We employed a standard backcross design for these studies utilizing P. maniculatus bairdii stock derived from Washtenaw Co. MI (BW) and P. polionotus stock derived from Ocala Nat'l forest in Florida (PO). While neither population is completely inbred, both originated from a limited number of founders and have been maintained as closed colonies. Thus, identifying fixed differences between the two (e.g., SNPs) was typically not difficult.

Map construction for P. maniculatus was conducted using both the Rattus and Mus maps as references 1516 and using assays designed to span rat-mouse synteny breakpoints. We use the term breakpoint when referring to a break between linkage groups as defined by Pevzner and Tesler 13. In all, we have genotyped 103 Type I gene markers from 18 different Rattus chromosomes on our backcross panels. Table 1 presents the complete list of markers used in our mapping study, their respective locations in the mouse and rat genomes, and the source of the primer sequences. The figures presented here though, focus on the markers around the breakpoint regions between Mus and Rattus genomes that are informative for this comparative analysis.

Our backcross panels facilitated linkage of markers at distances ranging from 1.2 cM to 35.8 cM. There are a few instances, however, where marker co-segregation occurs. These may be due to recombination "cold-spots", segmental inversions between the BW and PO strains, or simply the interval distance may be below our mapping panel resolution.

Our previous analysis of the Peromyscus genome using loci from Mus Chr 11 17 indicated that there are two separate deer mouse linkage groups. ZOO-FISH data by Mlynarski et al. (submitted, BMC Evolutionary Biology) also supported this conclusion. These linkage groups correspond to Rattus Chrs 10 and 14 and the resulting chromosomal breakpoint is shared with the Rattus genome relative to the Mus genome (Figure 1). This breakpoint is also shared in other species, including human, chimpanzee, dog, and pig 1118. This conserved similarity led us to consider whether the deer mouse genome might share a higher degree of chromosomal similarity with Rattus than to Mus.

To explore this possibility, we expanded the existing linkage groups using loci whose orthologs lie on Rattus Chrs 10 and 14 but are not located on Mus Chr 11 (Figure 1). For the Rattus Chr 10 homology group, we generated markers for Btbd12 and Gbl, which correspond to regions of Mus Chr 16 and 17, respectively. Single loci for each segment were sufficient because the Mus Chrs 16 and 17 segments are small and most of Rattus Chr 10 is homologous with Mus Chr 11. We found both markers to be clearly linked to the terminal locus from Rattus Chr 10, Xbp1, with very high LOD scores (>30) indicating rat genome homology. However, all three markers co-segregated. At a distance of only 1.9 Mb in the Rattus genome, these markers are likely to be closer in the deer mouse than our panel is able to resolve.

Using a similar strategy, we also expanded the Rattus Chr 14 homology group using two loci from Mus Chr 5 that have orthologs on Rattus Chr 14, Afp and Hip2. Similar to both Rattus and Mus genomes, Afp and Hip2 are linked in the deer mouse at a distance of 33.6 cM (LOD = 4.4) (Figure 1). Similar only to the Rattus genome though, we found linkage in the deer mouse between Hip2 and the Mus Chr 11 marker Xbp1 at a distance of 19.0 cM (LOD = 11.1) (Figure 1). The marker intervals for this group were larger than those of the Rattus Chr 10 markers and allowed us to map 83.0 Mb (~82%) of Rattus Chr 14 in the deer mouse with only a few markers. Our results again show a greater similarity between Peromyscus genome organization and the Rattus genome than either to the Mus genome.

The high degree of Rattus genome similarity that we found for these two deer mouse linkage groups warranted examination of additional informative regions where synteny breakpoints occur between the Mus and Rattus genomes. While not comprehensive for every chromosome, this strategy accelerated our examination of chromosome evolution for the deer mouse genome and helped determine the best reference genome for future deer mouse genome mapping.

