James W. Olmstead

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, β-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is worthwhile.

Generating Genomic Tools for Blueberry Improvement

Because of their recognized health benefits, there has been increased demand and consumption of blueberries in recent years. Great strides have been made in blueberry cultivar development since its domestication using traditional breeding approaches. However, genomic tools that could be used to hasten improvement are lacking. The aim of our Specialty Crop Research Initiative project, funded at the end of 2008, is to develop genomic tools for molecular breeding and assessing genetic diversity of blueberry. Marker-assisted breeding would be particularly useful for combining traits for climatic adaptation with those for improved fruit and nutritional quality in highbush blueberry (Vaccinium corymbosum). Genomic resources being developed include expressed sequence tag libraries, expressed sequence tag-based molecular markers, and genetic linkage maps. Transcriptome sequences have been generated from fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by 454 sequencing. About 600,000 sequences have been assembled into approximately 15,000 contigs. Markers derived from expressed sequence tags (simple sequence repeats and expressed sequence tag-polymerase chain reaction markers) are being used to identify quantitative trait loci associated with cold hardiness, chilling requirement, and fruit quality traits, in studies of genetic diversity, spatial genetic structure, and gene flow in the wild lowbush blueberry (V. angustifolium), and to construct a phylogenetic tree of Vaccinium species in the section Cyanococcus. Availability of these genomic tools will allow future advances, such as the development of a blueberry microarray to facilitate studying gene expression and the use of marker-assisted breeding.

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

We developed an R package for autopolyploids to construct the relationship matrix. We estimated the level and effect of double reduction in blueberry. The package is unique as it can create A‐matrices for different levels of ploidy. The package can create A‐matrices for different levels of double reduction

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.

The Use of Inter-Sectional Hybrids in Blueberry Breeding

The tetraploid species in Vaccinium section Cyanococcus and tetraploid V. uliginosum in section Vaccinium are autotetraploid. The same is probably true of the tetraploid species in other sections of the genus. Chromosome pairing at metaphase I in these species is normally regular and bivalent, but each gamete contains two homologous chromosomes for each of the 12 basic chromosome types (x = 12 in Vaccinium). Thus, F1 hybrids between tetraploid plants from different Vaccinium sections can have regular bivalent chromosome pairing during meiosis and high fertility, even though diploid hybrids involving the same sections are highly sterile. Such fertile tetraploid hybrids are called amphidiploids. Amphidiploidy can give rise to new species; several important crop species are domesticated amphidiploids. The conditions for amphidiploid formation are narrow. The two species that hybridize must be divergent enough to insure faithful homologous bivalent chromosome pairing in the hybrid, where each bivalent consists of chromosomes derived from the same parent species. However, the parent species must be closely enough related to permit formation of vigorous hybrids. The first indication that amphidiploidy could be useful in blueberry breeding was a report by Rousi in 1963 of vigorous, fertile hybrids between tetraploid V. uliginosum (section Vaccinium) and tetraploid V. corymbosum (section Cyanococcus). In Florida, crosses of colchicine-induced tetraploid V. arboreum (section Batodendron) with tetraploid highbush cultivars and with tetraploid Florida V. myrsinites (section Cyanococcus) indicate that Vaccinium sections Batodendron and Cyanococcus also have the right degree of divergence to produce vigorous, fertile tetraploid hybrids. The feasibility of producing other intersectional tetraploid combinations in Vaccinium, and the vigor and fertility of the hybrids, can best be determined by trial and error.

Estimation of genetic parameters and prediction of breeding values in an autotetraploid blueberry breeding population with extensive pedigree data

The strategy for breeding blueberries has been based on phenotypic selection without accounting for pedigree correlation information. A central premise of classical quantitative genetics is that through knowledge of the relationships among individuals in a population, we can make inferences about breeding values (BVs), estimate genetic parameters, and support phenotypic selection. Genetic evaluations using best linear unbiased prediction (BLUP) and restricted maximum likelihood (REML) are founded on pedigree information and have been the standard method used in livestock and forest breeding. Despite theoretical and practical benefits, their application in blueberry breeding programs remains unexploited. The objective of this study was to evaluate the use of REML/BLUP in the University of Florida blueberry breeding program in order to estimate genetic parameters and predict BVs of primary selection traits. To do this, we collected phenotypic data for eight selectable traits (yield, flower bud density, fruit weight, fruit firmness, fruit diameter, fruit soluble solids, fruit pH, and fruit scar diameter) from 1996 individuals. The highest narrow-sense heritabilities were found for fruit weight, scar diameter, and yield. The majority of genetic correlations between traits were under 0.20. The largest genetic gain was achieved when selections from early stages were used as parents and the top 5% of the population was selected. Compared to traditional phenotypic selection, our results are evidencing of the importance of implementing REML/BLUP to estimate genetic parameters that help and support the breeding strategy and maximize genetic gains.

