Eastern Filbert Blight Resistance Research Articles

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 haplotype-resolved chromosome-level assembly and annotation of European hazelnut (C. avellana cv. Jefferson) provides insight into mechanisms of eastern filbert blight resistance.

European hazelnut (Corylus avellana L.) is an important tree nut crop. Hazelnut production in North America is currently limited in scalability due to Anisogramma anomala, a fungal pathogen that causes Eastern Filbert Blight (EFB) disease in hazelnut. Successful deployment of EFB resistant cultivars has been limited to the state of Oregon, where the breeding program at Oregon State University (OSU) has released cultivars with a dominant allele at a single resistance locus identified by classical breeding, linkage mapping, and molecular markers. C. avellana cultivar "Jefferson" is resistant to the predominant EFB biotype in Oregon and has been selected by the OSU breeding program as a model for hazelnut genetic and genomic research. Here, we present a near complete, haplotype-resolved chromosome-level hazelnut genome assembly for "Jefferson". This new assembly is a significant improvement over a previously published genome draft. Analysis of genomic regions linked to EFB resistance and self-incompatibility confirmed haplotype splitting and identified new gene candidates that are essential for downstream molecular marker development, thereby facilitating breeding efforts.

New sources of eastern filbert blight resistance in European hazelnut (Corylus avellana L.) mapped to linkage groups 2, 6, and 7

New sources of eastern filbert blight resistance in European hazelnut (<i>Corylus avellana</i> L.) mapped to linkage groups 2, 6, and 7

Segregation of Eastern Filbert Blight Disease Response and Single Nucleotide Polymorphism Markers in Three European–American Interspecific Hybrid Hazelnut Populations

The perennial stem canker disease eastern filbert blight (EFB), caused by Anisogramma anomala, is devastating to most trees of European hazelnut (Corylus avellana), as genetic resistance is rare in the species. The pathogen is harbored by the wild American hazelnut (Corylus americana) found throughout much of eastern North America. Wild American hazelnut is generally resistant or tolerant to EFB, and is fully cross compatible with C. avellana, the species grown commercially for its nuts, making it a valuable resource for disease resistance breeding. The objective of this study was to identify quantitative trait loci (QTLs) associated with EFB resistance and tolerance in these two species. Three unrelated EFB-resistant C. americana selections [Oregon State University (OSU) 533.069 from Pennsylvania, OSU 403.040 from Nebraska, and OSU 557.122 from Wisconsin] were crossed with C. avellana ‘Tonda di Giffoni’ (TdG), a cultivar from Italy known to be tolerant of EFB. Their progenies, each containing 124 trees, were exposed to A. anomala through field inoculations and natural spread over 7 years, then each tree was evaluated for cumulative disease response. Results showed that disease response of all three populations exhibited a roughly normal distribution, indicating that resistance/tolerance was under multigenic control. An average of 2869 total markers were used to construct each population’s linkage map following genotyping, which included an average of 121 published simple sequence repeat markers to anchor linkage groups (LGs) to those of previous studies. Linkage maps were constructed for each parent of each population and used to map QTLs associated with EFB response. The subsequent analysis resolved five EFB-related QTLs across the three populations, highlighting three genic regions. Unexpectedly, only one QTL was identified from one of the three resistant C. americana parents, located on LG11 of the map of OSU 403.040, whereas three QTLs were found in a similar region on LG10 across the three maps of TdG, and a fifth QTL was found on LG6 of one TdG map. The lack of strong QTLs identified from the three EFB-resistant C. americana parents suggests that their resistance may be highly quantitative and not resolved within the constraints of this study. In contrast, tolerance from TdG appears to be conferred by a limited number of genes with relatively strong effects. Based on prior mapping work in European and American hazelnut where R genes have been located on LG2, LG6, and LG7, the QTLs associated with resistance/tolerance on LG10 and LG11 represent novel resistance regions. These QTLs present new targets for marker aided breeding, especially when pyramiding EFB resistance genes is a goal.

