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A. Levi and L.J. Rowland

Fifteen highbush (or highbush hybrid) blueberry cultivars (Vaccinium corymbosum Linnaeus), two rabbiteye blueberry cultivars (V. ashei Reade), and one southern lowbush (V. darrowi Camp) selection from the wild were examined using seventeen 10-base RAPD and seven 15- to 18-base SSR-anchored primers (primers comprised of SSR motifs) in polymerase chain reactions (PCRs). Fifteen RAPD and three SSR markers resulting from these reactions were chosen to construct a DNA fingerprinting table to distinguish among the genotypes included in this study. Similarity values were calculated based on 132 RAPD and 51 SSR bands, and a dendrogram was constructed based on the similarity matrix. The V. ashei cultivars and V. darrowi selection grouped out separately from the V. corymbosum cultivars as expected. However, estimates of relative genetic similarity between genotypes within the V. corymbosum group did not agree well with known pedigree data and, thus, indicated that RAPD and SSR data did not accurately assess the genetic relationships of cultivars within this species.

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Lisa J. Rowland and Elizabeth L. Ogden

Conditions for improving the efficiency of shoot regeneration from leaf sections of highbush blueberry (Vaccinium corymbosum L.) were investigated. Effectiveness of tissue culture medium supplemented with the cytokinin conjugate zeatin riboside or the cytokinin zeatin at 10, 20, or 30 μm was compared with medium supplemented with the optimum 2iP concentration of 15 μm. Use of 20 μm zeatin riboside resulted in the most shoots per leaf section, » 6-fold higher than the number of shoots produced on 2iP medium. The number of shoots produced on medium supplemented with zeatin was not significantly higher than the number of shoots produced on 2iP medium. Consequently, we concluded that the cytokinin conjugate zeatin riboside was more effective than either of the free cytokinins, 2iP or zeatin, in promoting shoot regeneration from leaf sections of highbush blueberry. Chemical names used: 6-(y,y-dimethylallylamino)-purine (2iP); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin).

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Anik L. Dhanaraj*, Janet P. Slovin and Lisa J. Rowland

To gain a better understanding of changes in gene expression associated with cold stress in the woody perennial blueberry (Vaccinium spp.), a genomics approach based on the analysis of expressed sequence tags (ESTs) was undertaken. Two cDNA libraries were constructed using RNA from cold acclimated (mid winter conditions when the plants are cold stressed) and non-acclimated (before they received any chilling) floral buds of the blueberry cultivar Bluecrop. About 600 5'-end ESTs were generated from each of the libraries. Putative functions were assigned to 57% of the cDNAs that yielded high quality sequences based on homology to other genes/ESTs from Genbank, and these were classified into 14 functional categories. From a contig analysis, which clustered sequences derived from the same or very similar genes, 430 and 483 unique transcripts were identified from the cold acclimated and non-acclimated libraries, respectively. Of the total unique transcripts, only 4.3% were shared between the libraries, suggesting marked differences in the genes expressed under the two conditions. The most highly abundant cDNAs that were picked many more times from one library than from the other were identified as representing potentially differentially expressed transcripts. Northern analyses were performed to examine expression of eight selected transcripts and seven of these were confirmed to be preferentially expressed under either cold acclimating or non-acclimating conditions. Only one of the seven transcripts, encoding a dehydrin, had been found previously to be up-regulated during cold stress of blueberry. This study demonstrates that analysis of ESTs is an effective strategy to identify candidate cold-responsive transcripts in blueberry.

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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.

