The Pan-Genome of Brachypodium distachyon, Capturing the Full Genetic Complement of a Plant Species

The genetic diversity of a species is the sum of the diversity found in all individuals of that species. Many studies have attempted to estimate the diversity of plant species by resequencing diverse accessions and aligning the reads to a reference genome. While this approach readily identifies SNPs and small indels with respect to the reference genome, it underestimates total genomic diversity because highly divergent regions align poorly to the reference and, of course, any sequence not found in the reference will be missed entirely. Thus, the true extent of diversity within plant species is largely unknown. De-novo genome assemblies and annotation can be used to more accurately estimate the true genomic diversity within a species. We applied this approach using 54 Brachypodium distachyon accessions to create a pan-genome that contains all the diversity found in the accessions sequenced. Analysis of this data yields a high-confidence B. distachyon pan-genome that includes 13,408 core gene clusters found in all lines, 7,283 soft-core genes cluster absent from a few lines, and 17,195 shell gene clusters found in 3 to 52 lines. We find 30,691 gene clusters represented by the reference genome/reference control and a varying number of other genomes. In addition, we identify 7,135 gene clusters not represented in the reference line or controls but present in multiple divergent lines. We show that non-core genes are expressed at lower levels, have narrower and more variable expression across accessions, are evolving faster, have reduced orthology to related grasses and are less likely to have a homeologous gene retained from the ancient genome duplication in the grass lineage. We evaluate the relationship between the number of sequenced lines and their phylogenetic position in relation to the addition of both genic and non-coding sequence. We describe the physical chromosome position of non-core and non-reference genes and its relation to transposable elements. This analysis suggests possible mechanisms by which dispensable genes are eliminated and also barriers to their removal.