<i>KPOK3A</i> polymorphisms.

Gamete killers are genetic loci that distort segregation in the progeny of hybrids because the killer allele promotes the elimination of the gametes that carry the sensitive allele. They are widely distributed in eukaryotes and are important for understanding genome evolution and speciation. We had previously identified a pollen killer in hybrids between two distant natural accessions of Arabidopsis thaliana. This pollen killer involves three genetically linked genes, and we previously reported the identification of the gene encoding the antidote that protects pollen grains from the killer activity. In this study, we identified the two other genes of the pollen killer by using CRISPR-Cas9 induced mutants. These two genes are necessary for the killer activity that we demonstrated to be specific to pollen. The cellular localization of the pollen killer encoded proteins suggests that the pollen killer activity involves the mitochondria. Sequence analyses reveal predicted domains from the same families in the killer proteins. In addition, the C-terminal half of one of the killer proteins is identical to the antidote, and one amino acid, crucial for the antidote activity, is also essential for the killer function. Investigating more than 700 worldwide accessions of A. thaliana, we confirmed that the locus is subject to important structural rearrangements and copy number variation. By exploiting available de novo genomic sequences, we propose a scenario for the emergence of this pollen killer in A. thaliana. Furthermore, we report the co-occurrence and behavior of killer and sensitive genotypes in several local populations, a prerequisite for studying gamete killer evolution in the wild. This highlights the potential of the Arabidopsis model not only for functional studies of gamete killers but also for investigating their evolutionary trajectories at complementary geographical scales.配子杀手（gamete killer）是一类可导致杂交后代发生偏分离的基因座：其携带的杀伤性等位基因会选择性清除带有敏感等位基因的配子。该类因子广泛分布于真核生物中，对于解析基因组进化与物种形成机制具有重要意义。我们此前在两份远缘拟南芥（Arabidopsis thaliana）自然种质的杂交后代中鉴定到一类花粉杀手（pollen killer），该因子由三个遗传连锁的基因构成，且此前我们已报道了编码保护花粉免受该杀伤活性侵害的解毒因子的基因的鉴定结果。本研究中，我们借助CRISPR-Cas9诱导产生的突变体，鉴定出了该花粉杀手的另外两个基因。实验证实，这两个基因是该花粉杀伤活性的必需组分，且该活性仅特异性作用于花粉。花粉杀手编码蛋白的细胞定位结果提示，其杀伤活性与线粒体相关。序列分析显示，这些杀伤蛋白均带有同一蛋白家族的预测结构域。此外，其中一款杀伤蛋白的C端半区与该解毒因子完全一致，且一个对解毒因子活性至关重要的氨基酸残基，同样也是杀伤功能发挥所必需的。通过分析超过700份全球范围内的拟南芥种质资源，我们证实该基因座存在显著的结构重排与拷贝数变异。我们利用已公开的从头组装基因组序列，为拟南芥中该花粉杀手的起源提出了一套演化假说。此外，我们还报道了多个本地种群中杀伤性与敏感性基因型的共存情况及其行为特征，这为在野生环境中研究配子杀手的演化提供了必要前提。本研究进一步凸显了拟南芥模型的应用潜力：其不仅可用于配子杀手的功能研究，还可在互补的地理尺度上探究其演化轨迹。