associations between bovine spongiform encephalopathy (BSE) susceptibility in cattle and frequencies of insertion/deletion (ins/del; indel) polymorphisms within the bovine prion protein gene (PRNP) have been reported. In this study, we investigated the frequencies of indel polymorphisms within two variable sites, a 23-bp indel polymorphism in the promoter region (23indel) and a 12-bp indel polymorphism in intron 1 region (12indel), in the PRNP in 206 Vietnamese dairy cattle and seven Japanese BSE-affected cattle. In Vietnamese dairy cattle, the frequency distributions of del allele and del/del genotypic polymorphisms in the 23indel site, which are thought to be associated with BSE susceptibility, were significantly higher, whereas the frequencies of del allelic and del/del genotypic polymorphisms in the 12indel site, which have been reported to confer BSE susceptibility, were significantly lower. We have provided evidence that Vietnamese dairy cattle have a unique genetic background in the PRNP gene in comparison with cattle or sires previously reported in other countries.     

Gene and haplotype polymorphisms of the Prion gene (PRNP) in Japanese Brown, Japanese native and Holstein cattle

Polymorphisms in the prion protein gene ( PRNP ) are known to be associated with transmissible spongiform encephalopathies in human, sheep and goats. There is tentative association between PRNP promoter polymorphism and bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, we genotyped for six bovine PRNP polymorphic sites including a 23-bp indel in the promoter, a 12-bp indel in the intron 1, two nonsynonymous single nucleotide polymorphisms (SNPs), octapeptide repeats in the coding region and a 14-bp indel in the 3'-untranslated region in 178 animals representing Japanese Brown, Kuchinoshima feral, Mishima, Japanese Shorthorn and Holstein. In 64 Japanese Brown cattle, three indel sites were polymorphic. All of the six sites were monomorphic in Kuchinoshima. The 23-bp and 12-bp indel sites were polymorphic in Mishima cattle. The 23-bp and 14-bp indel sites were polymorphic in Japanese Shorthorn cattle. Both SNP sites were monomorphic in all cattle examined in this study. At the 23-bp indel site, the genotype frequencies of Japanese Brown and Holstein breeds were similar to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A '23-12-K6S14+' haplotype was the major haplotype in all populations, whose frequencies ranged from 0.50 to 1.00.     

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.     

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.     

Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene

The aim of the present study was to identify and characterize polymorphisms within the 5′ flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental × Nelore (n = 30), Canchim × Nelore (n = 30) and Angus × Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648 , AY662649 and AY662650 , respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837 ), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652 , respectively). The allele sequences were compared with GenBank sequences ( M57764 , AF007750 and AH009106 ) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.     

中国美利奴羊OLA-DQB1基因exon2遗传多态性与布鲁氏菌病易感性的关联分析

本试验采用PCR-SSCP方法对148只布鲁氏菌阴性和60只布鲁氏菌阳性中国美利奴羊白细胞表面抗原DQB1(OLA-DQB1)基因exon 2单核苷酸多态性(SNPs)进行了检测,之后挑选不同等位基因进行PCR产物测序,旨在确定该基因的多态性位点,并对每个SNP位点的等位基因频率、基因型频率进行统计分析,从而分析其多态性与布鲁氏菌病易感性的相关性.测序结果表明,在270 bp的序列内共检测到43个SNPs,其中G196A位点的等位基因频率在病例组和对照组中的分布存在极显著差异(P< 0.01),其基因型频率存在显著差异(P< 0.05);C211T位点的等位基因频率在病例组和对照组中存在显著差异(P< 0.05).由此表明,OLA-DQB1基因exon 2多态性与中国美利奴羊布鲁氏菌病易感性呈显著相关.     

