Known and New Emerging Viruses Infecting Blueberry.

Blueberry (Vaccinium spp.) plants are exposed to existing and emerging viruses as a result of expanding acreage of blueberry plantations across the world, primarily in North America. Since blueberry is cultivated in areas where there are wild Vaccinium spp., there is increasing risk of virus movement between wild and cultivated blueberries. This is theoretically possible because viruses can spread from commercial cultivars to native species and vice versa causing the spread of existing and new viruses. The occurrence of these viruses in blueberry can be devastating to the industry considering the cost for cultivation and production of this perennial crop. However, the advent of high-throughput sequencing and bioinformatic sequence analysis have allowed for rapid identification of known and novel viruses in any crop including blueberry, thus facilitating proper intervention in response to serious viral diseases. In this paper, we aim to focus on the current status of known and novel viruses emerging in blueberry worldwide, which may impact the blueberry industry.

Discovery of Known and Novel Viruses in Wild and Cultivated Blueberry in Florida through Viral Metagenomic Approaches.

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families (Amalgaviridae, Caulimoviridae, Endornaviridae, Ophioviridae, Phenuiviridae, and Virgaviridae). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach.

Comprehensive evaluation of RNA-seq analysis pipelines in diploid and polyploid species.

Background: The usual analysis of RNA sequencing (RNA-seq) reads is based on an existing reference genome and annotated gene models. However, when a reference for the sequenced species is not available, alternatives include using a reference genome from a related species or reconstructing transcript sequences with de novo assembly. In addition, researchers are faced with many options for RNA-seq data processing and limited information on how their decisions will impact the final outcome. Using both a diploid and polyploid species with a distant reference genome, we have tested the influence of different tools at various steps of a typical RNA-seq analysis workflow on the recovery of useful processed data available for downstream analysis. Findings: At the preprocessing step, we found error correction has a strong influence on de novo assembly but not on mapping results. After trimming, a greater percentage of reads could be used in downstream analysis by selecting gentle quality trimming performed with Skewer instead of strict quality trimming with Trimmomatic. This availability of reads correlated with size, quality, and completeness of de novo assemblies and with number of mapped reads. When selecting a reference genome from a related species to map reads, outcome was significantly improved when using mapping software tolerant of greater sequence divergence, such as Stampy or GSNAP. Conclusions: The selection of bioinformatic software tools for RNA-seq data analysis can maximize quality parameters on de novo assemblies and availability of reads in downstream analysis.

Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq.

BACKGROUND: Blueberries are one of the few horticultural crops adapted to grow in acidic soils. Neutral to basic soil pH is detrimental to all commonly cultivated blueberry species, including Vaccinium corymbosum (VC). In contrast, the wild species V. arboreum (VA) is able to tolerate a wider range of soil pH. To assess the molecular mechanisms involved in near neutral pH stress response, plants from pH-sensitive VC (tetraploid) and pH-tolerant VA (diploid) were grown at near neutral pH 6.5 and at the preferred pH of 4.5. RESULTS: Transcriptome sequencing of root RNA was performed for 4 biological replications per species x pH level interaction, for a total of 16 samples. Reads were mapped to the reference genome from diploid V. corymbosum, transforming ~55% of the reads to gene counts. A quasi-likelihood F test identified differential expression due to pH stress in 337 and 4867 genes in VA and VC, respectively. Both species shared regulation of genes involved in nutrient homeostasis and cell wall metabolism. VA and VC exhibited differential regulation of signaling pathways related to abiotic/biotic stress, cellulose and lignin biosynthesis, and nutrient uptake. CONCLUSIONS: The specific responses in VA likely facilitate tolerance to higher soil pH. In contrast, response in VC, despite affecting a greater number of genes, is not effective overcoming the stress induced by pH. Further inspection of those genes with differential expression that are specific in VA may provide insight on the mechanisms towards tolerance.

Farm to Sensory Lab: Taste of Blueberry Fruit by Children and Adults.

The average American child eats fewer fruits than recommended. Although taste is the primary motivator for food intake among children, little research has systematically measured children's liking of fruit and determined whether their preferences differ from adults. We phenotyped 49 children and their mothers to determine: (1) their liking of the taste of 3 blueberry cultivars ("Arcadia," "Keecrisp," and "Kestrel") from 2 harvests for which total soluble solids were determined using a handheld Brix refractometer; (2) the association between liking and blueberry sugar content; and (3) the most preferred level of fructose, one of the primary sugars in blueberry fruit. Multiple methods, identical for all participants, assessed which cultivar they liked best. Dietary intake, determined via 24-h dietary recall, revealed most children (73%) and adults (92%) did not meet dietary guidelines for fruit intake. We found that during the 1st harvest, Keecrisp was sweeter by 4° Brix than either Arcadia or Kestrel and was the cultivar most preferred by both children and adults. For the 2nd harvest, mothers liked each of the cultivars equally, but children preferred Arcadia, which was 2° Brix sweeter than the other 2 cultivars. Like other sugars, children's most preferred concentration of fructose was significantly higher than that of adults. In sum, children appear to be more sensitive to smaller variations in sweetness than are adults. Identifying drivers of fruit preference and assessing children's liking for whole fruits are important steps in developing strategies to increase fruit consumption among children.

