There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.

The recent advances in high throughput RNA sequencing (RNA-Seq) have generated huge amounts of data in a very short span of time for a single sample. These data have required the parallel advancement of computing tools to organize and interpret them meaningfully in terms of biological implications, at the same time using minimum computing resources to reduce computation costs. Here we describe the method of analyzing RNA-seq data using the set of open source software programs of the Tuxedo suite: TopHat and Cufflinks. TopHat is designed to align RNA-seq reads to a reference genome, while Cufflinks assembles these mapped reads into possible transcripts and then generates a final transcriptome assembly. Cufflinks also includes Cuffdiff, which accepts the reads assembled from two or more biological conditions and analyzes their differential expression of genes and transcripts, thus aiding in the investigation of their transcriptional and post transcriptional regulation under different conditions. We also describe the use of an accessory tool called CummeRbund, which processes the output files of Cuffdiff and gives an output of publication quality plots and figures of the user's choice. We demonstrate the effectiveness of the Tuxedo suite by analyzing RNA-Seq datasets of Arabidopsis thaliana root subjected to two different conditions.

There is an increasing understanding that variation in gene presence-absence plays an important role in the heritability of agronomic traits; however, there have been relatively few studies on variation in gene presence-absence in crop species. Hexaploid wheat is one of the most important food crops in the world and intensive breeding has reduced the genetic diversity of elite cultivars. Major efforts have produced draft genome assemblies for the cultivar Chinese Spring, but it is unknown how well this represents the genome diversity found in current modern elite cultivars. In this study we build an improved reference for Chinese Spring and explore gene diversity across 18 wheat cultivars. We predict a pangenome size of 140 500 ± 102 genes, a core genome of 81 070 ± 1631 genes and an average of 128 656 genes in each cultivar. Functional annotation of the variable gene set suggests that it is enriched for genes that may be associated with important agronomic traits. In addition to variation in gene presence, more than 36 million intervarietal single nucleotide polymorphisms were identified across the pangenome. This study of the wheat pangenome provides insight into genome diversity in elite wheat as a basis for genomics-based improvement of this important crop. A wheat pangenome, GBrowse, is available at http://appliedbioinformatics.com.au/cgi-bin/gb2/gbrowse/WheatPan/, and data are available to download from http://wheatgenome.info/wheat_genome_databases.php.

SummaryHomoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

SummaryAs an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor‐bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor‐bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.