6. Impact of 4.3kb structural variant insertion on the expression of nearby genes.
To assess potential effect of this structural variant on the expression of nearby genes (CYP6P5, CYP6P9a, and CYP6P9b ), crosses between field samples (Elende, fully homozygous for the 4.3kb SV) and the fully susceptible FANG lab strain (4.3kb SV completely absent) were intercrossed to the F3 generation. Quantitative real-time PCR performed on pools of each of the 3 genotypes (SV+/SV+, SV+/SV- and SV-/SV-) relative to FANG revealed increased expression ofCYP6P9a (downstream) and CYP6P9b (immediately downstream) but not of CYP6P5 (upstream) in the SV+/SV+ pool only (Figure 4A). CYP6P9a was most expressed in SV+/SV+ with fold change (FC) of 18.7 (P-value = 0.008), while SV+/SV- and SV-/SV- genotypes showed no differential expression. Similarly, CYP6P9b’s expression was higher in the SV+/SV+ genotype (FC=16.0, P value = 0.002) and low in the SV+/SV- and SV-/SV- genotypes. This could indicate that possessing 2 copies of this 4.3kb SV further enhances the expression of these genes. For CYP6P5 , there was no difference in the expression level for the different genotypes. Screening for transcription factors known to regulate detoxification genes in the 4.3kb SV using CiiiDER software identified Ahr, ARNT and MAF binding sites. For Ahr/Arnt, 4 binding sites were identified at positions 183-188, 3449-3454 and 4283-4288, while for 14 MAF binding sites were determined with 3 for MAFG, 2 for MAFF and 9 for MAFb.