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

Abstract

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.

Achievements and Challenges in Improving Temperate Perennial Forage Legumes

“…This is predominantly due to a high proportion of putative SNP markers in silico arising from conflation of orthologous sequence within homoeologous pairs (Cogan et al, 2007). Reference sequence from progenitor species (Ellison et al, 2006) partially overcomes this limitation (Hand et al, 2008). A recent report (Nagy et al, 2013) demonstrates a novel approach using sequence data of diverse genotypes combined with sequence analysis and haplotyping of two inbreds as a pseudo-double haploid to identify over 70,000 putative SNPs in white clover, without reliance on a progenitor reference sequence (Nagy et al, 2013).…”

Adaptive gains through repeated gene loss: parallel evolution of cyanogenesis polymorphisms in the genusTrifolium(Fabaceae)

“…Proposed progenitors within Trifoliastrum have included T. occidentale, T. nigrescens ssp. petrisavii, T. pallescens and an unknown lineage [37,39,45,46].…”

Development of Genomic Resources in the Species of Trifolium L. and Its Application in Forage Legume Breeding

“…In addition progress has been made in sub-genome differentiation making robust SNP identification easier [24]. A preliminary study of marker-assisted selection (MAS) in white clover has been described by Barrett et al [25].…”

“…Its origin is considered quite recent, as suggested by the chromosome number being a multiple of the genus basic number [60,61]. An age of 4.2 million years has been estimated by Hand et al [24] by comparing synonymous substitutions between homoeologous genes from the putative ancestral species. However, measurement was carried out on a limited number of genes rather than a large collection, such as an EST library.…”

“…The work of Ellison et al [8] using ITS nrDNA and cpDNA to construct a phylogenetic tree of 218 Trifolium species strongly indicates T. pallescens as the female ancestor. Following SNP comparison in eight stress-related genes from T. repens, T. occidentale and T. pallescens, Hand et al [24] showed a close relation between T. occidentale and one of T. repens sub-genomes, while the T. pallescens sequences were more loosely related and somewhat less informative. This led to the argument that the real ancestor might be an extinct or yet to be discovered species related to, but different from T. pallescens.…”