We next chose markers that spanned Rattus Chr 1, focusing our efforts within the regions surrounding the breakpoints between Mus Chrs 7, 17, 10, and 19 (Figure 2). Within the Mus Chr 17 homology segment, we established linkage between Tcp10b and Pacrg at 3.6 cM and between Pacrg and Mas1 at 2.3 cM with LOD scores of 19.4 and 21.8, respectively. These distances are concordant with those for both the Rattus and Mus genomes. We had also previously shown linkage between Tcp10b and Mas1 using the deer mouse radiation hybrid panel 17. We then genotyped the Mus Chr 10 marker Plagl1 to test for linkage to the Mus Ch17 markers and found Mas1 to be linked at a distance of 21.2 cM (LOD = 7.1). This linkage conservation is again consistent with the Rattus genome but not with Mus.

We next tested for linkage between the Mus Chr 17 and Mus Chr 7 homology segments of Rattus Chr 1 and found no linkage in Peromyscus between any Mus Chr 17 markers and the Mus Chr 7 marker Usp29, which is only 17.8 Mb away on Rattus Chr 1. We made no further efforts to extend the Mus Chr 7 linkage group, as Usp29 is only 5.93 Mb from the centromeric end of Mus Chr 7 and additional markers in this region were unlikely to yield a different result. We also tested for linkage between the Mus Chr 17 marker Mas1 and the Mus Chr 7 markers Usp29 and Myod1 on a PO × PO/BW backcross panel. This was to ensure that our negative linkage results were not a result of aberrant interspecific chromosomal recombination in the BW × BW/PO panel. Again, Mas1 did not link to either locus. The lack of linkage between Mus Chr 17 and Mus Chr 7 homology segments in the deer mouse genome constituted the first example of a common breakpoint between the deer mouse and Mus genomes when compared to Rattus.

Although uninformative for our comparative rearrangement analyses, we established linkage homology in the deer mouse for the large (~130 Mb) Mus Chr 7 section of Rattus Chr 1 using five markers: Usp29, Q8C5Y2, Ube3a, Rab6ip1, and H19. Althoughthe RIKEN cDNA marker Q8C5Y2 has not been accurately mapped in either Rattus or Mus, BLAST results indicated intervals between Q8C5Y2 and Usp29 at 26.4 Mb for the Rattus genome and 37.3 Mb in the Mus genome. In support of our placement, the Mus Chr 7/Rattus Chr 1 marker Ube3a co-segregated with Q8C5Y2 in the deer mouse. Our data for these five Mus Chr 7 markers showed high conservation of linkage and gene order with both Rattus and Mus genomes (Figure 2) with LOD scores all well above the 3.0 threshold. Our results for the Mus Chr 7 region were also concordant with a previous study 19 from which we utilized two of the same markers, Usp29 and H19.

We also found genome homology between Rattus and the deer mouse genome by markers spanning the breakpoint between the Mus Chr 7 and Chr 19 regions of Rattus Chr 1. The Mus Chr 7 marker H19 is linked to the Mus Chr 19 marker Prdx5 at a distance of 6.5 cM (LOD = 12.2). Prdx5 is also linked to a second Mus Chr 19 marker Prpf19 at a distance 4.9 cM (LOD = 13.4 cM).

Overall, our data for Rattus Chr 1 loci show that the two deer mouse linkage groups span two Mus genome breakpoints but only one Rattus genome breakpoint, which shows a continued bias towards similarity with the Rattus genome. Our results also imply that the Mus genome has been more rearranged in this region.

To broaden the scope of the deer mouse map and help reduce bias resulting from any localized phenomenon such as segmental inversions, we acquired data for markers from multiple Rattus chromosomes. Rattus Chrs 4 and 6 were priority candidates because of the simple breakpoint arrangements and well-conserved gene orders between Rattus and Mus.