New Simple Sequence Repeat Markers on Linkage Groups 2 and 7, and Investigation of New Sources of Eastern Filbert Blight Resistance in Hazelnut

Eastern filbert blight (EFB), caused by Anisogramma anomala, is a fungal disease threatening the european hazelnut (Corylus avellana) industry in the Willamette Valley of Oregon. The pathogen is endemic to the eastern United States where it causes little damage to the wild Corylus americana but causes severe cankers on most cultivars of the commercially important european hazelnut. The host genetic resistance in ‘Gasaway’ is conferred by a dominant allele at a single locus on linkage group 6 (LG6), and resistance from several other sources has been mapped to the same region. Some fungal isolates can overcome ‘Gasaway’ resistance, prompting a search for other sources of resistance. Resistance from other sources has been mapped to LG2 and LG7, for which additional simple sequence repeat (SSR) markers would facilitate marker-assisted selection (MAS). In this study, an in silico approach was used to develop new polymorphic SSR markers in the EFB resistance regions on LG2 and LG7. Starting with a search of 17 contigs of the ‘Jefferson’ genome sequence, 45 new polymorphic SSR markers were developed, characterized, and placed on the linkage map. The new SSR markers had an average of 10.18 alleles per locus, and average values for expected heterozygosity, observed heterozygosity, polymorphism information content, and frequency of null alleles of 0.72, 0.65, 0.68, and 0.068, respectively. Of the 42 new polymorphic SSRs segregating in the mapping population, 24 were on LG2, 12 were on LG7, and six were placed on other LGs. The new and previously developed SSR markers were used to study six new sources of EFB resistance, four from Russia and two from Crimea. Six resistant selections were crossed with susceptible selections, resulting in 7 progenies. Phenotyping for disease response revealed that segregation in progenies of the two Moscow selections (#2 and #27), one Russian selection (OSU 1187.101), and one Crimean selection (H3R12P62) fit the 1:1 segregation ratio expected for control of resistance by a dominant allele at a single locus; but in progenies of the other Russian selection (OSU 1166.123) and the other Crimean selection (H3R07P11), there was an excess of resistant seedlings. Correlation of disease scores and alleles at SSR loci indicated that resistance from three Russian selections (Moscow selections #2 and #27 and OSU 1166.123) and the Crimean selection H3R12P62 was on LG7, while resistance from Russian selection OSU 1187.101 was on LG2. Resistance from Crimean selection H3R07P11 was not correlated with markers on LG6, or LG2, or LG7. These sources and new SSR markers will be useful in MAS and the pyramiding of resistance genes in the breeding of new EFB-resistant cultivars.

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.

Using genotyping-by-sequencing derived SNPs to examine the genetic structure and identify a core set of Corylus americana germplasm

The American hazelnut (Corylus americana) is native to a broad range of the eastern United States and southern Canada. It is the endemic host of the fungus Anisogramma anomala, which causes eastern filbert blight (EFB) disease and limits European hazelnut (C. avellana) production in eastern North America. While C. americana has thick-shelled, tiny nuts not suited for commercial production, it is cold hardy, highly tolerant of EFB, and phenotypically diverse. Previous studies with simple sequence repeat markers show that it is also genetically diverse. Further, the species is cross-compatible with C. avellana and is thus a valuable donor of EFB resistance and climate adaptability traits. However, only a narrow set of C. americana parents has been used in interspecific hybridizations, and current germplasm availability likely does not fully represent the species’ genetic diversity, given its vast range. A new collection of open-pollinated C. americana seed was assembled at Rutgers University to broaden available genetic resources. Here, we study the genetic diversity and population structure of 272 of these individuals, which represent 55 seedlots from across 15 states of the species’ native range. We use multivariate analyses to examine the distribution of genetic variation within the collection and to support the identification of a core set. A genotyping-by-sequencing (GBS) approach yielded 2653 single nucleotide polymorphisms and subsequent analyses revealed a collection with high estimates of heterozygosity (HE = 0.276, HO = 0.280), moderate differentiation (FST = 0.108) and low inbreeding (FIS = −0.136). Bayesian model-based and neighbor-joining (NJ) clustering corroborate an uppermost grouping of K = 3, with the NJ dendrogram depicting many small subgroups equally distant from common ancestry. Discriminant analysis of principal components (DAPC) reveals between-subgroup variation (K = 15) within the NJ dendrogram and allows the identification of 19 consensus subgroups. In general, our results support the assembly of a genetically diverse collection where a majority of the variation is explained at the genotype and subgroup levels, which aligns with previous studies of C. americana. Fifty-one accessions were identified that represent 95% of the observed allelic variation. These genotypes are suggested for inclusion in a core collection, which, when coupled to concurrent phenotypic evaluations, will aid in genetic resource assembly that preserves unique phenotypes and retains genetic variation.

