Double-fluorescent proteins enable robust maternal haploid identification in wheat

Doubled haploid (DH) technology is crucial for accelerating crop breeding. While the functional conservation of MATRILINEAL (MTL) in cereals enables haploid induction (HI) in wheat (Triticum aestivum), distinguishing haploid from diploid seeds remains a major bottleneck. Here, we developed an efficient haploid identification (HID) system for wheat by seamlessly integrating a two-fluorescent protein-based HID toolbox with an MTL mutant HI wheat line. This system enables efficient HI and utilizes dual-fluorescence screening, offering high accuracy while being independent of the genetic background of the recipient material. Our work demonstrates that engineered intraspecific HI is achievable in self-pollinating crops such as wheat, paving the way for applications that shorten the breeding cycle, facilitate quantitative genetic studies, and accelerate the fixation of desirable alleles. With further optimization and development, this system holds promise as a commercially viable pipeline for haploid production to facilitate wheat breeding.