BACKGROUND
cucumis melo  is an important crop worldwide.
it belongs to the cucurbitaceae family, which also includes cucumber, watermelon, pumpkin and squash, and whose economic importance, among horticulture crops, is second only to solanaceae.
melon has 2n =  <dig> chromosomes and its haploid genome contains  <dig>  ×  <dig> bp, only three times larger than the arabidopsis genome and similar in size to the rice genome  <cit> .
melon has high intra-specific genetic variation and morphologic diversity  <cit>  that, together with its small genome size, make it suitable for a great variety of molecular and genetic studies that can lead to the development of tools for crop improvement.
genomics approaches to melon breeding have already been successfully applied to the molecular characterization of important agronomic traits such as pathogen resistances  <cit> .
recent research has increased the genetic and genomic resources for melon  <cit> , such as the sequencing of ests  <cit> , the construction of bac libraries  <cit> , the development of an oligo-based microarray  <cit> , the production of melon mutation libraries for tilling analyses  <cit>  or the development of a collection of near isogenic lines   <cit> .
several genetic maps have also been reported for melon and a consensus genetic map, obtained by merging available maps using a common set of ssrs as anchor markers, has recently been obtained by the international cucurbit genomics initiative   <cit> .
the melonomics project, aimed at the sequencing of the complete melon genome following a whole-genome shotgun strategy that makes use of  <dig> sequencing data, has recently been started in spain.
a double-haploid line  population from the cross between the korean accession pi  <dig> and the inodorus type 'piel de sapo' t <dig> was the basis for the construction of 1) a bac library with an average insert size of  <dig> kb, representing  <dig>  genome equivalents of the c. melo haploid genome and 2) a genetic map with around  <dig> markers, of which more than  <dig> are gene-based markers   <cit> .
a fraction of the genetic markers in the melon genetic map has been mapped at low resolution using the bin-mapping strategy  <cit> .
these tools are essential for the construction of a melon physical map, a resource that greatly facilitates assembly and refinement of whole genome sequencing data and that can also be used to define a minimum tilling path of bac clones in bac-to-bac genome sequencing strategies.
the utility of physical maps has been reported by several classical genome sequencing projects such as those of human  <cit> , arabidopsis  <cit>  and rice  <cit> .
these first physical maps were constructed using restriction enzyme digestion of bac dna and agarose gel electrophoresis, with the restriction patterns analyzed by fingerprinted contigs  software to obtain contigs of bac clones  <cit> .
as an alternative to agarose gel-based fingerprinting methods, which are time-consuming and can often lead to poor maps due to the need for manual band calling and the comparatively low resolution of agarose gels, fluorescence-labeled capillary electrophoresis methods have been developed to produce larger, more accurate contigs using larger sets of bac clones  <cit> .
the evaluation of five different fingerprinting methods lead to the conclusion that the high-information-content fingerprinting method  with five-enzyme digestion plus snapshot labeling is the most effective  <cit> .
hicf methods have already been applied to the development of physical maps of several species such as catfish, apple, grape, wheat, b. rapa, peach, papaya, trout, brachypodium or maize but no physical maps have as yet been developed for any cucurbitaceae species  <cit> .
however, for physical maps to be useful, bac contigs need to be anchored to genetic maps in order to establish the relative genomic position of the maximum number of physical map fragments.
the anchored contigs can then be used as seed points for bidirectional chromosome sequencing, a crucial resource to fill gaps and refine assemblies of genome draft sequencing data.
pcr-based methods combined with adequate pooling of bac dna samples have proved to be very cost- and time-effective for the anchoring of bac libraries to genetic maps  <cit> .
here we report the construction of the first physical map of a cucurbitaceae species described so far, using a  <dig> × bac library and a genetic map previously developed in our laboratories.
hicf was carried out on  <dig>  bac clones, digesting with five restriction enzymes and snapshot labeling, followed by contig assembly with fpc software.
anchoring the bac library to the genetic map has also allowed the genetic positioning of  <dig> physical contigs/singletons to chromosomal loci.
the melon physical map fpc database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.
