A) Single-locus two-allele model illustration of the mating design used to perform a test of hypothesis for paternal transgenerational genetic effects in isogenic mice. Where males heterozygous AA’ are mated to AA females. Resulting offspring are genotyped and only those with an AA genotype are compared to a control offspring on the right. B) Schematic representation of the cross performed for each B6.A/J–<N> CSS. Cross begins at F0 where a single male homozygous for each of chromosomes 15, 17, 19, and X, was mated to a single B6 female, and a single B6 control male is mated in parallel. The resulting males from the F1 were subsequently backcrossed with multiple B6 females, and N2 offspring genotyped for 236 SNP polymorphic between A/J and B6 to identify isogenic mice that inherit a complete B6 chromosome. Isogenic B6.A-<N> N2 B6/B6 are then compared to the control contemporary group of B6/B6 purebred mice. A persistence N3 cross was performed by selecting an isogenic B6.A-<N> N2 B6/B6 male mice and mated to multiple B6/B6 females and compared to a contemporary group of B6/B6 males. The only exception was B6.A-X in which B6.A-X F1 B6/B6 isogenic mice are obtained within a single generation.

Number of significant differentially expressed genes in discovery and validation data sets

Differential expression analysis of RNA-Seq across five tissues comparing B6.A-<N> B6/B6 and B6 controls. A) QQ-plots differential expression -log10 (p-values) of all genes compared across three cohorts, the Original N2 backcross (2012), a second independent Replicate N2 backcross (2013), and a persistence backcross N3 (2013). Within the N2 backcross, differential expression was measured three times. RNA-Seq of 50 libraries each composed of 4 mouse tissues, qRT-PCR of the same 200 mice for 53 genes with FDR ≤ 0.05 genes from RNA-Seq, and qRT-PCR of technically replicating genes on isogenic littermates from the same cohort. The latter set of technically replicating genes was measured in the Replicate N2, and Persistence N3 backcrosses. Colours represent the sire lineage. P-values were calculated by comparing each B6.A-<N> N2 B6/B6 isogenic lineage to B6 Controls. Except for Mid1, from left to right our p-values show a loss of replication based on the significance and the directionality of the effects across all differentially expressed genes. B) Number of genes significant per B6.A–<N> N2 B6/B6 vs B6.C comparison across the five tested tissues for each of the four tested CSS. B6.A-15 N2 B6/B6 vs B6.C had the highest amount of differentially expressed genes, particularly in the liver. B6.A-19 N2 B6/B6 vs B6.C had the second, totalling 14 genes, of which Mid1 was consistently differentially expressed across all 5 tissues.

A) Segmented copy number profiles from the sex chromosomes of the B6.A-19 F0 founder male. At the distal end of mouse chromosome (MMU) X – left panel–, results show a duplication on the XY pseudo-autosomal region (PAR) highlighted with a circle. However, no duplication is observed in the MMU Y PAR highlighted in a circle (right panel). B) A closer view of the distal ends of MMU X and Y pseudo-autosomal boundaries (PAB), reveals the duplication event spans the Mid1 gene, explaining the significant differential over-expression of B6.A-19 N2 B6/B6 isogenic mice when compared to their B6 contemporary group.