Development and Validation of Red Root Marker-Based Haploid Inducers in Maize

One of the critical limitations for the in vivo production of doubled haploid (DH) lines in maize (Zea mays L.) is the inability to effectively identify haploids in a significant proportion of induction crosses due to the possibility of complete or partial inhibition of the currently used R1-nj (Navajo) color marker. In this study, we demonstrate that the R1-nj marker could result in a high proportion of false positives among the haploids identified, besides being ineffective in germplasm with natural anthocyanin expression in pericarp tissue. To address these limitations, we developed haploid inducer lines with triple anthocyanin color markers, including the expression of anthocyanin coloration in the seedling roots and leaf sheaths, in addition to the Navajo marker on the seed. Although these inducers show acceptable haploid induction rates ranging from 8.6 to 10.2%, they exhibited relatively poor agronomic performance compared with tropicalized haploid inducers within tropical environments. The addition of the red root marker more accurately identified haploids among the germinating seedlings, including four tropical inbred lines and eight breeding populations that showed complete inhibition of R1-nj. We also demonstrate that the red root marker can be used for haploid identification in germplasm with natural anthocyanin expression in the pericarp. A survey of 546 tropical inbreds and 244 landraces showed that anthocyanin accumulation in the roots of germinating seedlings is very rare compared with anthocyanin accumulation in the seed and leaf sheath tissues. As a result, the red root marker can serve as a highly complementary marker to R1-nj to enable effective identification of haploids within a wide range of tropical maize germplasm.