The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut.

BACKGROUND: The ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome. RESULTS: The genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors. CONCLUSIONS: This study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen's life cycle and a solid foundation for studying EFB.

Genetic Diversity Analysis of Anisogramma anomala in the Pacific Northwest and New Jersey.

Anisogramma anomala, a biotrophic ascomycete, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). EFB is endemic in eastern North America, preventing the commercial production of European hazelnut (C. avellana L.). In contrast, the historic absence of A. anomala in the Pacific Northwest (PNW) supported the development of a robust hazelnut industry. Circa 1960, A. anomala was inadvertently introduced into southwestern Washington, causing orchard devastation. Distribution of the pathogen in the PNW has been hypothesized to be the result of a single-point introduction. This study aimed to investigate the single-point introduction hypothesis of A. anomala by comparing the genetic diversity of A. anomala samples from the PNW and New Jersey (NJ). Specimens from the main PNW production region (n = 60) and an area within the pathogen's native range, NJ (n = 151), were genotyped using 15 simple sequence repeat (SSR) markers. The following were used to assess genetic diversity and population structure: allelic summary statistics, discriminant analysis of principal components, network median-joining tree, analysis of multilocus genotypes, and allelic population diversity analysis. Analyses separated the samples into one cluster containing all the PNW isolates, and five clusters of NJ isolates. The PNW samples were nearly genetically uniform, and the NJ isolates were diverse. These findings support the hypothesis that A. anomala in the PNW was derived from a single-point introduction and corroborate previous studies that have shown A. anomala is very diverse in NJ. This indicates that maintaining restrictions on the movement of Corylus into the PNW is important to prevent the introduction of new populations of A. anomala, thus protecting the PNW hazelnut industry.

New Sources of Eastern Filbert Blight Resistance and Simple Sequence Repeat Markers on Linkage Group 6 in Hazelnut (Corylus avellana L.).

Commercial production of hazelnut (Corylus avellana) in Oregon's Willamette Valley is threatened by eastern filbert blight (EFB), a serious canker disease caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller. The fungus also prevents the establishment of hazelnut orchards in eastern North America. Genetic resistance is considered the most effective way to control the disease. A high level of EFB resistance was first discovered in 'Gasaway'. This resistance is conferred by a dominant allele at a single locus on linkage group 6 (LG6). Resistance from several additional sources has been assigned to the same chromosomal region. In this study, new simple sequence repeat (SSR) markers were developed for the resistance region on LG6 and new sources of resistance were investigated. Forty-two new SSR markers were developed from four contigs in the genome sequence of 'Jefferson' hazelnut, characterized, and nine of them were placed on LG6 of the genetic map. Accessions representing 12 new sources of EFB resistance were crossed with susceptible selections resulting in 18 seedling populations. Segregation ratios in the seedling populations fit the expected 1:1 ratio for 10 sources, while one source showed an excess of resistant seedlings and another showed an excess of susceptible seedlings. Based on correlation of disease response and scores of SSR markers in the 'Gasaway' resistance region in the seedlings, eight resistance sources were assigned to LG6. Linkage maps were constructed for each progeny using SSR markers. The LG6 resistance sources include two selections (#23 and #26) from the Russian Research Institute of Forestry and Mechanization near Moscow, four selections from southern Russia, one selection (OSU 1185.126) from Crimea, one selection (OSU 533.129) from Michigan, Corylus heterophylla 'Ogyoo' from the South Korea, and the interspecific hybrid 'Estrella #1'. These new LG6 resistance sources and SSR markers should be useful in breeding new cultivars, including the pyramiding of resistance genes. For the other four resistance sources (Moscow #37, hybrid selection OSU 401.014, C. americana 'Winkler' and C. americana OSU 366.060), SSR marker scores on linkage groups 6, 7 and 2 were not correlated with disease response and merit further investigation.