PrP genotype frequencies of Quebec sheep breeds determined by real-time PCR and molecular beacons

The allele and genotype frequencies of the prion protein gene (PrP), known to have an impact on scrapie susceptibility, were determined by real-time PCR for 500 Quebec purebred rams. Molecular beacons were very efficient in discriminating the 5 alleles investigated. Polymorphisms at coding positions 136, 154, and 171 of the PrP gene were analyzed using 3 separate real-time PCR reactions and a total of 7 molecular beacons. A total of 4 different alleles (ARQ, ARR, AHR, and VRQ) were observed at different frequencies among the 7 breeds of sheep investigated. Results show that more than 50% of the rams in every breed carried at least one ARR allele, which is considered the most resistant to scrapie. The susceptibility ARQ allele was also present in every breed and together with the ARR allele, they were the most frequent alleles found in Quebec rams. The VRQ allele associated with the highest susceptibility to scrapie occurred in 5 of the 7 breeds, although at low frequencies. Overall, the results indicate that the frequencies of PrP alleles and genotypes in common breeds of sheep in Quebec make it feasible to reduce scrapie risk by selective breeding.     

Selection for scrapie resistance and simultaneous restriction of inbreeding in the rare sheep breed "Mergellander"

Scrapie is a fatal infectious neurodegenerative disease for which susceptibility is associated with polymorphisms in the ovine prion protein (PrP) gene. Scrapie-eradication programmes are based on eliminating the susceptible VRQ allele and/or breeding for the resistant ARR allele. In rare breeds or breeds with a low frequency of the ARR allele this can lead to unacceptably high inbreeding rates with associated increased risk of genetic defects and inbreeding depression. The conservation status of populations with inbreeding rates (DeltaF) above 1% is considered critical. In the Dutch rare sheep breed the Mergellander animals carrying ARR alleles are closely related to one another, and could reach 1.53% when only ARR/ARR animals are used as parents. Inbreeding rates can be reduced by selecting the set of parents according to their average co-ancestry. We minimised inbreeding rates by calculating the optimal contribution of each ram and selection of ewes. This resulted in inbreeding rates of -0.17% with exclusive use of homozygous ARR rams, and -0.38% if use of heterozygous rams was allowed as well. Thus sophisticated breeding programs can prevent unacceptably high inbreeding rates when breeding for scrapie resistance.     

Trends in genotype frequency resulting from breeding for resistance to classical scrapie in Belgium (2006~2011)

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.     

Genetic diversity loss due to selection for scrapie resistance in the rare Spanish Xalda sheep breed

The effect of selection for scrapie resistance on genetic variability in the rare Xalda sheep breed was studied. Pedigree information comprised 1851 animals (1444 alive) at the moment of sampling. A total of 304 reproductive (or selected for reproduction) Xalda individuals were sampled and genotyped for 14 microsatellites. Genetic variability was assessed using: gene diversity (1 − average kinship, GD), mean average relatedness (AR) and self-coancestry (c_i) at the genealogical level; and expected heterozygosity (H_e), molecular mean kinship (Mk), molecular self-coancestry (s_i) and rarefacted average number of alleles per locus (A) at the molecular level. Two breeding strategies were evaluated: a) use of only young rams with genotype ARR/ARR and young ewes with low to moderate risk (risk groups R1 to R3); b) breeding without selection for PrP genotypes. The major cause of losses of genetic variability in the Xalda breed is the drift that occurs when a new group of reproductive individuals is selected. The loss of genetic variability is small in females compared to the males, where it is considerable. However, losses at the molecular level for young females with respect to adult females were above 5%. Young male individuals also retained most of the genetic variability assessed in adult rams. Selection against susceptibility to scrapie produced additional losses of more than 2% for H_e and A when rYF individuals are considered. As regards males, the situation becomes critical because of the scant number of available ARR/ARR young rams. The consequences for the management of the Xalda breed are discussed.     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