Eastern Filbert Blight Resistance in American and Interspecific Hybrid Hazelnuts

Eastern filbert blight (EFB), caused by the fungus Anisogramma anomala, is a primary limitation to european hazelnut (Corylus avellana) cultivation in eastern North America. American hazelnut (Corylus americana) is the endemic host of A. anomala and, despite its tiny, thick-shelled nuts, is a potentially valuable source of EFB resistance and climatic adaptation. Interspecific hybrids (Corylus americana × C. avellana) have been explored for nearly a century as a means to combine EFB resistance with wider adaptability and larger nuts. Although significant progress was made in the past, the genetic diversity of the starting material was limited and additional improvements are needed for expansion of hazelnut (Corylus sp.) production outside of Oregon, where 99% of the U.S. crop is currently produced. Our objective was to determine if C. americana can be a donor of EFB resistance. We crossed 29 diverse EFB-resistant C. americana accessions to EFB-susceptible C. avellana selections (31 total progenies) to produce 2031 F1 plants. In addition, new C. americana germplasm was procured from across the native range of the species. The new collection of 1335 plants from 122 seed lots represents 72 counties and 22 states. The interspecific hybrid progenies and a subset of the American collection (616 trees from 62 seed lots) were field planted and evaluated for EFB response following field inoculations and natural disease spread over seven growing seasons. EFB was rated on a scale of 0 (no EFB) to 5 (all stems containing cankers). Results showed that progeny means of the interspecific hybrids ranged from 0.96 to 4.72. Fourteen of the 31 progenies were composed of at least one-third EFB-free or highly tolerant offspring (i.e., ratings 0–2), transmitting a significant level of resistance/tolerance. Several corresponding C. americana accessions that imparted a greater degree of resistance to their hybrid offspring were also identified. In addition, results showed that 587 (95.3%) of the 616 C. americana plants evaluated remained completely free of EFB. These findings confirm reports that the species rarely expresses signs or symptoms of the disease and should be robustly studied and exploited in breeding.

Identification and Mapping of Eastern Filbert Blight Resistance Quantitative Trait Loci in European Hazelnut Using Double Digestion Restriction Site Associated DNA Sequencing

European hazelnut (Corylus avellana L.) is an economically important edible nut producing species, which ranked sixth in world tree nut production in 2016. European hazelnut production in the United States is primarily limited to the Willamette Valley of Oregon, and currently nonexistent in the eastern United States because of the presence of a devastating endemic disease, eastern filbert blight (EFB) caused by Anisogramma anomala (Peck) E. Muller. The primary commercial means of control of EFB to date is through the development and planting of genetically resistant european hazelnut cultivars, with an R-gene introduced from the obsolete, late-shedding pollinizer ‘Gasaway’. Although the ‘Gasaway’ resistance source provides protection against EFB in the Pacific northwestern United States (PNW), recent reports have shown that it is not effective in parts of the eastern United States. This may be in part because the identification and selection of ‘Gasaway’ and ‘Gasaway’-derived cultivars occurred in an environment (PNW) with limited genetic diversity of A. anomala. The objectives of the current research were to develop a genetic linkage map using double digestion restriction site associated DNA sequencing (ddRADseq) and identify quantitative trait loci (QTL) markers associated with EFB resistance from the resistant selection Rutgers H3R07P25 from southern Russia. A mapping population composed of 119 seedling trees was evaluated in a geographic location (New Jersey) where the EFB fungus is endemic, exhibits high disease pressure, and has a high level of genetic diversity. The completed genetic linkage map included a total of 2217 markers and spanned a total genetic distance of 1383.4 cM, with an average marker spacing of 0.65 cM. A single QTL region associated with EFB resistance from H3R07P25 was located on european hazelnut linkage group (LG) 2 and was responsible for 72.8% of the phenotypic variation observed in the study. Based on its LG placement, origin, and disease response in the field, this resistance source is different from the ‘Gasaway’ source located on LG6. The current results, in combination with results from previous research, indicate that the H3R07P25 source is likely exhibiting resistance to a broader range of naturally occurring A. anomala isolates. As such, H3R07P25 will be important for the development of new european hazelnut germplasm that combines EFB resistance from multiple sources in a gene pyramiding approach. Identification of EFB resistance in high disease pressure environments representing a diversity of A. anomala populations is likely a requirement for identifying plants expressing durable EFB resistance, which is a precursor to the development of a commercially viable european hazelnut industry in the eastern United States.

