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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.

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and their benefits to human health, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice used in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock–scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests. info:eu-repo/semantics/publishedVersion

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and in the prevention of various diseases, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice use in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock–scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests and of the possible increase of their aggressiveness. They will also have to offer the premises of economic production, respectively yield and the quality, according to multiple destinations of nuts in the current consumption and food industry, but also the increasing exigencies of the consumer market and the profile industry.

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.

Eastern filbert blight (EFB) is a hazelnut disease caused by Anisogramma anomala, a fungus endemic to North America where it can be found associated with the wild hazelnut Corylus americana. While C. americana is resistant to EFB, the commercial hazelnut C. avellana is highly susceptible and typically dies within 5 years from exposure. A hazelnut breeding program was initiated in 1996 at Rutgers University in New Brunswick, New Jersey. The early objective was to identify sources of resistance to EFB for use in breeding regionally adapted, EFB-resistant plants to support the establishment of a hazelnut industry in eastern USA. By working with Oregon State University (OSU) and international colleagues, germplasm collections were made in Russia, Ukraine, Poland, Georgia, Latvia, Lithuania, Estonia, Italy, and Turkey. Over 5,000 seedlings were grown and evaluated for response to EFB. While most plants succumbed to EFB, about three percent were found to be resistant. Interestingly, the resistant plants spanned nearly all collection locations and molecular markers have shown them to be highly diverse. Today, we have access to over 100 EFB-resistant accessions selected from more than 60 locations, which represents a significant pool of germplasm to support breeding. Recent studies have shown that most resistance seems to be controlled by only one or a few major genes; R-gene mapping projects are underway at Rutgers as well as Oregon State University. We continue to study new sources of resistance for use in a focused cultivar improvement program.

Corylus americana is native to a wide area of land in eastern North America, bounded by the Rocky Mountains in the west and the Atlantic Ocean in the east, and ranging from southern Canada to the southeastern US. The species has been shown to be highly genetically diverse and is adapted to a variety of climates and soils. It is also resistant to the disease eastern filbert blight (EFB) caused by the fungus Anisogramma anomala. Unfortunately, C. americana has thick-shelled, tiny nuts that make it unsuitable for commercial production. However, it is cross-compatible with the commercial hazelnut C. avellana and can serve as a donor for genes for disease resistance, early nut maturity, cold tolerance, etc. As part of the Hybrid Hazelnut Consortium consisting of Rutgers University, Oregon State University, the University of Nebraska-Lincoln, and the Arbor Day Foundation, we have been successfully collecting C. americana germplasm since 2009 with the help of partners, colleagues, and the interested public around the USA. Today, we have a planting established in the field at Rutgers University in New Jersey that holds 1,899 seedlings obtained from 128 individual seed lots that span 23 states and one Canadian province. These bushes are being evaluated for EFB response, other diseases and insects, flowering and vegetative bud break phenology, cold hardiness, nut yield, and nut and kernel characteristics. The genetic diversity and population structure of the new collection is also being evaluated using several approaches, including genotyping by sequencing and subsequent SNP analysis, SSR allelic variation, and chloroplast haplotype determination across the large population. Using this multi-faceted approach, we hope that superior, diverse selections can be identified for use in systematic breeding efforts to develop new cultivars adapted to the eastern USA, especially in respect to consistent cropping in regions with highly variable year-to-year climates.

The hazelnut breeding program at Oregon State University (OSU) is developing new cultivars with high nut yield, few defects, and good kernel quality. Every year, approximately 4500 seedlings are planted and evaluated for seven years. The very best of these seedlings are put through a second period of testing in replicated yield trials to determine their potential as cultivars. The fungal disease eastern filbert blight (EFB) is now widespread in the Willamette Valley, so current trials include only EFB-resistant selections. In this paper, we summarize data for three recent releases. ‘York’ (OSU 878.048) is a widely compatible mid-season pollinizer with nuts and kernels that blend well with other recent OSU releases. ‘Wepster’ (OSU 894.030) and ‘McDonald’ (OSU 880.027) were released as main crop cultivars. Trees of ‘Wepster’, ‘McDonald’, and ‘York’ have rounded, moderately upright growth habits, and mature nuts 7-14 days before ‘Barcelona’. ‘Wepster’ nuts and kernels are small, similar in size to ‘Yamhill’, and have very few defects. The percent kernel of ‘Wepster’ is only 44% on average, but total nut yields are very high. ‘McDonald’ nuts are medium to small in size and are suitable for the kernel market. Nuts and kernels of ‘York’ are of good quality, but trees yield less than what is desirable for a main cultivar. ‘York’ has incompatibility alleles S(2) and S(21), but only the uncommon S(21) is expressed in the pollen. It is an excellent mid-season pollinizer, producing large quantities of highly viable pollen over a period of several weeks, and is compatible on most other genotypes. ‘Wepster’, ‘McDonald’ and ‘York’ are fully cross-compatible and are recommended to be planted together. All three are protected with US Plant Patents.

