Genetic map-based location of the red clover (Trifolium pratense L.) gametophytic self-incompatibility locus

Red clover is a hermaphroditic allogamous diploid (2n = 2x = 14) with a homomorphic gametophytic self-incompatibility (GSI) system (Trifolium pratense L.). Red clover GSI has long been studied, and it is thought that the genetic control of GSI constitutes a single locus. Although GSI genes have been identified in other species, the genomic location of the red clover GSI-locus remains unknown. The objective of this study was to use a mapping-based approach to identify simple sequence repeats (SSR) that were closely linked to the GSI-locus. Previously published SSR markers were used in this effort (Sato et al. in DNA Res 12:301–364, 2005). A bi-parental cross was initiated in which the parents were known to have one self-incompatibility allele (S-allele) in common. S-allele genotypes of 100 progeny were determined through test crosses and pollen compatibility. Pseudo F₁ linkage analysis isolated the GSI-locus on red clover linkage-group one within 2.5 cM of markers RCS5615, RCS0810, and RCS3161. A second 256 progeny mapping testcross population of a heterozygous self-compatible mutant revealed that this specific self-compatible mutant mapped to the same location as the GSI-locus. Finally, 82 genotypes were identified whose parents putatively shared one S-allele in common from maternal halfsib families derived from two random mating populations in which paternal identity was determined using molecular markers. Unique S-allele identity in the two random mating populations was tentatively inferred based on haplotypes of two highly allelic linkage-group one SSR (RCS0810 and RCS4956), which were closely linked to each other and the GSI-locus. Paternally derived pollen haplotype linkage analysis of RCS0810 and RCS4956 SSR and the GSI-locus again revealed tight linkage at 2.5 and 4.7 cM between the GSI-locus and RCS0810 and RCS4956, respectively. The map-based location of the GSI-locus in red clover has many immediate applications to red clover plant breeding and could be useful in helping to sequence the GSI-locus.