Unveiling the evolutionary history of lingonberry (Vaccinium vitis-idaea L.) through genome sequencing and assembly of European and North American subspecies.

Lingonberry (Vaccinium vitis-idaea L.) produces tiny red berries that are tart and nutty in flavor. It grows widely in the circumpolar region, including Scandinavia, northern parts of Eurasia, Alaska, and Canada. Although cultivation is currently limited, the plant has a long history of cultural use among indigenous communities. Given its potential as a food source, genomic resources for lingonberry are significantly lacking. To advance genomic knowledge, the genomes for 2 subspecies of lingonberry (V. vitis-idaea ssp. minus and ssp. vitis-idaea var. 'Red Candy') were sequenced and de novo assembled into contig-level assemblies. The assemblies were scaffolded using the bilberry genome (Vaccinium myrtillus) to generate a chromosome-anchored reference genome consisting of 12 chromosomes each with a total length of 548.07 Mb [contig N50 = 1.17 Mb, BUSCO (C%) = 96.5%] for ssp. vitis-idaea and 518.70 Mb [contig N50 = 1.40 Mb, BUSCO (C%) = 96.9%] for ssp. minus. RNA-seq-based gene annotation identified 27,243 and 25,718 genes on the respective assembly, and transposable element detection methods found that 45.82 and 44.58% of the genome were repeats. Phylogenetic analysis confirmed that lingonberry was most closely related to bilberry and was more closely related to blueberries than cranberries. Estimates of past effective population size suggested a continuous decline over the past 1-3 MYA, possibly due to the impacts of repeated glacial cycles during the Pleistocene leading to frequent population fragmentation. The genomic resource created in this study can be used to identify industry-relevant genes (e.g. anthocyanin production), infer phylogeny, and call sequence-level variants (e.g. SNPs) in future research.

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.

Genomic-environmental associations in wild cranberry (Vaccinium macrocarpon Ait.).

Understanding the genetic basis of local adaptation in natural plant populations, particularly crop wild relatives, may be highly useful for plant breeding. By characterizing genetic variation for adaptation to potentially stressful environmental conditions, breeders can make targeted use of crop wild relatives to develop cultivars for novel or changing environments. This is especially appealing for improving long-lived woody perennial crops such as the American cranberry (Vaccinium macrocarpon Ait.), the cultivation of which is challenged by biotic and abiotic stresses. In this study, we used environmental association analyses in a collection of 111 wild cranberry accessions to identify potentially adaptive genomic regions for a range of bioclimatic and soil conditions. We detected 126 significant associations between SNP marker loci and environmental variables describing temperature, precipitation, and soil attributes. Many of these markers tagged genes with functional annotations strongly suggesting a role in adaptation to biotic or abiotic conditions. Despite relatively low genetic variation in cranberry, our results suggest that local adaptation to divergent environments is indeed present, and the identification of potentially adaptive genetic variation may enable a selective use of this germplasm for breeding more stress-tolerant cultivars.

Chromobacterium alticapitis sp. nov. and Chromobacterium sinusclupearum sp. nov. isolated from wild cranberry bogs in the Cape Cod National Seashore, USA.

Two non-pigmented strains in the genus Chromobacterium, MWU14-2602T and MWU13-2610T, were isolated from wild cranberry bogs in the Cape Cod National Seashore, USA. The isolates were characterized by genomic and phenotypic analyses, the results of which indicated that they represent two novel species. Based on total genome sequences, the closest relatives were in the Chromobacterium amazonense group, which includes the recently described Chromobacterium paludis. Whole genome sequences were compared by genome blast distance phylogeny, digital DNA-DNA hybridization and average nucleotide identity analyses with each other and with the type strains of their nearest species. MWU14-2602T and MWU13-2610T fell well below the accepted cutoff values for species relatedness, clearly indicating that they represent novel species. Although little is known about these organisms in situ, under laboratory conditions, MWU13-2610T produced a modest amount of HCN and was strongly positive for exoprotease activity, whereas MWU14-2602T did not produce HCN or exoproteases. The predominant fatty acids for both isolates were summed C16 : 1ω7cis/C16 : 1ω6cis. Both isolates produced siderophores and pyomelanin pigment on rich media, and neither was haemolytic on sheep blood agar. We propose the names Chromobacterium alticapitis sp. nov. (type strain MWU14-2602T=ATCC TSD 260T=CCOS 1979T) and Chromobacterium sinusclupearum sp. nov. (type strain MWU13-2610T=ATCC TSD-259T=CCOS 1981T) for these taxa.

