Shawn A. Mehlenbacher

Characterization of European hazelnut (Corylus avellana) cultivars using SSR markers

World hazelnut production is based primarily on selections from the wild. In this study, we used 21 pairs of simple sequence repeat (SSR) primers to investigate genetic diversity in 270 clonal accessions of European hazelnut (Corylus avellana) representing a wide geographic range. Of the 270 accessions, 198 had unique fingerprints while 72 were duplicates. Based on the 198 unique accessions, the number of detected alleles per locus averaged 9.81 and observed heterozygosity (Ho) averaged 0.67. Of the 206 total alleles amplified, 20 were unique to a single accession. A genetic similarity matrix was constructed and the resulting dendrogram revealed four major geographical groups: Central European, Black Sea, English and Spanish-Italian. SSR alleles indicated the parentage of 31 accessions. The fingerprints are publicly available through the Germplasm Resources Information Network (GRIN) database. The identification of duplicate and mislabeled accessions will improve management of hazelnut genebanks, and information on genetic variation in hazelnut will assist the international research community.

Eastern filbert blight of European hazelnut. It's becoming a manageable disease.

Hazelnut eastern filbert blight caused by Anisogramma anomala in Oregon, USA, is discussed. The subjects included are the infection biology, ascospores, disease management, host resistance and the prospects in the Oregon industry.

Development, characterization, segregation, and mapping of microsatellite markers for European hazelnut (Corylus avellana L.) from enriched genomic libraries and usefulness in genetic diversity studies

Eighty-six new microsatellite loci were developed for European hazelnut (Corylus avellana L.) by screening two genomic libraries enriched for dinucleotide repeats. The loci, 73 developed from a GA-enriched library and 13 from a CA-enriched library, showed a high level of polymorphism in 50 accessions. The number of alleles per locus ranged from five to 21, with a mean of 10.55. Mean values for expected heterozygosity, observed heterozygosity, and polymorphism information content were high, averaging 0.76, 0.69, and 0.73, respectively. In a mapping population, loci segregated 1:1, 1:2:1, and 1:1:1:1. Segregation distortion and null alleles were observed at some loci. Eighty-one of the 86 loci were assigned to linkage groups. A neighbor-joining dendrogram reflected great diversity among the 50 accessions and showed clustering by geographic origin.

Revised dominance hierarchy for S-alleles in Corylus avellana L

Abstract Pollen-stigma compatibility was studied in cultivars and more than 1800 seedlings of the European hazelnut (Corylus avellana L). Four new S-alleles were identified, bringing the total to 25 unique alleles within C. avellana. The new alleles are the recessive alleles in ‘Tonda di Giffoni’ and ‘Segorbe’ (S23), in ‘Neue Riesennuss’ (S25), in ‘Gasaway’ (S26), and a dominant allele in a seedling of Turkish origin (S24). Dominance relationships in 233 of the possible 300 pairs of alleles were determined in both pistil and pollen. All alleles exhibited independent action in the pistil, whereas in the pollen either dominance or codominance was exhibited. The dominance hierarchy of alleles in the pollen was revised in light of the new information obtained. All 25 alleles have been assigned to a level in the hierarchy that is linear and now has eight levels. S6 and S9 were reassigned to lower levels in the hierarchy. Thirteen of the alleles are on the level of S1, while S4, S6, S11, and S23 occupy unique positions in the hierarchy. Improved pollen tester clones were identified for several S-alleles. The alleles in 55 cultivars were determined. The alleles identified in ‘DuChilly’ (S10 S14) did not agree with previous reports. Four cultivars have the same alleles as ‘Römische Nuss’ (S10 S18) and are morphologically indistinguishable from it: ‘Frutto-grosso’, ‘Istarski Okrogloplodna’, ‘Payrone’, and ‘Romai’. ‘Belle di Giubilino’ and ‘Tonda di Biglini’ are both S1 S10 and appear to be synonyms for the same cultivar.

Susceptibility of European hazelnut clones to eastern filbert blight

In the springs of 1989 and 1990, 2- to 3-yr-old hazelnut trees representing 19 cultivars were exposed to ascospores of Anisogramma anomala, which causes eastern filbert blight. The trees were potted and randomly arranged under wire mesh platforms elevated 1.8 m from the ground. Diseased hazelnut branches were placed on top of the platforms in low, medium, or high numbers to provide three levels of inoculum. During periods of rain, ascospores of the pathogen were released from the diseased branches and deposited on the potted trees. External disease symptoms developed at 16 and 28 mo after initial exposure to inoculum, at which times disease responses were evaluated. Cultivars differed significantly in disease incidence, mortality, number of cankers per tree, proportion of wood cankered, and proportion of dead wood (.)

Dominance relationships among S-alleles in Corylus avellana L.

