BACKGROUND
the american cranberry  is native to north america and is a member of the ericaceae .
cranberry fruit was collected from the wild by american indians and used for a variety of purposes including as a preservative of fish and meat and medicinally as a poultice for dressing wounds  <cit> .
the crop was first domesticated in the early 1800s on cape cod, massachusetts.
today, cranberries are grown primarily in wisconsin, massachusetts, new jersey, in the pacific northwest in oregon and washington, and in five provinces in canada.
outside of north america, v. macrocarpon is cultivated in parts of europe and chile.
in  <dig>  us cranberry production was  <dig> million pounds, valued at over $ <dig> million .
the american cranberry and other species of vaccinium, such as highbush blueberry  are known for their phytochemicals that can benefit human health  <cit> .
cranberry, in particular, is high in polyphenolic antioxidants  <cit> , helps prevent urinary tract infections  <cit> , has some anticancer properties  <cit> , and may help prevent oral caries  <cit>  among other health benefits.
v. macrocarpon is taxonomically placed in the core eudicots, a diverse group of angiosperms.
it is a member of the asterid clade, which contains the orders cornales and ericales  <cit> .
the order ericales is quite diverse and includes  <dig> families.
key economically important plants in the ericales include tea  and edible fruits such as kiwi  and persimmon.
the ericaceae is an important family in the ericales.
members of this family tend to thrive in nutrient-poor acid soils.
the ericaceae includes  <dig> genera and about  <dig> species  <cit> .
despite the importance of ericaceous plants such as blueberry, huckleberry, bilberry, cranberry and rhododendron, whole-genome sequence data for this group are currently lacking.
cranberry is diploid  and self-fertile, allowing generation of inbred lines.
cranberry can hybridize with, and give rise to fertile offspring, when crossed to related species such as v. oxycoccus l.  <cit> , producing unique populations segregating for a host of horticultural and biochemical characteristics.
a major characteristic important for sustainability is disease resistance.
cultivated cranberry is susceptible to a variety of fungal pathogens  <cit> .
in fact, fruit rot is the most significant problem in cranberry production in the northeastern u.s. where, in the absence of an appropriate fungicide regime, the entire crop can be lost  <cit> .
disease resistance in plants is complex, involving many different pathways and mechanisms.
whereas some of the resistance genes identified in various plant species impart resistance to specific pathogens, others have been implicated in broad-spectrum resistance  <cit> .
identification of putative resistance genes and their mapping for marker-assisted breeding  would be facilitated by whole-genome sequence analysis.
whole-genome sequence data can be used for many other purposes such as identification of key biosynthetic pathway genes, genotyping, structure-function studies, and evolutionary studies.
the american cranberry has several characteristics including compact size, a relatively short generation interval , ease of asexual propagation , diploidy, self-fertility, and moderate genome size, that make it suitable for use as a model system, specifically for temperate woody perennial plants species.
we present here the results of gene discovery utilizing whole-genome and transcriptome sequence data from an inbred line of american cranberry.
the data were analyzed to validate the taxonomic position of cranberry, to identify synteny with other representative sequenced plant species, to predict the majority of the encoded genes, and to identify genes potentially associated with disease resistance.
RESULTS
genome and transcriptome sequencing and assembly
we sequenced the genome of v. macrocarpon using the illumina gaiix sequencer.
a total of  <dig> million paired-end reads  were generated, corresponding to  <dig>  gbp of genomic data.
the assembly resulted in  <dig>  contigs .
scaffolding slightly reduced the number of contigs to  <dig>  and raised the n <dig> to  <dig>  bp.
the assembled size was  <dig> mbp, with an average sequencing coverage of 20x, comparable to that reported for vitis vinifera .
the cranberry genome size was estimated to be about  <dig> mbp  <cit> .
we believe that this genome size is reasonably accurate  because 93% of the individual sequence reads mapped to the assembly .
this suggests that the assembly includes the vast majority of the sequenced data.
furthermore, repeated regions of the assembly  did not show extreme coverage values  suggesting that we are not underestimating genome size due to the co-assembly of distinct repeats into single contigs.
evaluation of the completeness of the v. macrocarpon draft genome assembly was done using cegma  <cit>  and showed that  <dig>  out of the  <dig> core eukaryotic genes  were present in the assembly.
further analysis of the data using the  <dig> highly conserved cegs showed that  <dig>  were present in the v. macrocarpon assembly with  <dig>  being complete, and the remaining  <dig> being partial.
a total of  <dig>  million reads of mrna-seq  data were generated on the illumina gaiix sequencer and assembled into  <dig>  cdnas longer than  <dig> bp and with average coverage >5x.
the cdna set was aligned to the genome assembly resulting in  <dig>  cdnas  mapping with 98% identity.
these assemblies and the mrna-seq reads were used to guide the gene prediction program.
the genome and transcriptome data have been deposited in ncbi as bioproject  and bioproject  respectively.
transposable elements
genome-wide sequence analysis has demonstrated that transposable elements  of different types are widespread in eukaryotes.
in flowering plants, transposons can account for large percentages of the total genome.
even in the relatively small  arabidopsis genome, transposons comprise about  <dig> % percent of the genome.
the larger  maize genome contains greater than 70% of the genome as transposons.
it is becoming clear as more plant genomes are analyzed that transposons contribute to the size and diversity of plant genomes.
although we found representatives of most classes of known transposons, the class  <dig> retrotransposons of the ltr  type are the most common, as has been found in other plants .
surprisingly though, despite the fact that the cranberry genome  is about  <dig> times that of arabidopsis, the percentage of transposons in cranberry was found to be only about  <dig> %.
it is unclear why the percentage in cranberry might be so low.
of this  <dig> %, it would be instructive to determine the percentage that might be active.
a lack of active transposition would limit expansion of the elements in the genome.
in maize, active transposition sometimes gives rise to obvious phenotypic changes.
