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Background: Copy number variations (CNVs) are structural variants consisting of large-scale insertions and deletions of genomic fragments. Exploring CNVs and estimating their effects on phenotypes are useful for genome selection but remain challenging in the livestock., Results: We identified 1043 CNV regions (CNVRs) from array comparative genomic hybridization (CGH) data of 47 Holstein bulls. Using a probe-based CNV association approach, we detected 87 CNVRs significantly (Bonferroni-corrected P value < 0.05) associated with at least one out of 41 complex traits. Within them, 39 CNVRs were simultaneously associated with at least 2 complex traits. Notably, 24 CNVRs were markedly related to daughter pregnancy rate (DPR). For example, CNVR661 containing CYP4A11 and CNVR213 containing CTR9, respectively, were associated with DPR and other traits related to reproduction, production, and body conformation. CNVR758 was also significantly related to DPR, with a nearby gene CAPZA3, encoding one of F-actin-capping proteins which play a role in determining sperm architecture and male fertility. We corroborated these CNVRs by examining their overlapped quantitative trait loci and comparing with previously published CNV results., Conclusion: To our knowledge, this is one of the first genome-wide association studies based on CNVs called by array CGH in Holstein cattle. Our results contribute substantial information about the potential CNV impacts on reproduction, health, production, and body conformation traits, which lay the foundation for incorporating CNV into the future dairy cattle breeding program.

Several studies have shown that computation of genomic estimated breeding values (GEBV) with accuracies significantly greater than parent average (PA) estimated breeding values (EBVs) requires genotyping of at least several thousand progeny-tested bulls. For all published analyses, GEBV computed from the selected samples of markers have lower or equal accuracy than GEBV derived on the basis of all valid single nucleotide polymorphisms (SNPs). In the current study, we report on four new methods for selection of markers. Milk, fat, protein, somatic cell score, fertility, persistency, herd life and the Israeli selection index were analyzed. The 972 Israeli Holstein bulls genotyped with EBV for milk production traits computed from daughter records in 2012 were assigned into a training set of 844 bulls with progeny test EBV in 2008, and a validation set of 128 young bulls. Numbers of bulls in the two sets varied slightly among the nonproduction traits. In EFF12, SNPs were first selected for each trait based on the effects of each marker on the bulls' 2012 EBV corrected for effective relationships, as determined by the SNP matrix. EFF08 was the same as EFF12, except that the SNPs were selected on the basis of the 2008 EBV. In DIFmax, the SNPs with the greatest differences in allelic frequency between the bulls in the training and validation sets were selected, whereas in DIFmin the SNPs with the smallest differences were selected. For all methods, the numbers of SNPs retained varied over the range of 300 to 6000. For each trait, except fertility, an optimum number of markers between 800 and 5000 was obtained for EFF12, based on the correlation between the GEBV and current EBV of the validation bulls. For all traits, the difference between the correlation of GEBV and current EBV and the correlation of the PA and current EBV was >0.25. EFF08 was inferior to EFF12, and was generally no better than PA EBV. DIFmax always outperformed DIFmin and generally outperformed EFF08 and PA. Furthermore, GEBV based on DIFmax were generally less biased than PA. It is likely that other methods of SNP selection could improve upon these results.

Based on pairwise identity-by-state (IBS) distances and whole-genome SNP data, kinship was investigated in the Israeli Holstein population. A total of 789 bulls, including most of the artificial insemination sires in service since 1987, were genotyped by the BovineSNP50 BeadChip. This sample included up to five generations. For each bull-by-bull combination, three states are possible for each marker: no match, a single match and both alleles match. Summing over all markers, the 932 598 IBS scores (three match frequencies*310 866 bull-by-bull combinations) were visualized using three-dimensional coordinates that corresponded to the frequencies of the three possible states. Results were reduced to two dimensions using the transformations x' = 0.7071(1 + freq1-freq2) and y' = 1.2247freq0. Bull-by-bull pairs were grouped according to their level of kinship, and canonical scores were calculated using discriminant analysis and the x' and y' features. Of the 474 pairs of recorded maternal grandsire-grandson with both individuals genotyped, the probability for 28 pairs to belong to this level of kinship was low (P < 0.05), suggesting an error rate of around 3% per generation in pedigree determination., (© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.)

