BACKGROUND
high-density linkage maps based on transcribed genes are valuable resources to characterize genome structure, gene space distribution and synteny in species for which reference genome sequences are not yet available.
dense gene maps can also support genome sequence assembly  <cit>  and the identification of genes underlying loci that control complex phenotypic traits  <cit> .
most methods used to date for linkage mapping genes in plants such as rflp, single-strand conformation polymorphism , cleaved amplified polymorphic sequence  and denaturing gradient gel electrophoresis have very limited throughput  <cit> .
high-throughput genotyping of single-nucleotide polymorphism  derived from genic sequences have allowed large-scale gene mapping.
however such efforts require relatively high up-front snp development, screening and validation costs, which have restricted this technology to the major crop plants  <cit>  and a few forest trees  <cit> .
furthermore, current snp assay methods based on allele-specific primer extension can be of variable robustness in highly heterozygous genomes such as those of forest trees with high nucleotide diversity, frequently above 1%  <cit> .
dna microarrays have been used to reliably detect genetic differences among individuals  <cit>  as shown initially in the simple genome of yeast  <cit>  and later in arabidopsis by borevitz et al.
<cit> , who termed these genetic variants single feature polymorphisms .
the principle of sfp genotyping is based on the disruption of the hybridization of labeled dna sequences that are genetically variable relative to a reference sequence used for design of probes on a microarray.
segregating sequence differences among individuals in a mapping population can be detected based on the variation of the hybridization signal  <cit> .
sfps have been detected by hybridizing total genomic dna to microarrays in yeast  <cit> , arabidopsis  <cit> , and rice  <cit> .
ronald et al.
<cit> , rostoks et al.
<cit>  and cui et al.
<cit>  expanded the scope of the method by hybridizing rna-derived cdna or crna to expression microarrays in yeast and barley, with the anticipation that polymorphisms in protein-coding dna sequences that are transcribed into messenger rna would also weakly hybridize to microarray probes.
while this approach reduces the complexity of the pool of sequences hybridized to the microarray, differences in signal intensity that arise as a consequence of sequence variation among genotypes can be confounded with differences in transcript abundance.
sfp mapping involves the detection of probes revealing putative sfp whose behavior as mendelian markers is then evaluated in segregating populations.
sfp have been largely developed for inbred, highly homozygous species.
in these studies, a putative sfp is identified based on signal differences between two parental lines.
candidate sfps are then tested for a bimodal distribution in hybridization signal detected in a segregating population of recombinant inbred lines or backcross progeny, and individuals are assigned to the expected genotypes defined using the signal intensity of the parents as references  <cit> .
using this strategy in arabidopsis, singer et al.
<cit>  built a genetic map with  <dig> genes by sfps detection using dna hybridization while west et al.
<cit>  used complementary rna  hybridization to map  <dig> genes based on sfp.
in the more complex genome of barley, luo et al.
<cit>  reported mapping  <dig>  and  <dig>  sfps using crna from leaf and embryo from  <dig> segregating doubled-haploid lines.
in the only study to date that attempted to genotype microarray markers in an outcrossing species, drost et al.
<cit>  mapped  <dig> sfps segregating 1: <dig> in a populus pseudo-backcross progeny involving  <dig> individuals.
in this study we report the detection and linkage mapping of sfps in a reference pedigree of eucalyptus grandis × e. urophylla, two highly heterozygous tree species.
detection of robust sfps was improved by an initial sfp-discovery step based on the pseudo-testcross strategy followed by using a selective mapping approach to simultaneously optimize linkage mapping and microarray costs.
a total of  <dig>  genes were successfully mapped,  <dig> of them positioned with high likelihood support on a framework genetic map.
the experimental approach employed is cost efficient and should be readily applicable to large scale mapping of genes in highly heterozygous genomes of several plant species providing a useful resource for downstream applications such as gene discovery, association genetics and assembly of reference genome sequences..
RESULTS
screening for candidate sfps
sfp screening, selection, genotyping and mapping involved a sequence of analytical steps summarized in a flowchart for easy visualization .
the sfp-discovery microarray included  <dig>  unique probes representing  <dig>  unigenes.
analysis of the screening data resulted in a set of  <dig>  probes  representing  <dig>  genes  detecting expressed transcripts in at least four progeny individuals of the discovery set of  <dig> individuals.
the number of probes detecting transcripts varied considerably across genes  and only for  <dig>  genes of the  <dig>  assayed  all the probes in the probe-set had signal consistently above the negative control threshold in all  <dig> individuals.
note that in a perfect scenario an expressed transcript would hybridize to all the probes of its corresponding probe set.
this indicates that detectable sequence polymorphisms between the short oligonucleotide probes and the hybridized transcripts are the rule rather than the exception.
following the sfp-discovery step, probes with the lowest mean and standard deviation  in the signal intensity were removed.
low signal and limited variation in signal intensity in the discovery set was taken as an indication that these probes were less likely to reveal robust sfps.
a new microarray with  <dig>  selected probes was designed representing  <dig>  genes, with a variable number of probes per probe-set .
this new microarray containing selected probes was used to genotype an additional set of  <dig> individuals drawn from the mapping population using a selective mapping approach.
this procedure identified the most informative individuals in terms of complementary recombination breakpoints from each parent, optimizing the linkage information to be extracted from these individuals and thus reducing the costs of the sfp genotyping step for map construction.
sfp genotyping in the mapping population
the averaged, log <dig> transformed, quantile-normalized probe signal detected for each individual was analyzed as described by drost et al.
<cit>  to simultaneously identify and genotype sfps.
briefly, for each probe the signal intensity measured in each individual offspring was assigned to either one of two distinct clusters using the k-means clustering algorithm.
using a chi-square test , probes showing a 1: <dig> pseudo-testcross segregation, as well as probes segregating as dominant  markers were selected.
a total of  <dig>  probes out of the  <dig>  tested followed a mendelian segregation ratio, with  <dig>  segregating 1: <dig> and  <dig>  segregating 3: <dig> , representing a total of  <dig>  genes.
the degree of separation between the two clusters for a probe was measured by calculating the probability of individuals assigned to one cluster being a member of the other cluster through a modified normal deviate zi .
individuals with zi equal to or less than  <dig>   are likely to overlap with the other cluster and were assigned as missing data to avoid genotype miscalls.
when displaying more than 10% missing data, the probe was discarded from further analysis.
after this stringent selection step,  <dig>  probes representing  <dig>  unigenes survived with a slightly larger number of probes segregating in a 3: <dig> than a 1: <dig> ratio  .
for  <dig> genes, two or more probes revealed sfps.
in those cases, the probe with fewer miscalls and segregating 3: <dig> was selected over the alternative.
in spite of their dominant behavior sfp markers segregating in a 3: <dig> ratio segregate from both parents, provide alellic bridges between the parental maps and when linked in coupling at small recombination distances have essentially the same information content as 1: <dig> backcross markers.
the resulting  <dig>  selected sfps,  <dig>  segregating 1: <dig> and  <dig>  segregating 3: <dig> were used in the subsequent linkage analysis.
