Simple Sequence Repeat and Single Nucleotide Polymorphism Marker-Based Detection and Quantification of Fine Fescues (Festuca spp.) in a Mixed Stand.

The fine fescue species [Chewings fescue (Festuca rubra ssp. fallax), hard fescue (Festuca brevipila), sheep fescue (Festuca ovina), strong creeping red fescue (Festuca rubra ssp. rubra), and slender creeping red fescue (Festuca rubra ssp. litoralis)] have been shown to perform well in low input environments in temperate climates. Fine fescues grow well in the shade or sun, have reduced mowing requirements, and possess good drought tolerance. There are often, however, differences in performance between these species for traits such as disease resistance and traffic tolerance. For that reason, turfgrass researchers have investigated the use of fine fescue mixtures to enhance overall turf performance. One complication with these mixture studies is that final community composition is often difficult to ascertain due to morphological similarities between the fine fescue species. Polymerase Chain Reaction (PCR) assays should allow for the detection and quantification of specific grass species in a mixed turf stand. The objective of this project was to develop an economical, efficient method for the rapid identification and quantification of fine fescue species in a mixed sample. For species identification, Festuca EST sequences, and hard and strong creeping red fescue de novo transcriptomes were used for Simple Sequence Repeat (SSR) mining and marker development. For quantification, species-specific primers for each of the five fine fescue species were developed using sequence polymorphism of chloroplast trn intergenic region. The actin2 gene was used to correlate the chloroplast genome to the plant nucleus genome for content calibration and percentage calculation. We were able to obtain SSR markers that could be used for species identification. We also found that some trn gene copies are shared by more than one fine fescue species; therefore, we were only able to calculate Chewings fescue, sheep fescue, and slender creeping red fescue species percentage from fine fescue mixtures. While somewhat limiting, this still allows for an increased understanding of species composition in a mixed fine fescue stand. Following this research, more species specific primers will be developed using other polymorphic genes to provide a comprehensive screening protocol.