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.

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.

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.

“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.