Genetic relationships among Spanish sheep using microsatellites

Five indigenous Spanish breeds of sheep, Churra, Latxa, Manchega, Rasa–Aragonesa and Merino, with Awassi sheep as a reference breed were genotyped for 19 DNA microsatellites. Allele frequencies and mean heterozygosities revealed the greatest genetic variation in Merino sheep and the lowest in Awassis. Differences in variability were not great in the other breeds studied. The dendrograms obtained based on genetic distances showed a large differentiation between Awassi sheep and the Spanish breeds, as was to be expected from their distinct genetic origin. Merinos appeared separated from the other four breeds, of which, according to a classification based on the fleece characteristics, Churra and Latxa belong to the churro type and Manchega and Rasa–Aragonesa to the so called entrefino type, though no clear separation was evident between the two types. These results suggest that morphological data alone are insufficient for determining relationships between breeds and that studies involving genetic markers may be of great assistance.     

Genetic diversity in the Churra tensina and Churra lebrijana endangered Spanish sheep breeds and relationship with other Churra group breeds and Spanish mouflon

The aim of the present study was to estimate the genetic intra-breed variability of Churra tensina and Churra lebrijana endangered breeds and to establish genetic relationships with Churra, Latxa and Merino breeds, as well as Spanish mouflon, by using 28 microsatellite markers, to provide useful information for their conservation. Allele frequencies and heterozygosity revealed high genetic variation in the two endangered breeds despite their small population size. Estimates of inbreeding coefficient (F_IS) were significant for all breeds studied, except for Churra lebrijana breed. The highest inbreeding coefficient (F_IS = 0.143) was found in the Spanish mouflon. Genetic differentiation tests (F_ST = 0.121) and assignment of individuals to populations indicated the existence of defined breed populations, and low genetic flow between these breeds. The highest pairwise Reynolds distance (D_R) values were observed between Mouflon and the domestic sheep breeds. Considering only domestic sheep breeds, the Churra lebrijana breed showed the highest pairwise D_R values_. The lowest values were found between Latxa and the other domestic sheep, except for Churra lebrijana. Results of pairwise D_R values, as well as phylogenetic tree and bottleneck analysis showed an important genetic isolation of the Churra lebrijana breed from the other Churra types, and genetic signatures of a demographic bottleneck. Finally, structure analysis of populations detected a population subdivision in the Latxa sheep breed. In conclusion, this study presents valuable insight into the existing genetic variability of two Spanish endangered breeds, as well as the first study in Spanish mouflon based on microsatellite analysis. The high degree of variability demonstrated in Churra tensina and Churra lebrijana implies that these populations are rich reservoirs of genetic diversity.     

The role of bovine causal genes underlying dairy traits in Spanish Churra sheep

In dairy cattle, quantitative trait nucleotides (QTNs) underlying quantitative trait loci (QTL) for milk production traits have been identified in bovine DGAT1, GHR and ABCG2 genes. The SPP1 gene has also been proposed to be a regulator of lactation. In sheep, QTL underlying milk production traits have been reported only recently, and no proven QTN has been identified. Taking into account the close phylogenetic relationship between sheep and cattle, this study examined the possible effects of the aforementioned genes on sheep milk production traits. We first studied the genetic variability of the DGAT1, GHR, ABCG2 and SPP1 genes in 15 rams of the Spanish Churra dairy sheep breed. Second, we performed an association analysis between SNPs identified in these genes and three milk production traits recorded in a commercial population of Churra sheep. This analysis revealed only three significant associations at the nominal level (P-value <0.05) involving allelic variants of the ABCG2 gene, whereas no significant association was found for the DGAT1, GHR and SPP1 genes. When the Bonferroni correction was applied to take into account the multiple tests performed, none of the associations identified at the nominal level remained significant. Nevertheless, taking into account the high level of false-negative findings that can arise when applying the stringent Bonferroni correction, we think that our results provide a valuable primary assessment of strong candidate genes for milk traits in sheep.     

