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.