Plant & Animal Genome IX Conference

Workshop: Forest Tree Genome Mapping
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MOLECULAR CHARACTERIZATION OF A MUTANT ALLELE OF CAD IN LOBLOLLY PINE.

Institute of Paper Science and Technology, 500 10th Street N.W., Atlanta, GA 30318, USA

A mutant recessive allele (cad-n1) of the gene encoding the enzyme cinnamyl alcohol dehydrogenase (CAD) was discovered in loblolly pine. The cad-n1 allele causes a severe deficiency of CAD enzyme, resulting in the synthesis of lignin with altered composition and structure. The genetic lesion responsible for this phenotype has been linked to the cad gene locus, however, the nature of the lesion and its molecular basis have remained elusive. A survey of DNA polymorphism by direct sequencing of PCR amplified products from several alleles of cad revealed extensive sequence variability in this gene. PCR amplified DNA polymorphisms were verified by segregation analysis. Surprisingly, the mutant cad-n1 allele was identical to one of the wild type alleles. The intriguing homology between the mutant cad-n1 allele and one wild type allele was verified over a 4.5 kb region encompassing most of the transcription unit and 600 bp of the 5' upstream flanking region. We used RFLP analysis to search for DNA sequence polymorphisms in a broader region surrounding the cad gene. This approach revealed a polymorphic site now being localized to several kilobases upstream of the gene. Molecular characterization of this mutant may shed light on genome evolution and the nature of allelic diversity in trees, which in turn could impact gene discovery and mapping, and molecular breeding.