Ophir et al. BMC Genomics 2010, 11:269 http://www.biomedcentral.com/1471-2164/11/269 Page 3 of 10 Figure 1 Genetic variation between two melon accessions. A scat- ter plot of moderated-t vs. average signal of four samples (two differ- ent plant DNA pools from each accession), where moderated-t is the Bayesian correction of the t-statistic calculated from the comparison between two 'Dulce' biological replicates and two PI414723 biological replicates. Black dots are statistically insignificant differences, i.e., non- SFPs, and blue (adjusted-p < 0.05) and green (B ≥ 1.5) dots are statisti- cally significant differences, i.e., putative SFPs. from both plant cultivars. Identification and validation of a SFP is demonstrated in Figure 2. The sequences were compared against the information in the melon database using Vector NTI software (Invitrogen, USA). Most of the SFPs (81%) contained one or two SNPs, and the rest con- tained either more than two SNPs (13%) or indels (6%) (Additional file 1). Next, we counted the number of true and false positives (TP and FP, respectively) found by sequencing (summarized in Table 1). We found, the most reliable SFPs to be those that appear once per gene. We calculated the positive predictive value (PPV = TP/ [TP+FP]) for each number of SFPs per gene category, since the proportion of the genes according to SFP num- ber was biased in our sequencing sample. Our PPV was found to be 79%, similar to that found in rice [29], higher than what has been found in some other SFP-discovery studies, but significantly lower than that which has been found in some studies of Arabidopsis and yeast [5,30]. Analysis based on melon EST databases [23] showed that the frequency of variation discovered by our SFP array was 10-fold higher than that found in the partial tran- scriptome. Another indication that the nucleotide poly- morphisms in the detected SFP are biologically true is their polymorphism patterns. Transition nucleotide sub- stitutions are known to be more frequent than transver- sions [31]. Table 2 summarizes the genotyping as detected by sequencing. The transition-to-transversion ratios in the PI414723 and 'Dulce' populations were 1.6 and 2.5, respectively. Indeed, PI414723, which is expected to be genetically more variable than the domestic cultivar, had a total of 3.23-fold more nucleotide substitutions than 'Dulce' (relative to the database that is composed from different cultivars). Based on the distribution of SFPs per gene (Figure 3), most of the genes (~90%) accumulating SFPs possessed one or two SFPs per gene. One might postulate that this is because most of the UniGenes, from which the probes present on the array were designed, are short or partial, and therefore the real number of SFPs per gene cannot be determined. However, the length of the UniGenes is vari- able and cannot explain the SFP/UniGene variation. For each SFP density category (one SFP per UniGene to 18 SFPs per UniGene), the mean UniGene length was approximately constant at 800 ± 300 bp (Figure 4). In other words, length medians do not increase linearly with SFP per gene category as it would be expected if number of SFPs that were found were due to UniGene length. Therefore, it is reasonable to assume that the SFP/gene distribution was the actual distribution, and it was con- sistent with the SFP density that has been previously reported for Arabidopsis [7]. Can SFPs be used in different mapping populations? The ability of SFPs to function as markers in different genetic backgrounds is crucial for their use in MAS, asso- ciation studies and genetic mapping. As plants host large genetic diversity, we decided to test the use of a set of SFPs across different melon cultivars. We tested this question using parents of another melon mapping popu- lation, 'Piel de Sapo' and PI161375 [18]. We performed hybridizations of two biological replicates (pool of 10 plants) from each genotype on two arrays. The SFPs, i.e., statistically significant signal ratios, that were indepen- dently found in both comparisons--'Piel De Sapo' vs. PI161375 and 'Dulce' vs. PI414723 were designated as shared SFPs. In comparisons between the Spanish map- ping population's parents, PI161375 and 'Piel de Sapo', and the corresponding genotypes PI414723 and 'Dulce', the number of SFPs was 6598 and 6184 in 3820 and 3849 genes, respectively (Figure 5). Using this set of genetic markers between the two cultivars ('Piel de Sapo' and 'Dulce') and the wild accessions PI414723 and PI161375, we defined the shared SFP set of mapping markers, i.e. the intersection between the 6598 SFPs that differentiate 'Piel de Sapo' from PI161375 and the 6184 SFPs that dif- ferentiate 'Dulce' from PI414723. This subset included 2213 SFPs in 1548 UniGenes. An example of five genes from this intersecting subset is illustrated in Figure 6. From the data presented in Figures 5 and 6, we concluded