* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Supplemental Material
Gene desert wikipedia , lookup
Non-coding DNA wikipedia , lookup
Polymorphism (biology) wikipedia , lookup
Genetic engineering wikipedia , lookup
DNA barcoding wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Skewed X-inactivation wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Transposable element wikipedia , lookup
Ridge (biology) wikipedia , lookup
Genomic library wikipedia , lookup
Human genome wikipedia , lookup
Hybrid (biology) wikipedia , lookup
Public health genomics wikipedia , lookup
Metagenomics wikipedia , lookup
History of genetic engineering wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Genomic imprinting wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Gene expression profiling wikipedia , lookup
Pathogenomics wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Gene expression programming wikipedia , lookup
Minimal genome wikipedia , lookup
Helitron (biology) wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Genome editing wikipedia , lookup
Koinophilia wikipedia , lookup
Y chromosome wikipedia , lookup
Designer baby wikipedia , lookup
Genome evolution wikipedia , lookup
X-inactivation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Neocentromere wikipedia , lookup
Supplemental Text for Polytene Chromosomal Maps of 11 Drosophila species: The order of genomic scaffolds inferred from genetic and physical maps Tables of Contents 1. Supplemental Materials and Methods 2. Supplemental Results 3.Notes on Chimeric Assembly Scaffolds 4. File Formats for Supplemental Tables 5. List of Supplemental Tables 6. Supplemental Literature Cited …………………………………..1 …………………………………..7 …………………………………11 …………………………………12 …………………………………15 …………………………………17 1. Supplemental Materials and Methods. General Strategy for Mapping Scaffolds to Polytene Chromosomes. A variety of approaches were used to map genome scaffolds to polytene chromosomes. The simplest approach involved the four members of the melanogaster subgroup (D. simulans, D. sechellia, D. erecta and D. yakuba). Prior investigation of these species revealed that the banding pattern of the polytene chromosomes was sufficiently conserved that the inversion complexes that characterize differences in gene order among these species were apparent even when it was not possible to recover F1 hybrids (ASHBURNER and LEMEUNIER 1976; LEMEUNIER and ASHBURNER 1976; LEMEUNIER et al. 1978). Thus, a simple alignment of the D. melanogaster orthologs in the scaffolds of these species allows one to not only unequivocally assign a majority of scaffolds to chromosome arm but also to order and orient the scaffolds within the arm. Interestingly, these alignments were largely congruent with the older purely cytological observations confirming the basic accuracy of the earlier observations (ibid). For the more distantly related species, this was not possible because, while the syntenic relationships of the Muller elements could be used to assign scaffolds to arms, their order and orientation could not be as easily determined due to the number of overlapping inversions that rearranged genes within the arms. In some cases, this difficulty was overcome by prior mapping of clones to the polytene chromosomes of the non-melanogaster group species. Examples of this are the mapping of P1 clones in D. virilis (LOZOVSKAYA et al. 1993; VIEIRA et al. 1997) and the position of transposon-induced mutations in D. ananassae (MATSUBAYASHI et al. 1992). When this was not possible, for example in D. willistoni and D. mojavensis, probes were synthesized based on the sequence of the scaffolds and in situ hybridizations were performed. A unique approach was adopted for the Hawaiian species D. grimshawi. In this case, there were several prior localizations of individual genes, but these were done on Hawaiian species other than D. grimshawi (DAVIS et al. 1998). However, the phylogeny of the Drosophila species endemic to the Hawaiian chain is known and well established (see POWELL 1997). Moreover, one of the data sets used to establish the phylogeny is the set of inversion polymorphisms within and between species that are associated with their evolution (CARSON 1992; CARSON et al. 1992). Using the various localizations in non-D. grimshawi species and the inversion genealogy from those to D. grimshawi, it was possible to deduce the positions of the scaffolds in this species. These varied approaches have informed the organization of the major scaffolds on the chromosome maps of all 11 species and these analyses of the assembled genome sequences have provided novel insights into possible underlying causes and mechanisms of chromosomal evolution in the genus. D. melanogaster Species Group Chromosome Map Preparation. All of the analyses reported used the CAF1 assemblies of D. simulans, D. sechellia, D. erecta and D. yakuba and version 4.3 of the D. melanogaster assembled and annotated genome. The annotated versions of the four melanogaster group species were retrieved from FlyBase. These were derived from the community assemblies and annotations posted on the