Distribution of prion protein genotypes in breeds of sheep in New Zealand

AIM: To use an established high through-put genotyping procedure to gain an estimate of the frequency of alleles of the prion protein (PrP) gene in some common sheep breeds in New Zealand. METHODS: Using a genotyping procedure based on matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF), DNA samples from 3,024 sheep from New Zealand, including breeds such as Romney, Texel, Coopworth, Merino and mixed breed, were isolated, genotyped and the results analysed. RESULTS: The 15 scrapie genotypes commonly reported, and derived from the five commonly reported allelic variants (ARR, ARQ, AHQ, ARH and VRQ), were all observed in the samples analysed. The estimates were indicative of the frequencies in the population of alleles present in breeds of sheep in New Zealand. There was a significant difference between the frequencies of alleles between breeds, but the ARQ, followed by the ARR allele, were, except in Carwell sheep, the most common alleles present. CONCLUSION: This study gave an indication of the percentages of PrP gene alleles in sheep in New Zealand, including data previously unreported from breeds in this country. It is of interest because of the relatively large size of the sheep population in New Zealand compared with many countries, and it provides some useful information on the genetic susceptibility or resistance of the sheep population in New Zealand to scrapie. The frequencies of the alleles can be different for an individual breed compared between countries.     

Frequencies of PrP genotypes in 38 breeds of sheep sampled in the National Scrapie Plan for Great Britain

Between October 2001 and January 2003 the prion protein (PrP) genotypes of over 250,000 sheep were determined through the operation of the National Scrapie Plan (NSP); the results for 38 breeds were analysed to provide an estimate of the underlying PrP genotype distribution of the British sheep population. Although there was marked variability among the genotype profiles of the different breeds, several trends emerged. A comparison of the allele frequencies demonstrated that the breeds could be grouped into three categories: breeds dominated by ARR and ARQ in which the frequency of ARR exceeded the frequency of ARQ; breeds dominated by ARR and ARQ in which the frequency of ARQ exceeded the frequency of ARR; and breeds with significant levels of either AHQ, ARH or VRQ. Hill breeds were more likely to have a lower proportion of animals at low risk of scrapie (NSP type 1) and a higher proportion of animals at an intermediate risk of scrapie (NSP type 3) than other breeds. Most breeds had a small proportion of animals at high risk of scrapie (NSP type 5). The frequency of ARR/VRQ (NSP type 4) was variable.     

Prion protein genetic polymorphisms and breeding strategies in Portuguese breeds of sheep

Polymorphisms at the prion protein locus were studied in 965 animals, representative of 13 native and 3 exotic sheep breeds in Portugal. In general, ARQ was the predominant allele in most breeds, but the desirable ARR allele was present in all breeds studied. A different pattern was found in Ile de France, where ARR was predominant and VRQ had the highest frequency among the breeds analysed. In most of the breeds, especially in those of the coarse-wool type, the major hurdle in selecting for genetic resistance to clinical scrapie was the elimination of ARQ rather than VRQ alleles. Breeding schemes aiming at the creation of ARR-homozygous selection nuclei were simulated for representative breeds, and results indicate that, with intense selection, it would take between 4 years in Ile de France and 11 years in Mondegueira to obtain homozygosity in the nucleus, and the length of time needed for fixation was not affected by the type of mating (random or assortative) used. Nevertheless, intense selection for the PrP genotype alone would have undesirable consequences in terms of inbreeding, and correlated responses in production and adaptation traits should be evaluated before such a scheme is adopted.     

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.     

Distribution of prion protein genotypes in breeds of sheep in New Zealand

AIM: To use an established high through-put genotyping procedure to gain an estimate of the frequency of alleles of the prion protein (PrP) gene in some common sheep breeds in New Zealand.

METHODS: Using a genotyping procedure based on matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF), DNA samples from 3,024 sheep from New Zealand, including breeds such as Romney, Texel, Coopworth, Merino and mixedbreed, were isolated, genotyped and the results analysed.

RESULTS: The 15 scrapie genotypes commonly reported, and derived from the five commonly reported allelic variants (ARR, ARQ, AHQ, ARH and VRQ), were all observed in the samples analysed. The estimates were indicative of the frequencies in the population of alleles present in breeds of sheep in New Zealand. There was a significant difference between the frequencies of alleles between breeds, but the ARQ, followed by the ARR allele, were, except in Carwell sheep, the most common alleles present.

