Context. Red clover (Trifolium pratense L.) is an important legume forage in temperate agricultural zones. Evaluation of self- and cross-pollination fertility is important for setting up an effective breeding-program scale. However, the outcrossing rate of red clover under open-pollination conditions is not certain. Development of a reliable and time-saving marker system is needed to quantify and characterise outcrossing rates.

Aim. We aimed to develop a duplex PCR-based protocol based on a genome-wide simple sequence repeat (SSR) screen, and to determine the outcrossing rate of red clover under open-pollination environments.

Methods. We screened 209 SSR markers with pooled DNA samples of 60 plants from 20 red clover accessions, and selected 185 SSR markers that produced clear scorable bands for testing with 24 individual DNA samples to determine polymorphism. We selected 70 primer pairs, and then assembled a core set of 24 loci into 12 sets of duplex markers, which were used for outcrossing behaviour analysis of 60 maternal parents and their respective 22 half-sib progenies.

Key results. Mean polymorphic information content (PIC) for the 70 markers was 0.490 (range 0.117–0.878). Minimum, mean and maximum PIC values for the 24 markers constituting the 12 duplexes were 0.226, 0.594 and 0.781, respectively. The outcrossing rate was identified as 99.4% for red clover in a natural environment.

Conclusion. We successfully developed a duplex SSR-based PCR protocol consisting of 24 markers. This SSR system was applied to determine the outcrossing rate of red clover in a natural environment.