BMC Genomics 2004, 5:65 http://www.biomedcentral.com/1471-2164/5/65 DNA samples were the same and had not undergone sam- ple mix-up. Finally, a human ortholog sequence was iden- tified that was in agreement with the RH data for 15 (15/ 24) of these clones. end-sequences should align in a head-to-head orienta- tion. With this additional condition, the probability of scoring pairs that contain spurious alignments falls to less than 0.01%. Although we have no clear explanation for these discrep- ancies, it is of note that the two mapping methods used different DNA sequences to define the same marker. In the RH method, a pair of 25-mer oligonucleotides defin- ing only one end of the dog insert DNA is used. By com- parison, in the FISH analysis, the whole BAC encompassing approximately 150 Kb is hybridized. Since the DNA for the FISH localization was prepared from cul- tures initiated from single bacterial colonies, these anom- alies suggest that at least a subset of the clones may be chimeric. An additional explanation is that the clones are hybridizing to genomic regions containing one member of a multi-gene family. Whatever the explanation for these discrepancies, it is noteworthy that the total number of discrepancies between the FISH and RH data represent 3% of the total data. This is in agreement with reports that the frequency of chimeric clones in this BAC library is very low [27]. Thus, in most cases, the co-linearity of the FISH and RH maps is perfect, i.e. the order of the BACs deter- mined by multi-color FISH is identical to that derived from analysis of genotyping data using primers defining single BAC-ends. In addition to the conflicting results described above, in some cases, we note that the order for two closely posi- tioned markers is inverted between the FISH and the RH data. However, for all of these cases the distance between the two markers is estimated to be less than 50–100 Kb, as these clones overlap in interphase nuclei. Thus, they are well within the 600 Kb resolution limit of the RH5000 panel. Such minor inaccuracies in marker order will not be detrimental to gene mapping, as the integrated map has fixed positions every 3.42 Mb with support from FISH data. The example of CFA 1 is provided (Figure 1 and 2). Please refer to SOM and websites for all other chromosomes. Synteny and conserved segments Two criteria were used to assign dog BAC clones to orthol- ogous locations of the human genome. First, when com- pared to the complete human genome, each component of the paired end-sequences had greatest similarity to locations that are separated by 50–500 Kb. Although most BAC clones have inserts of 100–200 Kb, minor differences between the two genomes such as local duplication or loss of specific genes should be accommodated by the 50–500 Kb range. If one considers a single aligned end-sequence, the probability of the paired end-sequence aligning spuri- ously within 500 Kb in the human genome is approxi- mately 0.03%. The second criterion was that each pair of In the most recent version of the map [1], 75 conserved segments (CS) were detected within the 38 canine auto- somes. In addition, two CS corresponding to the sex chro- mosomes and nine singletons were detected. These 77 CS were identified through the analysis of 820 canine mark- ers having an unambiguous ortholog in the human sequence. In the present map, that number is more than doubled with 2233 markers having an ortholog in the human sequence. These newer data confirm all previously described CS, and incorporate two singletons into two novel CS for a new total of 79 CS. For the sake of clarity, the remaining singletons were not reported in this new version of the map. Interestingly, while the number of CS did not change sig- nificantly compared to the previous iteration of the RH map, their nature and composition were altered, as shown for CFA 1 (Figure 1). Four CS were identified for CFA1 that correspond, in order from centromere to telomere, to human chromosomes (HSA) 18, 6, 9 and 19. From the density of markers for which an ortholog can be identi- fied, it is clear that the order of markers is consistent between CFA 1 and HSA 18. Thus, this CS would be con- sidered a "conserved and ordered segment" (CSO). For the CS corresponding to HSA 6, however, two sub-seg- ments are noted which are caused by an inversion. In each of these two sub-segments, the order of markers is compa- rable to the syntenic portion of HSA 6, and each canine segment would independently be considered a CSO. In addition to identification of some previously unknown CSO, the increased number of markers for which a human ortholog could be identified unambiguously allowed for a substantial increase in the size of many previously iden- tified CS. At present, as shown in Figure 1 and SOM, CS in the dog are highly contiguous with the human genome. Indeed, only a few markers identified as human orthologs fall outside of a CS. This significantly refines our knowl- edge of evolutionary breakpoints between the dog and human genomes. Discussion The 4106 markers that constitute this new map occupy 3090 distinct positions, with an average of 1.3 markers per position. This reflects the fact that with 4106 geno- typed markers, we are approaching the saturation level of 4500 markers predicted on the basis of size of the canine genome, and resolving power of the 5000 rad panel [6]. A detailed analysis of the co-localized markers indicates that in the majority of cases markers are co-positioned with Page 6 of 11 (page number not for citation purposes)