Strong selection in the Israeli Holstein dairy cattle population over the last three decades should have left clear signatures of selection. Two experimental approaches were applied to detect evidence of contemporary selection based on the 54K BeadChip genotypes of ~1000 Israeli Holstein bulls: (i) the long-range haplotype test, which searches for structural evidence resulting from selective sweep, and (ii) direct analysis of the changes in haplotypes frequencies over time combined with linkage disequilibrium blocks haplotype-based association analysis. Ten traits were analyzed: the PD07 Israeli selection index, milk, milk fat, % fat, milk protein, % protein, somatic cell score, female fertility, milk production persistency and herd life. The long-range haplotype test detected ~15% of the 3288 haplotypes that showed significant positive frequency trends (P < 0.05) and was significantly correlated with the substitution effects of the haplotypes and the selection intensities for the different traits. Thirty signatures of recent selection, which correspond to both approaches and affect the Israeli PD07 selection index, were identified on 17 of the 29 autosomes. The second experimental approach also was used to estimate the selection intensity of the different traits. The correlation between the selection intensities for the traits analyzed, derived from changes in haplotype frequencies in the population of bulls, and those derived from trait-based analysis of the cow population was 0.93 over all traits. Thus, the changes in haplotypes frequencies in the bulls' population accurately estimate genetic trends in the general cow population and can be used to detect signatures of recent selection., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Two-plated self-piercing eartags were first developed in the 19th century, but information on their retention rates is scarce. A method is presented that facilitates estimation of eartag retention rate by using a random sample of cows that initially had 2 tags (1 on each ear) placed for identification and at least 1 of which survived. Striving to adopt the European Union standard for cattle ear tagging, the Israeli veterinary service conducted a field test to evaluate the performance of plastic eartags under the conditions of a typical Israeli dairy farm. The initial sample (n=900 cows) was tagged on a single farm. Retention rates were estimated based on the ratio between the observed numbers of cows with 1 or 2 eartags in the surviving group (n=97 cows). Based on this long-term (>3 yr) field test, the highest yearly retention of flag eartags (0.89±0.03) was lower than expected (0.98). Tag design and on-farm management were key factors affecting tag retention. A better design of the feedline yoke system in the feeding area, avoiding slits that can entangle the eartags, would help increase tag retention., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A single nucleotide polymorphism in the intergenic region upstream of the ZNF496 gene on Bos taurus chromosome 7 displayed significant population-wide linkage disequilibrium with milk protein percentage in the Israeli Holstein population. The frequency of the allele associated with increased protein concentration was 10%. This single nucleotide polymorphism was located in the promoter region from which a 10-exon transcript of the bovine and the ovine ZNF496 genes are transcribed. The gene architecture was similar to the mouse ortholog Zkscan17. A 5-exon murine antisense transcript was complementary to the 5' untranslated Zkscan17 region that included a sequence domain conserved between mouse and ruminants, suggesting a regulatory function. In the bovine ZNF496 chromosomal region, segregation of a quantitative trait locus (QTL) for milk protein percentage was confirmed in a daughter design sire family. Concordance was not obtained between QTL status of bulls and any of the polymorphisms in the functional elements of ZNF496. This excludes these variations as the causative polymorphism under the assumption of no epigenetic effect for this locus. However, ZNF496 variants were differentially expressed in bovine ovaries, and only the paternal variant was expressed in liver and kidney in a sheep family with polymorphic ZNF496 sequence. Thus, the search for the mutation underlying the minor QTL allele, which is a top economically favorable allele in Israeli Holstein cattle, may be complicated by the presence of an imprinting center in this QTL confidence interval., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Incorrect paternity assignment in cattle can have a major effect on rates of genetic gain. Of the 576 Israeli Holstein bulls genotyped by the BovineSNP50 BeadChip, there were 204 bulls for which the father was also genotyped. The results of 38 828 valid single nucleotide polymorphisms (SNPs) were used to validate paternity, determine the genotyping error rates and determine criteria enabling deletion of defective SNPs from further analysis. Based on the criterion of >2% conflicts between the genotype of the putative sire and son, paternity was rejected for seven bulls (3.5%). The remaining bulls had fewer conflicts by one or two orders of magnitude. Excluding these seven bulls, all other discrepancies between sire and son genotypes are assumed to be caused by genotyping mistakes. The frequency of discrepancies was >0.07 for nine SNPs, and >0.025 for 81 SNPs. The overall frequency of discrepancies was reduced from 0.00017 to 0.00010 after deletion of these 81 SNPs, and the total expected fraction of genotyping errors was estimated to be 0.05%. Paternity of bulls that are genotyped for genomic selection may be verified or traced against candidate sires at virtually no additional cost.