Inheritance and Linkage Mapping of Eastern Filbert Blight Disease Resistance in ‘Uebov’ Hazelnut

Eastern filbert blight (EFB) is a serious fungal disease of european hazelnut (Corylus avellana) in North America. The causal agent is the pyrenomycete Anisogramma anomala, which is native in the eastern United States where it occasionally produces small cankers on the wild american hazelnut (C. americana). However, most commercial cultivars of european hazelnut are susceptible. Infection leads to perennial cankers, girdling of branches, and premature tree death. Cultural practices including scouting, pruning out infected branches, and fungicide applications are recommended to slow disease spread but are expensive and not completely effective. EFB resistance from ‘Gasaway’ is conferred by a dominant allele at a single locus and has been extensively used in the Oregon State University hazelnut breeding program, but there is concern that this resistance could be overcome by isolates now present in the eastern United States or that a new race of the pathogen could arise in Oregon. Segregation for EFB resistance from ‘Uebov’, a new source from Serbia, was studied in three progenies by a combination of structure exposure and greenhouse inoculation. The frequency of resistant seedlings following structure exposure was about 20% in all three progenies. The ratios failed to fit the expected 1:1 ratio but did fit a ratio of 1 resistant:3 susceptible, which would be expected if resistance were conferred by dominant alleles at two independent loci. Seedlings from a cross of susceptible selection OSU 741.105 and ‘Uebov’ were used to study correlation of disease response and presence of alleles at microsatellite marker loci. Resistance was highly correlated with the presence of alleles at marker loci on linkage group 6 (LG6), and these markers also showed segregation distortion. We conclude that EFB resistance from ‘Uebov’ maps to a single locus on LG6 in the same region as resistance from ‘Gasaway’, although only about 20% of the seedlings are resistant because of segregation distortion. ‘Uebov’ has large, well-filled, round nuts and is suitable as a parent in breeding for the in-shell market, but its low nut yields and a high frequency of shells with split sutures are the drawbacks. Its use would expand options for breeding and ‘Uebov’ resistance could be combined with other resistance alleles with an expectation of more durable EFB resistance. Durable resistance would not only sustain the hazelnut industry in Oregon but would also allow expansion of plantings to new areas.

High-Resolution Genetic and Physical Mapping of the Eastern Filbert Blight Resistance Region in 'Jefferson' Hazelnut (Corylus avellana L.).

Eastern filbert blight (EFB), caused by the pyrenomycete (Peck) E. Müller, is a devastating disease of European hazelnut ( L) in the US Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer 'Gasaway' confers a high level of resistance to EFB. To identify the gene responsible for resistance, we initiated map-based cloning efforts in a population of 1488 seedlings that segregated for resistance. Chromosome walking was initiated using primers designed from eight previously identified random amplified polymorphic DNA markers linked to resistance. The bacterial artificial chromosome (BAC) library was screened using the primer pairs in a polymerase chain reaction-based pooling and subpooling strategy. Here, we report construction of a high-resolution genetic map and a physical map of the resistance region. Further, we sequenced BACs in the resistance region and identified and annotated the coding sequences. In seven contigs <1 cM from the resistance locus, 233 genes were predicted. The putative genes were compared with sequences in GenBank using a BLASTP search. Fifty-one markers were placed on the high-resolution genetic map, including markers newly developed from the BACs. Segregation in the mapping population placed the resistance locus in a single contig of three BACs (43F13, 66C22, and 85B7). Two of the putative genes are in the p-loop NTPase and F-box super-families localized in a 135-kb BAC, which have previously been shown to have disease-resistance properties. Further mapping, complementation, and expression tests of the genes in these BACs is essential to confirm which confer resistance to EFB.

Eastern filbert blight disease resistance from Corylus americana ‘Rush’ and selection ‘Yoder #5’ maps to linkage group 7