SummaryPollen-stigma compatibility relationship were studied in 50 cultivars and more than 800 seedlings of the European hazelnut (Corylus avellana L.). A total of 22 unique S-alleles have been identified. Dominance relationships in 75 of the possible 231 pairs of alleles have been determined in both pistil and pollen. In the pistil, all alleles exhibited independent action, whereas in the pollen, alleles exhibited either dominance or codominance. The dominance relationship was linear with 7 levels of dominance.

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

Abstract Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S1S12)×VR6-28 (S2S26) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S1 S2, S1 S26, S2 S12, and S12 S26. ‘Super bulks’ of 12 trees each for S1, S2, S12, and S26 were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S2 (OPI07750) and S1 (OPJ141700). OPJ141700 was identified in 13 of 14 S1 individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S1 individual. This same marker was not effective outside the original cross, identifying 4 of 5 S1 progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S1 individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07750 served as an excellent marker for the S2 allele and was linked closely to this allele, identifying 12 of 13 S2 individuals in the OSU23×VR6 population with no false positives. OPI07750 was found in 4 of 4 S2 individuals from ‘Will’×VR and 7 of 7 S2 individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S2 individuals of the cross OSU 296.082 (S1S8)×VR8-32 (S2S26), with only 1 false positive individual out of 21 progeny. OPI07750 was also present in 5 of 5 cultivars carrying the S2 allele, with no false-positive bands in non-S2 cultivars, and correctly identified all but 2 S2 individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ141700 and the S1 allele was 7.6% and between the OPI07750 marker and the S2 allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S1 and S2 markers were successfully cloned and 18 bp primers yielded the sole OPJ141700 product, while 24-bp primers yielded OPI07750 as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined.

Partial self-compatibility in ‘Tombul’ and ‘Montebello’ hazelnuts

SummarySelf-pollination of hazelnut (Corylus avellana L.) cultivars in 1988 and 1990 revealed the existence of partial self-compatibility in ‘Tombul’ and ‘Montebello’. Percent cluster set in these cultivars averaged 44 and 20%, respectively, but was less than 10% in 8 other cultivars investigated. Percent cluster set from pollination with ‘Segorbe’ averaged 62 and 41% in 1988 and 1990, respectively. Self-pollination produced 40% fewer nuts per cluster and twice as many blanks as cross-pollination. All cultivars and selections have an active sporophytic incompatibility system.Evaluation of self-compatibility in seedlings from the cross ‘Montebello’ × ‘Compton’ revealed that the partial self-compatibility of the maternal parent was transmitted to some of the progeny. Self-pollination resulted in greater than 10% cluster set in two selections, OSU 41.134 and OSU 43.025, in both years, but only in 1988 in OSU 42.089 and ‘Willamette’. Three other selections had very low set in both years. Results of incompatible crosses with standard testers were generally in agreement with those of self-pollination, except that the S2 tester induced greater set on 3 genotypes in 1988 and the S1 tester on 2 genotypes in 1990 than self-pollination. The partial self-compatibility of ‘Montebello’, OSU 41.134, and OSU 43.025 appears to be due to a failure of their stigmas to prohibit pollen tube growth in incompatible crosses. There is no evidence of a pollen-part mutation in ‘Montebello’, nor is there evidence that partial self-compatibility is due to the interaction of S-alleles, as ‘Barcelona’, which has the same alleles as these three genotypes, failed to set nuts in all incompatible crosses.

Nuclear and chloroplast microsatellite markers to assess genetic diversity and evolution in hazelnut species, hybrids and cultivars

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.

Use of ELISA to rapidly screen hazelnut for resistance to Eastern filbert blight.

A rapid and accurate screening system was developed to more rapidly identify resistance to eastern filbert blight based on an indirect enzyme-linked immunosorbent assay (ELISA) of greenhouse-inoculated hazelnut. Polyclonal antibodies were obtained from rabbits following immunization with antigens from pure cultures of Anisogramma anomala, the pathogen. One-thousand-fold dilution of the antiserum produced positive reactions to 1.7 x 10 5 dilutions of A. anomala-infected hazelnut tissue extracts, but did not react to 1.7 x 10 2 dilutions of healthy hazelnut tissue extracts. Symptomless plants infected by A. anomala were detected by the indirect ELISA 3 to 5 months after inoculation, an improvement over the 13- to 27-month incubation period required for susceptible genotypes to develop external symptoms of infection (cankers). ELISA was more sensitive and efficient than conventional microscopic assays (i.e., visualizing A. anomala mycelium in hand-sectioned plant tissue) in both healthy and infected extracts. The screening system was tested on selected progenies from populations segregating for a single, dominant resistance gene. ELISA detected 100% of the infections while microscopic examination detected only 36% of the infected samples. ELISA rapidly and reliably identifies hazelnut progeny with a gene conferring a high level of resistance derived from the cv. Gasaway.