*unigenes derived from a consensus sequence involving ests from different species are indicated as contigs, while singletons are listed by species.
**when several probes were selected per probe set, the number of unique genes represented is shown between parentheses.
construction of a gene-rich linkage map for eucalyptus
from the  <dig>  sfps that showed mendelian segregation, a total of  <dig>  sfps  were successfully grouped at a lod ≥  <dig>  and could be ordered along the linkage groups together with  <dig> microsatellites, providing a total map with  <dig>  markers with a relaxed marker order support.
details of this full map with all the ordered linkage information of the  <dig>  sfps and  <dig> microsatellites including map position, original unigene sequence from which the probe was designed, sequence of the probe revealing the sfp, best hit against genbank non-redundant database and annotation description generated using blast2go are presented in a table format .
to provide a gene map with high likelihood support for gene order, a framework map was built using the round  <dig> function of joinmap that removes markers that contribute to unstable order.
in the framework map  <dig> sfps were mapped together with  <dig> microsatellites thus resulting in a robust genetic linkage map with  <dig>  makers .
comparative statistics of the full and framework maps is presented .
linkage
inter-marker distances for the full map is not reported as such estimates might be ambiguous due to the low support for order
from the total number of sfps mapped in the framework map,  <dig> segregated in a 1: <dig> ratio, and  <dig> in a 3: <dig> ratio, identified by the acronym bc and f <dig>  respectively.
although no statistical test for clustering was carried out, markers seemed evenly distributed along the linkage groups with no obvious clustering except for some cases on the edge of linkage groups,.
some linkage groups  had more genes mapped than others  .
the sfp/microsatellite framework map had an average density of  <dig>  cm with  <dig> % of the intermarker distances smaller than  <dig> cm.
for only five of the  <dig>  intervals the distance was greater than  <dig> cm, with a maximum of  <dig>  cm.
even though the number of mapped markers increased more than five times when compared to the microsatellite-only map, the total genetic length was estimated at  <dig>  cm  which is within the expected range for eucalyptus  <cit> .
to evaluate the relative contribution of each class of sfp  to the quality of the genetic map, two separate maps were constructed including microsatellites and either sfps segregating 1: <dig> or 3: <dig>  the map that includes only the sfps segregating 1: <dig> decreased overall map quality, as illustrated by the expansion of total map length to  <dig>  cm based on the generally adopted premise that a shorter map is the most likely hypothesis to explain the data .
conversely, the length of the genetic map remained similar  when only the sfps segregating 3: <dig> were included.
thus the detection and use of sfps segregating 3: <dig> not only increases the total number of detectable sfps and consequently mappable genes, but also contributes to a higher quality map possibly due to their role as providers of allelic bridges when linked in coupling.
early sfp detection using a mixed-model analysis of variance
the signal intensity data of the  <dig> individuals of the sfp discovery set was analyzed by a mixed-model anova using the full probe-set for each gene separately to evaluate the correspondence between the putative sfps detected based on this procedure with those that were linkage mapped by the standard procedure adopted of sfp selection, genotyping and mapping .
after correcting the significance threshold of the f tests for multiple testing ,  <dig>  genes had significant genotype by probe interaction, corresponding to a putative sfp,.
these  <dig>  genes, all of them also included in the set of genes represented on the sfp-mapping microarray, encompassed  <dig>  of the  <dig>   that were mapped with lower confidence for marker order and  <dig> of the  <dig>  that were framework mapped by the standard sfp mapping procedure .
specific candidate sfp probes within probe-sets were then searched using the k-means clustering analysis and chi square segregation test for mendelian proportions.
a total of  <dig>  probes, representing  <dig>  genes, were selected in this analysis.
this set included probes for  <dig>  of the  <dig>  genes  that were mapped at a relaxed ordering threshold and  <dig> of the  <dig>  that were framework mapped by the standard procedure .
these results indicate that by testing each probe-set separately for probes that interact with genotypes, instead of  <dig>  probes, only  <dig>  would have been necessary on the sfp-mapping array to map close to 90% of the genes that were eventually mapped.
efficiency in detecting mappable sfp at the gene level  and probe level by the anova in comparison to the conventional linkage mapping approach
* in parentheses are indicated the percentage of genes or probes in common with those mapped by conventional linkage mapping considered as the benchmark approach
impact of the number of probes per probe-set on sfp detection
a total of  <dig> out of the  <dig>   genes for which five probes were used in the probe-set had at least one sfp discovered and were ultimately mapped.
in contrast, for the  <dig>  genes for which  <dig> probes were used, the efficiency was almost doubled, with  <dig> probes eventually mapping, an increase to  <dig> % a chi-square test of homogeneity with yates correction confirmed this difference to be highly significant  .
*the total number of mapped sfps used in the test  is lower than the total of sfp mapped  because numbers of probes different from  <dig> or  <dig> were used for some genes and these were removed from this analysis.
sfp validation on the eucalyptus draft genome
the linkage relationship of sfps to microsatellites was validated by using the  <dig> x draft genome sequence of e. grandis.
the location of  <dig> mapped microsatellites on the supercontigs was confidently determined using wu-blast and served to anchor supercontigs to linkage groups.
probes corresponding to  <dig> of the  <dig> sfps were mapped to scaffolds that contained one or more of the  <dig> anchor mapped microsatellites.
the remaining sfp probes mapped to supercontigs, but these were not anchored by any of the  <dig> microsatellites.
of these  <dig> sfps, only  <dig> were not genetically mapped to the same linkage group as the associated microsatellite, suggesting that they are trans-regulated gems .
for the remaining  <dig> sfp's  probes and microsatellites were correctly anchored to the same linkage group located within a  <dig> cm distance, on average, on the genetic map.
some of these probes could represent cis-regulated gems instead of sfps, but mapping of multiple probes within a probe-set to the same genetic position would have been expected in that case.
instead, probe-sets with multiple probes  showing mendelian segregation were only observed for  <dig> out of the  <dig> genes positioned on the framework map.
discussion
microarray-based sfp genotyping is an accessible approach for gene mapping
in this work, large numbers of sfp displaying mendelian behavior were readily detected using short  oligonucleotide probe arrays in a complex, highly heterozygous plant genome.
these sfps, in turn, allowed the construction of a gene-enriched linkage map providing valuable positional information for hundreds of transcribed genes.
the  <dig> -probe sfp-discovery oligonucleotide array used in this study was built from a relatively small est data set for current standards:  <dig>  phred- <dig> filtered 5'-sanger-sequenced ests assembled in  <dig>  unique sequences.
currently, much larger and more diverse est datasets can be rapidly generated at a fraction of the cost using next-generation sequencing technologies as demonstrated for uncharacterized plant genomes including eucalyptus  <cit> .
several millions of ests can be generated from pooled samples of rna for a couple of thousand us dollars providing a very rich source of sequence information to design a similar or much higher density oligonucleotide array.
costs of custom-made high-density microarrays have also dropped significantly and different formats and probe densities are available allowing optimized designs of experiments with multiple samples.
