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Background: Mastitis is a major disease of dairy cattle occurring in response to environmental exposure to infective agents with a great economic impact on dairy industry. Somatic cell count (SCC) and its log transformation in somatic cell score (SCS) are traits that have been used as indirect measures of resistance to mastitis for decades in selective breeding. A selective DNA pooling (SDP) approach was applied to identify Quantitative Trait Loci (QTL) for SCS in Valdostana Red Pied cattle using the Illumina Bovine HD BeadChip., Results: A total of 171 SNPs reached the genome-wide significance for association with SCS. Fifty-two SNPs were annotated within genes, some of those involved in the immune response to mastitis. On BTAs 1, 2, 3, 4, 9, 13, 15, 17, 21 and 22 the largest number of markers in association to the trait was found. These regions identified novel genomic regions related to mastitis (1-Mb SNP windows) and confirmed those already mapped. The largest number of significant SNPs exceeding the threshold for genome-wide significant signal was found on BTA 15, located at 50.43-51.63 Mb., Conclusions: The genomic regions identified in this study contribute to a better understanding of the genetic control of the mastitis immune response in cattle and may allow the inclusion of more detailed QTL information in selection programs.

A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ(9) -desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ(9) -desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents., (© 2014 Stichting International Foundation for Animal Genetics.)

Mastitis is an important and common dairy cattle disease affecting milk yield, quality, and consumer safety as well as cheese yields and quality. Animal welfare and residues of the antibiotics used to treat mastitis cause public concern. Considerable genetic variation may allow selection for increased resistance to mastitis. Because of high genetic correlation to milk somatic cell score (SCS), SCS can serve as a surrogate trait for mastitis resistance. The present study intended to identify quantitative trait loci (QTL) affecting SCS in Israeli and Italian Holstein dairy cattle (IsH and ItH, respectively), using selective DNA pooling with single and multiple marker mapping. Milk samples of 4,788 daughters of 6 IsH and 7 ItH sires were used to construct sire-family high- and low-tail pools, which were genotyped at 123 (IsH) and 133 (ItH) microsatellite markers. Shadow correction was used to obtain pool allele frequency estimates. Frequency difference between the tails and empirical standard error of D, SE(D), were used to obtain P-values. All markers significant by single marker mapping were also significant by multiple marker mapping, but not vice versa. Combining both populations, 22 QTL on 21 chromosomes were identified; all corresponded to previous reports in the literature. Confidence intervals were set by chi-squared drop method. Heterozygosity of QTL was estimated at 44.2%. Allele substitution effects ranged from 1,782 to 4,930 cells/mL in estimated breeding value somatic cell count units. Most (80%) of the observed variation in estimated breeding value somatic cell score could be explained by the QTL identified under the stringent criteria. The results found here can be used as a basis for further genome-wide association studies for the same trait., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Great interest was aroused by reports, based on microsatellite markers, of high levels of statistically significant long-range and nonsyntenic linkage disequilibrium (LD) in livestock. Simulation studies showed that this could result from population family structure. In contrast, recent SNP-based studies of livestock populations report much lower levels of LD. In this study we show, on the basis of microsatellite data from four cattle populations, that high levels of long-range LD are indeed obtained when using the multi-allelic D' measure of LD. Long-range and nonsyntenic LD are exceedingly low, however, when evaluated by the standardized chi-square measure of LD, which stands in relation to the predictive ability of LD. Furthermore, specially constructed study populations provided no evidence for appreciable LD resulting from family structure at the grandparent level. We propose that the high statistical significance and family structure effects observed in the earlier studies are due to the use of large sample sizes, which accord high statistical significance to even slight deviations from asymptotic expectations under the null hypothesis. Nevertheless, even after taking sample size into account, our results indicate that microsatellites testify to the presence of usable LD at considerably wider separation distances than SNPs, suggesting that use of SNP haplotypes may considerably increase the usefulness of a given fixed SNP array.

To fine map a quantitative trait locus (QTL) affecting milk production traits previously associated with microsatellite RM188, we implemented an interval mapping analysis by using microsatellite markers in a large Israeli Holstein half-sib sire family, and linkage disequilibrium (LD) mapping in a large set of US Holstein bulls. Interval mapping located the target QTL to the near vicinity of RM188. For the LD mapping, we identified 42 single nucleotide polymorphisms (SNP) in 15 genes in a 12-Mb region on bovine chromosome 4. A total of 24 tag SNP were genotyped in 882 bulls belonging to the University of California Davis archival collection of Holstein bull DNA samples with predicted transmitted ability phenotypes. Marker-to-marker LD analysis revealed 2 LD blocks, with intrablock r(2) values of 0.10 and 0.46, respectively; outside the blocks, r(2) values ranged from 0.002 to 0.23. A standard additive/dominance model using the generalized linear model procedure of SAS and the regression module of HelixTree software were used to test marker-trait associations. Single nucleotide polymorphism 9 on ARL4A, SNP10 on XR_027435.1, SNP12 on ETV1, SNP21 on SNX13, and SNP24 were significantly associated with milk production traits. We propose the interval encompassing ARL4A and SNX13 genes as a candidate region in bovine chromosome 4 for a concordant QTL related to milk protein traits in dairy cattle. Functional studies are needed to confirm this result.

