COMBINED SEM ULTRASTRUCTURE STUDIES AND PCR WITH INDIVIDUAL DIATOM CELLS

Abstract

Perforation of frustules of individual cells of the diatom Aulacoseira skvortzowii Edlund, Stoermer & Taylor with an extended glass needle controlled by a micro-manipulator was used to liberate the protoplast. The isolated total DNA was used in subsequent amplification with specific primers and sequencing of a fragment of the rbcL gene. Comparison of the sequence of the amplified rbcL gene fragment from A. skvortzowii, with those known for other plants, revealed that it is closest to the sequence of rbcL from the marine diatom Skeletonema costatum (12 amino acid substitutions over a fragment of 130 amino acid residues). Features of the ultra-structure of A. skvortzowii that are important for species identification (form and density of areolae, form of suture spine, etc.) remained intact when using this technique. The combined approach promises a breakthrough in the application of DNA sequencing to diatom research.