Abstract
The wheat stripe rust fungus (Puccinia striiformis f. sp. tritici, PST) is responsible for significant yield losses in wheat production worldwide. In spite of its economic importance, the PST genomic sequence is not currently available. Fortunately Next Generation Sequencing (NGS) has radically improved sequencing speed and efficiency with a great reduction in costs compared to traditional sequencing technologies. We used Illumina sequencing to rapidly access the genomic sequence of the highly virulent PST race 130 (PST-130). We obtained nearly 80 million high quality paired-end reads (>50x coverage) that were assembled into 29,178 contigs (64.8 Mb), which provide an estimated coverage of at least 88% of the PST genes and are available through GenBank. Extensive micro-synteny with the Puccinia graminis f. sp. tritici (PGTG) genome and high sequence similarity with annotated PGTG genes support the quality of the PST-130 contigs. We characterized the transposable elements present in the PST-130 contigs and using an ab initio gene prediction program we identified and tentatively annotated 22,815 putative coding sequences. We provide examples on the use of comparative approaches to improve gene annotation for both PST and PGTG and to identify candidate effectors. Finally, the assembled contigs provided an inventory of PST repetitive elements, which were annotated and deposited in Repbase. The assembly of the PST-130 genome and the predicted proteins provide useful resources to rapidly identify and clone PST genes and their regulatory regions. Although the automatic gene prediction has limitations, we show that a comparative genomics approach using multiple rust species can greatly improve the quality of gene annotation in these species. The PST-130 sequence will also be useful for comparative studies within PST as more races are sequenced. This study illustrates the power of NGS for rapid and efficient access to genomic sequence in non-model organisms.
Highlights
Wheat stripe rust caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (PST), is one of the most devastating diseases of wheat worldwide [1,2]
Illumina sequencing and de novo contig assembly The sequence reads from race PST race 130 (PST-130) together with the resulting assemblies are available from NCBI (Whole Genome Shotgun project: AEEW00000000; Sequence Read Archive: SRP002642)
In order to identify DNA sequences resulting from contamination during spore collection from infected wheat plants we queried the PST-130 assemblies against the GenBank nt database, 30,696 wheat contigs (37.8 Mb) assembled from a 454 transcriptome study of hexaploid wheat flag leaves [9], the complete Triticeae Repeat Sequence Database (TREP; http://wheat.pw.usda.gov/ITMI/Repeats) and the 454 sequence reads of the hexaploid wheat genome
Summary
Wheat stripe rust caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (PST), is one of the most devastating diseases of wheat worldwide [1,2]. The understanding of pathogenicity and virulence factors, and of their evolution, is critical to the development of more effective breeding strategies for durable resistance. Progress in these areas has been hampered by the lack of PST genome sequence information. Tritici (PGTG; causal agent of wheat stem rust) has recently been published [5] and the genome of P. triticina (PTTG; causal agent of wheat leaf rust) is currently being fully sequenced and annotated (http://www.broadinstitute.org/annotation/genome/ puccinia_group/Info.html). A genome sequence for PST is not currently available (a project is in progress at the Broad Institute to sequence and annotate race PST-78). Tritici, PST) is responsible for significant yield losses in wheat production worldwide. We used Illumina sequencing to rapidly access the genomic sequence of the highly virulent PST race 130 (PST-130)
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