Assessing Structural Variation at the Subspecies Level within Trifolium Subterraneum L. — Towards a Trifolium Pangenome

Forage legumes are highly valued feed for extensive livestock production. Symbiotic nitrogen fixation in legumes leads to high protein fodder content and rejuvenated soils for a sustainable feed system. Subterranean clover (Trifolium subterraneum L.) makes the greatest contribution to livestock feed production and soil improvement in terms of total worldwide usage among annual clovers, particularly in Australia, where it is sown over 29 mill. ha. Subterranean clover is a diploid (2n = 2x = 16), predominantly inbreeding, annual species with a relatively small genome size of 540 Mbp that can be readily hybridised, and exhibits wide diversity for both qualitative and quantitative agronomic and morphological characters Within the genus Trifolium, subterranean clover is established as a reference species for genetic and genomic studies. We reported a de novo draft genome assembly of the subterranean clover (based on cv. Daliak) representing 86% of the estimated 556.6 Mb total genome.

The intraspecific taxonomy of T. subterraneum L. has long been disputed. The most commonly used classification describes three subspecies in T. subterraneum - subterraneum, yanninicum, and brachycalycinum based on plant morphology, genetic and cytogenetic data. These three subspecies are different morphologically, allowing them to be readily distinguished, and have different soil preferences. Both ssp. subterraneum and ssp. yanninicum are best suited to moderately acidic soils, with ssp. subterraneum adapted to well-drained soils and ssp. yanninicum to poorly drained soils or those with high water-holding capacity. Subspecies brachycalycinum, on the other hand, is best adapted to well-drained, neutral–alkaline soils, preferably containing cracks or stones for burr development. Subspecies subterraneum is both the most common and widely distributed of the three. Ssp. brachycalycinum is more confined to areas adjacent to the Mediterranean Sea; while ssp. yanninicum tends to be confined to sites prone to winter waterlogging.

In the present study we generated BioNano optical maps for representative cultivars of the three subspecies to assess structural variation at the subspecies level within T. subterraneum. We demonstrate an on-going speciation process, with ssp. yanninicum and ssp. subterraneum being genetically closer to each other than the more distant ssp. brachycalycinum. Having the structural information and gene mining for the three subspecies has important implications for pre-breeding and molecular marker development. Presently, a panel comprising 97 accessions of the world core collection and 28 elite Australian cultivars has been extensively genotyped to construct a PanGenome which will serve as a powerful tool enabling identification of the genes and genetic variation for important economic and morphological traits.