The Correlation Analysis between Chinese Merino Sheep OLA-DQB1 Gene Exon 2 Genetic Polymorphism and Brucellosis Susceptibility

The single nucleotide polymorphisms (SNPs) of ovine lymphocyte antigen DQB1 (OLA-DQB1) gene exon 2 was amplified by PCR-SSCP method from 148 healthy and 60 infected with Brucella Chinese Merino sheep and then PCR products of different alleles were sequenced to determine the polymorphism loci of the gene.The differences in gene frequency and genotype frequency of each SNP loci were analyzed statistically to analyze its correlation with brucellosis susceptibility.The sequencing result showed that 43 SNPs were detected in 270 bp DNA sequence,the gene frequencies of G196A allele had extremely significant difference in case and control samples (P< 0.01),and its genotype frequencies presented significant difference (P< 0.05).Similarly,C211T allele was significantly different in case and control samples (P< 0.05).The results showed that the polymorphism of OLA-DQB1 gene exon 2 might be a significant association gene with brucellosis susceptibility.     

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.     

布莱凯特黑牛心脏脂肪酸结合蛋白基因单核苷酸多态性的研究

利用PCR-SSCP方法测定96头布莱凯特黑牛H-FABP基因外显子2和内含子2的部分序列多态性,并对含有多态序列的片段进行了序列分析。结果表明,引物1、3的扩增序列不存在多态性,引物2、4的扩增序列存在多态性;引物2在内含子第323位点处存在G→A突变,在群体中产生了AA、AB、BB 3种基因型,基因型频率分别为0.448、0.146、0.406,多态信息含量(PIC)为0.375,属于中度多态(0.25χ²检验该位点基因型频率不处于Hardy-Weinberg平衡状态;引物4在内含子2第872位点处存在C→G的突变,产生了HH、Hh、hh3种基因型,基因型频率分别为0.104、0.417、0.479,多态信息含量(PIC)为0.337,为中度多态(0.25χ²检验该位点基因型频率达到Hardy-Weinberg平衡状态。     

Genotypes and allele frequencies of buried SNPs in a bovine single‐nucleotide polymorphism array in Japanese Black cattle

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding; thus, data derived from SNP arrays have accumulated on a large scale nationwide. Commercial SNP arrays contain a considerable number of unassigned SNPs on the chromosome/position on the genome; these SNPs are excluded in subsequent analyses. Notably, the position‐unassigned SNPs, or “buried SNPs” include some of the markers associated with genetic disease. In this study, we identified the position of buried SNPs using the Basic Local Alignment Search Tool against the surrounding sequences and characterized the relationship between SNPs and genetic diseases in Online Mendelian Inheritance in Animals based on the genomic position. We determined the position of 285 buried SNPs on the genome and surveyed the genotype and allele frequencies of these SNPs in 5,955 individual Japanese Black cattle. Eleven SNPs associated with genetic disease, which contained five buried SNPs, were found in the population with the risk allele frequency ranging from 0.00008396 to 0.46. These results indicate that buried SNPs in the bovine SNP array can be utilized to identify associations with genetic disorders from large scale accumulated SNP genotype data in Japanese Black cattle.     

Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor genes with blood serum insulin-like growth factor I concentration and growth traits in Angus cattle

This study was conducted to identify polymorphisms in the promoter and coding regions of the bovine growth hormone and growth hormone receptor genes and to study association of polymorphisms identified in these genes with growth traits and serum insulin-like growth factor-I (IGF-I) concentration. The denaturing gradient gel electrophoresis method and sequencing were utilized to identify three new single nucleotide polymorphisms in the promoter region of the growth hormone gene in Angus cattle. Polymerase chain reaction-based restriction fragment length polymorphism procedures were developed for rapid determination of the single nucleotide polymorphism genotypes in the growth hormone and the growth hormone receptor genes among Angus calves from lines divergently selected for high or low blood serum IGF-I concentration. The IGF-I concentration and growth traits were analyzed using animal models. The single nucleotide polymorphism in the promoter region of the growth hormone receptor gene was associated with serum IGF-I concentration on d 42 of the postweaning test and with mean IGF-I concentration. The associated effects of the markers need to be verified in other populations.     

Polymorphisms in the canine glutamate transporter-1 gene: identification and variation among five dog breeds

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.     