Rattus Chr 4 is represented by the entirety of Mus Chr 6 and approximately 30 Mb of the centromeric end of Mus Chr 5. To test for a conserved organization in the deer mouse genome, we typed two markers from each side of the breakpoint on our backcross panel (Figure 3). We found the two Mus Chr 5 markers, Srpk2 and Sri, are linked to each other at 1.2 cM (LOD = 22.0) and the two Mus Chr 6 markers, Ccdc132 and 1700016G05Rik, are linked to each other at a distance of 7.6 cM (LOD = 14.8). Spanning the breakpoint, we found that Sri and Ccdc132 are linked to each other at a distance of 13.3 cM (LOD = 10.3). Overall, our results span about 30% of Rattus Chr 4 and show conservation of the Rattus gene order. However, the linkage between Sri and Srpk2 was shorter than expected and may be due to a recombination cold-spot, an interspecific inversion, or a deletion.

Our mapping of Rattus Chr 6 loci consisted of ten markers: Sos1, Ppp1cb, Preb, Dnmt3a, Allc, 493504H06Rik, Pygl, Clec14a, Pcnx, and 1700001K19Rik. These markers span Mus chromosomes 17, 5, and 12 and our results yielded two separate linkage groups (Figure 4). The deer mouse linkage group homologous to the centromeric end of Rattus Chr 6 consists of five loci and represents three different chromosomes in Mus. This group is conserved in both synteny and gene order with the Rattus genome. From Mus Chr 17, Sos1 is linked to the Mus Chr 5 marker Ppp1cb at a distance of 11.0 cM (LOD = 12.6). Within the Mus Chr 5 segment, Ppp1cb is linked to Preb at a distance of 6.1 cM (LOD = 16.8). A second Mus breakpoint is spanned by the linkage between Preb and the Mus Chr 12 marker Dnmt3a at a distance of 5.2 cM (LOD = 16.4). Also from Mus Chr 12, Allc represents the terminal marker and links to Dnmt3a at an interval of 16.5 cM (LOD = 7.6).

The five remaining markers from Mus Chr 12 form a second linkage group in Peromyscus. Although synteny with both Rattus Chr 6 and Mus Chr 12 is conserved, we discovered an inversion with respect to both Mus and Rattus involving markers Clec14a and Pygl (Figure 4). Defining this inversion, Pygl is linked to 493504H06Rik at a distance of 4.2 cM (LOD = 9.2) while Clecl4a is linked to the more telomeric marker Pcnx at a distance of 18.2 cM (LOD = 6.3). We also found that Clec14a and Pygl have smaller distance interval at 1.3 cM (LOD = 21.2) than would have been expected from the physical intervals in Mus (11.9 Mb) and Rattus (13.4 Mb). Forming the end of the linkage group, Pcnx was linked to 1700001K19Rik at a distance of 25.3 cM (LOD = 3.9).

The spanning of two Mus genome breakpoints by the deer mouse linkage groups again indicates a more Rattus-like genome organization. However, the breakpoint between the two Peromyscus linkage groups flanked by the two Mus Chr 12 markers Allc and 493504H06Rik represents a unique rearrangement that differs from both the Rattus and Mus genomes. ZOO-FISH results from Mlynarski et al. (submitted, BMC Evolutionary Biology) also concur that Rattus Chr 6 is indeed represented by two separate chromosomes in the deer mouse.

To avoid possible bias towards finding only Rattus genome similarity, we also selected markers to span Rattus synteny breakpoints in relation to the Mus genome. This involved markers that span approximately 89% of Mus Chr 1 but are located separately in Rattus on Chrs 5, 9, and 13 (Figure 5). With exception of the Rattus Chr 9 marker Col9a1, we found linkage between all of the markers within their Rattus chromosome homology groups but not between them, indicating a bias toward Rattus genome similarity.

For the Rattus Chr 5 region, we detected non-segregating linkage between Oprk and Tram in the deer mouse with a strong LOD score of 20.8. To further investigate Rattus genome homology, we employed a third Rattus Chr 5 marker, Ube2j1, which is located on Mus Chr 4. Consistent with Rattus genome organization, Ube2j1 linked strongly (LOD = 23.5) but without segregation to Oprk1 and Tram1, despite a distance 35.3 Mb in the Rattus genome between Ube2j1 and Oprk1.

