A "public bulk" strategy identifies the causal gene for male sterility in ms1 soybean

As soybean is an extremely photoperiod-sensitive and self-pollinated crop which lacks any functionally confirmed male sterility genes, the scope of genetic diversity that soybean breeders have harnessed to date is much narrower than in other major crops. The ms1 mutant was the first genic male sterile mutant discovered for soybean and this mutant has been deployed in breeding, but its causal locus remains unknown. Here, by using a "control dominant homozygous bulk" or "public bulk" that we prepared in silico from public resequencing datasets in combination with sequencing data for a small ms1 recurrent breeding population, we found that a deletion fragment containing a soy homolog of AtNACK2, a microtubule motor kinesin-7 family protein, is the causal locus for male sterility in ms1 soybean materials. We used CRISPR/Cas9 gene editing technology to edit multiple deletion alleles of this 'GmMS1' (Glyma.13G114200) locus into an elite soybean cultivar, and found that the GmMS1 knockout plants phenocopy ms1 materials. Thus, beyond illustrating how an easy-to-implement in silico bulking strategy can be used to rapidly identify (potentially any) casual loci for recessive male sterility traits in a recurrent population, our study opens the door for large-scale hybridization breeding in soybean, the world's most economically important eudicot crop.

大豆是光周期敏感性极强的自花授粉作物，且尚未发现经功能验证的雄性不育基因，因此迄今为止大豆育种者可利用的遗传多样性范围远窄于其他主要农作物。ms1突变体是首个被发现的大豆细胞核雄性不育突变体，该突变体已应用于育种研究，但其导致雄性不育的因果位点仍未明确。本研究将我们从公共重测序数据集中通过计算机模拟（in silico）构建的"对照显性纯合混合池"（即公共混合池public bulk），与小型ms1轮回育种群体的测序数据相结合，发现一段包含拟南芥NACK2（AtNACK2）的大豆同源基因的缺失片段——AtNACK2属于微管动力蛋白驱动蛋白-7（kinesin-7）家族，正是ms1大豆材料雄性不育的因果位点。本研究利用CRISPR/Cas9基因编辑技术，在优良大豆品种中编辑了该GmMS1（Glyma.13G114200）位点的多个缺失等位基因，结果发现GmMS1敲除植株的表型与ms1材料完全一致。综上，本研究不仅阐明了如何通过易于实施的计算机模拟混合池分析策略，快速鉴定轮回群体中隐性雄性不育性状的潜在因果位点，还为全球经济价值最高的真双子叶作物——大豆的大规模杂交育种打开了大门。