Awad et al. Genetics Selection Evolution 2011, 43:8 http://www.gsejournal.org/content/43/1/8 and PY1, and between 121.800 Mb and 122.200 Mb (i.e. 0.400 Mb) for the QTL affecting only PY1. There were two additional peaks with LRT values over 15 in regions around the positions 115.650 and 116.300 Mb, but they were not included in the 97% confidence interval for PY1 and were not supported by the highly correlated MY1 trait. The two identified peaks (located between 118.107 Mb and 118.606 Mb and at 122.115 Mb, respectively) may be due to either the presence of more than one QTL, or the presence of one QTL with carryover effects to another region. Thus, a multiple-QTL analysis was per- formed. Two-QTL analyses using pedigree SNPPED421 for MY1 and PY1 fitting a QTL at position 118.202 Mb revealed a single QTL affecting only MY1 at this loca- tion and an additional QTL affecting PY1 at position 122.115 Mb (P = 0.019). However, two-QTL analyses accounting for the QTL at position 122.115 Mb did not rule out a possible second QTL affecting PY1 at position 118.202 Mb (P = 0.019). Discussion The aim of this study was to refine the position of a previously mapped QTL by increasing the marker den- sity in the region, target sampling of additional families and adapting fine mapping methods. According to our previous results [24] and to results from the initial part of this study, we hypothesized the presence of a minor QTL allele with a strong effect, but at a very low fre- quency, in the Fleckvieh dual-purpose cattle breed. In such a situation, random sampling of additional families for confirmation and fine-mapping purposes can result in an increased frequency of the common QTL allele in the mapping design Thus, the capacity to differentiate between genetic background noise and the initially tar- geted QTL will be decreased. The reduced accuracy of QTL position estimates when using all genotyped ani- mals (pedigrees MSPED2089 or SNPPED723) compared to a subset of animals (pedigrees MSPED1038 or SNPPED421) is counterintuitive to the general notion that the use of more information should result in better estimates. To further explore this unexpected result, we have investigated several possible explanations, including the effects of the haplotype distribution and the possibi- lity of additional QTL. To study the haplotype distribu- tion in the Fleckvieh population, 485 animals were genotyped with the Illumina 50 K SNP chip. Of these, a subset of 144 animals were not progeny-tested and not relevant for QTL mapping, but were very informative for the study of haplotype distribution. In particular, considering the putative QTL affecting MY1 and PY1 located within the 97% CI (between 117.962 Mb and 119.018 Mb), a haplotype of 25 markers (A0H1) covering this region was detected in 89 of 485 animals. This Page 9 of 11 haplotype A0H1, most probably carrying the minor QTL allele, could be traced back to the ancestor A0 in all 89 cases (Figure 1). The alternative haplotype A0H2, most probably carrying the common QTL allele, was found in 13 cases but was traced back to the ancestor A0 only in three. A perfect LD between the minor QTL allele and A0H1 (and only A0H1) would result in a relatively low allele frequency (0.137) of the minor QTL allele in phe- notyped animals of pedigree SNPPED723, and in a fre- quency about double (0.254) in pedigree SNPPED421. The mapping results did reflect this difference too. In contrast, consider the six markers located within the 97% CI (between 121.800 Mb and 122.200 Mb) of the putative QTL region affecting only PY1. Ancestor A0 is homozygous for a very long segment of this region i.e. from positions 118.266 Mb to 123.347 Mb (three SNP telomeric to the main peak of QTL affecting MY1 and PY1). This segment of 5.080 Mb includes 109 informative markers in the Fleckvieh population. Comparison of map- ping results from pedigrees SNPPED723(Figures 4), SNPPED421 (Figure 6),and SNPPED308 (Figure 5) revealed a highly significant QTL allele affecting PY1 only when the pedigree included families segregating for haplo- type A0H1 (see comparison between Figures 4 and 6). Excluding these families yielded LRT values below 3.99 (P > 0.045) for all three milk yield traits and for the com- plete investigated region (Figure 5, between 113.500 Mb and 123.700 Mb). We therefore mainly used the linkage information in the SNPPED421 pedigree (A0H1 always traceable to A0), to map a QTL affecting both MY1 and PY1 in a 97% CI of 1 Mb. Haplotype and LDL analyses by microsatellite markers (Figures 2 and 3) and SNP (Figures 4 and 6) clearly sug- gest that the minor QTL allele associated with the puta- tive QTL around the physical position 118 Mb (97% CI between 117.962 Mb to 119.018 Mb) has been intro- duced by ancestor A0 into the mapping population. The explanation of the second possible QTL that maps to the physical position 122.115 Mb and affects only PY1 is different. First, this QTL should also be associated with ancestor A0 haplotypes, i.e. absence of effect in the smaller SNPPED308 pedigree (Figure 5). Second, both ancestor haplotypes at the physical position 122.115 Mb are most probably identical by descent (i.e. homozygous for a 5.080 Mb segment with 109 informative SNP). Therefore, ancestor A0 is most probably homozygous for the putative QTL at this position too. Third, this part of the haplotype is not unique to A0, but also seg- regates in other families, i.e. there is LD information for mapping, too. The relatively sharp LRT peak at position 122.115 Mb and homozygosity of A0 suggest an essen- tial contribution of LD to this mapping result. Fourth, analyses with the two-QTL model did not rule out the possibility of a second QTL affecting PY1 within the