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James Polashock and Nicholi Vorsa

We have used RAPDs (Randomly Amplified Polymorphic DNAs) to successfully fingerprint cranberry. Although this method is simple and inexpensive, disadvantages include limited reproducibility in other labs and it is not easily computer-analyzed. RAPDs can also be labor-intensive because multiple primers are required to adequately fingerprint a single sample. As an alternative, we have utilized a method called SCARs (Sequence Characterized Amplified Regions). Clear polymorphic RAPD markers were cloned and sequenced. Primers were designed to amplify each polymorphic band and contained the original 10-mer RAPD primer sequence and 10 to 12 additional “clone-specific” bases. Primer sets were tested on eight common cranberry cultivars to determine if the desired polymorphic marker was amplified. The success rate of developing ëgoodí primer sets was ≈25%. The most common problem was loss of polymorphism, suggesting that selectivity was contained within the original 10-mer RAPD primer. The amplification of many similarly sized markers, suggesting the primer set amplified a repeat region, was another problem. Useful primer sets were multiplexed in PCR reactions to establish a “fingerprint.” The SCARs system we developed to fingerprint cranberry is powerful enough to distinguish individual clones in both crosses and selfed progeny. To further simply the system, computer automation for detection and analysis using fluorescently labeled primers is underway. One problem we are addressing is reduced product in the labeled multiplex reactions. Reduced product yield is presumably because the dye molecule (Cy5) is very large and may reduce primer binding and/or polymerization efficiency. This problem has been somewhat alleviated using a patented form of Taq DNA polymerase.

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Nicholi Vorsa and James J. Polashock

The flavonoids of american cranberry (Vaccinium macrocarpon Ait.) are documented to be beneficial for human health. Among their benefits is a high antioxidant potential, with anthocyanin glycosides being the main contributors. Flavonoid glucose conjugates are reported to be more bioavailable than those with other sugar conjugates. The anthocyanin glycosides of V. macrocarpon fruit are mainly galactosides and arabinosides of the aglycones, cyanidin and peonidin, with less than 8% glucosides. In contrast, the fruit anthocyanins of another cranberry species, V. oxycoccus L. were found to be largely glucosides of cyanidin and peonidin. Interspecific hybrids between these two species were intermediate to the parental species in the proportion of fruit anthocyanin glucosides. About half the progeny (1:1 segregation) in a backcross population (to V. macrocarpon) maintained the relatively high anthocyanin glucoside ratio. In this study, we demonstrate the genetic manipulation of anthocyanin glycosylation in cranberry using interspecific hybridization, resulting in dramatically increased glucose-conjugated anthocyanins.

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James J. Polashock and Matthew Kramer

Stem diseases of blueberry (Vaccinium spp.) can cause significant crop loss as well as loss of entire bushes. Stem diseases are also more difficult to control with fungicides than foliar or fruit diseases. A screening program was initiated to test blueberry cultivars for resistance to two pathogenic fungi: botryosphaeria stem blight and phomopsis twig blight. An attached stem assay was developed to compare the host response with both fungi. The relative resistance of 50 blueberry cultivars was assessed using stem lesion lengths, analyzed on a log scale, taken at 4 weeks postinoculation. For Botryosphaeria stem blight, mean lesion length ranged from about 10 mm in resistant cultivars to about 140 mm in susceptible cultivars. The half-high cultivars Northsky, Northblue, and Chippewa, and the lowbush cultivar Putte were among the most resistant. Phomopsis twig blight lesions ranged in mean length from about 18 to 98 mm. Similar to results for Botryosphaeria stem blight, resistance was limited to half-high (`Northsky' and `Chippewa') and lowbush (`Blomidon', `Chignecto', and `Cumberland') cultivars. Individual cultivars resistant to one pathogen were not necessarily resistant to the other; although, overall, the resistances were correlated. Approximate 95% confidence intervals were established for all cultivars to predict mean performance across years. The cultivars tested varied in resistance, but the largest single factor affecting lesion length was the fungal isolate used for inoculations. These data enable us to identify cultivars resistant to both diseases that can be used for planting in problem areas, as well as selection of parental material for breeding cultivars with improved resistance.