CONCLUSION: This study gave an indication of the percentages of PrP gene alleles in sheep in New Zealand, including data previously unreported from breeds in this country. It is of interest because of the relatively large size of the sheep population in New Zealand compared with many countries, and it provides some useful information on the genetic susceptibility or resistance of the sheep population in New Zealand to scrapie. The frequencies of the alleles can be different for an individual breed compared between countries.     

Effect of selection for scrapie resistance on genetic diversity in a rare and locally adapted sheep breed: The case of Sambucana

In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. The impact of this selection on genetic diversity was assessed on Sambucana, an autochtonous sheep breed reared in southern Piedmont, by analysing the evolution of allele frequencies at different levels: PRNP (prion protein) gene, microsatellite loci on OAR13 (where PRNR maps), and microsatellite loci on other chromosomes, not subjected to selection for scrapie resistance. A total of 147 young rams, 80 born in 2004 and 67 in 2008–2009 were analysed. Evidence of diversity loss was observed for PRNP gene as a consequence of the directional selection. Diversity was affected in the immediate vicinity of PRNP but the effect on more distant loci on the same chromosome was trivial. With regard to neutral markers, lack of heterozygosis with no changeover of allele frequencies was observed suggesting an increase of inbreeding. Mating policies would be sufficient to solve these problems. A selection scheme based on genotyping rams and eliminating carriers of both susceptible and high susceptible alleles is the best way to improve natural resistance to scrapie with low costs and minimal problems in the current conservation programmes targeting rare breeds.     

10个云南地方猪种微卫星遗传多样性分析

为阐明保山猪、迪庆藏猪、高贡黎山猪、丽江猪、明光小耳猪、滇南小耳猪、撒坝猪、大河猪、昭通猪、大河乌猪10个云南省优良地方猪种的群体遗传多样性和遗传结构,本试验参考ArkDB数据库中家猪14条染色体上的15对微卫星引物,对549个云南地方猪个体的耳组织样品进行了检测分析,计算各微卫星座位等位基因数（Na）、有效等位基因数（Ne）、Shannon's信息指数（I）、表观杂合度（Ho）、期望杂合度（He）、F-统计量（Fis、Fit、Fst）、基因流（Nm）、群体遗传距离等相关参数及多态信息含量（PIC）,采用NTsys 2.10软件构建聚类树,并采用STRUCTRUE 2.3.3软件评估群体遗传结构。结果显示：15个微卫星座位共检测到293个等位基因,各座位等位基因数在5~38个之间,平均等位基因数为19个,平均有效等位基因数8.3682个;10个云南地方猪群体的Shannon多样性、表观杂合度、期望杂合度和多态信息含量分别在1.4374~2.0317、0.6389~0.7756、0.7021~0.8281和0.6647~0.7993之间。各座位单个群体内近交系数（Fis）、总群体近交系数（Fit）、群体间遗传分化系数（Fst）的变化范围分别为-0.0678~0.4594、0.0618~0.5567和0.0713~0.1801;群体间Fst平均值为0.1101,处于中度遗传分化状态,有11.01%的遗传变异来自于群体间,大部分遗传变异来自于群体内;每个座位基因流程度较高,变化范围在1.6392~3.2551之间,平均值为2.0214。基于Nei氏遗传距离构建UPGMA系统发生树显示,高黎贡山猪与迪庆藏猪聚为一支,并与丽江猪、保山猪和明光小耳猪聚为一大支,其结果得到了STRUCTURE分析的验证。综上所述,10个云南地方猪种遗传多样性丰富,群体内有近交现象,群体间处于中度遗传分化,存在着一定的基因交流。     

利用微卫星标记分析江苏三个地方鸡品种的遗传多样性

利用30个微卫星DNA标记对3个江苏地方鸡品种进行遗传多样性分析，通过计算等位基因频率、多态信息含量、平均杂合度、有效等位基因数和遗传距离，评估各品种内遗传变异和各品种间遗传相关。结果表明：江苏的3个品种在30个微卫星上共发现196个等位基因，其中96个等位基因为三个品种共有，47个等位基因为各品种所特有；3个地方鸡品种的平均杂合度：狼山鸡为0．655，鹿苑鸡为0．681，溧阳鸡为0．687；狼山鸡与溧阳鸡的遗传距离较小，而两鸡与鹿苑鸡的遗传距离较远。     