by using a two-step sfp discovery and mapping approach combined with selective mapping, one can envisage the construction of a high-density linkage map with thousands of genes for less than  <dig>  us dollars.
sfp-based gene mapping should therefore prove a very accessible and valuable experimental approach for several plant species.
recently, a high density diversity arrays technology  microarray for genome-wide genotyping has been developed for eucalyptus providing over  <dig>  polymorphic markers at the population level and an average of  <dig>  segregating polymorphic markers per pedigree  <cit> .
when compared to the dart technology, sfp requires rna preparation and has a slightly higher cost, while dart uses dna and a clever genome complexity reduction method.
nevertheless sfps have a clear advantage when it comes to specifically mapping genes.
the microarray probe design is based on previously known and possibly annotated gene sequences while probes for a dart array are predetermined and only about 35% of them correspond to coding regions .
these two technologies should therefore be considered as highly complementary and distinctively valuable depending on the objectives of the experiment and envisaged downstream applications of the results.
mapping genes by sfp discovery and linkage analysis would compare well cost-wise to gene mapping by the currently used snp genotyping platforms, although snp genotyping costs are rapidly falling.
snp development also requires a transcriptome or sequence database analogous to the one used for sfp probe design.
although targeted single-base assays do require a deeper sequence coverage to adequately select polymorphic snps in silico, sequencing costs have dropped drastically so that no major difference in cost between sfp and snp would happen there.
on the other hand, upfront development costs are necessary to screen snp markers for robustness of the assay and informative polymorphism in a single pedigree.
in a recent snp development study only about  <dig> out of  <dig> snps  assayed in transcribed genes by the golden gate technology segregated and mapped in the exact same mapping population used in this sfp study .
similar yields of polymorphic segregating snps have been found when mapping snps in other forest trees without any prior selection of polymorphic snps between the parents  <cit> .
given the costs of snp genotyping on current illumina chip platforms, the cost of snp-based mapping the same number of genes mapped by sfps in this study would be approximately 50% higher.
it is important to point out, however, that sfp would not be the method of choice when the same markers have to be assayed repeatedly such as in downstream qtl, association mapping or molecular breeding applications.
in such cases optimized snp platforms would be more adequate.
finally, recent developments in genotyping-by-sequencing methods in plants might soon radically change this scenario by providing orders of magnitude more markers for orders of magnitude lower costs, i.e.
tens of thousands of markers for tens of dollars  <cit> .
pseudo-testcross candidate sfp probe screening increases gene mapping efficiency
sfp discovery and mapping benefited from two key experimental approaches frequently used in linkage mapping studies: pseudo-testcross marker screening  <cit>  and selective mapping  <cit> .
in addition, the flexibility of custom designing oligonucleotide arrays allowed a cost-efficient transition between the sfp-discovery and sfp-mapping steps.
screening for polymorphic sfps in species for which inbred lines are available have typically relied on comparison of signal intensity between parental lines  <cit> .
in highly heterozygous species, on the other hand, as sfps are dominant markers, discovery and mapping sfps becomes significantly more efficient if parents and a small set of progeny individuals are genotyped to reveal pseudo-testcross segregating markers as originally shown with the rapd technology  <cit> .
in our experiment the parents were not available.
however we used  <dig> individuals and a cut-off of at least four individuals displaying signal intensity above background to declare a putatively segregating sfp in the sfp-discovery step.
only eight progeny individuals would have sufficed to screen for putatively 1: <dig> segregating sfp with >99% probability .
however by using a larger set of progeny individuals we were also able to target sfps segregating 3: <dig> increasing the number of mappable genes and improving the quality of the final linkage map, possibly due to their role as locus bridges and to the fact that most of them were positioned at small distances from other markers therefore providing similar information content for linkage as the 1: <dig> backcross markers  <cit> .
sfps segregating 3: <dig> could potentially be further tested for a 1:2: <dig> segregation ratio if evidences existed for differential hybridization and detection of the two alleles.
although some sfps apparently followed such a behavior, we did not attempt to resolve these cases because the putative clusters would be too close to each other therefore resulting in an unacceptable separation statistics and therefore an excessive number of missing data.
by genotyping the parents and using them as references for heterozygous genotypes, together with alternative statistical methods, it might be possible to consistently call at least some sfps in a co-dominant fashion.
drost et al.
<cit>  pioneered sfps mapping in a highly heterozygous genomes.
they selected candidate sfps for mapping by comparing signal intensity between the two heterozygous parents of an interspecific populus pedigree using 60-mer oligonucleotide probes.
longer probes were used in an attempt to satisfy a second objective of identifying optimal probes to map gems  and measure gene expression in the progeny.
from a total of  <dig>  probes designed for  <dig>  gene models obtained from the genome sequence, screening resulted in one candidate sfp probe for  <dig>  genes.
later, in the mapping phase, only  <dig>  sfp probes  fitted 1: <dig> mendelian expectations and a limited final number of  <dig> sfps were mapped.
using only the parents for sfp screening not only ruled out the possibility of selecting sfps segregating 3: <dig> but also led to the selection of several candidate sfps that, in spite of being polymorphic between the two parents, did not segregate in the mapping population.
in addition, the use of longer probes, although allowed the identification of  <dig> gems, likely reduced the power to detect sfps.
in our study we focused exclusively on mapping sfps over gene expression markers  and thus used shorter 25-mer probes.
an initial set of  <dig>  probes for  <dig>  genes derived from ests was used and  <dig>  were selected in the pseudo-testcross sfp-discovery step.
this initial screening for sfps using the progeny, resulted in a much larger proportion of candidate sfp probes  surviving after testing for mendelian expectations and  <dig>  sfps were eventually mapped .
although comparisons with the populus study are not straightforward due to differences in species, level of polymorphism, gene prediction and especially probe length, these results suggest that screening for sfps using not only the parents but also the progeny is a better approach to be considered for future sfp mapping experiments.
the highest density transcript linkage map for a forest tree
to the best of our knowledge our eucalyptus  <dig>  gene map is the most highly enriched map for transcriptional information for any forest tree species to date.
as for the vast majority of plant species, linkage maps for forest trees including eucalyptus have rarely included transcriptional information.
preference has been for more accessible and polymorphic markers such as microsatellites, rapd, aflps and more recently snps  <cit> .
in populus, the most gene-dense linkage maps reported had  <dig> genes mapped by sfps  <cit>  or  <dig> unique genes mapped by snps  <cit> .
in oak   <dig> transcribed genes were mapped by exploiting microsatellites mined in their sequences  <cit> .
in loblolly pine, a linkage map with  <dig>  snps representing  <dig>  genes was recently published  <cit> , while in white and black spruce   <dig> genes were mapped using snp markers  <cit> .
eucalyptus gene mapping effort to date positioned  <dig> cambium-specific expressed sequence tags  and  <dig> known function genes in a e. globulus pedigree using rflp  <cit>  and eight lignification genes using sscp  <cit> .
