One-step strategy for developing wheat haploid inducer lines with efficient markers for haploid identification

Wheat ( Triticum aestivum L.) is one of the most important staple crops globally. Doubled haploid technology enables rapid development of pure lines and has been extended from maize to several other crop species. A key step in DH breeding is the identification of haploids from diploids, which requires accurate and convenient phenotypic markers. In this study, we generated two wheat haploid inducers carrying different markers by a one-step strategy. One harbored a dual fluorescent marker system consisting of eGFP and TagRFP, the other carried a RUBY reporter. Both markers enabled near 100% accuracy of haploid identification at the immature embryo, mature embryo, and germinating seedling stages. Moreover, both lines consistently exhibited a high and stable haploid induction rate (∼20%). This study not only provides efficient wheat haploid inducers but also establishes a convenient pipeline for developing haploid induction systems in other crop species.