Table_2_Overexpression of Modified CENH3 in Maize Stock6-Derived Inducer Lines Can Effectively Improve Maternal Haploid Induction Rates.XLSX

Maize (Zea mays) doubled haploid (DH) breeding is a technology that can efficiently generate inbred lines with homozygous genetic backgrounds. Haploids are usually produced through in vivo induction by haploid inducer lines in maize. Currently, two approaches are usually used to develop maize haploid inducer lines. One is through the conventional breeding improvement based on the Stock6 germplasm, and this strategy is extensively used to induce maternal haploids in commercial maize DH breeding. Another strategy, newly developed but less utilized so far, is by genetic manipulation of the Centromeric Histone3 (CENH3) in regular lines. However, whether both approaches can be combined to develop the haploid inducer line with higher maternal haploid induction rate (HIR) has not been reported. In this study, we manipulated the Stock6-derived inducer lines by overexpressing maize CENH3 fused with different fluorescent protein tags and found that the engineered Stock6-derived lines showed an obvious increase in the maternal HIR. Intriguingly, this above strategy could be further improved by substituting a tail-altered CENH3 for the full-length CENH3 in the tagged expression cassette, resulting in a maternal HIR up to 16.3% that was increased by ~6.1% than Stock6-derived lines control. These results suggested that integration of two in vivo haploid induction methods could rapidly and effectively improve the maternal HIRs of maize Stock6-derived inducer lines, and provided a potentially feasible solution for further optimizing the process of commercial maize DH breeding.

玉米（Zea mays）双单倍体（doubled haploid，DH）育种是一种可高效获得具有纯合遗传背景的自交系（inbred lines）的技术。目前玉米单倍体通常通过单倍体诱导系（haploid inducer lines）进行活体诱导产生。当前开发玉米单倍体诱导系主要有两种途径：其一为基于Stock6种质的常规育种改良策略，该方法已广泛应用于商业化玉米DH育种中的母本单倍体诱导；其二为近年来新兴但目前应用较少的策略，即对常规自交系的着丝粒组蛋白3（Centromeric Histone3，CENH3）进行遗传操纵。然而，目前尚无关于将这两种途径结合以开发更高母本单倍体诱导率（maternal haploid induction rate，HIR）的单倍体诱导系的相关报道。本研究通过过表达融合不同荧光蛋白标签（fluorescent protein tags）的玉米CENH3对Stock6来源的诱导系进行改造，发现工程化改造后的Stock6来源诱导系的母本HIR显著提升。有趣的是，若在带标签的表达盒（expression cassette）中用尾部修饰型CENH3替代全长CENH3，可进一步优化该策略，最终使母本HIR达到16.3%，较Stock6来源的对照诱导系提升约6.1%。上述结果表明，整合两种活体单倍体诱导方法可快速且有效地提升玉米Stock6来源诱导系的母本HIR，为进一步优化商业化玉米DH育种流程提供了潜在可行的解决方案。