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.
James Polashock and Nicholi Vorsa
Nicholi Vorsa and Richard Novy
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.
Mark Ehlenfeldt and Nicholi Vorsa
Mark K. Ehlenfeldt and Nicholi Vorsa
Forty-one half-sib families (>3000 individuals) segregating for parthenocarpic fruit production were evaluated under field conditions. Within these families, ≈280 parthenocarpic individuals were identified. In general, three categories of segregants were observed: normal-seeded types, small/low-seeded types, and parthenocarpic types. Inheritance patterns suggested that the trait is recessive, but did not fit simple tetrasomic recessive or incomplete dominance models. Lack of fit may be attributable to environmental interactions or incomplete penetrance of this trait. Further evaluations are underway with small/low-seeded types with the expectation that offspring of these plants may express the trait at higher frequencies than in F1 × F1 crosses. The reduced vigor in many of the parthenocarpic segregants suggests that this germplasm will be most useful, initially, in improving fruit quality in the intermediate expression types, which have better fruit set and reduced seed development.
Richard G. Novy and Nicholi Vorsa
Cranberry (Vaccinium macrocarpon Ait.) has few qualitative, morphological characteristics that can be used to reliably distinguish among cultivars. Fifty-two silver-stained random amplified polymorphic DNAs (ssRAPDs) were used to assess genetic heterogeneity and relatedness within accessions of four major cranberry cultivars (`Early Black', `Howes', `McFarlin', and `Searles'). Rather than being represented by one genotype, as might be expected in an asexually propagated crop, each cultivar was represented by multiple genotypes, which in many cases did not appear to be closely related to one another. The intracultivar heterogeneity was often so extreme that clonal representatives of a cultivar would group with representatives of other cultivars following cluster analysis. Of the total ssRAPD variation, 9.7% could be attributed to variation among the four cultivar groups and 90.3% to variation within the cultivars. `Howes' was the only cultivar in which a consensus DNA fingerprint among regional representatives could be identified.
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.
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.
Joan R. Davenport and Nicholi Vorsa
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.
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.
Elissa M. Novy and Nicholi Vorsa
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.