A Tale of Four Genomes: de novo Genome Sequencing of Allotetraploid White Clover (Trifolium repens L.) and Its Progenitors

Polyploidy, a feature common to many crop species, impedes assembly when developing de novo reference genomes based on short-read sequencing platforms.  White clover (Trifolium repensL.), an allotetraploid, is a principal legume in temperate pastoral systems.  It produces high quality herbage, and fixes atmospheric nitrogen into plant available forms through bacterial symbiosis.  Pastoral Genomics, a New Zealand Industry:Government-funded research consortium, is sequencing white clover in collaboration with researchers at Aarhus University, Denmark, to facilitate understanding and improvement of the species. 

White clover has a relatively compact genome (≈1 Gb), but presents sequencing challenges as an allogamous species (2n=4x=32) comprising genetically similar O and P’ homoeologues derived from diploid progenitors T. occidentale and T. pallescens, respectively.  To reduce intra-homoeologous diversity and facilitate sequence assembly, we focused on an inbred (S₉) white clover genotype using various platforms (Illumina HiSeq, Roche 454 GS FLX Titanium) and libraries to ≈200x coverage.  These data were supplemented with transcriptome sequence from multiple tissues.  To aid sequence assignment to homoeologous sets, an individual of inbred T. occidentale and of T. pallescens (≈0.5 Gb each) were sequenced as for white clover; and to resolve assembly conflation, the white clover short-read data were augmented by Illumina TruSeq Synthetic Long-Read sequencing. 

Final annotated assemblies of allotetraploid white clover and its diploid progenitors provide insights into sequence assembly challenges and strategies in high-diversity polyploid species; homoeologue contribution to allopolyploid gene expression; and routes via sequenced model legumes to elucidating conservation and variation in genome structure and function within family Fabaceae.