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Chaikam V, Nair SK, Babu R, Martinez L, Tejomurtula J and Prasanna BM (2015) 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. Theor Appl Genet (2015) 128: 159.DOI: 10.1007/s00122-014-2419-3.

Abstract

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.

Chaikam V,  Martinez L, Melchinger A, Schipprack W and Prasanna BM(2016) Development and validation of red root marker-based haploid inducers  in maize, Crop Science, 56(4), 1678-1688. DOI: 10.2135/cropsci2015.10.0653

Abstract

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.

Hu, H., Schrag, T., Peis, R., Unterseer, S., Schipprack, W., Chen, S., . . . Melchinger, A. (2016). The Genetic Basis of Haploid Induction in Maize Identified with a Novel Genome-Wide Association Method. Genetics, 202(4). DOI: 10.1534/genetics.115.184234

Abstract

In vivo haploid induction (HI) triggered by pollination with special intraspecific genotypes, called inducers, is unique to Zea mays L. within the plant kingdom and has revolutionized maize breeding during the last decade. However, the molecular mechanisms underlying HI in maize are still unclear. To investigate the genetic basis of HI, we developed a new approach for genome-wide association studies (GWAS), termed conditional haplotype extension (CHE) test that allows detection of selective sweeps even under almost perfect confounding of population structure and trait expression. Here, we applied this test to identify genomic regions required for HI expression and dissected the combined support interval (50.34 Mb) of the QTL qhir1, detected in a previous study, into two closely linked genomic segments relevant for HI expression. The first, termed qhir11 (0.54 Mb), comprises an already fine-mapped region but was not diagnostic for differentiating inducers and noninducers. The second segment, termed qhir12 (3.97 Mb), had a haplotype allele common to all 53 inducer lines but not found in any of the 1482 noninducers. By comparing resequencing data of one inducer with 14 noninducers, we detected in the qhir12 region three candidate genes involved in DNA or amino acid binding, however, none for qhir11. We propose that the CHE test can be utilized in introgression breeding and different fields of genetics to detect selective sweeps in heterogeneous genetic backgrounds.