microsatellites derived from ests have been developed for eucalyptus  <cit>  and over  <dig> of them have been recently mapped  although throughput is clearly unacceptable to map large numbers of genes.
in our study, sfp detection and mapping was done on a segregating population derived from an interspecific cross between two elite genotypes of e. urophylla and e. grandis.
both species are highly heterozygous as revealed by microsatellite surveys  <cit> .
high levels of nucleotide sequence diversity have been reported for e. grandis   <cit>  and even higher when a larger sampling was carried out in e. camaldulensis   <cit> .
we predict that equivalent sfp and gene mapping efficiencies will be attainable in other eucalyptus species and pedigrees given the generally high levels of nucleotide diversity seen in species of this genus.
genes revealing spfs are likely to be more variable at the dna sequence level.
perhaps these genes are less constrained by selection or, in the present case, display a higher level of inter specific differentiation given the hybrid nature of the cross.
although speculative at this point, there may be some evolutionary significance associated with the detection of sfps and consequently with the genes mapped by this method.
gene-rich linkage maps of sfp markers can therefore provide an excellent framework to carry out genus wide high-density comparative mapping in eucalyptus and related genera in the myrtaceae and possibly derive some appealing evolutionary hypotheses.
the  <dig>  transcribed gene map reported in this study represents a significant improvement beyond the number of genes previously positioned on eucalyptus genetic maps.
to generate preliminary functional annotation of the  <dig>  genes mapped, we employed gene ontology  mapping using blast2go   <cit> .
this automated tool designed for non-model species extracts and maps go terms to the sequences to generate candidate annotations.
this was done using a locally installed instance of b2g using the output of a blastx against genebank  as starting point.
a total of  <dig>  unigenes had a significant blast hit and, from these, b2g annotated go terms to  <dig> genes and  <dig> genes had annotation ultimately mapped after applying b2g default filters.
details of this annotation for both the full and framework maps are available in additional files  <dig> and  <dig>  respectively.
an improved annotation of these mapped genes will have to wait for the annotated version of the eucalyptus grandis genome.
nevertheless, this gene map not only offers a coarse first glance at the still undefined gene space in eucalyptus, but also provides a useful resource for improving the assembly of the e. grandis genome, currently in progress  <cit> .
in poplar, even a relatively modest  <dig> sfp map allowed localizing over  <dig> million base pairs of previously unplaced whole-genome shotgun  scaffold sequence to putative locations in the genome of populus trichocarpa  <cit> .
out of the  <dig>  genes successfully mapped,  <dig> of them were positioned with high likelihood support for gene order .
robust marker ordering for all  <dig>  genes, if desired, could be obtained by increasing the number of progeny individuals by selective mapping.
the  <dig> genes  mapped with a relaxed order support are somewhat equivalent to bin mapped markers, i.e.
markers that are assigned to specific map segments  typically using less than  <dig> individuals and loosely assigning them to map bins  <cit> .
a much higher mapping precision than bin-mapping was attained in our study, however, by using  <dig> individuals chosen by selective mapping plus the  <dig> used in the sfp screening step.
possibly several hundreds more sfps and corresponding genes that were left out in the linkage and ordering analysis could be assigned to linkage groups by using such a coarse bin-mapping approach to provide approximate positions.
bin-mapping using only  <dig> individuals or less would be a very appealing approach to map large numbers of genes by sfp genotyping in the vast majority of species that suffer from limited resources providing an initial sense of the gene space and candidate positional genes for further genomics undertakings.
sfp detection and mapping is improved by using larger probe-sets and anova based sfp-discovery
our results showed that two procedures improve the overall efficiency of sfp detection and mapping.
the first one is to use a mixed-model anova on the raw signal intensity data of the sfp discovery set data to analyze the full probe-set for each gene in a separate model.
a retrospective analysis of the discovery set data showed that this approach allows early detection of high quality sfps and, thus, could reduce experimental costs by optimizing the selection of probes to populate the final sfp-mapping array .
had we used this additional analytical procedure on the discovery set data early on, we would have successfully detected informative probe-sets and specific probes within probe-sets for the vast majority of the genes that were eventually mapped by conventional linkage mapping.
instead of using probe-sets for  <dig>  genes in the sfp-mapping array, only  <dig>  genes that showed a significant genotype by probe-set interaction would have been represented on the array.
furthermore, instead of  <dig>  probes, only  <dig>  would have been used on the final sfp-mapping array, significantly reducing overall experimental costs while keeping a very similar final gene mapping efficiency .
the second procedure that improves sfp detection and mapping is to use a large number of probes per probe-set.
on average, five unique non-overlapping 25-mer probes were designed and used to represent each unigene on the microarray.
however, for a subset of  <dig>  genes a probe-set of  <dig> probes was used to assess the impact of using a larger number of probes on the likelihood of detecting a mappable sfp.
considering a similar mutation rate along the coding region, we anticipated that doubling the number of screened probes would result in an equally proportional increase in the detection of probes with sfps.
an almost two-fold increase from  <dig> % to  <dig> % was observed and highly significant .
using more probes per unigene does result in a significantly increased likelihood of discovering a segregating sfp that will allow mapping the corresponding gene.
this result is relevant when considering mapping specific genes using sfp markers.
in such a scenario, the recommendation would be to design the largest possible number of non-overlapping probes so as to scan the whole extension of the target gene for detectable sequence polymorphisms.
this approach would be particularly useful to map a large number of putative candidate genes in an attempt to co-localize such genes and mapped qtls for species that still lack a reference genome sequence.
a protocol to optimize gene mapping by sfp genotyping
results of our study allow us to propose an experimental procedure to build high-density transcript linkage maps in less characterized plant species with no reference genome yet available with a concurrent objective of reducing microarray synthesis and hybridization costs:  a large est collection of several tens of millions of reads is quickly constructed at a relatively low cost by pooling and sequencing rna samples from several different individuals and tissues using next generation sequencing technologies;  a high-density microarray with large numbers  of probes for each one of all available unique genes identified in the est database is designed to be used as a sfp-discovery array; if particular genes are targeted for mapping, the number of probes designed for them should be maximized;  rna from the two parents and sixteen offspring individuals of a mapping population is hybridized to this sfp-discovery array; candidate sfp probes within probe-sets interacting with genotypes, representing genes with a significant expression signal difference between the parents and/or segregating in the progeny are identified by a combined anova and clustering analysis ; selected probes are then used to populate a sfp-mapping array;  rna from a larger progeny set of a few tens of individuals , drawn from a larger mapping population using selective mapping is used for final sfp genotyping, segregation analysis and linkage mapping using the sfp-mapping array.
the efficiency of this protocol can be enhanced by carrying out the above steps in more than one pedigree simultaneously, thus increasing the probability of detecting segregating sfps for a larger number of genes and eventually mapping them.