some of these, such as color change in the aleurone layer of kernels, led to their discovery  <cit> .
these types of changes, such as variegated leaves or color variation in the fruit epidermis, have not been observed in cranberry in cultivars that have been domesticated for over  <dig> years, e.g., ‘early black’ and ‘ben lear’, as is common in apple  <cit> .
as such, cranberry lacks the phenotypic evidence of active transposition.
however, although the class ii te represented only about 1% of the genome , similar to apple and cucumber, the hobo-activator represented  <dig> % of the cranberry genome with a copy number  <dig> .
class ii tes are well recognized as promoting chromosomal rearrangements, including reciprocal translocations  <cit> .
reciprocal translocation heterozygotes have been identified in cranberry  <cit>  suggesting class ii tes may be or have been active in cranberry.
arabidopsis
the percent of the genome for each type are shown relative to other selected plant species.
cranberry is reported to have gone through a severe genetic bottleneck, possibly during the pleistocene  <cit> .
as a result, wild populations were found to lack phenotypic variability and have relatively low genetic heterogeneity  <cit> .
cranberry is self-fertile and the proposed genetic bottleneck would result in at least some level of inbreeding.
in our studies, a 5th generation inbred line of cranberry was selected for sequencing and it is not known how this level of inbreeding might affect the transposon complement in the cranberry genome.
in maize, where inbred lines are used routinely for breeding, high levels of transposons are maintained.
gene prediction
annotation of the v. macrocarpon genome assembly was done using augustus  <cit>  which was trained to be v. macrocarpon specific .
a set of  <dig>  genes was predicted, out of which  <dig>  genes had alignments larger than 30% to a te protein database  <cit> ; these genes were filtered out.
the remaining  <dig>  genes were used as query for a blastp  search against the ncbi nr database.
combining the genes that had a blast hit  with those that had not and were larger than  <dig> amino acids in length  resulted in a set of  <dig>  genes that was used for further downstream analysis.
the average coding sequence size was calculated to be  <dig> bp and the mean number of exons per gene was  <dig> ; the gene density was calculated to be  <dig>  genes per  <dig> kbp.
comparison of v. macrocarpon mrna-seq data to the set of  <dig>  gene models showed that 36%  of the genes had transcriptome evidence supporting the exons.
to determine whether some of the gene models in v. macrocarpon may be artifacts associated with predicted regions in repeats, we re-ran the gene prediction using an assembly that had been masked for de novo repeats .
this procedure resulted in the prediction of  <dig>  gene models.
to test whether the prediction made without masking repeats was significantly different, we compared the two sets of gene predictions  to each other.
using blastp with a cut-off < 1e- <dig> for this comparison, 97% of the new models were found to be present in the more extensive set of predictions.
given this evidence for the accuracy of the gene prediction with respect to potential biases introduced by repeats, we used the larger set of gene models for downstream analyses.
taxonomic assessment
to verify the taxonomic placement of cranberry, two chloroplast- and one mitochondrial-encoded protein were concatenated and aligned with those of  <dig> other plant species including four asterids ,  <dig> rosids , one basal eudicot , and one monocot .
the complete chloroplast genome of cranberry has been published  <cit> .
a maximum likelihood  tree was built and the results of  <dig> bootstrap replicates were used to determine branch support in an unrooted phylogeny .
most branches in the tree were well supported and of the species compared, cranberry was clearly located in the asterid group.
more specifically, cranberry is in the ericaceae  in the section oxycoccus.
within this section are at least two species, vaccinium macrocarpon ait.
and v. oxycoccus l.   <cit> .
although v. oxycoccus occurs at the diploid, tetraploid and hexaploid levels, and older literature often splits some of these into different species, the section oxycoccus is fairly well defined.
in contrast, section cyanococcus contains many blueberry species, the boundaries of which are less clearly defined  <cit> .
traditional methods coupled with molecular data such as the chloroplast rbcl and matk genes and nuclear sequences such as its, have been used to study phylogenetic relationships within the blueberry tribe   <cit>  and for wider placement in the ericaceae  <cit> .
the use of the matr mitochondrial gene has been proposed for phylogenetic studies in rosids, but not in the order ericales  <cit> .
as the taxonomy of higher plants continues to advance, it is likely that more molecular data will be needed and utilized.
thus, the genomic data presented here can be mined for various gene sequences or molecular markers that can be used for this purpose.
conserved orthologous set  markers
single or low copy nuclear dna markers are useful for phylogenetic studies and comparative genomic analyses.
a cos is defined as a gene conserved in sequence and copy number that shares a common ancestor by descent, while paralogs are duplicated sequences resulting in gene duplications or polyploidization events  <cit> .
cos markers have been successfully developed and used for phylogenetic inference among species in the solanaceae and poaceae families  <cit> .
the determination of cos markers in vaccinium macrocarpon is of importance to resolve the phylogenetic relationship of the cranberry and wild relative species in the ericaceae family.
the comparison between arabidopsis and sunflower transcriptomes yielded  <dig> single copy orthologous genes, while the comparison against arabidopsis and lettuce detected  <dig> genes.
there were  <dig> putative cosii markers in common between the two datasets, of which  <dig> had known function .
microsatellite detection
a total of  <dig>  perfect ssrs were detected in the sequenced and assembled genome.
a set of  <dig>  and  <dig>  ssrs corresponded to  <dig>  assembled genome scaffolds and  <dig>  unigene sequences, respectively.
the most frequent motif length repeated in the genome scaffolds corresponded to di-nucleotides  .
the maximum repeat length was  <dig> and the average length of the ssrs was  <dig> nucleotides.
the nuclear scaffolds showed di- and tetra-nucleotides to be the most abundant motifs representing  <dig> and 21% of the ssrs, respectively.
the ga and aaat motifs were the most abundant corresponding to  <dig>  and  <dig> % of the total detected ssrs, respectively.
