Detection of prion gene promoter and intron1 indel polymorphisms in Anatolian water buffalo (Bubalus bubalis)

Bovine spongiform encephalopathy (BSE) is a fatal disease caused by miss folded prion protein. Studies in the cattle, comparing genetic data from BSE diseased and healthy animals have shown that indel polymorphisms in the promoter and intron 1 of PRNP gene were associated with disease susceptibility. Several studies were conducted to find out allele and genotypic frequencies of indel polymorphisms in promoter and intron 1 of the cattle PRNP gene. Unlike domestic cattle and bison, no indel polymorphisms of the PRNP promoter and intron 1 were examined in any population of the water buffalo (Bubalus bubalis). Aim of this study was to analyse frequencies of allele, genotype, and haplotype of the indel polymorphisms (23 bp indel in promoter and 12 bp indel in intron 1) in prion protein coding gene (PRNP) of water buffalo. Therefore a PCR based procedure, previously used in cattle to detect indel polymorphisms of PRNP promoter and intron 1 locus, was applied to 106 Anatolian water buffalo DNAs. Our results have revealed high frequency of in variants and in23/in12 haplotype for PRNP promoter and intron 1 indel polymorphisms in water buffalo. The results of the study have demonstrated that frequencies of allele, genotype, and haplotype of the indel polymorphisms in PRNP gene of the Anatolian water buffalo are significantly different those from cattle and bison PRNP indel polymorphisms.     

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.     

Genetic mapping of the prion protien gene (PRPN) on bovine chromosome 13

The bovine prion protein gene (PRNP) potentially plays a key role in the development of bovine spongiforme encephalopathy (BSE). In species other than cattle, expression of BSE is clearly dependent on polymorphisms in this gene. PRNP has previously been assigned to the bovine chromosome 13 (BTA13). The present study is an attempt to embed PRNP into a grid of published marker maps. A genetic mapping panel consisting of 266 animals has been genotyped with 19 microsatellites and a polymerase chain reaction‐amplified polymorphism within the PRNP coding region. The linear locus order and the relative distances of these loci are presented. Our linkage map spans 111.6c m of BTA13. The results suggest PRNP to be located telomeric of the microsatellite BMS1580 and centromeric of BM9248 with a log‐likelihood of 2. Our findings further characterize the vicinity of PRNP on BTA13.     

Genetic variability of the prion protein gene (PRNP) in wild ruminants from Italy and Scotland

The genetics of the prion protein gene (PRNP) play a crucial role in determining the relative susceptibility to transmissible spongiform encephalopathies (TSEs) in several mammalian species. To determine the PRNP gene variability in European red deer (Cervus elaphus), roe deer (Capreolus capreolus) and chamois (Rupicapra rupicapra), the PRNP open reading frame from 715 samples was analysed to reveal a total of ten single nucleotide polymorphisms (SNPs). In red deer, SNPs were found in codons 15, 21, 59, 78, 79, 98, 136, 168 and 226. These polymorphisms give rise to 12 haplotypes, and one of which is identical to the PRNP of American wapiti (Rocky Mountain elk, Cervus elaphus nelsoni). One silent mutation at codon 119 was detected in chamois and no SNPs were found in roe deer. This analysis confirmed that European wild ruminants have a PRNP genetic background that is compatible with TSE susceptibility, including chronic wasting disease.     

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.     

Genetic variability following selection for scrapie resistance in six autochthonous sheep breeds in the province of Bolzano (northern Italy)

Scrapie is an ovine transmissible spongiform encephalopathy, and its susceptibility is associated with polymorphisms in the prion protein gene (PRNP). Genetic selection is currently the most effective mean for eradication of the susceptible VRQ allele in favour of resistant ARR allele. Maintenance of genetic diversity should be one of the major objectives in breeding programmes, especially in endangered breeds, and genetic information are an excellent alternative to pedigree data where these information are missing. The aim of our study was to determine changes of genetic variability in six native sheep breeds from autonomous province of Bolzano, northern Italy, following simulation of scrapie selection scenarios. A total of 684 rams were investigated for PRNP polymorphisms and for 10 microsatellite loci to estimate genetic variability. Across all loci, a total of 163 alleles were detected with a mean of 10.4 alleles per locus. Average observed (Ho) and unbiased expected (uHe) heterozygosity overall loci were 0.74 and 0.78, respectively, showing a statistically significant deviation from Hardy–Weinberg equilibrium (HWE) in all breeds. This heterozygosity deficit was confirmed by a positive fixation index (Fis), determining a moderate inbreeding in each breed. Simulating a soft selection, where only rams having at least a VRQ allele should be excluded from reproduction, Ho, uHe and Fis values remained almost unchanged, indicating that genetic variability should not be affected by the removal of these individuals. With a mild selection scenario, considering only rams with at least one ARR allele, we observed a decrease in the mean alleles per breed (8.9) and the maintenance of heterozygosity deficiency, except for two breeds, where it was any longer significant. These results showed that selection strategies allowing use of heterozygous as well homozygous ARR rams might be the right compromise to improve resistance to scrapie and to do not dramatically affect genetic variability of these breeds.     