CONCLUSIONS
our study shows that starting from a relatively modest est resource, sfp discovery and mapping using high-density oligonucleotide arrays is a powerful approach to quickly position several hundreds or thousands of genes on a reference map for any organism.
using this experimental approach in eucalyptus we generated the most highly enriched map for transcriptional information for any forest tree species to date with  <dig>  genes.
the large number of genes mapped by sfp detection provides useful information for several genomic applications and offers a preliminary glimpse at the eucalyptus gene space.
gene-rich maps represent a useful resource for comparative genomics and gene discovery for plant and animal species for which reference genomes are still to come.
when used in combination with qtl and association mapping data, the co-localization of mapped genes with high resolution qtl mapping data could rapidly lead to positional candidate genes for which specific snps could be developed and used in association genetics studies.
additionally, gene mapping information could increase the relative weight of specific gene-rich regions in predictive genomic selection models to assist forest tree breeding programs for multiple complex quantitative traits  <cit> .
finally, gene-rich high-density linkage maps will be especially valuable when time comes to assemble reference genome sequences, a likely task for several plant and animal species in the very near future.

 high-density linkage maps based on transcribed genes are valuable resources to characterize genome structure, gene space distribution and synteny in species for which reference genome sequences are not yet available. dense gene maps can also support genome sequence assembly and the identification of genes underlying loci that control complex phenotypic traits. most methods used to date for linkage mapping genes in plants such as rflp, single-strand conformation polymorphism, cleaved amplified polymorphic sequence and denaturing gradient gel electrophoresis have very limited throughput. high-throughput genotyping of single-nucleotide polymorphism derived from genic sequences have allowed large-scale gene mapping. however such efforts require relatively high up-front snp development, screening and validation costs, which have restricted this technology to the major crop plants and a few forest trees. furthermore, current snp assay methods based on allele-specific primer extension can be of variable robustness in highly heterozygous genomes such as those of forest trees with high nucleotide diversity, frequently above 1%. dna microarrays have been used to reliably detect genetic differences among individuals as shown initially in the simple genome of yeast and later in arabidopsis by borevitz et al., who termed these genetic variants single feature polymorphisms. the principle of sfp genotyping is based on the disruption of the hybridization of labeled dna sequences that are genetically variable relative to a reference sequence used for design of probes on a microarray. segregating sequence differences among individuals in a mapping population can be detected based on the variation of the hybridization signal. sfps have been detected by hybridizing total genomic dna to microarrays in yeast, arabidopsis, and rice. ronald et al., rostoks et al. and cui et al. expanded the scope of the method by hybridizing rna-derived cdna or crna to expression microarrays in yeast and barley, with the anticipation that polymorphisms in protein-coding dna sequences that are transcribed into messenger rna would also weakly hybridize to microarray probes. while this approach reduces the complexity of the pool of sequences hybridized to the microarray, differences in signal intensity that arise as a consequence of sequence variation among genotypes can be confounded with differences in transcript abundance. sfp mapping involves the detection of probes revealing putative sfp whose behavior as mendelian markers is then evaluated in segregating populations. sfp have been largely developed for inbred, highly homozygous species. in these studies, a putative sfp is identified based on signal differences between two parental lines. candidate sfps are then tested for a bimodal distribution in hybridization signal detected in a segregating population of recombinant inbred lines or backcross progeny, and individuals are assigned to the expected genotypes defined using the signal intensity of the parents as references. using this strategy in arabidopsis, singer et al. built a genetic map with genes by sfps detection using dna hybridization while west et al. used complementary rna hybridization to map genes based on sfp. in the more complex genome of barley, luo et al. reported mapping and sfps using crna from leaf and embryo from segregating doubled-haploid lines. in the only study to date that attempted to genotype microarray markers in an outcrossing species, drost et al. mapped sfps segregating 1: in a populus pseudo-backcross progeny involving individuals. in this study we report the detection and linkage mapping of sfps in a reference pedigree of eucalyptus grandis × e. urophylla, two highly heterozygous tree species. detection of robust sfps was improved by an initial sfp-discovery step based on the pseudo-testcross strategy followed by using a selective mapping approach to simultaneously optimize linkage mapping and microarray costs. a total of genes were successfully mapped, of them positioned with high likelihood support on a framework genetic map. the experimental approach employed is cost efficient and should be readily applicable to large scale mapping of genes in highly heterozygous genomes of several plant species providing a useful resource for downstream applications such as gene discovery, association genetics and assembly of reference genome sequences..  screening for candidate sfps sfp screening, selection, genotyping and mapping involved a sequence of analytical steps summarized in a flowchart for easy visualization. the sfp-discovery microarray included unique probes representing unigenes. analysis of the screening data resulted in a set of probes representing genes detecting expressed transcripts in at least four progeny individuals of the discovery set of individuals. the number of probes detecting transcripts varied considerably across genes and only for genes of the assayed all the probes in the probe-set had signal consistently above the negative control threshold in all individuals. note that in a perfect scenario an expressed transcript would hybridize to all the probes of its corresponding probe set. this indicates that detectable sequence polymorphisms between the short oligonucleotide probes and the hybridized transcripts are the rule rather than the exception. following the sfp-discovery step, probes with the lowest mean and standard deviation in the signal intensity were removed. low signal and limited variation in signal intensity in the discovery set was taken as an indication that these probes were less likely to reveal robust sfps. a new microarray with selected probes was designed representing genes, with a variable number of probes per probe-set. this new microarray containing selected probes was used to genotype an additional set of individuals drawn from the mapping population using a selective mapping approach. this procedure identified the most informative individuals in terms of complementary recombination breakpoints from each parent, optimizing the linkage information to be extracted from these individuals and thus reducing the costs of the sfp genotyping step for map construction. sfp genotyping in the mapping population the averaged, log transformed, quantile-normalized probe signal detected for each individual was analyzed as described by drost et al. to simultaneously identify and genotype sfps. briefly, for each probe the signal intensity measured in each individual offspring was assigned to either one of two distinct clusters using the k-means clustering algorithm. using a chi-square test, probes showing a 1: pseudo-testcross segregation, as well as probes segregating as dominant markers were selected. a total of probes out of the tested followed a mendelian segregation ratio, with segregating 1: and segregating 3:, representing a total of genes. the degree of separation between the two clusters for a probe was measured by calculating the probability of individuals assigned to one cluster being a member of the other cluster through a modified normal deviate zi. individuals with zi equal to or less than are likely to overlap with the other cluster and were assigned as missing data to avoid genotype miscalls. when displaying more than 10% missing data, the probe was discarded from further analysis. after this stringent selection step, probes representing unigenes survived with a slightly larger number of probes segregating in a 3: than a 1: ratio. for genes, two or more probes revealed sfps. in those cases, the probe with fewer miscalls and segregating 3: was selected over the alternative. in spite of their dominant behavior sfp markers segregating in a 3: ratio segregate from both parents, provide alellic bridges between the parental maps and when linked in coupling at small recombination distances have essentially the same information content as 1: backcross markers. the resulting selected sfps, segregating 1: and segregating 3: were used in the subsequent linkage analysis. *unigenes derived from a consensus sequence involving ests from different species are indicated as contigs, while singletons