for the microsatellites located in the unigene sequences, tri- and di-nucleotides were the most abundant motifs accounting for 37% and 35%, respectively, and the longest ssr was  <dig> nucleotides.
the most frequent di-, tri-, and tetra-nucleotide motifs were ga, aag and aaat accounting for  <dig>   <dig>  and  <dig> %, respectively, of the transcriptome ssrs.
this is the first whole genome microsatellite search in cranberry and provides the largest data set available until now of easy-to-use markers for molecular-based studies in cranberry and other vaccinium crops.
snp distribution
we used a 5th generation inbred accession  of cranberry to lower heterozygosity and facilitate assembly.
assuming ‘ben lear’ has a coefficient of inbreeding  =  <dig>  the estimated heterozygosity of the clone sequenced would be  <dig> %.
however, ‘ben lear’ was reported to have  <dig> of  <dig> ssr loci being homozygous, the highest among cultivars analyzed  <cit> , suggesting f >  <dig>  the bivariate snp distribution showed a total of  <dig>  snps that occur at a minimum frequency of 20% across reads that provide >10x genome coverage.
the total number of snps detected represents a level of heterozygosity of about  <dig> %, in our  <dig> mbp assembly.
to determine the ‘starting’ level of homozygosity, we compared these results with those from the ‘ben lear’ parent.
using the cranberry transcriptome as reference to search for common snps, comparisons between ‘ben lear’  and cnj99-125- <dig> yielded  <dig>  versus  <dig>  snps, respectively .
cnj99-125- <dig> possessed fewer multi-nucleotide variants  and single-nucleotide variants , than ‘ben lear’ .
additionally, cnj99-125- <dig> showed a reduction in indels  when compared with ‘ben lear’  .
overall, cnj99-125- <dig> showed a general reduction in variability based on all snp types observed.
mnv = multi-nucleotide variants, snv = single-nucleotide variants.
many different marker systems have been used for cultivar identification in cranberry including rapds  <cit> , scars  <cit> , and ssrs  <cit> .
ssrs were also used for mapping and qtl analysis  <cit> .
breeding and selection in cranberry, as with most woody perennials, is a lengthy process, requiring at least 3- <dig> years from seed to flowering progeny.
the progeny are then typically cloned and planted in small field plots for evaluation and selection, which might take an additional 3- <dig> years.
the cranberry breeding program would benefit tremendously from molecular markers that can be used for marker-assisted selection .
this approach has gained in popularity in many crop systems  <cit> .
until very recently, ssrs were the marker of choice for higher plant breeding and although these markers will probably be used for some time, the use of snps is likely to become more widespread.
the utilization of snps for mas has been propelled by the tremendous capability of next generation genome sequencing  <cit> .
similar to ssrs, snps also have the advantage of being abundant in plant genomes.
many detection methods are available and continue to be developed for snp detection, including high throughput platforms such as snp microarrays.
association of certain snps with common diseases has already been described in humans  <cit>  and this technology is now being applied to plant research.
for example, snps were used for genome-wide association study of  <dig> phenotypes in arabidopsis <cit> .
many common alleles with major effect were detected, offering many candidates for follow up studies.
although generally limited to major crops and model systems, this technology will expand into specialty crops such as cranberry.
transporters
transport proteins are integral to the movement of materials across membranes - into and out of cells and subcellular compartments, and between cells via plasmodesmata.
we identified a total of  <dig>  putative transporters in  <dig> families.
given the fragmented nature of our assembly, we recognize that this number is likely to be an over-estimate because some genes may be fragmented into more than one contig and thus may be mistakenly identified as independent transporter genes.
this potential bias is also applicable to all other genes and gene families identified in v. macrocarpon .
if we consider only those families that are better represented , there are  <dig>  in  <dig> families.
by far, the single most abundant group  is the plant plamodesmata family .
the relative numbers in each family were similar between grape and cranberry .
the universality of transporters in eukaryotic systems offers the opportunity for phylogenetic studies and prediction of function  <cit> .
transporters in plants are also critical to uptake of water and mineral nutrients in the roots.
as the global climate changes, plants are increasingly subjected to stresses such as drought, and those grown on more marginal soils may experience salt build up due to irrigation.
a better understanding of these proteins will be critical to sustaining agricultural crops.
percent shown is based on the total number found in each species.
the difference is between cranberry vs. grape.
transcription factors
we identified  <dig>  transcription factors , grouped into  <dig> families, within the v. macrocarpon genome , compared to  <dig>  in apple,  <dig>  in arabidopsis,  <dig>  in grape, and  <dig>  in rice.
thus, the level  found in cranberry is much lower than in these other plants.
the most common tfs in cranberry, as in most plants, are in the myb superfamily , the bhlh  family  and the ap2/erebp  family .
together, these accounted for about 21% of the total tfs found.
the zim family, which is a unique tf containing a gata-type zinc-finger domain  <cit> , appears to be absent from cranberry, apple, and grape.
the numbers found in cranberry for each family as compared to other selected species.
transcription factors are important regulators of gene expression.
most transcription factors reported are predicted based on their dna-binding domain and can be subdivided based on a variety of characteristics such as the number of repeats  <cit> .
although many have been predicted in plants, experimental characterization to document function is lacking for most.
prediction of biological function is further complicated by the fact that within a family, the origin is probably from gene duplication but divergence has resulted in potentially very different functions  <cit> .
disease resistance genes
a total of  <dig> putative r-genes were identified in the v. macrocarpon genome and classified using the prgdb  <cit>  .
the total number was similar to that found in grape and arabidopsis.
of these,  <dig> were classified as cytoplasmic proteins that function using the canonical resistance domains like the nucleotide-binding site , toll/interleukin- <dig> receptor  and the leucine-rich repeat  domains.
moreover,  <dig> putative v. macrocarpon r-genes were found to be transmembrane receptors, of which  <dig> were classified as receptor-like kinases  and  <dig> as receptor-like proteins .
