Quantitative Trait Loci Analysis and Marker Development for Fruit Rot Resistance in Cranberry Shows Potential Genetic Association with Epicuticular Wax.

Fruit rot is a fungal disease complex that threatens cranberry yields in North American growing operations. Management of fruit rot is especially difficult because of the diversity of the infecting fungal species, and although infections take place early in the season, the pathogens usually remain latent in the ovary until the fruit ripen. Control methods heavily rely on fungicide applications, a practice that may be limited in viability long term. Breeding for fruit rot resistance (FRR) is essential for sustainable production. It is likely that field resistance is multifaceted and involves a myriad of traits that fortify cranberry plants against the biotic and abiotic stresses contributing to fruit rot. In this study, we identified quantitative trait loci (QTL) for FRR in a segregating population. Interestingly, a QTL associated with resistance was found to overlap with one associated with fruit epicuticular wax (ECW). A single-nucleotide polymorphism genotyping assay successfully identified accessions that exhibit the desired phenotypes (i.e., less rot and more ECW), thus making it a useful tool for marker-assisted selection. Candidate genes that may contribute to FRR and ECW were also identified. This work will expedite breeding for improved cranberry fruit quality.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures), as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium-a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Blueberry and cranberry pangenomes as a resource for future genetic studies and breeding efforts.

Domestication of cranberry and blueberry began in the United States in the early 1800s and 1900s, respectively, and in part owing to their flavors and health-promoting benefits are now cultivated and consumed worldwide. The industry continues to face a wide variety of production challenges (e.g. disease pressures) as well as a demand for higher-yielding cultivars with improved fruit quality characteristics. Unfortunately, molecular tools to help guide breeding efforts for these species have been relatively limited compared with those for other high-value crops. Here, we describe the construction and analysis of the first pangenome for both blueberry and cranberry. Our analysis of these pangenomes revealed both crops exhibit great genetic diversity, including the presence-absence variation of 48.4% genes in highbush blueberry and 47.0% genes in cranberry. Auxiliary genes, those not shared by all cultivars, are significantly enriched with molecular functions associated with disease resistance and the biosynthesis of specialized metabolites, including compounds previously associated with improving fruit quality traits. The discovery of thousands of genes, not present in the previous reference genomes for blueberry and cranberry, will serve as the basis of future research and as potential targets for future breeding efforts. The pangenome, as a multiple-sequence alignment, as well as individual annotated genomes, are publicly available for analysis on the Genome Database for Vaccinium - a curated and integrated web-based relational database. Lastly, the core-gene predictions from the pangenomes will serve useful to develop a community genotyping platform to guide future molecular breeding efforts across the family.

Assessment and comparison of rhizosphere communities in cultivated Vaccinium spp. provide a baseline for study of causative agents in decline.

It has long been recognized that the community of organisms associated with plant roots is a critical component of the phytobiome and can directly or indirectly contribute to the overall health of the plant. The rhizosphere microbial community is influenced by a number of factors including the soil type, the species of plants growing in those soils, and in the case of cultivated plants, the management practices associated with crop production. Vaccinium species, such as highbush blueberry and American cranberry, are woody perennials that grow in sandy, acidic soils with low to moderate levels of organic matter and a paucity of nutrients. When properly maintained, fields planted with these crops remain productive for many years. In some cases, however, yields and fruit quality decline over time, and it is suspected that degenerating soil health and/or changes in the rhizosphere microbiome are contributing factors. Determining the assemblage of bacterial and fungal microorganisms typically associated with the rhizosphere of these crops is a critical first step toward addressing the complex issue of soil health. We hypothesized that since blueberry and cranberry are in the same genus and grow in similar soils, that their associated rhizosphere microbial communities would be similar to each other. We analyzed the eukaryotic (primarily fungal) and bacterial communities from the rhizosphere of representative blueberry and cranberry plants growing in commercial fields in New Jersey. The data presented herein show that while the bacterial communities between the crops is very similar, the fungal communities associated with each crop are quite different. These results provide a framework for examining microbial components that might contribute to the health of Vaccinium spp. crops in New Jersey and other parts of the northeastern U.S.