RESULTS
bac fingerprinting and contig assembly
a bac library from the double-haploid melon line 'pit92' had been previously constructed in our laboratory with an average insert size of  <dig> kb and representing  <dig>  genome equivalents of the c. melo haploid genome   <cit> .
all  <dig>  bac clones, of which 80% are estimated to be non-empty clones, were fingerprinted by digestion with five restriction enzymes and posterior labeling using the snapshot kit.
the labeled fragments were sized using an abi <dig> dna sequencer and the fpb software used to remove background, poor quality fingerprints, vector bands and fingerprints with less than  <dig> bands in the  <dig> bp- <dig> bp range  <cit> .
the resulting  <dig>  clones  had an average of  <dig> valid bands per fingerprint , which where subsequently subjected to contig assembly using the fpc software with tolerance  <dig>  bp  <cit> .
number of contigs with q clones
number of fpc contigs and singletons linked to
a/: contigs/singletons
b contigs or singletons anchored to markers with conflicting genetic positions are not considered
c length of physical contigs and singletons anchored to each chromosome appears in parenthesis
d contigs and singletons anchored to markers that map loci in two chromosomes
an initial test to determine the optimal cutoff value, minimizing the contig number while avoiding a large number of questionable clones , was performed with several cutoff values in the 1e-35/1e- <dig> range.
lower cutoff values mean more stringent assembly conditions that prevent chimeric contig assemblies but at the possible cost of breaking true contigs, increasing the number of contigs and of unassembled clones .
based on the test results , a sulston score of 1e- <dig> was chosen for the first automatic assembly.
the physical map was built according to a standard iterated procedure with the initial cutoff stringency sufficient to give valid contigs, which are then gradually merged at successively greater cutoff values.
details of the procedure are described in the methods section and a summary of the successive physical map assemblies is given in table  <dig>
tests for
assembly
dqer
aan initial test to determine the optimal cutoff value was performed with several cutoff values in the 1e-35/1e- <dig> range
bnumber of contigs containing ≤/> than 5% of q-clones
ca cutoff value of 1e- <dig> was used for the initial contig assembly.
consecutive dqer , end-merge and singleton-merge steps were performed to minimize the number of singletons, contigs and q-clones
ddistribution according to the number of clones belonging to each contig
the final physical map  had  <dig>  contigs and  <dig> singletons, with an estimated physical length of  <dig> mb .
the longest contig was  <dig>  mb long; the average contig length,  <dig> kb; 40% of the contigs were made up of the assembly of more than  <dig> clones; 84% of the contigs contained between  <dig> and  <dig> clones, and  <dig> contigs contained less than the maximum number of q-clones, 5%.
this represents a small percentage of q-contigs and was the result of the forced breaking of all contigs with more than 5% of q clones in the first stage of construction.
this was achieved by reducing the cutoff to values as low as 1e- <dig> where necessary.
although this increased the number of contigs and unassembled clones, the reliability of the resulting contigs was greatly improved.
these figures are comparable to those of other plant physical maps recently described.
for example, the physical map of b. rapa, a species with a genome size of  <dig> mb, similar to that of melon, was built by fingerprinting  <dig>  bac clones .
it has  <dig>  contigs of average length  <dig> kb, 57% of which are made up of more than  <dig> clones, and the estimated genomic coverage of the map is  <dig>  ×   <cit> .
however, the number of singletons  is more than  <dig> times higher than that in our map, even though the iterated procedures used to build both maps were similar.
the fact that the b. rapa library represents  <dig>  times more genomic equivalents than the melon could partially explain the differences in singleton number.
the analysis of the number of q clones after each round of construction at different cutoffs suggests that clones remaining as singletons in the b. rapa map may not just be due to low quality fingerprints but may come from regions of low coverage in the bac libraries used  <cit> .
this means that the differences in number of singletons on these physical maps could also be due to differences in the representation of the b. rapa and c. melo genomes in the different libraries.
as another example, the physical map of papaya, a species with a genome size of  <dig> mb, slightly smaller than that of melon, was built by fingerprinting clones representing  <dig>  genomic equivalents.
it has  <dig> contigs, 59% of which are made up of more than  <dig> clones, with an estimated genomic coverage of  <dig>  ×  <cit> .