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala, is a serious threat to the hazelnut industry in the Pacific Northwest. EFB is endemic in eastern North America where it occasionally produces small cankers on the wild American hazelnut (Corylus americana). In contrast, most cultivars of European hazelnut (Corylus avellana) are susceptible. Genetic resistance is the most promising disease control method and is an objective of the Oregon State University hazelnut breeding program. ‘Gasaway’ resistance, which is governed by a dominant allele at a single locus, has been extensively used in the program. However, ‘Gasaway’ and some of its offspring have been infected by EFB isolates from New Jersey, Minnesota, and Michigan. Efforts to create new cultivars with durable EFB resistance include identifying and studying new resistance sources. In this study, resistant accessions C. americana ‘Rush’ and interspecific hybrid selection ‘Yoder #5’ were crossed with susceptible C. avellana selections and the resulting segregating seedling populations were inoculated by either exposure of potted trees under a structure topped with diseased branches or field exposure supplemented by tying diseased branches to each tree. Disease response was scored when cankers were visible 20 months after inoculation. Resistance from both sources segregated in a 1:1 ratio, indicating control by a single locus and a dominant allele for resistance. DNA extracted from the seedlings was amplified with previously mapped microsatellite markers. Resistance from both C. americana ‘Rush’ and ‘Yoder #5’ was placed on linkage group 7 in the same position as resistance from C. avellana ‘Ratoli.’ Linked microsatellite markers B753, GB372, and B509 will be useful for marker-assisted selection and the pyramiding of genes for durable EFB resistance. Assessing response to EFB is challenging, whether the plants are inoculated under a structure topped with diseased wood or in a humidity chamber in the greenhouse, or by exposure in the field. The pathogen has a 2-year life cycle, and there is a 15-month wait between inoculation and symptom expression. A small number of escapes is commonly encountered, and resistant plants occasionally develop small cankers. Our approach of studying segregation ratios and then mapping with microsatellite markers should be a useful approach for disease resistance studies in many tree crops.

Eastern Filbert Blight Resistance in Hazelnut Accessions ‘Culplà’, ‘Crvenje’, and OSU 495.072

European hazelnut (Corylus avellana L.) is a significant crop in Oregon, where 99% of United States hazelnuts are produced. Eastern filbert blight (EFB) caused by Anisogramma anomala (Peck) E. Müller is an important disease that infects the trees, reduces yield, and causes premature death. Managing the disease through cultural methods and fungicide applications is laborious and expensive, and genetic host resistance is considered the most viable option for control. Genetic resistance from ‘Gasaway’ has been used to develop resistant cultivars including Yamhill and Jefferson, but concern about the durability of this single resistance gene stimulated a search for additional sources of resistance. This study used three recently identified sources of EFB resistance: ‘Culplà’ from Spain, ‘Crvenje’ from Serbia, and OSU 495.072 from southern Russia. RAPD markers linked to resistance from ‘Gasaway’ were absent in all three accessions. Disease response was noted in segregating progenies following greenhouse or structure inoculation, and the resistance loci were mapped using microsatellite markers. In only four of the nine progenies did segregation for disease response fit the ratio of 1 resistant:1 susceptible expected for a single locus, a heterozygous resistant parent, and a dominant allele for resistance. Three progenies showed an excess of resistant seedlings while two showed a deficiency of resistant seedlings. The reciprocal translocations reported in several leading hazelnut cultivars may be present in the parents of the studied progenies, and affecting the segregation ratios. Microsatellite marker A614, previously mapped to linkage group (LG) 6, was closely linked to resistance from all three sources. Maps were constructed for LG6 for each resistant parent using microsatellite markers. The three resistance loci mapped to the same region on LG6 where resistance from ‘Gasaway’ and OSU 408.040 are located. The resistance alleles in all five accessions may be the same, or more likely are a cluster of different resistance genes in the same region. Markers LG628, LG610, and LG696 will be useful to breed new hazelnut cultivars with resistance from Culplà, Crvenje, and OSU 495.072.

CURRENT STATUS OF EASTERN FILBERT BLIGHT RESISTANCE SOURCES AND MAPPING

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. MA¼ller, is a devastating disease of European hazelnut (Corylus avellana L.) in the USA’s Pacific Northwest. Complete resistance from ‘Gasaway’, controlled by a dominant allele at a single locus, has been extensively used in the hazelnut breeding program at Oregon State University (OSU) since its discovery in 1975. Most advanced selections and the cultivars ‘Santiam’, ‘Yamhill’ and ‘Jefferson’ carry resistance from ‘Gasaway’. Concern over the breakdown of a single resistance gene prompted a continuing search for new sources of resistance. Greenhouse inoculation studies identified several sources of qualitative resistance, including accessions of C. avellana, other Corylus species and interspecific hybrids. The challenges of breeding for resistance to a pathogen absent in Corvallis, state quarantine regulations to slow its spread, its two-year life cycle, and difficulties in determining disease response stimulated a search for molecular markers linked to resistance. We have used the robust RAPD markers 152-800 and 268-580 in marker-assisted selection for ‘Gasaway’ resistance for more than a decade. Molecular markers have also been identified linked to resistance from ‘Ratoli’, OSU 759.010 and OSU 408.040. Using simple sequence repeat (SSR) markers as anchor loci, we assigned resistance from ‘Gasaway’, ‘Ratoli’, OSU 759.010, OSU 408.040 and ‘Culpla’ to linkage groups 6, 7, 2, 6 and 6, respectively. In this paper, we summarize the current status of the EFB resistance sources being used in the OSU hazelnut breeding program. For resistance loci on different linkage groups, we intend to use pyramiding with an expectation that the combined resistance will be more durable. Molecular markers linked to each source of resistance will facilitate pyramiding.