Cranberry fruit epicuticular wax benefits and identification of a wax-associated molecular marker.

BACKGROUND: As the global climate changes, periods of abiotic stress throughout the North American cranberry growing regions will become more common. One consequence of high temperature extremes and drought conditions is sunscald. Scalding damages the developing berry and reduces yields through fruit tissue damage and/or secondary pathogen infection. Irrigation runs to cool the fruit is the primary approach to controlling sunscald. However, it is water intensive and can increase fungal-incited fruit rot. Epicuticular wax functions as a barrier to various environmental stresses in other fruit crops and may be a promising feature to mitigate sunscald in cranberry. In this study we assessed the function of epicuticular wax in cranberries to attenuate stresses associated with sunscald by subjecting high and low epicuticular wax cranberries to controlled desiccation and light/heat exposure. A cranberry population that segregates for epicuticular wax was phenotyped for epicuticular fruit wax levels and genotyped using GBS. Quantitative trait loci (QTL) analyses of these data identified a locus associated with epicuticular wax phenotype. A SNP marker was developed in the QTL region to be used for marker assisted selection. RESULTS: Cranberries with high epicuticular wax lost less mass percent and maintained a lower surface temperature following heat/light and desiccation experiments as compared to fruit with low wax. QTL analysis identified a marker on chromosome 1 at position 38,782,094 bp associated with the epicuticular wax phenotype. Genotyping assays revealed that cranberry selections homozygous for a selected SNP have consistently high epicuticular wax scores. A candidate gene (GL1-9), associated with epicuticular wax synthesis, was also identified near this QTL region. CONCLUSIONS: Our results suggest that high cranberry epicuticular wax load may help reduce the effects of heat/light and water stress: two primary contributors to sunscald. Further, the molecular marker identified in this study can be used in marker assisted selection to screen cranberry seedlings for the potential to have high fruit epicuticular wax. This work serves to advance the genetic improvement of cranberry crops in the face of global climate change.

Molecular and behavioral studies reveal differences in olfaction between winter and summer morphs of Drosophila suzukii.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is a major economic pest of several fruit crops in Europe, North and South America, and other parts of the world because it oviposits in ripening thin-skinned fruits. This vinegar fly exhibits two distinct morphotypes: a summer and a winter morph. Although adaptations associated with the winter morph enhance this invasive pest's capacity to survive in cold climates, winter is still a natural population bottleneck. Since monitoring early spring populations is important for accurate population forecasts, understanding the winter morph's response to olfactory cues may improve current D. suzukii management programs. In this study, a comparative transcriptome analysis was conducted to assess gene expression differences between the female heads of the two D. suzukii morphs, which showed significant differences in 738 genes (p ≤ 0.0001). Out of twelve genes related to olfaction determined to be differentially expressed in the transcriptome, i.e., those related to location of food sources, chemosensory abilities, and mating behavior, nine genes were upregulated in the winter morph while three were downregulated. Three candidate olfactory-related genes that were most upregulated or downregulated in the winter morph were further validated using RT-qPCR. In addition, behavioral assays were performed at a range of temperatures to confirm a differing behavioral response of the two morphs to food odors. Our behavioral assays showed that, although winter morphs were more active at lower temperatures, the summer morphs were generally more attracted to food odors. This study provides new insights into the molecular and behavioral differences in response to olfactory cues between the two D. suzukii morphs that will assist in formulating more effective monitoring and physiological-based control tools.

There and back again; historical perspective and future directions for Vaccinium breeding and research studies.

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops' relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.

Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding.

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3 s per read on a Mac Mini with strain-level resolution for sequences with >84% identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95 ± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

Application of Plant Defense Elicitors Fails to Enhance Herbivore Resistance or Mitigate Phytoplasma Infection in Cranberries.

