Divergence of duplicated genes in maize: evolution of contrasting targeting information for enzymes in the porphyrin pathway.

The divergence of sequence and expression pattern of duplicated genes provides a means for genetic innovation to occur without sacrificing an essential function. The cpx1 and cpx2 genes of maize are a singular example of duplicated genes that have diverged by deletion and creation of protein targeting information. The cpx genes encode coproporphyrinogen III oxidase ('coprogen oxidase'), which catalyzes a step in the synthesis of chlorophyll and heme. In plants, this enzyme has been found exclusively in the plastids. The cpx1 and cpx2 genes encode almost identical, catalytically active enzymes with distinctive N-terminal peptide sequences. The cpx1 gene encodes the expected plastid transit peptide, but this region is deleted from the cpx2 gene. While the 5' regions of both messenger RNAs are highly similar, the cpx2 gene has an open-reading frame that could encode a new targeting signal. GFP fused with CPX1 localized to the plastids. In contrast, the GFP fusion with CPX2 did not target plastids and appeared to localize to mitochondria. Both cpx genes are expressed ubiquitously but, based on mutant phenotype, they seem to have discrete biological roles. Seedlings homozygous for a null mutation in the cpx1 gene completely lack chlorophyll and develop necrotic lesions in the light. However, the mutant seedlings and callus cultures will grow in tissue culture in the dark, implying that they retain a capacity to produce heme. We discuss models for the evolution of the cpx genes and possible roles of mitochondrion-localized coprogen oxidase activity in maize.

Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis.

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

Activator mutagenesis of the pink scutellum1/viviparous7 locus of maize.

The transposable elements Activator/Dissociation (Ac/Ds) were first discovered in maize, yet they have not been used extensively in their native host for gene-tagging experiments. This can be attributed largely to the low forward mutation rate and the propensity for closely linked transpositions associated with Ac and its nonautonomous deletion derivative Ds. To overcome these limitations, we are developing a series of nearly isogenic maize lines, each with a single active Ac element positioned at a well-defined location. These Ac elements are distributed at 10- to 20-centimorgan intervals throughout the genome for use in regional mutagenesis. Here, we demonstrate the utility of this Ac-based gene-tagging approach through the targeted mutagenesis of the pink scutellum1/viviparous7 (ps1/vp7) locus. Using a novel PCR-based technique, the Ps1 gene was cloned and Ac elements positioned precisely in each of the seven alleles recovered. The Ps1 gene is predicted to encode lycopene beta-cyclase and is necessary for the accumulation of both abscisic acid and the carotenoid zeaxanthin in mature maize embryos. This study demonstrates the utility of an Ac mutagenesis program to efficiently generate allelic diversity at closely linked loci in maize.

Fine mapping in tomato using microsynteny with the Arabidopsis genome: the Diageotropica (Dgt) locus.

BACKGROUND: The Arabidopsis thaliana genome sequence provides a catalog of reference genes applicable to comparative microsynteny analysis of other species, facilitating map-based cloning in economically important crops. We have applied such an analysis to the tomato expressed sequence tag (EST) database to expedite high-resolution mapping of the Diageotropica (Dgt) gene within the distal end of chromosome 1 in tomato (Lycopersicon esculentum). RESULTS: A BLAST search of the Arabidopsis database with nucleotide sequences of markers that flank the tomato dgt locus revealed regions of microsynteny between the distal end of chromosome 1 in tomato, two regions of Arabidopsis chromosome 4, and one on chromosome 2. Tomato ESTs homeologous to Arabidopsis gene sequences within those regions were converted into co-dominant molecular markers via cleaved amplified polymorphic sequence (CAPS) analysis and scored against an informative backcross mapping population. Six new microsyntenic EST (MEST) markers were rapidly identified in the dgt region, two of which further defined the placement of the Dgt gene and permitted the selection of a candidate tomato bacterial artificial chromosome clone for sequence analysis. CONCLUSIONS: Microsynteny-based comparative mapping combined with CAPS analysis of recombinant plants rapidly and economically narrowed the dgt mapping region from 0.8 to 0.15 cM. This approach should contribute to developing high-density maps of molecular markers to target-specific regions for positional cloning and marker-assisted selection in a variety of plants.