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Key Message: Increased efficiencies achieved in different steps of DH line production offer greater benefits to maize breeding programs. Doubled haploid (DH) technology has become an integral part of many commercial maize breeding programs as DH lines offer several economic, logistic and genetic benefits over conventional inbred lines. Further, new advances in DH technology continue to improve the efficiency of DH line development and fuel its increased adoption in breeding programs worldwide. The established method for maize DH production covered in this review involves in vivo induction of maternal haploids by a male haploid inducer genotype, identification of haploids from diploids at the seed or seedling stage, chromosome doubling of haploid (D 0 ) seedlings and finally, selfing of fertile D 0 plants. Development of haploid inducers with high haploid induction rates and adaptation to different target environments have facilitated increased adoption of DH technology in the tropics. New marker systems for haploid identification, such as the red root marker and high oil marker, are being increasingly integrated into new haploid inducers and have the potential to make DH technology accessible in germplasm such as some Flint, landrace, or tropical material, where the standard R1-nj marker is inhibited. Automation holds great promise to further reduce the cost and time in haploid identification. Increasing success rates in chromosome doubling protocols and/or reducing environmental and human toxicity of chromosome doubling protocols, including research on genetic improvement in spontaneous chromosome doubling, have the potential to greatly reduce the production costs per DH line.

Key Message: Among the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion. In vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region. To characterize the effects of each sub-region of qhir1 on haploid induction rate, F 2 recombinants segregating for one of the sub-regions and fixed for the other were identified in a cross between CML269 (non-inducer) and a tropicalized haploid inducer TAIL8. To quantify the haploid induction effects of qhir11 and qhir12, selfed progenies of recombinants between these sub-regions were genotyped. F 3 plants homozygous for qhir11 and/or qhir12 were identified, and crossed to a ligueless tester to determine their haploid induction rates. The study revealed that only the qhir11 sub-region has a significant effect on haploid induction ability, besides causing significant segregation distortion and kernel abortion, traits that are strongly associated with maternal haploid induction. The results presented in this study can guide fine mapping efforts of qhir1 and in developing new inducers efficiently using marker assisted selection.

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.

The partially dominant, autoactive maize disease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on leaves and stems in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each derived from a cross between the maize line B73 and one of 25 diverse inbred lines. By crossing a line carrying the Rp1-D21 gene with lines from three of these subpopulations and assessing the F(1) progeny, we were able to map several novel loci that modify the maize HR, using both single-population quantitative trait locus (QTL) and joint analysis of all three populations. Joint analysis detected QTL in greater number and with greater confidence and precision than did single population analysis. In particular, QTL were detected in bins 1.02, 4.04, 9.03, and 10.03. We have previously termed this technique, in which a mutant phenotype is used as a "reporter" for a trait of interest, Mutant-Assisted Gene Identification and Characterization (MAGIC).