Synthetic elicitors of the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways can be used to increase crop protection against herbivores and pathogens. In this study, we tested the hypothesis that elicitors of plant defenses interact with pathogen infection to influence crop resistance against vector and nonvector herbivores. To do so, we employed a trophic system comprising of cranberries (Vaccinium macrocarpon), the phytoplasma that causes false blossom disease, and two herbivores-the blunt-nosed leafhopper (Limotettix vaccinii), the vector of false blossom disease, and the nonvector gypsy moth (Lymantria dispar). We tested four commercial elicitors, including three that activate mainly SA-related plant defenses (Actigard, LifeGard, and Regalia) and one activator of JA-related defenses (Blush). A greenhouse experiment in which phytoplasma-infected and uninfected plants received repeated exposure to elicitors revealed that both phytoplasma infection and elicitor treatment individually improved L. vaccinii and L. dispar mass compared to uninfected, untreated controls; however, SA-based elicitor treatments reduced L. vaccinii mass on infected plants. Regalia also improved L. vaccinii survival. Phytoplasma infection reduced plant size and mass, increased levels of nitrogen (N) and SA, and lowered carbon/nitrogen (C/N) ratios compared to uninfected plants, irrespective of elicitor treatment. Although none of our elicitor treatments influenced transcript levels of a phytoplasma-specific marker gene, all of them increased N and reduced C/N levels; the three SA activators also reduced JA levels. Taken together, our findings reveal positive effects of both phytoplasma infection and elicitor treatment on the performance of L. vaccinii and L. dispar in cranberries, likely via enhancement of plant nutrition and changes in phytohormone profiles, specifically increases in SA levels and corresponding decreases in levels of JA. Thus, we found no evidence that the tested elicitors of plant defenses increase resistance to insect herbivores or reduce disease incidence in cranberries.

Molecular and ecological plant defense responses along an elevational gradient in a boreal ecosystem.

Plants have the capacity to alter their phenotype in response to environmental factors, such as herbivory, a phenomenon called phenotypic plasticity. However, little is known on how plant responses to herbivory are modulated by environmental variation along ecological gradients. To investigate this question, we used bilberry (Vaccinium myrtillus L.) plants and an experimental treatment to induce plant defenses (i.e., application of methyl jasmonate; MeJA), to observe ecological responses and gene expression changes along an elevational gradient in a boreal system in western Norway. The gradient included optimal growing conditions for bilberry in this region (ca. 500 m a.s.l.), and the plant's range limits at high (ca. 900 m a.s.l.) and low (100 m a.s.l.) elevations. Across all altitudinal sites, MeJA-treated plants allocated more resources to herbivory resistance while reducing growth and reproduction than control plants, but this response was more pronounced at the lowest elevation. High-elevation plants growing under less herbivory pressure but more resource-limiting conditions exhibited consistently high expression levels of defense genes in both MeJA-treated and untreated plants at all times, suggesting a constant state of "alert." These results suggest that plant defense responses at both the molecular and ecological levels are modulated by the combination of climate and herbivory pressure, such that plants under different environmental conditions differentially direct the resources available to specific antiherbivore strategies. Our findings are important for understanding the complex impact of future climate changes on plant-herbivore interactions, as this is a major driver of ecosystem functioning and biodiversity.

Transcriptome analysis identifies genes related to the waxy coating on blueberry fruit in two northern-adapted rabbiteye breeding populations.

