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We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP., (© 2012 The Authors, Animal Genetics © 2012 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.)

Selective DNA pooling (SDP) is a cost-effective means for an initial scan for linkage between marker and quantitative trait loci (QTL) in suitable populations. The method is based on scoring marker allele frequencies in DNA pools from the tails of the population trait distribution. Various analytical approaches have been proposed for QTL detection using data on multiple families with SDP analysis. This article presents a new experimental procedure, fractioned-pool design (FPD), aimed to increase the reliability of SDP mapping results, by "fractioning" the tails of the population distribution into independent subpools. FPD is a conceptual and structural modification of SDP that allows for the first time the use of permutation tests for QTL detection rather than relying on presumed asymptotic distributions of the test statistics. For situations of family and cross mapping design we propose a spectrum of new tools for QTL mapping in FPD that were previously possible only with individual genotyping. These include: joint analysis of multiple families and multiple markers across a chromosome, even when the marker loci are only partly shared among families; detection of families segregating (heterozygous) for the QTL; estimation of confidence intervals for the QTL position; and analysis of multiple-linked QTL. These new advantages are of special importance for pooling analysis with SNP chips. Combining SNP microarray analysis with DNA pooling can dramatically reduce the cost of screening large numbers of SNPs on large samples, making chip technology readily applicable for genomewide association mapping in humans and farm animals. This extension, however, will require additional, nontrivial, development of FPD analytical tools.

Many large, half-sib sire families are an integral component of chicken genetic improvement programs. These family structures include a sufficient number of individuals for mapping quantitative trait loci (QTL) at high statistical power. However, realizing this statistical power through individual or selective genotyping is yet too costly to be feasible under current genotyping methodologies. Genotyping costs can be greatly reduced through selective DNA pooling, involving densitometric estimates of marker allele frequencies in pooled DNA samples. When using dinucleotide microsatellite markers, however, such estimates are often confounded by overlapping "shadow" bands and can be confounded further by differential amplification of alleles. In the present study a shadow correction procedure provided accurate densitometric estimates of allele frequency for dinucleotide microsatellite markers in pools made from chicken purified DNA samples, fresh blood samples, and frozen-thawed blood samples. In a retrospective study, selective DNA pooling with thawed blood samples successfully identified two QTL previously shown by selective genotyping to affect resistance in chickens to Marek's disease. It is proposed that use of selective DNA pooling can provide relatively low-cost mapping and use in marker-assisted selection of QTL that affect production traits in chickens.

Selective DNA pooling was employed in a daughter design to screen all bovine autosomes for quantitative trait loci (QTL) affecting estimated breeding value for milk protein percentage (EBVP%). Milk pools prepared from high and low daughters of each of seven sires were genotyped for 138 dinucleotide microsatellites. Shadow-corrected estimates of sire allele frequencies were compared between high and low pools. An adjusted false discovery rate (FDR) method was employed to calculate experimentwise significance levels and empirical power. Significant associations with milk protein percentage were found for 61 of the markers (adjusted FDR = 0.10; estimated power, 0.68). The significant markers appear to be linked to 19--28 QTL. Mean allele substitution effects of the putative QTL averaged 0.016 (0.009--0.028) in units of the within-sire family standard deviation of EBVP% and summed to 0.460 EBVP%. Overall QTL heterozygosity was 0.40. The identified QTL appear to account for all of the variation in EBVP% in the population. Through use of selective DNA pooling, 4400 pool data points provided the statistical power of 600,000 individual data points.

Somatic cells in milk were used as a source of DNA and as a substrate for the polymerase chain reaction. Successful DNA extraction required a minimum total of 17 x 10(6) cells in the milk sample. The DNA yield per cell was highly variable but comparable, on average, with the yield from peripheral blood leukocytes. In all cases, direct polymerase chain reaction on milk samples, using a variety of primer pairs, yielded amplified products of correct size that were identical to those obtained by polymerase chain reaction of purified DNA extracted from milk or blood. Milk and DNA extracted from milk served as substrates for direct sequencing of part of the bovine growth hormone gene. Milk samples with added preservative were stored over 200 d without effect on the polymerase chain reaction. Thus, milk can often substitute for blood as a source of DNA for Southern blot analysis and is a preferred substrate for the polymerase chain reaction. The technical convenience of milk as a source of DNA can be expected to increase the field of application of marker-based methods for genetic analysis and genetic improvement of economic traits in dairy cattle.