AGHmatrix: R Package to Construct Relationship Matrices for Autotetraploid and Diploid Species: A Blueberry Example.

Progress in the rate of improvement in autopolyploid species has been limited compared with diploids, mainly because software and methods to apply advanced prediction and selection methodologies in autopolyploids are lacking. The objectives of this research were to (i) develop an R package for autopolyploids to construct the relationship matrix derived from pedigree information that accounts for autopolyploidy and double reduction and (ii) use the package to estimate the level and effect of double reduction in an autotetraploid blueberry breeding population with extensive pedigree information. The package is unique, as it can create A-matrices for different levels of ploidy and double reduction, which can then be used by breeders to fit mixed models in the context of predicting breeding values (BVs). Using the data from this blueberry population, we found for all the traits that tetrasomic inheritance creates a better fit than disomic inheritance. In one of the five traits studied, the level of double reduction was different from zero, decreasing the estimated heritability, but it did not affect the prediction of BVs. We also discovered that different depths of pedigree would have significant implications on the estimation of double reduction using this approach. This freely available R package is available for autopolyploid breeders to estimate the level of double reduction present in their populations and the impact in the estimation of genetic parameters as well as to use advanced methods of prediction and selection.

Identifying Breeding Priorities for Blueberry Flavor Using Biochemical, Sensory, and Genotype by Environment Analyses.

Breeding for a subjective goal such as flavor is challenging, as many blueberry cultivars are grown worldwide, and identifying breeding targets relating to blueberry flavor biochemistry that have a high degree of genetic control and low environmental variability are priorities. A variety of biochemical compounds and physical characters induce the sensory responses of taste, olfaction, and somatosensation, all of which interact to create what is perceived flavor. The goal of this study was to identify the flavor compounds with a larger genetic versus environmental component regulating their expression over an array of cultivars, locations, and years. Over the course of three years, consumer panelists rated overall liking, texture, sweetness, sourness, and flavor intensity of 19 southern highbush blueberry (Vaccinium corymbosum hybrids) genotypes in 30 sensory panels. Significant positive correlations to overall liking of blueberry fruit (P<0.001) were found with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). Sourness had a significantly negative relationship with overall liking (R2 = 0.55). The relationship between flavor and texture liking was also linear (R2 = 0.73, P<0.0001) demonstrating interaction between olfaction and somatosensation. Partial least squares analysis was used to identify sugars, acids, and volatile compounds contributing to liking and sensory intensities, and revealed strong effects of fructose, pH, and several volatile compounds upon all sensory parameters measured. To assess the feasibility of breeding for flavor components, a three year study was conducted to compare genetic and environmental influences on flavor biochemistry. Panelists could discern genotypic variation in blueberry sensory components, and many of the compounds affecting consumer favor of blueberries, such as fructose, pH, ß-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is worthwhile.

Correlation between sensory and instrumental measurements of standard and crisp-texture southern highbush blueberries (Vaccinium corymbosum L. interspecific hybrids).

BACKGROUND: Fruit texture is a primary selection trait in southern highbush blueberry (SHB) breeding to increase fresh fruit postharvest quality and consumer acceptance. A novel crisp fruit texture has recently been identified among SHB germplasm. In this study, we developed a common set of descriptors that align sensory evaluation of blueberry fruit texture with instrumental measures that could be used for quantitative measurements during pre- and postharvest evaluation. RESULTS: Sensory and instrumental characteristics were measured in 36 and 49 genotypes in 2010 and 2011, respectively. A trained sensory panel evaluated fresh fruit based on five common textural attributes in 2010 and 2011: bursting energy, flesh firmness, skin toughness, juiciness and mealiness. Instrumental measures of compression and bioyield forces were significantly different among cultivars and correlated with sensory scores for bursting energy, flesh firmness and skin toughness (R > 0.7, except skin toughness in 2011), but correlations with sensory scores for juiciness and mealiness were low (R < 0.4). CONCLUSION: The results of sensory and instrumental measures supported the use of both compression and bioyield force measures in distinguishing crisp from standard-texture genotypes, and suggest that crisp texture in SHB is related to the sensory perception of bursting energy, flesh firmness and skin toughness.