Identification of a new Y chromosome haplogroup in Spanish native cattle.

The aim of this work was to perform a thorough analysis of the diversity of Y-haplotypes in Spanish cattle. A total of 207 Bos taurus males were sampled across 25 European breeds, with a special focus on rare, local Spanish populations. Animals were genotyped with five Y-specific microsatellites (INRA189, UMN0103, UMN0307, BM861 and BYM1), two indels (ZFY10 and USP9Y) and one SNP (UTY19). A new haplogroup, distinct from those described by Götherström et al. (2005), was identified and named Y1.2. Samples representing the three B. taurus Y-haplogroups were genotyped for four additional Y chromosome SNPs (rs121919254, rs121919281, rs121919323 and rs137049553). Among these SNPs, only rs121919281 was informative in B. taurus and helped to confirm the new Y1.2 haplogroup. Analysis of a larger dataset of standardized haplotypes for 1507 individuals from 57 populations from Spain, other European countries and Africa showed the new Y1.2 haplogroup to be found exclusively in Spanish breeds. This finding reinforces the importance of local Spanish cattle as reservoirs of genetic diversity as well as the importance of the Iberian Peninsula in the history of cattle.

Contribution of Lidia cattle breed historical castes to the paternal genetic stock of Spain.

The main objective of this work was to determine whether the five founding castes defined in the Lidia cattle breed actually have an important contribution to the Spanish paternal genetic stock as well as to the paternal genetic origin support. A total of 1300 Bos taurus male individuals were genotyped for five microsatellites (INRA189, UMN0103, UMN0307, BM861 and BYM1) and one indel (ZFY10). Microsatellite and indel alleles were combined into haplotypes, identifying a total of 38 haplotypes, 11 of them belonging to haplogroup Y1 and 27 to haplogroup Y2. Ten different haplotypes were found in the Lidia cattle breed, with five being exclusive to this breed. Our results agree with different male genetic stocks in the Lidia breed: one hypothetically representing the ancient Iberian bovine genetic stock (Gallardo, Navarra and Cabrera castes and some encastes from Vistahermosa) and a second one that is the result of the more recent breeding strategy of choosing the most aggressive individuals from traditional herds (including some Vistahermosa encastes and the Vazqueña caste). In terms of conservation, it would be better to not consider this breed as a unit but to consider the caste, or even better the encaste, as the target of putative conservation efforts.

Mitochondrial DNA and Y-chromosome diversity in East Adriatic sheep.

Variation in mitochondrial DNA (mtDNA) and Y-chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon (Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt-3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt-3 respectively. In contrast, Y-chromosome analyses revealed extraordinary paternal allelic richness: HY-6, 89.3%; HY-8, 5.0%; HY-18, 3.1%; HY-7, 1.3%; and HY-5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY-18 (A-oY1/135-SRYM18), identified here for the first time, provides a link between the haplotype HY-12 (A-oY1/139-SRYM18) found in a few rams in Turkey and haplotype HY-9 (A-oY1/131-SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt-55), and all were paternally monomorphic for haplotype HY-6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y-chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to 'upgrade' local populations using rams with different paternal origins.

Multiple paternal origins of domestic cattle revealed by Y-specific interspersed multilocus microsatellites.

In this study, we show how Y-specific interspersed multilocus microsatellites, which are loci that yield several amplified bands differing in size from the same male individual and PCR reaction, are a powerful source of information for tracing the history of cattle. Our results confirm the existence of three main groups of sires, which are separated by evolutionary time and clearly predate domestication. These three groups are consistent with the haplogroups previously identified by Götherström et al. (2005) using five Y-specific segregating sites: Y1 and Y2 in taurine (Bos taurus) cattle and Y3 in zebu (Bos indicus) cattle. The zebu cattle cluster clearly originates from a domestication process that was geographically and temporally separated from that of taurine clusters. Our analyses further suggest that: (i) introgression of wild sire genetic material into domesticated herds may have a significant role in the formation of modern cattle, including the formation of the Y1 haplogroup; (ii) a putative domestication event in Africa probably included local Y2-like wild sires; (iii) the West African zebu cattle Y-chromosome may have partially originated from an ancient introgression of humped cattle into Africa; and (iv) the high genetic similarity among Asian zebu sires is consistent with a single domestication process.