Protein yield (PY) is currently the major economic product of the dairy herd. Genome-wide scans for quantitative trait loci (QTL) affecting milk yield (MY) and milk protein percentage (PP) suggest that of the loci affecting the 2 traits, about 1/4 exclusively affect MY, 1/4 exclusively affect PP, and half affect both traits. Because PY is the product of MY and PP, it is of interest to evaluate the expected effects on PY of marker-assisted selection (MAS) applied to these 3 classes of QTL. It is clear that selection for the appropriate allele at QTL exclusively affecting MY or PP will have a positive effect on PY. The question arises as to the effect of MAS directed at QTL affecting both MY and PP. Because the observed genetic correlation of about -0.5 between MY and PP must be generated by these loci, and because they comprise about half the total number of loci affecting the 2 traits, it can be inferred that the genetic correlation between MY and PP at loci affecting both traits is close to -1.0. This seems to imply that generally such loci would be neutral in their effects on PY. In the present study, biometrical expressions originally developed to describe the relationships of MY, fat percentage, and fat yield were adapted to describe the relationships of MY, PP, and PY. The resultant expressions were validated by showing that they correctly predicted the observed phenotypic standard deviation and heritability of PY, and the vastly different genetic correlations of PY with MY (very high positive) and of PY with PP (very low positive). Contrary to initial impressions, further biometrical analysis of the projected effects on PY of MAS at the loci affecting both traits, showed that even under the assumption that the genetic correlation between MY and PP at these loci is -1.0, selection for the allele favoring MY will have a strong positive effect on PY, whereas selection for the allele favoring PP will have an equal but opposite negative effect on PY. These diametrically opposed effects are due to the lower genetic coefficient variation of PP compared with MY. It is speculated that the reduced coefficient of variation of PP may be because of more stringent homeostatic buffering of milk composition compared with milk yield.

Fibroblast growth factor 2 (FGF2) is expressed in the bovine mammary gland and may play a role in the development and reorganization of the mammary gland. It is also expressed by the uterine endometrium throughout the estrous cycle and early pregnancy. The FGF2 was chosen for this study because it regulates the expression of interferon-tau, a key member of the signal transduction pathway involved in milk production. In previous studies, we reported the association of several genes in this pathway with milk production and health traits in dairy cattle. The objective of this study was to examine the association of FGF2 polymorphisms with milk composition, somatic cell score, and productive life in 3 Holstein cattle populations from the United States and Israel. The pooled DNA sequencing approach was used to identify single nucleotide polymorphisms (SNP) in FGF2. Sequencing of a total of 6.4 kb including 3 exons of the gene revealed only one SNP (A/G) in intron 1 at position 11646. This SNP was investigated for association with production traits in 2,773 individuals from 3 Holstein populations: the granddaughter-design Cooperative Dairy DNA Repository and the daughter-design University of Wisconsin populations from the United States and a daughter-design population from Israel. For both the Israeli and the UW populations, FGF2 variants were associated with fat yield and percentage, somatic cell score, and productive life with significant dominance and complete dominance effects. For the Cooperative Dairy DNA Repository population, no significant associations were observed for the examined traits. Given that FGF2 was chosen for this study because of its role in the interferon-tau signal transduction pathway and was found to be associated with production traits, results suggest that the candidate pathway could be an attractive strategy to search for candidate quantitative trait genes.

Although numerous quantitative trait loci (QTL) mapping studies involving milk protein percent (PP), milk yield (MY), and protein yield (PY) have been carried out, there has not been any systematic evaluation of the effects of individual QTL on these 3 interrelated traits. Consequently, the aim of the present study was to investigate the effects on MY and PY of QTL for PP previously mapped in various laboratories. The study, based on selective DNA pooling of milk samples, included 10 Israeli Holstein artificial insemination bulls, each the sire of 1,800 or more milk-recorded daughters. For each sire-trait combination across the 10 sires, milk samples of the highest and lowest daughters with respect to estimated breeding values for PP, PY, and MY were collected for pooling. A total of 134 dinucleotide microsatellites distributed over 25 bovine autosomes were used. An empirical standard error for marker-QTL linkage testing was calculated based on the variation among split samples within the same tail. Threshold comparison-wise error rate P-values were set to control proportion of false positives at P = 0.10 level for declaring significant effects at the marker-trait level. Estimates of the number of true null hypotheses for each trait were obtained from the histogram of marker comparison-wise error rate P-values. Based on these estimates, effective power of the experiment at the marker-trait level was estimated as 0.75, 0.41, and 0.73 for PP, PY, and MY. The proportion of heterozygosity at the QTL was estimated as 0.46, 0.39, and 0.40, respectively. After correcting for incomplete power and proportion of false positives, it was estimated that 38.7 and 37.5% of the markers affecting PP and MY, respectively, also affected PY. Of the markers affecting PY, 68.9 and 76.5%, respectively, also affected PP and MY. Apparently, none of the significant markers affected PY exclusively, and only 6.5 and 16.0%, respectively, affected PP or MY exclusively. Thus, almost all significant markers, and by inference almost all QTL, had effects on at least 2 of the 3 traits.