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James J. Polashock and Nicholi Vorsa

Most varieties of the American cranberry (Vaccinium macrocarpon) cultivated today were selected from native selections or breeding progeny between the late 1800s and mid-1900s. We have previously shown using RAPDs that contamination, i.e., a mixture of genotypes, is common in commercial bogs. One source of contamination could be establishment of selfed progeny. The purpose of this study was to determine how effective RAPDs would be in distinguishing selfed progeny from the parent. Results suggest that the number of scorable polymorphic bands is low compared to outcrossed or unrelated progeny. Thus, five to nine primers were used as compared to the three primers normally required to separate outcrossed and unrelated clones. Segregation of some RAPD bands was not consistent with expected mendelian ratios. However, using 9 to 12 polymorphic bands, only 3% to 5% of the selfed progeny had fingerprints identical to the parent. Additional primers should further reduce this percentage. It was also noted that certain cultivars exhibited a large number of non-parental bands. The origin of the non-parental bands has not yet been determined.

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James J. Polashock and Nicholi Vorsa

DNA fingerprinting has been useful for genotypic classification of American cranberry (Vaccinium macrocarpon Ait.). Polymerase chain reaction (PCR) based methodologies including randomly amplified polymorphic DNA (RAPD) markers are relatively easy to use, and inexpensive as compared to other methods. However, RAPD markers have some limitations including seamless interlaboratory transferability and susceptibility to certain types of error. An alternative method, sequence characterized amplified regions (SCARs), was developed for cranberry germplasm analysis. Nine primer sets were designed from RAPD-identified polymorphic markers for use in two multiplex PCR reactions. These primer sets generated 38 markers across a cranberry germplasm collection. Estimates of genetic relatedness deduced from employment of the RAPD and SCAR methods were compared among 27 randomly chosen cranberry germplasm accessions. Although both methods produced comparable results above 0.90 coefficient of similarity, branches below this level exhibited variation in clustering. SCAR and RAPD markers can be employed for identifying closely related genotypes. However, the inferences of more distant genetic relationships are less certain. SCAR marker reactions provided more polymorphic markers on a per reaction basis than RAPD marker reactions and as such more readily separated closely related progeny. When SCAR primers were fluorescent dye-labeled for computerized detection and data collection, reduced marker intensity relative to unlabeled reactions was one problem encountered.

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Mark K. Ehlenfeldt and James J. Polashock

The primary gene pool of Vaccinium species used by blueberry breeders has traditionally been the North American Vaccinium species of section Cyanococcus. Blueberries in commercial production represent three primary Vaccinium species and two ploidy levels. Significant use has been made of the secondary gene pool of Vaccinium, especially in the development of southern highbush blueberry (Vaccinium ×corymbosum) cultivars. Section Hemimyrtillus species are distantly related and are best considered part of the tertiary gene pool of Vaccinium. Vaccinium padifolium, a member of section Hemimyrtillus and native to the Madeira Islands, Portugal, has features of notable value to conventional blueberry development, among these: upright structure, strong growth, abundant flowering and fruiting, good self-fertility, inflorescence structure suited to mechanical harvesting, and indeterminate/repeat flowering. Our objective was to incorporate germplasm from this section into cultivated materials and transfer the desirable traits these species possess for commercial production. We used V. padifolium as a female in crosses with V. corymbosum and generated two highly fertile hybrids. These hybrids are intermediate in morphology, phonological, and their hybridity has been confirmed through DNA testing. These hybrids were used in further crosses to a variety of section Cyanococcus selections and have generated numerous second-generation hybrids. We have also determined by flow cytometry the ploidy levels of the hybrids and several previously unevaluated section Hemimyrtillus species.

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Luping Qu, James Polashock and Nicholi Vorsa

Putative transgenic cranberry plants have been achieved via Agrobacterium-mediated transformation. Leaf explants were transformed with a supervirulent Agrobacterium tumefaciens strain EHA 105, harboring the binary vector P35SGUSint and nptII selectable marker genes. Inoculation of precultured explants (≈10 days on regeneration medium) coupled with sonicasion improved transformation efficiency significantly. Adventitious shoots were directly regenerated from explants. Putative transformed shoots were identified by being kanamycin-resistant and GUS-positive. Stable GUS gene expression (turning blue) could be detected within 1 h of incubation at 37 °C. Confirmation of transformation by molecular analysis is in progress. Eight putative transgenic cranberry plants were obtained. All appeared morphologically normal. This appears to be the first success in achieving cranberry transformed plants by Agrobacterium-mediated method. Optimizing the transformation system is ongoing.