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.

Selection of reference genes for normalization of cranberry (Vaccinium macrocarpon Ait.) gene expression under different experimental conditions.

Real-time fluorescent quantitative PCR (qRT-PCR) is often chosen as an effective experimental method for analyzing gene expression. However, an appropriate reference gene as a standard is needed to obtain accurate gene expression data. To date, no internal reference genes have been reported for research on cranberries. Expanding the selection of internal reference genes for cranberry will enable reliable gene expression analysis, and, at the same time, can also lay a solid foundation for revealing the biological characteristics of cranberry. Here, we selected ten candidate reference gene families and used three statistical software tools-geNorm, NormFinder and BestKeeper-to evaluate their expression stability under the influence of different experimental factors. The results showed that protein phosphatase 2A regulatory subunit (PP2A) or RNA helicase-like 8 (RH 8) was the best choice for an internal reference gene when analyzing different cranberry cultivars. In two sample sets comprising different cranberry organs and three abiotic stress treatments, sand family protein (SAND) was the best choice as a reference gene. In this study, we screened genes that are stably expressed under the influence of various experimental factors by qRT-PCR. Our results will guide future studies involving gene expression analysis of cranberry.

Pacbio Sequencing Reveals Identical Organelle Genomes between American Cranberry (Vaccinium macrocarpon Ait.) and a Wild Relative.

Breeding efforts in the American cranberry (Vaccinium macrocarpon Ait.), a North American perennial fruit crop of great importance, have been hampered by the limited genetic and phenotypic variability observed among cultivars and experimental materials. Most of the cultivars commercially used by cranberry growers today were derived from a few wild accessions bred in the 1950s. In different crops, wild germplasm has been used as an important genetic resource to incorporate novel traits and increase the phenotypic diversity of breeding materials. Vaccinium microcarpum (Turcz. ex Rupr.) Schmalh. and V. oxycoccos L., two closely related species, may be cross-compatible with the American cranberry, and could be useful to improve fruit quality such as phytochemical content. Furthermore, given their northern distribution, they could also help develop cold hardy cultivars. Although these species have previously been analyzed in diversity studies, genomic characterization and comparative studies are still lacking. In this study, we sequenced and assembled the organelle genomes of the cultivated American cranberry and its wild relative, V. microcarpum. PacBio sequencing technology allowed us to assemble both mitochondrial and plastid genomes at very high coverage and in a single circular scaffold. A comparative analysis revealed that the mitochondrial genome sequences were identical between both species and that the plastids presented only two synonymous single nucleotide polymorphisms (SNPs). Moreover, the Illumina resequencing of additional accessions of V. microcarpum and V. oxycoccos revealed high genetic variation in both species. Based on these results, we provided a hypothesis involving the extension and dynamics of the last glaciation period in North America, and how this could have shaped the distribution and dispersal of V. microcarpum. Finally, we provided important data regarding the polyploid origin of V. oxycoccos.

Genetic diversity and cultivar variants in the NCGR cranberry (Vaccinium macrocarpon Aiton) collection.