AAA web site (http://rana.lbl.gov/drosophila/). The orthology calls made by V. Iyer and M. Eisen, which were also posted at the AAA site, were also used in this analysis. The FlyBase inferred cytological map locations were assigned to all of the orthologs called in the four species. These associations were then ordered and sorted according to their scaffold assignments and molecular coordinates for each species. These simple alignments based on a correlation of D. melanogaster cytology, scaffold linkage and gene order by molecular coordinate proved remarkably congruent and allowed ready alignment of the major scaffolds to the polytene chromosome maps. An added feature resulting from the alignments was that the known inversion constellations that differentiate the four species from D. melanogaster were easily discerned and mapped to the sequence of the assembled scaffolds. D. ananassae Chromosome Map Preparation. The stock of D. ananassae (AABBg1) was maintained at 23o C in a corn meal-yeast-glucose and agar medium. Well-fed larvae ready for pupation were dissected in a solution containing lactic acid: distilled water: acetic acid (1:2:3). Salivary glands were immediately transferred to the same solution and were squashed after 10 minutes for chromosome preparation. The photographic maps (TOBARI et al. 1993) were used to anchor assembles scaffolds to the cytological map. The positions of the genetic and physical markers within the assembled scaffolds were obtained from the Synpipe output (BHUTKAR et al. 2006). The cytological position of each molecular marker was determined by in situ hybridization to the polytene chromosomes with minor modification of the procedures described in Biemont et al. (2004). The DNA fragments of PCR product were labeled with digoxigenein-11-dUTP (PCR DIG Labeling Mix, Roche) as a probe for the hybridization. The linkage maps of morphological mutants constructed by Hinton (unpublished in 1991 in TOBARI 1993) were used for the analysis. D. pseudoobscura Species Group Chromosome Map Preparation. The stocks of D. pseudoobscura (MV2-25, 14011-0121.94) and D. persimilis (MSH3, 14011-0111.49) were maintained at 18o C in a corn meal molasses agar culture medium. Third instar larvae were collected and placed in Drosophila Ringers solution for 5 minutes. Salivary glands were dissected from larvae and squashed according to the procedure developed by Harshman (1977). This technique helped to obtain chromosomes that tended to be linearized. A 700 gram weight was set on the coverslip to aid in flattening the chromosomes (BALLARD and BEDO 1991). The chromosomes were viewed at 1,000x and digital images of linear chromosomes were collected for the six chromosomal arms. Adobe Photoshop was used to build mosaic images of each of the six chromosomes. We minimized the number of different polytene chromosomes that were used to build the mosaic images, but in all cases, we made sure that the different chromosomal sections blended in the mosaic were of similar scales. Section designations were available for each of the six chromosomes except for XR (Muller A•D). Sub-sections were assigned using the approach of Bridges (1935). We made every effort to begin each sub-section at an easily recognizable landmark such as a band or boundary to a puff. New ideograms for the six chromosomes of the two species were drawn by tracing the original maps developed by Tan (1936; 1937) in Adobe Illustrator. These new ideograms are superior to the old reproductions because they are drawn in vector graphics and allow infinite scalability in web based applications. There was not an ideogram available for Muller A•D so the photomicrograph image of this chromosome was used to develop the representation for this map. We used the locations of previously mapped genetic markers (ANDERSON 1993; BECKENBACH 1981; DONALD 1936; KOVACEVIC and SCHAEFFER 2000; LANCEFIELD 1922; LEVINE and LEVINE 1955; NOOR et al. 2000; ORR 1995; ORTIZ-BARRIENTOS et al. 2006; PRAKASH 1974; STURTEVANT and NOVITSKI 1941; STURTEVANT and TAN 1937; TAN 1936; YARDLEY 1974) and physical markers (AQUADRO et al. 1991; BABCOCK and ANDERSON 1996; BONDINAS et al. 2002; DOBZHANSKY and STURTEVANT 1938; HAMBLIN and AQUADRO 1999; MACHADO et al. 2002; MOORE and TAYLOR 1986; PAPACEIT et al. 2006; SCHAEFFER and AQUADRO 1987; SCHAEFFER et al. 2003; SEGARRA and AGUADE 1992; SEGARRA et al. 1996) to anchor the assembled scaffolds to the cytological map. The positions of the genetic and physical markers within the assembled scaffolds were obtained from the Synpipe output (BHUTKAR et al. 2006). D. willistoni Chromosome Map Preparation. The stocks of D. willistoni were maintained in 25o C incubators on the culture medium of Marques et al. (1966). Salivary gland cells of third instar, well-nourished larvae were prepared using a modification of the technique of Ashburner (1967), the glands were fixed with acetic acid (45%) and stained with acetic orcein (2%). The Gd-H4-1 strain of D. willistoni from Guadeloupe (16° 15’ N 61° 35’ W) was chosen for genomic sequencing from a set of six strains because it was chromosomally