Colour-sidedness in Gloucester cattle is associated with a complex structural variant impacting regulatory elements downstream of KIT.

Colour-sidedness is a striking coat colour pattern found in a number of cattle breeds, typically characterised by a white stripe that extends along the back, head and belly of the animal. This dominant phenotype is caused by two related translocations (Cs6 and Cs29 ) that alter a region downstream of the KIT gene. Gloucester cattle are native to the UK and are known for an unusual colour-sided pattern that does not extend to the head. We carried out whole-genome sequencing of two Gloucester bulls as well as colour-sided Irish Moiled, British White and Pustertaler Sprinzen for comparison. We found that the Gloucester cattle also have a complex structural variant downstream of KIT, which overlaps the regions involved in Cs6 and Cs29 . All three alleles potentially disrupt a number of putative regulatory elements downstream of KIT. These results complement and expand on the recently published work focused on the Pinzgauer breed from Austria, a carrier of the same colour-sided pattern as seen in Gloucester cattle.

Genome-Wide Association Study of Acute Renal Graft Rejection.

Acute renal rejection is a major risk factor for chronic allograft dysfunction and long-term graft loss. We performed a genome-wide association study to detect loci associated with biopsy-proven acute T cell-mediated rejection occurring in the first year after renal transplantation. In a discovery cohort of 4127 European renal allograft recipients transplanted in eight European centers, we used a DNA pooling approach to compare 275 cases and 503 controls. In an independent replication cohort of 2765 patients transplanted in two European countries, we identified 313 cases and 531 controls, in whom we genotyped individually the most significant single nucleotide polymorphisms (SNPs) from the discovery cohort. In the discovery cohort, we found five candidate loci tagged by a number of contiguous SNPs (more than five) that was never reached in iterative in silico permutations of our experimental data. In the replication cohort, two loci remained significantly associated with acute rejection in both univariate and multivariate analysis. One locus encompasses PTPRO, coding for a receptor-type tyrosine kinase essential for B cell receptor signaling. The other locus involves ciliary gene CCDC67, in line with the emerging concept of a shared building design between the immune synapse and the primary cilium.

Genetic variation in PLAG1 associates with early life body weight and peripubertal weight and growth in Bos taurus.

Variation at the pleiomorphic adenoma gene 1 (PLAG1) locus has recently been implicated in the regulation of stature and weight in Bos taurus. Using a population of 942 outbred Holstein-Friesian dairy calves, we report confirmation of this effect, demonstrating strong association of early life body weight with PLAG1 genotype. Peripubertal body weight and growth rate were also significantly associated with PLAG1 genotype. Growth rate per kilogram of body weight, daily feed intake, gross feed efficiency and residual feed intake were not significantly associated with PLAG1 genotype. This study supports the status of PLAG1 as a key regulator of mammalian growth. Further, the data indicate the utility of PLAG1 polymorphisms for the selection of animals to achieve enhanced weight gain or conversely to aid the selection of animals with lower mature body weight and thus lower maintenance energy requirements.

Identifying cows with subclinical mastitis by bulk single nucleotide polymorphism genotyping of tank milk.

Mastitis remains the most important health issue in dairy cattle. Improved methods to identify cows developing subclinical mastitis would benefit farmers. We herein describe a novel method to determine the somatic cell counts (SCC) of individual cows by bulk genotyping a sample of milk from the milk tank with panels of genome-wide single nucleotide polymorphisms (SNP). We developed a simple linear model to estimate the contribution of individual cows to the genomic DNA present in the tank milk from 1) the known genotypes of individual cows for the interrogated SNP and 2) the ratio of SNP alleles in the tank milk. Using simulations, we estimate that 3,000, 50,000, and 700,000 SNP are sufficient to accurately (R(2)>0.98) estimate individual SCC in tanks containing milk from 25, 100, and 500 cows, respectively. Using actual data, we demonstrate that the SCC of 21 cows can be estimated with a coefficient of determination of 0.60 using approximately 9,000 SNP. The proposed method increases the value of the proposition of SNP genotyping individual cows for genomic selection purposes.