the difference in genomic coverage of the libraries could again explain the higher number of singletons,  <dig> , ten times higher than in our map.
regarding the internal structure of the c. melo fpc contigs, the comparison of ordered lists of contigs, based on the contig physical length or the contig size , revealed a high proportion of 'stacked' contigs, that is, contigs containing regions where the depth far exceeds the estimated coverage of the library used , possibly due to the non-randomness affecting all libraries constructed by one enzyme restriction of genomic dna.
this poses a problem in that many clones in these stacked contigs do not contribute to extending the physical length of the contig and their fingerprints carry no new information.
also, the visual inspection of contigs revealed some assembly artifacts affecting several contigs.
for example, of three contigs containing more than  <dig> clones, the largest  most probably contains a large proportion of wrongly assembled clones.
the sequence of several bac-ends  of clones from this contig revealed tandem repeat sequences of dna in the form of ribosomal rna genomic regions .
similar results have been obtained with other repetitive sequences, such as retrotransposons, analyzing bes from other problematic contigs.
anchorage of the bac library to the genetic map
a total of  <dig> genetic markers  were used to anchor  <dig> bac clones from our genomic library to the genetic map .
genetic markers were selected from previous versions of the pi  <dig> × t <dig> melon genetic map, mainly rflps  <cit>  and snps  <cit> .
selected genetic markers were homogeneously distributed along the melon genetic map.
a bac pooling strategy and pcr-based library screening, using pairs of oligonucleotides designed for each marker, were used to identify positive bac clones.
a complete list of all markers analyzed together with their bibliographical references is in additional file  <dig> table s <dig>  when used to screen all bac superpools from the library,  <dig> pairs of primers  failed to amplify, even though they produced amplification bands when tested against melon genomic dna.
this points to the existence of genomic regions poorly, or not represented in the bac library used.
a total of  <dig> markers  could be linked to  <dig> bac clones, with  <dig> of these markers linked to a single clone while  <dig> markers linked to more than one clone.
this gave a total of  <dig> bac clones grouped in  <dig> sets of overlapping clones containing one common marker  .
a markers that failed to amplify when tested in the bac library even though pcr amplification bands were detected when parental
genomic dna was used as a positive control
the average number of bacs amplified per marker was  <dig> .
this is lower than expected based on the estimated genomic coverage of our library .
this is a further indication of the absence of several genomic loci or overestimation of representation in the library used.
a distribution of the number of positive bacs found per marker shows a two-zone distribution with about 79% of markers evenly distributed in the 1- <dig> bac/marker range while the remaining markers are linked on average to 9- <dig> bac/marker, probably reflecting the fact that about 20% of the primers used amplified duplicated or closely related genomic sequences .
in fact, while only 15% of the snp markers were linked to contigs of more than six bac clones, 26% of all anchored rflps were linked to between  <dig> and  <dig> clones.
this indicates that the rflp-derived primers were twice as prone to amplifying duplicated sequences as the snp-derived primers.
while an average of  <dig>  clones were linked to rflp markers, only  <dig>  were linked to snps.
based on these results, it can be tentatively assumed that the genomic coverage of the library is somewhere in the 4- <dig> range.
integration of the physical and genetic maps
the anchorage of bac clones to the genetic map was used to establish a link between the fingerprint-based physical map and the genetic linkage map.
to this end, information regarding anchored genetic markers and their chromosome locations was introduced in the fpc database for all anchored bac clones successfully fingerprinted.
as a result,  <dig> genetic markers were anchored to the physical map, with  <dig> of them linked to fpc contigs of several lengths while  <dig> linked only to singletons .
figure  <dig> shows an example of an fpc contig anchored to a melon linkage group using information derived from the genetic map.
one hundred and fifty-eight of the anchored fpc contigs were linked to just one marker,  <dig> to two markers, and two contigs to three markers.
all but  <dig> genetic markers mapped a single fpc contig or singleton:  <dig> markers mapped two separate contigs/singletons,  <dig> markers were each linked to three fpc contigs/singletons and one marker to four contigs.
