Characterization of Four Members of the Alpha-Tubulin Gene Family in Ceratopteris richardii

—Four members of the alpha-tubulin gene family were examined in Ceratopteris richardii. Genetic linkage mapping based on a population of nearly 500 Doubled Haploid Lines was able to position three or four members of this gene family on linkage groups 17, 24, and 28, respectively (two of the four observed polymorphic restriction fragments containing alpha-tubulin genes are either identical or map too close to each other on linkage group 17 to be distinguishable in map distance). Non-mapable monomorphic bands observed on probed Southern blots suggest that the alpha-tubulin gene family in this species is large. Four alpha-tubulin genes from C. richardii were sequenced and found to be fairly similar to each other in terms of their amino acid sequences, with their greatest diversity at the carboxy-terminal ends. BLAST comparisons found each of these four amino acid sequences more similar to an alpha-tubulin from a dicot, gymnosperm, or alga species than it was to any other alpha-tubulin sequence presently known from Ceratopteris or from the fern Anemia phyllitidis or the moss Physcomitrella patens. Bayesian phylogenetic analysis of nucleotide sequences placed three of the four Ceratopteris alpha-tubulin gene copies in a clade with copies from Pseudotsuga and Anemia, consistent with a history of two gene duplication events, one following and one preceding the divergence of ferns and seed plants. The fourth copy is robustly separated from the preceding three and placed in a clade of algal alphatubulin genes, suggesting its divergence from the ancestor of the other three before the divergence of algae and land plants. As characterized thus far, the alpha-tubulin gene family of C. richardii is relatively large as compared to the six copies known from fully sequenced Arabidopsis thaliana, a condition that may be correlated with the large genome size and diverse life history constraints of this homosporous fern species. These findings suggest several new opportunities for research into the evolution, function, and regulation of the alpha-tubulin gene family in Ceratopteris. This report describes the application of DNA sequencing and genetic linkage mapping to the alpha-tubulin genes of Ceratopteris richardii and shows how such studies can further enhance the utility of this model system. Initial successes with Ceratopteris were in the areas of cytogenetics (e.g., Hickok, 1976, 1977a, 1977b, 1978, 1979a, 1979b; Hickok and Klekowski, 1973, 1974), physiology and development, and Mendelian genetics (reviewed in Hickok, 1987; Hickok et al., 1987, 1995). More recent studies have focused on the molecular genetics of Ceratopteris (Munster et al., 1997; Hasebe et al., 1998; Aso et al., 1999; Stout et al., 2003; Rutherford et al., 2004; Salmi et al., 2005). These recent studies that employ robust molecular methods are especially encouraging, since the lack of a technique to induce stable genetic transAmerican Fern Journal 97(2):47–65 (2007)