Incompatibility in European hazelnut (Corylus avellana L.) is sporophytic and under the control of a single locus on linkage group 5 between markers G05-510 and AU02-1350. In this study, two rounds of marker development and a population of 192 seedlings with known S-alleles that showed recombination between the flanking markers were used for fine mapping. Using the sequences of random amplified polymorphic DNA and simple sequence repeat (SSR) markers and bacterial artificial chromosome end sequences, 36 contigs from the genome sequence of “Jefferson” hazelnut were identified for pursuit. Di-nucleotide SSR markers in those contigs were developed, characterized, and mapped. This reduced the size to a region of 500 kb that contained the S-locus and 50 predicted genes, in which single-nucleotide polymorphism and additional SSR markers were developed. When the new markers were used in fine mapping, they fully exploited all recombination in the fine mapping population and reduced the region to 193.5 kb containing 18 genes. This 193.5-kb region most likely contains the S1 haplotype. A second region, 2 Mbp away from the first in the “Jefferson” genome (V3), is predicted to represent the S3 haplotype based on SSR marker allele sizes and the ratio of parental reads that align to them. Although they appear side-by-side in the “Jefferson” genome (V3), the mapped markers appear in both sequences in the same order. Gene annotations in the S1 and S3 haplotypes are highly similar and include five probable leucine-rich repeat receptor-like protein kinases with homology to Arabidopsis thaliana genes At1g35710 and three probable receptor-like serine/threonine protein kinases with homology to At5g15080 (PIX7).

Hazelnut is an economically important tree nut whose production is mostly destined to the confectionery industry with a demand that currently exceeds supply. Its cultivation remains substantially based on named selections from local, wild vegetation. Public breeding programs were not initiated until the 1960s and only two, both in the USA, are in operation today that are relatively large. Oregon State University has produced new cultivars with Gasaway resistance to the fungus Anisogramma anomala, causal agent of eastern filbert blight (EFB), a major disease in North America; these cultivars are being widely planted. In China, cold-hardy hybrid cultivars from Corylus heterophylla and C. avellana were recently released and are planted in northeastern China. In the past 25 years, molecular markers have facilitated a much better understanding of genetic diversity in the genus Corylus, aided the construction of linkage maps and allowed for marker-assisted selection for disease resistance. The genome of C. avellana was sequenced and assembled, and DNA markers identified from the transcriptome, providing the basis for the isolation of important genes, including those related to nut quality and adaptive and phenological traits. Many new genotypes expressing eastern filbert blight (EFB) resistance have been identified in the germplasm, and subsequent linked DNA markers developed, allowing new approaches to breeding for durable resistance. Micropropagation is routinely used in the USA, Chile and Italy for multiplication, but work with other in vitro techniques is less advanced. Genetic engineering has not been developed in hazelnut due to regeneration difficulties from somatic tissues but recent advances have established a protocol for organogenesis. More research is being carried out to assemble a high-quality hazelnut genome and achieve somatic embryogenesis. The results from this research will provide knowledge and tools enabling the isolation of genes and molecular markers, and the application of genome editing techniques to hazelnut.