Genetic variations in blood proteins within and between Spanish dairy sheep breeds

Eleven flocks of three Spanish sheep breeds have been studied: four Churra, four Lacha and three Manchega. Genetic variations between flocks of the same and different breeds have been analysed, using eight genetic blood systems and a wide range of statistical methods. The results show that the Churra, Lacha and Manchega breeds present significant differences between each other. Significant intraracial variations have been observed in Churras and Lachas, although they were almost non-existent in Manchegas. In some cases the genetic distances between flocks of the same breed were greater than the distances between flocks of different breeds. The data indicate that the closest breeds genetically are Lacha and Manchega.     

Relationship between cell age, glutathione and cation concentrations in sheep erythrocytes with a normal and a defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-related decreases in GSH and K+ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of gamma-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was paralleled by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.     

Erythrocyte oxidized glutathione in Australian Merino sheep

The concentration of GSSG was determined in the erythrocytes of Merino sheep. These sheep were grouped according to erythrocyte potassium type, haemoglobin type, and GSH type. It was found that haemoglobin and potassium type were not correlated with GSSG concentration; however, GSSG concentration was found to be significantly correlated with GSH concentration. This relationship may explain previously reported differences in ATPase activity and may reflect further metabolic differences in the erythrocytes of GSH-high and GSH-low type Merino sheep.     

Erythrocyte oxidized glutathione in Australian Merino sheep.

The concentration of GSSG was determined in the erythrocytes of Merino sheep. These sheep were grouped according to erythrocyte potassium type, haemoglobin type, and GSH type. It was found that haemoglobin and potassium type were not correlated with GSSG concentration; however, GSSG concentration was found to be significantly correlated with GSH concentration. This relationship may explain previously reported differences in ATPase activity and may reflect further metabolic differences in the erythrocytes of GSH-high and GSH-low type Merino sheep.     

Replication and refinement of a quantitative trait locus influencing milk protein percentage on ovine chromosome 3

A previous genome scan that was conducted in Spanish Churra sheep identified a significant quantitative trait locus (QTL) for milk protein percentage (PP) on chromosome 3 (OAR3), between markers KD103 and OARVH34. The aim of this study was to replicate these results and to refine the mapped position of this QTL. To accomplish this goal, we analysed 14 new half‐sib families of Spanish Churra sheep including 1661 ewes from 29 different flocks. These animals were genotyped for 21 microsatellite markers mapping to OAR3. In addition to a classical linkage analysis (LA), a combined linkage disequilibrium and linkage analysis (LDLA) was performed with the aim of enhancing the resolution of the QTL mapping. The LA that was performed in this sheep population identified the presence of a highly significant QTL for PP near marker KD103 (P_c < 0.001; P_exp < 0.001). The phenotypic variance that was owing to the QTL was 2.74%. Two segregating families for the target QTL were identified in this population with QTL effect estimates of 0.47 and 0.95 SD. The LDLA identified the same QTL as the previous analyses with a high level of statistical significance (P = 9.184 E‐11) and narrowed the confidence interval (CI) to a 13 cM region. These results confirm the segregation of the previously identified OAR3 QTL that influences PP in Spanish Churra sheep. Future research will aim to increase the marker density across the refined CI and to analyse the corresponding candidate genes to identify the allelic variant or variants that underlie this genetic effect.     

Genetic parameters for resistance to trichostrongylid infection in dairy sheep

In sheep, the traditional chemical control of gastrointestinal nematode (GIN) parasites with anthelmintics has led to the widespread development of anthelmintic resistance. The selection of sheep with enhanced resistance to GIN parasites has been suggested as an alternative strategy to develop sustainable control of parasite infections. Most of the estimations of the genetic parameters for sheep resistance to GIN parasites have been obtained from young animals belonging to meat- and/or wool-specialised breeds. We present here the estimated genetic parameters for four parasite resistance traits studied in a commercial population of adult Spanish Churra dairy ewes. These involved two faecal egg counts (FECs) (LFEC0 and LFEC1) and two serum indicator traits, the anti-Teladorsagia circumcincta fourth stage larvae IgA (IgA) and the pepsinogen (Peps) levels. In addition, this study has allowed us to identify the environmental factors influencing parasite resistance in naturally infected Spanish Churra sheep and to quantify the genetic component of this complex phenotype. The heritabilities estimated for the two FECs analysed (0.12 for LFEC0 and 0.09 for LFEC1) were lower than those obtained for the examined serum indicators (0.19 for IgA and 0.21 for Peps). The genetic correlations between the traits ranged from 0.43 (Peps-IgA) to 0.82 (LFEC0-LFEC1) and were higher than their phenotypic counterparts, which ranged between 0.07 and 0.10. The heritabilities estimated for the studied traits were lower than previously reported in lambs. This may be due to the differences in the immune mechanisms controlling the infection in young (antibody reactions) and adult (hypersensitivity reactions) animals/sheep. In summary, this study demonstrates the presence of heritable variation in parasite resistance indicator traits in the Churra population studied, which suggests that genetic improvement is feasible for this complex trait in this population. However, further studies in which the experimental variables are controlled as much as possible are needed to identify the best trait that could be measured routinely in adult sheep as an indicator of parasite resistance.     

