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Alfalfa ( Medicago sativa  L.) is one of the most important and widely cultivated forage crops. It is commonly used as a vegetable and medicinal herb because of its excellent nutritional quality and significant economic value. Based on Illumina, Nanopore and Hi-C data, we assembled a chromosome-scale assembly of  Medicago sativa  spp . caerulea  (voucher PI464715), the direct diploid progenitor of autotetraploid alfalfa. The assembled genome comprises 793.2 Mb of genomic sequence and 47,202 annotated protein-coding genes. The contig N50 length is 3.86 Mb. This genome is almost twofold larger and contains more annotated protein-coding genes than that of its close relative,  Medicago truncatula  (420 Mb and 44,623 genes). The more expanded gene families compared with those in  M. truncatula  and the expansion of repetitive elements rather than whole-genome duplication (i.e., the two species share the ancestral Papilionoideae whole-genome duplication event) may have contributed to the large genome size of  M. sativa  spp . caerulea . Comparative and evolutionary analyses revealed that  M. sativa  spp . caerulea  diverged from  M. truncatula  ~5.2 million years ago, and the chromosomal fissions and fusions detected between the two genomes occurred during the divergence of the two species. In addition, we identified 489 resistance ( R ) genes and 82 and 85 candidate genes involved in the lignin and cellulose biosynthesis pathways, respectively. The near-complete and accurate diploid alfalfa reference genome obtained herein serves as an important complement to the recently assembled autotetraploid alfalfa genome and will provide valuable genomic resources for investigating the genomic architecture of autotetraploid alfalfa as well as for improving breeding strategies in alfalfa.

A chromosome-scale genome assembly of a diploid alfalfa, the progenitor of autotetraploid alfalfa