therefore, 18% of all markers anchored to the physical map were linked to more than one fpc contig/singleton, a figure in accordance to the above estimation of the number of primers amplifiying duplicated or closely related genomic regions.
in all,  <dig>  fingerprinted bac clones, distributed in  <dig> contigs/singletons and representing  <dig> mb or  <dig> % of the melon genome, have been positioned in unique loci of the genetic map .
on average,  <dig> bac clones,  <dig> contigs and  <dig>  mb have been anchored to unique loci for every c. melo linkage group.
all information regarding contig estimated length, contig clone number and chromosome location of all fpc contigs or singletons anchored to genetic markers can be found in additional file  <dig> table s <dig>  a representation of the anchored genetic map together with information on which markers are linked to fpc contigs is shown in figure 3& <dig>  while more detailed information regarding linkage group distribution of the type and number of genetic markers analyzed and linked to both physical and anchored genetic maps is given in table  <dig>  the resulting genetic-anchored melon physical map fcp database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.
validation of physical map contigs
the comparison between pcr and fpc contigs served as a quality control of the physical map building.
column  <dig> in additional file  <dig> table s <dig> shows the number of clones of every pcr contig anchored to any genetic marker while column  <dig> shows how many of those clones are also present in the fpc contigs anchored to the correspondent marker.
bearing in mind that an estimated 20% of all primer pairs tested produced positive bacs belonging to two or even three separate genomic regions and that, accordingly, 18% of all fpc-anchored markers are linked to more than one fpc contig or singleton, the degree of coincidence between pcr and fpc contigs has been computed using data from markers linked to a single fpc contig/singleton.
as an average, 75% of those clones belonging to the same pcr contig are predicted to overlap according to the fpc information.
therefore, as 20% of the library clones were not successfully fingerprinted and so are absent from the physical map, we estimate that the degree of coincidence between pcr and fpc contigs is around 80%.
however, when distinguishing duplicated or closely related genomic regions or gene families, the pcr screening procedure used to anchor the bac library to the genetic map - i.e.
pcr amplification of regions 100- <dig>  bp long - should be much less efficient than fingerprinting whole bac genomic regions.
this can account for several of those clones that belong to pcr contigs but are absent from the correspondent fpc contig and, if so, the degree of coincidence between our physical and anchored genetic maps would be higher than the above estimation.
as another way to validate the physical map, the linkage group position of those markers linked to a common fpc contig was compared.
as described above, of all fpc contigs/singletons linked to genetic markers, only  <dig> were anchored to more than one marker each.
of these, ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig>  ctg <dig> and singleton cm19_g <dig>  are each linked to two or three markers that have the same position in the genetic map , which validates these fpc contigs as good assemblies.
two additional contigs, ctg <dig> and ctg <dig>  were each linked to pairs of markers with conflicting genetic positions.
an analysis of the construction of each contig revealed the merging of previous smaller contigs during the final steps of the autobuild process at greater cutoffs/lower stringency , which explains the presence of incompatible markers in these contigs.
another contig, ctg <dig>  is also linked to a set of two incompatible genetic markers.
however, in this case, the clones belonging to the corresponding pcr contig coincide, and so they probably map two separate genomic regions that share some degree of sequence identity.
the last two contigs, ctg <dig> and ctg  <dig>  are also linked to conflicting sets of genetic markers , but this cannot be explained by low stringency automerge.
CONCLUSIONS
here we describe the physical map of cucumis melo, the first example of a cucurbitaceae physical map so far developed.
the map fpc database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.
as melon is a species with an average sized genome , the  <dig> enzyme/snapshot hicf method is the natural choice to build a physical map providing significant coverage of the genome.
using a bac library representing about five genomic equivalents, the estimated physical length of the map is  <dig> mb .
the anchorage of the bac library to the available genetic map has allowed the genetic positioning of  <dig> physical contigs that represent an estimated 12% of the melon genome.
the data presented is already helping to improve the quality of the available genomic sequence of this species, with considerable research effort to obtain a complete genomic sequence of the melon currently being carried out in spain, adopting a whole-genome shotgun approach based on new generation massive sequencing data.