From the genome sequence of hazelnut (Corylus avellana), 192 new polymorphic simple sequence repeat (SSR) markers were developed, characterized, and used to investigate genetic diversity in 50 accessions. Next-generation sequencing allows inexpensive sequencing of plant genomes and transcriptomes, and efficient development of polymorphic SSR markers, also known as microsatellite markers, at low cost. A search of the genome sequence of ‘Jefferson’ hazelnut identified 9094 fragments with long repeat motifs of 4, 5, or 6 base pairs (bp), from which polymorphic SSR markers were developed. The repeat regions in the ‘Jefferson’ genome were used as references to which genomic sequence reads of seven additional cultivars were aligned in silico. Visual inspection for variation in repeat number among the aligned reads identified 246 as polymorphic, for which primer pairs were designed. Polymerase chain reaction (PCR) amplification followed by agarose gel separation indicated polymorphism at 195 loci, for which fluorescent forward primers were used to amplify the DNA of 50 hazelnut accessions. Amplicons were post-PCR multiplexed for capillary electrophoresis, and allele sizes were determined for 50 accessions. After eliminating three, 192 were confirmed as polymorphic, and 169 showed only one or two alleles in each of the 50 cultivars, as expected in a diploid. At these 169 SSRs, a total of 843 alleles were found, for an average of 4.99 and a range of 2 to 17 alleles per locus. The mean observed heterozygosity, expected heterozygosity, polymorphism information content, and the frequency of null alleles were 0.51, 0.53, 0.47, and 0.03, respectively. An additional 25 primer pairs produced more than two bands in some accessions with an average of 6.8 alleles. The UPGMA dendrogram revealed a wide genetic diversity and clustered the 50 accessions according to their geographic origin. Of the new SSRs, 132 loci were placed on the linkage map. These new markers will be useful for diversity and parentage studies, cultivar fingerprinting, marker-assisted selection, and aligning the linkage map with scaffolds of the genome sequence.

The disease eastern filbert blight (EFB), caused by Anisogramma anomala, severely limits hazelnut (Corylus avellana) production in North America. In the 1970s, ‘Gasaway’, a late-flowering pollenizer, was found to be resistant to EFB in the Pacific northwestern U.S., and to transmit its resistance in a predictable Mendelian pattern. Its subsequent use in breeding at Oregon State University led to the release of a number of EFB-resistant cultivars, including Jefferson, Yamhill, Dorris, Wepster, and McDonald, which are now being widely planted across the Willamette Valley of Oregon. However, some cultivars carrying the ‘Gasaway’ R-gene develop EFB in New Jersey. Thus, the utility of this resistance source for breeding plants adapted to the eastern U.S has not been fully evaluated. In this study, we examined the EFB response of seedlings from 31 different full-sib progenies expected to segregate for the ‘Gasaway’ R-gene. Trees were exposed to the disease over at least 5 years and evaluated on a scale of 0 to 5 (0 = resistant, 5 = highly susceptible). In general, we found the number of seedlings free of EFB (class 0) to be less than expected based on prior work in Oregon, but resistant individuals were identified in each progeny, indicating the presence of heritable resistance. When disease response classes 0, 1, and 2 were combined (resistant through highly tolerant) and considered as “resistant”, the expected ratio of resistant to susceptible trees was recovered, signifying that the single dominant allele alone continues to provide a predictable level of at least tolerance. Modifying genes/factors appear to play a role in the final disease response of the individual seedling. It is thus hypothesized that some cultivars selected as resistant in Oregon that develop cankers in New Jersey may lack the necessary modifying genetic factors for full protection in the east. Until these modifying genes are elucidated, long-term field evaluation remains necessary to identify selections that carry the ‘Gasaway’ R-gene and express a high level of resistance to EFB in the eastern U.S.

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.

Inheritance of resistance to eastern filbert blight, caused by Anisogramma anomala, in European hazelnut (Corylus avellana) was evaluated in the progeny of seven cultivars crossed in 12 combinations. The progeny were subjected to inoculation with A. anomala in the greenhouse and in the field. Three disease responses were measured: disease incidence, number of cankers, and proportion of wood diseased. In both the greenhouse and the field, progeny produced by crossing VR6-28 with three susceptible cultivars segregated 1:1 for complete resistance to eastern filbert blight, confirming a previous report that VR6-28 is heterozygous for a single, dominant resistance gene. Histograms of disease responses in progeny of the remaining six parents showed continuous distributions for all crosses examined. Consequently, these parents were analyzed for general and specific combining abilities for each disease response. In the field, general and specific combining ability were both significant (P < 0.05) for all disease responses, with general combining ability having twice the magnitude of specific combining ability. These results suggest these disease responses are controlled by additive gene action in the cultivars examined, with nonadditive gene action being of some importance. Based on general combining ability values, high levels of partial resistance were transmitted by the pollen parents, Gem and Tonda di Giffoni, and the seed parent, Willamette. Heritability of disease incidence, number of cankers, and proportion of wood diseased were calculated to be 0.21, 0.39, and 0.47, respectively, for this set of nine crosses after the first exposure period in the field. This suggests that it will be possible to use partially resistant parents to breed for hazelnuts exhibiting fewer and smaller cankers.