Characterization of Eastern Filbert Blight-resistant Hazelnut Germplasm Using Microsatellite Markers

The development of new cultivars resistant to the disease eastern filbert blight (EFB), caused by Anisogramma anomala, is of primary importance to hazelnut (Corylus sp.) breeders in North America. Recently, a large number of EFB-resistant cultivars, grower selections, and seedlings from foreign germplasm collections were identified. However, for a significant number of these, little is known of their origin, relationships, or genetic background. In this study, 17 microsatellite markers were used to investigate the genetic diversity and population structure of 323 unique accessions, including EFB-resistant and tolerant germplasm of uncertain origins, in comparison with a panel of known reference accessions representing a wide diversity of Corylus cultivars, breeding selections, and interspecific hybrids. The resulting allelic data were used to construct an unweighted pair group method using arithmetic averages (UPGMA) dendrogram and STRUCTURE diagram to elucidate relationships among the accessions. Results showed 11 consensus groups with EFB-resistant or tolerant accessions in all, providing strong evidence that EFB resistance is relatively widespread across the genus Corylus. Furthermore, open-pollinated seedlings tended to group together with reference accessions of similar geographic origins, providing insight into their genetic backgrounds. The results of this study add to the growing body of knowledge of hazelnut genetic resources and highlight recently introduced EFB-resistant seedling germplasm as new, unrelated genetic pools of resistance.

De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance

Bacterial artificial chromosome (BAC) libraries are widely used in map-based cloning of plant genes. Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Muller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer “Gasaway” confers complete resistance. Our map-based cloning efforts use a BAC library for “Jefferson” hazelnut, which is heterozygous for resistance. Screening the library with primer pairs designed from RAPD markers closely linked to the EFB resistance locus identified 38 BACs. We sequenced 28 of these BACs using Illumina technology, by multiplexing with barcoded adapters. De novo sequence assembly using the programs Velvet and SOPRA and further alignment using CodonCode Aligner generated contigs whose length ranged from 393 to 108,194 bp. The number of contigs per BAC ranged from 1 to 19, and estimated coverage of assembled BACs ranged from 64 % to 100 %. Preliminary analysis of the sequences identified 779 simple sequence repeats (SSRs), from which we developed 23 markers. Of these, 17 were assigned to linkage group 6 adjacent to the disease resistance locus, five were placed on other linkage groups, and one could not be assigned to a linkage group. The BAC sequences and new SSR markers will be useful for our efforts at map-based cloning of the disease resistance gene.

Eastern Filbert Blight Susceptibility of American × European Hazelnut Progenies

Eastern filbert blight (EFB), caused by Anisogramma anomala, is a devastating disease of Corylus avellana, the European hazelnut of commerce, and is considered the primary limiting factor of production in eastern North America. Conversely, C. americana, the wild American hazelnut, is generally highly tolerant of EFB, although it lacks many horticultural attributes necessary for commercial nut production. Hybrids of C. americana and C. avellana combine the EFB resistance of the wild species with the improved nut quality of the European species. However, inheritance of EFB resistance from C. americana remains unclear with existing hybrids derived from a very limited selection of parents. To investigate this topic, C. americana and advanced-generation C. americana × C. avellana hybrids were crossed with susceptible C. avellana and the resulting seedlings exposed to EFB through field inoculations and natural disease spread. In the winter after their fifth growing season, plants were rated for the presence of EFB using an index of 0 (no disease) through 5 (all stems containing cankers). The three progeny related to C. americana ‘Rush’ segregated for resistance in a ratio of one resistant to one susceptible, suggesting the presence of a single dominant R gene. A wide array of disease responses was observed for the other progenies with some expressing little EFB resistance or tolerance and others showing a distribution of disease phenotypes typical of control by multiple genes. Overall, the results indicate that both qualitative and quantitative resistance is present in C. americana. They also suggest that the choice of C. americana parent as well as the C. avellana parent will play a significant role in obtaining useful levels of EFB resistance in hybrid offspring, although the degree of disease expression in the parents may not be a useful predictor of progeny performance. Thus, more research is needed to understand inheritance of resistance, especially in advanced-generation backcrosses to susceptible C. avellana.