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Mark K. Ehlenfeldt, Lisa J. Rowland, Elizabeth L. Ogden and Bryan T. Vinyard

Cold injury to plants can occur by early fall freezes before cold acclimation, by severe midwinter freezes that exceed the limits of the plant's tolerance, or by hard freezes in late winter or early spring after partial or complete deacclimation. Ideally, blueberry (Vaccinium L.) cultivars for temperate regions should acclimate to cold quickly in the fall, have a high midwinter-hardiness, and deacclimate late and/or slowly during spring or during unseasonably warm spells in winter, and do all of this without adversely delaying time of fruiting. Until recently, only limited evaluations have been done on the acclimation and deacclimation process in blueberry, although it is an integral part of flower bud survival and, thus, is directly related to potential yield. In this study, we have measured the timing and rate of acclimation and deacclimation in seven blueberry genotypes with different amounts of diverse species germplasm in their backgrounds. Primary differences observed among the seven genotypes were differences in maximum hardiness levels and the date at which they were reached, and differences in the date at which maximum acclimation levels were no longer sustained and deacclimation started. Highbush cultivars Bluecrop and Legacy (V. corymbosum L.), rabbiteye cultivar Tifblue [V. ashei Reade (= V. virgatum Aiton)], and two rabbiteye hybrid derivatives (US 1043 and US 1056) all reached maximum or near maximum cold-hardiness by late December with temperatures causing 50% lethality (LT50) in a range from –22 to –27 °C. The half-high, ‘Northsky’, and a hybrid of V. constablaei Gray × V. ashei ‘Little Giant’ both achieved cold acclimation of –28 °C or below (the lowest value we could measure) by the end of November. After reaching their maximum hardiness in late December, ‘Legacy’, ‘Tifblue’, and US 1043 began a sustained and relatively linear deacclimation, whereas US 1056, ‘Bluecrop’, ‘Northsky’, and ‘Little Giant’ sustained their acclimation for longer intervals. ‘Bluecrop’ and US 1056 did not begin to deacclimate until early March, and ‘Little Giant’ and ‘Northsky’ had no LT50 values higher (warmer) than –25 °C until late March. As concerns about climate change increase, knowledge of the ability of breeding germplasm to tolerate greater temperature extremes and fluctuations will prove increasingly valuable.

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M. Faust, A. Erez, L.J. Rowland, S.Y. Wang and H.A. Norman

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Stan C. Hokanson, Kelvin G. Grant, Elizabeth L. Ogden and Lisa J. Rowland

Commercial strawberry plantings in the mid-Atlantic region are often quickly infected with one or more aphid-transmitted viruses, resulting in the loss of plant vigor, stunting, lowered yields, etc. To produce virus-free plant material for the strawberry industry and for cultivar development programs, heat therapy and/or meristem tip culture protocols are generally employed. One of the problems associated with meristem culturing is the potential for somaclonal mutations to occur in the meristem or surrounding proliferating tissue. As a result, distinct “bud lines” displaying functionally insignificant to distressingly high levels of phenotypic variation can arise from individual meristems. It would be desirable to differentiate these off-types by genetic fingerprinting to maintain trueness-to-type. Randomly amplified polymorphic DNA (RAPD) markers were evaluated for the potential to differentiate six pairs of strawberry bud lines that exhibit slight to fairly extreme levels of phenotypic variation. Reproducible RAPD marker profiles were generated using 10 primers in amplification reactions with genomic DNA obtained from multiple extractions. While five of the bud line pairs remained indistinguishable, three primers distinguished two variants of the Mohawk cultivar that are now in existence in the strawberry industry. Results suggest that typical somaclonal variation produced in the meristem culture process is of a magnitude that is not readily detectable with the RAPD protocol. The two Mohawk lines were probably produced by a higher magnitude mutation event than generally occurs or a cultivar mix-up.