Molnar, T. J., Walsh, E., Capik, J. M., Sathuvalli, V., Mehlenbacher, S. A., Rossman, A. Y., and Zhang, N. 2013. A real-time PCR assay for early detection of eastern filbert blight. Plant Dis. 97:813-818. 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.

European hazelnut (Corylus avellanaL.) is of global agricultural and economic significance, with genetic diversity existing in hundreds of accessions. Breeding efforts have focused on maximizing nut yield and quality and reducing susceptibility to diseases such as Eastern filbert blight (EFB). Here we present the first sequenced genome among the order Fagales, the EFB-resistant diploid hazelnut accession ‘Jefferson’ (OSU 703.007). We assembled the highly heterozygous hazelnut genome using an Illumina only approach and the final assembly has a scaffold N50 of 21.5kb. We captured approximately 91 percent (345 Mb) of the flow-cytometry-determined genome size and identified 34,910 putative gene loci. In addition, we identified over 2 million polymorphisms across seven diverse hazelnut accessions and characterized t heir effect on coding sequences. We produced t wo high-density genetic maps with 3,209 markers from an F1 hazelnut population, representing a five-fold increase in marker density over previous maps. These genomic resources will aide in the discovery of molecular markers linked to genes of interest for hazelnut breeding efforts, and are available to the community athttps://www.cavellanagenomeportal.com/.

The growing area of European hazelnut (Corylus avellana L.) is increasing, as well as the number of producing countries, and there is a pressing need for new improved cultivars. Hazelnut conventional breeding process is slow, due to the length of juvenile phase and the high heterozygosity level. The development of genetic linkage maps and the identification of molecular markers tightly linked to QTL (quantitative trait loci) of agronomic interest are essential tools for speeding up the selection of seedlings carrying desired traits through marker-assisted selection. The objectives of this study were to enrich a previous linkage map and confirm QTL related to time of leaf budburst, using an F1 population obtained by crossing Tonda Gentile delle Langhe with Merveille de Bollwiller. Genotyping-by-Sequencing was used to identify a total of 9,999 single nucleotide polymorphism markers. Well saturated linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. A reciprocal translocation was detected in Tonda Gentile delle Langhe between two non-homologous chromosomes. Applying a bioinformatic approach, we were able to disentangle 'pseudo-linkage' between markers, removing markers around the translocation breakpoints and obtain a linear order of the markers for the two chromosomes arms, for each linkage group involved in the translocation. Twenty-nine QTL for time of leaf budburst were identified, including a stably expressed region on LG_02 of the Tonda Gentile delle Langhe map. The stability of these QTL and their coding sequence content indicates promise for the identification of specific chromosomal regions carrying key genes involved in leaf budburst.

Eleven simple sequence repeat (SSRs) markers were used to investigate population structure of 582 accessions representing hazelnut cultivars grown in different geographical regions of the world. SSR profiles for 229 accessions were assessed for the first time, and their allele sizes were combined with those of 353 accessions previously characterized and published, for an analysis of genetic diversity and population structure. The combined data for all 582 accessions was used to construct an UPGMA (unweighted pair group method of arithmetic averages) dendrogram. The UPGMA tree showed multiple groups with clustering partly based on their geographic origin. The STRUCTURE analysis indicated that the most likely number of sub-populations is three to seven. Taken together, the results showed that cultivated hazelnuts consist of multiple genetic groups and cultivars were likely selected from local wild populations rather than originating in one or two domestication centers.

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.

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

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.

