Awad et al. Genetics Selection Evolution 2011, 43:8 http://www.gsejournal.org/content/43/1/8 single generation separating parents and progeny. Typi- cally, the size of confidence intervals for the most likely QTL positions ranges between 20 and 40 cM. Fine-mapping approaches have been developed to reduce these confidence intervals e.g. [3-5], leading in some instances to the identification of the underlying causal mutation [6-9]. These approaches are usually based on the addition of new families, new markers and the use of statistical methods combining linkage-disequi- librium and linkage (LDL) analysis. In general, the mar- ker density is increased by adding a few tens of new markers (microsatellite markers or single nucleotide polymorphism (SNP)) identified within the QTL region or candidate gene. At present, high-throughput SNP analysis provides the opportunity to genotype many animals for hundreds or even thousands of SNP per bovine chromosome [10-12]. Therefore, the limiting factors in QTL fine-mapping studies have now switched partly from marker density to the applied methods and designs. Use of linkage- disequilibrium (LD) information increases the precision of QTL mapping because it exploits the entire number of recombinations accumulated since the original muta- tion generating the new QTL allele occurred [13]. The degree of LD in livestock populations has attracted much attention because it provides useful information regarding the possibility of fine-mapping QTL and the potential to use marker-assisted selection. In cattle, previous reports using a low density microsatellite map (10 cM interval on average) and Hedrick’s normalized measure of LD [14] D’ have shown that LD extends over several tens of centimorgans [10,15,16]. However, an exceedingly low long-range and non-syntenic LD has been estimated [17] when evaluated by the standardized chi-square measure of LD, which is related to the predic- tive ability of LD. Nevertheless, the extent of LD in cattle [18] is greater than in humans [19] but smaller than in dog [20]. Combined linkage disequilibrium and linkage (LDL) analysis [3] makes it possible to exploit recombinations occurring both within and outside the pedigree and gen- otyped population. It also gives a clearer signal for QTL positions compared with LA or LD mapping alone [3]. Additionally, the LDL approach reduces the risk of false-positive QTL identification caused by accidental marker-phenotype associations when LA and LD are used separately, and also increases the power and reso- lution of QTL mapping by combining all available infor- mation [21]. In dairy cattle, several studies have reported the pre- sence of one or more QTL affecting milk production traits on BTA5 e.g. [22-25], but the results differ among studies with respect to the number of QTL detected, Page 2 of 11 their positions, and the extent to which the milk traits are affected by the QTL. The present study aimed at refining the previously detected QTL affecting milk yield (MY1), milk protein yield (PY1) and milk fat yield (FY1) during first lactation in the distal part of BTA5 in the Fleckvieh dual-purpose cattle breed [24], and to define the candidate region for high-throughput sequencing. To achieve this, we sampled additional families carrying the low frequency allele of the putative QTL (minor QTL allele) and geno- typed additional markers covering the most likely QTL region on BTA5. These new families were identified by combining results from QTL-mapping based on micro- satellites and haplotype analysis based on SNP in a com- plex pedigree. Single- and multiple-QTL analyses based on the LDL method were performed in different sam- ple-sets, in order to allocate the minor QTL allele to specific families and to use the increased frequency of the minor QTL allele for refined mapping. Methods Animals and phenotype In this study, we analysed the same nine granddaughter (GD) families used in our previous study [24], in which we identified three GD families (G01, G02 and G03) as heterozygous for a QTL located in the distal region of BTA5. The grandsires of these three GD families are designated as G01, G02 and G03, respectively. Grand- sires G01 and G02 are half-sibs and have inherited the same haplotype in the distal region of BTA5 from their common ancestor A0 [24]. By target sampling (see hap- lotyping section, below), we introduced two additional GD families; family G10 with 85 sons, and family G11 with 47 sons. Grandsire G10 (grandsire of family G10), was connected through his dam to A0. Grandsire G11 (grandsire of family G11) is a son of grandsire G02. In addition, we identified all available progeny-tested maternal grandsons of grandsires G01, G02, G10 and G11 to add more, possibly recombinant, A0 haplotypes into the mapping population. In this way, we created three maternal grandsire (MGS) families, M02 with 21 grandsons, M10 with 32 grandsons and M11 with 33 grandsons, descendants of grandsires G02, G10 and G11, respectively. Samples of maternal grandsons were not available for grandsire G01. Thus, the analysis included 11 GD families: G01 to G11 and three MGS families (M02, M10 and M11). Figure 1 shows the rela- tionships of all families included in this study. In some cases, mapping analyses were carried out on 173 addi- tional animals available from other projects that are not descended from ancestor A0. Estimated breeding values (EBV) of the Fleckvieh bulls for milk production traits MY1, PY1, and FY1, (along with their reliability values)