Single nucleotide polymorphisms (SNPs) are amenable to automation and therefore have become the marker of choice for DNA profiling. SNaPshot, a primer extension-based method, was used to multiplex 25 SNPs that have been previously validated as useful for identity control. Detection of extended products was based on four different fluorochromes and extension primers with oligonucleotide tails of differing lengths, thus controlling the concise length of the entire chromatogram to 81 bases. Allele frequencies for Holstein, Simmental, Limousin, Angus, Charolais and Tux Cattle were estimated and significant positive Pearson-correlation coefficients were obtained among the analysed breeds. The probability that two randomly unrelated individuals would share identical genotypes for all 25 loci varied from 10(-8) to 10(-10) for these breeds. For parentage control, the exclusion power was found to be 99.9% when the genotypes of both putative parents are known. A traceability test of duplicated samples indicated a high genotyping precision of >0.998. This was further corroborated by analysis of 60 cases of parent-sib pairs and trio families. The 25-plex SNaPshot assay is adapted for low- and high-throughput capacity and thus presents an alternative for DNA-based traceability in the major commercial cattle breeds.

The lack of conventions for confirming the discovery of quantitative trait nucleotides in livestock was evidenced by the proposals of mutations in two different genes (SPP1 and ABCG2) as the underlying functional mutation for a major quantitative trait locus (QTL) for milk concentration on bovine chromosome 6 (BTA6). Of these conflicting candidates, SPP1 was excluded by follow-up studies and by the data described here. A simple test for concordance of the zygosity state between QTL segregation status and the candidate polymorphism was shown, in this case, to be a critical step towards establishing the proof. If a given sample effectively represents the genetic variation across the QTL region, haplotype-based concordance may further enhance the functionality and resolution power of this test, allowing identification of the causative gene.

Twinning rate was analyzed in the Israeli Holstein dairy cattle population by the multiple-trait animal model, and a daughter design genome scan for quantitative trait loci was performed. Each parity was considered a separate trait. Heritabilities of twinning rate were very low, but increased by parity from 0.01 in first parity to 0.03 in fifth parity. All genetic correlations among parities were >0.77, but all environmental correlations were <0.07. Genetic correlations between twinning rate and female fertility (measured as the inverse of the number of inseminations to conception) in the first 3 parities were negative for all 9 parity-by-trait combinations. All environmental correlations were very small, but generally negative. The overall genetic trend since 1985 was positive at 0.02% twinning/yr, whereas the phenotypic trends were positive for parities 3 and 4 and negative for the other parities, but all trends were quite small. A total of 5,221 cows, daughters of 11 sires, were genotyped for 73 markers spanning all 29 autosomes. There were 9 markers with significant effects on twinning rate at P < 0.05, for a false discovery rate of 0.4; thus, about 5 of these probably represent true effects. Significant effects were found on chromosomes 1, 6, 7, 8, 14, 15, and 23. Of these, 3 effects were significant at P < 0.01, for a false discovery rate of 0.24. All 11 families were analyzed by interval mapping of chromosome 7. Only 2 families showed nominally significant effects, but chromosome-wise significance at P < 0.05 was not obtained for either family. Suggestive evidence of quantitative trait loci near the beginning of the chromosome and near position 50 cM were found in both families. Sire 3070 also had a significant effect for female fertility near the beginning of the chromosome. There was also evidence for a third quantitative trait loci at the end of the chromosome for sire 2357.

