THE CINNAMYL ALCOHOL-DEHYDROGENASE GENE STRUCTURE IN PICEA-ABIES (L.)KARST - GENOMIC SEQUENCES, SOUTHERN HYBRIDIZATION, GENETIC-ANALYSIS AND PHYLOGENETIC-RELATIONSHIPS

Based on PCR technologies, we have isolated three genomic cinnamyl alc ohol dehydrogenase (CAD) clones from Norway spruce, Picea abies (L.) K arst., revealing about 99% identity within their protein coding region s. All clones contain five introns with an identity of 97-100% for int ervening sequences II, III and IV, whereas intron V sequences revealed only 87-89% identity. Intron I sequences share an identity of 85-98% among all three clones. Intron IV is only present in Norway spruce and not found in published genomic CAD sequences of angiosperms. Tandem r epeats between 24 and 49 bp were discovered within intervening sequenc es I and V. Southern hybridization of seedling DNA and PCR-based intro n analyses using diploid leaf buds and haploid megagametophytes indica te the existence of a small CAD gene family within the spruce genome, consisting of at least two loci. Evolutionary analyses of CAD encoding sequences using distance matrix- and parsimony-based methods revealed that CADs from angiosperms form a clade distinct from those of gymnos perms. Confirmed by maximal bootstrap values of 100%, a gene duplicati on gave rise to two different groups of angiospermous CADs and this du plication may have occurred in an early stage of angiosperm radiation, certainly before the separation of the Dilleniidae and Rosidae lineag es. Phylogenetic investigations suggest angiosperm CAD II sequences to have evolved more rapidly than angiosperm CAD I genes. On the other h and, CAD gene evolution appears to be significantly slower in conifers than in angiosperms.