Optimization and validation of a simple sequence repeat (SSR)-PCR system in wheatgrass

Using wheatgrass DNA as a template, both single-factor and orthogonal-design methods were used to optimize the concentrations of five components (dNTPs, Mg²⁺, DNA template, primers, and Taq DNA polymerase) that influence marker amplification in simple sequence repeat (SSR)-PCR systems. The optimal SSR-PCR system for wheatgrass included 250 μmol·L^–1 dNTP, 2.25 mmol·L^–1 Mg²⁺, 0.60 mmol·L^–1 for each primer, 40 ng·μL^–1 DNA, and 0.75 U Taq DNA polymerase, with a total reaction volume of 20 μL. This optimization, which could improve the definition and reliability of SSR bands, establishes a foundation for genetic mapping in wheatgrass, as well as for the localization of important QTLs and, thus, molecular marker-assisted breeding.