Relative cis-regulatory activity is independent of trans-regulatory variation. (A) X, second, and third chromosome genotypes of flies crossed to generate different classes of F₁ hybrids are shown. “In” indicates chromosomes from the In(1)AB strain and “zhr” indicates chromosomes from the zhr strain. For each of the three hybrid classes, two pools, each containing 14 adult female flies (7–10 days old) were analyzed. RNA and genomic DNA were sequentially extracted from each pool. cDNA was synthesized and used to measure expression of the eight autosomal genes using pyrosequencing. Allele abundance in genomic DNA samples was also measured and used to normalize measurements as described in Landry et al. (2005). After normalization, the log₂ ratios of allelic expression were fitted to the mixed linear model with a fixed effect of genotype (B, H, and R) and a random effect of replicate fly pools using proc MIXED in SAS v.8.2. (B) Least-squares means with their standard errors from this model are shown. In all cases, no significant difference was observed among the three hybrid genotypes (P > 0.2).

trans-regulatory variation affects levels of gene expression. (A) X, second, and third chromosome genotypes present in the F and I pools are shown, with “In” indicating chromosomes from the In(1)AB strain and “zhr” indicating chromosomes from the zhr strain. Note that these genotypic combinations differ only by the presence or absence of the FM7 X chromosome. For both the heterozygous (F) and homozygous (I) X chromosome genotypes, the relative abundance of autosomal transcripts from the zhr and In(1)AB alleles was measured in four replicate pools of adult flies. Each pool contained seven adult females (7–10 days old) from the zhr line and seven females from the In(1)AB line, either with (F) or without (I) the FM7 X chromosome. After sequentially extracting RNA and genomic DNA from each pool, duplicate cDNA pools were synthesized and used to measure expression of eight autosomal genes using pyrosequencing. Allele abundance in the genomic DNA samples was also measured in duplicate and used to normalize measurements as described in Landry et al. (2005). After normalization, the log₂ expression ratios of the In(1)AB allele to the zhr allele were fitted to a mixed linear model with a fixed effect of genotype (F and I) and a random effect of replicate fly pools using “proc MIXED” in SAS v.8.2 (Cary, NC). (B) Least-squares means and significance tests from this model are shown. Asterisks indicate cases where P < 0.06 and error bars indicate standard errors. The selection of the nontraditional α = 0.06 is explained in the main text.