A novel DNA-based diagnostic test for the detection of annual and intermediate ryegrass contamination in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) is a preferred choice for the turf grass industry due to its ability to provide a durable turf cover. Genetic or physical contamination of annual (L. multiflorum Lam.) or intermediate (L. hybridum) ryegrass species in perennial ryegrass is one of the major problems affecting the grass seed industry. At present, seedling root fluorescence (SRF), a biochemical marker, is used for the detection of annual ryegrass contamination. Due to the unreliability of the SRF test, the seed industry is seeking an alternative, more reliable and accurate detection method. Currently, there are no DNA tests available in ryegrass for detecting contamination with annual and intermediate ryegrass types. We developed a novel quantitative polymerase chain reaction (Q-PCR)-based DNA test for the detection of annual and/or intermediate ryegrass types in perennial ryegrass. This DNA test was designed using an insertion/deletion (InDel) site in the LpVRN2_2 (Vernalization 2) gene, which is one of the several genes controlling vernalization in ryegrass. The new DNA test is more reliable, accurate and cost-effective in detecting contamination, with a high sensitivity of 0.04% in a sample size of 5,000 seeds. Use of larger sample sizes (12.5-fold higher compared to SRF test) provided additional accuracy in detecting the level of contamination. The method has produced consistent results in 68 perennial, 26 annual and 14 intermediate ryegrass lines.