3个品种兔微卫星标记的遗传多样性研究

为分析福建黄兔、新西兰兔、日本大耳白兔群体内的遗传变异情况和群体间的遗传相关性,本试验选取3个品种兔各25只,分别耳静脉采血并采用试剂盒抽提全血基因组,运用15个微卫星标记,结合荧光PCR技术和毛细管电泳方法获得目的片段,通过计算等位基因数、有效等位基因数、杂合度、多态信息含量和遗传距离,分析3个品种兔的遗传多样性。结果显示,3个品种兔在15个座位共检测到110个等位基因,平均等位基因数为7.3个,其中福建黄兔、新西兰兔和日本大耳白兔分别检测到95、95和88个等位基因,且分别在11、12和11个微卫星座位表现为高度多态,表明3个品种兔在筛选的15个微卫星座位均呈现较丰富的遗传多态信息。Hardy-Weinberg遗传平衡检验结果表明,福建黄兔、新西兰兔和日本大耳白兔分别有8、8和6个座位处于Hardy-Weinberg不平衡状态。Nei氏遗传距离分析显示,福建黄兔与新西兰兔、福建黄兔与日本大耳白兔、新西兰兔与日本大耳白兔的Nei氏遗传距离分别为0.1761、0.2347和0.0432。遗传距离越小时,相似度越高,亲缘关系越近,因此,新西兰兔与日本大耳白兔间的亲缘关系最近,福建黄兔与日本大耳白兔间的亲缘关系最远。     

Evaluating different PrP genotype selection strategies for expected severity of scrapie outbreaks and genetic progress in performance in commercial sheep

Stochastic computer simulations were used for quantifying the effect of selecting on prion protein (PrP) genotype on the risk of major outbreaks of classical scrapie and the rate of genetic progress in performance in commercial sheep populations already undergoing selection on performance. The risk of a major outbreak on a flock was measured by the basic reproduction ratio (R₀). The effectiveness of different PrP selection strategies for reducing the population risk was assessed by the percentage of flocks with R₀ < 1. When compared with the scenario where there was no selection on PrP genotype, selection against the VRQ allele had a minimal impact on genetic progress for performance traits. However, this strategy was not sufficient to eliminate the population risk after 15 years of selection when the initial frequency of the ARR allele was relatively low. More extreme PrP selection strategies aimed at increasing the frequency of the ARR allele and decreasing the frequency of the VRQ allele led to decreases in the rate of genetic progress for performance but reduced the population risk to very low values. The reduction in genetic progress was only large when the initial ARR frequency was low and, in general, the risk of major epidemics was very small when the frequency of this allele reached 0.7.     

PrP genotypes of rare breeds of sheep in Great Britain

In total 31,669 blood samples were collected from 1187 flocks of 27 rare breeds of sheep in the UK, and their genotype profiles at the prion protein locus were determined. The frequencies of the five alleles varied widely among the breeds and some had only two of the alleles and others had all five; the average was three. The average allele frequencies across all 27 breeds were 49.7 per cent for ARR, 4.4 per cent for AHQ, 2.7 per cent for ARH, 37.4 per cent for ARQ and 5.8 per cent for VRQ. The highest frequencies for each allele were 90.7 per cent for ARR in the Leicester Longwool, 24.7 per cent for AHQ in the Hebridean, 68.7 per cent for ARH in the Manx Loghtan, 98.7 per cent for ARQ in the North Ronaldsay and 28.4 per cent for VRQ in the Boreray. All 27 breeds had the ARR allele, 21 had AHQ, 11 had ARH, 26 had ARQ and 20 had VRQ.     

Merino sheep breed's genetic resistance to scrapie: genetic structure and comparison of five eradication strategies

Three thousand one hundred and ninety-three Merino-breeding males were genotyped for Scrapie-resistance gene. They showed a high frequency of ARQ allele while VRQ allele frequency, linked to the highest susceptibility, was close to 0. Alleles linked to Scrapie-susceptibility frequencies have been studied in different Merino flocks. Most of the variability is intraflock, not interflock. In addition, there is an excess of heterozygotes due to crossbreeding or Wahlund effect.

