limited our analysis to transcripts that were expressed in at least 1/ 3 of the samples within each sex, using this liberal cut off so as not to omit transcripts that expressed in only one of the parental strains. The female sample had 21,798 expressed transcripts, with 17,986 (82%) of these transcripts expressed in at least 75% of the female samples. Due to presence of multiple probes for some genes, these 21,798 transcripts represent only 11,531 unique genes. We used the gene expression of each of these transcripts as a phenotype, along with a high density genome scan composed of 1,967 informative SNPs on a mouse SNP chip [15], to identify eQTLs associated with the expression level of each transcript in a genome-wide method of generating LOD plots for expression of each transcript across the mouse genome. We used an initial suggestive LOD cutoff of $3.0. With 2 degrees of freedom, this suggestive LOD threshold corresponds to a nominal p-value of 161023 [16]. We calculated a genome wide p-value of 0.25 for this LOD threshold by 1000 permutations each of 10 randomly selected female eQTLs with a LOD score of 3.00. We identified a total of 9,308 eQTLs in the female mice that met this suggestive LOD threshold, and applying our genome wide p-value at this threshold, we expect ,7000 of these eQTLs to be authentic. We characterized as cis eQTLs those in which the eQTL mapped on the same chromosome and within 20 Mb of the transcript location on the mouse genome. All of the remaining eQTLs were identified as either trans eQTLs (eQTL maps at a different locus than the transcript), or ambiguous eQTLs for which the Affymetrix probe target sequence matched to more than one genomic location. There were 1,859 cis eQTLs in the female mice, representing 20% of the total eQTLs, and their average LOD score was 8.77. There were 6,117 trans eQTLs in the female mice, representing 66% of the total eQTLs, and their average LOD score was 3.55. We also identified 1332 ambiguous eQTLs, representing 14% of the total eQTLs, with an average LOD score was 5.08. Table 1 provides a summary of the eQTL findings, and Supplemental Table S1 gives the details of each of the 9,308 female eQTLs, arranged by the genomic location of the eQTL. A similar eQTL analysis was performed for the male cohort with 21,733 expressed transcripts (representing 11,557 unique genes), with 17,632 (81% of these transcripts) expressed in at least 75% of the male samples. For the 19 autosomes and the X chromosome, we identified 12,361 eQTLs with a LOD score of $3.0. We calculated a genome wide p-value of 0.25 for this LOD threshold by 1000 permutations each of 10 male eQTLs with a LOD score of 3.00. There were 1,990 cis eQTLs, representing Sex Specific Gene Regulation 16% of the total male eQTLs, and their average LOD score was 9.46. There were 8,625 trans eQTLs, representing 70% of the total eQTLs, and their average LOD score was 3.64. There were also 1,746 ambiguous eQTLs, representing 14% of the total, with an average LOD score of 5.07. As our strain intercross used males from both strains, we also looked for eQTLs due to the grandparental Y chromosome, and we detected 1145 that met the LOD.3.0 threshold. 90% of these were trans eQTLs, 10% were ambiguous eQTLs, with only 4 potentially cis eQTLs, associated with probes mapping to the Y chromosome (Table 1). Supplemental Table S2 provides the details of each of the male eQTLs. Overall, as seen in prior eQTL studies using a liberal LOD threshold [2,12], there were many more trans eQTLs observed in both the female and male cohorts, but these had lower LOD scores than the cis eQTLs, presumably due to the more direct effect of cis variation in regulatory or transcribed regions on gene expression or mRNA stability. eQTL Hotspots We examined the distribution of these suggestive eQTLs over the mouse genome in partially overlapping 20 Mb bins. There were non-random distributions in both female and male cohorts. In the female samples there were 11 hotspots of eQTLs, each having over 200 eQTLs (.2% of all female eQTLs), with the largest peak on chromosome 7 in a bin that had 339 eQTLs (Figure 1A). There were 2 hotspots on the X chromosome in the females, and overall there were 861 eQTLs on the X chromosome (11 cis, 698 trans, and 170 ambiguous). In the males there were 13 hotspots with over 200 eQTLs (Figure 1B). The male data yielded three super hotspots each containing between 8.5 to 11.5% of all the male eQTLs, one near the proximal end of chromosome 1 containing 1316 eQTLs, one on chromosome 16 containing 1434 eQTLs, and one for the entire Y chromosome containing 1145 eQTLs (Fig 1B). Gene ontology analysis did not detect over representation in any functional group of the transcripts in the chromosome 1 and 16 eQTL hotspots compared to all male eQTLs. For the transcripts associated with the Y chromosome eQTL hotspot compared to all eQTL associated transcripts on the autosomes and the X chromosome, the gene ontology analysis found several over represented classes including the chromosome and extracellular cellular components, the cell cycle M phase and DNA metabolic biological processes, and the microtubule motor activity molecular function (see Supplemental Table S3 for full list and P values). There was only limited overlap of the eQTL . ... Table 1. eQTL summary in female and male F2 mice .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Females Males (autosomes, X Chr) Males Y Chr . .... Count Ave. LOD Count Ave. LOD Count Ave. LOD . ... LOD 3.0 .... Total eQTLs 9,308 4.81 12,361 4.78 1,145 4.06 ... Cis eQTLs 1,859 (20.0%) 8.77 1,990 (16.1%) 9.46 4 (0.35%) 5.71 . ... Trans eQTLs 6,117 (65.7%) 3.55 8,625 (69.8%) 3.64 1,026 (89.6%) 4.07 . ... Ambiguous eQTLs 1,332 (14.3%) 5.08 1,746 (14.1%) 5.07 115 (10.0%) 3.96 . .. LOD 4.3 . ... Total eQTLs 2,177 9.38 2,988 8.95 334 5.30 . ... Cis eQTLs 1,321 (60.7%) 10.89 1,445 (48.4%) 11.67 2 (0.60%) 7.86 .... Trans eQTLs 526 (24.2%) 5.14 1,086 (36.4%) 5.06 309 (92.5%) 5.29 ... Ambiguous eQTLs 330 (15.1%) 10.13 457 (15.3%) 9.6 23 (6.9%) 5.34 . . . .. doi:10.1371/journal.pone.0001435.t001 PLoS ONE | www.plosone.org 2 January 2008 | Issue 1 | e1435