Quantitative trait loci (QTL) mapping projects have been implemented mainly in the Holstein dairy cattle breed for several traits. The aim of this study is to map QTL for milk yield (MY) and milk protein percent (PP) in the Brown Swiss cattle populations of Austria, Germany, and Italy, considered in this study as a single population. A selective DNA pooling approach using milk samples was applied to map QTL in 10 paternal half-sib daughter families with offspring spanning from 1,000 to 3,600 individuals per family. Three families were sampled in Germany, 3 in Italy, 1 in Austria and 3 jointly in Austria and Italy. The pools comprised the 200 highest and 200 lowest performing daughters, ranked by dam-corrected estimated breeding value for each sire-trait combination. For each tail, 2 independent pools, each of 100 randomly chosen daughters, were constructed. Sire marker allele frequencies were obtained by densitometry and shadow correction analyses of 172 genome-wide allocated autosomal markers. Particular emphasis was placed on Bos taurus chromosomes 3, 6, 14, and 20. Marker association for MY and PP with a 10% false discovery rate resulted in nominal P-values of 0.071 and 0.073 for MY and PP, respectively. Sire marker association tested at a 20% false discovery rate (within significant markers) yielded nominal P-values of 0.031 and 0.036 for MY and PP, respectively. There were a total of 36 significant markers for MY, 33 for PP, and 24 for both traits; 75 markers were not significant for any of the traits. Of the 43 QTL regions found in the present study, 10 affected PP only, 8 affected MY only, and 25 affected MY and PP. Remarkably, all 8 QTL regions that affected only MY in the Brown Swiss, also affected MY in research reported in 3 Web-based QTL maps used for comparison with the findings of this study (http://www.vetsci.usyd.edu.au/reprogen/QTL_Map/; http://www.animalgenome.org/QTLdb/cattle.html; http://bovineqtl.tamu.edu/). Similarly, all 10 QTL regions in the Brown Swiss that affected PP only, affected only PP in the databases. Thus, many QTL appear to be common to Brown Swiss and other breeds in the databases (mainly Holstein), and an appreciable fraction of QTL appears to affect MY or PP primarily or exclusively, with little or no effect on the other trait. Although QTL information available today in the Brown Swiss population can be utilized only in a within family marker-assisted selection approach, knowledge of QTL segregating in the whole population should boost gene identification and ultimately the implementation and efficiency of an individual genomic program.

SSCP analysis of the bovine growth hormone (bGH) gene in Israel Holstein dairy cattle uncovered five intragenic haplotypes, denoted A to E. Of these, Haplotype E differed from the others at six fragments; one of which corresponded to the polymorphic MspI site in intron III, at which haplotype E carried the disabled MspI (-) allele. Haplotype E was observed in a single sire only, carrying haplotype A as the second bGH allele. In 523 daughters of this sire genotyped for the MspI polymorphism, heterozygous (+/-) as compared to homozygous (+/+) daughters, showed a significant increasing effect on protein percentage and kg protein per year; and a decreasing effect (P < 0.10) on milk somatic cell counts (MSSC). None of the daughters were homozygous (-/-), indicating that the frequency of this allele in the general population was essentially zero. Calculated skewness (g1) values for the two daughter groups differed significantly with (+/-) daughters showing negative skewness (in the direction of lower protein percentage), and (+/+) daughters positive skewness (in the direction of higher protein percentage). The direction of skewness in each group is indicative of the presence of a QTL having an increasing effect on milk protein percentage in coupling linkage with the MspI (-) allele in this sire, but at some distance from it. Maximum likelihood estimates of the proportion of recombination (r) between the putative QTL and bGH, and the allele substitution effect at the QTL (d), were r = 0.33, a = 0.07% protein, with standard errors 0.058 and 0.009% protein, respectively.

"Selective DNA pooling" accomplishes quantitative trait locus (QTL) mapping through densitometric estimates of marker allele frequencies in pooled DNA samples of phenotypically extreme individuals. With poly(TG) microsatellites, such estimates are confounded by "shadow" ("stutter") bands. A correction procedure was developed on the basis of an observed linear regression between shadow band intensity and allele TG repeat number. Using this procedure, a selective DNA pooling study with respect to milk protein percentage was implemented in Israel-Holstein dairy cattle. Pools were prepared from milk samples of high and low daughters of each of seven sires and genotyped with respect to 11 markers. Highly significant associations with milk protein percentage were found for 5 of the markers; 4 of these markers confirmed previous reports. Selective DNA pooling accessed 80.6 and 48.3%, respectively, of the information that would have been available through individual selective genotyping or total population genotyping. In effect, the statistical power of 45,600 individual genotypings was obtained from 328 pool genotypings. This methodology can make genome-wide mapping of QTL accessible to moderately sized breeding organizations.