Quantitative trait loci analysis for five milk production traits on chromosome six in the Dutch Holstein-Friesian population.

Twenty Dutch Holstein-Friesian families, with a total of 715 sires, were evaluated in a granddaughter experiment design for marker-QTL associations. Five traits-milk, fat and protein yield and fat and protein percent-were analyzed. Across-family analysis was undertaken using multimarker regression principles. One and two QTL models were fitted. Critical values for the test statistic were calculated empirically by permuting the data. Individual trait distributions of permuted test statistics differed and, thus distributions, had to be calculated for each trait. Experimentwise critical values, which account for evaluating marker-QTL associations on all 29 autosomal-bovine chromosomes and for five traits, were calculated. A QTL for protein percent was identified in one, and two QTL models and was significant at the 1 and 2% level, respectively. Extending the multimarker regression approach to an analysis including two QTL was limited by families not being informative at all markers, which resulted in singularity. Below average heterozygosity for the first and last marker lowered information content for the first and last marker bracket. Highly informative markers at the ends of the mapped chromosome would overcome the decrease in information content in the first and last marker bracket and singularity for the two QTL model.

A rank-based nonparametric method for mapping quantitative trait loci in outbred half-sib pedigrees: application to milk production in a granddaughter design.

We describe the development of a multipoint nonparametric quantitative trait loci mapping method based on the Wilcoxon rank-sum test applicable to outbred half-sib pedigrees. The method has been evaluated on a simulated dataset and its efficiency compared with interval mapping by using regression. It was shown that the rank-based approach is slightly inferior to regression when the residual variance is homoscedastic normal; however, in three out of four other scenarios envisaged, i.e., residual variance heteroscedastic normal, homoscedastic skewed, and homoscedastic positively kurtosed, the latter outperforms the former one. Both methods were applied to a real data set analyzing the effect of bovine chromosome 6 on milk yield and composition by using a 125-cM map comprising 15 microsatellites and a granddaughter design counting 1158 Holstein-Friesian sires.

Pig plasma alpha-protease inhibitors PI2, PI3 and PI4 are members of the antichymotrypsin family.

Three related alpha-protease inhibitors, PI2 I, PI3 C and PI4 C2, of blood serum of the pig (Sus scrofa) were isolated. PI2 I inhibited both trypsin and chymotrypsin; PI3 C and PI4 C2 strongly inhibited chymotrypsin, but did not significantly inhibit trypsin. By using SDS-PAGE, the three proteins were found to be composed of single polypeptide chains, and molecular weights were 63,000 for PI2 I, 58,000 for PI3 C and 64,000 for PI4 C2. All three proteins were shown to be glycoproteins. In PI3 C, eight sialic acid residues were found, and in PI4 C2 (similarly as in PI2 F) 10-11 residues were found. Amino acid composition as well as N-terminal sequences of the three proteins were very similar, indicating close homology. Comparison of these partial amino acid sequences with the cDNA-deduced amino acid sequence of pig alpha-antichymotrypsin (AACT; Buchman, 1989, GenBank, Accession No. M29508) revealed great similarities, the sequence of PI2 I being virtually identical with the pig AACT. On the basis of all available results, PI2 is proposed to be pig AACT, an orthologue of human AACT.

Measuring the extent of linkage disequilibrium in commercial pig populations.

To evaluate the extent of linkage disequilibrium in domestic pigs, we genotyped 33 and 44 unrelated individuals from two commercial populations for 29 and five microsatellite markers located on chromosomes 15 and 2 respectively. A high proportion of marker pairs up to 40 cM apart exhibited significant linkage disequilibrium in both populations. Pair-wise r(2) values averaged between 0.15 and 0.50 (depending on chromosome and population) for markers <1 cM apart and declined to values of 0.05 for more distant syntenic markers. Our results suggest that both populations underwent a bottleneck approximately 20 generations ago, which reduced the effective population size from thousands to <200 animals.

