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Nahla Bassil, Kim Hummer, and Joseph Postman

Simple Sequence Repeat (SSR) markers developed in apple and pear were used to determine genetic relationships among heritage apple and pear cultivars from Portugal's Azore Islands, and to develop identity fingerprints for European and Asian pear accessions at the USDA–ARS National Clonal Germplasm Repository (NCGR). We used 11 SSR markers (six from apple and five from pear) to examine 18 heritage apple and 9 heritage pear cultivars from the Azores. Eight additional Portuguese and economically important cultivars of apple and eight of pear were used as standards. Cluster analysis separated the apple and pear accessions into two distinct groups. Among apple genotypes, 12 unique accessions and five groups of synonyms were identified, while, in pear, seven unique genotypes and three pairs of synonyms were found. None of the accessions obtained from the Azores corresponded to widely grown standard Portuguese apple or pear cultivars. To examine 144 NCGR pear accessions, we used nine polymorphic SSR loci that were developed from GenBank sequences. Cluster analysis identified five sets of synonyms (four in P. communis L. and one in P. ussuriensis Maxim.) and four pairs of homonyms (three in P. communis and one in P. pyrifolia Burm. f. Nakai), and confirmed three clonal sets. Morphological evaluations and additional SSR markers will be used to confirm these results, and to genetically document the identities of pear genotypes. SSR markers will greatly assist the management of ex situ pome fruit germplasm collections by helping to eliminate duplicate accessions and expanding the genetic diversity represented.

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Peter Boches, Nahla V. Bassil, and Lisa Rowland

Sixty-nine accessions representing wild and domesticated highbush blueberry (Vaccinium corymbosum L.) germplasm were genotyped using 28 simple sequence repeats (SSRs). A total of 627 alleles was detected and unique fingerprints were generated for all accessions. Suspected duplicate accessions of `Coville' and `Ivanhoe' had DNA fingerprints that were identical to `Coville' and `Ivanhoe', respectively. Genetic similarity measures placed wild and cultivated blueberries in separate groups. Northern highbush blueberries grouped among ancestral clones that were used extensively in blueberry breeding such as `Rubel' and `Stanley'. Southern highbush blueberries formed a separate group from northern highbush blueberries. The microsatellite markers used here show excellent promise for further use in germplasm identification, in genetic studies of wild Vaccinium L. populations, and for constructing linkage maps.

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William M. Proebsting, Nahla V. Bassil, and David A. Lightfoot

Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.

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William M. Proebsting, Nahla V. Bassil, and David A. Lightfoot

Propagation of Corylus avellana stem cuttings may be limited by either root initiation or bud abscission. We divided juvenile shoots of 3 varieties growing in layering beds in mid-July into 4 or 5 3-node cuttings with leaves at the upper two nodes, except that terminal cuttings had one expanded leaf. Cuttings were treated with 5 mM IBA in 50% EtOH, a mixture of A. rhizogenes strains A7 + 22 or left untreated. IBA and bacteria stimulated rooting of cuttings from all shoot positions. Rooting of the terminal cuttings (<50%) was less than that of the sub-terminal cuttings (>80%). Bud retention was <50% on terminal cuttings, nearly 100% on sub-terminal cuttings. Using juvenile stock plants of various varieties, sub-terminal cuttings treated with Agrobacterium or 5 mM IBA may yield 70-90% cuttings with both roots and buds, Agravitropic roots, characteristic of genetic transformation, were observed on Agrobacterium-treated cuttings. Dot blots probed for TL-DNA were negative, however.

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Peter Boches, Lisa J. Rowland, Kim Hummer, and Nahla V. Bassil

Microsatellite markers for blueberry (Vaccinium L.) were created from a preexisting blueberry expressed sequence tag (EST) library of 1305 sequences and a microsatellite-enriched genomic library of 136 clones.

Microsatellite primers for 65 EST-containing simple sequence repeats (SSRs) and 29 genomic SSR were initially tested for amplification and polymorphism on agarose gels. Potential usefulness of these SSRs for estimating species relationships in the genus was assessed through cross-species transference of 45 SSR loci and cluster analysis using genetic distance values from five highly polymorphic EST-SSR loci. Cross-species amplification for 45 SSR loci ranged from 17% to 100%, and was 83% on average in nine sections. Cluster analysis of 59 Vaccinium species based on genetic distance measures obtained from 5 EST-SSR loci supported the concept of V. elliotii Chapm. as a genetically distinct diploid highbush species and indicated that V. ashei Reade is of hybrid origin. Twenty EST-SSR and 10 genomic microsatellite loci were used to determine genetic diversity in 72 tetraploid V. corymbosum L. accessions consisting mostly of common cultivars. Unique fingerprints were obtained for all accessions analyzed. Genetic relationships, based on microsatellites, corresponded well with known pedigree information. Most modern cultivars clustered closely together, but southern highbush and northern highbush cultivars were sufficiently differentiated to form distinct clusters. Future use of microsatellites in Vaccinium will help resolve species relationships in the genus, estimate genetic diversity in the National Clonal Germplasm Repository (NCGR) collection, and confirm the identity of clonal germplasm accessions.