Novel SNPs in IL-17F and IL-17A genes associated with somatic cell count in Chinese Holstein and Inner-Mongolia Sanhe cattle

Background: Bovine mastitis is the most common and costly disease of lactating cattle worldwide. Apart from milk somatic cell count(SCC) and somatic cell score(SCS), serum cytokines such as interleukin-17(IL-17) and interleukin-4(IL-4) may also be potential indicators for bovine mastitis. The present study was designed to investigate the effects of single nucleotide polymorphisms(SNPs) in bovine IL-17 F and IL-17 A genes on SCC, SCS and serum cytokines in Chinese Holstein and Inner-Mongolia Sanhe cattle, and to compare the m RNA expression variations of the cows with different genotypes.Results: A total of 464 lactating cows(337 Holstein and 127 Inner-Mongolia Sanhe cattle) were screened for SNPs identification and the data were analyzed using fixed effects of herd, parity, season and year of calving by general linear model procedure. The results revealed that SNP g.24392436 C T in IL-17 F and SNP g.24345410 A G in IL-17 A showed significant effects on SCC and IL-4 in Holstein(n = 337) and on IL-17 and IL-4 in Sanhe cattle(n = 127). The homozygous GG genotype of SNP g.24345410 A G had significantly higher m RNA expression compared with the heterozygous AG genotype.Conclusions: The results indicate that IL-17 F and IL-17 A could be powerful candidate genes of mastitis resistance and the significant SNPs might be useful genetic markers against mastitis in both dairy and dual purpose cattle.     

Serotonin receptor 1B (HTR1B) genotype associated with milk production traits in cattle

Serotonin receptor 1B (HTR1B) is one of the 14 different identified serotonin receptors which are involved in the regulation of behaviors such as sleep, fear, aggression, mood, and feeding. The aims of this study were to characterize polymorphisms in the 5' coding and the 3' flanking regions of the bovine HTR1B gene among Chinese Holsteins and to identify the association of HTR1B polymorphisms with milk production performance. SSCP was used to examine the polymorphisms at four loci and the fragments with different SSCP patterns were sequenced. A total of three single nucleotide polymorphisms (SNPs) were detected, among which the 205G>T mutation was found to cause a predicted amino acid change: from Ala to Ser at position 69. The polymorphism of G205T was identified to be associated with milk yield trait. Furthermore, the H1-C genotype was found to be associated with a significant increase in milk yield of 489kg vs. the H1-A genotype (P<0.05).     

DGAT1, GH, GHR, PRL and PRLR polymorphism in water buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1‐exon8 K232A, GH‐intron3 MspI, GH‐exon5 AluI, GHR‐exon8 F279Y, PRL‐exon3 RsaI and PRLR‐exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili‐ravi, Murrah‐Nili‐Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR–Single‐strand conformation polymorphism and sequencing. Twenty‐eight inter‐specific single‐nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra‐specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1‐exon8, MspI⁺ restriction site(c nucleotide) and AluI⁺ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI^? restriction site at PRL‐exon3/exon4 and N allele genotype of S18N mutation at PRLR‐exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra‐specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL‐exon3/exon4 and 43rd bp (c/t) in PRLR‐exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.     

Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.     

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).     

西南地方品种黄牛MyoD基因部分编码序列PCR-SSCP分析

采用PCR-SSCP技术分析了MyoD基因在3个西南地方黄牛品种的遗传多态性。结果表明:MyoD的第2外显子内不存在遗传多态性;第1外显子在3个牛品种中检测到了AA、AB和BB基因型,而且A等位基因为3个牛群体的优势等位基因,分布较高。3个品种中,盘江牛AA基因型频率最高,达到0.5365,而巴山牛和昭通牛则相对较低,分别为0.4465和0.2564。对第1外显子的多态片段测序分析表明:位于MyoD基因第782bp处发生碱基G→A的突变,并导致了氨基酸的突变,使甘氨酸变为丝氨酸。