Y-specific microsatellites reveal an African subfamily in taurine (Bos taurus) cattle.

Five cattle Y-specific microsatellites, totalling six loci, were selected from a set of 44 markers and genotyped on 608 Bos taurus males belonging to 45 cattle populations from Europe and Africa. A total of 38 haplotypes were identified. Haplogroups (Y1 and Y2) previously defined using single nucleotide polymorphisms did not share haplotypes. Nine of the 27 Y2-haplotypes were only present in African cattle. Network and correspondence analyses showed that this African-specific subfamily clustered separately from the main Y2-subfamily and the Y1 haplotypes. Within-breed genetic variability was generally low, with most breeds (78%) showing haplotypes belonging to a single haplogroup. AMOVA analysis showed that partitioning of genetic variation among breeds can be mainly explained by their geographical and haplogroup assignment. Between-breed genetic variability summarized via Principal Component Analysis allowed the identification of three principal components explaining 94.2% of the available information. Projection of principal components on geographical maps illustrated that cattle populations located in mainland Europe, the three European Peninsulas and Mediterranean Africa presented similar genetic variation, whereas those breeds from Atlantic Europe and British Islands (mainly carrying Y1 haplotypes) and those from Sub-Saharan Africa (belonging to Y2-haplogroup) showed genetic variation of a different origin. Our study confirmed the existence of two large Y-chromosome lineages (Y1 and Y2) in taurine cattle. However, Y-specific microsatellites increased analytical resolution and allowed at least two different Y2-haplotypic subfamilies to be distinguished, one of them restricted to the African continent.

Female segregation patterns of the putative Y-chromosome-specific microsatellite markers INRA124 and INRA126 do not support their use for cattle population studies.

Here we tested the segregation and paternal compatibility of markers INRA124 and INRA126 on female DNA in 10 different cattle families, in order to clarify the usefulness of these microsatellites for the study of male-mediated population processes in cattle. Their performance was compared with that of four microsatellites located in the PAR-BTAY (UMN0108, UMN0803, UMN0929 and UMN0905) and another one male-specific microsatellite (INRA189). INRA124 and INRA126 amplified the same sized fragment in both sexes. Same size alleles were sequenced and the high homology found allowed us to rule out non-specific female amplification. INRA124 showed full parental compatibility, whilst the locus INRA126 showed 55% parental incompatibility. Based on these observations, it is recommended that markers INRA124 and INRA126 should not be used in studies to characterize male-mediated genetic events in cattle.

Analysis of mitochondrial DNA diversity in Burkina Faso populations confirms the maternal genetic homogeneity of the West African goat.

To date, no comprehensive study has been performed on mitochondrial genetic diversity of the West African goat. Here, we analysed a 481-bp fragment of the HVI region of 111 goats representing four native West African populations, namely the three main Burkina Faso breeds, zoo-farm kept Dwarf goats and endangered Spanish goat breeds used as the outgroup. Analyses gave 83 different haplotypes with 102 variable sites. Most haplotypes (65) were unique. Only three haplotypes were shared between populations. Haplotypes were assigned to cluster A except for H45 (belonging to the Spanish Bermeya goat) which was assigned to cluster C. amova analysis showed that divergence between groups (Phi(CT)) was not statistically significant regardless of whether the partition in two hierarchical levels that was fitted included Spanish samples or not. The West African goat scenario shown here is consistent with that previously reported for the species: haplogroup A is predominant and has a very high haplotype diversity regardless of the geographic area or sampled breed. The large phenotypic differences observable between the West African Dwarf and Sahelian long-legged goat populations are not detectable with mitochondrial markers. Moreover, a previously suggested introgression of Sahelian goat southwards because of desertification could not be assessed using mtDNA information.