BACKGROUND: Blueberry is of high economic value. Most blueberry varieties selected for the fresh market have an appealing light blue coating or "bloom" on the fruit due to the presence of a visible heavy epicuticular wax layer. This waxy layer also serves as natural defense against fruit desiccation and deterioration. RESULTS: In this study, we attempted to identify gene(s) whose expression is related to the protective waxy coating on blueberry fruit utilizing two unique germplasm populations that segregate for the waxy layer. We bulked RNA from waxy and non-waxy blueberry progenies from the two northern-adapted rabbiteye hybrid breeding populations ('Nocturne' x T 300 and 'Nocturne' x US 1212), and generated 316.85 million RNA-seq reads. We de novo assembled this data set integrated with other publicly available RNA-seq data and trimmed the assembly into a 91,861 blueberry unigene collection. All unigenes were functionally annotated, resulting in 79 genes potentially related to wax accumulation. We compared the expression pattern of waxy and non-waxy progenies using edgeR and identified overall 1125 genes in the T 300 population and 2864 genes in the US 1212 population with at least a two-fold expression difference. After validating differential expression of several genes by RT-qPCR experiments, a candidate gene, FatB, which encodes acyl-[acyl-carrier-protein] hydrolase, emerged whose expression was closely linked to the segregation of the waxy coating in our populations. This gene was expressed at more than a five-fold higher level in waxy than non-waxy plants of both populations. We amplified and sequenced the cDNA for this gene from three waxy plants of each population, but were unable to amplify the cDNA from three non-waxy plants that were tested from each population. We aligned the Vaccinium deduced FATB protein sequence to FATB protein sequences from other plant species. Within the PF01643 domain, which gives FATB its catalytic function, 80.08% of the amino acids were identical or had conservative replacements between the blueberry and the Cucumis melo sequence (XP_008467164). We then amplified and sequenced a large portion of the FatB gene itself from waxy and non-waxy individuals of both populations. Alignment of the cDNA and gDNA sequences revealed that the blueberry FatB gene consists of six exons and five introns. Although we did not sequence through two very large introns, a comparison of the exon sequences found no significant sequence differences between the waxy and non-waxy plants. This suggests that another gene, which regulates or somehow affects FatB expression, must be segregating in the populations. CONCLUSIONS: This study is helping to achieve a greater understanding of epicuticular wax biosynthesis in blueberry. In addition, the blueberry unigene collection should facilitate functional annotation of the coming chromosomal level blueberry genome.

Transcriptional profiling of methyl jasmonate-induced defense responses in bilberry (Vaccinium myrtillus L.).

BACKGROUND: Bilberry (Vaccinium myrtillus L.) is one of the most abundant wild berries in the Northern European ecosystems. This species plays an important ecological role as a food source for many vertebrate and invertebrate herbivores. It is also well-recognized for its bioactive compounds, particularly substances involved in natural defenses against herbivory. These defenses are known to be initiated by leaf damage (e.g. chewing by insects) and mediated by activation of the jasmonic acid (JA) signaling pathway. This pathway can be activated by exogenous application of methyl jasmonate (MeJA), the volatile derivative of JA, which is often used to stimulate plant defense responses in studies of plant-herbivore interactions at ecological, biochemical, and molecular organismal levels. As a proxy for herbivore damage, wild V. myrtillus plants were treated in the field with MeJA and changes in gene expression were compared to untreated plants. RESULTS: The de novo transcriptome assembly consisted of 231,887 unigenes. Nearly 71% of the unigenes were annotated in at least one of the databases interrogated. Differentially expressed genes (DEGs), between MeJA-treated and untreated control bilberry plants were identified using DESeq. A total of 3590 DEGs were identified between the treated and control plants, with 2013 DEGs upregulated and 1577 downregulated. The majority of the DEGs identified were associated with primary and secondary metabolism pathways in plants. DEGs associated with growth (e.g. those encoding photosynthesis-related components) and reproduction (e.g. flowering control genes) were frequently down-regulated while those associated with defense (e.g. encoding enzymes involved in biosynthesis of flavonoids, lignin compounds, and deterrent/repellent volatile organic compounds) were up-regulated in the MeJA treated plants. CONCLUSIONS: Ecological studies are often limited by controlled conditions to reduce the impact of environmental effects. The results from this study support the hypothesis that bilberry plants, growing in natural conditions, shift resources from growth and reproduction to defenses while in a MeJA-induced state, as when under insect attack. This study highlights the occurrence of this trade-off at the transcriptional level in a realistic field scenario and supports published field observations wherein plant growth is retarded and defenses are upregulated.

Genotypic Variation and Phenotypic Plasticity in Gene Expression and Emissions of Herbivore-Induced Volatiles, and their Potential Tritrophic Implications, in Cranberries.