Microsatellite markers, also known as simple sequence repeat (SSR) markers, are useful in genetics and plant breeding for marker-assisted selection, cultivar fingerprinting, and kinship studies. They were previously developed for European hazelnut (Corylus avellana L.) from enriched libraries, inter-simple sequence repeat (ISSR) fragments, and bacterial artificial chromosome (BAC) sequences. This study utilized the ‘Jefferson’ transcriptome sequence to develop microsatellite markers for hazelnut. Microsatellites were mined from the transcriptome sequence and aligned with the corresponding genomic sequence using a BLASTN search, and primers were designed from the genomic sequence. From a total of 1432 identified microsatellites, 382 primer pairs were designed for repeats with motifs of three base pairs or longer and at least five repeats, and a few di-repeat sequences. The primers were initially screened for polymorphism with a set of 24 C. avellana accessions, and those that were polymorphic were characterized using a set of 63 accessions. One hundred nine polymorphic microsatellite markers that generated one or two fragments per accession were characterized, with AAG, AGC, and ACT being the most common motifs. Two additional SSR primer pairs generated three PCR products in a few accessions. Of the 109 loci, 53 were placed on the linkage map of the mapping population OSU 252.146 × OSU 414.062, and 22 were assigned to linkage groups using alternate segregating populations. A dendrogram constructed from the SSR fingerprints at the 109 new marker loci mostly showed clustering of accessions from similar geographic origins and confirmed the tremendous amount of genetic diversity present within C. avellana. These markers will be useful for fingerprinting, marker-assisted selection, and genetic studies in hazelnut.

Pollen-stigma incompatibility in hazelnut is sporophytic and under the control of a single locus with multiple alleles. To date, 33 alleles have been identified at the S-locus, and a designated pollen tester for each preserved in our collection. Hazelnut is diploid, and nearly all cultivars are heterozygous at the S-locus. Over the past 25 years, we have enlarged our collection by importing cultivars from several countries as scions. As the trees began to flower, we identified their S-alleles using florescence microscopy. In December, we bagged emasculated branches on trees growing in the field to protect the female inflorescences from wind-borne pollen. In January, we collected pollen from our tester trees. In February, females were collected from bagged branches and brought to the lab in petri dishes. The females were pollinated in the afternoon, one or two per tester pollen. The following day, the styles were squashed in aniline blue and observed under UV light. In compatible pollinations, pollen germination was excellent and tubes could be seen growing parallel down the style. In incompatible pollinations, germination was often reduced, pollen tubes were short and did not penetrate the stigmatic surface, and the tubes often ended in bulbs. Each pollination was scored as compatible or incompatible, and the testing of a tree required 1-3 years for completion. In this paper, we report the alleles of 170 cultivars for the first time, and summarize the alleles of 112 cultivars previously typed. Cultivars with different names often have identical microsatellite marker fingerprints and S-alleles. To reduce confusion, a list of cultivars with identical fingerprints is also included.

Pollen–stigma incompatibility in european hazelnut (Corylus avellana L.) is of the sporophytic type and under the control of a single locus with multiple alleles (haplotypes). The S-locus was previously assigned to linkage group 5 (LG5) and linked DNA markers were identified. The loci that control leaf color and style color are linked to the S-locus. We investigated segregation for leaf and style color and S-alleles in two progenies, mapped the loci, and compared the two new maps with the LG5 reference map using simple sequence repeat (SSR) markers. Segregation for color, S-alleles and SSR markers fit expectations. The color loci and the S-locus mapped to LG5 between SSR markers B028 and B774. The three maps aligned and the SSR markers were collinear. The SSR markers closest to the S-locus are KG819, KG847, and BR259. In progeny 05050, which segregated for style and leaf color, no recombination was observed between the two traits. Recombination between the S-locus and the style color locus was 5.4 cM in progeny 05050 and 10.1 cM in progeny 00064. The style color locus was placed very close to SSR marker B028 in both progenies. On the reference map, random amplified polymorphic DNA (RAPD) markers 564-500M, 345-1050dF, and 204-950dF and intersequence simple sequence repeat (ISSR) marker 815-540dF are very close to the S-locus. The identification of closely linked markers will facilitate the map-based cloning of the S-locus and color loci in hazelnut.