Microsatellite analysis characterizes Burkina Faso as a genetic contact zone between Sahelian and Djallonke sheep.

A total of 123 sheep belonging to the Djallonke, Mossi, and Burkina-Sahel breeds, along with 41 Spanish Xalda sheep were genotyped for 27 microsatellites. The pair Djallonke-Mossi had the highest between breeds molecular coancestry. Admixture analysis informed on the parental role of the Burkina-Sahel and Djallonke breeds. The Mossi breed was a hybrid population nearer to the Djallonke breed. Only half of the Mossi individuals were correctly assigned to their breed. The Burkina-Sahel and Djallonke breeds can be considered ancestrally different genetic entities. Differentiation between the Djallonke and Mossi breeds may be due to introgression of Sahelian sheep.

Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep: evidence from the rare Xalda sheep breed.

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100_104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.

Retinoid receptor-specific agonists regulate bovine in vitro early embryonic development, differentiation and expression of genes related to cell cycle arrest and apoptosis.

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 microM, 1 microM and 10 microM), ATRA 0.7 microM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 microM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 microM, while a dose-dependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r=0.93; p<0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation.

Lambs are Susceptible to Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist.

Vaccination against Louping Ill Virus Protects Goats from Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus.

First Confirmation of Schmallenberg Virus in Cattle in Spain: Tissue Distribution and Pathology.

Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field.

Genetic relationships and admixture among sheep breeds from Northern Spain assessed using microsatellites.

Although many research papers have studied diversity and differentiation within livestock species, genetic relationships among neighboring populations remain poorly understood. Here we apply recent methodologies to analyze the polymorphism of 14 microsatellites in 238 unrelated individuals belonging to six sheep breeds from Northern Spain to ascertain their historical relationships and the relative genetic contributions existing between populations. Individual genotypes were analyzed to assess the existence of an underlying genetic structure. Long-term and recent migration rates were estimated to identify patterns of relative genetic contribution among breeds. The complete data set showed a strong population structure derived from both different ancestral origins and some geographical patterns of recent gene flow. Two of the analyzed breeds (Black-faced Latxa and Churra) had a marked genetic background, supporting the hypothesis that, regardless of their phenotypical similarities, they have different ancestral origins. Some of the more presumably related breeds had negative long-term admixture coefficients, showing that they diverged only recently. In addition, we show how methodologies for estimation of long-term gene flow and recent patterns of migration are complementary, providing information about migration rates on different timescales.

Artificial intelligence techniques point out differences in classification performance between light and standard bovine carcasses.

The validity of the official SEUROP bovine carcass classification to grade light carcasses by means of three well reputed Artificial Intelligence algorithms has been tested to assess possible differences in the behavior of the classifiers in affecting the repeatability of grading. We used two training sets consisting of 65 and 162 examples respectively of light and standard carcass classifications, including up to 28 different attributes describing carcass conformation. We found that the behavior of the classifiers is different when they are dealing with a light or a standard carcass. Classifiers follow SEUROP rules more rigorously when they grade standard carcasses using attributes characterizing carcass profiles and muscular development. However, when they grade light carcasses, they include attributes characterizing body size or skeletal development. A reconsideration of the SEUROP classification system for light carcasses may be recommended to clarify and standardize this specific beef market in the European Union. In addition, since conformation of light and standard carcasses can be considered different traits, this could affect sire evaluation programs to improve carcass conformation scores from data from markets presenting a great variety of ages and weights of slaughtered animals.

Usefulness of molecular-based methods for estimating effective population size in livestock assessed using data from the endangered black-coated Asturcón pony.