 cucumis melo is an important crop worldwide. it belongs to the cucurbitaceae family, which also includes cucumber, watermelon, pumpkin and squash, and whose economic importance, among horticulture crops, is second only to solanaceae. melon has 2n = chromosomes and its haploid genome contains × bp, only three times larger than the arabidopsis genome and similar in size to the rice genome. melon has high intra-specific genetic variation and morphologic diversity that, together with its small genome size, make it suitable for a great variety of molecular and genetic studies that can lead to the development of tools for crop improvement. genomics approaches to melon breeding have already been successfully applied to the molecular characterization of important agronomic traits such as pathogen resistances. recent research has increased the genetic and genomic resources for melon, such as the sequencing of ests, the construction of bac libraries, the development of an oligo-based microarray, the production of melon mutation libraries for tilling analyses or the development of a collection of near isogenic lines. several genetic maps have also been reported for melon and a consensus genetic map, obtained by merging available maps using a common set of ssrs as anchor markers, has recently been obtained by the international cucurbit genomics initiative. the melonomics project, aimed at the sequencing of the complete melon genome following a whole-genome shotgun strategy that makes use of sequencing data, has recently been started in spain. a double-haploid line population from the cross between the korean accession pi and the inodorus type'piel de sapo' t was the basis for the construction of 1) a bac library with an average insert size of kb, representing genome equivalents of the c. melo haploid genome and 2) a genetic map with around markers, of which more than are gene-based markers. a fraction of the genetic markers in the melon genetic map has been mapped at low resolution using the bin-mapping strategy. these tools are essential for the construction of a melon physical map, a resource that greatly facilitates assembly and refinement of whole genome sequencing data and that can also be used to define a minimum tilling path of bac clones in bac-to-bac genome sequencing strategies. the utility of physical maps has been reported by several classical genome sequencing projects such as those of human, arabidopsis and rice. these first physical maps were constructed using restriction enzyme digestion of bac dna and agarose gel electrophoresis, with the restriction patterns analyzed by fingerprinted contigs software to obtain contigs of bac clones. as an alternative to agarose gel-based fingerprinting methods, which are time-consuming and can often lead to poor maps due to the need for manual band calling and the comparatively low resolution of agarose gels, fluorescence-labeled capillary electrophoresis methods have been developed to produce larger, more accurate contigs using larger sets of bac clones. the evaluation of five different fingerprinting methods lead to the conclusion that the high-information-content fingerprinting method with five-enzyme digestion plus snapshot labeling is the most effective. hicf methods have already been applied to the development of physical maps of several species such as catfish, apple, grape, wheat, b. rapa, peach, papaya, trout, brachypodium or maize but no physical maps have as yet been developed for any cucurbitaceae species. however, for physical maps to be useful, bac contigs need to be anchored to genetic maps in order to establish the relative genomic position of the maximum number of physical map fragments. the anchored contigs can then be used as seed points for bidirectional chromosome sequencing, a crucial resource to fill gaps and refine assemblies of genome draft sequencing data. pcr-based methods combined with adequate pooling of bac dna samples have proved to be very cost- and time-effective for the anchoring of bac libraries to genetic maps. here we report the construction of the first physical map of a cucurbitaceae species described so far, using a × bac library and a genetic map previously developed in our laboratories. hicf was carried out on bac clones, digesting with five restriction enzymes and snapshot labeling, followed by contig assembly with fpc software. anchoring the bac library to the genetic map has also allowed the genetic positioning of physical contigs/singletons to chromosomal loci. the melon physical map fpc database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.  bac fingerprinting and contig assembly a bac library from the double-haploid melon line'pit92' had been previously constructed in our laboratory with an average insert size of kb and representing genome equivalents of the c. melo haploid genome. all bac clones, of which 80% are estimated to be non-empty clones, were fingerprinted by digestion with five restriction enzymes and posterior labeling using the snapshot kit. the labeled fragments were sized using an abi dna sequencer and the fpb software used to remove background, poor quality fingerprints, vector bands and fingerprints with less than bands in the bp- bp range. the resulting clones had an average of valid bands per fingerprint, which where subsequently subjected to contig assembly using the fpc software with tolerance bp. number of contigs with q clones number of fpc contigs and singletons linked to a/: contigs/singletons b contigs or singletons anchored to markers with