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Luping Qu, James Polashock and Nicholi Vorsa

A very efficient adventitious regeneration (shoot organogenesis) system for cranberry (Vaccinium macrocarpon Ait.) leaves was developed. A basal medium consisting of Anderson's rhododendron salts and Murashige and Skoog's (MS) organics, supplemented with 10.0 μm thidiazuron (TDZ) and 5.0 μm 2ip, was effective for adventitious regeneration from leaves for the five cranberry cultivars tested: `Early Black', `Pilgrim', `Stevens', `Ben Lear', and `No. 35'. Parameters examined included: 1) varying combinations of three plant growth regulators (TDZ, 2ip, and NAA); 2) explant orientation (adaxial vs. abaxial side in contact with the medium); and 3) leaf position relative to the apical meristem from the source plant. Cultivars varied in regeneration frequency, but cultivar × growth regulator interaction was nonsignificant. With optimal treatment conditions, regeneration occurred on more than 95% of the explants, with `Early Black' and `Pilgrim' producing as many as 100 shoot meristems per explant. At all concentrations tested, NAA (as low as 0.1 μm) increased callus formation and significantly reduced regeneration. Emerging adventitious shoots were always observed on the adaxial side of the leaves regardless of explant orientation on the medium. Regeneration was much greater when the abaxial side was in contact with the medium, and was not related to leaf position on the source plants. Elongation of adventitious shoots began ≈2 weeks after transfer to the basal medium without growth regulators. Cuttings of elongated shoots rooted 100% both in vitro in the basal medium and ex vitro in shredded sphagnum moss. The high regeneration efficiency achieved by using this system will be very useful in the application of techniques, such as Agrobacterium- and particle bombardment-mediated transformation. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ); N6-(γ-γ-dimethyallylamino) purine (2ip); α-naphthaleneacetic acid (NAA).

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Luping Qu, James Polashock and Nicholi Vorsa

We have established a very efficient cranberry regeneration (shoot organogenesis) system from leaf explants using a basal medium consisting of Anderson's salts and Murashige and Skoog (MS) organics supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) and N6-(-??-dimethyallylamino) purine) (2ip). Characteristics examined include combinations of varying levels of three plant growth regulators (TDZ, 2ip, and naphthaleneacetic acid (NAA), explant orientation (adaxial or abaxial side in contact with the media), and leaf position relative to the distal end of the shoot. Genotypes (`Early Black', `Pilgrim', `Stevens', `Ben Lear', and US#35) differed significantly in regeneration capacity, and there were no genotype by treatment interaction effects. Regeneration occurred on more than 95% of the explants with `Early Black' and `Pilgrim' producing as many as 100 shoot tips per explant with one particular treatment. Emerging adventitious shoots were always observed on the adaxial side of the leaves regardless of explant orientation. However, regeneration was much greater when the adaxial side was in contact with the media. Regeneration efficiency was not significantly affected by leaf position (10 leaves). Elongation of shoot tips began about 2 weeks after the regenerating explants were transferred to the basal medium without hormones and continued for several months. Elongated shoot cuttings rooted readily.

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James J. Polashock, Robert A. Saftner and Matthew Kramer

Fruit of highbush blueberry (Vaccinium corymbosum L.) produce antimicrobial volatiles, including trans-2-hexenal, that may confer resistance to anthracnose fruit rot, an important postharvest disease caused by Colletotrichum acutatum J.H. Simmonds. To investigate whether aromatic volatiles in highbush blueberry fruit are associated with postharvest fruit rot resistance, we compared volatiles emitted from whole fruit and extracts from fruit kept in air at 20 °C for 0 to 6 days postharvest from cultivars having a wide range of resistance to anthracnose. Antimicrobial volatiles detected included the aldehydes, trans-2-hexenal and hexanal; the monoterpenes, limonene, linalool, 8-hydroxylinalool, α-terpineol, and terpinyl acetate; and the sesquiterpenes, cadinene, caryophyllene, and α-farnesene. There were significant correlations between some detected volatiles and these differed in whole fruit and extracts. Hexanal (in fruit extracts), trans-2-hexenal, terpinyl acetate, and cadinene emissions increased in most cultivars when fruit were kept in air at 20 °C for various times postharvest. Volatile emissions from whole fruit and extracts varied widely among the cultivars with early ripening cultivars generally showing higher volatile emissions than later ripening cultivars. Although the cultivars tested differed in quantities, and in some cases, the types of volatiles produced, these differences were not related to pedigree (i.e., species composition) nor to known anthracnose resistance ratings. Except for the confounded emissions of terpinyl acetate and cadinene, more than 80% of the variation observed for each volatile was attributable to the cultivar (genetic), year (environmental), and cultivar–by-year interaction. The results suggest that, although antimicrobial aldehydes and terpenes emitted from fully ripe highbush blueberry fruit and extracts might be important flavor and aroma components, they do not significantly contribute to disease resistance against anthracnose fruit rot.