are listed by species. **when several probes were selected per probe set, the number of unique genes represented is shown between parentheses. construction of a gene-rich linkage map for eucalyptus from the sfps that showed mendelian segregation, a total of sfps were successfully grouped at a lod ≥ and could be ordered along the linkage groups together with microsatellites, providing a total map with markers with a relaxed marker order support. details of this full map with all the ordered linkage information of the sfps and microsatellites including map position, original unigene sequence from which the probe was designed, sequence of the probe revealing the sfp, best hit against genbank non-redundant database and annotation description generated using blast2go are presented in a table format. to provide a gene map with high likelihood support for gene order, a framework map was built using the round function of joinmap that removes markers that contribute to unstable order. in the framework map sfps were mapped together with microsatellites thus resulting in a robust genetic linkage map with makers. comparative statistics of the full and framework maps is presented. linkage inter-marker distances for the full map is not reported as such estimates might be ambiguous due to the low support for order from the total number of sfps mapped in the framework map, segregated in a 1: ratio, and in a 3: ratio, identified by the acronym bc and f respectively. although no statistical test for clustering was carried out, markers seemed evenly distributed along the linkage groups with no obvious clustering except for some cases on the edge of linkage groups,. some linkage groups had more genes mapped than others. the sfp/microsatellite framework map had an average density of cm with % of the intermarker distances smaller than cm. for only five of the intervals the distance was greater than cm, with a maximum of cm. even though the number of mapped markers increased more than five times when compared to the microsatellite-only map, the total genetic length was estimated at cm which is within the expected range for eucalyptus. to evaluate the relative contribution of each class of sfp to the quality of the genetic map, two separate maps were constructed including microsatellites and either sfps segregating 1: or 3: the map that includes only the sfps segregating 1: decreased overall map quality, as illustrated by the expansion of total map length to cm based on the generally adopted premise that a shorter map is the most likely hypothesis to explain the data. conversely, the length of the genetic map remained similar when only the sfps segregating 3: were included. thus the detection and use of sfps segregating 3: not only increases the total number of detectable sfps and consequently mappable genes, but also contributes to a higher quality map possibly due to their role as providers of allelic bridges when linked in coupling. early sfp detection using a mixed-model analysis of variance the signal intensity data of the individuals of the sfp discovery set was analyzed by a mixed-model anova using the full probe-set for each gene separately to evaluate the correspondence between the putative sfps detected based on this procedure with those that were linkage mapped by the standard procedure adopted of sfp selection, genotyping and mapping. after correcting the significance threshold of the f tests for multiple testing, genes had significant genotype by probe interaction, corresponding to a putative sfp,. these genes, all of them also included in the set of genes represented on the sfp-mapping microarray, encompassed of the that were mapped with lower confidence for marker order and of the that were framework mapped by the standard sfp mapping procedure. specific candidate sfp probes within probe-sets were then searched using the k-means clustering analysis and chi square segregation test for mendelian proportions. a total of probes, representing genes, were selected in this analysis. this set included probes for of the genes that were mapped at a relaxed ordering threshold and of the that were framework mapped by the standard procedure. these results indicate that by testing each probe-set separately for probes that interact with genotypes, instead of probes, only would have been necessary on the sfp-mapping array to map close to 90% of the genes that were eventually mapped. efficiency in detecting mappable sfp at the gene level and probe level by the anova in comparison to the conventional linkage mapping approach * in parentheses are indicated the percentage of genes or probes in common with those mapped by conventional linkage mapping considered as the benchmark approach impact of the number of probes per probe-set on sfp detection a total of out of the genes for which five probes were used in the probe-set had at least one sfp discovered and were ultimately mapped. in contrast, for the genes for which probes were used, the efficiency was almost doubled, with probes eventually mapping, an increase to % a chi-square test of homogeneity with yates correction confirmed this difference to be highly significant. *the total number of mapped sfps used in the test is lower than the total of sfp mapped because numbers of probes different from or were used for some genes and these were removed from this analysis. sfp validation on the eucalyptus draft genome the linkage relationship of sfps to microsatellites was validated by using the x draft genome sequence of e. grandis. the location of mapped microsatellites on the supercontigs was confidently determined using wu-blast and served to anchor supercontigs to linkage groups. probes corresponding to of the sfps were mapped to scaffolds that contained one or more of the anchor mapped microsatellites. the remaining sfp probes mapped to supercontigs, but these were not anchored by any of the microsatellites. of these sfps, only were not genetically mapped to the same linkage group as the associated microsatellite, suggesting that they are trans-regulated gems. for the remaining sfp's probes and microsatellites were correctly anchored to the same linkage group located within a cm distance, on average, on the genetic map. some of these probes could represent cis-regulated gems instead of sfps, but mapping of multiple probes within a probe-set to the same genetic position would have been expected in that case. instead, probe-sets with multiple probes showing mendelian segregation were only observed for out of the genes positioned on the framework map. discussion microarray-based sfp genotyping is an accessible approach for gene mapping in this work, large numbers of sfp displaying mendelian behavior were readily detected using short oligonucleotide probe arrays in a complex, highly heterozygous plant genome. these sfps, in turn, allowed the construction of a gene-enriched linkage map providing valuable positional information for hundreds of transcribed genes. the -probe sfp-discovery oligonucleotide array used in this study was built from a relatively small est data set for current standards: phred- filtered 5'-sanger-sequenced ests assembled in unique sequences. currently, much larger and more diverse est datasets can be rapidly generated at a fraction of the cost using next-generation sequencing technologies as demonstrated for uncharacterized plant genomes including eucalyptus. several millions of ests can be generated from pooled samples of rna for a couple of thousand us dollars providing a very rich source of sequence information to design a similar or much higher density oligonucleotide array. costs of custom-made high-density microarrays have also dropped significantly and different formats and probe densities are available allowing optimized designs of experiments with multiple samples. by using a two-step sfp discovery and mapping approach combined with selective mapping, one can envisage the construction of a high-density linkage map with thousands of genes for less than us dollars. sfp-based gene mapping should therefore prove a very accessible and valuable experimental approach for several plant species. recently, a high density diversity arrays technology microarray for genome-wide genotyping has been developed for eucalyptus providing over polymorphic markers at the population level and an average of segregating polymorphic markers per pedigree. when compared to the dart technology, sfp requires rna preparation and has a slightly higher cost, while dart uses dna and a clever genome complexity reduction method. nevertheless sfps have a clear advantage when it comes to specifically mapping genes. the microarray probe design is based on previously known and possibly annotated gene sequences while probes for a dart array are predetermined and only about 35% of them correspond to coding regions. these two technologies should therefore be considered as highly complementary and distinctively valuable depending on the objectives of the experiment and envisaged downstream applications of the results. mapping genes by sfp discovery and linkage analysis would compare well cost-wise to gene mapping by the currently used snp genotyping platforms, although snp genotyping costs are rapidly falling. snp development also requires a transcriptome or sequence database analogous to the one used for sfp probe design. although targeted single-base assays do require a deeper sequence coverage to adequately select polymorphic snps in silico, sequencing costs have dropped drastically so that no major difference in cost between sfp and snp would happen there. on