Genetic relationships between Spanish Assaf (Assaf.E) and Spanish native dairy sheep breeds

At present, the Assaf is the main dairy sheep in Spain. The Spanish Assaf (Assaf.E) was formed by male-mediated absorption of native Spanish sheep. Here we assess the genetic relationships among the Assaf.E and major native Spanish dairy breeds using microsatellites to contribute to the knowledge of the formation and within-population genetic variability of the breed. Blood samples from 44 unrelated Assaf.E individuals from 23 different Assaf.E flocks spread throughout 6 different Spanish provinces were obtained and genotyped using 14 microsatellites. Up to 312 additional samples belonging to the Awassi and Milchschaf sheep breeds and to six native Spanish dairy sheep breeds (Castellana, Churra, Latxa, Manchega, and Rubia de El Molar) as well as samples from Merino individuals to be used as the outgroup were also analysed observed (H_o) and expected (H_e) heterozygosity, rarefacted number of alleles per locus and distances based on molecular coancestry information were computed. Probabilities of assignment of the Assaf.E individuals to native Spanish dairy sheep breeds and cryptic genetic structure in the whole dataset were also assessed. It can be concluded that the Assaf.E breed has low genetic variability and high genetic distance with respect native Spanish dairy sheep breeds. From our results, the formation of the Assaf.E breed basically occurred via the absorption of individuals belonging to the Entrefino type, particularly to the Castellana and Manchega populations. Furthermore, Churra individuals may have participated in the formation of the Assaf.E breed at an early moment of the introduction of the breed into Spain.     

The importance of selenium in the prenatal and postnatal development of calves and lambs.

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH Px, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadequate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

Genetic relationships between Spanish Assaf (Assaf.E) and Spanish native dairy sheep breeds

At present, the Assaf is the main dairy sheep in Spain. The Spanish Assaf (Assaf.E) was formed by male-mediated absorption of native Spanish sheep. Here we assess the genetic relationships among the Assaf.E and major native Spanish dairy breeds using microsatellites to contribute to the knowledge of the formation and within-population genetic variability of the breed. Blood samples from 44 unrelated Assaf.E individuals from 23 different Assaf.E flocks spread throughout 6 different Spanish provinces were obtained and genotyped using 14 microsatellites. Up to 312 additional samples belonging to the Awassi and Milchschaf sheep breeds and to six native Spanish dairy sheep breeds (Castellana, Churra, Latxa, Manchega, and Rubia de El Molar) as well as samples from Merino individuals to be used as the outgroup were also analysed observed (H_o) and expected (H_e) heterozygosity, rarefacted number of alleles per locus and distances based on molecular coancestry information were computed. Probabilities of assignment of the Assaf.E individuals to native Spanish dairy sheep breeds and cryptic genetic structure in the whole dataset were also assessed. It can be concluded that the Assaf.E breed has low genetic variability and high genetic distance with respect native Spanish dairy sheep breeds. From our results, the formation of the Assaf.E breed basically occurred via the absorption of individuals belonging to the Entrefino type, particularly to the Castellana and Manchega populations. Furthermore, Churra individuals may have participated in the formation of the Assaf.E breed at an early moment of the introduction of the breed into Spain.     

