Use of Real-Time PCR for Determining Species Proportions in a Mixed Fine Fescue Turfgrass Community.

The fine fescue species [Chewings fescue (Festuca rubra ssp. falax), 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 lower input environments in temperate climates. These grasses tend to do well in the shade or sun, have reduced mowing requirements compared to other commonly-used cool-season turfgrasses, and possess good drought tolerance. There are often, however, differences in performance between these species for certain 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. In these studies, seed is planted with a known mixture composition and the turf is then evaluated for various stress tolerances and overall quality. Because the fine fescues are very similar morphologically to one another, quantifying the species composition in a mixed fine fescue stand is very difficult. Fortunately, the TRN gene, which is a chloroplast gene that is highly polymorphic across plant species, will enable us to design species specific primers for fine fescues species identification. Our overall goal is to use real-time PCR to quantify final fine fescue species composition in clippings samples. The objective of this project was to develop an economical, efficient methodology for rapid quantification of fine fescue species in a mixed sample.

TRN genes of the five fine fescues subspecies were cloned and sequenced. Species specific primers were developed based on the TRN gene polymorphic region. The Actin2 gene was used to correlate the chloroplast genome to the plant core genome for content calibration and percentage calculation. We found that some TRN gene copies are shared by more than one fine fescue species; therefore, we are 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.