At the telomeric end of Mus Chr 1, we detected linkage between the Rattus Chr 13 markers Acbd3 and Glul at a distance of 21.2 cM (LOD = 9.6) and between Glul and Bcl2 at 35.8 cM (LOD = 3.8) (Figure 5). However, we did not detect linkage between Bcl2 and the Mus Chr 9 marker Fn1, as would be expected by Mus genome homology.

Amongst the Rattus Chr 9 markers, we found Col3a1 and Fn1 were linked at a distance of 18.8 cM (LOD = 10.3). However, Col3a1 is surprisingly not linked to Col9a1, which represents a deviation from the Rattus genome. To confirm these results, we also tested Mut, a marker that is closely linked to Col9a1 on Rattus Chr 9 (Figure 6). Mut is located 7.13 Mb from Col9a1 on Rattus Chr 9 but in Mus is located separately on Chr 17. Mut did not link to Col3a1 but did co-segregate with Col9a1 (LOD = 12.0), thus identifying a linkage similarity between the Rattus and deer mouse genomes. The breakpoints present in the deer mouse and Mus maps for this region are offset and may represent breakpoint area re-usage and a rearrangement hotspot 13. However, more detailed mapping using markers located between Col9a1 and Col3a1 on Rattus Chr 9 are needed to refine the breakpoint location. We discovered additional Rattus Chr 9 similarity using the marker Lama, which represents a second and separate region of Mus Chr 17 than that of Mut (Figure 6). We found Lama1 and Fn1 are linked in the deer mouse at a distance of 32.7 cM (LOD = 4.7).

As another test of Peromyscus genome homology to Mus, we performed an analysis using six loci from Mus Chr 8 that form two linkage groups in the Rattus genome (Figure 7). We selected markers in each group that are less than 20.Mb apart in the Rattus genome to facilitate deer mouse linkage detection. Additionally, the two markers that flank the breakpoint, 9130404H06Rik and Elmod2, are about 16.0 Mb apart in Mus, which is well within the range of our mapping panel.

As with the Mus Chr 1 analysis, we found linkage with highly significant LOD scores but only within the individual Rattus chromosomal groups, not between them. For the Rattus Chr 16 segment, Adam3 and Mtus1 are linked at distance of 8.4 cM (LOD = 14.8) and Mtus1 is linked to Spata4 at a distance of 16.9 cM (LOD = 9.3). Spata4 is also linked to the terminal marker 9130404D08Rik at a distance of 7.0 cM (LOD = 16.4). The two markers from Rattus Chr 19, Elmod2 and Pmfbp1, are linked 10.3 cM (LOD = 12.5).

Based on suggestions from ZOO-FISH results (Mlynarski et al., submitted BMC Evolutionary Biology), we also examined an additional chromosomal segment for linkage. Representing Rattus Chr 17 and Mus Chr 2, Sec61a2 links to the Rattus Chr 19/Mus Chr 8 marker Pmfbp1 at a distance of 30.1 cM (LOD = 3.7). This linkage indicates a clear deviation from both the Rattus and Mus genomes by the deer mouse genome and highlights the benefit of performing cytogenetic analyses in tandem with meiotic linkage mapping.

We performed additional tests for Mus genome similarity within the deer mouse using loci from Mus Chrs 17, 5, and 13 (Figures 8, 9, and 10). These results also came out negative for Mus genome similarity. Mus chromosomes 17 and 5 are two of the most rearranged chromosomes in the Mus genome compared to the Rattus genome. Mus Chr 17 and has seven different regions representing five different Rattus chromosomes (Figure 8) and Mus Chr 5 has four major regions representing four Rattus chromosomes and three very small segments representing three additional Rattus chromosomes (Figure 9). Positive linkage results for these highly rearranged chromosomes in the deer mouse would have been a strong indicator of Mus homology.