Investigation on the Profile of Phenolic Acids and Flavonoids withAntioxidant Capacity in Florida Highbush (Vaccinium corymbosum L.)and Rabbiteye (Vaccinium virgatum) Blueberries

It is well documented that blueberry phenolic compositions, such as phenolic acids and flavonoids differ based on species and cultivars. However, phenolic compositions in Florida blueberries have been little explored. Information on Florida blueberries are of interest as they are harvested earlier than other areas of the United States, which may result in compositional differences due to shorter daylight hours and cooler temperatures. Samples were harvested from University of Florida grower-cooperator farm near Gainesville, FL. After liquid-liquid extraction and C-18 SPE cartridge purification, concentrations of phenolic acids and flavonoids of twenty two blueberry cultivars (20 highbush and 2 rabbiteye) were measured using HPLC. Five phenolic acids (gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid and ferulic acid), two flavan-3-ols (catechin and epicatechin) and five flavonols (quercetin-3-galactoside or quercetin-3-glucoside, quercetin-3-arabinoside, myricetin, quercetin and kaempferol) were identified and quantified. Chlorogenic acid was the major component in highbush blueberries. Flavonoid and phenolic acid composition were profoundly different among Florida blueberries and these differences have not been previously reported. The first two components of PCA explained 95% of the variation totally; it showed clear differentiation of blueberry cultivars based on phenolic composition. The majority of the variation between the cultivars was due to variation in quercetin-3-galactoside or glucoside, quercetin-3-arabinoside and chlorogenic acid, which are most likely due to genetic and maturity differences. Complete-linkage clustering analysis displayed five significantly different (p<0.05) clusters of blueberries, which were in agreement with PCA results, although some small differences were noted. Furthermore, Florida blueberries can be differentiated based on phenolic composition between highbush and rabbiteye species and varieties. Low flavanol content in rabbiteye blueberries may be related to maturity as these compounds typically decrease as berries ripen. Although the concentrations for phenolic compositions were lower than found in other reports, Florida blueberries contained a greater range of phenolic compounds. This information is valuable for blueberry breeding programs and growers for development of higher phenolic composition cultivar.

Insights Into the Genetic Basis of Blueberry Fruit-Related Traits Using Diploid and Polyploid Models in a GWAS Context

Abstract Polyploidization is an ancient and recurrent process in plant evolution, impacting the diversification of natural populations and plant breeding strategies. Polyploidization occurs in many important crops; however, its effects on inheritance of many agronomic traits are still poorly understood compared with diploid species. Higher levels of allelic dosage or more complex interactions between alleles could affect the phenotype expression. Hence, the present study aimed to dissect the genetic basis of fruit-related traits in autotetraploid blueberries and identify candidate genes affecting phenotypic variation. We performed a genome-wide association study (GWAS) assuming diploid and tetraploid inheritance, encompassing distinct models of gene action (additive, general, different orders of allelic interaction and the corresponding diploidized models). A total of 1,575 southern highbush blueberry individuals from a breeding population of 117 full-sib families were genotyped using sequence capture and next-generation sequencing, and evaluated for eight fruit-related traits. For the diploid allele calling, 77,496 SNPs were detected; while 80,591 SNPs were obtained in tetraploid, with a high degree of overlap (95%) between them. A linear mixed model that accounted for population and family structure was used for the GWAS analyses. By modeling tetraploid genotypes, we detected 15 SNPs significantly associated with five fruit-related traits. Alternatively, seven significant SNPs were detected for only two traits using diploid genotypes, with two SNPs overlapping with the tetraploid scenario. Our results showed that the importance of tetraploid models varied by trait and that the use of diploid models has hindered the detection of SNP-trait associations and, consequently, the genetic architecture of some commercially important traits in autotetraploid species. Furthermore, 14 SNPs co-localized with candidate genes, five of which lead to non-synonymous amino acid changes. The potential functional significance of these SNPs is discussed.

