Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression.

KEY MESSAGE: R1-nj anthocyanin marker inhibition is highly frequent in tropical maize germplasm considerably affecting efficiency of haploid identification. Molecular markers reliably differentiating germplasm with anthocyanin color inhibitor have been identified in this study. The R1-Navajo (R1-nj) color marker facilitates easy and quick identification of haploid kernels at the seed stage during in vivo haploid induction process in maize. However, the Navajo phenotype can be completely suppressed or poorly expressed in some germplasm, making it impossible or inefficient to identify haploids at the seed stage. In this study, we characterized the expression of R1-nj marker in a large array of tropical/subtropical inbred lines, breeding populations and landraces by crossing with the R1-nj-based tropicalized haploid inducer. There was a high frequency of inhibition of the Navajo phenotype in the maize inbred lines, which are used in tropical breeding programs. Genome-wide association mapping showed that the C1 anthocyanin regulatory locus is the most significant genetic factor influencing inhibition of the Navajo phenotype. Molecular marker assays were designed based on polymorphism in the C1 vs C1-I alleles. Analysis of a set of 714 inbred lines demonstrated that a combination of two gene-specific markers--8 bp C1-I InDel and C1-I SNP--could predict with high accuracy the presence of anthocyanin color inhibition in the germplasm analyzed. Information generated in this study aids in making informed decisions on the constitution of source populations for doubled haploid (DH) line development in tropical germplasm, particularly those derived from elite maize lines from CIMMYT. The C1-I gene-specific molecular markers identified and validated will facilitate high-throughput and cost-effective evaluation of a large pool of germplasm for the presence of the dominant color inhibitor in maize germplasm.