Leveraging genetically simple traits to identify small-effect variants for complex phenotypes.

BACKGROUND: Polymorphisms underlying complex traits often explain a small part (less than 1 %) of the phenotypic variance (σ2P). This makes identification of mutations underling complex traits difficult and usually only a subset of large-effect loci are identified. One approach to identify more loci is to increase sample size of experiments but here we propose an alternative. The aim of this paper is to use secondary phenotypes for genetically simple traits during the QTL discovery phase for complex traits. We demonstrate this approach in a dairy cattle data set where the complex traits were milk production phenotypes (fat, milk and protein yield; fat and protein percentage in milk) measured on thousands of individuals while secondary (potentially genetically simpler) traits are detailed milk composition traits (measurements of individual protein abundance, mineral and sugar concentrations; and gene expression). RESULTS: Quantitative trait loci (QTL) were identified using 11,527 Holstein cattle with milk production records and up to 444 cows with milk composition traits. There were eight regions that contained QTL for both milk production and a composition trait, including four novel regions. One region on BTAU1 affected both milk yield and phosphorous concentration in milk. The QTL interval included the gene SLC37A1, a phosphorous antiporter. The most significant imputed sequence variants in this region explained 0.001 σ2P for milk yield, and 0.11 σ2P for phosphorus concentration. Since the polymorphisms were non-coding, association mapping for SLC37A1 gene expression was performed using high depth mammary RNAseq data from a separate group of 371 lactating cows. This confirmed a strong eQTL for SLC37A1, with peak association at the same imputed sequence variants that were most significant for phosphorus concentration. Fitting any of these variants as covariables in the association analysis removed the QTL signal for milk production traits. Plausible causative mutations in the casein complex region were also identified using a similar strategy. CONCLUSIONS: Milk production traits in dairy cows are typical complex traits where polymorphisms explain only a small portion of the phenotypic variance. However, here we show that these mutations can have larger effects on secondary traits, such as concentrations of minerals, proteins and sugars in the milk, and expression levels of genes in mammary tissue. These larger effects were used to successfully map variants for milk production traits. Genetically simple traits also provide a direct biological link between possible causal mutations and the effect of these mutations on milk production.

Genotype by environment interaction for fertility, survival, and milk production traits in Australian dairy cattle.

The existence of a genotype x environment interaction (G x E) for fertility traits, survival, and milk yield traits was examined by considering performance recorded in different calving systems (seasonal, split, and year round) or regions as different traits. For fertility traits and survival, G x E were also investigated by applying a random regression model using continuous environmental variables, such as level of herd milk production, temperature-humidity index, and herd size as environmental descriptors. The traits considered were calving interval, calving to first service interval (CFS), 25-d nonreturn rate at first service, pregnancy rate, survival, milk yield, fat yield, and protein yield and percentage. Data on Holstein-Friesian cows that calved between 1997 and 2005 were analyzed. The number of cows included in the analyses ranged from approximately 21,000 for pregnancy rate to approximately one-half million for survival. For all traits, heterogeneity in additive and phenotypic variances was observed. For example, for CFS the additive genetic and phenotypic variance in seasonal calving herds was only 9 and 15% of that in year-round calving herds, respectively. Genetic correlations among calving systems for milk yield traits were greater than 0.96. For calving interval, the lowest genetic correlation, of 0.83, was between split and year-round calving herds, but for CFS and pregnancy rate, genetic correlations as low as 0.37 were observed, although these estimates were associated with large standard errors. Genetic correlations between traits recorded in different Australian regions were greater than 0.89. Heritability and phenotypic variance for milk yield traits were the greatest in region 1, which consisted of Queensland, West Australia, South Australia, and New South Wales, and were least in region 3, which included Gippsland and Tasmania, in accordance with mean milk yield levels. Genetic correlations as low as 0.5 for some fertility traits between the 5th and 95th percentile of the distribution of the environmental descriptors, such as herd size and average herd milk production, were also observed. However, these estimates had large standard errors. Regardless of the environmental descriptor used, there was no evidence for the presence of a large G x E that resulted in economically significant reranking of bulls.

Genetic parameters for growth traits of Australian beef cattle from a multibreed selection experiment.

Estimates of covariance components and genetic parameters were obtained for birth, weaning, 200-d, and 400-d weight for a herd of Polled Herefords and a herd of a multibreed synthetics, the so-called Wokalups. Data originated from an experiment in Western Australia selecting for preweaning growth rate. Analyses were carried out by REML fitting an animal model including genetic and permanent environmental maternal effects. Wokalups showed consistently more phenotypic variation, partly due to a scale effect, and higher direct and lower maternal heritabilities than Herefords. Maternal environmental effects were more than twice as important in Herefords than in Wokalups. Estimates of both genetic and environmental correlations among weaning and subsequent weights were essentially unity, identifying maternal effects found for postweaning weights as a "carry over" of those on weaning weight.

