An Estimation of the Outcrossing Rate in Sagittaria trifolia Using SSR Fluorescence Markers

The estimation of mating systems in self-compatible plants can help us understand the reproductive status of plants, the evolutionary trajectories between outcrossing and selfing, and the natural history or selective pressure on specific populations. Sagittaria trifolia is a self-compatible species, with both sexual and clonal reproduction. Male and female flowers sometimes co-bloom among and within inflorescences, therefore creating a chance of geitonogamy. In this study, we used SSR markers to estimate the outcrossing rate of S. trifolia in natural populations. Our experiment emphasized the polymorphism of microsatellite loci in different populations and highlighted the accuracy and advantage of the methodology of fluorescence quantification in visualizing PCR products. Results showed that 3 out of 28 primer pairs had high polymorphism, with the number of alleles at 5, 6, and 6 combining multiple populations. With the use of the three selected primer pairs, we estimated the outcrossing rate for 31 fruits from six plants in a natural population of S. trifolia, which was 92.87%±2.5%. Our study indicated that the mating system in S. trifolia was predominately outcrossing in natural populations and the contribution of clonal reproduction to offspring was very limited. We also compared two alternative methodologies in analyzing PCR products, namely Native-PAGE imaging and fluorescence quantification. The latter yielded a higher estimation on allele polymorphism, heterozygosity and therefore a more accurate outcrossing rate. After considering the cost, manpower and time collectively, we recommend employing fluorescence quantification.