Genetic mapping of opaque2 modifier genes
--Maurício A. Lopes, Glóverson L. Moro and Brian Larkins

Although the opaque2 (o2) mutation causes an increase in the lysine content of the grain, the pleiotropic effects of the mutation prevented its widespread agronomic utilization. The soft, chalky endosperm of o2 kernels is less dense and highly susceptible to insect and mechanical damage. The identification of genes with the ability to overcome these negative effects of the mutation, while maintaining its high nutritional value, provided new hope for the development of commercially attractive high lysine genotypes. These genes, designated o2 modifiers, were subsequently used by breeders at the International Maize and Wheat Improvement Center (CIMMYT, Mexico) and University of Natal (South Africa) to develop modified o2 genotypes called Quality Protein Maize (QPM). QPMs are o2 varieties with normal, vitreous appearance and hardness, but with an enhanced percentage of lysine.

QPMs have not reached widespread commercial utilization, partially because the mechanisms controlling the modification of o2 endosperm are not fully understood. Our lab has been working on the biochemical, genetic and molecular characterization of the modification and a considerable amount of information regarding this process is now available. The formation of vitreous endosperm is highly correlated with an increased accumulation of the 27-kD (-zein, suggesting the involvement of this protein in the endosperm modification. However, the precise mechanism by which this happens is unclear. The modifiers, which act in a semi-dominant fashion, seem to affect (-zein expression through a post-transcriptional mechanism. Although o2 modifiers were considered to be genetically complex, segregation analysis suggests that there are probably no more than two or three loci with a major effect.

To determine the genetic map location of the modifier loci, we used bulked segregant analysis to identify linked RFLPs. Segregating progenies from crosses of W64A o2 and W22 o2 by Pool 33 QPM were analyzed. Approximately 90 RFLP markers covering the entire genome were used in this study. A single locus contributing to the phenotypic variation was identified in each cross. For W22o2 x Pool 33 QPM a modifier locus was mapped near the telomere of chromosome 7L. For W64Ao2 x Pool 33 QPM, a second modifier locus was mapped at the 27-kD (-zein locus, near the centromere on 7L.

The role of (-zein locus in endosperm modification is unclear. The 27-kD (-zein locus may contain one (Ra) or two (A and B) genes. Analysis of 44 recombinant inbred lines developed from the cross between W64Ao2 (Ra allele) x Pool 33 (AB allele) shows that modification is associated with the presence of the AB allele. Similar analysis of 37 recombinant inbred lines between W22o2 x Pool 33 (both AB allele) showed that some inbreds with modified phenotype carry the AB allele from W22o2. This suggests that the allele of Pool 33 per se is not necessary for modification. Sequence analysis of A and B genes from o2 and modified o2 genotypes showed very few nucleotide differences in either the promoter or coding sequences.

We are extending the sequence analysis of the (-zein locus to identify cis-acting regulatory elements. We are also extending the mapping analysis of o2 modifier genes using QPM lines from South Africa. These materials were developed independently from those at CIMMYT, and they may help further resolve loci involved in endosperm modification. 

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