Identification of the endangered Australian orchid Microtis angusii using an allele-specific PCR assay

The endangered orchid, Microtis angusii, was recently described from a single population consisting of approximately 100 plants. This species is morphologically very similar to close relatives, and taxonomic difficulties have hindered attempts to identify further populations for protection. Here we present a rapid, economical, PCR-based assay for the effective identification of this species based on rDNA sequence variation. Using two single nucleotide substitutions in the internal transcribed spacer (ITS) region of the ribosomal DNA that are diagnostic for M. angusii, we developed an allele-specific PCR that can be easily visualized on a standard agarose gel, avoiding the use of expensive restriction enzymes and DNA sequencing reagents and equipment. Using PCR primer pairs for both the M. angusii, and the alternate allele, we also detected an individual heterozygous for the two alleles, indicating a need for further detailed genetic study. We performed a 'blind trial' to confirm the utility of this assay. Microtis angusii samples were successfully discriminated from amongst several congeners, and a further, previously unknown, population of the species was identified.