out of the  <dig> putative r-genes,  <dig> were classified as ‘others’, including genes which have been described as conferring resistance through different molecular mechanisms.
within this class  <dig>   <dig> and  <dig> genes showed homology to the rice pid <dig> <cit> , the tomato pto <cit>  and the wheat lr <dig> genes, respectively.
the best characterized r-genes impart some resistance to biotrophic pathogens.
for many small fruits, including cranberry, the majority of the fruit rot pathogens are necrotrophs  <cit> .
using  <dig> genes implicated in arabidopsis immune responses to necrotrophic pathogens  <cit>  for reference, cranberry was found to have about half as many , while grape , rice , apple , and melon  were found to have more, but still fewer than arabidopsis.
because the actions of these genes impact susceptibility to pathogens, they may be under positive selection, resulting in sequence divergence that complicates the identification of homologues.
arabidopsis
the number found in each class as compared to other selected species.
mitochondrial genome comparison
intact nuclei are typically prepared for genomic dna isolation and subsequent high throughput sequencing to reduce organellar contamination.
the number of extranuclear genomes per cell varies depending on the species, cell type and age of the tissue.
for example, land plants can have high numbers of chloroplasts  in the leaf cells.
even though precautions were taken to prevent such small genome contamination, some assembled scaffolds showed high similarity to the sequenced mitochondrial genome after mapping using mummer <dig>  it was not possible to reconstruct these sequences into a single scaffold representing the whole mitochondrial genome .
a set of  <dig> scaffolds showed high similarity to the cranberry mitochondrial genome  <cit> , with one scaffold suggesting a rearrangement or misassembly.
paired end reads that mapped against the mt genome from cnj99-125- <dig> were selected for reassembly by using abyss, obtaining a set of  <dig> mitochondrial contigs, showing coverage of the complete mitochondrial genome .
the scaffold that showed the rearrangement appeared as three separate contigs, rejecting the possibility of misassembly.
even though the mitochondrial genome was not recovered as a single scaffold, after the comparison against that of ‘hyred’, we annotated the mitochondrial genes in the assembled scaffolds, including the trna-sec and a selenocysteine insertion sequence  element, which were notable findings in the cranberry mitochondrial genome derived from ‘hyred’  <cit> .
metabolic pathways
many kegg biosynthetic pathways were generated as part of this project.
cranberry is rich in plant secondary metabolites, particularly polyphenolics synthesized from phenylalanine, that not only benefit plant health , but also potentially benefit humans.
these phytochemicals are in several different biochemical classes, but among the most important are the phenols including the flavonoids.
the major flavonoids in cranberry are the proanthocyanidins, anthocyanins and flavonols.
all three classes are being intensely investigated for their potential benefit to human health.
for example; proanthocyanidins are thought to help in maintaining urinary tract health  <cit> , anthocyanins are important as antioxidants  <cit>  and flavonols are implicated in anti-atherogenic, anti-inflammatory, and anti-cancer bioactivities, among others  <cit> .
considering the importance of the flavonoids, we show the kegg reference pathway for flavonoid biosynthesis .
the enzymes found in our cranberry sequence are compared to those reported to be found in grape.
our data show that essentially all of those found in grape are also present in cranberry with the exception of two enzymes, flavonoid 3’,5’ hydroxylase  and leucoanthocyanidin reductase .
the flavonoid 3’, 5’ hydroxylase  catalyzes hydroxylation of the b-ring of dihydrokaempferol to form dihydromyricetin.
dihydromyricetin is then converted to blue-colored delphinidins  <cit> .
thus, f35h catalyzes a key step leading to the biosynthesis of blue pigment in flowers and fruits and is sometimes referred to as the ‘blue’ gene  <cit> .
ripe cranberries contain primarily the galactosides and arabinosides of cyanidin and peonidin, with small amounts of the glucosides  <cit> , resulting in their brilliant red color.
fruit of v. macrocarpon are virtually devoid of delphinidin, although interspecific hybrids with v. oxycoccus contained trace amounts  <cit> .
it is therefore expected that this enzyme  might be lacking in american cranberry, but present in purple-blue varieties of grape.
the leucoanthocyanidin reductase catalyzes the synthesis of catechin, catechin-4beta-ol  as well as the flavan-3-ols afzelechin and gallocatechin.
these compounds and others are important precursors to the biosynthesis of condensed tannins.
although cranberry does contain epicatechin and condensed tannins produced by alternate pathways, catechin, gallocatechin, or afzelechin have not been reported  <cit> .
because cranberry fruit lacks catechin, it is not surprising that a gene encoding the enzyme  for its biosynthesis was not found.
flavonoid biosynthesis is tissue specific, developmentally regulated and can be induced by a variety of environmental factors, including light, uv radiation, fungal infection, interaction with microorganisms, wounding, etc.
many of the ‘core’ structural genes involved in plant flavonoid biosynthesis are known .
the structural genes and the regulatory genes in this pathway are of interest in cranberry as these affect the temporal and spatial flavonoid biosynthesis as well as the specific flavonoids produced.
these genes can be targeted for manipulation of flavonoid biosynthesis through various means or used as markers for selection of desirable flavonoid profiles through breeding.
for example, an important aspect of anthocyanins as antioxidants is the specific aglycone, as well as the glycoside, as this affects the both the antioxidant potential and bioavailability  <cit> .
interspecific hybridization was demonstrated to alter anthocyanin glycosylation in cranberry, but the gene involved were not identified  <cit> .
kegg pathway analysis offers the opportunity to identify these candidate genes, such as the glycosyltransferases, for further study.
another important class of secondary compounds is the terpenes.
terpenes are a diverse group of compounds that are the primary components of plant essential oils.
their tremendous structural diversity requires a diverse array of enzymes for their synthesis.
terpenes are synthesized via two major pathways; the acetate-mevalonate pathway, which operates in the plant cell cytoplasm and mitochondria, and the non-mevalonate which operates in the plastids  <cit> .
we generated kegg pathways for biosynthesis of terpenes thought to be involved in plant-insect interactions .
selected genes encoding key enzymes were identified and used to design real-time pcr primers.
these primers were then used to monitor expression of those genes in response to insect feeding  <cit> .
the volatile terpenes produced, as a result of the demonstrated up-regulation of certain genes, deterred further feeding and attracted parasites of the herbivores  <cit> .