A total of 5,459 Israeli Holstein cows, daughters of 11 sires, were genotyped for 29 microsatellites spanning chromosome 7 and analyzed by the daughter design for 9 economic traits: milk, fat, and protein yield, fat and protein percentage, somatic cell score, female fertility, herd life, and milk persistency. Quantitative trait loci at chromosome-wise 0.05 significance were obtained for fat and protein yield, fat percentage, somatic cell score, and female fertility. Peak F-values were obtained at 29 cM for fat and protein yield and fat percentage, at 60 cM for somatic cell score, at 74 cM for herd life, and at 11 cM for female fertility. The 0.95 confidence intervals for quantitative trait loci locations were 20 cM for kilograms of fat, 27 cM for fertility, and 51 cM for somatic cell score. Two loci affecting fertility at opposite ends of the chromosome are apparently segregating in the population. A quantitative trait locus for fertility near the centromere was confirmed by application of the modified granddaughter design to a single family. Estimated frequency of the economically favorable allele in the Israeli Holstein cattle was less than 0.5. Significant genetic gain for fertility seems possible by marker-assisted selection.

Background: Many studies have found segregating quantitative trait loci (QTL) for milk production traits in different dairy cattle populations. However, even for relatively large effects with a saturated marker map the confidence interval for QTL location by linkage analysis spans tens of map units, or hundreds of genes. Combining mapping and arraying has been suggested as an approach to identify candidate genes. Thus, gene expression analysis in the mammary gland of genes positioned in the confidence interval of the QTL can bridge the gap between fine mapping and quantitative trait nucleotide (QTN) determination., Results: We hybridized Affymetrix microarray (MG-U74v2), containing 12,488 murine probes, with RNA derived from mammary gland of virgin, pregnant, lactating and involuting C57BL/6J mice in a total of nine biological replicates. We combined microarray data from two additional studies that used the same design in mice with a total of 75 biological replicates. The same filtering and normalization was applied to each microarray data using GeneSpring software. Analysis of variance identified 249 differentially expressed probe sets common to the three experiments along the four developmental stages of puberty, pregnancy, lactation and involution. 212 genes were assigned to their bovine map positions through comparative mapping, and thus form a list of candidate genes for previously identified QTLs for milk production traits. A total of 82 of the genes showed mammary gland-specific expression with at least 3-fold expression over the median representing all tissues tested in GeneAtlas., Conclusion: This work presents a web tool for candidate genes for QTL (cgQTL) that allows navigation between the map of bovine milk production QTL, potential candidate genes and their level of expression in mammary gland arrays and in GeneAtlas. Three out of four confirmed genes that affect QTL in livestock (ABCG2, DGAT1, GDF8, IGF2) were over expressed in the target organ. Thus, cgQTL can be used to determine priority of candidate genes for QTN analysis based on differential expression in the target organ.

We present a simple algorithm for reconstruction of haplotypes from a sample of multilocus genotypes. The algorithm is aimed specifically for analysis of very large pedigrees for small chromosomal segments, where recombination frequency within the chromosomal segment can be assumed to be zero. The algorithm was tested both on simulated pedigrees of 155 individuals in a family structure of three generations and on real data of 1149 animals from the Israeli Holstein dairy cattle population, including 406 bulls with genotypes, but no females with genotypes. The rate of haplotype resolution for the simulated data was >91% with a standard deviation of 2%. With 20% missing data, the rate of haplotype resolution was 67.5% with a standard deviation of 1.3%. In both cases all recovered haplotypes were correct. In the real data, allele origin was resolved for 22% of the heterozygous genotypes, even though 70% of the genotypes were missing. Haplotypes were resolved for 36% of the males. Computing time was insignificant for both data sets. Despite the intricacy of large-scale real pedigree genotypes, the proposed algorithm provides a practical rule-based solution for resolving haplotypes for small chromosomal segments in commercial animal populations.