Screening for Susceptibility to Anthracnose Stem Lesions in Southern Highbush Blueberry

‘Flicker’ is a southern highbush blueberry (SHB, Vaccinium corymbosum) cultivar frequently selected by growers in Central and South Florida. In 2014, several growers in Central Florida experienced issues with anthracnose stem lesions and twig dieback on ‘Flicker’, resulting in a reduction in new plantings and the removal of many existing plantings. The objective of this study was to determine the level of anthracnose susceptibility of certain commercially available SHB cultivars, which information can be used to limit further use of susceptible cultivars in the University of Florida blueberry breeding program. The screening was performed using a spray inoculation of a virulent Colletotrichum gloeosporioides isolate onto whole V. corymbosum plants, followed by measurement of incidence and severity of disease over time. In repeated experiments, ‘Flicker’ and two other cultivars had a significantly higher mean number of lesions and area under the disease progress curve (AUDPC) than any other tested cultivar, and in both experiments, the observed lesions were similar in many respects to those previously reported on northern highbush blueberry (also V. corymbosum). Although the results of these experiments may ultimately indicate that Flicker has a unique genetic susceptibility to this form of anthracnose among SHB cultivars commercially grown in Florida, screening of additional cultivars must be performed for confirmation. Anthracnose is a group of diseases incited by fungal pathogens including those in the genus Colletotrichum. Colletotrichum is distributed primarily in tropical and subtropical regions, although it can also be found in more temperate regions (Cannon et al., 2012). The disease is characterized by dark, sunken, subcircular, or angular necrotic lesions, as shown in Fig. 1, with salmon or pink conidial masses erupting from the lesions in later stages. Lesions typically enlarge, coalesce, and can result in significant dieback (Freeman et al., 1998; Jeffries et al., 1990). Anthracnose is typically observed as fruit rots, leaf spots, or stem lesions. Stem lesions can be incited by different species of Colletotrichum, and many crops grown throughout the world are susceptible to one or more species. Certain Colletotrichum species have been found to cause stem lesion anthracnose on cassava (Fokunang et al., 2002), mango (Gupta et al., 2015), and dragon fruit (Vijaya et al., 2015), all grown primarily in tropical regions. There have been a few reported incidences of anthracnose stem lesions on blueberry. Kim et al. (2009) reported C. gloeosporioides infecting stems on highbush blueberry in Gochang, South Korea. The infected stems turned dark brown, then became gray and died. Of five stem isolates taken, all were identified as C. gloeosporioides based on morphological and cultural characteristics. Although no cultivar names were provided, it is assumed the infected plants were northern highbush because of the climate in Gochang, South Korea, which is located at latitude 35 N. In a separate study, stem blight was observed on highbush blueberry in Japan, with the previous year’s shoots turning brown and then blighted, along with the death of adjacent floral buds. In addition, small red or brown leaf spots were observed near the blighted stems. Isolates from stem tissue were identified as Colletotrichum acutatum based solely on morphological and cultural characteristics (Yoshida and Tsukiboshi, 2002). In addition, in 2010 lesions on green northern highbush blueberry canes were reported in Michigan. The lesions were described as dark brown to black, circular or oval, with light brown to gray centers, and salmon-pink masses of spores. The pathogen was identified as C. acutatum by morphological characteristics. This report also noted that anthracnose stem lesions had been observed on northern highbush blueberry in Ontario, Canada, and Michigan in 2003 and 2004 (Schilder, 2010). Finally, in 2013, stem lesions and leaf spots on highbush blueberry caused by anthracnose were reported in Liaoning, China. It is assumed the symptomatic plants were northern highbush, consistent with the authors’ use of the term ‘‘highbush blueberry’’ (not ‘‘southern highbush blueberry’’) and the location of Liaoning near latitude lat. 42 N, the same latitude as Michigan. Symptoms were described as yellow to red irregularly shaped lesions on stems and leaves, which expanded and turned dark brown, surrounded by a red halo. Isolates were identified as C. gloeosporioides based on morphological and cultural characteristics, which was later confirmed through molecular methods (Xu et al., 2013). Until recently, there have been no peerreviewed reports of anthracnose stem lesions on SHB. ‘Flicker’ is an SHB cultivar frequently selected by growers in Central and South Florida. Characteristics favoring production of ‘Flicker’ in this region are that it has very low chilling requirements, can be grown in an evergreen management system, and tends to ripen early. In 2014, several blueberry farms in Central Florida experienced issues with anthracnose stem lesions and twig dieback on ‘Flicker’, and to a lesser extent on ‘Scintilla’, a progeny of ‘Flicker’ (Harmon, 2014a). Using molecular methods, isolates taken from infected plants on four farms in this area were confirmed to be C. gloeosporioides (Velez-Climent and Harmon, 2016). Further compounding the disease issue, isolates of this pathogen collected on these farms have been found to be resistant to fungicides in the quinone outside inhibitor class (also called strobilurins), including azoxystrobin and pyraclostrobin (Harmon, 2014b). This outbreak of disease has resulted in a reduction in new plantings of ‘Flicker’ and ‘Scintilla’ and the removal of some existing plantings (P.F. Harmon, personal communication). Because of the early success of ‘Flicker’, Received for publication 20 Feb. 2018. Accepted for publication 5 May 2018. Corresponding author. E-mail: p.munoz@ufl.edu. This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). 