For Mus Chr 17, six markers (Tcp10b, Mas1, and Pacrg from Rattus Chr 1; Gbl from Rattus Chr 10; Lama1 from Rattus Chr 9; and Sos1 from Rattus Chr 6) were tested for linkage in all possible arrangements despite having already been assigned to other deer mouse linkage groups. No linkage was found other than that which already existed amongst the Rattus Chr 1 markers Tcp10b, Pacrg, and Mas1 (Figure 8).

We conducted a similar test for several markers from Mus Chr 5 (Figure 9). Eleven markers representing five Rattus chromosomes were tested for linkage. As in the Mus Chr 17 analysis, linkage was found only between markers located on the same Rattus chromosomes.

Two Mus Chr 13 markers, Clptml1 and Spz, also failed to show linkage despite being only about 19.6 Mb apart in the Mus genome, thus further reinforcing the linkage group disparities between the deer mouse and Mus genomes. Clptml1 is located in Rattus Chr 1 and Spz1 is on Rattus Chr 2 (Figure 10).

Development and availability of multiple mapping tools is essential for accurate and timely exploration of any species' genome. Three methods have already been employed in mapping small portions of the deer mouse genome in the form of cytogenetics 1420, meiotic segregation analysis 172122232425, and a whole-genome radiation hybrid cell panel 17. These tools are most powerful when used in combination, as exemplified by Rowe et al. 2 for Mus and by Menotti et al. 26 for the cat.

Here we significantly expand the Peromyscus meiotic segregation mapping data using two P. maniculatus × P. polionotus interspecific backcross panels and present the most comprehensive comparative linkage mapping data for the deer mouse to date using Type I gene markers. In addition to providing an important genetic tool for Peromyscus research, we tested whether the deer mouse genome displayed organizational homology to that of Mus musculus, Rattus norvegicus, or a combination of both. Our results indicate a large degree of gene order and synteny conservation by the deer mouse genome with that of Rattus.

Our analysis was done by establishing linkage over approximately 35% of the deer mouse genome using gene markers that predominantly spanned junctions of large-scale genome rearrangements between the Rattus and Mus genomes. While using the Rattus genome as the reference, we tested 13 Mus genome breakpoints. Ten of the 13 breakpoints spanned by the Rattus genome were similarly linked in the deer mouse genome. In contrast, only one of 12 Rattus genome breakpoints that we examined while using the Mus genome as a reference closely coincided with any linkage breakpoints that we found in deer mouse genome. These data demonstrate that the organization of the deer mouse and Rattus genomes are more similar to each other than either is to Mus.

There are three instances, however, where the deer mouse map differs from both the Rattus and Mus maps. Two examples are located between markers Allc and 493504H06Rik (Figure 4) and between markers Col9a1 and Col3a1 (Figure 6). Approximately 21 Mb separates the latter pair in both Rattus and Mus, so additional markers will need to be applied to the deer mouse panel to better pinpoint the location of this breakpoint. The third instance is the unique deer mouse linkage of Sec61a2 to Pmfbp1. Collaborative efforts have also helped to inform, as well as confirm, some of these data using additional tools such as ZOO-FISH analyses using flow-sorted whole chromosome probes (Mlynarski et al., submitted BMC Evolutionary Biology). The strong organizational similarity of the deer mouse genome with the Rattus genome rather than the more morphologically similar Mus musculus suggests that a significant amount of rearrangement occurred in the Mus genome after the divergence of the cricetid and murid lineages. Concomitantly, our results suggest that the genomic organizations of Rattus and Peromyscus are more representative of the ancestral muroid genome than the Mus genome, which is in agreement with previous literature that indicated a higher rate of genome rearrangement for Mus 56. Most eutherian genomes have 30 to 40 blocks of homology with the human genome while the Mus genome is extraordinary with approximately 200 homology blocks. However, the Mus genome is not unique in having higher relative rearrangement rates, as the canine and gibbon genomes have approximately twice the average number of homology blocks 27.