The importance of selenium in the prenatal and postnatal development of calves and lambs

Selenium deficiency is responsible for Zenker type muscle degeneration in calves, lambs, and foals in the prenatal and postnatal stages of development. Investigations have shown that the selenium GSH P_x, and vitamin E content of the maternal and fetal parts of the placenta in cattle are different. Similarly, low concentrations of selenium are present in milk from cows and sheep. In addition to an inadquate supply of selenium and vitamin E as a contributory cause of fetal nutritive muscular dystrophy (FNMD), it is assumed that a placental transport block and/or impaired selenium metabolism in the placenta are also responsible. Postnatal nutritive muscular dystrophy, however, is attributed to either acute selenium and vitamin E deficiency in basic feed or impaired plant absorption of selenium as a result of antagonistic elements, such as sulphur.     

Estimations of linkage disequilibrium,effective population size and ROH‐based inbreeding coefficients in Spanish Churra sheep using imputed high‐density SNP genotypes

In this study, the availability of the Ovine HD SNP BeadChip (HD‐chip) and the development of an imputation strategy provided an opportunity to further investigate the extent of linkage disequilibrium (LD) at short distances in the genome of the Spanish Churra dairy sheep breed. A population of 1686 animals, including 16 rams and their half‐sib daughters, previously genotyped for the 50K‐chip, was imputed to the HD‐chip density based on a reference population of 335 individuals. After assessing the imputation accuracy for beagle v4.0 (0.922) and fimpute v2.2 (0.921) using a cross‐validation approach, the imputed HD‐chip genotypes obtained with beagle were used to update the estimates of LD and effective population size for the studied population. The imputed genotypes were also used to assess the degree of homozygosity by calculating runs of homozygosity and to obtain genomic‐based inbreeding coefficients. The updated LD estimations provided evidence that the extent of LD in Churra sheep is even shorter than that reported based on the 50K‐chip and is one of the shortest extents compared with other sheep breeds. Through different comparisons we have also assessed the impact of imputation on LD and effective population size estimates. The inbreeding coefficient, considering the total length of the run of homozygosity, showed an average estimate (0.0404) lower than the critical level. Overall, the improved accuracy of the updated LD estimates suggests that the HD‐chip, combined with an imputation strategy, offers a powerful tool that will increase the opportunities to identify genuine marker‐phenotype associations and to successfully implement genomic selection in Churra sheep.     

Copper deficiency and tissue glutathione concentration in the rat

Copper deficiency in rats increased renal vein and arterial (heart) plasma GSH concentration by approximately 50%. There was no change in plasma GSSG concentration. Renal vein plasma GSSG/GSH ratio was decreased in copper deficiency, which is consistent with previous reports showing a copper-dependent thiol oxidase activity in the renal basement membrane. No change occurred in arterial plasma GSSG/GSH ratio. Hepatic GSH concentrations were also elevated by 50% in copper deficiency, GSSG concentrations were unaffected, but GSSG/GSH ratio was depressed. Renal and cardiac tissue GSH and GSSG were unaffected by copper deficiency. The decreased SOD activity and GSH-Px activity observed in copper deficiency may contribute to increased hepatic and plasma GSH concentrations.     

Identification of a 31-bp Deletion in the RELN Gene Causing Lissencephaly with Cerebellar Hypoplasia in Sheep

Lissencephaly is an inherited developmental disorder in which neuronal migration is impaired. A type of lissencephaly associated with cerebellar hypoplasia (LCH) was diagnosed in a commercial flock of Spanish Churra sheep. The genotyping of 7 affected animals and 33 controls with the OvineSNP50 BeadChip enabled the localization of the causative mutation for ovine LCH to a 4.8-Mb interval on sheep chromosome 4 using genome-wide association and homozygosity mapping. The RELN gene, which is located within this interval, was considered a strong positional and functional candidate because it plays critical roles in neuronal migration and layer formation. By performing a sequencing analysis of this gene’s specific mRNA in a control lamb, we obtained the complete CDS of the ovine RELN gene. The cDNA sequence from an LCH-affected lamb revealed a deletion of 31 bp (c.5410_5440del) in predicted exon 36 of RELN, resulting in a premature termination codon. A functional analysis of this mutation revealed decreased levels of RELN mRNA and a lack of reelin protein in the brain cortex and blood of affected lambs. This mutation showed a complete concordance with the Mendelian recessive pattern of inheritance observed for the disease. The identification of the causal mutation of LCH in Churra sheep will facilitate the implementation of gene-assisted selection to detect heterozygous mutants, which will help breeders avoid at-risk matings in their flocks. Moreover, the identification of this naturally occurring RELN mutation provides an opportunity to use Churra sheep as a genetically characterized large animal model for the study of reelin functions in the developing and mature brain.     

Close linkage between the C blood group locus and the locus controlling amino acid transport in sheep erythrocytes

Data from 838 Finnish Landrace or Finnish Landrace crossbred sheep showed a highly significant correlation between phenotypes of the C blood group system and erythrocyte amino acid transport variants. Erythrocytes with normal amino acid transport properties (GSH high, Ly- type) were Cb-positive or Cb-negative. Erythrocytes with the amino acid transport lesion (GSH low, Ly +) were never Cb-negative. Sheep erythrocytes homozygous for C^bshowed stronger lysis reactions with anti-Cb than heterozygous cells. Ly + sheep were nearly always homozygous for C^b, whereas most Ly- sheep were heterozygous or Cb-negative. Inheritance studies provided strong evidence that this association is due to close genetic linkage.     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

Quantitative trait loci underlying milk production traits in sheep

Improvement of milk production traits in dairy sheep is required to increase the competitiveness of the industry and to maintain the production of high quality cheese in regions of Mediterranean countries with less favourable conditions. Additional improvement over classical selection could be reached if genes with significant effects on the relevant traits were specifically targeted by selection. However, so far, few studies have been undertaken to detect quantitative trait loci (QTL) in dairy sheep. In this study, we present a complete genome scan performed in a commercial population of Spanish Churra sheep to identify chromosomal regions associated with phenotypic variation observed in milk production traits. Eleven half-sib families, including a total of 1213 ewes, were analysed following a daughter design. Genome-wise multi-marker regression analysis revealed a genome-wise significant QTL for milk protein percentage on chromosome 3. Eight other regions, localized on chromosomes 1, 2, 20, 23 and 25, showed suggestive significant linkage associations with some of the analysed traits. To our knowledge, this study represents the first complete genome scan for milk production traits reported in dairy sheep. The experiment described here shows that analysis of commercial dairy sheep populations has the potential to increase our understanding of the genetic determinants of complex production-related traits.     

GWA Analysis for Milk Production Traits in Dairy Sheep and Genetic Support for a QTN Influencing Milk Protein Percentage in the LALBA Gene

In this study, we used the Illumina OvineSNP50 BeadChip to conduct a genome-wide association (GWA) analysis for milk production traits in dairy sheep by analyzing a commercial population of Spanish Churra sheep. The studied population consisted of a total of 1,681 Churra ewes belonging to 16 half-sib families with available records for milk yield (MY), milk protein and fat yields (PY and FY) and milk protein and fat contents (PP and FP). The most significant association identified reached experiment-wise significance for PP and FP and was located on chromosome 3 (OAR3). These results confirm the population-level segregation of a previously reported QTL affecting PP and suggest that this QTL has a significant pleiotropic effect on FP. Further associations were detected at the chromosome-wise significance level on 14 other chromosomal regions. The marker on OAR3 showing the highest significant association was located at the third intron of the alpha-lactalbumin (LALBA) gene, which is a functional and positional candidate underlying this association. Sequencing this gene in the 16 Churra rams of the studied resource population identified additional polymorphisms. One out of the 31 polymorphisms identified was located within the coding gene sequence (LALBA_g.242T>C) and was predicted to cause an amino acid change in the protein (Val27Ala). Different approaches, including GWA analysis, a combined linkage and linkage disequilibrium study and a concordance test with the QTL segregating status of the sires, were utilized to assess the role of this mutation as a putative QTN for the genetic effects detected on OAR3. Our results strongly support the polymorphism LALBA_g.242T>C as the most likely causal mutation of the studied OAR3 QTL affecting PP and FP, although we cannot rule out the possibility that this SNP is in perfect linkage disequilibrium with the true causal polymorphism.