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Originally published as Genetics Published Articles Ahead of Print on May 5, 2008.

Genetics, Vol. 179, 517-526, May 2008, Copyright © 2008
doi:10.1534/genetics.107.080366

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

Kenneth M. Olsen1, Shih-Chung Hsu and Linda L. Small

Department of Biology, Washington University, St. Louis, Missouri 63130-4899

1 Corresponding author: Department of Biology, Campus Box 1137, Washington University, 1 Brookings Dr., St. Louis, MO 63130-4899.
E-mail: kolsen{at}wustl.edu

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT–PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.