The American cranberry (Vaccinium macrocarpon) is an endemic domesticated species that has become an economically important commercial fruit crop. The USDA-ARS National Clonal Germplasm Repository (NCGR) houses the national Vaccinium collection, which includes representatives of historical cranberry cultivars and wild-selected germplasm. The objective of this study wasto examine the genotypes of 271 cranberry plants from 77 accessions representing 66 named cultivars using 12 simple-sequence repeats to assess clonal purity and cultivar relatedness. Using principal components analysis and neighbour-joining based on estimated genetic distances between individuals, we identified 64 unique genotypes and observed that intracultivar variants (i.e. subclones) existed in the germplasm collection and in the commercial bogs where some accessions originated. Finally, through a comparison of the genotypes of this study with the previous studies, pedigree analysis and the study of the geographic distribution of cranberry diversity, we identified consensus genotypes for many accessions and cultivars. We highlight the important role that the NCGR collection playsfor ex situ conservation of cranberry germplasm for future breeders and researchers. The NCGR continues to search for historically relevant cultivars absent from the collection in an effort to preserve these genotypes before they are lost and no longer commercially grown.

Massive phenotyping of multiple cranberry populations reveals novel QTLs for fruit anthocyanin content and other important chemical traits.

Because of its known phytochemical activity and benefits for human health, American cranberry (Vaccinium macrocarpon L.) production and commercialization around the world has gained importance in recent years. Flavonoid compounds as well as the balance of sugars and acids are key quality characteristics of fresh and processed cranberry products. In this study, we identified novel QTL that influence total anthocyanin content (TAcy), titratable acidity (TA), proanthocyanidin content (PAC), Brix, and mean fruit weight (MFW) in cranberry fruits. Using repeated measurements over the fruit ripening period, different QTLs were identified at specific time points that coincide with known chemical changes during fruit development and maturation. Some genetic regions appear to be regulating more than one trait. In addition, we demonstrate the utility of digital imaging as a reliable, inexpensive and high-throughput strategy for the quantification of anthocyanin content in cranberry fruits. Using this imaging approach, we identified a set of QTLs across three different breeding populations which collocated with anthocyanin QTL identified using wet-lab approaches. We demonstrate the use of a high-throughput, reliable and highly accessible imaging strategy for predicting anthocyanin content based on cranberry fruit color, which could have a large impact for both industry and cranberry research.

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.

Phenolics of Selected Cranberry Genotypes (Vaccinium macrocarpon Ait.) and Their Antioxidant Efficacy.

Free, esterified, and bound phenolic fractions of berries from five different cranberry genotypes and two market samples were evaluated for their total phenolic, flavonoid, and monomeric anthocyanin contents as well as their antioxidant efficacy using TEAC, ORAC, DPPH radical, reducing power, and ferrous ion chelation capacity assays. HPLC-MS/MS analysis was performed for two of the rich sources (Pilgrim and wild clone NL2) of phenolics and high antioxidant activity. Among the genotypes, Pilgrim showed the highest phenolic and flavonoid contents and wild clones NL3 and NL2 showed the highest monomeric anthocyanin and proanthocyanidin content, respectively. Protocatechuic and syringic acids were detected only in Pilgrim, whereas luteolin 7-O-glucoside, quercetin 3-O-rhamnoside, quercetin 3-O-galactoside, proanthocyanidin B-type, and myricetin 3-O-galactoside were found in wild clone NL3 genotype. Moreover, proanthocyanin trimer A-type and dimer B-type predominated in the wild clone NL2, whereas proanthocyanidin dimer B and trimer A were predominant in Pilgrim.

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).

Fragman: an R package for fragment analysis.