conflicting genetic positions are not considered c length of physical contigs and singletons anchored to each chromosome appears in parenthesis d contigs and singletons anchored to markers that map loci in two chromosomes an initial test to determine the optimal cutoff value, minimizing the contig number while avoiding a large number of questionable clones, was performed with several cutoff values in the 1e-35/1e- range. lower cutoff values mean more stringent assembly conditions that prevent chimeric contig assemblies but at the possible cost of breaking true contigs, increasing the number of contigs and of unassembled clones. based on the test results, a sulston score of 1e- was chosen for the first automatic assembly. the physical map was built according to a standard iterated procedure with the initial cutoff stringency sufficient to give valid contigs, which are then gradually merged at successively greater cutoff values. details of the procedure are described in the methods section and a summary of the successive physical map assemblies is given in table tests for assembly dqer aan initial test to determine the optimal cutoff value was performed with several cutoff values in the 1e-35/1e- range bnumber of contigs containing ≤/> than 5% of q-clones ca cutoff value of 1e- was used for the initial contig assembly. consecutive dqer, end-merge and singleton-merge steps were performed to minimize the number of singletons, contigs and q-clones ddistribution according to the number of clones belonging to each contig the final physical map had contigs and singletons, with an estimated physical length of mb. the longest contig was mb long; the average contig length, kb; 40% of the contigs were made up of the assembly of more than clones; 84% of the contigs contained between and clones, and contigs contained less than the maximum number of q-clones, 5%. this represents a small percentage of q-contigs and was the result of the forced breaking of all contigs with more than 5% of q clones in the first stage of construction. this was achieved by reducing the cutoff to values as low as 1e- where necessary. although this increased the number of contigs and unassembled clones, the reliability of the resulting contigs was greatly improved. these figures are comparable to those of other plant physical maps recently described. for example, the physical map of b. rapa, a species with a genome size of mb, similar to that of melon, was built by fingerprinting bac clones. it has contigs of average length kb, 57% of which are made up of more than clones, and the estimated genomic coverage of the map is ×. however, the number of singletons is more than times higher than that in our map, even though the iterated procedures used to build both maps were similar. the fact that the b. rapa library represents times more genomic equivalents than the melon could partially explain the differences in singleton number. the analysis of the number of q clones after each round of construction at different cutoffs suggests that clones remaining as singletons in the b. rapa map may not just be due to low quality fingerprints but may come from regions of low coverage in the bac libraries used. this means that the differences in number of singletons on these physical maps could also be due to differences in the representation of the b. rapa and c. melo genomes in the different libraries. as another example, the physical map of papaya, a species with a genome size of mb, slightly smaller than that of melon, was built by fingerprinting clones representing genomic equivalents. it has contigs, 59% of which are made up of more than clones, with an estimated genomic coverage of ×. the difference in genomic coverage of the libraries could again explain the higher number of singletons,, ten times higher than in our map. regarding the internal structure of the c. melo fpc contigs, the comparison of ordered lists of contigs, based on the contig physical length or the contig size, revealed a high proportion of'stacked' contigs, that is, contigs containing regions where the depth far exceeds the estimated coverage of the library used, possibly due to the non-randomness affecting all libraries constructed by one enzyme restriction of genomic dna. this poses a problem in that many clones in these stacked contigs do not contribute to extending the physical length of the contig and their fingerprints carry no new information. also, the visual inspection of contigs revealed some assembly artifacts affecting several contigs. for example, of three contigs containing more than clones, the largest most probably contains a large proportion of wrongly assembled clones. the sequence of several bac-ends of clones from this contig revealed tandem repeat sequences of dna in the form of ribosomal rna genomic regions. similar results have been obtained with other repetitive sequences, such as retrotransposons, analyzing bes from other problematic contigs. anchorage of the bac library to the genetic map a total of genetic markers were used to anchor bac clones from our genomic library to the genetic map. genetic markers were selected from previous versions of the pi × t melon genetic map, mainly rflps and snps. selected genetic markers were homogeneously distributed along the melon genetic map. a bac pooling strategy and pcr-based library screening, using pairs of oligonucleotides designed for each marker, were used to identify positive bac clones. a complete list of all markers analyzed together with their bibliographical references is in