De novo Genome Sequencing of White Clover (Trifolium repens L.)

White clover (Trifolium repens L.), a principal legume in temperate pastoral systems, produces high quality herbage, offers compatibility and persistence in mixed swards, and through bacterial symbiosis fixes atmospheric nitrogen into plant available forms.  Pastoral Genomics, a New Zealand Industry:Government-funded research consortium, is sequencing the white clover genome to complement existing genetic and genomic resources and facilitate understanding and improvement of the species.  Although white clover has a relatively compact genome (≈ 960 Mb), it presents sequencing challenges as an allogamous allotetraploid (2n=4x=32; comprising O and P’ homoeologues) species exhibiting high levels of sequence diversity.  To better enable sequence assembly, we focused on an inbred (S₉) white clover genotype using various platforms (Illumina HiSeq2000, Roche 454 GS FLX Titanium) and libraries (shotgun; 500 bp insert pair-end; 3 Kb, 5 Kb, 8 Kb, and 20 Kb insert mate-pair) to approximately 200x coverage.  These data were supplemented with 454 transcriptome sequence from leaf, root, stolon and floral tissue.  To aid correct assignment of sequence to homoeologous sets, a genotype of T. occidentale (≈ 500 Mb) - an inbred diploid progenitor which contributed the O homoeologous set to modern clover – was sequenced using Illumina HiSeq2000 with 500 bp insert pair-end; 3 Kb, 5 Kb, and 8 Kb mate-pair libraries to 180x coverage.  These data have been used to generate draft assemblies of T. occidentale and white clover, of which the most recent metrics will be presented, and provide insights into sequence assembly challenges and strategies in high diversity polyploidy species.