Isolation of rfk-2 (UV) , a mutation that blocks spore killing by Neurospora Spore killer-3

Neurospora Spore killer-3 ( Sk-3 ) is a selfish genetic element that kills spores to achieve gene drive.  Here, to help identify Sk-3’s killer, we performed a genetic screen for required for killing (rfk) mutations (see methods). The genetic screen uses Sk‑3 rskΔ × SkS crosses, which abort development before the production of viable ascospores (Hammond et al. 2012; Harvey et al. 2014). We isolated a few candidate rfk mutations with our genetic screen and chose the most promising candidate, rfk-2UV, for additional analysis. As demonstrated in Figure 1 (A and B), rfk-2UV disrupts spore killing and gene drive. To determine the approximate genomic location of rfk-2UV, we performed two sets of three-point crosses (Figure 1C). Recombination analysis of 213 offspring from the first set of crosses (rfk-2UV hphA × mus-52Δ) indicates that rfk-2UV islocated 2.8 cM from hphA and 15.0 cM from mus-52Δ (Figure 1D). For the second set of crosses (rfk-2UV hphB × mus-52Δ), recombination analysis of 186 offspring indicates that rfk-2UV is located 7.5 cM from hphB and 16.1 cM from mus-52Δ (Figure 1E).  The significance of this discovery with respect to Sk-3 evolution is discussed.

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