CONCLUSIONS
the use of an inbred genotype derived from five generations of selfing,  where 97% or greater of the alleles are identical by descent, allowed a preliminary assembly of the genome and the transcriptome, and the identification of key genes and gene families in the american cranberry.
the data generated not only allow for a myriad of studies of cranberry and related species, but also contribute to the mounting information available on higher plants.
these data provide a genomic database of this recently domesticated north american temperate crop, offering facilitation for genetic enhancement, and the study of biotic and abiotic stresses that will be encountered with the changing climate.

 the american cranberry is native to north america and is a member of the ericaceae. cranberry fruit was collected from the wild by american indians and used for a variety of purposes including as a preservative of fish and meat and medicinally as a poultice for dressing wounds. the crop was first domesticated in the early 1800s on cape cod, massachusetts. today, cranberries are grown primarily in wisconsin, massachusetts, new jersey, in the pacific northwest in oregon and washington, and in five provinces in canada. outside of north america, v. macrocarpon is cultivated in parts of europe and chile. in us cranberry production was million pounds, valued at over $ million. the american cranberry and other species of vaccinium, such as highbush blueberry are known for their phytochemicals that can benefit human health. cranberry, in particular, is high in polyphenolic antioxidants, helps prevent urinary tract infections, has some anticancer properties, and may help prevent oral caries among other health benefits. v. macrocarpon is taxonomically placed in the core eudicots, a diverse group of angiosperms. it is a member of the asterid clade, which contains the orders cornales and ericales. the order ericales is quite diverse and includes families. key economically important plants in the ericales include tea and edible fruits such as kiwi and persimmon. the ericaceae is an important family in the ericales. members of this family tend to thrive in nutrient-poor acid soils. the ericaceae includes genera and about species. despite the importance of ericaceous plants such as blueberry, huckleberry, bilberry, cranberry and rhododendron, whole-genome sequence data for this group are currently lacking. cranberry is diploid and self-fertile, allowing generation of inbred lines. cranberry can hybridize with, and give rise to fertile offspring, when crossed to related species such as v. oxycoccus l., producing unique populations segregating for a host of horticultural and biochemical characteristics. a major characteristic important for sustainability is disease resistance. cultivated cranberry is susceptible to a variety of fungal pathogens. in fact, fruit rot is the most significant problem in cranberry production in the northeastern u.s. where, in the absence of an appropriate fungicide regime, the entire crop can be lost. disease resistance in plants is complex, involving many different pathways and mechanisms. whereas some of the resistance genes identified in various plant species impart resistance to specific pathogens, others have been implicated in broad-spectrum resistance. identification of putative resistance genes and their mapping for marker-assisted breeding would be facilitated by whole-genome sequence analysis. whole-genome sequence data can be used for many other purposes such as identification of key biosynthetic pathway genes, genotyping, structure-function studies, and evolutionary studies. the american cranberry has several characteristics including compact size, a relatively short generation interval, ease of asexual propagation, diploidy, self-fertility, and moderate genome size, that make it suitable for use as a model system, specifically for temperate woody perennial plants species. we present here the results of gene discovery utilizing whole-genome and transcriptome sequence data from an inbred line of american cranberry. the data were analyzed to validate the taxonomic position of cranberry, to identify synteny with other representative sequenced plant species, to predict the majority of the encoded genes, and to identify genes potentially associated with disease resistance.  genome and transcriptome sequencing and assembly we sequenced the genome of v. macrocarpon using the illumina gaiix sequencer. a total of million paired-end reads were generated, corresponding to gbp of genomic data. the assembly resulted in contigs. scaffolding slightly reduced the number of contigs to and raised the n to bp. the assembled size was mbp, with an average sequencing coverage of 20x, comparable to that reported for vitis vinifera. the cranberry genome size was estimated to be about mbp. we believe that this genome size is reasonably accurate because 93% of the individual sequence reads mapped to the assembly. this suggests that the assembly includes the vast majority of the sequenced data. furthermore, repeated regions of the assembly did not show extreme coverage values suggesting that we are not underestimating genome size due to the co-assembly of distinct repeats into single contigs. evaluation of the completeness of the v. macrocarpon draft genome assembly was done using cegma and showed that out of the core eukaryotic genes were present in the assembly. further analysis of the data using the highly conserved cegs showed that were present in the v. macrocarpon assembly with being complete, and the remaining being partial. a total of million reads of mrna-seq data were generated on the illumina gaiix sequencer and assembled into cdnas longer than bp and with average coverage >5x. the cdna set was aligned to the genome assembly resulting in cdnas mapping with 98% identity. these assemblies and the mrna-seq reads were used to guide the gene prediction program. the genome and transcriptome data have been deposited in ncbi as bioproject and bioproject respectively. transposable elements genome-wide sequence analysis has demonstrated that transposable elements of different types are widespread in eukaryotes. in flowering plants, transposons can account for large percentages of the total genome. even in the relatively small arabidopsis genome, transposons comprise about % percent of the genome. the larger maize genome contains greater than 70% of the genome as transposons. it is becoming clear as more plant genomes are analyzed that transposons contribute to the size and diversity of plant genomes. although we found representatives of most classes of known transposons, the class retrotransposons of the ltr type are the most common, as has been found in other plants. surprisingly though, despite the fact that the cranberry genome is about times that of arabidopsis, the percentage of transposons in cranberry was found to be only about %. it is unclear why the percentage in cranberry might be so low. of this %, it would be instructive to determine the percentage that might be active. a lack of active transposition would limit expansion of the elements in the genome. in maize, active transposition sometimes gives rise to obvious phenotypic changes. some of these, such as color change in the aleurone layer of kernels, led to their discovery. these types of changes, such