While genome assembly projects have been successful in a number of haploid or inbred species, one of the main current challenges is assembling non-inbred or rearranged heterozygous genomes. To address this critical need, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble Single Molecule Real-Time (SMRT®) Sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We demonstrate the quality of this approach by assembling new reference sequences for three heterozygous samples, including an F1 hybrid of the model species Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata that have challenged short-read assembly approaches. The FALCON-based assemblies were substantially more contiguous and complete than alternate short or long-read approaches. The phased diploid assembly enabled the study of haplotype structures and heterozygosities between the homologous chromosomes, including identifying widespread heterozygous structural variations within the coding sequences.

New races of Puccinia striiformis f. sp. tritici (Pst), the causal pathogen of wheat stripe rust, show high virulence to previously deployed resistance genes and are responsible for large yield losses worldwide. To identify new sources of resistance we performed a genome-wide association study (GWAS) using a worldwide collection of 1000 spring wheat accessions. Adult plants were evaluated under field conditions in six environments in the western United States, and seedlings were tested with four Pst races. A single-nucleotide polymorphism (SNP) Infinium 9K-assay provided 4585 SNPs suitable for GWAS. High correlations among environments and high heritabilities were observed for stripe rust infection type and severity. Greater levels of Pst resistance were observed in a subpopulation from Southern Asia than in other groups. GWAS identified 97 loci that were significant for at least three environments, including 10 with an experiment-wise adjusted Bonferroni probability < 0.10. These 10 quantitative trait loci (QTL) explained 15% of the phenotypic variation in infection type, a percentage that increased to 45% when all QTL were considered. Three of these 10 QTL were mapped far from previously identified Pst resistance genes and QTL, and likely represent new resistance loci. The other seven QTL mapped close to known resistance genes and allelism tests will be required to test their relationships. In summary, this study provides an integrated view of stripe rust resistance resources in spring wheat and identifies new resistance loci that will be useful to diversify the current set of resistance genes deployed to control this devastating disease.

Structural variants (SVs) affect plant phenotypes, but they are a largely unexplored feature of plant genomes. Little is known about the type and size of SVs, their distribution among individuals or their evolutionary dynamics. Here we identify SVs and study their evolutionary dynamics in clonally propagated grapevine cultivars and their outcrossing wild relatives. To catalog SVs, we assembled the highly heterozygous Chardonnay genome, for which one in seven genes is hemizygous. Using genomic inference as the standard, we extended SV detection to population samples. We found that negative selection acts against SVs, but particularly against inversion and translocation events. SVs nonetheless accrue as recessive heterozygotes in clonal lineages. They also define outlier regions of genomic divergence between wild and cultivated grapevines, suggesting roles in domestication. Outlier regions include the sex determination region and the berry color locus, where independent large, complex inversions drive convergent phenotypic evolution..

BackgroundWheat yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (PST) is one of the most devastating diseases of wheat worldwide. To design effective breeding strategies that maximize the potential for durable disease resistance it is important to understand the molecular basis of PST pathogenicity. In particular, the characterisation of the structure, function and evolutionary dynamics of secreted effector proteins that are detected by host immune receptors can help guide and prioritize breeding efforts. However, to date, our knowledge of the effector repertoire of cereal rust pathogens is limited.ResultsWe re-sequenced genomes of four PST isolates from the US and UK to identify effector candidates and relate them to their distinct virulence profiles. First, we assessed SNP frequencies between all isolates, with heterokaryotic SNPs being over tenfold more frequent (5.29 ± 2.23 SNPs/kb) than homokaryotic SNPs (0.41 ± 0.28 SNPs/kb). Next, we implemented a bioinformatics pipeline to integrate genomics, transcriptomics, and effector-focused annotations to identify and classify effector candidates in PST. RNAseq analysis highlighted transcripts encoding secreted proteins that were significantly enriched in haustoria compared to infected tissue. The expression of 22 candidate effector genes was characterised using qRT-PCR, revealing distinct temporal expression patterns during infection in wheat. Lastly, we identified proteins that displayed non-synonymous substitutions specifically between the two UK isolates PST-87/7 and PST-08/21, which differ in virulence to two wheat varieties. By focusing on polymorphic variants enriched in haustoria, we identified five polymorphic effector candidates between PST-87/7 and PST-08/21 among 2,999 secreted proteins. These allelic variants are now a priority for functional validation as virulence/avirulence effectors in the corresponding wheat varieties.ConclusionsIntegration of genomics, transcriptomics, and effector-directed annotation of PST isolates has enabled us to move beyond the single isolate-directed catalogues of effector proteins and develop a framework for mining effector proteins in closely related isolates and relate these back to their defined virulence profiles. This should ultimately lead to more comprehensive understanding of the PST pathogenesis system, an important first step towards developing more effective surveillance and management strategies for one of the most devastating pathogens of wheat.

SignificanceWe generated genomic data to estimate the population history of grapes, the most economically important horticultural crop in the world. Domesticated grapes experienced a protracted, 22,000-y population decline prior to domestication; we hypothesize that this decline reflects low-intensity cultivation by humans prior to domestication. Domestication altered the mating system of grapes. The sex determination region is detectable as a region of heightened genetic divergence between wild and cultivated accessions. Based on gene expression analyses, we propose candidate genes that alter sex determination. Finally, grapes contain more deleterious mutations in heterozygous states than do their wild ancestors. The accumulation of deleterious mutations is due in part to clonal propagation, which shelters deleterious recessive mutations.