BACKGROUND: Determination of microsatellite lengths or other DNA fragment types is an important initial component of many genetic studies such as mutation detection, linkage and quantitative trait loci (QTL) mapping, genetic diversity, pedigree analysis, and detection of heterozygosity. A handful of commercial and freely available software programs exist for fragment analysis; however, most of them are platform dependent and lack high-throughput applicability. RESULTS: We present the R package Fragman to serve as a freely available and platform independent resource for automatic scoring of DNA fragment lengths diversity panels and biparental populations. The program analyzes DNA fragment lengths generated in Applied Biosystems® (ABI) either manually or automatically by providing panels or bins. The package contains additional tools for converting the allele calls to GenAlEx, JoinMap® and OneMap software formats mainly used for genetic diversity and generating linkage maps in plant and animal populations. Easy plotting functions and multiplexing friendly capabilities are some of the strengths of this R package. Fragment analysis using a unique set of cranberry (Vaccinium macrocarpon) genotypes based on microsatellite markers is used to highlight the capabilities of Fragman. CONCLUSION: Fragman is a valuable new tool for genetic analysis. The package produces equivalent results to other popular software for fragment analysis while possessing unique advantages and the possibility of automation for high-throughput experiments by exploiting the power of R.

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

BACKGROUND: Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. RESULTS: In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. CONCLUSIONS: Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

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.

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

BACKGROUND: The 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. RESULTS: The 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%). CONCLUSIONS: Shotgun 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.

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.

Impact of early season apical meristem injury by gall inducing tipworm (Diptera: Cecidomyiidae) on reproductive and vegetative growth of cranberry.

Larvae of cranberry tipworm, Dasineura oxycoccana Johnson, disrupt early season growth of cranberry (Vaccinium macrocarpon Aiton) uprights or shoots by feeding on apical meristem tissue. A 2-yr field study was carried out at three different locations to determine the impact of tipworm feeding injury on the reproductive and vegetative growth of two cranberry cultivars ('Howes' and 'Stevens') in Massachusetts. In addition to tipworm-injured and intact control uprights, an artificial injury treatment simulating tipworm feeding was also included. Individual uprights of cranberry exhibited tolerance to natural (tipworm) and simulated apical meristem injury in the current growing season (fruit production) and results were corroborated by a greenhouse study. In the field study, weight of fruit was higher in tipworm-injured uprights as compared with intact control uprights at the sites with Howes. However, majority of injured uprights (tipworm and simulated) did not produce new growth from lateral buds (side-shoots) before the onset of dormancy. In the next growing season, fewer injured uprights resumed growth and produced flowers as compared with intact uprights at two of the three sites. We suggest that multiple-year studies focusing on whole plant response to tipworm herbivory will be required to determine the costs of chronic feeding injury over time.

The first genetic map of the American cranberry: exploration of synteny conservation and quantitative trait loci.

The first genetic map of cranberry (Vaccinium macrocarpon) has been constructed, comprising 14 linkage groups totaling 879.9 cM with an estimated coverage of 82.2 %. This map, based on four mapping populations segregating for field fruit-rot resistance, contains 136 distinct loci. Mapped markers include blueberry-derived simple sequence repeat (SSR) and cranberry-derived sequence-characterized amplified region markers previously used for fingerprinting cranberry cultivars. In addition, SSR markers were developed near cranberry sequences resembling genes involved in flavonoid biosynthesis or defense against necrotrophic pathogens, or conserved orthologous set (COS) sequences. The cranberry SSRs were developed from next-generation cranberry genomic sequence assemblies; thus, the positions of these SSRs on the genomic map provide information about the genomic location of the sequence scaffold from which they were derived. The use of SSR markers near COS and other functional sequences, plus 33 SSR markers from blueberry, facilitates comparisons of this map with maps of other plant species. Regions of the cranberry map were identified that showed conservation of synteny with Vitis vinifera and Arabidopsis thaliana. Positioned on this map are quantitative trait loci (QTL) for field fruit-rot resistance (FFRR), fruit weight, titratable acidity, and sound fruit yield (SFY). The SFY QTL is adjacent to one of the fruit weight QTL and may reflect pleiotropy. Two of the FFRR QTL are in regions of conserved synteny with grape and span defense gene markers, and the third FFRR QTL spans a flavonoid biosynthetic gene.

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.).

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar 'HyRed'. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540-590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2-15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.