920 HORTSCIENCE VOL. 53(7) JULY 2018 it has been used as a parent in the University of Florida blueberry breeding program in the past, raising concerns regarding potential susceptibility of offspring from these crosses. In addition, there is a concern about whether other commercial cultivars may be susceptible to this fungicide-resistant form of anthracnose. This could potentially lead to similar problems as those experienced with ‘Flicker’ and ‘Scintilla’, including a possible rejection by growers of further use of such cultivars and the costly removal of existing plantings. The objective of this study was to determine the level of anthracnose susceptibility of certain commercially available SHB cultivars, so that University of Florida (UF) personnel could use any identified susceptibility to limit the use of susceptible cultivars in the UF blueberry breeding program and communicate the findings to growers for their use in making decisions on which cultivars to plant. Materials and Methods Plant material. Plant material used in the evaluation of anthracnose susceptibility consisted of 10 cultivars of interspecific crosses of V. corymbosum (SHB), all of which are commercially grown in Florida. These included ‘Chickadee’, ‘Emerald’, ‘Farthing’, ‘Flicker’, ‘Jewel’, ‘Kestrel’, ‘Rebel’, ‘San Joaquin’, ‘Springhigh’, and ‘Star’. All plants were purchased from Fall Creek Nursery in Lowell, OR, with the exception of ‘Flicker’, which was purchased from AgriStarts in Apopka, FL. Both of these nurseries propagate plants from tissue culture, and they were selected to minimize any issues with latent plant infections. The plants were 10 cm tall, in 38-cell trays. All of the cultivars with the exception of ‘Rebel’ were developed by the University of Florida blueberry breeding program. The plants were transplanted into 15-cm Kord plastic pots (HC Companies, Middlefield, OH), filled with 100% Fafard peat (Sun Gro Horticulture, Agawam, MA), and allowed to grow for 4 months before inoculation. Plants were each placed 11.5 cm apart, within rows that were also 11.5 cm apart on greenhouse benches. The plants were watered regularly, and fertilized with a Peters Professional 20–20–20 fertilizer (Scotts, Marysville, OH) every 2 weeks. The experiments were conducted in a temperature-controlled greenhouse without supplemental lighting at the University of Florida in Gainesville, FL.Greenhouse temperature ranged between 16 and 36 C,measured using a HOBO temperature logger (HOBO U23-002; Onset Computer Corporation, Bourne, MA). Experimental design. The experiment was arranged in a randomized complete block design with 10 replications, one plant per cultivar per replication. The experiment was conducted twice, both during May 2016. The treatments included 10 different cultivars of V. corymbosum, all of which were inoculated with a single isolate of C. gloeosporioides. To increase the number of replication that would be inoculated with the pathogen, 50 ‘Flicker’ plants (known to be susceptible to anthracnose) were used as susceptible standards (sprayed with distilled water instead of the pathogen), instead of using an equal number of control replications for each cultivar. The results of spraying the susceptible ‘Flicker’ plants with distilled water were then compared with the pathogen-inoculated ‘Flicker’ plants to determine whether additional control replications (i.e., cultivars sprayed with distilled water instead of being inoculated with the pathogen) were required. Source of isolate and inoculum preparation. The pathogen used for inoculation was a single-conidium isolate of C. gloeosporioides (15–646), originally isolated in 2015 from naturally infected stems of ‘Flicker’ on a commercial blueberry farm in Central Florida (P.F. Harmon, personal communication). The isolate was incubated on autoclaved potato dextrose agar (PDA) unde

A cost-benefit analysis to select the most effective method for positional cloning: genotyping by sequencing versus allele-specific PCR

Next-generation sequencing technologies have enabled the rapid generation of high-resolution genetic maps to enable the identification of genomic regions associated with traits of interest. In certain cases these regions of interest need to be narrowed down via fine mapping to increase resolution to the level of the individual gene. In those cases, there is a choice of genotyping options, whereby cost, time, and information content need to be considered. We developed detailed, customizable models to compare the cost of genotyping by sequencing and allele-specific PCR for fine-mapping genomic regions of interest. The models were validated experimentally with data from a fine-mapping experiment designed to identify candidate genes within a 3.2-Mb disease resistance locus in the cereal crop sorghum (Sorghum bicolor (L.). Moench), which has a sequenced genome. The size of the mapping population, genetic diversity of the parental lines, choice of reagents, and labor cost were shown to influence the overall cost of the two methods, and, consequently, which method would be most cost-effective for a given experiment. The model can be easily customized to reflect experiments with different species, genetic populations, experimental and personnel costs to determine the most cost-effective procedure for a new positional cloning project.