and 1452349_x_at detect Ifi204 and Mnda transcripts in B6 but not in D2. The expression difference is robust enough to generate cis-QTLs with very high LOD scores (.40). This gene cluster has low expression in the CNS (Affymetrix declares this probe sets to be ‘‘not present’’), but high expression in tissues such as hematopoietic stem cells and kidney, in which the trans-effect of Qrr1 is not detected. The Ifi200 gene cluster was therefore excluded as a high priority candidate. cis-QTLs in Qrr1 Transcripts of 26 of the 35 selected candidate genes map as cis- QTLs (LOD$3) in the BXD CNS datasets (table 3). These Table 3. Candidate genes in Qrr1. Mb nsSNPa Expb BXDc B6C3HF2c CXBc LXSc Gene Fcgr3 Sdhc Pcp4l1 Apoa2 Fcer1g Ndufs2 Adamts4 B4galt3 Ppox Usp21 Ufc1 Dedd Nit1 Pfdn2 Usf1 Refbp2 Vangl2 Ncstn Copa Pex19 Wdr42a Pea15 Atp1a2 Igsf8 Kcnj9 Kcnj10 Tagln2 Dusp23 Dfy Igsf4b Fmn2 Grem2 Rgs7 Tomm40l 173.148 Arhgap30 173.319 4 172.981 2 173.059 2 173.103 173.155 173.160 173.165 2 173.181 1 173.201 173.207 173.212 173.219 173.260 173.272 1 173.276 173.342 173.434 2 173.935 173.996 174.013 1 174.057 1 174.078 174.127 174.202 174.243 174.251 174.430 174.561 175.262 175.264 174.271 1 176.419 3 176.764 176.989 8.2 12.3 8.7 9.67 7.2 8.5 13.6 8.1 9.5 7.8 9.0 10.8 9.7 9.8 12.8 7.6 7.5 9.7 7.6 8.5 12.7 9.9 10.3 14.1 15.4 12.1 9.1 11.2 8.8 7.4 10.3 10.6 10.4 8.2 11.5 cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis cis QTL Hotspot on Mouse Distal Chromosome 1 putatively cis-regulated genes are among the strongest candidates in the QTL interval. The D allele in Qrr1 has the positive effect on the expression of Sdhc, Ndufs2, Adamts4, Dedd, Pfdn2, Ltap, Pea15, Atp1a2, Kcnj9, Kcnj10, Igsf4b, and Grem2. Increase in expression caused by the D allele ranges from about 10% for Adamts4 to over 2-fold for Atp1a2. In contrast, the B allele has the positive effect on the expression of Pcp4l1, Fcer1g, B4galt3, Ppox, Ufc1, Nit1, Usf1, Copa, Pex19, Wdr42a, Igsf8, Dfy, Fmn2, and Rgs7. Increase in expression caused by the B allele ranges from about 7% for Usf1 to 40% for Pex19. Individual probes were screened to assess if the strong cis-effects are due to hybridization artifacts caused by SNPs in probe targets. Thirteen candidate genes with cis-QTLs were then selected for further analysis and validation of cis-regulation by measuring allele specific expression (ASE) difference [56]. This method exploits transcribed SNPs, and uses single base extension to assess expression difference in F1 hybrids. By means of ASE, we validated the cis-regulation of 10 candidate genes—Ndufs2, Nit1, Pfdn2, Usf1, Copa, Atp1a2, Kcnj9, Kcnj10, Dfy, and Fmn2 (table 4). Adamts4 and Igsf4b failed to show significant allelic expression difference. In the case of Ufc1, the polarity of the allele effect failed to agree with the ASE result (D positive at p-value = 0.02). High-Resolution cis-QTL Mapping The BXD CNS datasets were generated from a combined panel of conventional RI strains and advanced RI strains that were derived by inbreeding advanced intercross progeny. The advanced RIs have approximately twice as many recombinations compared to standard RIs and the merged panel offers over a 3-fold increase in mapping resolution [57]. This expanded RI set combined with the relatively high intrinsic recombination rate within Qrr1 [58] provides comparatively high mapping resolution. Mapping precision can be empirically determined by analyzing cis-QTLs in multiple large datasets, particularly the BXD Hippocampus Consortium, UMUTAffy Hippocampus, and Hamilton Eye datasets. These three datasets were selected because they have expression measurements from six BXD strains with recombina- tions in Qrr1. These strains—BXD8, BXD29, BXD62, BXD64, BXD68, and BXD84—collectively provide six sets of informative markers and divide Qrr1 into six non-recombinant segments, labeled as segments 1–6 (haplotype structures shown in figure 2). As cis-acting regulatory elements are usually located within a few kilobases of a gene’s coding sequence [59], we used the cis- QTLs as an internal metric of mapping precision by measuring the offset distance between a cis-QTL (position of peak QTL marker) and the parent gene (figure 3). For cis-QTLs with LOD scores between 3–4 (genome-wide p-value of 0.1–0.01) the mean gene-to- QTL peak distance is 900 kb. The offset decreases to a mean of 640 kb for cis-QTLs with LOD scores greater than 4 (p- value,0.001). Very strong cis-QTLs with LOD scores greater than 11 (p-value,1026) have a mean gene-to-QTL peak distance of only 450 kb. In all, 60% of cis-QTLs we examined have peak linkage on markers located precisely in the same non-recombinant segment as the parent gene, and 30% have peak linkage on markers in a segment adjacent to the parent gene (dataset S1). These cis-QTLs provide an empirical metric of mapping precision within Qrr1. Parsing trans-QTLs by High-Resolution Mapping and Gene Functions Mapping precision of cis-QTLs is comparatively higher in the BXD hippocampus dataset (average offset of only 410 kb), and we used this set to examine the trans-QTLs (LOD$3) at higher resolution. The trans-QTLs in Qrr1 were parsed into subgroups aNumber of missense mutations between B and D alleles. bMean expression signal of probe sets in BXD Hippocampus PDNN dataset; below 7 is considered to be below background. cCis-QTLs in BXD, B6C3HF2, CXB, and LXS crosses. doi:10.1371/journal.pgen.1000260.t003 PLoS Genetics | www.plosgenetics.org 5 November 2008 | Volume 4 | Issue 11 | e1000260