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  • Author or Editor: Nicholi Vorsa x
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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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Vaccinium darrowi (D) is a wild blueberry species with low chilling requirements for budbreak, and heat and drought tolerance. Breeding efforts to incorporate these desirable traits into cultivated blueberry (V. corymbosum) (C) would be facilitated with a better understanding of the genomic homology between the two species. An interspecific tetraploid hybrid (CCDD, 2n=4x=48) was used to evaluate genome homology and interspecific recombination. Pollen mother cells examined at diakinesis and early metaphase I exhibited an average of 4.6 chain bivalents, 11.4 ring bivalents, 1.0 chain quadrivalent, and 3.0 ring quadrivalents. This data most closely fits a chromosome pairing model in which there is a greater pairing affinity between homologues than homoeologues. An analysis of the inheritance of 14 RAPD markers unique to V. darrowi in 72 backcross progeny of the V. darrowi–corymbosum hybrid also supported the pairing model: Seven of the 14 markers deviated significantly from tetrasomic inheritance ratios, expected if chromosome pairing was totally random. On the basis of the cytogenetic and RAPD analyses, the genomes of V. darrowi and V. corymbosum are divergent from one another, with preferential pairing within genomes. This outcome suggests there may be difficulty in breaking undesirable linkages when introgressing desirable traits from V. darrowi to V. corymbosum.

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Cranberry (Vaccinium macrocarpon Ait.) has the opportunity to partition resources into sexual and/or asexual (stolons) modes of reproduction. Nitrogen status has been shown to impact the degree of stoloniferous growth. To determine whether there is a genotypic response to varying nitrogen levels, six hybrid and four native cultivars were treated with three annual rates of nitrogen fertilizer (17, 34, or 67 kg·ha-1) for 4 years. Fruit yield was determined each year and asexual vegetative growth (stolons) weight was removed and measured in all but the first year of the experiment. Cultivars exhibited different patterns of yield and stolon weight response over the three nitrogen rates. Not all cultivars exhibited significant yield decreases at the high N levels. Vegetative growth (stolon weight) generally increased with increasing N, however, not all cultivars responded similarly over three N rates. Partitioning between yield and stolon production favored fruit yield at the lower N rates in three of the four native cultivars studied (`Cropper', `Early Black', and `Howes'). Yield over N rates was more stable for four of the six hybrid cultivars, which may be the result of greater heterozygosity in hybrids than natives, and/or genetic gain from one breeding and selection cycle, offering increased tolerance to nitrogen stress. This study indicates that genetic variation exists for yield, yield stability, and stolon production relative to nitrogen level, and that genetic gain in cranberry is possible for these traits. Future studies involving cranberry physiology and nutrition should consider the genotypes used.

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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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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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Accurate estimates of yield and yield components for parental selection would facilitate cranberry breeding efforts. A study was designed to obtain value estimates for traits related to yield. Ten commonly-cultivated varieties grown in a replicated planting, were evaluated in 1991 and 1992 for fruit yield per unit area (FY), average berry weight (BW) and number of berries per unit area, or berry concentration (BC). Averaged over all varieties, FY was significantly greater in 1992. BC was responsible for higher yields in 1992. Regression analysis revealed that BC accounted for more of the variation in FY than did BW in both years. BW accounted for some variation, however, in 1991 when FY was lower. Varieties differed significantly in FY, BW and BC. Hybrid varieties bad significantly greater FY and BW than wild selections. Variation for yield components exists among varieties tested, suggesting genetic gain is possible for yield with additional breeding efforts. In particular, greater fruit set should be emphasized as a breeding objective.

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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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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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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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