James J. Polashock

Movement of a small mitochondrial double-stranded RNA element of Cryphonectria parasitica: ascospore inheritance and implications for mitochondrial recombination.

Abstract Movement via somatic fusion and inheritance of a small mitochondrial double-stranded (ds) RNA element was examined in Cryphonectria parasitica. The 2.7-kb dsRNA from the C. parasitica strain NB631 encodes a putative RNA-dependent RNA polylmerase when the mitochondrial code (UGA = Trp) is invoked. All progeny from asexual spores (conidia) of strain NB631 examined for dsRNA contained the 2.7-kb element. Unlike other C. parasitica dsRNAs, which are cytoplasmic, the dsRNA in strain NB631 was transmitted through the sexual cycle (ascospores) if the strain containing the element acted as the female in crosses. Movement of the 2.7-kb dsRNA was also observed through hyphal anastomosis. Transfer by anastomosis was accompanied by mitochondrial movement and recombination of the mitochondrial genome as determined by RFLP analysis. In control pairings between isolates lacking dsRNA, mitochondrial movement and recombination were also observed. Transfer by anastomosis allowed the generation of infected and uninfected isogenic lines, and permitted us to evaluate the effects of the dsRNA element on virulence of the host. Bark virulence assays on American chestnut suggest that NB631 dsRNA decreases the virulence of C. parasitica, but not to the level associated with members of the Hypoviridae.

The American cranberry: first insights into the whole genome of a species adapted to bog habitat

BackgroundThe American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear.ResultsThe genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%).ConclusionsShotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession

Culturable fungal population diversity and succession was investigated in developing cranberry ovaries of fruit rot-resistant and rot-susceptible cranberry selections, from flower through mature fruit. Fungi were recovered in culture from 1185 of 1338 ovary tissues collected from June to September, yielding 2255 isolates that represented 42 morphotaxa. During the season, species richness varied from 2 to 17 and 2 to 18 in rot-resistant and rot-susceptible selections, respectively, increasing from wk1 to wk10 and then gradually declining to wk14. Shannon-Wiener diversity index varied from 0.27 to 2.32 in rot-resistant and 0.18 to 2.38 in rot-susceptible, and Pielou’s evenness index varied from 0.11 to 0.63 and 0.06 to 0.64 in rot-resistant and rot-susceptible selections, respectively, confirming that diversity of fungi in developing ovaries was similar among rot-resistant and rot-susceptible selections, but varied among sampling time points. Principal component analysis grouped samples collected at the same sampling time point together regardless of rot susceptibility of the selections, and detected the predominant fungal species associated with each stage of development. Successional changes were observed in populations of endophytic, pathogenic and saprophytic fungi throughout the season as ovaries matured.

Cloning, sequencing, and promoter identification of Blueberry red ringspot virus, a member of the family Caulimoviridae with similarities to the “Soybean chlorotic mottle-like” genus

Summary. The double-stranded DNA genome of Blueberry red ringspot virus (BRRV), a member of the family Caulimoviridae, was cloned and sequenced. The genome organization and relationships of the 8303 nt sequence revealed BRRV to be a tentative member of the genus that has been provisionally named “Soybean chlorotic mottle-like viruses”, rather than a member of the genus Caulimovirus, in which it had been placed previously. Insertion of the putative 35S promoter homolog of BRRV into promoterless constructs carrying the UidA (β-glucuronidase) gene resulted in high-level transient expression from cranberry and stable expression from transgenic tobacco. Sequences of 5′-RACE clones derived from transcripts from transgenic tobacco were consistent with the map position of the promoter.

New and emerging viruses of blueberry and cranberry.

