The development of highly transferable intron-spanning markers for temperate forage grasses

Abstract

DNA markers have the potential to be a powerful tool for the molecular study and breeding of agronomic traits of temperate forage grasses, but some of these grasses have only limited sequence information available. We aimed to design highly transferable primer sets by using the abundant sequence information available for related crop species such as wheat and rice. The degree of similarity between the primer sequences of the wheat PCR-based landmark unique gene (PLUG) primer set and the corresponding sequences of rice orthologs was designated as the “universality index” (UI). We classified 359 PLUG primer pairs based on their UI values and found that primer pairs with high UI values showed higher successful amplification rates in Festuca and Lolium species than those with low UI values. Based on these results, we designed new primer sets, designated Conserved Three-prime-End Region (COTER) primers, with complete similarity to rice orthologs for eight bases at the 3′ end of each primer. COTER primer sets developed from both tall fescue and wheat showed high transferability in six temperate grasses (mean amplification rates of 95% for tall fescue primers; 79% for wheat primers). This strategy and primer information could also be useful for developing DNA markers for other grass species with little genome information available.