Results of a whole-genome quantitative trait locus scan for growth, carcass composition and meat quality in a porcine four-way cross.

A whole-genome quantitative trait locus (QTL) scan for 31 phenotypes related to growth, carcass composition and meat quality was conducted using 1187 progeny of a commercial four-way cross. Animals were genotyped for 198 microsatellite markers that spanned the entire porcine genome. QTL analysis was conducted to extract information from paternal and maternal meioses separately using a rank-based nonparametric approach for half-sib designs. Nine QTL exceeded genome-wide significance: one QTL affecting growth (average daily gain on SSC1), two QTL influencing carcass composition (fatness on SSC3 and muscle mass on SSC15) and six QTL influencing meat quality (tenderness on SSC4 and SSC14; colour on SSC5, SSC6 and SSCX; and conductivity on SSC16). All but one of these coincided with previously reported QTL. In addition, we present evidence for 78 suggestive QTL with a combined false discovery rate of 40%.

A 2.5-Mb contig constructed from Angus, Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1.

The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.

The effect of mixed selected and unselected samples on the power of QTL mapping.

It is known from previous work that selection by truncation can lead to substantial loss of power to detect linkage with a QTL We examine the effect of mixed selected and unselected samples. We show that even if a modest proportion of the sample originates from an unselected population, this loss of power is quite effectively neutralized. Moreover, we reach the unexpected conclusion that if the selection intensity in the selected subpopulation is high, the power to detect QTL may actually increase.

A hypervariable pig DNA fragment.

A new hypervariable tandem repeat was isolated from the pig genome and characterized by DNA sequence. The use of this DNA fragment as a probe in order to follow allelic segregation and DNA fingerprinting in pigs, horses and rabbits is documented.

The homology between the serum proteins PO2 in pig, Xk in horse and alpha 1B-glycoprotein in human.

1. Pig serum Po2 protein and horse Xk protein were purified by FPLC, non-denaturing 2D agarose-PAGE and 2D IPG-PAGE. 2. The separated fractions were electroblotted to poly(4-vinyl-N-methylpyridinium iodide) coated GF/C glass fiber sheets. 3. The partial amino acid sequences and amino acid compositions of different genetic variants of the proteins were determined. 4. The results proved that previously reported polymorphic serum post-albumins in each of these species were homologous to human plasma alpha 1B-glycoprotein.

Statistical power of QTL mapping methods applied to bacteria counts.

Most QTL mapping methods assume that phenotypes follow a normal distribution, but many phenotypes of interest are not normally distributed, e.g. bacteria counts (or colony-forming units, CFU). Such data are extremely skewed to the right and can present a high amount of zero values, which are ties from a statistical point of view. Our objective is therefore to assess the efficiency of four QTL mapping methods applied to bacteria counts: (1) least-squares (LS) analysis, (2) maximum-likelihood (ML) analysis, (3) non-parametric (NP) mapping and (4) nested ANOVA (AN). A transformation based on quantiles is used to mimic observed distributions of bacteria counts. Single positions (1 marker, 1 QTL) as well as chromosome scans (11 markers, 1 QTL) are simulated. When compared with the analysis of a normally distributed phenotype, the analysis of raw bacteria counts leads to a strong decrease in power for parametric methods, but no decrease is observed for NP. However, when a mathematical transformation (MT) is applied to bacteria counts prior to analysis, parametric methods have the same power as NP. Furthermore, parametric methods, when coupled with MT, outperform NP when bacteria counts have a very high proportion of zeros (70.8%). Our results show that the loss of power is mainly explained by the asymmetry of the phenotypic distribution, for parametric methods, and by the existence of ties, for the non-parametric method. Therefore, mapping of QTL for bacterial diseases, as well as for other diseases assessed by a counting process, should focus on the occurrence of ties in phenotypes before choosing the appropriate QTL mapping method.