additional file table s when used to screen all bac superpools from the library, pairs of primers failed to amplify, even though they produced amplification bands when tested against melon genomic dna. this points to the existence of genomic regions poorly, or not represented in the bac library used. a total of markers could be linked to bac clones, with of these markers linked to a single clone while markers linked to more than one clone. this gave a total of bac clones grouped in sets of overlapping clones containing one common marker. a markers that failed to amplify when tested in the bac library even though pcr amplification bands were detected when parental genomic dna was used as a positive control the average number of bacs amplified per marker was. this is lower than expected based on the estimated genomic coverage of our library. this is a further indication of the absence of several genomic loci or overestimation of representation in the library used. a distribution of the number of positive bacs found per marker shows a two-zone distribution with about 79% of markers evenly distributed in the 1- bac/marker range while the remaining markers are linked on average to 9- bac/marker, probably reflecting the fact that about 20% of the primers used amplified duplicated or closely related genomic sequences. in fact, while only 15% of the snp markers were linked to contigs of more than six bac clones, 26% of all anchored rflps were linked to between and clones. this indicates that the rflp-derived primers were twice as prone to amplifying duplicated sequences as the snp-derived primers. while an average of clones were linked to rflp markers, only were linked to snps. based on these results, it can be tentatively assumed that the genomic coverage of the library is somewhere in the 4- range. integration of the physical and genetic maps the anchorage of bac clones to the genetic map was used to establish a link between the fingerprint-based physical map and the genetic linkage map. to this end, information regarding anchored genetic markers and their chromosome locations was introduced in the fpc database for all anchored bac clones successfully fingerprinted. as a result, genetic markers were anchored to the physical map, with of them linked to fpc contigs of several lengths while linked only to singletons. figure shows an example of an fpc contig anchored to a melon linkage group using information derived from the genetic map. one hundred and fifty-eight of the anchored fpc contigs were linked to just one marker, to two markers, and two contigs to three markers. all but genetic markers mapped a single fpc contig or singleton: markers mapped two separate contigs/singletons, markers were each linked to three fpc contigs/singletons and one marker to four contigs. therefore, 18% of all markers anchored to the physical map were linked to more than one fpc contig/singleton, a figure in accordance to the above estimation of the number of primers amplifiying duplicated or closely related genomic regions. in all, fingerprinted bac clones, distributed in contigs/singletons and representing mb or % of the melon genome, have been positioned in unique loci of the genetic map. on average, bac clones, contigs and mb have been anchored to unique loci for every c. melo linkage group. all information regarding contig estimated length, contig clone number and chromosome location of all fpc contigs or singletons anchored to genetic markers can be found in additional file table s a representation of the anchored genetic map together with information on which markers are linked to fpc contigs is shown in figure 3& while more detailed information regarding linkage group distribution of the type and number of genetic markers analyzed and linked to both physical and anchored genetic maps is given in table the resulting genetic-anchored melon physical map fcp database is available for download at http://melonomics.upv.es/static/files/public/physical_map/. validation of physical map contigs the comparison between pcr and fpc contigs served as a quality control of the physical map building. column in additional file table s shows the number of clones of every pcr contig anchored to any genetic marker while column shows how many of those clones are also present in the fpc contigs anchored to the correspondent marker. bearing in mind that an estimated 20% of all primer pairs tested produced positive bacs belonging to two or even three separate genomic regions and that, accordingly, 18% of all fpc-anchored markers are linked to more than one fpc contig or singleton, the degree of coincidence between pcr and fpc contigs has been computed using data from markers linked to a single fpc contig/singleton. as an average, 75% of those clones belonging to the same pcr contig are predicted to overlap according to the fpc information. therefore, as 20% of the library clones were not successfully fingerprinted and so are absent from the physical map, we estimate that the degree of coincidence between pcr and fpc contigs is around 80%. however, when distinguishing duplicated or closely related genomic regions or gene families, the pcr screening procedure used to anchor the bac library to the genetic map - i.e. pcr amplification of regions 100- bp long - should be much less efficient than fingerprinting whole bac genomic regions. this can account for several of those clones that belong to pcr contigs but are absent from the correspondent fpc contig and, if so, the degree of coincidence between our physical and anchored genetic maps would be higher than the above estimation. as another way to validate the physical map, the linkage group position of those markers linked to a common fpc contig was compared. as described above, of all fpc contigs/singletons linked to genetic markers, only were anchored to more than one marker each. of these, ctg ctg ctg ctg ctg ctg ctg ctg and singleton cm19_g are each linked to two or three markers that have the same position in the genetic map, which validates these fpc contigs as good assemblies. two additional contigs, ctg and ctg were each linked to pairs of markers with conflicting genetic positions. an analysis of the construction of each contig revealed the merging of previous smaller contigs during the final steps of the autobuild process at greater cutoffs/lower stringency, which explains the presence of incompatible markers in these contigs. another contig, ctg is also linked to a set of two incompatible genetic markers. however, in this case, the clones belonging to the corresponding pcr contig coincide, and so they probably map two separate genomic regions that share some degree of sequence identity. the last two contigs, ctg and ctg are also linked to conflicting sets of genetic markers, but this cannot be explained by low stringency automerge.  here we describe the physical map of cucumis melo, the first example of a cucurbitaceae physical map so far developed. the map fpc database is available for download at http://melonomics.upv.es/static/files/public/physical_map/. as melon is a species with an average sized genome, the enzyme/snapshot hicf method is the natural choice to build a physical map providing significant coverage of the genome. using a bac library representing about five genomic equivalents, the estimated physical length of the map is mb. the anchorage of the bac library to the available genetic map has allowed the genetic positioning of physical contigs that represent an estimated 12% of the melon genome. the data presented is already helping to improve the quality of the available genomic sequence of this species, with considerable research effort to obtain a complete genomic sequence of the melon currently being carried out in spain, adopting a whole-genome shotgun approach based on new generation massive sequencing data. 
a project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. the anchoring of bac clones to genetic markers also allowed the genetic positioning of physical map contigs/singletons, representing mb of the melon genome, to individual chromosomal loci. the physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. melon has high intra-specific genetic variation, morphologic diversity and a small genome size, which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. a number of genetic and genomic resources have already been developed, such as several genetic maps and bac genomic libraries. a melon physical map was constructed using a × bac library and a genetic map previously developed in our laboratories. cucumis melo belongs to the cucurbitaceae family, whose economic importance among horticulture crops is second only to solanaceae. high-information-content fingerprinting was carried out on bac clones, digesting with five restriction enzymes and snapshot labeling, followed by contig assembly with fpc software. these tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. the data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in spain, adopting a whole genome shotgun approach based on sequencing data. however, no physical map of any cucurbitaceae has yet been developed. a bac-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced. the physical map has contigs and singletons, with an estimated physical length of mb and the longest contig being mb. here we report the construction of the first physical map of a cucurbitaceae species described so far.
 cucumis melo belongs to the cucurbitaceae family, whose economic importance among horticulture crops is second only to solanaceae. melon has high intra-specific genetic variation, morphologic diversity and a small genome size, which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. a number of genetic and genomic resources have already been developed, such as several genetic maps and bac genomic libraries. these tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. however, no physical map of any cucurbitaceae has yet been developed. a project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. a bac-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced.  a melon physical map was constructed using a × bac library and a genetic map previously developed in our laboratories. high-information-content fingerprinting was carried out on bac clones, digesting with five restriction enzymes and snapshot labeling, followed by contig assembly with fpc software. the physical map has contigs and singletons, with an estimated physical length of mb and the longest contig being mb. the anchoring of bac clones to genetic markers also allowed the genetic positioning of physical map contigs/singletons, representing mb of the melon genome, to individual chromosomal loci. the melon fpc database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.  here we report the construction of the first physical map of a cucurbitaceae species described so far. the physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. the data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in spain, adopting a whole genome shotgun approach based on sequencing data. 