Design of plant-specific PCR primers for the ETS region with enhanced specificity for tribe Bromeae and their application to other grasses (Poaceae)

Selective primers were developed for the amplification via polymerase chain reaction (PCR) of the external transcribed spacer (ETS) region in the nuclear ribosomal repeat unit. These primers were intended to be specific to the Poaceae tribe Bromeae but were found to function broadly across subfamily Pooideae. ETS primers previously developed for grasses were unable to amplify tribe Bromeae taxa because of ETS variability and several subrepeats. After detailed analysis, a region was chosen and four candidate primers were designed to amplify and sequence the ETS fragment. This fragment of approximately 800-900 bp is located in the 3' region of the ETS of 18S-26S nuclear ribosomal DNA. A preliminary study with these primers was conducted in eight samples of Bromus. The best two primers showed strong amplification and successful sequencing for all samples tested. We also tested the specificity of these two primers in samples belonging to all large taxa of Bromeae, including all three genera and five subgenera and samples of eight tribes and 15 subtribes of the subfamily Pooideae, and both worked for most of the taxa tested. Our results demonstrate that the ETS region is more informative for resolving relationships at different taxonomic levels than internal transcribed spacer region. Also, these results indicate the utility of these new primers for studying the ETS region in the tribe Bromeae as well as across the subfamily Pooideae.