the other hand, upfront development costs are necessary to screen snp markers for robustness of the assay and informative polymorphism in a single pedigree. in a recent snp development study only about out of snps assayed in transcribed genes by the golden gate technology segregated and mapped in the exact same mapping population used in this sfp study. similar yields of polymorphic segregating snps have been found when mapping snps in other forest trees without any prior selection of polymorphic snps between the parents. given the costs of snp genotyping on current illumina chip platforms, the cost of snp-based mapping the same number of genes mapped by sfps in this study would be approximately 50% higher. it is important to point out, however, that sfp would not be the method of choice when the same markers have to be assayed repeatedly such as in downstream qtl, association mapping or molecular breeding applications. in such cases optimized snp platforms would be more adequate. finally, recent developments in genotyping-by-sequencing methods in plants might soon radically change this scenario by providing orders of magnitude more markers for orders of magnitude lower costs, i.e. tens of thousands of markers for tens of dollars. pseudo-testcross candidate sfp probe screening increases gene mapping efficiency sfp discovery and mapping benefited from two key experimental approaches frequently used in linkage mapping studies: pseudo-testcross marker screening and selective mapping. in addition, the flexibility of custom designing oligonucleotide arrays allowed a cost-efficient transition between the sfp-discovery and sfp-mapping steps. screening for polymorphic sfps in species for which inbred lines are available have typically relied on comparison of signal intensity between parental lines. in highly heterozygous species, on the other hand, as sfps are dominant markers, discovery and mapping sfps becomes significantly more efficient if parents and a small set of progeny individuals are genotyped to reveal pseudo-testcross segregating markers as originally shown with the rapd technology. in our experiment the parents were not available. however we used individuals and a cut-off of at least four individuals displaying signal intensity above background to declare a putatively segregating sfp in the sfp-discovery step. only eight progeny individuals would have sufficed to screen for putatively 1: segregating sfp with >99% probability. however by using a larger set of progeny individuals we were also able to target sfps segregating 3: increasing the number of mappable genes and improving the quality of the final linkage map, possibly due to their role as locus bridges and to the fact that most of them were positioned at small distances from other markers therefore providing similar information content for linkage as the 1: backcross markers. sfps segregating 3: could potentially be further tested for a 1:2: segregation ratio if evidences existed for differential hybridization and detection of the two alleles. although some sfps apparently followed such a behavior, we did not attempt to resolve these cases because the putative clusters would be too close to each other therefore resulting in an unacceptable separation statistics and therefore an excessive number of missing data. by genotyping the parents and using them as references for heterozygous genotypes, together with alternative statistical methods, it might be possible to consistently call at least some sfps in a co-dominant fashion. drost et al. pioneered sfps mapping in a highly heterozygous genomes. they selected candidate sfps for mapping by comparing signal intensity between the two heterozygous parents of an interspecific populus pedigree using 60-mer oligonucleotide probes. longer probes were used in an attempt to satisfy a second objective of identifying optimal probes to map gems and measure gene expression in the progeny. from a total of probes designed for gene models obtained from the genome sequence, screening resulted in one candidate sfp probe for genes. later, in the mapping phase, only sfp probes fitted 1: mendelian expectations and a limited final number of sfps were mapped. using only the parents for sfp screening not only ruled out the possibility of selecting sfps segregating 3: but also led to the selection of several candidate sfps that, in spite of being polymorphic between the two parents, did not segregate in the mapping population. in addition, the use of longer probes, although allowed the identification of gems, likely reduced the power to detect sfps. in our study we focused exclusively on mapping sfps over gene expression markers and thus used shorter 25-mer probes. an initial set of probes for genes derived from ests was used and were selected in the pseudo-testcross sfp-discovery step. this initial screening for sfps using the progeny, resulted in a much larger proportion of candidate sfp probes surviving after testing for mendelian expectations and sfps were eventually mapped. although comparisons with the populus study are not straightforward due to differences in species, level of polymorphism, gene prediction and especially probe length, these results suggest that screening for sfps using not only the parents but also the progeny is a better approach to be considered for future sfp mapping experiments. the highest density transcript linkage map for a forest tree to the best of our knowledge our eucalyptus gene map is the most highly enriched map for transcriptional information for any forest tree species to date. as for the vast majority of plant species, linkage maps for forest trees including eucalyptus have rarely included transcriptional information. preference has been for more accessible and polymorphic markers such as microsatellites, rapd, aflps and more recently snps. in populus, the most gene-dense linkage maps reported had genes mapped by sfps or unique genes mapped by snps. in oak transcribed genes were mapped by exploiting microsatellites mined in their sequences. in loblolly pine, a linkage map with snps representing genes was recently published, while in white and black spruce genes were mapped using snp markers. eucalyptus gene mapping effort to date positioned cambium-specific expressed sequence tags and known function genes in a e. globulus pedigree using rflp and eight lignification genes using sscp. microsatellites derived from ests have been developed for eucalyptus and over of them have been recently mapped although throughput is clearly unacceptable to map large numbers of genes. in our study, sfp detection and mapping was done on a segregating population derived from an interspecific cross between two elite genotypes of e. urophylla and e. grandis. both species are highly heterozygous as revealed by microsatellite surveys. high levels of nucleotide sequence diversity have been reported for e. grandis and even higher when a larger sampling was carried out in e. camaldulensis. we predict that equivalent sfp and gene mapping efficiencies will be attainable in other eucalyptus species and pedigrees given the generally high levels of nucleotide diversity seen in species of this genus. genes revealing spfs are likely to be more variable at the dna sequence level. perhaps these genes are less constrained by selection or, in the present case, display a higher level of inter specific differentiation given the hybrid nature of the cross. although speculative at this point, there may be some evolutionary significance associated with the detection of sfps and consequently with the genes mapped by this method. gene-rich linkage maps of sfp markers can therefore provide an excellent framework to carry out genus wide high-density comparative mapping in eucalyptus and related genera in the myrtaceae and possibly derive some appealing evolutionary hypotheses. the transcribed gene map reported in this study represents a significant improvement beyond the number of genes previously positioned on eucalyptus genetic maps. to generate preliminary functional annotation of the genes mapped, we employed gene ontology mapping using blast2go. this automated tool designed for non-model species extracts and maps go terms to the sequences to generate candidate annotations. this was done using a locally installed instance of b2g using the output of a blastx against genebank as starting point. a total of unigenes had a significant blast hit and, from these, b2g annotated go terms to genes and genes had annotation ultimately mapped after applying b2g default filters. details of this annotation for both the full and framework maps are available in additional files and respectively. an improved annotation of these mapped genes will have to wait for the annotated version of the eucalyptus grandis genome. nevertheless, this gene map not only offers a coarse first glance at the still undefined gene space in eucalyptus, but also provides a useful resource for improving the assembly of the e. grandis genome, currently in progress. in poplar, even a relatively modest sfp map allowed localizing over million base pairs of previously unplaced whole-genome shotgun scaffold sequence to putative locations in the genome of populus trichocarpa. out of the genes successfully mapped, of them were positioned with high likelihood support for gene order. robust marker ordering for all genes, if desired, could be obtained by increasing the number of progeny individuals by selective mapping. the genes mapped with a relaxed order support are somewhat equivalent to bin mapped markers, i.e. markers that are assigned to specific map segments typically using less than individuals and loosely assigning them to map bins. a much higher mapping precision than bin-mapping was attained in our study, however, by using individuals chosen by selective mapping plus the used in the sfp screening step. possibly several hundreds more sfps and corresponding genes that were left out in the linkage and ordering analysis could be assigned to linkage groups by using such a coarse bin-mapping approach to provide approximate positions. bin-mapping using only individuals or less would be a very appealing approach to map large numbers of genes by sfp genotyping in the vast majority of species that suffer from limited resources providing an initial sense of the gene space and candidate positional genes for further genomics undertakings. sfp detection and mapping is improved by using larger probe-sets and anova based sfp-discovery our results showed that two procedures improve the overall efficiency of sfp detection and mapping. the first one is to use a mixed-model anova on the raw signal intensity data of the sfp discovery set data to analyze the full probe-set for each gene in a separate model. a retrospective analysis of the discovery set data showed that this approach allows early detection of high quality sfps and, thus, could reduce experimental costs by optimizing the selection of probes to populate the final sfp-mapping array. had we used this additional analytical procedure on the discovery set data early on, we would have successfully detected informative probe-sets and specific probes within probe-sets for the vast majority of the genes that were eventually mapped by conventional linkage mapping. instead of using probe-sets for genes in the sfp-mapping array, only genes that showed a significant genotype by probe-set interaction would have been represented on the array. furthermore, instead of probes, only would have been used on the final sfp-mapping array, significantly reducing overall experimental costs while keeping a very similar final gene mapping efficiency. the second procedure that improves sfp detection and mapping is to use a large number of probes per probe-set. on average, five unique non-overlapping 25-mer probes were designed and used to represent each unigene on the microarray. however, for a subset of genes a probe-set of probes was used to assess the impact of using a larger number of probes on the likelihood of detecting a mappable sfp. considering a similar mutation rate along the coding region, we anticipated that doubling the number of screened probes would result in an equally proportional increase in the detection of probes with sfps. an almost two-fold increase from % to % was observed and highly significant. using more probes per unigene does result in a significantly increased likelihood of discovering a segregating sfp that will allow mapping the corresponding gene. this result is relevant when considering mapping specific genes using sfp markers. in such a scenario, the recommendation would be to design the largest possible number of non-overlapping probes so as to scan the whole extension of the target gene for detectable sequence polymorphisms. this approach would be particularly useful to map a large number of putative candidate genes in an attempt to co-localize such genes and mapped qtls for species that still lack a reference genome sequence. a protocol to optimize gene mapping by sfp genotyping results of our study allow us to propose an experimental procedure to build high-density transcript linkage maps in less characterized plant species with no reference genome yet available with a concurrent objective of reducing microarray synthesis and hybridization costs: a large est collection of several tens of millions of reads is quickly constructed at a relatively low cost by pooling and sequencing rna samples from several different individuals and tissues using next generation sequencing technologies; a high-density microarray with large numbers of probes for each one of all available unique genes identified in the est database is designed to be used as a sfp-discovery array; if particular genes are targeted for mapping, the number of probes designed for them should be maximized; rna from the two parents and sixteen offspring individuals of a mapping population is hybridized to this sfp-discovery array; candidate sfp probes within probe-sets interacting with genotypes, representing genes with a significant expression signal difference between the parents and/or segregating in the progeny are identified by a combined anova and clustering analysis; selected probes are then used to populate a sfp-mapping array; rna from a larger progeny set of a few tens of individuals, drawn from a larger mapping population using selective mapping is used for final sfp genotyping, segregation analysis and linkage mapping using the sfp-mapping array. the efficiency of this protocol can be enhanced by carrying out the above steps in more than one pedigree simultaneously, thus increasing the probability of detecting segregating sfps for a larger number of genes and eventually mapping them.  our study shows that starting from a relatively modest est resource, sfp discovery and mapping using high-density oligonucleotide arrays is a powerful approach to quickly position several hundreds or thousands of genes on a reference map for any organism. using this experimental approach in eucalyptus we generated the most highly enriched map for transcriptional information for any forest tree species to date with genes. the large number of genes mapped by sfp detection provides useful information for several genomic applications and offers a preliminary glimpse at the eucalyptus gene space. gene-rich maps represent a useful resource for comparative genomics and gene discovery for plant and animal species for which reference genomes are still to come. when used in combination with qtl and association mapping data, the co-localization of mapped genes with high resolution qtl mapping data could rapidly lead to positional candidate genes for which specific snps could be developed and used in association genetics studies. additionally, gene mapping information could increase the relative weight of specific gene-rich regions in predictive genomic selection models to assist forest tree breeding programs for multiple complex quantitative traits. finally, gene-rich high-density linkage maps will be especially valuable when time comes to assemble reference genome sequences, a likely task for several plant and animal species in the very near future. 
high-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with qtl and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species. a total of genes were mapped, with of them ordered with high likelihood support on a framework map anchored to microsatellites with average density of cm. a general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by sfp genotyping with a concurrent objective of reducing microarray costs. the eucalyptus gene map is the most highly enriched map for transcriptional information for any forest tree species to date. an sfp-mapping microarray with selected candidate sfp probes representing genes was subsequently designed and used to genotype sfps in a larger subset of the segregating population drawn by selective mapping. it represents a major improvement on the number of genes previously positioned on eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. sfp discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. we report the construction of a high-density linkage map of expressed genes for the heterozygous genome of eucalyptus using single feature polymorphism markers. in silico validation showed that 87% of the sfps map to the expected location on the x draft sequence of the eucalyptus grandis genome.
 technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. high-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. we report the construction of a high-density linkage map of expressed genes for the heterozygous genome of eucalyptus using single feature polymorphism markers.  sfp discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. sfp genotyping was carried out by hybridizing complementary rna prepared from year-old trees xylem to an sfp array containing 25-mer oligonucleotide probes representing unigenes derived from a modest size expressed sequence tags collection. an sfp-mapping microarray with selected candidate sfp probes representing genes was subsequently designed and used to genotype sfps in a larger subset of the segregating population drawn by selective mapping. a total of genes were mapped, with of them ordered with high likelihood support on a framework map anchored to microsatellites with average density of cm. using more probes per unigene increased by two-fold the likelihood of detecting segregating sfps eventually resulting in more genes mapped. in silico validation showed that 87% of the sfps map to the expected location on the x draft sequence of the eucalyptus grandis genome.  the eucalyptus gene map is the most highly enriched map for transcriptional information for any forest tree species to date. it represents a major improvement on the number of genes previously positioned on eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. a general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by sfp genotyping with a concurrent objective of reducing microarray costs. high-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with qtl and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species. 