Having a precise and thorough structural gene annotation is essential for any pan-genome study. Especially for large and highly repetitive genomes like the wheat genome, an erroneous gene annotation will lead to wrong or incomplete conclusions. Here we present an enhanced protocol developed for the 10+ wheat genome project to unify individual gene predictions from all sequenced accessions. Based on the already established annotation process published as part of the IWGCS 2018 wheat reference genome paper additional methods were refined to counteract possible annotation errors. To aid the initial structural annotation RNAseq data derived from 5 different tissues/developmental stages, as well as ISOseq data from root and shoot tissue were generated for each wheat accession. Combined with protein homology and ab initio gene predictions we were able to define accurate gene models for each accession. A supplementary consolidation step that overcomes fuzzy limitations in gene calling on individual genomes ensures a uniform gene annotation across all genomes. The consolidation method is based on whole genome alignments to identify syntenic regions, cross-map gene models of each of the lines and rectify wrong or missing gene models.

Along with the availability of reference-quality genome sequences for 10 agriculturally important wheat accessions unified gene annotations will leverage any further downstream genomic and breeding-related analyses.