3. Design of a genotyping assay for detection of the 4.3kb SV and delimitation of its geographical distribution.
The availability of the 4.3kb structural variant sequence and its flanking region facilitated the design of a simple PCR for genotyping its presence or absence in laboratory and field samples. This assay consists of three primers, two flanking this SV and one within it ( Figure 1D), as previously designed for a nearby 6.5kb SV betweenCYP6P9a and b genes (Mugenzi et al., 2020). The assay was initially tested on Uganda (Tororo, 2014) and Cameroon (Mibellon, 2021) samples containing the insertion and showed a band at 780bp. Further genotyping of Uganda (Tororo 2014 and Mayuge, 2017) mosquito samples confirmed its presence and at a high frequency approaching fixation of the 4.3kb SV in Uganda mosquitoes with 100% frequency in Tororo and 97.83% in Mayuge with no mosquito found homozygous for the wild SV- allele (Figure 2A). Similar results were obtained for the Cameroon mosquito populations collected in Elon (2021), Mibellon (2021), Gounougou (2021) and Elende (2020), with SV+ allele frequencies of 94%, 100%, 98% and 94%, respectively (Figure 2A). Exploring its distribution in other localities across Africa revealed that it was absent from Ghana (West Africa) in 2014 and Mozambique (southern Africa) in 2015 and present at a very low frequency in East African Tanzania (3% SV+) in 2018 (Figure 2A). Genotyping of the An. funestusFUMOZ and FANG lab colonies showed that this structural variant was absent from those colonies.