Molecular evolution of the regulatory locus opaque2 from Zea --Lazzaroni, N, Hartings, H The evolution of the opaque-2 (o2) locus within members of the grass family was analyzed at the molecular level. For this purpose, a segment of the locus ranging from the third to the sixth exon was purified by means of PCR amplification and subjected to sequence analysis. Eleven accessions covering all Zea taxa and one Tripsacum dactyloides accession were assayed, identifying 21 haplotypes.

Our analyses reveal little evidence of selection at silent sites across the stretch of the o2 locus considered. First, the distribution of polymorphic silent sites between coding and intron regions was uniform, as was the distribution of polymorphic synonymous sites across the exons examined. Second, the distribution of observed nucleotide frequencies at synonymous polymorphic sites did not deviate significantly from the predicted frequency distributions under a model of selective neutrality. Third, Tajima�s D was not significantly different from zero. Selective pressure was measured by means of a sliding windows approach, which revealed a region with a significantly negative D value. This region, in the fourth exon, encodes the basic domain of the O2 protein, which is responsible for DNA binding and is involved in the relocation of the O2 protein into the nucleus. The preservation of this structure is, therefore, of fundamental importance in order to keep a functional gene product.

The pattern of non-synonymous site variation in the region of the o2 locus analyzed suggests the operation of natural selection. First, the number of observed nonsynonymous polymorphic sites is significantly lower than expected from synonymous site polymorphism. Second, the distribution of nonsynonymous polymorphism is not uniform across exons. The exons encoding the two functional parts of the active domain of the O2 protein, the basic domain, encoded by the fourth exon, and the zipper domain, encoded by the fifth exon, show an evident reduction of nonsynonymous polymorphic sites with respect to the other exons considered. Third, the use of a sliding windows approach to determine Tajima�s D statistic, identified three regions with negative D values residing within the third, fourth and fifth exon, respectively. These results indicate that selection is operating on nonsynonymous sites of the region of the o2 locus taken into consideration. This selection is most marked within the fourth and fifth exon, the two exons encoding the active domain of the O2 polypeptide. The sixth exon, which exhibits a higher than average density of nonsynonymous polymorphic sites, was found to contain a region showing a positive D value. Since two alternative polypeptide sequences have been identified, the C-terminal region of the O2 protein is probably not exposed to strict structural constraints.

Upper bounds for the time of speciation within Zea were recently determined using synonymous polymorphism at the alcohol dehydrogenase1 (adh1) locus. These measures of speciation time can be used to estimate the synonymous site substitution rate at the o2 locus. Synonymous site substitution rates of 2.2-3.6 x 10-8 per site per year were obtained. With the synonymous substitution rate at the o2 locus, it becomes possible to derive �Ne the effective population size of Zea, which was estimated at 3.9-6.4 x 105 by using the equation �Ne = �q/4�µ. This value is in good agreement with estimates (8.1 x 105) based on adh1 synonymous polymorphism.

In view of the fact that introgression between teosinte and maize is believed to occur infrequently, the relatively close relationship between the o2 loci of maize, teosinte, and T. dactyloides, as inferred from our molecular analyses, indicates a shared evolutionary history. It is, moreover, probably safe to assume that the variation at the o2 locus predates the speciation events leading to Z. perennis, Z. diploperennis, Z. luxurians, and Z. mays.
 
 

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