Our results also show that genome rearrangement can act independently from other forms of genome evolution, such as sequence mutation. Although rodent sequence mutation rates are higher compared to other mammals, such measurements of genome evolution show Mus and Rattus shared a common ancestor significantly more recently than either have with Peromyscus. Our data does not propose to change this phylogeny but rather merely highlights that DNA sequence variation and chromosome rearrangement are independent processes and greater understanding of both processes can provide different insights into the evolution of the structure and function of the eukaryotic genome.

We chose interspecific backcross analysis in order to maximize genetic polymorphism and for the ease of linkage analysis 28. The two species used in the cross were the deer mouse (P. maniculatus bairdii; BW) and the old field mouse (P. polionotus subgriseus; PO) and were obtained from the Peromyscus Genetic Stock Center at the University of South Carolina 29. We set up the initial crosses in only one direction, BW females × PO males, to generate interspecific hybrid F1's. The direction of this cross is essential, as the reciprocal cross results in lethal overgrowth of the offspring 30.

We created two separate backcross panels, BX116 and BX2, for the linkage analysis. For the BX116 panel, we bred twelve hybrid (plt BW × PO) F1 animals with 12 unrelated plt BW animals to generate 116 backcross progeny. Backcrosses to BW can be performed using both female and male F1 hybrid animals, as both matings will give viable offspring. However, all but one of the matings used for this panel were F1 × BW (♀ × ♂).

We used a similar strategy for the BX2 panel but the plt BW stock was substituted with wild-type BW stock. The plt allele originated in a different subspecies of P. maniculatus than the BW stock to which it was crossed. This additional backcross panel was designed to circumvent intraspecific SNP variation and possible recombination problems due to chromosomal inversions that are known to exist within some P. maniculatus sub-species.

To create the BX2 panel, we crossed four F1 males from separate unrelated ♀ BW × ♂ PO matings to non-sibling BW females, which were generated from separate matings. This resulted in four unrelated backcross families. We obtained twenty-two ♀ BW × ♂ F1 offspring from each of three of these backcross matings and 20 offspring were obtained from the fourth for a total of 86 backcross animals. These were grouped in a 96-well tray along with the eight parentals and a positive and a negative control. We employed this strategy to minimize variation within families while maximizing information between families. Similarly, the strategy of crossing F1 hybrid males with BW females minimized variation due to gender-based differences in recombination frequency.

We extracted genomic DNA from all backcross parents and progeny for the BX116 panel from 1.0 cm tail snips with the Qiagen DNEasy Tissue Kit using the manufacturers protocol (Qiagen, Inc.). Genomic DNA for the BX2 panel animals was extracted using a phenol/chloroform/isoamyl alcohol extraction method to increase yields.

We obtained primer sequences for some Type I markers from published sets of orthologous gene markers. These are termed UMPS, CATS, and TOASTs 313233. Primer sequences for Il23a, Mas1, H19, Sos1, and Grb10 were developed or obtained from published Peromyscus data 343536. Trp53 primers were developed from P. maniculatus sequence and were kindly provided by Michael McLachlan. We developed all other primers from P. maniculatus EST sequences (Weston Glenn et al., submitted BMC Genomics). Deer mouse EST clones were used for marker design because of greater PCR amplification success (>80% versus ~60% for the published sets).

We designed all the markers to be ~400 bp–1500 bp and to span an intron to increase polymorphism detection. This was done by aligning deer mouse DNA sequences to Mus musculus genomic sequences using cross-species megaBLAST (NCBI). Some critical markers however, spanned larger introns and resulted in amplicons slightly larger than the ideal size parameters.