as variegated leaves or color variation in the fruit epidermis, have not been observed in cranberry in cultivars that have been domesticated for over years, e.g., ‘early black’ and ‘ben lear’, as is common in apple. as such, cranberry lacks the phenotypic evidence of active transposition. however, although the class ii te represented only about 1% of the genome, similar to apple and cucumber, the hobo-activator represented % of the cranberry genome with a copy number. class ii tes are well recognized as promoting chromosomal rearrangements, including reciprocal translocations. reciprocal translocation heterozygotes have been identified in cranberry suggesting class ii tes may be or have been active in cranberry. arabidopsis the percent of the genome for each type are shown relative to other selected plant species. cranberry is reported to have gone through a severe genetic bottleneck, possibly during the pleistocene. as a result, wild populations were found to lack phenotypic variability and have relatively low genetic heterogeneity. cranberry is self-fertile and the proposed genetic bottleneck would result in at least some level of inbreeding. in our studies, a 5th generation inbred line of cranberry was selected for sequencing and it is not known how this level of inbreeding might affect the transposon complement in the cranberry genome. in maize, where inbred lines are used routinely for breeding, high levels of transposons are maintained. gene prediction annotation of the v. macrocarpon genome assembly was done using augustus which was trained to be v. macrocarpon specific. a set of genes was predicted, out of which genes had alignments larger than 30% to a te protein database; these genes were filtered out. the remaining genes were used as query for a blastp search against the ncbi nr database. combining the genes that had a blast hit with those that had not and were larger than amino acids in length resulted in a set of genes that was used for further downstream analysis. the average coding sequence size was calculated to be bp and the mean number of exons per gene was; the gene density was calculated to be genes per kbp. comparison of v. macrocarpon mrna-seq data to the set of gene models showed that 36% of the genes had transcriptome evidence supporting the exons. to determine whether some of the gene models in v. macrocarpon may be artifacts associated with predicted regions in repeats, we re-ran the gene prediction using an assembly that had been masked for de novo repeats. this procedure resulted in the prediction of gene models. to test whether the prediction made without masking repeats was significantly different, we compared the two sets of gene predictions to each other. using blastp with a cut-off < 1e- for this comparison, 97% of the new models were found to be present in the more extensive set of predictions. given this evidence for the accuracy of the gene prediction with respect to potential biases introduced by repeats, we used the larger set of gene models for downstream analyses. taxonomic assessment to verify the taxonomic placement of cranberry, two chloroplast- and one mitochondrial-encoded protein were concatenated and aligned with those of other plant species including four asterids, rosids, one basal eudicot, and one monocot. the complete chloroplast genome of cranberry has been published. a maximum likelihood tree was built and the results of bootstrap replicates were used to determine branch support in an unrooted phylogeny. most branches in the tree were well supported and of the species compared, cranberry was clearly located in the asterid group. more specifically, cranberry is in the ericaceae in the section oxycoccus. within this section are at least two species, vaccinium macrocarpon ait. and v. oxycoccus l.. although v. oxycoccus occurs at the diploid, tetraploid and hexaploid levels, and older literature often splits some of these into different species, the section oxycoccus is fairly well defined. in contrast, section cyanococcus contains many blueberry species, the boundaries of which are less clearly defined. traditional methods coupled with molecular data such as the chloroplast rbcl and matk genes and nuclear sequences such as its, have been used to study phylogenetic relationships within the blueberry tribe and for wider placement in the ericaceae. the use of the matr mitochondrial gene has been proposed for phylogenetic studies in rosids, but not in the order ericales. as the taxonomy of higher plants continues to advance, it is likely that more molecular data will be needed and utilized. thus, the genomic data presented here can be mined for various gene sequences or molecular markers that can be used for this purpose. conserved orthologous set markers single or low copy nuclear dna markers are useful for phylogenetic studies and comparative genomic analyses. a cos is defined as a gene conserved in sequence and copy number that shares a common ancestor by descent, while paralogs are duplicated sequences resulting in gene duplications or polyploidization events. cos markers have been successfully developed and used for phylogenetic inference among species in the solanaceae and poaceae families. the determination of cos markers in vaccinium macrocarpon is of importance to resolve the phylogenetic relationship of the cranberry and wild relative species in the ericaceae family. the comparison between arabidopsis and sunflower transcriptomes yielded single copy orthologous genes, while the comparison against arabidopsis and lettuce detected genes. there were putative cosii markers in common between the two datasets, of which had known function. microsatellite detection a total of perfect ssrs were detected in the sequenced and assembled genome. a set of and ssrs corresponded to assembled genome scaffolds and unigene sequences, respectively. the most frequent motif length repeated in the genome scaffolds corresponded to di-nucleotides. the maximum repeat length was and the average length of the ssrs was nucleotides. the nuclear scaffolds showed di- and tetra-nucleotides to be the most abundant motifs representing and 21% of the ssrs, respectively. the ga and aaat motifs were the most abundant corresponding to and % of the total detected ssrs, respectively. for the microsatellites located in the unigene sequences, tri- and di-nucleotides were the most abundant motifs accounting for 37% and 35%, respectively, and the longest ssr was nucleotides. the most frequent di-, tri-, and tetra-nucleotide motifs were ga, aag and aaat accounting for and %, respectively, of the transcriptome ssrs. this is the first whole genome microsatellite search in cranberry and provides the largest data set available until now of easy-to-use markers for molecular-based studies in cranberry and other vaccinium crops. snp distribution we used a 5th generation inbred accession of cranberry to lower heterozygosity and facilitate assembly. assuming ‘ben lear’ has a coefficient of inbreeding = the estimated heterozygosity of the clone sequenced would be %. however, ‘ben lear’ was reported to have of ssr loci being homozygous, the highest among cultivars analyzed, suggesting f > the bivariate snp distribution showed a total of snps that occur at a minimum frequency of 20% across reads that provide >10x genome coverage. the total number of snps detected