We previously localized a quantitative trait locus (QTL) on chromosome 6 affecting milk fat and protein concentration to a 4-cM confidence interval, centered on the microsatellite BM143. We characterized the genes and sequence variation in this region and identified common haplotypes spanning five polymorphic sites in the genes IBSP, SPP1, PKD2, and ABCG2 for two sires heterozygous for this QTL. Expression of SPP1 and ABCG2 in the bovine mammary gland increased from parturition through lactation. SPP1 and all the coding exons of ABCG2 and PKD2 were sequenced for these two sires. The single nucleotide change capable of encoding a substitution of tyrosine-581 to serine (Y581S) in the ABCG2 transporter was the only polymorphism corresponding to the segregation status of all 3 heterozygous and 15 homozygous sires for the QTL in the Israeli and U.S. Holstein populations. The allele substitution fixed effects on the genetic evaluations of 335 Israeli sires were -341 kg milk, +0.16% fat, and +0.13% protein (F-value = 200). No other polymorphism gave significant effect for fat and protein concentration in models that also included Y581S. The allele substitution effects on the genetic evaluations of 670 cows, daughters of two heterozygous sires, were -226 kg milk, 0.09% fat, and 0.08% protein (F-value = 394), with partial dominance towards the 581S homozygotes. We therefore propose that Y581S in ABCG2 is the causative site for this QTL.

Twelve bull calves were produced by mating elite Israeli cows to "Glenhapton Enhancer", a Canadian Holstein bull. The frequency of umbilical hernia (UH) in the progeny of the sons ranged from 1 to 21%, consistent with the hypothesis that Enhancer is the carrier of major dominant or codominant gene with partial penetrance for UH. Five sons of Enhancer produced progeny with >10% frequency of UH including sire 3259, whereas progeny of three sons had <3% UH. A total of 116 grand-progeny of Enhancer, all progeny of 3259, were genotyped for 59 microsatellites spanning the 29 bovine autosomes. Of these offspring, 41 were affected. Significant differences in paternal allele frequencies between the affected and unaffected progeny groups were found for marker BMS1591 on bovine chromosome 8 (BTA8). The UH-associated paternal allele originated from Enhancer. The chromosomal segment associated with UH was more precisely mapped between UWCA47, on the centromeric end of BTA8 and RM321, 12 cM from the centromere. A maximum LOD score of 3.84 was obtained 2.5 cM from the centromere with a support interval of 8 cM. Haplotype analysis of eight sons of Enhancer suggested that the UH gene is located in the centromeric end of BTA8 beyond ARO71/ARO72. Thus, by integrating the results from progeny of sire 3259 and sons of Enhancer the location of the UH gene was further refined to the BTA8 segment between ARO71/ARO72 and UWCA47.

A cluster of genes coding for proteins of the extracellular matrix (ECM) containing sequence motifs essential for integrin-receptor interactions is located on HSA4q21 and on BTA6, within the critical region of a quantitative trait locus (QTL) affecting milk protein production. Genes within this cluster are involved in the formation of bone and lobuloalveolar structures in mammary gland and in kidney function. We cloned a bovine gene neighboring this ECM cluster, termed FAM13A1, the first member of a novel gene family (FAM13). A short predominant 5.1-kb mRNA variant capable of encoding 697 amino acids is transcribed from 18-exon orthologous genes in human and mouse. All putative protein orthologs contained a bipartite nuclear-localization signal and two coil-coiled domains. We detected two other FAM13 paralogs, C5ORF5 and FAM13C1, on HSA5q31 and HSA10q21, respectively. All FAM13 paralogs produce transcripts that are complementary to adjacent genes, suggesting that antisense transcription may regulate their functions. The structure of a longer 5.9-kb variant, FAM13A1&#95;v2, that has an extra 7-exon putative RhoGAP domain at the N-terminus provides further clues to FAM13 function. Analysis of 400 bulls revealed a population-wide linkage disequilibrium between FAM13A1 polymorphisms and the QTL.

Eleven Israeli Holstein families including 5221 cows were analyzed by a daughter design for eight economic traits: milk, fat and protein production, fat and protein percentage, somatic cell score (SCS), herd-life, and female fertility. The cows were genotyped for 73 microsatellites with maximum spacing between markers of 53 cM. There were 86,304 informative genotypes. Preliminary analysis was by ANOVA of each trait, with the marker effect nested within sire. Significance was determined by controlling the false discovery rate at 0.4, after excluding markers with genome-wide significance for at least a single trait, and traits without any significant effects at this level. Thus, four markers on chromosomes 6 and 14 and female fertility were excluded. There remained 40 significant marker-trait combinations, and it is expected that 24 of these are true effects. To perform interval mapping for the families with significant contrasts, 21 additional markers were genotyped on chromosomes 2, 7, and 27. The bootstrap confidence intervals for gene effect did not include zero for protein percent on chromosome 2 and fat yield, protein yield, and SCS on chromosome 7. Quantitative trait locus heterozygosity was 33%, which is consistent with the hypothesis that only two alleles are segregating with unequal allele frequency.

