A high throughput single nucleotide polymorphism multiplex assay for parentage assignment in New Zealand sheep.

Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs) are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development--firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) are assigned. An 84 "parentage SNP panel" was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams) was absent, highlighting the SNP test's suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.

Fatty acid synthase effects on bovine adipose fat and milk fat.

A quantitative trait locus (QTL) was identified by linkage analysis on bovine Chromosome 19 that affects the fatty acid, myristic acid (C14:0), in subcutaneous adipose tissue of pasture-fed beef cattle (99% level: experiment-wise significance). The QTL was also shown to have significant effects on ten fatty acids in the milk fat of pasture-fed dairy cattle. A positional candidate gene for this QTL was identified as fatty acid synthase (FASN), which is a multifunctional enzyme with a central role in the metabolism of lipids. Five single nucleotide polymorphisms (SNPs) were identified in the bovine FASN gene, and animals were genotyped for FASN SNPs in three different cattle resource populations. Linkage and association mapping results using these SNPs were consistent with FASN being the gene underlying the QTL. SNP substitution effects for C14:0 percentage were found to have an effect in the opposite direction in adipose fat to that in milk fat. It is concluded that SNPs in the bovine FASN gene are associated with variation in the fatty acid composition of adipose fat and milk fat.

Establishment of a pipeline to analyse non-synonymous SNPs in Bos taurus.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are an abundant form of genetic variation in the genome of every species and are useful for gene mapping and association studies. Of particular interest are non-synonymous SNPs, which may alter protein function and phenotype. We therefore examined bovine expressed sequences for non-synonymous SNPs and validated and tested selected SNPs for their association with measured traits. RESULTS: Over 500,000 public bovine expressed sequence tagged (EST) sequences were used to search for coding SNPs (cSNPs). A total of 15,353 SNPs were detected in the transcribed sequences studied, of which 6,325 were predicted to be coding SNPs with the remaining 9,028 SNPs presumed to be in untranslated regions. Of the cSNPs detected, 2,868 were predicted to result in a change in the amino acid encoded. In order to determine the actual number of non-synonymous polymorphic SNPs we designed assays for 920 of the putative SNPs. These SNPs were then genotyped through a panel of cattle DNA pools using chip-based MALDI-TOF mass spectrometry. Of the SNPs tested, 29% were found to be polymorphic with a minor allele frequency >10%. A subset of the SNPs was genotyped through animal resources in order to look for association with age of puberty, facial eczema resistance or meat yield. Three SNPs were nominally associated with resistance to the disease facial eczema (P < 0.01). CONCLUSION: We have identified 15,353 putative SNPs in or close to bovine genes and 2,868 of these SNPs were predicted to be non-synonymous. Approximately 29% of the non-synonymous SNPs were polymorphic and common with a minor allele frequency >10%. Of the SNPs detected in this study, 99% have not been previously reported. These novel SNPs will be useful for association studies or gene mapping.

Reduction in adiposity affects the extent of afferent projections to growth hormone-releasing hormone and somatostatin neurons and the degree of colocalization of neuropeptides in growth hormone-releasing hormone and somatostatin cells of the ovine hypothalamus.

Various neuropeptides and neurotransmitters affect GH secretion by acting on GHRH and somatostatin (SRIF) cells. GH secretion is also affected by alteration in adiposity, which could be via modulation of GHRH and SRIF cells. We quantified colocalization of neuropeptides in GHRH and SRIF cells and afferent projections to these cells in lean (food restricted) and normally fed sheep (n=4/group). The number of GHRH-immunoreactive (IR) cells in the arcuate nucleus was higher in lean animals, but the number of SRIF-IR cells in the periventricular nucleus was similar in the two groups. A subpopulation of GHRH-IR cells colocalized neuropeptide Y in lean animals, but this was not seen in normally fed animals. GHRH/galanin (GAL) colocalization was higher in lean animals with no difference in numbers of GHRH/tyrosine hydroxylase or GHRH/GAL-like peptide cells. SRIF/enkephalin colocalization was lower in lean animals. The percentage of GHRH neurons receiving SRIF input was similar in lean and normally fed animals, but more GHRH cells received input from enkephalin afferents in normally fed animals. The percentage of SRIF cells receiving GHRH, neuropeptide Y, GAL, and orexin afferents was higher in lean animals. These findings provide an anatomical evidence of central mechanism(s) by which appetite-regulating peptides and dopamine could regulate GH secretion. Increased input to SRIF cells in lean animals may be inhibitory and permissive of increased GH. The appearance of NPY in GHRH cells of lean animals may be a mechanism for regulation of increasing GH secretion with reduced adiposity.