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Perennial ryegrass ( Lolium perenne L.) displays a two-locus gametophytic self-incompatibility ( SI) system that remains intact at the tetraploid level. Two models are plausible for SI in autotetraploids. In Model I: both alleles at the S locus and both at the Z locus in diploid pollen matching the female genotype results in incompatibility. In Model II: only one allele at S and one at Z locus in diploid pollen matching the female results in incompatibility. The goals were to determine which of the models best explains SI in our autotetraploid ryegrass population and to evaluate the efficiency of high-resolution melting ( HRM) genotyping for discriminating different iso-allelic genotypes. The progeny of a cross between two autotetraploids was characterized with three HRM-based markers co-segregating with Z. Segregation ratios were used to make inferences about the mode of action of the SI system. The observed segregation differed significantly (P < 0.001) from the expected under Model I, but not from the expected under Model II (P = 0.463). Thus, Model II explains SI in this population, and HRM is an efficient tool to distinguish different iso-allelic genotypic classes. [ABSTRACT FROM AUTHOR]