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.

Population Differentiation Within Anisogramma anomala in North America.

Anisogramma anomala, a biotrophic ascomycete in the order Diaporthales, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). Until recently, little has been documented on its genetic diversity and population structure. In this study, 18 simple sequence repeat markers were used to fingerprint 182 accessions of the fungus originating from across North America. Our results, based on summary statistics of the allelic data, a discriminant analysis of principal components (DAPC) scatterplot, an unweighted pair group method with arithmetic mean (UPGMA) dendrogram, and analysis of multilocus genotypes, show that A. anomala exhibits considerable genetic diversity across multiple populations. Eleven clusters were resolved from the DAPC scatterplot, five of which were validated by statistically supported clusters in the UPGMA dendrogram. The 11 DAPC clusters were statistically significant via an analysis of molecular variance. Dendrogram topology and DAPC scatterplot groups showed some correlation with collection origin; samples collected in proximity tended to cluster together and be genetically similar. However, some locations held populations that were diverse and some populations with a high degree of similarity had disparate origins, suggesting movement by humans. Overall, the results demonstrate the presence of multiple, genetically distinct populations of A. anomala in North America and serve as a reference to assist in understanding and managing EFB.

Development of Genomic Resources for the Powdery Mildew, Erysiphe pulchra.

Powdery mildews (PMs) are important plant pathogens causing widespread damage. Here, we report the first draft genome of Erysiphe pulchra, the causative agent of PM of flowering dogwood, Cornus florida. The assembled genome was 63.5 Mbp and resulted in formation of 19,442 contigs (N50 = 11,686 bp) that contained an estimated 6,860 genes with a genome coverage of 62×. We found 102 candidate secreted effector proteins (CSEPs) in E. pulchra similar to E. necator genes that are potentially involved in disease development. This draft genome is an initial step for understanding the evolutionary history of the PMs and will also provide insight into evolutionary strategies that led to the wide host expansion and environmental adaptations so effectively employed by the PM lineages.

Genetic diversity analysis of big-bracted dogwood (Cornus florida and C. kousa) cultivars, interspecific hybrids, and wild-collected accessions using RADseq.

Big-bracted dogwoods are popular ornamental trees known for their beautiful spring blooms with showy bracts and four-season appeal. The two most widely grown species are Cornus florida and Cornus kousa, native to Eastern North America and East Asia. Despite their horticultural prominence, there is little information available regarding genetic diversity, population structure, relatedness, and subspecies origins of dogwood cultivars. In this study, 313 cultivars, wild-collected plants, and Rutgers University breeding selections, focusing on C. florida, C. kousa, and interspecific hybrids, were genotyped using restriction-site associated DNA sequencing (RADseq) generating thousands of single nucleotide polymorphism (SNP) and insertion deletion (Indel) markers. The research results showed high genetic diversity among C. florida and C. kousa wild-collected plants and cultivars. For C. florida, pink-bracted plants formed a distinct clade from those with white-bracts with the Mexican C. florida ssp. urbiniana forming an outgroup. For C. kousa, Chinese-collected plants (ssp. chinensis) were a distinct subspecies with clear separation from Japanese and Korean accessions (ssp. kousa) and cultivars were designated as ssp. chinensis, ssp. kousa, or ssp. hybrid. Using this information, a Kompetitive allele specific PCR (KASP) assay genotyping panel was designed to determine C. kousa trees' subspecies makeup. Results revealed many cases of genetically identical cultivars being sold under different names, especially for pink-bracted cultivars of both species. Additionally, reported parent-progeny relationships were evaluated and either validated or discredited. Finally, the hybrid germplasm analysis validated pedigrees of interspecific F1 hybrids and found many of the recent Rutgers breeding selections contain small regions of pacific dogwood (C. nuttallii) DNA introgressed into C. kousa backgrounds. This diversity study elucidates origins, diversity, and relationships of a large population of big-bracted dogwoods. The results can inform plant breeders, arboreta, and the ornamental plant industry, as most modern cultivars and popular historic cultivars are represented.

Variable response of eastern filbert blight resistance sources in New Jersey.

Eastern filbert blight (EFB), caused by Anisogramma anomala, is the primary limiting factor for hazelnut (Corylus sp.) production in the United States. In this study, 82 cultivars and selections shown to be resistant or tolerant to EFB in Oregon were field planted in New Jersey in 2017 and 2019 and evaluated for their EFB response under high disease pressure. The trees carry known single resistance (R) genes with most mapped to their respective linkage groups (LG), including LG2, LG6, and LG7, or they express quantitative resistance (QR, horizontal resistance). Disease incidence and severity was documented, stem cankers counted and measured, and the proportion of diseased wood calculated. The EFB disease response of some cultivars/selections varied considerably between New Jersey and Oregon while others were consistent. Trends were observed in relation to resistance source origin and LGs, which provide insight into durability and usefulness of resistance. In striking contrast to Oregon, nearly all selections with R-genes mapped to LG6, including those carrying the 'Gasaway' resistance allele, exhibited severe EFB infections (232 of 266 [87%]). This finding is of consequence since the U.S. hazelnut industry currently relies solely on LG6 resistance for EFB resistance. Further, for the first time, EFB was observed on several selections carrying LG7 resistance, specifically offspring of 'Ratoli' from Spain. Interestingly, selections carrying LG7 resistance from origins other than 'Ratoli' remained free of EFB, with one exception, all selections carrying LG2 (n=9) resistance also remained free from EFB. Interestingly, the EFB responses of selections expressing QR (n=26) more closely resembled the disease phenotypes they exhibited in Oregon. Overall, the divergence in EFB response between Oregon and New Jersey, where pathogen populations differ, supports the presence of pathogenic variation in A. anomala and highlights potential limitations of using single R-genes to manage the disease. Results also suggest trees expressing QR may be more stable across pathogenic populations.

A Real-Time PCR Assay for Early Detection of Eastern Filbert Blight.

Eastern filbert blight (EFB) is a devastating disease of European hazelnut, Corylus avellana, which causes economic losses in Oregon, where 99% of the U.S. crop is produced. The causal fungus, Anisogramma anomala, is native to eastern North America, where it is found associated with the American hazelnut (C. americana). Although C. americana is tolerant, EFB causes cankers, branch dieback, and death of C. avellana. Detection and identification of A. anomala is time consuming using conventional methods because the fungus can only be cultured from sporulating perithecia and the disease symptoms and signs only show 12 to 16 months after infection. In this study, a TaqMan real-time polymerase chain reaction (PCR) assay based on a ribosomal DNA internal transcribed spacer was developed for A. anomala. The assay was validated with multiple isolates of A. anomala, closely related species, common environmental microorganisms, and over 100 C. avellana samples. The real-time PCR assay detected as low as 0.12 pg of A. anomala genomic DNA, and positively diagnosed EFB on 82% of asymptomatic plants as early as 15 weeks from infection. The real-time PCR assay is more sensitive and faster than traditional diagnostic methods. It can facilitate hazelnut breeding and disease management by early and accurate diagnosis of EFB.

Genome-wide microsatellite identification in the fungus Anisogramma anomala using Illumina sequencing and genome assembly.

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the "Seq-to-SSR" approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel "Seq-Assembly-SSR" approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the "Seq-Assembly-SSR" approach has proven to be a successful one for microsatellite discovery.