A molecular identification key for economically important thrips species (Thysanoptera: Thripidae) using direct sequencing and a PCR-RFLP-based approach

Abstract

1. Treatments against pathogens or pests are often very specific and, as a fundamental first step, require the ability to identify taxa correctly and unambiguously. We used PCR amplification techniques to successfully establish a molecular identification key for economically important thrips species. 2. A PCR amplified 433 bp long fragment of the mitochondrial COI coding gene was analysed by automated direct sequencing and RFLP. Sequencing of 264 individual thrips representing 10 named species detected 17 haplotypes. Variation within species was low, whereas among species variation was high resulting in an average sequence divergence of 18.6% and an average pairwise species differentiation (calculated as FST-value) of 0.9896. 3. Two restriction enzymes (AluI, Sau3AI) produced patterns that allowed unambiguous identification of all thrips species. 4. Statistical support for the quality of the key was given by (i) a highly significant permutation approach, assigning individual haplotypes to the correct species groups and (ii) a hierarchical NJ cluster analysis in which all conspecific individual sequences clustered together with maximal (100%) bootstrap support. 5. This study has shown that the use of genetic markers represents a valuable alternative for situations, such as epidemiological research, in which correct identification with classical morphological methods is either very difficult and time consuming or virtually impossible.