Genetic diversity,population structure,and correlations between locally adapted zebu and taurine breeds in Brazil using SNP markers

Genetic diversity is one of the most important issues in studies on conservation of cattle breeds and endangered species. The objective of this study was to estimate the levels of genetic differentiation between locally adapted taurine (Bos taurus taurus) and zebu (Bos taurus indicus) breeds in Brazil, which were genotyped for more than 777,000 SNPs. The fixation index (F _ST), principal component analysis (PCA), and Bayesian clustering were estimated. The F _ST highlighted genetic differentiation between taurine and zebu breeds. The taurine lines, Caracu and Caracu Caldeano, had significant genetic differentiation (F _ST close to 5%) despite their recent selection for different uses (meat and milk). This genetic variability can be used for conservation of locally adapted animals, as well as for breeding programs on zebu breeds. Introgression of zebu in locally adapted breeds was identified, especially in Curraleiro Pé-Duro breed. The Gyr breed, however, had low breed purity at genomic level due to its very heterogeneous mixing pattern.     

Frequency of bovine Nramp1 (Slc11a1) alleles in Holstein and Zebu breeds

Natural resistance against brucellosis in cattle is linked to the Nramp1 gene, which encodes a divalent cation transporter that localizes in the phagolysosome membrane in macrophages. Nramp1 gene in mouse plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphisms at the bovine Nramp1 3' untranslated region (3'UTR), detectable by Single Strand Conformational Analysis (SSCA), are associated with natural resistance against brucellosis. Such polymorphisms are associated with variation in the number of GT repeats. This study compared the frequency of Nramp1 3'UTR polymorphisms between Zebu and European bovine breeds. Eighty-one Holsteins (Bos taurus taurus) and 167 Zebu (Bos taurus indicus), including the following breeds: Nelore (n=95), Guzerá (n=37), and Gir (n=35), totaling 248 pure breed cattle studied. DNA extraction was performed using the guanidium protocol and genotyping was performed by SSCA. DNA from cattle considered genotypically resistant to brucellosis resulted in a single band (homozygous) with 175bp, corresponding to the 3'UTR with 13 GT pairs (GT13), whereas DNA from genotypically susceptible cattle generated one single band with 177bp (homozygous GT14) or double bands with both 175 and 177bp, or 175 and 179bp (heterozygous GT13/GT14 or GT13/GT15, respectively). A marked difference in the frequency of alleles was detected between the Zebu and Holstein cattle. Holsteins had an extremely homogeneous genotype, with 100% of the individuals with a GT13 genotype. In sharp contrast the Nelore breed had the most heterogeneous genotype with four allelic combinations, namely, homozygous GT13, homozygous GT14, heterozygous GT13/GT14, and heterozygous GT13/GT15. When the Zebu breeds were compared to each other, the only significant difference observed was the frequencies of the genotypes GT13 and GT14 between the Nelore and Guzerá breeds. The knowledge of allelic frequencies in different breeds of cattle may prove to be very useful in the future for planning breeding strategies for selection of resistant cattle.     

BSE‐associated polymorphisms in the prion protein gene: an investigation

The aim of this study was to determine the frequency of the 12‐bp and 23‐bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12‐bp indel frequency between the BSE animals and control group. For the 23‐bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The ? ? / ? ? genotype frequency in the BSE animals was not significantly higher when compared with the control animals. One ? allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12‐bp indel and by a factor of 2.10 for the 23‐bp indel. When both indels are considered, one ? allele increased the risk of BSE by a factor of 1.54.     

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.     

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).     

Development of discrimination markers between Japanese domestic and imported beef

In the meat industry, correct labeling of beef origins or breed is required to assure quality and safety. This paper describes the development of discrimination markers between Japanese domestic and imported beef from the United States (US) and Australia (AUS) based on a bovine 50K single nucleotide polymorphism (SNP) array using a total of 110 samples: Japanese Black (n = 50), Japanese Holstein (n = 50) and US cattle (n = 10). Genotyping information revealed 1081 SNPs as candidate markers that were polymorphic only in US cattle. The genotyping results by PCR – restriction length polymorphism in Japanese Black (n = 300) and Holstein cattle (n = 146) revealed that 11 SNPs had alleles specific to US cattle. Their allelic frequencies in US cattle (n = 108) ranged from 0.097 to 0.250 with an average of 0.178 and the combined identification probability of US cattle was 0.987. In addition, we also verified the applicability of these US‐specific markers to AUS cattle. Their allelic frequencies in AUS cattle (n = 280) ranged from 0.063 to 0.224 with an average of 0.137 and the combined identification probability of AUS cattle was 0.963. In conclusion, a set of these markers could be useful for discriminating between Japanese domestic and imported beef and would contribute to identify origins and prevent falsified labeling of beef.     

Characterization of the prion protein gene (PRNP) region in Swiss sheep breeds

Polymorphisms in the PRNP gene have both an influence on the progress of scrapie and also of bovine spongiform encephalopathy (BSE) in sheep. In the light that sheep are potentially susceptible to BSE the goal of this project was to characterize 50 animals of each of the four major Swiss sheep breeds, namely Swiss Oxford Down, Swiss Black‐Brown Mountain, Valais Blacknose and Swiss White Alpine with respect to polymorphisms in the PRNP region. A 628‐bp fragment of exon 3 of PRNP comprising codons 112–211 was amplified by PCR and analysed by direct sequencing. Heterozygous genotypes were further verified by restriction fragment length polymorphism analyses. Polymorphisms could be observed at codons 112, 136, 137, 141, 143, 154 and 171 but not at codons 138, 151 and 211. One of the scrapie cases in Switzerland, a sheep of Swiss White Alpine, was also genotyped. Based on the polymorphisms observed at codons 136, 154 and 171 all four sheep breeds should be considered potentially susceptible to scrapie and BSE.     

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.     

MC1R c.310G>‐ and c.871G > A determine the coat color of Kumamoto sub‐breed of Japanese Brown cattle

Coat color is one of the important factors characterizing breeds for domestic animals. Melanocortin 1 receptor (MC1R) is a representative responsible gene for this phenotype. Two single‐nucleotide polymorphisms (SNPs) in bovine MC1R gene, c.296T > C and c.310G>‐, have been well characterized, but these SNPs are not enough to explain cattle coat color. As far as we know, MC1R genotypes of Kumamoto sub‐breed of Japanese Brown cattle have not been analyzed. In the current study, genotyping for c.296T > C and c.310G>‐ was performed to elucidate the role of MC1R in determining the coat color of this sub‐breed. As a result, most animals were e/e genotype, suggesting the coat color of this sub‐breed is derived from the e allele of MC1R gene. However, we found six animals with E/e genotype, which coat color would be black theoretically. Subsequently, sequence comparison was performed with these animals to identify other polymorphisms affecting coat color, elucidating that these animals possessed the A allele of c.871G > A commonly. c.871G > A was a non‐synonymous mutation in the seventh transmembrane domain, suggesting alteration of the function and/or the structure of MC1R protein. Our data indicated that the A allele of c.871G > A might be a loss‐of‐function mutation.     

Allele frequencies of gene polymorphisms related to economic traits in Bos taurus and Bos indicus cattle breeds

Allele frequencies of 10 representative polymorphisms for beef and milk traits were investigated for a total of 240 animals from Bos taurus and Bos indicus breeds, including two Japanese groups (Japanese Black and Japanese Brown), two East Asian groups (Korean and Mongolian), three European groups (Holstein, Angus and Hereford) and a Bos indicus group in South Asia (Myanmar, Laos and Cambodia). The Japanese Black revealed unique genetic construction in GH, FASN and SREBP‐1 and the other Asian populations show intermediate frequencies between European and Japanese populations. The Bos indicus group showed low favorable allele frequencies in most of the genes. The study showed the variability and distribution of 10 genes affecting economic traits among world representative cattle breeds. The genetic information would contribute to elucidating the genetic background for worldwide cattle breeds and the possibility of improvement using the markers.     

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.     

Nucleotide and octapeptide-repeat variations of the prion protein coding gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Anatolian,Murrah, and crossbred water buffaloes

Resistance to bovine spongiform encephalopathy (BSE) that is significantly associated with insertion/deletion (indel) polymorphisms at two loci (putative promoter and intron 1) on the prion protein gene (PRNP) in cattle has been well documented. Studies suggest that the insertion alleles are related to BSE resistance. Until recently, BSE has never been reported in water buffaloes (unlike cattle). Previous studies have demonstrated that the PRNP gene in water buffalo consists mostly of insertion alleles at both loci; nevertheless, whether or not water buffaloes are genetically resistant to BSE and the role of indel polymorphisms in their resistance status is not clear. We examined the coding region of PRNP to determine the nucleotide and octapeptide-repeat (octarepeats) variations of Anatolian, Murrah and Murrah × Anatolian (M?×?A) water buffaloes. Three synonymous single nucleotide polymorphisms (SNP) at positions 126, 234, and 285, and a non-synonymous SNP at position 322 (G108S) were detected. Triplet G/A/T base substitutions were observed at position 126 and two additional genotypes, T/A and T/G, at this position were determined. We also found six octarepeats that indicated the presence of the wild-type PRNP6 allele in the coding region. To the best of our knowledge, this is the first report of the T/A and T/G genotypes in water buffaloes.     

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.