Progress in Sequencing the Genome of Tetraploid Alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is an autotetraploid (2n=4x=32), out-crossing perennial forage legume crop species with an estimated haploid (1x) genome size of 830-860 Mbp. We have generated 180x genome coverage with Illumina reads from short-insert paired-end libraries (read length of 2x100-nt) and long-insert paired-end libraries (2x50-nt and 2x54-nt) to provide ~30-50x genome coverage.  Additionally, PacBio sequencing generated 1.4 million reads for a total of 9.5 Gbp and a N50 value of 10,268. The read length of the libraries exceeds 20,000 with a mean sequence length of 12,000 bp.  Combining sequences from Illumina, PacBio and Whole Genome Profiling as a hybrid assembly will result in fewer but longer contig scaffolds, enabling the development of a draft genome assembly of tetraploid alfalfa.  Initial alignment with the M. truncatula genome (Mt 4.0) includes over 600,000 mapped reads on all eight chromosomes, with the highest synteny observed in the genic areas.  Sequence variations between M. truncatula and M. sativa include insertions and deletions, although the majority of variants are due to sequence substitutions. Ongoing efforts include additional sequencing to increase the depth of sequence coverage to facilitate the assembly of this highly heterozygous autotetraploid genome.  We will also develop tools to manage and visualize the draft genome in the context of other legume genomes.  The draft genome will also serve as the backbone of the Alfalfa Breeder’s Toolbox, facilitating basic and applied research on alfalfa, including the implementation of molecular breeding strategies to improve key traits including yield, biotic and abiotic stress tolerances, and nutritive value.