Five control and prevention strategies were studied: first genotyping males and females and eliminating VRQ carriers and ARQ/ARQ rams; second genotyping males and females and eliminating VRQ carriers; third genotyping males and eliminating VRQ carriers and ARQ/ARQ rams; fourth genotyping males and using only as breeders ARR/ARR, ARR/AHQ, AHQ/AHQ and ARQ/ARQ rams; fifth genotyping males and using only as breeders ARR/ARR, ARR/AHQ and AHQ/AHQ rams. They were simulated from allelic and genotypic frequencies to decide which animals had to be genotyped and which animals had to be eliminated due to their genotypes and risk levels. The third option, to genotype rams and to eliminate ARQ/ARQ and VRQ carriers, would be the best strategy to improve the resistance and would cause minimal cost and loss of animals.     

The impact of the genotype on the prevalence of classical scrapie at population level

ABSTRACT: Total number and genotypes of animals in holdings selected for the genotype & cull option in the Compulsory Scrapie Flock Scheme (CSFS) in Great Britain were extracted from the National Scrapie Plan data warehouse. The association between various genotype-related measures and scrapie prevalence infection was tested using zero-inflated negative binomial models with the counts of positive cases as dependent variable, and country, number of flocks in the scheme, flock size, surveillance source and the following genotype-related measurements: the centered-log ratios (clr) oof the 15 genotypes, of the proportions of the 5 alleles at codons 136, 154 and 171, of the proportions of the 5 NSP types, and two flock-susceptibility risk indicators, as explanatory variables. A total of 319341 genotyped animals from 168 holdings were included in the analysis. An increased proportion of the ARR/ARR genotype corresponded to a decrease in the number of scrapie cases. ARR/AHQ, AHQ/VRQ, ARH/VRQ and ARQ/VRQ genotypes, NSP type V, ARH, ARQ, AHQ and VRQ alleles and the low and high-susceptibility risk indicators are all associated with an increase risk in the number of scrapie cases.Regardless the management practices; the increased susceptibility that the non-ARR alleles confer on an individual could be extrapolated at the population level. Increasing prevalence of ARR allele reduces the overall risk of scrapie at population level. At genotype level, the VRQ/VRQ genotype, present a very low frequency in the study population, seems to play a residual effect in the overall risk of scrapie in a flock.     

8个微卫星座位在海北金银滩藏羊群体中的多态性分析

研究旨在分析金银滩藏羊的微卫星DNA多态性,以期了解该品种的遗传多样性,为该品种的保种选育和品质的进一步提高提供有益资料。利用8对微卫星引物,以金银滩藏羊为研究对象,通过计算基因频率、多态性信息含量、有效等位基因数和杂合度,评估其品种内的遗传变异。结果表明：在8个微卫星座位中,共检测到83个等位基因,每个座位平均10.38个等位基因;平均杂合度0.893;平均有效等位基因数9.06;平均多态性信息含量0.871。结果提示,金银滩藏羊群体存在丰富的遗传多样性,所选微卫星标记可用于绵羊遗传多样性评估。     

PrP genotype progression in flocks participating in the National Scrapie Plan for Great Britain

The National Scrapie Plan (NSP) for Great Britain is a voluntary scheme that, through PrP genotype testing and restricted breeding, aims to reduce the risk of scrapie in the national sheep flock. To gauge the progress in member flocks and within breeds, the genotype profile of successive crops of ram lambs was monitored between 2002 and 2004. In each of the 11 most frequently sampled breeds, the proportion of ram lambs testing in the most resistant genotype category (NSP type 1: ARR/ARR) increased, and there was a reduction in the proportion of genotypes associated with the highest disease risk, that is X/VRQ, where X is an allele other than ARR. Changes in the proportion of ram lambs testing for ARR-heterozygous genotypes (NSP type 2: ARR/X, where X is not VRQ) appeared to be influenced by whether the sheep were hill breeds or non-hill breeds. In each of six frequently sampled hill breeds these genotypes expanded, in relative terms, whereas they declined in four of five prominent lowland/crossing breeds. The proportion of ram lambs that carried neither ARR nor VRQ (NSP type 3) declined consistently in the top 11 breeds, but there was little change in the ARR/VRQ genotype (NSP type 4). Among individual flocks that had ram lambs tested in all three years 2002 to 2004, the majority experienced an increase in the proportion testing ARR/ARR, and of those that had VRQ ram lambs in 2002, most recorded a decrease in their frequency by 2004.