Empirical evidence of the usefulness of different molecular-based methods to estimate the effective population size (N(e)) for conservation purposes in endangered livestock populations is reported. The black-coated Asturcón pony pedigree (1,981 individuals) was available. Additionally, a total of 267 Asturcón individuals born in 1998, 2002, and 2008 were typed for 15 microsatellites. These yearly cohorts (cohort(1998, 2002, 2008)) included almost all individuals kept for reproduction at the end of the corresponding foaling season. The genealogical realized N(e) was estimated for each cohort by using the individual increase in inbreeding. Molecular N(e) was computed by using 1) linkage disequilibrium [N(e)(()(D)())], 2) a temporal method based on F-statistics [N(e)(()(T)())], 3) an unbiased temporal method [N(e)(()(JR)())], and 4) a Bayesian temporal method [N(e)(()(B)())]. Estimates of increased from cohort(1998) (18.8 ± 5.1) to cohort(2008) (24.9 ± 5.2), illustrating the history of the population and its breeding policy of avoiding matings between close relatives. The estimates of N(e)(()(D)()) were highly biased upward, with the maximum N(e)(()(D)()) value obtained for cohort(2002) (137.0). The estimates of N(e)(()(T)()), N(e)(()(JR)()), and N(e)(()(B)()) showed similar performance. However, N(e)(()(JR)()) estimates were very consistent across cohorts, varying from 14.9 to 15.5 after correcting for the effect of overlapping generations. When the drift signal was not strong (pair cohort(1998)-cohort(2002)), estimates of N(e)(()(T)()) and N(e)(()(B)()) were not realistic. Estimates of N(e)(()(B)()) tended to be biased downward (being 9.0 or below for the sampling pairs including cohort(2008)). Results of N(e)(()(D)()) are more likely to be estimates of the effective number of breeders producing the sample, rather than the effective size for a generation. The temporal methods were strongly affected by a weak drift signal, particularly when samplings were not spaced a sufficient number of generations or a sufficient time apart. The use of molecular-based estimates of N(e) is not straightforward, and their use in livestock conservation programs should be carried out with caution. Sampling strategies (including sampling sizes, sampling periods, and the age structure of the sampled individuals) must be carefully planned to ensure that robust estimates of N(e) are obtained.

Quantifying diversity losses due to selection for scrapie resistance in three endangered Spanish sheep breeds using microsatellite information.

The effect of selection for scrapie resistance on genetic variability in three endangered Spanish sheep breeds (Colmenareña, Mallorquina and Rubia de El Molar) was studied using two different criteria for quantifying contributions to genetic variability: (a) molecular coancestry or genetic identity; and (b) average number of alleles per locus or allelic richness. A total of 236 (81 Colmenareña, 76 Mallorquina and 79 Rubia de El Molar) individuals were genotyped for the PrP gene and for 22 microsatellite markers. The analyses assumed a selective policy aimed at the elimination of the VRQ allele and the reduction of the frequency of the ARQ/ARQ genotype. These goals are approached by rejecting for breeding those individuals with the highest susceptibility for scrapie (risk groups R4 and R5) in a genetic scenario with no previous selection programmes considering the PrP gene polymorphism carried out. When all the individuals classified into risk groups R4 and R5 were removed from the dataset, the total molecular coancestry slightly increased in the Colmenareña breed illustrating that the carriers of undesirable PrP genotypes are not essential to maintain its overall gene diversity. When the allelic richness was considered, the removal of the R4 and R5 individuals gave high losses in the Rubia de El Molar breed. The analyses carried out considering the sex of the individuals informed that most increases in genetic identity in the Colmenareña breed resulted from the removal of the R4 and R5 males while in the Mallorquina breed resulted from the removal of the undesirable females. Losses of diversity in the Rubia de El Molar breed were basically independent of the sex of the individuals due to the balanced contributions to diversity of both sexes. As a general recommendation, not all the individuals of undesirable risk groups should be rejected for reproduction at the same time to avoid irretrievable loses of genetic diversity but according to the sex of the individuals.

Sry-negative XX true hermaphroditism in a roe deer.

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.