Herbivorous insects are important problems in cranberry (Vaccinium macrocarpon Ait.) production. The use of chemical pesticides is common practice, but beneficial insects such as natural enemies of herbivores (e.g. predators and parasitoids) could be affected as well. Therefore, we studied the defensive mechanisms that cranberry plants use to combat pests, focusing on herbivore-induced plant volatiles (HIPVs), which can be used to recruit predators and parasitoids foraging for prey or hosts. Then, we used synthetic HIPVs to test the attraction of natural enemies. In a greenhouse, we assessed nine cranberry genotypes for expression of genes involved in HIPV biosynthesis and/or emission of HIPVs. In an experimental field, we assessed whether baiting traps with individual or combinations of HIPVs increased attractiveness to natural enemies. The results showed that different cranberry genotypes vary in their emission of monoterpenes and sesquiterpenes but not in their expression of two genes associated with terpene biosynthesis, α-humulene/ß-caryophyllene synthase and (3S,6E)-nerolidol/R-linalool synthase. Induction with methyl jasmonate or herbivore (gypsy moth, Lymantria dispar L.) feeding increased the expression of these genes and emission of HIPVs. The HIPV methyl salicylate (MeSA), alone or in combination with other HIPVs, increased syrphid attraction by 6-fold in the field, while (Z)-3-hexenyl acetate and MeSA repelled ladybeetles and megaspilids, respectively. Linalool and ß-caryophyllene elicited no behavioral responses of natural enemies. Elucidating the mechanisms of pest resistance, as well as experimentally augmenting plant defenses such as HIPVs, may contribute to the development of more sustainable pest management practices in crops, including cranberries.

Multivariate GBLUP Improves Accuracy of Genomic Selection for Yield and Fruit Weight in Biparental Populations of Vaccinium macrocarpon Ait.

The development of high-throughput genotyping has made genome-wide association (GWAS) and genomic selection (GS) applications possible for both model and non-model species. The exploitation of genome-assisted approaches could greatly benefit breeding efforts in American cranberry (Vaccinium macrocarpon) and other minor crops. Using biparental populations with different degrees of relatedness, we evaluated multiple GS methods for total yield (TY) and mean fruit weight (MFW). Specifically, we compared predictive ability (PA) differences between univariate and multivariate genomic best linear unbiased predictors (GBLUP and MGBLUP, respectively). We found that MGBLUP provided higher predictive ability (PA) than GBLUP, in scenarios with medium genetic correlation (8-17% increase with corg~0.6) and high genetic correlations (25-156% with corg~0.9), but found no increase when genetic correlation was low. In addition, we found that only a few hundred single nucleotide polymorphism (SNP) markers are needed to reach a plateau in PA for both traits in the biparental populations studied (in full linkage disequilibrium). We observed that higher resemblance among individuals in the training (TP) and validation (VP) populations provided greater PA. Although multivariate GS methods are available, genetic correlations and other factors need to be carefully considered when applying these methods for genetic improvement.

Construction of a High-Density American Cranberry (Vaccinium macrocarpon Ait.) Composite Map Using Genotyping-by-Sequencing for Multi-pedigree Linkage Mapping.

The American cranberry (Vaccinium macrocarpon Ait.) is a recently domesticated, economically important, fruit crop with limited molecular resources. New genetic resources could accelerate genetic gain in cranberry through characterization of its genomic structure and by enabling molecular-assisted breeding strategies. To increase the availability of cranberry genomic resources, genotyping-by-sequencing (GBS) was used to discover and genotype thousands of single nucleotide polymorphisms (SNPs) within three interrelated cranberry full-sib populations. Additional simple sequence repeat (SSR) loci were added to the SNP datasets and used to construct bin maps for the parents of the populations, which were then merged to create the first high-density cranberry composite map containing 6073 markers (5437 SNPs and 636 SSRs) on 12 linkage groups (LGs) spanning 1124 cM. Interestingly, higher rates of recombination were observed in maternal than paternal gametes. The large number of markers in common (mean of 57.3) and the high degree of observed collinearity (mean Pair-wise Spearman rank correlations >0.99) between the LGs of the parental maps demonstrates the utility of GBS in cranberry for identifying polymorphic SNP loci that are transferable between pedigrees and populations in future trait-association studies. Furthermore, the high-density of markers anchored within the component maps allowed identification of segregation distortion regions, placement of centromeres on each of the 12 LGs, and anchoring of genomic scaffolds. Collectively, the results represent an important contribution to the current understanding of cranberry genomic structure and to the availability of molecular tools for future genetic research and breeding efforts in cranberry.

