Identification of homologous, homoeologous and paralogous sequence variants in an outbreeding allopolyploid species based on comparison with progenitor taxa

The combination of homologous, homoeologous and paralogous classes of sequence variation presents major challenges for SNP discovery in outbreeding allopolyploid species. Previous in vitro gene-associated SNP discovery studies in the allotetraploid forage legume white clover (Trifolium repens L.) were vulnerable to such effects, leading to prohibitive levels of attrition during SNP validation. Identification of T. occidentale and T. pallescens as the putative diploid progenitors of white clover has permitted discrimination of the different sequence variant categories. Amplicons from selected abiotic stress tolerance-related genes were obtained using mapping family parents and individuals from each diploid species. Following cloning, progenitor comparison allowed tentative assignment of individual haplotypes to one or other sub-genome, as well as to gene copies within sub-genomes. A high degree of coincidence and identity between SNPs and HSVs was observed. Close similarity was observed between the genome of T. occidentale and one white clover sub-genome, but the affinity between T. pallescens and the other sub-genome was weaker, suggesting that a currently uncharacterised taxon may be the true second progenitor. Selected validated SNPs were attributed to individual sub-genomes by assignment to and naming of homoeologous linkage groups, providing the basis for improved genetic trait-dissection studies. The approach described in this study is broadly applicable to a range of allopolyploid taxa of equivocal ancestry.