A population-wide linkage disequilibrium on bovine chromosome 14 between microsatellite ILSTS039 and DGAT1, a putative quantitative trait locus affecting milk production traits, was found in the Israeli Holstein population. A total of 394 bulls were genotyped for both DGAT1 and ILSTS039, and 1747 cows were genotyped for ILSTS039. The ILSTS039 allele termed "225," and the DGAT1 K allele (substitution of a lysine residue with alanine), were associated with decreased milk production, and increased fat production and fat and protein percent. The number of 225 ILSTS039 and K DGAT1 alleles per individual were the same for 80% of the bulls genotyped. From the effects associated with cows homozygous for the 225 allele, the effect of the quantitative trait locus appears to be approximately codominant. The substitution effect was 0.16% fat. Genotype probabilities for the quantitative gene were determined for the entire Israeli Holstein milk-recorded population, including 507,725 cows and 1442 bulls, using segregation analysis. Overall frequency of the allele that increased fat percent was 8.9% in cows and 15.5% in bulls. The frequency of this allele decreased from 1981 until 1990, from 15 to 5%, and since has increased to 10%. The effects estimated on the population-wide analyses of both cows and bulls were similar to the effect associated with DGAT1 in the daughters of genotyped bulls. Modified animal model evaluations were computed for the entire population with the effect of this gene included in the model. The correlations between the modified and standard animal model evaluations for all traits were > 0.99.

Blood is the standard source for DNA analysis, but requires venipuncture of cows by veterinarian and tedious and costly DNA extraction. A procedure was developed for sampling of vaginal cells from cows, establishing a cell lysis protocol using robotics, and applying fluorescent analysis of genetic markers. Two insemination technicians collected vaginal cells from 254 elite Israeli Holstein cows located in 152 herds using commercial Catch-All sample collection brushes. Cells were lysed in a 400-microliters solution, and 5 microliters was used as template for polymerase chain reaction (PCR). Sensitivity of the PCR was enhanced using only 1 microliter of lysed cells. Eight markers of the International Society of Animal Genetics paternity panel were amplified in four separate PCR. ILSTS039, a marker for a quantitative trait loci on BTA14, was amplified in a separate reaction. Genotypes from one to nine genetic markers were obtained for 253 out of 254 samples, and 244 cows had genotypes for at least three markers (96%). Cows that did not inherit either paternal allele for at least two loci were considered not to be daughters of the sire listed. Fifteen cows met this criterion, for a paternity misidentification rate of 6.25%. The frequency of allele 225 of ILSTS039, which was associated with increased milk fat content, was 11.1% in the bull-dam population, similar to the 12% found in the cow population in Israel. The use of vaginal cells for genetic analysis is accurate, as demonstrated by replicated analysis and by comparison to individual and population analysis based on DNA derived from blood.

Nine Israeli Holstein sire families with 2978 daughters were analyzed for quantitative trait loci effects on chromosome 6 for five milk production traits by a daughter design. All animals were genotyped for 2 markers. The three families with significant effects were genotyped for up to 10 additional markers spanning positions 0-122 cM of BTA6. Two sires were segregating for a locus affecting protein and fat percentage near position 55 cM with an estimated substitution effect of 0.18% protein, which is equivalent to one phenotypic standard deviation. This locus was localized to a confidence interval of 4 cM. One of these sires was also heterozygous for a locus affecting milk, fat, and protein production near the centromere. The hypothesis of two segregating loci was verified by multiple regression analysis. A third sire was heterozygous for a locus affecting milk and protein percentage near the telomeric end of the chromosome. Possible candidates for the major quantitative gene near position 55 cM were determined by comparative mapping. IBSP and SSP1 were used as anchors for the orthologous region on human chromosome 4. Twelve genes were detected within a 2-Mbp sequence. None of these genes have been previously associated with lactogenesis.