Exploiting genotyping by sequencing to characterize the genomic structure of the American cranberry through high-density linkage mapping.

BACKGROUND: The application of genotyping by sequencing (GBS) approaches, combined with data imputation methodologies, is narrowing the genetic knowledge gap between major and understudied, minor crops. GBS is an excellent tool to characterize the genomic structure of recently domesticated (~200 years) and understudied species, such as cranberry (Vaccinium macrocarpon Ait.), by generating large numbers of markers for genomic studies such as genetic mapping. RESULTS: We identified 10842 potentially mappable single nucleotide polymorphisms (SNPs) in a cranberry pseudo-testcross population wherein 5477 SNPs and 211 short sequence repeats (SSRs) were used to construct a high density linkage map in cranberry of which a total of 4849 markers were mapped. Recombination frequency, linkage disequilibrium (LD), and segregation distortion at the genomic level in the parental and integrated linkage maps were characterized for first time in cranberry. SSR markers, used as the backbone in the map, revealed high collinearity with previously published linkage maps. The 4849 point map consisted of twelve linkage groups spanning 1112 cM, which anchored 2381 nuclear scaffolds accounting for ~13 Mb of the estimated 470 Mb cranberry genome. Bin mapping identified 592 and 672 unique bins in the parentals and a total of 1676 unique marker positions in the integrated map. Synteny analyses comparing the order of anchored cranberry scaffolds to their homologous positions in kiwifruit, grape, and coffee genomes provided initial evidence of homology between cranberry and closely related species. CONCLUSIONS: GBS data was used to rapidly saturate the cranberry genome with markers in a pseudo-testcross population. Collinearity between the present saturated genetic map and previous cranberry SSR maps suggests that the SNP locations represent accurate marker order and chromosome structure of the cranberry genome. SNPs greatly improved current marker genome coverage, which allowed for genome-wide structure investigations such as segregation distortion, recombination, linkage disequilibrium, and synteny analyses. In the future, GBS can be used to accelerate cranberry molecular breeding through QTL mapping and genome-wide association studies (GWAS).

Interactions between cranberries and fungi: the proposed function of organic acids in virulence suppression of fruit rot fungi.

Cranberry fruit are a rich source of bioactive compounds that may function as constitutive or inducible barriers against rot-inducing fungi. The content and composition of these compounds change as the season progresses. Several necrotrophic fungi cause cranberry fruit rot disease complex. These fungi remain mostly asymptomatic until the fruit begins to mature in late August. Temporal fluctuations and quantitative differences in selected organic acid profiles between fruit of six cranberry genotypes during the growing season were observed. The concentration of benzoic acid in fruit increased while quinic acid decreased throughout fruit development. In general, more rot-resistant genotypes (RR) showed higher levels of benzoic acid early in fruit development and more gradual decline in quinic acid levels than that observed in the more rot-susceptible genotypes. We evaluated antifungal activities of selected cranberry constituents and found that most bioactive compounds either had no effects or stimulated growth or reactive oxygen species (ROS) secretion of four tested cranberry fruit rot fungi, while benzoic acid and quinic acid reduced growth and suppressed secretion of ROS by these fungi. We propose that variation in the levels of ROS suppressive compounds, such as benzoic and quinic acids, may influence virulence by the fruit rot fungi. Selection for crops that maintain high levels of virulence suppressive compounds could yield new disease resistant varieties. This could represent a new strategy for control of disease caused by necrotrophic pathogens that exhibit a latent or endophytic phase.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

Development and validation of 697 novel polymorphic genomic and EST-SSR markers in the American cranberry (Vaccinium macrocarpon Ait.).

The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the p < 0.05 level, and contained moderate to high levels of polymorphism with a polymorphic information content >0.25. This comprehensive collection of developed and validated microsatellite loci represents a substantial addition to the molecular tools available for geneticists, genomicists, and breeders in cranberry and Vaccinium.