Blueberry and cranberry are fruit crops native to North America and they are well known for containing bioactive compounds that can benefit human health. Cultivation is expanding within North America and other parts of the world raising concern regarding distribution of existing viruses as well as the appearance of new viruses. Many of the known viruses of these crops are latent or asymptomatic in at least some cultivars. Diagnosis and detection procedures are often non-existent or unreliable. Whereas new viruses can move into cultivated fields from the wild, there is also the threat that devastating viruses can move into native stands of Vaccinium spp. or other native plants from cultivated fields. The aim of this paper is to highlight the importance of blueberry and cranberry viruses, focusing not only on those that are new but also those that are emerging as serious threats for production in North America and around the world.

Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco

Abstract A clone representing a fragment of the dihydroflavonol-4-reductase ( DFR ) gene from cranberry was isolated from a genomic DNA library using the tomato DFR gene as a probe. Sequence analysis of the clone confirmed homology to published DFR gene sequences. 3′ and 5′ RACE (rapid amplification of cDNA ends) reactions from cranberry leaf total RNA were used to obtain the entire cDNA sequence. The sequence information was used to amplify a full-length clone by RT-PCR. Sequencing analysis to confirm the identity of the full-length DFR cDNA identified a putative second allele. Segregation analysis suggested that the two sequences are not allelic, but multi-locus. Nucleotide sequence homology of the full-length clones was highest to published DFR sequence from Camellia sinensis (about 80% identity) followed by Forsythia x intermedia , Antirrhinum majus , Rosa hybrida and Petunia hybrida . When expressed using the CaMV 35S promoter, the corolla of flowers of transgenic tobacco plants were much darker pink than the controls. Some flower parts not normally highly pigmented, such as the filaments, were also dark pink. These data confirm the identity and function of the cranberry clones and further suggest that overexpression of the cranberry DFR could be used to increase anthocyanin production in transgenic plants.

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants.

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.

Discrimination of American Cranberry Cultivars and Assessment of Clonal Heterogeneity Using Microsatellite Markers

Cranberries (Vaccinium macrocarpon Ait.) are an economically important fruit crop derived from a North American native species. We report the application of 12 simple sequence repeats (SSR) or microsatellite markers to assess the genetic diversity of cranberry cultivars. We studied 164 samples of 21 different cranberry cultivars, 11 experimental hybrids, and 6 representative accessions of wild species. Genetic cluster analysis, based on 117 SSR alleles, differentiated the major cranberry cultivars. However, some cranberry cultivar subclone variants and mislabeled samples were observed. Consensus genetic profiles identified the most likely clonal representatives of several important cranberry cultivars (e.g., “Ben Lear,” “Howes,” and “Stevens”). The markers were further used to confirm putative parents of several hybrid progenies. The long-term goal of our studies is to identify, preserve, and utilize unique genetic materials to breed improved cranberries. Attaining this goal will help growers maintain sustainability under changing economic and environmental conditions.

Anthracnose Fruit Rot Resistance in Blueberry Cultivars

Anthracnose fruit rot (causal agent, Colletotrichum acutatum) is an important disease in most blueberry growing regions of North America. Losses caused by the disease are usually seen as a postharvest rot with orange spore masses appearing on the surface of affected fruit. One hundred cultivars/selections of blueberry were screened for resistance to fruit rot between 1993 and 2003 by inoculating container-grown plants bearing green fruit. Visible rot symptoms on ripe fruits were evaluated after a 1-week incubation at room temperature. Our analyses revealed that infection levels were affected by mean May temperatures in New Jersey, generally increasing as temperatures increased; however, this effect was not consistent among all cultivars. A generalized linear mixed model was developed to predict resistance at the historic mean May temperature, conservatively explaining 59% of the variance in resistance. Percent infection ranged from 9 to 91% with a mean of 51% across all cultivars. Results for common cultivars corresponded well with field reports of their relative susceptibilities. An estimate of narrow-sense heritability of 0.32 suggested additive inheritance of resistance. Since very high inoculum loads were used in this study, cultivars exhibiting a low percentage of fruit rot are predicted to show superior field resistance to the disease and will be incorporated into an ongoing breeding program.