represents a level of heterozygosity of about %, in our mbp assembly. to determine the ‘starting’ level of homozygosity, we compared these results with those from the ‘ben lear’ parent. using the cranberry transcriptome as reference to search for common snps, comparisons between ‘ben lear’ and cnj99-125- yielded versus snps, respectively. cnj99-125- possessed fewer multi-nucleotide variants and single-nucleotide variants, than ‘ben lear’. additionally, cnj99-125- showed a reduction in indels when compared with ‘ben lear’. overall, cnj99-125- showed a general reduction in variability based on all snp types observed. mnv = multi-nucleotide variants, snv = single-nucleotide variants. many different marker systems have been used for cultivar identification in cranberry including rapds, scars, and ssrs. ssrs were also used for mapping and qtl analysis. breeding and selection in cranberry, as with most woody perennials, is a lengthy process, requiring at least 3- years from seed to flowering progeny. the progeny are then typically cloned and planted in small field plots for evaluation and selection, which might take an additional 3- years. the cranberry breeding program would benefit tremendously from molecular markers that can be used for marker-assisted selection. this approach has gained in popularity in many crop systems. until very recently, ssrs were the marker of choice for higher plant breeding and although these markers will probably be used for some time, the use of snps is likely to become more widespread. the utilization of snps for mas has been propelled by the tremendous capability of next generation genome sequencing. similar to ssrs, snps also have the advantage of being abundant in plant genomes. many detection methods are available and continue to be developed for snp detection, including high throughput platforms such as snp microarrays. association of certain snps with common diseases has already been described in humans and this technology is now being applied to plant research. for example, snps were used for genome-wide association study of phenotypes in arabidopsis. many common alleles with major effect were detected, offering many candidates for follow up studies. although generally limited to major crops and model systems, this technology will expand into specialty crops such as cranberry. transporters transport proteins are integral to the movement of materials across membranes - into and out of cells and subcellular compartments, and between cells via plasmodesmata. we identified a total of putative transporters in families. given the fragmented nature of our assembly, we recognize that this number is likely to be an over-estimate because some genes may be fragmented into more than one contig and thus may be mistakenly identified as independent transporter genes. this potential bias is also applicable to all other genes and gene families identified in v. macrocarpon. if we consider only those families that are better represented, there are in families. by far, the single most abundant group is the plant plamodesmata family. the relative numbers in each family were similar between grape and cranberry. the universality of transporters in eukaryotic systems offers the opportunity for phylogenetic studies and prediction of function. transporters in plants are also critical to uptake of water and mineral nutrients in the roots. as the global climate changes, plants are increasingly subjected to stresses such as drought, and those grown on more marginal soils may experience salt build up due to irrigation. a better understanding of these proteins will be critical to sustaining agricultural crops. percent shown is based on the total number found in each species. the difference is between cranberry vs. grape. transcription factors we identified transcription factors, grouped into families, within the v. macrocarpon genome, compared to in apple, in arabidopsis, in grape, and in rice. thus, the level found in cranberry is much lower than in these other plants. the most common tfs in cranberry, as in most plants, are in the myb superfamily, the bhlh family and the ap2/erebp family. together, these accounted for about 21% of the total tfs found. the zim family, which is a unique tf containing a gata-type zinc-finger domain, appears to be absent from cranberry, apple, and grape. the numbers found in cranberry for each family as compared to other selected species. transcription factors are important regulators of gene expression. most transcription factors reported are predicted based on their dna-binding domain and can be subdivided based on a variety of characteristics such as the number of repeats. although many have been predicted in plants, experimental characterization to document function is lacking for most. prediction of biological function is further complicated by the fact that within a family, the origin is probably from gene duplication but divergence has resulted in potentially very different functions. disease resistance genes a total of putative r-genes were identified in the v. macrocarpon genome and classified using the prgdb. the total number was similar to that found in grape and arabidopsis. of these, were classified as cytoplasmic proteins that function using the canonical resistance domains like the nucleotide-binding site, toll/interleukin- receptor and the leucine-rich repeat domains. moreover, putative v. macrocarpon r-genes were found to be transmembrane receptors, of which were classified as receptor-like kinases and as receptor-like proteins. out of the putative r-genes, were classified as ‘others’, including genes which have been described as conferring resistance through different molecular mechanisms. within this class and genes showed homology to the rice pid, the tomato pto and the wheat lr genes, respectively. the best characterized r-genes impart some resistance to biotrophic pathogens. for many small fruits, including cranberry, the majority of the fruit rot pathogens are necrotrophs. using genes implicated in arabidopsis immune responses to necrotrophic pathogens for reference, cranberry was found to have about half as many, while grape, rice, apple, and melon were found to have more, but still fewer than arabidopsis. because the actions of these genes impact susceptibility to pathogens, they may be under positive selection, resulting in sequence divergence that complicates the identification of homologues. arabidopsis the number found in each class as compared to other selected species. mitochondrial genome comparison intact nuclei are typically prepared for genomic dna isolation and subsequent high throughput sequencing to reduce organellar contamination. the number of extranuclear genomes per cell varies depending on the species, cell type and age of the tissue. for example, land plants can have high numbers of chloroplasts in the leaf cells. even though precautions were taken to prevent such small genome contamination, some assembled scaffolds showed high similarity to the sequenced mitochondrial genome after mapping using mummer it was not possible to reconstruct these sequences into a single scaffold representing the whole mitochondrial genome. a set of scaffolds showed high similarity to the cranberry mitochondrial genome, with one scaffold suggesting a rearrangement or misassembly. paired end reads that mapped against the mt genome from cnj99-125- were selected for reassembly by using abyss, obtaining a set of mitochondrial contigs, showing coverage of the complete mitochondrial genome. the scaffold that showed the rearrangement appeared as three separate contigs, rejecting the possibility of misassembly. even though the mitochondrial genome was not recovered as a single scaffold, after the comparison against that of ‘hyred’, we annotated the mitochondrial genes in the assembled scaffolds, including the trna-sec and a selenocysteine insertion sequence element, which were notable findings in the cranberry mitochondrial genome derived from ‘hyred’. metabolic pathways many kegg biosynthetic pathways were generated as part of this project. cranberry is rich in plant secondary metabolites, particularly polyphenolics synthesized from phenylalanine, that not only benefit plant health, but also potentially benefit humans. these phytochemicals are in several different biochemical classes, but among the most important are the phenols including the flavonoids. the major flavonoids in cranberry are the proanthocyanidins, anthocyanins and flavonols. all three classes are being intensely investigated for their potential benefit to human health. for example; proanthocyanidins are thought to help in maintaining urinary tract health, anthocyanins are important as antioxidants and flavonols are implicated in anti-atherogenic, anti-inflammatory, and anti-cancer bioactivities, among others. considering the importance of the flavonoids, we show the kegg reference pathway for flavonoid biosynthesis. the enzymes found in our cranberry sequence are compared to those reported to be found in grape. our data show that essentially all of those found in grape are also present in cranberry with the exception of two enzymes, flavonoid 3’,5’ hydroxylase and leucoanthocyanidin reductase. the flavonoid 3’, 5’ hydroxylase catalyzes hydroxylation of the b-ring of dihydrokaempferol to form dihydromyricetin. dihydromyricetin is then converted to blue-colored delphinidins. thus, f35h catalyzes a key step leading to the biosynthesis of blue pigment in flowers and fruits and is sometimes referred to as the ‘blue’ gene. ripe cranberries contain primarily the galactosides and arabinosides of cyanidin and peonidin, with small amounts of the glucosides, resulting in their brilliant red color. fruit of v. macrocarpon are virtually devoid of delphinidin, although interspecific hybrids with v. oxycoccus contained trace amounts. it is therefore expected that this enzyme might be lacking in american cranberry, but present in purple-blue varieties of grape. the leucoanthocyanidin reductase catalyzes the synthesis of catechin, catechin-4beta-ol as well as the flavan-3-ols afzelechin and gallocatechin. these compounds and others are important precursors to the biosynthesis of condensed tannins. although cranberry does contain epicatechin and condensed tannins produced by alternate pathways, catechin, gallocatechin, or afzelechin have not been reported. because cranberry fruit lacks catechin, it is not surprising that a gene encoding the enzyme for its biosynthesis was not found. flavonoid biosynthesis is tissue specific, developmentally regulated and can be induced by a variety of environmental factors, including light, uv radiation, fungal infection, interaction with microorganisms, wounding, etc. many of the ‘core’ structural genes involved in plant flavonoid biosynthesis are known. the structural genes and the regulatory genes in this pathway are of interest in cranberry as these affect the temporal and spatial flavonoid biosynthesis as well as the specific flavonoids produced. these genes can be targeted for manipulation of flavonoid biosynthesis through various means or used as markers for selection of desirable flavonoid profiles through breeding. for example, an important aspect of anthocyanins as antioxidants is the specific aglycone, as well as the glycoside, as this affects the both the antioxidant potential and bioavailability. interspecific hybridization was demonstrated to alter anthocyanin glycosylation in cranberry, but the gene involved were not identified. kegg pathway analysis offers the opportunity to identify these candidate genes, such as the glycosyltransferases, for further study. another important class of secondary compounds is the terpenes. terpenes are a diverse group of compounds that are the primary components of plant essential oils. their tremendous structural diversity requires a diverse array of enzymes for their synthesis. terpenes are synthesized via two major pathways; the acetate-mevalonate pathway, which operates in the plant cell cytoplasm and mitochondria, and the non-mevalonate which operates in the plastids. we generated kegg pathways for biosynthesis of terpenes thought to be involved in plant-insect interactions. selected genes encoding key enzymes were identified and used to design real-time pcr primers. these primers were then used to monitor expression of those genes in response to insect feeding. the volatile terpenes produced, as a result of the demonstrated up-regulation of certain genes, deterred further feeding and attracted parasites of the herbivores.  the use of an inbred genotype derived from five generations of selfing, where 97% or greater of the alleles are identical by descent, allowed a preliminary assembly of the genome and the transcriptome, and the identification of key genes and gene families in the american cranberry. the data generated not only allow for a myriad of studies of cranberry and related species, but also contribute to the mounting information available on higher plants. these data provide a genomic database of this recently domesticated north american temperate crop, offering facilitation for genetic enhancement, and the study of biotic and abiotic stresses that will be encountered with the changing climate. 
the american cranberry is one of only three widely-cultivated fruit crops native to north america- the other two are blueberry and native grape. the candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance. in addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20x, allowed efficient assembly and gene calling. of the predicted genes, were assigned to candidate genes based on homology. the genome size of v. macrocarpon has been estimated to be about mb. genes supported by transcriptome data totaled.
 the american cranberry is one of only three widely-cultivated fruit crops native to north america- the other two are blueberry and native grape. 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 organisms is heterozygosity. cranberry has the advantage of being diploid and self-fertile. to minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype derived from five generations of selfing originating from the cultivar ben lear.  the genome size of v. macrocarpon has been estimated to be about mb. genomic sequences were assembled into scaffolds representing mbp with 20x average coverage. the number of predicted genes was and represents % of the assembled genome. of the predicted genes, were assigned to candidate genes based on homology. genes supported by transcriptome data totaled.  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. vaccinium macrocarponericaceaetranscriptomecosiipolyphenolicsresistance genesssrssnpsinbred 