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Lisa J. Rowland, Elizabeth L. Ogden, Mark K. Ehlenfeldt and Rajeev Arora

Cold hardiness in woody perennials is determined by complex interacting factors: the timing and rate of cold acclimation; the maximum level of cold tolerance attained; the maintenance of cold tolerance during the winter; and the rate of loss of cold tolerance or deacclimation on resumption of spring growth. For highbush blueberry, the degree of winter freezing tolerance and susceptibility to spring frosts have been identified as the most important genetic limitations of current cultivars. Depending on the winter and the location, both winter freezes and spring frosts can cause damage to floral buds or flowers resulting in substantial losses in yield. To identify genotypes that are particularly slow or late to deacclimate and thus may be useful in breeding for spring frost-tolerant cultivars, we compared deacclimation kinetics under controlled laboratory and field conditions among several blueberry genotypes with diverse genetic backgrounds. Clear genotypic differences in timing and rate of deacclimation were found. In the field study, the species Vaccinium constablaei Gray was identified as particularly late to deacclimate, and ‘Little Giant’ (50:50 hybrid of V. constablaei and V. ashei Reade) was nearly as late to deacclimate as 100% V. constablaei. Recently, we extended our cold tolerance measurements from October through midwinter comparing acclimation kinetics and maximum cold tolerance levels among genotypes. Although all genotypes appeared to reach maximum cold tolerance about mid-December under the study conditions, genotypic differences were detected in other aspects, including initial cold tolerance, rate of acclimation, maximum cold tolerance, and length of the plateau. ‘Little Giant’ and ‘Northsky’ (75:25 hybrid of V. corymbosum L. and V. angustifolium Ait.) were very early to acclimate and were hardier than the other genotypes both initially and when maximum cold tolerance was reached. Understanding how cold tolerance levels change throughout the dormant period should help us to develop cultivars better suited to their environments.

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Lisa J. Rowland, Anik L. Dhanaraj, Dhananjay Naik, Nadim Alkharouf, Ben Matthews and Rajeev Arora

To gain a better understanding of changes in gene expression associated with cold acclimation in the woody perennial blueberry (Vaccinium corymbosum L.) and ultimately use this information to develop more freeze-tolerant cultivars, a genomics approach based on the analysis of expressed sequence tags (ESTs) and microarrays was undertaken. Initially, two standard cDNA libraries, constructed using RNA from cold-acclimated (CA) and nonacclimated (NA) floral buds of the blueberry cultivar Bluecrop, were used for the generation of ≈2400 ESTs, half from each library. Putative functions were assigned to cDNAs based on homology to other genes/ESTs from GenBank. From contig analyses, 796 and 865 unique transcripts were identified from the CA and NA libraries, respectively. The most highly abundant cDNAs, that were picked many more times from one library than from the other, were identified as representing potentially differentially expressed transcripts. A cDNA microarray was constructed and used to study gene expression under cold-acclimating conditions in the field and cold room. Results indicated that the abundance of transcripts of numerous blueberry genes change during cold acclimation, including genes not found previously to be cold-responsive in Arabidopsis, and, interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Finally, forward and reverse subtracted cDNA libraries were prepared from ‘Bluecrop’ RNA to enrich for transcripts that are expressed at higher levels in floral buds at 400 h and at 0 h of low-temperature exposure, respectively. Many genes encoding putative transcription factors and other proteins related to signal transduction were identified from both libraries.

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Mark K. Ehlenfeldt, Lisa J. Rowland, Elizabeth L. Ogden and Bryan T. Vinyard

From 2004 to 2006, cold hardiness assays were performed to evaluate the relative winterhardiness of flower buds in selections of pure Vaccinium ashei Reade and V. constablaei Gray as well as in selections/families composed of various combinations of V. ashei and V. constablaei germplasm. Significant differences were observed among entries with LT50 values ranging from −17.2 to −28.4 °C. An analysis of LT50 versus percent V. constablaei yielded a regression of LT50 (°C) = (−0.08 × V. constablaei percentage) – 21.57. Families or selections with 50% (or greater) V. constablaei and some with 25% V. constablaei had LT50 values equivalent to or better than ‘Bluecrop’. Based on this information, a 25% V. constablaei constitution appears suitable to develop northern-adapted rabbiteye types if proper parents are selected and if sufficient selection pressure for winterhardiness is exercised.