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.
James J. Polashock and Nicholi Vorsa
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.
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.
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.
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.
Ebrahiem M. Babiker, Stephen J. Stringer, Hamidou F. Sakhanokho, Barbara J. Smith, and James J. Polashock
Species of Botryosphaeria and Neofusicoccum are major pathogens of blueberry worldwide. Accurate identification of these species is essential for developing effective management practices. A multigene sequencing strategy was used to distinguish between six isolates of stem blight pathogens collected from two different regions of the United States. The temperature growth study revealed that the optimal temperature for growth of five of the tested isolates ranged from 25 to 30 °C, although no significant difference was detected for the growth of Neofusicoccum spp. isolate SD16-86 at 20, 25, 30, and 35 °C. In vitro fungicide assays showed four fungicides, cyprodinil + fludioxonil, propiconazole, pyraclostrobin + boscalid, and azoxystrobin, were effective against the tested isolates with isolate SD16-86 being less sensitive compared with the other isolates. In a detached stem assay, none of 39 blueberry accessions displayed immunity or a high level of resistance to the two tested isolates, and no significant difference in lesion length was detected among the seven tested Vaccinium species inoculated with the two isolates.
Daniel J. Bell, Lisa J. Rowland, James J. Polashock, and Frank A. Drummond
Little is known of the genetic structure and variability of wild fields, or of the dramatic differences in yield among clones (genetic individuals), of lowbush blueberry (Vaccinium angustifolium Ait.), Maine's most economically important fruit crop. Express sequence tag-polymerase chain reaction (EST-PCR) markers that were originally developed for genetic mapping purposes in highbush blueberry (Vaccinium corymbosum L.) are shown here to be valuable for genetic fingerprinting and relationship studies in the related species, V. angustifolium. As part of an interspecific genetic relationship study, 14 genotypes, including at least two specimens of each of four closely related Vaccinium L. species (V. pallidum Ait., V. corymbosum, V. boreale Hall & Aald., and V. myrtilloides Michx.) and the only four pedigreed cultivars of V. angustifolium, grouped out as expected in a genetic similarity dendrogram (matrix “r” correlation = 0.91). This work is ultimately aimed at using these markers in exploring how genetic relationship affects yield among proximal and distant breeding individuals via controlled field hand crosses. To help address this issue, a separate group of six individuals of V. angustifolium from two managed fields were also genotyped using the EST-PCR markers. The markers were very effective at intraspecific discrimination of individuals within the same field.
Lisa J. Rowland, Anik L. Dhanaraj, James J. Polashock, and Rajeev Arora
Expressed sequence tag-polymerase chain reaction (EST-PCR) markers for DNA fingerprinting and mapping in blueberry (Vaccinium sp.) had previously been developed from expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants. Because EST-PCR markers are derived from gene coding regions, they are more likely to be conserved across populations and species than markers derived from random regions of DNA, such as randomly amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers. In this study, we tested whether many of the EST-PCR primer pairs developed for blueberry are capable of amplifying DNA fragments in other members of the family Ericaceae. In addition, we cloned and sequenced a selection of 13 EST-PCR fragments to determine if they showed homology to the original blueberry cDNA clones from which the EST-PCR primer pairs were derived. Closely related cranberry genotypes (two wild selections of V. oxycoccus L. and two cultivars of V. macrocarpon Aiton, `Early Black' and `Stevens') and more distantly related rhododendron genotypes (one wild selection each of Rhododendron arboreum Marsh, R. maximum L., and R. ponticum L. and three complex species hybrids, `Sonata', `Grumpy Yellow', and `Roseum elegans') were used. Of 26 primer pairs tested in cranberry, 23 (89%) resulted in successful amplification and eight of those (35%) amplified polymorphic fragments among the cranberry genotypes. Of 39 primer pairs tested in rhododendron, 29 (74%) resulted in successful amplification and 21 of those (72%) amplified polymorphic fragments among the rhododendron genotypes. Approximately 50% of the 13 sequenced EST-PCR fragments were found to be homologous to the original blueberry cDNA clones. These markers should be useful for DNA fingerprinting, mapping, and assessing genetic diversity within cranberry and rhododendron species. The markers which are shown to be homologous to the blueberry cDNA clones by DNA sequencing should also be useful for comparative mapping and genetic diversity studies between some genera of the family Ericaceae.
James J. Polashock, Rajeev Arora, Yanhui Peng, Dhananjay Naik, and Lisa J. Rowland
Highbush blueberry (Vaccinium corymbosum) is susceptible to winter freezing injury and frost damage in the spring. As part of an ongoing project to understand the process of cold acclimation, we isolated a C-repeat binding factor (CBF) transcriptional activator gene-coding region from the highbush blueberry cultivar Bluecrop. Expression of the highbush blueberry CBF gene was compared in floral buds of the cold-tolerant northern highbush cultivar Bluecrop and the more cold-sensitive southern rabbiteye (V. virgatum) blueberry cultivar Tifblue. Relative gene expression was higher in ‘Bluecrop’ than in ‘Tifblue’. Expression in both cultivars was highest at the earliest time point in the fall (coincident with the first stage of cold acclimation), declined during the later fall and winter, and, in ‘Bluecrop’, increased again as buds deacclimated, when temperatures tend to fluctuate. To confirm the putative identity of the gene as a member of the CBF gene family, and to determine if expression in a heterologous system could enhance freezing tolerance, the blueberry gene coding sequence was overexpressed in transgenic Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Transgenic plants expressing the putative blueberry CBF gene exhibited induced expression of the A. thaliana cold-regulated (COR) genes COR78 and COR6.6, under non-inducing conditions (i.e., 23 °C); however, expression of two other COR genes was unaffected. Transgenic plants also exhibited enhanced freezing tolerance under non-acclimated conditions, but not to the level of acclimated control plants. Thus, the expression pattern in floral buds and the ability of the isolated gene to turn on a subset of COR genes and increase freezing tolerance in a heterologous system suggest it is a functional member of the CBF gene family in blueberry.