Identification and Mapping of DNA Markers Linked to Eastern Filbert Blight Resistance from OSU 408.040 Hazelnut

Eastern filbert blight (EFB) of European hazelnut (Corylus avellana L.), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a major disease problem and production constraint in Oregon’s Willamette Valley. Host genetic resistance is viewed as the most economical means of controlling this disease. Marker-assisted selection has been extensively used for ‘Gasaway’ resistance in the hazelnut breeding program at Oregon State University (OSU). Concern over potential breakdown of this single resistance gene prompted a search for new sources of resistance. Selection OSU 408.040 showed no signs or symptoms of the fungus after a series of disease inoculations, and resistance was transmitted to half of its offspring, indicating control by a dominant allele at a single locus. In this study, we identified six random amplified polymorphic DNA (RAPD) and 11 simple sequence repeat (SSR) markers linked to EFB resistance from OSU 408.040. The new markers supplement the previously identified amplified fragment length polymorphism (AFLP) markers. A linkage map constructed in the progeny OSU 245.098 × OSU 408.040 spanned a distance of 19.5 cM with the resistance locus cosegregating with AFLP marker A8-150 and located between SSR markers LG675 and LG682. Using SSR markers as anchor loci, OSU 408.040 resistance was assigned to linkage group 6 (LG6). Comparison with the previously mapped ‘Gasaway’ resistance locus showed that resistance from OSU 408.040 maps to the same location.

Assessment of Host (Corylus sp.) Resistance to Eastern Filbert Blight in New Jersey

One hundred ninety clonal accessions of Corylus, including species and various interspecific hybrids of C. avellana, C. americana, C. heterophylla, C. colurna, and C. fargesii, were assessed for their response to field exposure to the eastern filbert blight (EFB) pathogen, Anisogramma anomala, in New Jersey, where the fungus is native. Plants were obtained from the U.S. Department of Agriculture Agricultural Research Service National Clonal Germplasm Repository and Oregon State University, the University of Nebraska, Lincoln, and the National Arbor Day Foundation. Additional plant material was acquired from the Morris and Holden Arboreta and from private nurseries in Amherst, NY, and Niagara-on-the-Lake, Ontario, Canada. The accessions were chosen based on their resistance to EFB in Oregon, a region where A. anomala is not native, or anecdotal reports and grower observations of tolerance or resistance to the disease. Trees were planted in the field from 2002 through 2009 in New Jersey where they were exposed to EFB yearly through field inoculations and natural spread. In Jan. 2012, they were visually evaluated for the presence of EFB. The cankers were measured, and the proportion of diseased wood was calculated for susceptible trees. Nearly all accessions reported to be resistant to EFB in Oregon maintained at least a useful level of tolerance in New Jersey with a number remaining free of cankers. However, several accessions developed small to medium-sized cankers and showed branch dieback, including offspring of C. avellana ‘Gasaway’. Most C. americana and C. heterophylla accessions remained free of EFB, although variation in EFB response was found in hybrids of these species with C. avellana, ranging from no signs or symptoms to severe EFB. Nearly half of the C. colurna × C. avellana hybrids developed cankers, whereas each of the C. fargesii accessions and most grower selections developed in eastern North America remained free of EFB. The results document the existence of a wide diversity of Corylus germplasm that expresses resistance or a high level of tolerance to EFB in New Jersey and confirms previous reports that C. americana is highly resistant to the disease. Interestingly, most C. heterophylla and the C. fargesii were also found to be resistant despite originating in Asia where A. anomala has not been found. The various interspecific hybrids show the potential for incorporating EFB resistance from wild species through breeding. The results provide further evidence of differences in disease expression in Oregon and New Jersey, where isolates differ and disease pressure may be higher.

A bacterial artificial chromosome library for ‘Jefferson’ hazelnut and identification of clones associated with eastern filbert blight resistance and pollen–stigma incompatibility

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci.