A genome-screen experiment to detect quantitative trait loci affecting resistance to facial eczema disease in sheep.

Facial eczema (FE) is a secondary photosensitization disease arising from liver cirrhosis caused by the mycotoxin sporidesmin. The disease affects sheep, cattle, deer and goats, and costs the New Zealand sheep industry alone an estimated NZ$63M annually. A long-term sustainable solution to this century-old FE problem is to breed for disease-resistant animals by marker-assisted selection. As a step towards finding a diagnostic DNA test for FE sensitivity, we have conducted a genome-scan experiment to screen for quantitative trait loci (QTL) affecting this trait in Romney sheep. Four F(1) sires, obtained from reciprocal matings of FE resistant and susceptible selection-line animals, were used to generate four outcross families. The resulting half-sib progeny were artificially challenged with sporidesmin to phenotype their FE traits measured in terms of their serum levels of liver-specific enzymes, namely gamma-glutamyl transferase and glutamate dehydrogenase. In a primary screen using selective genotyping on extreme progeny of each family, a total of 244 DNA markers uniformly distributed over all 26 ovine autosomes (with an autosomal genome coverage of 79-91%) were tested for linkage to the FE traits. Data were analysed using Haley-Knott regression. The primary screen detected one significant and one suggestive QTL on chromosomes 3 and 8 respectively. Both the significant and suggestive QTL were followed up in a secondary screen where all progeny were genotyped and analysed; the QTL on chromosome 3 was significant in this analysis.

Fine mapping of genes on sheep chromosome 1 and their association with milk traits.

On the basis of comparative mapping between cattle/sheep and human for milk trait quantitative trait loci (QTL) on BTA3/OAR1, annexin A9 (ANXA9) and solute carrier family 27 (fatty acid transporter), member 3 (SLC27A3) were selected as candidate genes for fat content (FC) in sheep milk. Two other genes in the same region, cingulin (CGN) and acid phosphatase 6, lysophosphatidic (ACP6), were also considered. DNA fragments of 1931 and 2790 bp corresponding to ANXA9 and SLC27A3 respectively were isolated, and 14 and 6 single nucleotide polymorphisms (SNPs) respectively were found in each gene. ANXA9, SLC27A3, CGN and ACP6 were localized to chromosome 1 between INRA006 and AE57 by linkage mapping using the International Mapping Flock. Across-family analyses of a daughter design comprising 13 sire families revealed significant sire and SLC27A3 genotype-nested-within-sire effects for FC. Within-family analyses indicated significant regression coefficients for FC in four of six heterozygous sires. These results could reflect the existence of a QTL for FC linked to SLC27A3 in sheep.

Positional and functional characterisation of apoptosis related genes belonging to the BCL2 family in sheep.

Apoptosis is a process whereby cells die in a controlled manner and it is involved in animal development, tissue homeostasis and a variety of diseases. The B-cell lymphoma 2 family proteins are central regulators of intracellular apoptotic signalling cascades. This work describes the isolation of cDNA and genomic fragments from five sheep BCL2 related genes: BCL2, BCL2L1, BCL2L2, BAX and MCL1. Transcript sequences showed a high homology with BCL2 related genes from other species. Three cattle BAC probes containing the homologous BCL2, BCL2L1 and BCL2L2 genes were identified and used for comparative FISH mapping in sheep. BCL2 was localised in OAR23q27, BCL2L1 in OAR13q22 and BCL2L2 in OAR7q15-->q21. Intron polymorphisms were used for linkage mapping of BAX and MCL1, which were mapped on OAR14 and OAR1 respectively. Moreover, a BCL2L1 pseudogene was also identified and linkage mapped on OAR2. The expression of these genes was analysed in mammary gland, ovary, intestine and brain which are target tissues for sheep pathological processes where apoptosis is involved.

Ovine alpha-amylase genes: isolation, linkage mapping and association analysis with milk traits.

On the basis of comparisons between cattle and sheep genome mapping information the ovine alpha-amylase gene was examined as a possible genetic marker for milk traits in sheep. The objective of the present study was to isolate, map and determine whether this gene is a candidate gene for milk traits. DNA fragments (832 and 2360 bp) corresponding to two different AMY genes were isolated, and one SNP in intron 3 and one GTG deletion in exon 3 of the 2360 bp DNA fragment were found. The 2360 bp ovine AMY DNA fragment was located on chromosome 1 by linkage mapping using the International Mapping Flock. No association was found between estimated breeding values for milk yield, protein and fat contents and AMY genotypes in a daughter design comprising 13 Manchega families with an average of 29 daughters (12-62) per sire.

Bone density in sheep: genetic variation and quantitative trait loci localisation.

Bone density (BD) is an important factor in osteoporotic fracture risk in humans. However, BD is a complex trait confounded by environmental influences and polygenic inheritance. Sheep provide a potentially useful model for studying differences in BD, as they provide a means of circumventing complex environmental factors and are a similar weight to humans. The aims of this study were to establish whether there is genetic variation in BD in sheep and then to localise quantitative trait loci (QTLs) associated with this variation. We also aimed to evaluate the relationship between fat and muscle body components and BD in sheep. Results showed that there was significant (P < 0.01) genetic variation among Coopworth sheep sires for BD. This genetic difference was correlated (P < 0.01) with body weight and muscle mass. A number of QTLs exceeding the suggestive threshold were identified (nine in total). Of these, two (chromosomes 1, P < 0.05; chromosome 24, P < 0.01) were significant using genome-wide permutation significance thresholds (2000 iterations). The position of the QTL on chromosome 24 coincided with a number of other body composition QTLs, indicating possible pleiotropic effects or the presence of multiple genes affecting body composition at that site. This study shows that sheep are potentially a useful model for studying the genetics of BD.

Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.

Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

Mapping the Horns (Ho) locus in sheep: a further locus controlling horn development in domestic animals.

The presence or absence of horns in Merino sheep is under the genetic control of the autosomal Horns (Ho) locus. Sheep chromosome OOV1 is a candidate region for the Ho locus because it shows conserved synteny with cattle chromosome BBO1 where the cattle polled locus has been located. We demonstrate that the Ho locus in sheep is excluded from sheep chromosome OOV1 and we identified linkage between the Ho locus and markers from sheep chromosome OOV10. These data suggest that there are at least two loci affecting the presence or absence of horns in sheep and cattle. The orthologous regions to OOV10 are likely to be on cattle, human, and mouse chromosomes BBO12, HSA13, and MMU14.

An autosomal genetic linkage map of the sheep genome.

We report the first extensive ovine genetic linkage map covering 2070 cM of the sheep genome. The map was generated from the linkage analysis of 246 polymorphic markers, in nine three-generation full-sib pedigrees, which make up the AgResearch International Mapping Flock. We have exploited many markers from cattle so that valuable comparisons between these two ruminant linkage maps can be made. The markers, used in the segregation analyses, comprised 86 anonymous microsatellite markers derived from the sheep genome, 126 anonymous microsatellites from cattle, one from deer, and 33 polymorphic markers of various types associated with known genes. The maximum number of informative meioses within the mapping flock was 222. The average number of informative meioses per marker was 140 (range 18-209). Linkage groups have been assigned to all 26 sheep autosomes.