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Nahla V. Bassil, R. Botta, and S.A. Mehlenbacher

Three microsatellite-enriched libraries of the european hazelnut (Corylus avellana L.) were constructed: library A for CA repeats, library B for GA repeats, and library C for GAA repeats. Twenty-five primer pairs amplified easy-to-score single loci and were used to investigate polymorphism among 20 C. avellana genotypes and to evaluate cross-species amplification in seven Corylus L. species. Microsatellite alleles were estimated by fluorescent capillary electrophoresis fragment sizing. The number of alleles per locus ranged from 2 to 12 (average = 7.16) in C. avellana and from 5 to 22 overall (average = 13.32). With the exception of CAC-B110, di-nucleotide SSRs were characterized by a relatively large number of alleles per locus (≥5), high average observed and expected heterozygosity (Ho and He > 0.6), and a high mean polymorphic information content (PIC ≥ 0.6) in C. avellana. In contrast, tri-nucleotide microsatellites were more homozygous (Ho = 0.4 on average) and less informative than di-nucleotide simple sequence repeats (SSRs) as indicated by a lower mean number of alleles per locus (4.5), He (0.59), and PIC (0.54). Cross-species amplification in Corylus was demonstrated. These microsatellite markers were highly heterozygous and polymorphic and differentiated among genotypes of C. avellana irrespective of geographical origin. They will aid in fingerprinting genotypes of the european hazelnut and other Corylus species, genome mapping, and genetic diversity assessments.

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Nahla V. Bassil, B.J. Rebhuhn, David W.S. Mok, and Machteld C. Mok

Development of optimum protocols for micropropagation of C. avellana is particularly important due to the threat of Eastern Filbert Blight and the need for rapid increase of resistant varieties and advanced selections. Therefore, efforts were directed at in vitro establishment, multiplication and rooting, starting with buds from mature trees. The frequency of shoot formation from buds was highest in August but varied with the genotype. Medium containing high Ca levels was more effective in preventing bud necrosis than MS medium. Multiplication rates of 4-7 new shoots/propagule were obtained over a 6-week culture period. Rooting of some genotypes could be accomplished by inclusion of 1 or 3 μM β- indolebutyric acid (IBA) in the medium. Other genotypes responded better to a dip of shoot bases in 1-10 mM IBA for 10 sec., followed by a passage on auxin-free medium. Large numbers of healthy plantlets have been produced for transfer to soil.

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Nahla V. Bassil, William M. Proebsting, Larry W. Moore, and David A. Lightfoot

Hazelnut (Corylus avellana L.) softwood cuttings of the cultivars Ennis and Casina were propagated under mist during June and July 1987 and 1988. Rooting of stem cuttings was stimulated by both Agrobacterium and IBA treatment; however, IBA caused nearly complete bud abscission. Better rooting and bud retention were observed in `Casina' than in `Ennis' in 1988. Bud retention on Agrobacterium -inoculated cuttings improved as the cuttings approached the semi-hardwood stage. Six months after transplanting, Agrobacterium -inoculated hazelnut cuttings had an extensive root system, characteristic of hairy root. Although the mechanism remains unclear, strains of Agrobacterium rhizogenes are effective rooting agents in hazelnut and may cause less bud abscission than IBA. Chemical name used: 1 H -indole-3-butyric acid (IBA).

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David R. Sandrock, Anita N. Azarenko, Ruth M. Martin, and Nahla V. Bassil

The NRT1gene family encodes transport proteins with dual or low affinity for nitrate. The objectives of this experiment were to develop a system that could be used to compare the expression of the NRT1genes between species. This was accomplished by comparing sequences of NRT1homologues from various species and designing degenerate primers in regions of high homology. These primers were used to amplify a region of the NRT1gene from species of interest. A 635 bp PCR product was amplified from each species using the MD2-1 (5' ATGTTACCAAYWTGGGCMAC-3') and MD2-2 (5'-GCCAMWARCCARTAGAAAT-3') primers. The PCR products were cloned and sequenced. At the nucleotide level, CornussericeaL. `Kelseyi' and RhododendronL. `Unique' were 79.52% identical. Species-specific primers were designed and used for RT-PCR to compare NRT1expression in roots of hydroponically grown C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique'. The relative levels of NRT1expression, normalized using 18S rRNA as a standard, were ≈3.2 to 1.7 to 1.0 for C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique', respectively. This approach may eventually be used to examine nitrate uptake potential in different taxa of plants at different times during the growing season.