Genetic parameters for milk production of Australian beef cows and weaning weight of their calves.

Estimates of covariance components were obtained for milk production of beef cows, measured by the weigh-suckle-weigh method, and weaning weight and preweaning growth of their calves for a herd of Polled Herefords and a herd of a multibreed synthetics, so-called Wokalups. Analyses were carried out by restricted maximum likelihood fitting an animal model incorporating both direct and maternal genetic and permanent environmental effects and allowing for the direct-maternal covariances. Average 14-h milk yield was 3.6 kg for Herefords and 4.9 kg for Wokalups and heritabilities were .12 and .08, respectively. Treating preweaning gain of the calf as a trait of the cow, estimates of the additive genetic and permanent environmental correlations between preweaning gain and milk yield were higher for Herefords (.73 and 1.00, respectively) than for Wokalups (.53 and .77). Bivariate analyses of milk production (treated as a trait of the cow) and weaning weight (treated as a trait of the calf) showed virtually no direct additive genetic, direct permanent environmental, or phenotypic association between the two traits but identified strong correlations between direct effects for milk yield and maternal effects for weaning weight. The estimate of the direct-maternal genetic correlation between milk yield and weaning weight was .80 for both breeds, and the estimated correlation between direct, permanent environmental effects for milk yield and maternal, permanent environmental effects on weaning weight was unity for Herefords and .89 for Wokalups. Results indicate that milk production is the main determinant of maternal effects on the growth of beef calves with breed differences in the importance of maternal effects largely attributable to differences in milk production.

Freeze-thawed skeletal muscle autografts used for brachial plexus repair in the non-human primate.

An experimental study undertaken in the marmoset is reported. A defect in the lateral cord of the brachial plexus was repaired with a longitudinally aligned freeze-thawed skeletal muscle autograft. Recovery was assessed after one year using sensory and motor electrophysiological and also histological examination of the nerve. The results show that this is a satisfactory method of peripheral nerve repair in the marmoset. It is suggested that the technique may be applicable to repair of the human brachial plexus.

Inherited progressive spinal myelinopathy in Murray Grey cattle.

In a breeding experiment conducted to determine the mode of inheritance of progressive spinal myelinopathy, semen from a Murray Grey bull which had previously sired affected calves was used to inseminate 120 cows. Female progeny were then inseminated with semen from the same bull. Of the 51 calves born, six (11.8%) had spinal cord lesions consistent with progressive spinal myelinopathy. From analysis of pedigrees and the results of the breeding experiment it was concluded that the condition was inherited as an autosomal recessive condition in Murray Grey cattle.

Estimation of genetic parameters for milk fat depression in dairy cattle.

The objective of this study was to apply reaction norm models to milk recording data to investigate genetic variation in and environmental sensitivity of susceptibility to milk fat depression (MFD). Data comprised 556,276 test-day records of 80,493 heifers in 1043 herds. Breeding values and genetic variances for fat percentage and fat yield were estimated by applying random regression models to average herd-test-day fat percentage. Genetic and permanent environmental correlations between fat yield expressed in different environments ranged, respectively, from 0.83 to 1.00 and from 0.29 to 1.00. Genetic and permanent environmental correlations between fat percentage expressed in different environments ranged, respectively, from 0.87 to 1.00 and from -0.05 to 0.99. Two traits were defined for MFD. The first trait reflected variation of milk fat percentage of animals within lactation after correction for year-season, herd-test-day, age-at-calving, and stage-of-lactation. This trait had an estimated heritability of about 5% and a genetic correlation between the fifth and 95th percentile of the data of 0.50. The second trait reflected the deviation of an animal's fat percentage on a test-day from its expected fat percentage based on fat percentage on the first test-day. This trait had an estimated heritability of about 4% and a genetic correlation between the fifth and 95th percentile of the data of 0.43. The correlation between estimated breeding values of sires for the 2 MFD traits was -0.3. Our results suggest that genetic variation in susceptibility to MFD is present and that selection for reduced susceptibility to MFD is possible.

Distribution of complement protein C4 concentrations in bovine plasma.

Complement component C4 concentrations were measured in 40 pure bred Hereford cattle and 40 cattle from a mixed breed herd. Significant differences were not observed between the two groups studied nor between bulls and cows. However, the distribution of C4 concentrations was relatively disperse and appeared polymodal suggesting the presence of two isotypes of C4. Polyacrylamide gel electrophoresis of immunoprecipitated bovine C4 showed many samples to have two C4 alpha chains differing in relative molecular mass by about 1800. Isoelectric focusing of bovine plasma in agarose gels followed by immunofixation with specific anti-C4 antisera revealed two populations of native C4 differing in pI by about 0.3 pH unit. An association between the type of C4 alpha chain present and the pI of the native C4 molecule was observed. Collectively these findings indicate the presence of two structural C4 genetic loci in cattle.

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.