Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate “in vitro crosses” in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between the Mus musculus laboratory mouse and Mus spretus (∼1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine–guanine phosphoribosyltransferase (Hprt) in as few as 21 d through “flow mapping” by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids.

发现潜在物种差异的遗传变化是进化遗传学的中心目标。然而，哺乳动物中物种之间的杂交杂交经常遭受杂交不育，这极大地复杂了物种间性状变异的遗传图谱。在这里，我们描述了一种简单，可靠且无转基因的技术，该技术通过使用抑制DNA解旋酶Bloom综合征的药物ML216诱导随机有丝分裂交叉，从而在杂种小鼠胚胎干（ES）细胞中产生“体外杂交”。 BLM）。从小家鼠和老鼠之间的种间F1杂交ES细胞系开始（相距约150万年），我们将抗代谢药物噻鸟嘌呤的耐药性遗传基础定位在一个包含次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（Hprt）的区域）在短短21 d内通过“流图绘制”，将体外杂交与荧光激活细胞分选（FACS）结合使用。我们还展示了我们的平台如何通过重组胚胎干细胞系的贡献重新培养胚胎，从而能够直接研究发育变异。我们展示了体外杂交如何克服小鼠复杂性状遗传学的主要瓶颈，并解决了进化生物学中的基本问题，这些问题由于成本高，窝产小和/或杂种不育而无法通过传统育种来解决。为此，我们描述了一个实验平台，旨在研究小鼠以及潜在的人类和其他哺乳动物（包括跨物种杂种）中的进化系统生物学。