The pseudogenes of barley

Summary Pseudogenes have a reputation of being ‘evolutionary relics’ or ‘junk DNA’. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high‐quality genome reference assembly available for a Triticeae crop, we conducted a whole‐genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full‐length pseudogenes (11 015) have preferentially retained their exon–intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense‐response were found to be under‐represented in cultivated barley, we detected several defense‐related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non‐random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations. Significance Statement Despite ground‐breaking discoveries on the evolutionary and regulatory functions of pseudogenes in mammals, genome‐wide analyses in plants are lagging behind. We assessed the complete pseudogene complement of the first high‐quality reference genome of barley (5.1 Gb), exemplifying a continuum of functional and pseudogene states and indicating a potential reservoir for gene innovation.