PCR cycling conditions for all Type I markers were optimized for P. maniculatus and P. polionotus DNA using a MJ Research PTC-200 DNA Engine gradient thermal cycler and are defined as follows: 1) Standard: 95°C for 14.5 min followed by 35 cycles of (95°C for 30 s, 48–65°C for 30 s, 72°C for 30 s per 0.5 kb), 72°C for 10 min, 4°C hold. 2) Touchdown65 (TD65): 95°C for 14.5 min followed by 20 cycles of (95°C for 30 s, 65°C for 30 s minus 0.5°C/cycle for 20 cycles, 72°C for 30 s per 0.5 kb) followed by 15 cycles of (95°C for 30 s, 55°C for 30 s, 72°C for 30 s per 0.5 kb), 72°C for 10 min, 4°C hold. If no product was obtained using Touchdown65, the starting annealing temperature was changed to 60°C or 55°C, with the final annealing temperature remaining 10°C lower than the starting temperature.

PCR was performed using 20 ng of genomic DNA in a 10 μl reaction containing 1 μl 10× Qiagen HotStar buffer (1.5 mM MgCl₂), 200 μM each dNTP, 0.4 μM forward primer, 0.4 μM reverse primer, and 1 unit Qiagen HotStar Taq polymerase. Some difficult templates required the use of Qiagen Q-solution at either 1× or 0.5× strength. Four markers, Sparc, Xbp1, Grb10, and Ugp2 required 2.0 mM MgCl₂.

For all markers, 5 μL of each amplification product was visualized by gel electrophoresis. The remaining 5 μL portion of the PCR products was treated for sequencing with 5 units Exonuclease I and 0.75 units Shrimp Alkaline Phosphatase (SAP) and incubated at 37°C for 15 minutes followed by heat-inactivation at 80°C for 15 minutes. For sequencing reactions, 2.0μL of purified PCR product was direct sequenced with BigDye (v3.1) (ABI) on an ABI 3130 × l according to the manufacturer's protocol.

Sequence identities were verified by cross-species megaBLAST or BLASTN search to the Mus musculus genome. Any predicted simple size polymorphisms between BW and PO were tested by gel electrophoresis using amplification products from BW, PO, and BW/PO mix (equal ratio) DNAs. Markers not showing size polymorphisms were further analyzed for species-specific RFLPs by sequence comparison using Sequencher software (Gene Codes Corporation), the TCAG program available as part of the Biology Workbench software utilities provided at the San Diego Supercomputer Center 37, or with the SNP-RFLPing program 38. Candidate enzymes were chosen from those predicted by the software. RFLP tests for each marker and enzyme were conducted according to manufacturer's protocols on 10 μL PCR products from a template test panel consisting of DNA from BW, PO, a BW/PO mix (equal ratio), and a negative using only TE. All RFLP products were analyzed by gel electrophoresis. Any essential markers that could not be genotyped by either size polymorphism or RFLP were sequenced on all the backcross animals and their parents.

We tested the backcross panel parental mice with each marker prior to use on the backcross panel. We typed then typed the markers on all possible backcross animals whose parents exhibited the expected genotype. On the BX116 panel, no fewer than 73 animals were used for data to establish linkage between any two markers with exception of Gbl to Hba and Hba to Canx, of which both only used 39 animals due to the small usable data set for Hba. For the BX2 panel, no fewer than 60 animals were typed between any two markers with the exception of Mut and Col9a1, for which only 40 animals could be genotyped in common.

We performed backcross linkage analysis using Map Manager QTX software 39. A minimum LOD score of 3 was used to establish linkage. Ordering of markers typed on the backcross data was determined by subjecting the data to the "ripple test", which evaluated local permutations and selected the optimal order based on minimum breakage. Once linkage and gene order was established with high degree of confidence, omitted or unavailable genotypes could sometimes be inferred by Map Manager, as double crossovers between closely linked markers are rare. The procedure of inferring genotypes did not change any gene orders but typically tightened linkages slightly and raised LOD scores.