Strategies for amplification by polymerase chain reaction of the complete sequence of the gene encoding nuclear large...

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3'-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3'-end primer was constructed in tandem with the universal 5'-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non- Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5'-end of the IGS primer. Analysis of the 5'-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals.