A novel multivariate approach to phenotyping and association mapping of multi-locus gametophytic self-incompatibility reveals S, Z and other loci in a perennial ryegrass (Poaceae) population

Self-incompatibility (SI) is a mechanism that many flowering plants employ to preventfertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour.Although a number of single locus mechanisms have been characterised in detail, nomulti-locus systems have been fully elucidated. Historically, examples of the geneticanalysis of multi-locus SI, to make analysis tractable, are either made on the progenyof bi-parental crosses, where the number of alleles at each locus is restricted, or oncrosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass(Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophyticincompatibility system. A more universal, realistic proof of principle study was conductedin a perennial ryegrass population in which allelic and non-allelic diversity was notartificially restricted. A complex pattern of pollinations from a diallel cross was revealedwhich could not possibly be interpreted easily per se, even with an already establishedgenetic model. Instead, pollination scores were distilled into principal component scoresdescribed as Compatibility Components (CC1-CC3). These were then subjected toa conventional genome-wide association analysis. CC1 associated with markers onlinkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic regionon LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodiumphysical map revealed highly significantly associated markers with peak associationswith genes adjacent and four genes away from the chromosomal locations of candidateSI genes, S- and Z-DUF247, respectively. Further significant associations were found in aBrachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genomebetween B. distachyon and L. perenne. Significant associations with gene sequencesaligning with marker sequences on Lolium LGs 3 and 6 were also identified. We thereforedemonstrate the power of a novel association genetics approach to identify the genescontrolling multi-locus gametophytic SI systems and to identify novel loci potentiallyinvolved in already established SI systems.