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13 Pags.- 6 Figs. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder ; While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution. ; The work conducted by the US DOE Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract no. DE-AC02-05CH11231. D.P. W. and R.A. were funded in part by the National Science Foundation (grant no. IOS–1258126), and the Great Lakes Bioenergy Research Center (Department of Energy Biological and Environmental Research Office of Science grant no. DE– FCO2–07ER64494). TEJ and DLDM were supported by NSF PGRP grant IOS-0922457. P.C. and B.C.M. were funded by Spanish MINECO (CGL2012-39953-C02-01 and CGL2016-79790-P). B.C.M. was partially funded by DGA—Obra Social La Caixa (grant number GA-LC-059-2011) and Spanish MINECO (AGL2013-48756-R, CSIC13-4E-2490). PC was partially funded by Spanish Aragon Government-European Social Fund (Bioflora). ; Peer reviewed