The european hazelnut (Corylus avellana L.) is native to most of Europe and nearby areas in Asia Minor and the Caucasus Mountains. Cross-pollination is enforced by sporophytic incompatibility under the control of a single locus with multiple alleles (haplotypes). Fluorescence microscopy is routinely used to determine if a pollination is compatible or incompatible, and use of an array of known testers allows identification of the alleles of cultivars and selections. Both alleles are expressed in the stigmas, but often only one is expressed in the pollen because of dominance. Cultivars are highly heterozygous diploids (2n = 2x = 22) and clonally propagated. Most of the world's leading cultivars were selected from local wild populations near where they are now planted on a commercial scale. Genetic improvement efforts are recent and, although tremendous genetic variability is available, such efforts have had little impact outside of Oregon and France. Studies of genetic diversity using simple sequence repeat markers have placed most cultivars in one of the four main groups: Spanish-Italian, Central European, English, or Black Sea. This study presents 17 years of data on incompatibility in hazelnut, including the discovery of six new S-alleles and determination of the dominance relationships among 105 new pairs of alleles. The total number of alleles now stands at 33. The S-alleles of 284 cultivars, 13 interspecific hybrids, and 522 selections of diverse origin are presented. The S-alleles identified in hazelnut cultivars is information that should be useful to breeders in the planning of crosses, to curators of germplasm collections, and to growers and nurseries as they choose cultivars and pollenizers when designing orchards. Differences in S-allele frequency seen in the cultivars and selections are related to geographic origin. The most common alleles of cultivars in the major geographical groups are S2 in the Spanish-Italian group, S5 in the Central European group, S3 in the English group, and S4 in the Black Sea group. Most selections belonged to the Black Sea group, and S4 was by far the most common allele. Differences in allele frequency were also observed among seed lots within a country.

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.

European hazelnut (Corylus avellana L.) is of worldwide agricultural significance, with breeding efforts focused on combining high nut yield and nut quality with resistance to diseases such as eastern filbert blight (EFB), a cause of severe crop loss in much of the United States. Oregon State University recently released a resistant cultivar, 'Jefferson' (OSU 703.007), that was chosen for transcriptome sequencing to establish further genomic resources for C. avellana L. We used Illumina ribonucleic acid sequencing (RNA-seq) to characterize complementary DNA (cDNA) libraries from four hazelnut tissues, including leaves, catkins, bark, and whole seedlings. The 6.8 Gb of hazelnut transcriptome data was assembled de novo into 28,255 contigs with an average length of 532 bp and an N50 (the minimum contig length necessary such that all contigs of equal or greater length will equal half of the bases of the assembly) of 961 bp. Sequence comparisons using BLASTX and gene ontology (GO) classifications were used to generate automated descriptive function annotations. High similarity of the predicted proteins to sequences in related plants demonstrates the validity of the transcript contigs, with 80.8% having similarity to grape (Vitis vinifera L.), poplar (Populus trichocarpa Torr. & A. Gray), and castor bean (Ricinus communis L.) sequences in the public domain. A survey of GO terms enriched among tissue-specific transcripts further validates the assembly. A basic local alignment search tool (BLAST) portal and web resources (http:// hazelnut.cgrb.oregonstate.edu [accessed 8 Jan. 2010]) are available and will be of importance to breeders for marker-assisted breeding efforts.

The US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon, preserves more than 800 accessions of hazelnut (Corylus), including C. avellana cultivars and representatives of 10 other recognized shrub and tree species. Characterization and study of genetic diversity in this collection require cross-transferable markers, such as trinucleotide microsatellite or simple sequence repeat (SSR) markers and universal chloroplast SSR markers. We developed new SSR markers and evaluated 114 Corylus accessions representing 11 species and 44 interspecific hybrids. Eight of 23 SSRs generated easy-to-score alleles in all species and seven were highly polymorphic. For those seven, the average heterozygosity was moderate at 0.49, and mean allele number, genetic diversity and polymorphism information index were high at 11.71, 0.79 and 0.76, respectively. The three most polymorphic SSRs were CaC-C008, CaC-C040 and CaC-C118. Neighbor-joining (NJ) clustering and structure analysis agreed with classical taxonomic analysis and supported inclusion of C. maxima within the large polymorphic species, C. avellana. Analysis also indicated that C. californica is a distinct species rather than a botanical variety of C. cornuta. Six universal cpSSRs were polymorphic in Corylus and generated 21 distinct chlorotypes with an average of 3 alleles per locus. Diversity at these cpSSRs was high and ranged from 0.33 to 0.64, with an average of 0.54. Incongruence in NJ topologies between the nuclear and chloroplast markers could be attributed to chloroplast capture related to hybridization during the ancestral diversification of the genus, or to homoplasy. The phylogeographical relationships among the 21 chlorotypes in the 11 Corylus species support Asia as a refugium where several hazelnut lineages survived during glaciation and from which they continued to evolve after dispersal from Asia through the Mediterranean to Europe, and across the Atlantic and/or the Bering land bridge to North America.

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.