indicated as in Figure 1 from centromere to telomere. Common names for some genes are indicated in parentheses. (TIF) Figure S2 Comparative organization of the ultraconserved region CG15121–CG16894 in Diptera. (A) Chromosomal location (red arrowhead) of six protein-coding genes with reliable one-to- one orthologous relationships between D. melanogaster and A. gambiae [77]. The same numerical code as in Figure 1 is used to indicate the identity of the gene. (B) Conserved collinearity of the gene Toll-7 (blue) and Obp genes (red) across Diptera. Other intervening protein-coding genes are indicated in grey. Drosophila and Anopheles diverged ,250 mya [83], Anopheles and Aedes diverged ,150 mya [84], and the divergence time accumulated by the nine Drosophila species previously analyzed was ,381 my [22–24], so that the total divergence time between the Drosophila and mosquito species considered is ,970 my. Genes Obp56f and Obp56i are not indicated since they are not present in all Drosophila species examined [22,85]. Doted lines indicate orthologous relationships ([38] and this work). Gene sizes and distances are not to scale in D. melanogaster. (C) Phylogenetic relationships of the OBP protein sequences encoded by genes nearby Toll-7 between A. gambiae and A. aegypti. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches when higher than the cut- off value of 0.5. (TIF) Figure S3 Crossing scheme followed to generate the inversion In(2R)51F11-56E2. (A) Generation of individuals carrying two FRT-bearing elements in cis (REC) upon recombination in the F2. (B) Generation of strains with (INV) or without (SIM for SIMultaneous control) the inversion In(2R)51F11-56E2 using a heat-inducible flippase-recombinase. Notice that both strains derive from progeny generated from the same vial and therefore they have been exposed to the same experimental conditions. Grey box, mosaic flies for the inversion In(2R)51F11-56E2, both in the soma and the germ line. Carriers of the inversion are red-eyed (w+) and therefore readily identifiable. (C) Restoration of standard gene order via a heat-shock inducible reversion (REV for REVertant control). Only relevant chromosomes are indicated for the genotypes. Dotted box, genotype of flies subsequently made homozygous to construct the stocks to be used in further experiments. For clarity, the terminology used here in relation to FRT-bearing TEs and their derivatives in Table S4 is replaced by explicit indications on the number of FRTs and the state of the reporter gene. 2FRT, original TEs (Figures S4 and S5 for further details). (PDF) Figure S4 Overview of the chromosomal changes occurred during the generation of the inversion In(2R)51F11-56E2 and the corresponding eye phenotypes. Upon a crossing over event, two starting FRT-bearing TEs are placed on the same homolog. In the presence of a FLP recombinase source, recombination events are heat-shock induced between FRT sequences. The first FLP- mediated recombination event occurs between the two FRT sequences located within each TE leading to two recombined FRT sequences, one at each breakpoint, in opposite orientations. The second recombination event is mediated between these two resulting FRT sequences leading to the generation of the inversion. Phenotypic changes in the eye pigmentation of Drosophila adults are caused by alterations in the reporter gene mini-white carried by the TEs. These alterations occur as a result of the FLP-mediated FRT recombination events. Strains that carry a particular chromosome configuration are indicated. Details on the Collinearity Conservation in Eukaryotes precise structure of the TEs in each strain are provided in Figure S5. Sizes and distances are not to scale. (TIF) Figure S5 Schematic representation of the molecular configu- ration of the FRT-bearing TEs at the breakpoint regions of different strains obtained in the course of the generation of the inversion In(2R)51F11-56E2. Two TEs bearing FRT sites in opposite orientation, P{RS3}CB-0236-3 and P{RS5}5-HA-1995, were selected to generate a Type 1 Inversion according to the nomenclature in [42]. Notice that the two FRTs are flanking one of the exons of the modified reporter gene mini-white [86]. While the TEs are intact in the REC strain before the first heat-shock pulse, they undergo different kinds of molecular rearrangements during the rest of the protocol. Specifically, the recombination between the internal FRTs (yellow arrowhead) of each TE leads to the deletion of one of the exons of the reporter gene mini-white (orange and red boxes), which impairs its activity. If the second heat-shock pulse fails to induce a successful NAHR event between the single FRT present in each of the TEs, no rearrangement is generated and no reconstitution of the reporter gene occurs (SIM), which is associated with the w2 phenotype. On the contrary, if ectopic recombination occurs, the newly generated inverted arrangement (INV) will be characterized by the presence of a reconstituted reporter gene at one breakpoint (and therefore by the w+ phenotype) and one FRT at the other breakpoint [42]. A subsequent heat-shock pulse can lead to another successful ectopic recombination event restoring the original gene order (REV) and molecular organization at the breakpoint regions as before the inversion. The terminology used in for the FRT-bearing TEs and their derivatives follows that of Figure S4. Amplicons (A–H) used to confirm the molecular configuration of the breakpoint regions in all relevant strains are shown (Tables S5 and S6). Sizes and distances are not to scale. (TIF) Figure S6 Validation of strains carrying the FRT-bearing TEs used to generate the inversion In(2R)51F11-56E2. (A) Progeny size and (B) sex ratio (female to male) from low-density crosses of homozygous flies for each of the elements alone (5-HA-1995 and CB-0236-3), for both elements in cis (REC), and for flies with the same genetic background but carrying no transposable elements (w1118). No statistically significant difference was found among the strains (Kruskal-Wallis, d.f. = 3; progeny size, P = 0.0730; sex ratio, P = 0.8688; n = 5). Error bars indicate 95% CI. (TIF) Figure S7 Performance of the strains generated in low-density crosses with homozygous flies. (A) Progeny size and (B) sex ratio (female to male). Kruskal-Wallis test indicated that there are no statistically significant differences among strains both in progeny size (d.f. = 7; P = 0.2308) and in sex ratio (d.f. = 7; P = 0.0863). Pairwise contrasts confirmed that strains carrying the disrupted ultraconserved region CG15121–CG16894 did not show signifi- cantly lower values than strains with the ultraconserved region in its intact form (Tables S7 and S8). Error bars indicate 95% CI (n = 5). (TIF) Figure S8 Sequence alignment of the region surrounding the FRT-bearing TEs P{RS5}5-HA-1995 and P{RS3}CB-0236, and their derivatives across strains. (A) Outer (2R:11,260,347..11,261,062) and (B) inner breakpoint (2R:15,613,890..15,614,461), respectively, of the inversion In(2R)51F11-56E2. No major mutation was incidentally generated during the course of our experiments relative to the strain w1118, which was used by others to generate the strains PLoS Genetics | www.plosgenetics.org 10 February 2012 | Volume 8 | Issue 2 | e1002475