Dissecting an Allopolyploid Genome: De Novo Genome Sequencing of White Clover (Trifolium repens L.)

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 allopolyploid, 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 (high levels of sequence diversity) allotetraploid (2n=4x=32) comprising genetically similar O and P’ homoeologues derived from the 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 and used various platforms (Illumina HiSeq, Roche 454 GS FLX Titanium) and libraries (shotgun; 500 bp insert pair-end; 3, 5, and 8 Kb insert mate-pair) to ≈200x coverage.  These data were supplemented with transcriptome sequence from multiple tissues.  To aid sequence assignment to homoeologous sets, inbred T. occidentale(≈0.5 Gb) was sequenced as for white clover; and to resolve assembly conflation, the white clover short-read data were augmented by Illumina Long-Read sequencing. 

Draft assemblies of white clover and T. occidentale provide insights into sequence assembly challenges and strategies in high diversity polyploid species, and provide a route via sequenced model legumes to elucidating conservation and variation in genome structure within family Fabaceae.