Whole genome scan to detect quantitative trait loci for conformation and functional traits in dairy cattle.

A granddaughter design was used to locate quantitative trait loci determining conformation and functional traits in dairy cattle. In this granddaughter design, consisting of 20 Holstein Friesian grandsires and 833 sons, genotypes were determined for 277 microsatellite markers covering the whole genome. Breeding values for 27 traits, regarding conformation (18), fertility (2), birth (4), workability (2), and udder health (1), were evaluated in an across-family analysis using multimarker regression. Significance thresholds were determined using a permutation test. The across-family analysis suggested the presence of 61 quantitative trait loci when 27 (i.e., one for each trait) were expected by chance. The test statistic exceeded the genomewise significance threshold for the following traits and chromosomes: chest width on chromosome 2; gestation length on chromosome 4; stature, body capacity, and size on chromosome 5; dairy character on chromosome 6; angularity on chromosome 12; fore udder attachment on chromosome 13; and fore udder attachment and front teat placement on chromosome 19. The quantitative trait loci for size traits on chromosomes 2, 5, and 6 may also have an effect on calving ease. The quantitative trait loci for udder traits on chromosomes 13 and 19 may also affect somatic cell score and mastitis resistance. If there are no negative effects on other economically important traits, marker assisted selection using markers associated with these quantitative trait loci can be applied.

Convenient genotyping of six myostatin mutations causing double-muscling in cattle using a multiplex oligonucleotide ligation assay.

We herein describe a procedure that allows for simultaneous genotyping of six loss-of-function mutations in the bovine myostatin gene associated with the double-muscling phenotype. The proposed method relies on a multiplex oligonucleotide ligation assay and detection of the fluorescently labelled products using automatic sequencers.

Linkage maps of porcine chromosomes 3, 6, and 9 based on 31 polymorphic markers.

Linkage maps of porcine Chromosomes (Chrs) 3, 6, and 9, based on 31 polymorphic markers, are reported. The markers include 14 microsatellites, 12 RFLPs, three protein polymorphisms, and two blood group loci. The genetic interpretations of 11 RFLPs are documented. The markers were scored in a three-generation Wild Boar/Large White pedigree, and genetic maps were constructed on the basis of two-point and multi-point linkage analysis. Altogether the maps span a genetic distance of 216 cM, and previous physical assignments indicate that the linkage groups cover major parts of the three chromosomes. Significant differences in recombination rates between the sexes were observed for all three chromosomes. The recombination rate on the q arm of Chr 6 was markedly low. Sixteen loci are informative with regard to comparative mapping, that is, they have previously been mapped in the human and/or mouse genomes.

Non-parametric interval mapping in half-sib designs: use of midranks to account for ties.

In QTL analysis of non-normally distributed phenotypes, non-parametric approaches have been proposed as an alternative to the use of parametric tests on mathematically transformed data. The non-parametric interval mapping test uses random ranking to deal with ties. Another approach is to assign to each tied individual the average of the tied ranks (midranks). This approach is implemented and compared to the random ranking approach in terms of statistical power and accuracy of the QTL position. Non-normal phenotypes such as bacteria counts showing high numbers of zeros are simulated (0-80% zeros). We show that, for low proportions of zeros, the power estimates are similar but, for high proportions of zeros, the midrank approach is superior to the random ranking approach. For example, with a QTL accounting for 8% of the total phenotypic variance, a gain from 8% to 11% of power can be obtained. Furthermore, the accuracy of the estimated QTL location is increased when using midranks. Therefore, if non-parametric interval mapping is chosen, the midrank approach should be preferred. This test might be especially relevant for the analysis of disease resistance phenotypes such as those observed when mapping QTLs for resistance to infectious diseases.

Cloning and sequencing of the porcine complement factor B.

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes.