BACKGROUND
the family passifloraceae belongs to the order malpighiales, one of the largest orders of the flowering plants and a member of the clade rosids according to classical and molecular phylogenetic analyses including several different gene sequences.
the family consists of about  <dig> species of herbaceous or woody vines, shrubs, and trees, classified in around  <dig> genera, with almost all of its members belonging to the genus passiflora, popularly known as passion fruits  <cit> .
passifloraceae is widely distributed in tropical and warm-temperate regions of the neotropics and in africa.
only a small number of species  are found outside the neotropics, in the pacific and indomalesian region.
the hallmark of the family is the corona , more developed in insect-pollinated species.
it stores the scent and nectar and also acts as a landing platform for pollinators.
the passiflora species  are typically tendril-bearing vines with a non-pedunculate inflorescence and one or two sessile, pentamerous flowers, many of which are highly prized for their exotic, unusual flowers.
three characters, the corona, operculum, and limen, have historically been used as taxonomic characters for delimiting relationships within passiflora
<cit> .
more recently, the genus was divided into four subgenera, astrophea , decaloba  deidamioides , and passiflora   <cit> .
in the subgenus astrophea , shrubs or true trees tend to occur, distributed in lowland tropical south america.
species of decaloba include herbaceous vines with small flowers and fruits occurring throughout the entire distribution of the genus and referred to as the ‘n =  <dig> group’.
lianas or herbaceous vines are typical in the deidamioides subgenus , species of which are found throughout central and south america and mexico.
one species occurs in new zealand.
finally, the passiflora subgenus comprises herbaceous or woody lianas with exotic flowers and edible fruits, distributed throughout central and south america and the southern us.
the chromosome number led to the designation of this subgenus as ‘n =  <dig> group’  <cit> .
the wide range of floral colours, sizes, and shapes is related to pollination .
for instance, in subgenus astrophea, large bee and hummingbird pollination is common, while bat pollination has been documented in the night-blooming passionflower , a representative of subgenus decaloba.
interestingly, the genus has undergone changes in pollinators at various times.
p. roseorum is an example of hummingbird pollination within subgenus passiflora, in which bee pollination is predominant.
many new hybrids in subgenus passiflora are created annually as growers try to develop novel ornamentals, since most species have conspicuous, scented, purple-banded coronas.
almost all the flowers bloom for less than a day.
there is substantial variation of genome sizes  and flower diameters  in the genus  <cit> .
the difference between the largest and smallest genomes was reported as high as  <dig> times .
both genome size and flower diameter means were smaller in decaloba than in passiflora, the two subgenera to which the  <dig> diploid  species investigated belong.
furthermore, a previous study has shown a wide genome size variation in a sample of eight taxa, ranging from  <dig>  pg  to  <dig>  pg   <cit> .
note that most of passiflora nucleotide sequences available from public databases are microsatellite sequences used to develop genetic markers  <cit> , transcription factors and internal transcribed spacers, ribosomal rna genes, or partial gene sequences used in phylogenetic studies  <cit> .
in addition, information on genes involved in the flowering process of passiflora spp.
is accessible over the internet   <cit> .
as mentioned above, some passiflora species are popular ornamentals, whereas others are grown for their edible fruits, including the giant granadilla , the purple granadilla  and the yellow granadilla , all widely grown in latin america.
the purple passion fruit is also grown in australia and hawaii, and nowadays in kenya, south africa, israel, and in north america.
p. maliformis is the sweet calabash of the west indies.
commercial plantations of passionflowers are a somewhat recent phenomenon.
in brazil, the yellow passion fruit  is a crop of considerable economic importance both for juice production and consumption as fresh fruit.
the plant is also used in the cosmetics and phytotherapeutic industries  <cit> .
brazil is the largest producer and consumer, with a cropping area of around  <dig>  ha yielding some  <dig>  tons a year  <cit> .
despite its commercial importance, very little is known about the genome of the passion fruit.
therefore, improving our understanding of passion fruit genome is very important and to some degree a pre-requisite if its genetic resources are to be used more efficiently.
bacterial artificial chromosome  libraries have become an invaluable resource as a starting point for genomic research, and in particular genome sequencing, physical mapping, complex analysis of targeted genomic regions, and studies on gene structure and function .
many important plant species are reported to have their entire genomes inserted in a bac-based system , which remains the most commonly used large-insert cloning vector.
consequently, a very efficient and viable strategy for obtaining the initial insight into the content and complexity of a particular genome involves sequencing the terminal regions of a representative number of bac clones selected at random from this genomic library.
such sequences, known as bac-ends , can be used for generating partial physical maps and performing comparative analyses  <cit> .
bes data analysis provides an overview of genome composition, as well as information on gene density and the occurrence and distribution of transposable elements  and simple sequence repeat , essential data for the development of molecular markers .
in this study, we have constructed and characterized the first large-insert bac library for a passiflora species in order to facilitate genomic research.
the long-term objective of our work is to study the structure, organization, and content of the yellow passion fruit genome, using bes data as a major resource.
specifically, our aims were  to construct a representative bac library with six haploid genome equivalents using the estimation of  <dig>  mb genome size of p. edulis from souza et al.
<cit> ;  to characterize the clones in relation to their insert sizes, chloroplast and mitochondrial dna content;  to demonstrate the utility of the bac library in gene isolation and cytogenetic mapping;  to use bes to sequence approximately 15% of the bacs; and  to compare our genome data with other data on related plant species.
it is important to emphasize that our analysis, in terms of genome organization and sequence representation, is very preliminary and is mostly being used to assess the quality of the library.
RESULTS
bac library construction and characterization
the passiflora edulis bac library consists of  <dig>  individual bac stored clones in  <dig>  plates and is kept at the french plant genomic resource center .
the library has the following designation: ped-b-flav .
to evaluate the medium insert size,  <dig> randomly chosen bacs were digested with noti enzyme and analysed by pulsed field gel electrophoresis.
the resulting restricted dna typically showed the vector band and one or two insert bands per clone.
the inserts ranged in size from  <dig> to 196 kb and the average insert size for the library was 108 kb.
the insert size of most of the clones  was larger than 90 kb .
our data confirmed that partial digestion of high molecular weight dna with the restriction enzyme hindiii  was critical to produce fragments with a good size range, leading to a good quality of the bac library containing fragments with acceptable insert sizes, important for future whole genome study.figure  <dig>
insert features of the bac library.
insert size distribution in a random sample of clones from the library ; most of the clones had an insert size larger than 90 kb.
a typical agarose pulsed-field gel showing noti digests of clones.
the lambda ladder pfg marker  is in the lanes at far left and right.
marker bands are given in kb, and the size of the vector  is  <dig>  bp.
additionally, the library has been gridded onto macroarrays  in which  <dig>  clones were double-spotted on each nylon membranes of 22 × 22 cm.
subsequently, the level of organellar dna contamination was estimated by macroarray hybridization using radiolabeled mitochondrial and chloroplast gene probes.
hybridization results allowed the identification of  <dig> and  <dig> clones for mitochondrial and chloroplast genes, respectively .
our results are consistent with the generally low frequency of mtdna and cpdna contamination observed in other plant bac libraries such as in quercus robur
<cit> , camellia sinensis
<cit>  and pinus taeda
<cit> .
on the other hand, the identification of a number of bac clones  containing cpdna in the present study could provide a valuable basis for investigating the structure of the passiflora chloroplast dna .figure  <dig>
hybridization results of the
passiflora edulis
bac library screening using chloroplast gene probes,
psba,
psbb
and
ndhb.
to some extent, the above mentioned hybridization data confirmed the genome coverage of the library.
assuming that  <dig> clones contain organellar dna, the number of clones harbouring yellow passion fruit nuclear dna is  <dig> .
since the haploid genome content of p. edulis is reported to be ~ <dig>  pg or  <dig>  mb  <cit> , a library composed of  <dig>  clones with an average insert size of 108 kb affords coverage of ~ <dig> genome equivalents of nuclear dna .
the probability of isolating any single-copy dna sequence from the p. edulis library would be greater than  <dig> %, according to the poisson distribution.
to check the utility of our library in gene isolation, macroarray screening experiments were carried out with radiolabeled probes complementary to eight passiflora gene sequences available in public databases .
hybridization allowed us to identify  <dig> positive clones.
these clones were individually checked by pcr using the  <dig> paired primers of the genes chosen, and  <dig> of them  corresponded to the 1-aminocyclopropane-1-carboxylate oxidase  gene;  <dig> ,  <dig>  and  <dig> clones  corresponded to the sequences of glyceraldehyde-3-phosphate dehydrogenase , lipoxygenase  and nadp-dependent isocitrate dehydrogenase  genes.
approximately 10% of the clones were specific of the primer pairs complementary to the ethylene response sensor  gene, embryo defective  <dig>  gene, cyclin-dependent protein kinase regulator  gene and the myo-inositol 1-phosphate synthase  gene.
all genes have a multifamily organization and therefore these differences are possibly due to random effects.table  <dig>
passiflora
genes, codes, and primer sequences used for screening the library
bacs harbouring those eight genes were used individually in fluorescent in situ hybridization  on somatic metaphase p. edulis chromosomes.
unique signals were detected for four bacs in three chromosomes of the complement .
using the p. edulis karyotype defined previously as reference , the sequence complementary to the ers gene harboured by bac198h <dig> was mapped to the long arm of putative chromosome  <dig>  the aco <dig> gene sequence  was mapped to the short arm of chromosome  <dig>  and the cycd <dig> and g3pd gene sequences  were both mapped to the short arm of chromosome  <dig>  all in terminal positions .
the remaining bacs resulted in strong and dispersed signals located preferentially in subtelomeric or pericentromeric regions of most chromosomes, indicating they are enriched in repeated dna sequences.
the library’s potential for building a p. edulis physical map based on the bac-fish approach was clearly demonstrated.figure  <dig>
fluorescent
in situ
hybridization of
passiflora edulis
mitotic chromosomes using bac probes harbouring the following gene sequences:
ers
,
aco1
and
g3pd
.
slides were counterstained with dapi.
the corresponding karyogram  is shown in the bottom of the figure.
bac-end sequence analyses
in total, dna from  <dig>  bac clones were purified for subsequent sequencing using an abi 3500 × l automated platform.
of these bacs,  <dig>  were sequenced at both ends , and  <dig>  were sequenced in only one direction .
thus,  <dig>  sequencing reactions were performed, yielding  <dig>  good quality bac-end sequences , indicating efficiency of  <dig> %.
the average length of the sequences was 587 bp, ranging from the minimum acceptable size  to  <dig>  bp.
the redundancy in the sequenced set was  <dig> %.
to summarize, our bac-end sequencing effort generated approximately  <dig>  mb  of genomic information for p. edulis, covering around  <dig> % of its genome.
first, we searched in plant databases for mitochondrial and chloroplast sequences.
only  <dig> % of the bac-end sequences were similar to mitochondrial  and chloroplast  genome sequences.
next, the gc content of the passion fruit genome was estimated and found to be high  compared to other dicots.
though preliminary, it is interesting to see that the value is high even when compared to the gc content of phylogenetically close species, such as vitis vinifera   <cit> , populus trichocarpa   <cit>  and ricinus communis   <cit> , and is close to the value usually found in monocots  <cit> .
the gc content observed in the genic portion of the p. edulis genome was  <dig> %, supporting previous findings which have shown that genes generally have a higher gc content in contrast to the background gc content of the entire genome.
similarly, we reported a gc content of  <dig> % when examining an ssh library enriched for a set of sequences of p. edulis  expressed in response to xanthomonas axonopodis infection  <cit> .
the average gc content of plant genomes varies enormously and reflects significant compositional features of the genomes.
in dicots, for instance, the values generally range from 33% to 36% as reported for the genomes of carica papaya   <cit> , quercus robur   <cit> , and arabidopsis   <cit> .
monocots tend to have higher gc contents, generally between 43% and 47%.
the highest values are found in grasses, which may be explained by the proliferation of gc-rich retrotransposons and the presence of grass genes that are extremely gc-rich.
prospecting for retro- and transposable elements and microsatellite sequences
sequences from repbase were used to delineate the occurrence of different classes of repetitive elements within the p. edulis genome, screening out the fraction that corresponds only to sequences of viridiplantae.
repetitive elements were recognized in  <dig> % of all bes , covering approximately  <dig>  mb .
compared to other studies that used bes to provide an overview of the composition of plant genomes, the inference of the amount of repetitive elements in p. edulis is reasonable.
lower values were found in quercus robur   <cit> , cucumis melo   <cit>  and juglans regia   <cit>  but much higher estimates were made for dicots, citrus clementina   <cit>  and daucus carota   <cit> , and in the monocots phalaenopsis equestris   <cit>  and musa acuminata   <cit> .
evidently, part of this variation can be explained by technical factors, i.e.
the quality and quantity of sequences analysed.table  <dig>
classification and distribution of the repetitive elements detected among
passiflora edulis
bac-end sequences
class i retroelements
<dig>
401
<dig>
<dig>
329
<dig>
ltr elements
<dig>
<dig> ,137
<dig>
lines/sines
74
<dig>
<dig>
class ii dna transposons
38
<dig>
881
<dig>
unclassified elements
7
<dig>
<dig>
small rna
2
162
<dig>
satellites
1
41
<dig>
simple repeats
327
<dig>
514
<dig>
low complexity
<dig>
000
<dig>
760
<dig>
total repetitive dna
<dig>
<dig>
<dig>
879
<dig>
total number of nucleotides examined:  <dig>   <dig>   <dig>
on the other hand, compared to estimates reported for other dicot genomes already sequenced, including the more closely-related species populus trichocarpa   <cit>  and vitis vinifera   <cit>  as well as malus × domestica   <cit>  and carica papaya   <cit> , we found a much lower percentage of repetitive elements in p. edulis.
the elements annotated as class i were significantly more abundant than class ii .
there is a robust consensus in the literature, that the larger the plant genome, the greater the chance it contains many retrotransposons.
for example, the large genome of barley  comprises up to 70% retrotransposons  <cit> , while in the small rice genome  these elements represent only 17% of its total composition  <cit> .
in the present study, retrotransposons were identified in about  <dig> % of all bes.
proportionally, this value is not high, but we need to take into account the fact that p. edulis has a larger genome on average, than most dicots.
quantitatively, that is to say  <dig> % of a genome that can reach  <dig>  mb is a large amount of the transposable elements.
long terminal repeats , such as ty1-copia and ty3-gypsy retrotransposons, are enriched in pericentromeric regions, playing important roles in maintaining chromatin structures and centromere functions and regulating gene expression in the host genomes  <cit> .
a parameter commonly used in bac-end analyses is the proportion of ltr retrotransposons ty3-gypsy relative to ty1-copia.
these groups differ from each other in both their degree of sequence similarity and the order of encoding genes.
we observed an approximate ratio of 1: <dig>  indicating that the contributions of ty3-gypsy and ty1-copia to the p. edulis genomes were apparently equal .
very distinct proportions were reported to occur in plants: 1: <dig> in quercus robur
<cit> , 1: <dig> in both malus × domestica
<cit>  and vitis vinifera
<cit> , and 2: <dig> in saccharum spp.
<cit>  where ty1-copia outnumbered ty3-gypsy transposons.
ratios of 3: <dig> and 5: <dig> were found in oryza sativa
<cit>  and phalaenopsis equestris
<cit> , respectively.
in the present study, non-ltr elements included  <dig>  belonging to the line/sine category.
lines are sequences  able to transpose autonomously, while sines  depend on the reverse transcription machinery of other retrotransposons and both are of great importance in the evolutionary history of plant genomes.
class ii dna transposons were found infrequently  as were the remaining repetitive elements  consisting in approximately 1% of all repetitive elements.
similar rates were reported among walnut  bac-end sequences  <cit> .
it is worth noting that species of populus are predominantly dioecious, and therefore outcrossing and wind pollinated, resulting in high levels of gene flow and heterozygosity  <cit> .
similarly, p. edulis is an obligate outcrossing species based on self-incompatibility and insect-pollination.
however, there is evidence of low levels of allelic polymorphism or heterozygosity  <cit> , as already documented in passiflora alata, the sweet passion fruit  <cit> .
speculating about populus trichocarpa has a small genome   <cit>  and passiflora edulis a large genome   <cit> , but the p. edulis genome seems not to accumulate repetitive elements in contrast to p. trichocarpa which is described as a large repetitive genome.
before masking repetitive regions and transposable elements , sequences were subjected to a search for microsatellites or simple sequence repeat .
after running the automatic search, each chromatogram was manually checked to ascertain the quality and reliability of automatically annotated peaks.
in total,  <dig> microsatellite sequences were found in the  <dig> , <dig> bases analysed, an average of  <dig>  ssrs every 100 kb or one ssr every  <dig>  kb.
our estimate is close to the figures documented for other plants, such as brassica rapa   <cit>  and solanum tuberosum   <cit> .
however, it is low in comparison to the average density of microsatellites in two phylogenetically close species, v. vinifera   <cit>  and p. trichocarpa   <cit> .
ssrs were grouped according to motif, and we were able to identify all possible classes of repeats.
altogether,  <dig> % of the ssrs were mono-,  <dig> % di-,  <dig> % tri-,  <dig> % tetra-, and  <dig> % pentanucleotides.
only  <dig> % of the ssrs were hexanucleotides.
for comparison purposes, analyses were performed to identify microsatellites in data sets from bac-end sequences of other fruit species, vitis vinifera, carica papaya, cucumis melo, malus × domestica, as well as arabidopsis thaliana.
these public data were obtained from the ncbi website  using the same software and parameters used in the exploitation of microsatellites in bac-end sequences of p. edulis.
the results of these comparisons are shown in figure  <dig> figure  <dig>
comparison of the percentage of simple sequence repeats  detected in bac-end sequences of
passiflora edulis
,
vitis vinifera
,
carica papaya
,
cucumis melo
,
malus
×
domestica
and
arabidopsis thaliana
, according to the repeat motif .
among the mononucleotides, a/t exceeded by far the proportion of g/c repeats .
in fact, analyses carried out on arabidopsis thaliana, vitis vinifera and populus trichocarpa have shown that, in general, dicots have a high frequency of a/t-rich microsatellites.
the most frequent dinucleotides were at, followed by ag repeats.
none of the classes contained significant frequencies of gc-rich motifs.
this is consistent with studies on arabidopsis thaliana
<cit> , carica papaya
<cit> , and malus × domestica
<cit>  showing that at-rich sequences are much more prevalent in the genomes of higher plants.
in p. edulis, the motif distribution within repeat classes follows the same tendency observed in other plant bac-end sequences.
finally, the most abundant motif was the tetranucleotide aatt .
interestingly there is a high incidence of tetranucleotide motifs  compared to other classes of microsatellites in our data set.
this arrangement is unusual in dicots, where dinucleotides or mononucleotides are normally the most abundant repeats.
exploiting a microsatellite-enriched library of p. edulis, skewed for dinucleotides due to the probes used in its preparation, oliveira and co-authors also observed a higher proportion of tetranucleotides compared to other microsatellite classes  <cit> .
protein coding regions
with the aim of identifying protein-coding regions, the bac-end sequences were compared to the non-redundant protein data bank using the blastx search engine .
around  <dig> % of all bes  were found to have high levels of similarity  to genes of several plant species.
the vast majority of the top-hits made by our sequences were to populus trichocarpa , theobroma cacao , ricinus communis , vitis vinifera  and prunus persica .
this scenario was expected since these are species related to p. edulis on which there is an abundance of genomic data.
the list of species hits in descending order is shown in figure  <dig> figure  <dig>
list of species that produced significant hits with the bac-end sequences of
passiflora edulis
based upon sequence similarity to plant genes .
assuming that  <dig> % of the bes analysed had matching coding regions, we can estimate that ~ <dig> mbp of the passion fruit genome consist of coding sequences.
assuming an average gene length of  <dig>  kb, the same as vitis vinifera
<cit> , we can estimate that the total number of genes per haploid genome of p. edulis is  <dig> .
both the estimate of the coding portion and the number of genes are consistent with the figures proposed for phaleanopsis equestris, a species with a genome size very close to that of p. edulis
<cit> .
our approximation is within the range of the gene numbers estimated for the closest relatives vitis vinifera   <cit>  and populus trichocarpa   <cit> .
altogether  <dig>  ontological terms were assigned to  <dig> of the  <dig> sequences analysed, resulting in an average of  <dig>  terms per sequence.
up to  <dig> go terms were annotated for the sequences, and those related to molecular function were the most abundant.
of the total sequences with assigned ontological terms,  <dig> were assigned to at least one term related to molecular function,  <dig> to at least one term related to biological process, and  <dig> to at least one term related to cellular component.
any term was assigned to  <dig> of the sequences which contained matches with coding sequences, and  <dig> of the sequences were mapped but not annotated.
within the category molecular function, the most frequent terms were binding , catalytic activity  and transporter activity  .
within the category biological process, the most frequent terms were cellular process , metabolic process , single-organism process , and response to stimulus  .
finally, in the category cellular component, the most common terms were cell , organelle  and membrane   .figure  <dig>
distribution of go annotations of gene products predicted from the
passiflora edulis
bess for molecular function , biological process  and cellular component .
only major categories are presented.
comparative genome mapping
in total,  <dig>  bes were compared to the sequenced arabidopsis thaliana, vitis vinifera, and populus trichocarpa genomes for identifying microsyntenic regions .
p. trichocarpa and v. vinifera are among the plant genomes with the highest number of top hits for p. edulis .
although phylogenetically closer, the ricinus communis and prunus persica genomes are still in the early stages of their assembly and annotation, which enormously affects comparative mapping studies.
the same is true for the draft genome of theobroma cacao.
for obvious reasons, a. thaliana has been chosen as a default template.figure  <dig>
comparison of collinear microsyntenic regions identified in
passiflora edulis
bacs  and
arabidopsis thaliana
,
vitis vinifera
and
populus trichocarpa
chromosomes.
the matches  were classified into six categories based on the classification in rampant and co-authors  <cit> .
the ‘single end’  category refers to bac end pairs for which only one of the ends  matched a sequence in the reference genome; bes were assigned to the ‘paired end’  category when both end sequences matched the genome.
the pe category, in turn, is subdivided into ‘co-localized’  and ‘non co-localized’.
we considered all matches occurred at points  <dig> to 350 kb apart on the reference genome; either in the correct orientation with respect to each other  or rearranged with respect to each other .
when the distance between the paired matches was either smaller or larger than these limits, they were assigned to the ‘gapped’ category.
first, we identified  <dig> positive matches to a. thaliana genome,  <dig> ses and  <dig> pes.
four pes formed two ‘collinear’ alignments, composed of sequences pe85q4f <dig>  and pe69q4g <dig> , encompassing  <dig> and  <dig> genes, respectively.
the lengths of potential microsyntenic regions found in a. thaliana are close to the average insert sizes of the p. edulis bac library.
for instance, the arabidopsis region shared at its ends by the pe85q4f <dig> sequences is 92 kb in length; the respective p. edulis bac is 93 kb.
similarly, the arabidopsis region shared by the pe69q4g <dig> sequences is 80 kb in length; the respective p. edulis bac is 97 kb in length.
it is interesting to note that arabidopsis regions overlap each other covering a region of  <dig>  kb, from chromosome c  positions  <dig>  bp  to  <dig>  bp .
clearly, this kind of information can be applied to assemble the two p. edulis bacs that harbour the mentioned sequences.
in addition, the pair pe164q1g <dig>  was classified as ‘rearranged’.
it is noteworthy that the ends of the pe pe85q4f <dig> showed sequence similarity to a 30s subunit ribosomal rna  and to a membrane hydrolase .
additionally, the ends of the pe69q4g <dig> pe showed sequence similarity to an unspecified subunit of the nadh complex  and to a portion of subunit  <dig> of the nadh complex .
according to cellular component annotation analysis , the four sequences belong to ‘chloroplast’ group.
we are possibly facing a cpdna contamination not detected on the macroarrays.
comparing genome intervals shared by p. edulis and v. vinifera, we identifed  <dig> positive matches,  <dig> ses and  <dig> pes.
among the pes, there were two ‘non co-localized’ pairs , one ‘collinear’ pair , one ‘rearranged’ pair , and one ‘gapped’ pair .
the paired sequences pe164q2a <dig>  f/r were mapped to chromosome  <dig> of v. vinifera, the forward end beginning at position  <dig> ,417 bp of the grapevine genome and the reverse end terminating at position  <dig> ,226 bp, separated by around  <dig>  kb.
this region encompasses  <dig> genes, including small genes consisting of single exons, and large genes consisting of more than  <dig> exons.
compared to the p. edulis interval, the region in question is twice the length of the bac pe164q2a <dig> .
finally, comparative mapping between the p. edulis and p. trichocarpa genomes revealed a large number of microsyntenic regions.
altogether, positive matches were found in  <dig> p. edulis sequences, including  <dig> ses and  <dig> pes.
three paired sequences of p. edulis were ‘collinear’ to those of p. trichocarpa.
in addition, pe173q4a <dig>  was classified as ‘rearranged’ and pe164q1g <dig>  as ‘gapped’.
the first ‘collinear’ pair pe216q4a <dig>  is located on chromosome  <dig> of p. trichocarpa, over an interval consisting of ~217 kb of dna harbouring  <dig> genes.
the second pair, pe75q4b <dig>  is separated by ~176 kb, including around  <dig> genes, and the third, pe164q1f <dig>  is separated by a 306 kb interval in p. trichocarpa genome that contains  <dig> genes involved in a number of different physiological processes.
all these paired sequences were correctly orientated and separated by larger intervals in the p. trichocarpa genome, compared to p. edulis.
it is interesting to note that paired sequences pe75q4b <dig> and pe164q1f <dig> were both assigned to chromosome  <dig> in p. trichocarpa, making these bacs good candidates for a bac-by-bac sequencing project based on a bes database.
the pair consisting of pe173q4a <dig>  sequences was classified as ‘rearranged’ because of their incorrect orientation with respect to each other, and the pair consisting of pe164q1g <dig>  sequences was classified as ‘gapped’, since the distance between the sequences is around 6 mb, much larger than the maximum acceptable span.
this huge distance could be the result of a major chromosome event in an ancestral genome, for instance, involving massive insertion as the predominant event.
it is interesting that this pair was also mapped in the genomes of a. thaliana and v. vinifera, but spanning much smaller intervals.
the pes included  <dig> classified as ‘non co-localized’ .
these events are possibly due to macro chromosomal rearrangements that occurred since the split of p. trichocarpa
<cit> , a hypothesis that finds support in the fact that p. trichocarpa  has twice as many chromosomes as p. edulis .
the above results are summarized in additional file 1: table s <dig>  we expected to find a number of potentially microsyntenic regions between the p. edulis and p. trichocarpa genomes since the latter is evolutionarily closer evolutionarily to passiflora than v. vinifera and a. thaliana.
obviously ‘collinear’ relationships tend to erode as we move further away from the genus passiflora.
CONCLUSIONS
we explored approximately  <dig>  bac-end sequences  of the yellow passion fruit  genome after generating a bac library for the species.
this is the first large-insert library for a member of passifloraceae and a valuable tool for future sequencing and genome studies.
we were able to verify the quality of the library , its genome coverage  and utility for gene isolation.
this was done using exclusively the few passiflora gene sequences deposited in public databases as probes in macroarray hybridizations, and the corresponding bacs used in fish experiments allowed us to map four genes to three chromosomes of the species.
we were able to identify reads likely to contain repetitive mobile elements , simple sequence repeats and putative proteins.
we estimate a ~42% gc content  from the analysis of the reads.
approximately  <dig> bac end-sequences contained protein sequences, and ontological terms were assigned to  <dig> of them.
comparison analyses of the sequences generated were performed against sequence data from two phylogenetically close species p. trichocarpa and v. vinifera, as well as from a. thaliana.
around  <dig> positive matches or sequence similarities were detected.
finally, a number of bac-end pair sequences could be mapped to intervals of a. thaliana, v. vinifera and p. trichocarpa chromosomes, representing regions of potential microsynteny.

 the family passifloraceae belongs to the order malpighiales, one of the largest orders of the flowering plants and a member of the clade rosids according to classical and molecular phylogenetic analyses including several different gene sequences. the family consists of about species of herbaceous or woody vines, shrubs, and trees, classified in around genera, with almost all of its members belonging to the genus passiflora, popularly known as passion fruits. passifloraceae is widely distributed in tropical and warm-temperate regions of the neotropics and in africa. only a small number of species are found outside the neotropics, in the pacific and indomalesian region. the hallmark of the family is the corona, more developed in insect-pollinated species. it stores the scent and nectar and also acts as a landing platform for pollinators. the passiflora species are typically tendril-bearing vines with a non-pedunculate inflorescence and one or two sessile, pentamerous flowers, many of which are highly prized for their exotic, unusual flowers. three characters, the corona, operculum, and limen, have historically been used as taxonomic characters for delimiting relationships within passiflora. more recently, the genus was divided into four subgenera, astrophea, decaloba deidamioides, and passiflora. in the subgenus astrophea, shrubs or true trees tend to occur, distributed in lowland tropical south america. species of decaloba include herbaceous vines with small flowers and fruits occurring throughout the entire distribution of the genus and referred to as the ‘n =  group’. lianas or herbaceous vines are typical in the deidamioides subgenus, species of which are found throughout central and south america and mexico. one species occurs in new zealand. finally, the passiflora subgenus comprises herbaceous or woody lianas with exotic flowers and edible fruits, distributed throughout central and south america and the southern us. the chromosome number led to the designation of this subgenus as ‘n =  group’. the wide range of floral colours, sizes, and shapes is related to pollination. for instance, in subgenus astrophea, large bee and hummingbird pollination is common, while bat pollination has been documented in the night-blooming passionflower, a representative of subgenus decaloba. interestingly, the genus has undergone changes in pollinators at various times. p. roseorum is an example of hummingbird pollination within subgenus passiflora, in which bee pollination is predominant. many new hybrids in subgenus passiflora are created annually as growers try to develop novel ornamentals, since most species have conspicuous, scented, purple-banded coronas. almost all the flowers bloom for less than a day. there is substantial variation of genome sizes and flower diameters in the genus. the difference between the largest and smallest genomes was reported as high as times. both genome size and flower diameter means were smaller in decaloba than in passiflora, the two subgenera to which the diploid species investigated belong. furthermore, a previous study has shown a wide genome size variation in a sample of eight taxa, ranging from  pg to  pg. note that most of passiflora nucleotide sequences available from public databases are microsatellite sequences used to develop genetic markers, transcription factors and internal transcribed spacers, ribosomal rna genes, or partial gene sequences used in phylogenetic studies. in addition, information on genes involved in the flowering process of passiflora spp. is accessible over the internet. as mentioned above, some passiflora species are popular ornamentals, whereas others are grown for their edible fruits, including the giant granadilla, the purple granadilla and the yellow granadilla, all widely grown in latin america. the purple passion fruit is also grown in australia and hawaii, and nowadays in kenya, south africa, israel, and in north america. p. maliformis is the sweet calabash of the west indies. commercial plantations of passionflowers are a somewhat recent phenomenon. in brazil, the yellow passion fruit is a crop of considerable economic importance both for juice production and consumption as fresh fruit. the plant is also used in the cosmetics and phytotherapeutic industries. brazil is the largest producer and consumer, with a cropping area of around  ha yielding some tons a year. despite its commercial importance, very little is known about the genome of the passion fruit. therefore, improving our understanding of passion fruit genome is very important and to some degree a pre-requisite if its genetic resources are to be used more efficiently. bacterial artificial chromosome libraries have become an invaluable resource as a starting point for genomic research, and in particular genome sequencing, physical mapping, complex analysis of targeted genomic regions, and studies on gene structure and function. many important plant species are reported to have their entire genomes inserted in a bac-based system, which remains the most commonly used large-insert cloning vector. consequently, a very efficient and viable strategy for obtaining the initial insight into the content and complexity of a particular genome involves sequencing the terminal regions of a representative number of bac clones selected at random from this genomic library. such sequences, known as bac-ends, can be used for generating partial physical maps and performing comparative analyses. bes data analysis provides an overview of genome composition, as well as information on gene density and the occurrence and distribution of transposable elements and simple sequence repeat, essential data for the development of molecular markers. in this study, we have constructed and characterized the first large-insert bac library for a passiflora species in order to facilitate genomic research. the long-term objective of our work is to study the structure, organization, and content of the yellow passion fruit genome, using bes data as a major resource. specifically, our aims were to construct a representative bac library with six haploid genome equivalents using the estimation of  mb genome size of p. edulis from souza et al.; to characterize the clones in relation to their insert sizes, chloroplast and mitochondrial dna content; to demonstrate the utility of the bac library in gene isolation and cytogenetic mapping; to use bes to sequence approximately 15% of the bacs; and to compare our genome data with other data on related plant species. it is important to emphasize that our analysis, in terms of genome organization and sequence representation, is very preliminary and is mostly being used to assess the quality of the library.  bac library construction and characterization the passiflora edulis bac library consists of individual bac stored clones in plates and is kept at the french plant genomic resource center. the library has the following designation: ped-b-flav. to evaluate the medium insert size, randomly chosen bacs were digested with noti enzyme and analysed by pulsed field gel electrophoresis. the resulting restricted dna typically showed the vector band and one or two insert bands per clone. the inserts ranged in size from to 196 kb and the average insert size for the library was 108 kb. the insert size of most of the clones was larger than 90 kb. our data confirmed that partial digestion of high molecular weight dna with the restriction enzyme hindiii was critical to produce fragments with a good size range, leading to a good quality of the bac library containing fragments with acceptable insert sizes, important for future whole genome study.figure insert features of the bac library. insert size distribution in a random sample of clones from the library; most of the clones had an insert size larger than 90 kb. a typical agarose pulsed-field gel showing noti digests of clones. the lambda ladder pfg marker is in the lanes at far left and right. marker bands are given in kb, and the size of the vector is  bp. additionally, the library has been gridded onto macroarrays in which clones were double-spotted on each nylon membranes of 22 × 22 cm. subsequently, the level of organellar dna contamination was estimated by macroarray hybridization using radiolabeled mitochondrial and chloroplast gene probes. hybridization results allowed the identification of and clones for mitochondrial and chloroplast genes, respectively. our results are consistent with the generally low frequency of mtdna and cpdna contamination observed in other plant bac libraries such as in quercus robur, camellia sinensis and pinus taeda. on the other hand, the identification of a number of bac clones containing cpdna in the present study could provide a valuable basis for investigating the structure of the passiflora chloroplast dna.figure hybridization results of the passiflora edulis bac library screening using chloroplast gene probes, psba, psbb and ndhb. to some extent, the above mentioned hybridization data confirmed the genome coverage of the library. assuming that clones contain organellar dna, the number of clones harbouring yellow passion fruit nuclear dna is. since the haploid genome content of p. edulis is reported to be ~  pg or  mb, a library composed of clones with an average insert size of 108 kb affords coverage of ~ genome equivalents of nuclear dna. the probability of isolating any single-copy dna sequence from the p. edulis library would be greater than %, according to the poisson distribution. to check the utility of our library in gene isolation, macroarray screening experiments were carried out with radiolabeled probes complementary to eight passiflora gene sequences available in public databases. hybridization allowed us to identify positive clones. these clones were individually checked by pcr using the paired primers of the genes chosen, and of them corresponded to the 1-aminocyclopropane-1-carboxylate oxidase gene;, and clones corresponded to the sequences of glyceraldehyde-3-phosphate dehydrogenase, lipoxygenase and nadp-dependent isocitrate dehydrogenase genes. approximately 10% of the clones were specific of the primer pairs complementary to the ethylene response sensor gene, embryo defective gene, cyclin-dependent protein kinase regulator gene and the myo-inositol 1-phosphate synthase gene. all genes have a multifamily organization and therefore these differences are possibly due to random effects.table passiflora genes, codes, and primer sequences used for screening the library bacs harbouring those eight genes were used individually in fluorescent in situ hybridization on somatic metaphase p. edulis chromosomes. unique signals were detected for four bacs in three chromosomes of the complement. using the p. edulis karyotype defined previously as reference, the sequence complementary to the ers gene harboured by bac198h was mapped to the long arm of putative chromosome the aco gene sequence was mapped to the short arm of chromosome and the cycd and g3pd gene sequences were both mapped to the short arm of chromosome all in terminal positions. the remaining bacs resulted in strong and dispersed signals located preferentially in subtelomeric or pericentromeric regions of most chromosomes, indicating they are enriched in repeated dna sequences. the library’s potential for building a p. edulis physical map based on the bac-fish approach was clearly demonstrated.figure fluorescent in situ hybridization of passiflora edulis mitotic chromosomes using bac probes harbouring the following gene sequences: ers, aco1 and g3pd. slides were counterstained with dapi. the corresponding karyogram is shown in the bottom of the figure. bac-end sequence analyses in total, dna from bac clones were purified for subsequent sequencing using an abi 3500 × l automated platform. of these bacs, were sequenced at both ends, and were sequenced in only one direction. thus, sequencing reactions were performed, yielding good quality bac-end sequences, indicating efficiency of %. the average length of the sequences was 587 bp, ranging from the minimum acceptable size to  bp. the redundancy in the sequenced set was %. to summarize, our bac-end sequencing effort generated approximately  mb of genomic information for p. edulis, covering around % of its genome. first, we searched in plant databases for mitochondrial and chloroplast sequences. only % of the bac-end sequences were similar to mitochondrial and chloroplast genome sequences. next, the gc content of the passion fruit genome was estimated and found to be high compared to other dicots. though preliminary, it is interesting to see that the value is high even when compared to the gc content of phylogenetically close species, such as vitis vinifera, populus trichocarpa and ricinus communis, and is close to the value usually found in monocots. the gc content observed in the genic portion of the p. edulis genome was %, supporting previous findings which have shown that genes generally have a higher gc content in contrast to the background gc content of the entire genome. similarly, we reported a gc content of % when examining an ssh library enriched for a set of sequences of p. edulis expressed in response to xanthomonas axonopodis infection. the average gc content of plant genomes varies enormously and reflects significant compositional features of the genomes. in dicots, for instance, the values generally range from 33% to 36% as reported for the genomes of carica papaya, quercus robur, and arabidopsis. monocots tend to have higher gc contents, generally between 43% and 47%. the highest values are found in grasses, which may be explained by the proliferation of gc-rich retrotransposons and the presence of grass genes that are extremely gc-rich. prospecting for retro- and transposable elements and microsatellite sequences sequences from repbase were used to delineate the occurrence of different classes of repetitive elements within the p. edulis genome, screening out the fraction that corresponds only to sequences of viridiplantae. repetitive elements were recognized in % of all bes, covering approximately  mb. compared to other studies that used bes to provide an overview of the composition of plant genomes, the inference of the amount of repetitive elements in p. edulis is reasonable. lower values were found in quercus robur, cucumis melo and juglans regia but much higher estimates were made for dicots, citrus clementina and daucus carota, and in the monocots phalaenopsis equestris and musa acuminata. evidently, part of this variation can be explained by technical factors, i.e. the quality and quantity of sequences analysed.table classification and distribution of the repetitive elements detected among passiflora edulis bac-end sequences class i retroelements 401 329 ltr elements,137 lines/sines 74 class ii dna transposons 38 881 unclassified elements 7 small rna 2 162 satellites 1 41 simple repeats 327 514 low complexity 000 760 total repetitive dna 879 total number of nucleotides examined: on the other hand, compared to estimates reported for other dicot genomes already sequenced, including the more closely-related species populus trichocarpa and vitis vinifera as well as malus × domestica and carica papaya, we found a much lower percentage of repetitive elements in p. edulis. the elements annotated as class i were significantly more abundant than class ii. there is a robust consensus in the literature, that the larger the plant genome, the greater the chance it contains many retrotransposons. for example, the large genome of barley comprises up to 70% retrotransposons, while in the small rice genome these elements represent only 17% of its total composition. in the present study, retrotransposons were identified in about % of all bes. proportionally, this value is not high, but we need to take into account the fact that p. edulis has a larger genome on average, than most dicots. quantitatively, that is to say % of a genome that can reach  mb is a large amount of the transposable elements. long terminal repeats, such as ty1-copia and ty3-gypsy retrotransposons, are enriched in pericentromeric regions, playing important roles in maintaining chromatin structures and centromere functions and regulating gene expression in the host genomes. a parameter commonly used in bac-end analyses is the proportion of ltr retrotransposons ty3-gypsy relative to ty1-copia. these groups differ from each other in both their degree of sequence similarity and the order of encoding genes. we observed an approximate ratio of 1: indicating that the contributions of ty3-gypsy and ty1-copia to the p. edulis genomes were apparently equal. very distinct proportions were reported to occur in plants: 1: in quercus robur, 1: in both malus × domestica and vitis vinifera, and 2: in saccharum spp. where ty1-copia outnumbered ty3-gypsy transposons. ratios of 3: and 5: were found in oryza sativa and phalaenopsis equestris, respectively. in the present study, non-ltr elements included belonging to the line/sine category. lines are sequences able to transpose autonomously, while sines depend on the reverse transcription machinery of other retrotransposons and both are of great importance in the evolutionary history of plant genomes. class ii dna transposons were found infrequently as were the remaining repetitive elements consisting in approximately 1% of all repetitive elements. similar rates were reported among walnut bac-end sequences. it is worth noting that species of populus are predominantly dioecious, and therefore outcrossing and wind pollinated, resulting in high levels of gene flow and heterozygosity. similarly, p. edulis is an obligate outcrossing species based on self-incompatibility and insect-pollination. however, there is evidence of low levels of allelic polymorphism or heterozygosity, as already documented in passiflora alata, the sweet passion fruit. speculating about populus trichocarpa has a small genome and passiflora edulis a large genome, but the p. edulis genome seems not to accumulate repetitive elements in contrast to p. trichocarpa which is described as a large repetitive genome. before masking repetitive regions and transposable elements, sequences were subjected to a search for microsatellites or simple sequence repeat. after running the automatic search, each chromatogram was manually checked to ascertain the quality and reliability of automatically annotated peaks. in total, microsatellite sequences were found in the, bases analysed, an average of ssrs every 100 kb or one ssr every  kb. our estimate is close to the figures documented for other plants, such as brassica rapa and solanum tuberosum. however, it is low in comparison to the average density of microsatellites in two phylogenetically close species, v. vinifera and p. trichocarpa. ssrs were grouped according to motif, and we were able to identify all possible classes of repeats. altogether, % of the ssrs were mono-, % di-, % tri-, % tetra-, and % pentanucleotides. only % of the ssrs were hexanucleotides. for comparison purposes, analyses were performed to identify microsatellites in data sets from bac-end sequences of other fruit species, vitis vinifera, carica papaya, cucumis melo, malus × domestica, as well as arabidopsis thaliana. these public data were obtained from the ncbi website using the same software and parameters used in the exploitation of microsatellites in bac-end sequences of p. edulis. the results of these comparisons are shown in figure  figure comparison of the percentage of simple sequence repeats detected in bac-end sequences of passiflora edulis, vitis vinifera, carica papaya, cucumis melo, malus × domestica and arabidopsis thaliana, according to the repeat motif. among the mononucleotides, a/t exceeded by far the proportion of g/c repeats. in fact, analyses carried out on arabidopsis thaliana, vitis vinifera and populus trichocarpa have shown that, in general, dicots have a high frequency of a/t-rich microsatellites. the most frequent dinucleotides were at, followed by ag repeats. none of the classes contained significant frequencies of gc-rich motifs. this is consistent with studies on arabidopsis thaliana, carica papaya, and malus × domestica showing that at-rich sequences are much more prevalent in the genomes of higher plants. in p. edulis, the motif distribution within repeat classes follows the same tendency observed in other plant bac-end sequences. finally, the most abundant motif was the tetranucleotide aatt. interestingly there is a high incidence of tetranucleotide motifs compared to other classes of microsatellites in our data set. this arrangement is unusual in dicots, where dinucleotides or mononucleotides are normally the most abundant repeats. exploiting a microsatellite-enriched library of p. edulis, skewed for dinucleotides due to the probes used in its preparation, oliveira and co-authors also observed a higher proportion of tetranucleotides compared to other microsatellite classes. protein coding regions with the aim of identifying protein-coding regions, the bac-end sequences were compared to the non-redundant protein data bank using the blastx search engine. around % of all bes were found to have high levels of similarity to genes of several plant species. the vast majority of the top-hits made by our sequences were to populus trichocarpa, theobroma cacao, ricinus communis, vitis vinifera and prunus persica. this scenario was expected since these are species related to p. edulis on which there is an abundance of genomic data. the list of species hits in descending order is shown in figure  figure list of species that produced significant hits with the bac-end sequences of passiflora edulis based upon sequence similarity to plant genes. assuming that % of the bes analysed had matching coding regions, we can estimate that ~ mbp of the passion fruit genome consist of coding sequences. assuming an average gene length of  kb, the same as vitis vinifera, we can estimate that the total number of genes per haploid genome of p. edulis is. both the estimate of the coding portion and the number of genes are consistent with the figures proposed for phaleanopsis equestris, a species with a genome size very close to that of p. edulis. our approximation is within the range of the gene numbers estimated for the closest relatives vitis vinifera and populus trichocarpa. altogether ontological terms were assigned to of the sequences analysed, resulting in an average of terms per sequence. up to go terms were annotated for the sequences, and those related to molecular function were the most abundant. of the total sequences with assigned ontological terms, were assigned to at least one term related to molecular function, to at least one term related to biological process, and to at least one term related to cellular component. any term was assigned to of the sequences which contained matches with coding sequences, and of the sequences were mapped but not annotated. within the category molecular function, the most frequent terms were binding, catalytic activity and transporter activity. within the category biological process, the most frequent terms were cellular process, metabolic process, single-organism process, and response to stimulus. finally, in the category cellular component, the most common terms were cell, organelle and membrane.figure distribution of go annotations of gene products predicted from the passiflora edulis bess for molecular function, biological process and cellular component. only major categories are presented. comparative genome mapping in total, bes were compared to the sequenced arabidopsis thaliana, vitis vinifera, and populus trichocarpa genomes for identifying microsyntenic regions. p. trichocarpa and v. vinifera are among the plant genomes with the highest number of top hits for p. edulis. although phylogenetically closer, the ricinus communis and prunus persica genomes are still in the early stages of their assembly and annotation, which enormously affects comparative mapping studies. the same is true for the draft genome of theobroma cacao. for obvious reasons, a. thaliana has been chosen as a default template.figure comparison of collinear microsyntenic regions identified in passiflora edulis bacs and arabidopsis thaliana, vitis vinifera and populus trichocarpa chromosomes. the matches were classified into six categories based on the classification in rampant and co-authors. the ‘single end’ category refers to bac end pairs for which only one of the ends matched a sequence in the reference genome; bes were assigned to the ‘paired end’ category when both end sequences matched the genome. the pe category, in turn, is subdivided into ‘co-localized’ and ‘non co-localized’. we considered all matches occurred at points to 350 kb apart on the reference genome; either in the correct orientation with respect to each other or rearranged with respect to each other. when the distance between the paired matches was either smaller or larger than these limits, they were assigned to the ‘gapped’ category. first, we identified positive matches to a. thaliana genome, ses and pes. four pes formed two ‘collinear’ alignments, composed of sequences pe85q4f and pe69q4g, encompassing and genes, respectively. the lengths of potential microsyntenic regions found in a. thaliana are close to the average insert sizes of the p. edulis bac library. for instance, the arabidopsis region shared at its ends by the pe85q4f sequences is 92 kb in length; the respective p. edulis bac is 93 kb. similarly, the arabidopsis region shared by the pe69q4g sequences is 80 kb in length; the respective p. edulis bac is 97 kb in length. it is interesting to note that arabidopsis regions overlap each other covering a region of  kb, from chromosome c positions  bp to  bp. clearly, this kind of information can be applied to assemble the two p. edulis bacs that harbour the mentioned sequences. in addition, the pair pe164q1g was classified as ‘rearranged’. it is noteworthy that the ends of the pe pe85q4f showed sequence similarity to a 30s subunit ribosomal rna and to a membrane hydrolase. additionally, the ends of the pe69q4g pe showed sequence similarity to an unspecified subunit of the nadh complex and to a portion of subunit of the nadh complex. according to cellular component annotation analysis, the four sequences belong to ‘chloroplast’ group. we are possibly facing a cpdna contamination not detected on the macroarrays. comparing genome intervals shared by p. edulis and v. vinifera, we identifed positive matches, ses and pes. among the pes, there were two ‘non co-localized’ pairs, one ‘collinear’ pair, one ‘rearranged’ pair, and one ‘gapped’ pair. the paired sequences pe164q2a f/r were mapped to chromosome of v. vinifera, the forward end beginning at position,417 bp of the grapevine genome and the reverse end terminating at position,226 bp, separated by around  kb. this region encompasses genes, including small genes consisting of single exons, and large genes consisting of more than exons. compared to the p. edulis interval, the region in question is twice the length of the bac pe164q2a. finally, comparative mapping between the p. edulis and p. trichocarpa genomes revealed a large number of microsyntenic regions. altogether, positive matches were found in p. edulis sequences, including ses and pes. three paired sequences of p. edulis were ‘collinear’ to those of p. trichocarpa. in addition, pe173q4a was classified as ‘rearranged’ and pe164q1g as ‘gapped’. the first ‘collinear’ pair pe216q4a is located on chromosome of p. trichocarpa, over an interval consisting of ~217 kb of dna harbouring genes. the second pair, pe75q4b is separated by ~176 kb, including around genes, and the third, pe164q1f is separated by a 306 kb interval in p. trichocarpa genome that contains genes involved in a number of different physiological processes. all these paired sequences were correctly orientated and separated by larger intervals in the p. trichocarpa genome, compared to p. edulis. it is interesting to note that paired sequences pe75q4b and pe164q1f were both assigned to chromosome in p. trichocarpa, making these bacs good candidates for a bac-by-bac sequencing project based on a bes database. the pair consisting of pe173q4a sequences was classified as ‘rearranged’ because of their incorrect orientation with respect to each other, and the pair consisting of pe164q1g sequences was classified as ‘gapped’, since the distance between the sequences is around 6 mb, much larger than the maximum acceptable span. this huge distance could be the result of a major chromosome event in an ancestral genome, for instance, involving massive insertion as the predominant event. it is interesting that this pair was also mapped in the genomes of a. thaliana and v. vinifera, but spanning much smaller intervals. the pes included classified as ‘non co-localized’. these events are possibly due to macro chromosomal rearrangements that occurred since the split of p. trichocarpa, a hypothesis that finds support in the fact that p. trichocarpa has twice as many chromosomes as p. edulis. the above results are summarized in additional file 1: table s we expected to find a number of potentially microsyntenic regions between the p. edulis and p. trichocarpa genomes since the latter is evolutionarily closer evolutionarily to passiflora than v. vinifera and a. thaliana. obviously ‘collinear’ relationships tend to erode as we move further away from the genus passiflora.  we explored approximately bac-end sequences of the yellow passion fruit genome after generating a bac library for the species. this is the first large-insert library for a member of passifloraceae and a valuable tool for future sequencing and genome studies. we were able to verify the quality of the library, its genome coverage and utility for gene isolation. this was done using exclusively the few passiflora gene sequences deposited in public databases as probes in macroarray hybridizations, and the corresponding bacs used in fish experiments allowed us to map four genes to three chromosomes of the species. we were able to identify reads likely to contain repetitive mobile elements, simple sequence repeats and putative proteins. we estimate a ~42% gc content from the analysis of the reads. approximately bac end-sequences contained protein sequences, and ontological terms were assigned to of them. comparison analyses of the sequences generated were performed against sequence data from two phylogenetically close species p. trichocarpa and v. vinifera, as well as from a. thaliana. around positive matches or sequence similarities were detected. finally, a number of bac-end pair sequences could be mapped to intervals of a. thaliana, v. vinifera and p. trichocarpa chromosomes, representing regions of potential microsynteny. 
then, we explored bes and we identified reads likely to contain repetitive mobile elements, simple sequence repeats and putative proteins, and to estimate the gc content of the reads. the vast majority of the top-hits made by our sequences were to populus trichocarpa, theobroma cacao, ricinus communis, vitis vinifera and prunus persica. remarkably, a number of bac-end pair sequences could be mapped to intervals of the sequenced arabidopsis thaliana, v. vinifera and p. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified. therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. the library represents six haploid genome equivalents, and the average insert size was 108 kb. around % of all bes were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed. we generated the first large-insert library for a member of passifloraceae. the passion fruit is a tropical crop of economic importance both for juice production and consumption as fresh fruit. in this study, we have constructed a large-insert bac library and provided the first view on the structure and content of the passion fruit genome, using bac-end sequence data as a major resource. passiflorapassion fruitgenomicsbac-end sequencingrepetitive elementsgene contentmicrosyntenyfluorescent in situ hybridizationissue-copyright-statement© the author 2014 this bac library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. to check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight passiflora gene sequences available in public databases. the library consisted of clones and its levels of organellar dna were very low. bacs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four bacs in three chromosomes.
 the passion fruit is a tropical crop of economic importance both for juice production and consumption as fresh fruit. the juice is also used in concentrate blends that are consumed worldwide. however, very little is known about the genome of the species. therefore, improving our understanding of passion fruit genomics is essential and to some degree a pre-requisite if its genetic resources are to be used more efficiently. in this study, we have constructed a large-insert bac library and provided the first view on the structure and content of the passion fruit genome, using bac-end sequence data as a major resource.  the library consisted of clones and its levels of organellar dna were very low. the library represents six haploid genome equivalents, and the average insert size was 108 kb. to check its utility for gene isolation, successful macroarray screening experiments were carried out with probes complementary to eight passiflora gene sequences available in public databases. bacs harbouring those genes were used in fluorescent in situ hybridizations and unique signals were detected for four bacs in three chromosomes. then, we explored bes and we identified reads likely to contain repetitive mobile elements, simple sequence repeats and putative proteins, and to estimate the gc content of the reads. around % of all bes were found to have high levels of similarity to plant genes and ontological terms were assigned to more than half of the sequences analysed. the vast majority of the top-hits made by our sequences were to populus trichocarpa, theobroma cacao, ricinus communis, vitis vinifera and prunus persica.  we generated the first large-insert library for a member of passifloraceae. this bac library provides a new resource for genetic and genomic studies, as well as it represents a valuable tool for future whole genome study. remarkably, a number of bac-end pair sequences could be mapped to intervals of the sequenced arabidopsis thaliana, v. vinifera and p. trichocarpa chromosomes, and putative collinear microsyntenic regions were identified. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords passiflorapassion fruitgenomicsbac-end sequencingrepetitive elementsgene contentmicrosyntenyfluorescent in situ hybridizationissue-copyright-statement© the author 2014 