monomorphic. Gd-H4-1 contains the standard chromosomal order, except for two small fixed inversions in arm IIL and one inversion in XL. The genetic maps of morphological mutant alleles (SPASSKY and DOBZHANSKY 1950) and allozyme variants (LAKOVAARA and SAURA 1972) were used to anchor assembled scaffolds to the cytological map. These maps were developed using a strain of D. willistoni that was chromosomally monomorphic for standard gene arrangements. The method of Engels et al. (1986) was used for the in situ hybridization assays to physically map genes to the polytene chromosomes of D. willistoni. Probes were labeled with biotin-7-dATP by nick translation with the GIBCO BRL kit, the hybridizations were detected using the BCIP, SAP and NBT while chromosomes were stained with 0.1 % lacto-aceto-orcein. The positions of the genetic and physical markers within the assembled scaffolds were obtained from the Synpipe output (BHUTKAR et al. 2006). To construct the new photomap, we used as the standard X-chromosome order those patterns of the XL and XR arms with the widest geographical distribution within all the populations analyzed (according to ROHDE 2000). These standard gene arrangements (designed XL-A and XR-A) are fixed in an old laboratory population-WIP4, collected by A. Cordeiro and H. Winge in the 1960s in Ipitanga, Bahia Northeast State in Brazil and have the closest phylogenetic relationship with the remaining arrangements. Microscopic analysis of X chromosomes in offspring from crosses between the WIP4 population and southern Brazilian wild populations was used to confirm the gene arrangement on the X chromosome. The Xchromosome pattern of the WIP4 population probably corresponds to the Standard arrangement from the Belém population described by Dobzhansky (1950). Until recently, the status of the dot chromosome was unclear in D. willistoni (STURTEVANT and NOVITSKI 1941). Many authors considered that this small chromosome might be fused to another chromosome. Papaceit and Juan (1998) solved this mystery when they used probes for genes on the fourth chromosome of D. melanogaster and found that they hybridized to the most basal section of the third chromosome. Thus, the dot chromosome or Muller element F has apparently fused to the E element in D. willistoni. D. virilis Chromosome Map Preparation. The sequenced strain of D. virilis contains visible mutations at loci on each of the large autosomal elements. The original strain appears to have been constructed at The Institute for Developmental Biology (Moscow, USSR), and it was subsequently placed in the species collection currently maintained at the Tucson Drosophila Stock Center. A derivative of this original strain was inbred more than 14 generations by single pair sib-mating in the laboratories of Brian Charlesworth and Bryant McAllister, and it underwent two additional generations of sib-mating immediately prior to expansion for isolating genomic DNA for sequencing. Physically mapped positions in the genome of D. virilis were identified from Flybase records, published literature, and unpublished data. Cytological map positions determined by in situ hybridization are relative to the nomenclature of the standard photographic chromosome map (GUBENKO and EVGEN'EV 1984) and the corresponding graphic map (KRESS 1993). Loci on the linkage map, where a clear relationship exists between the locus and a reference DNA sequence of either D. virilis or D. melanogaster, were also identified (ALEXANDER 1976; GUBENKO and EVGEN'EV 1984). Reported positions of these markers within the physical and/or linkage maps, coupled with an associated DNA sequence, provided reference points to anchor the assembled genome sequence along the chromosomal arms. Most of the reported mapped positions within the genome of D. virilis were obtained through a series of analyses that used in situ hybridization to localize large-insert P1 clones along the chromosomes (LOZOVSKAYA et al. 1993; VIEIRA et al. 1997). End sequences from 593 of these P1 clones that map to unique sites within the genome were generated to anchor the assembly onto the polytene chromosome map. In cases where a reference sequence of D. virilis was available for the in situ localized probe, position of the sequence in the CAF1 assembly was determined using local alignment. Large sequences were localized using MEGABLAST (e < 1E-50), and small microsatellite loci were localized using the best score obtained from BLASTN searches. Otherwise, the transcript of the putative ortholog of D. melanogaster was used as a query in a BLASTN search (e < 1E10). Identification of each mapped position, its associated reference sequence, and its identified position in the CAF1 assembly is included in Supplemental Table 24. Overall organization of the assembled genome sequence on the chromosomal arms of D. virilis was inferred through a comparison of the order and orientation of scaffolds indicated by the anchored positions in the physical and linkage maps and scaffold joins identified from the Synpipe analysis of conserved syntenic blocks of orthologous genes. Position and orientation of scaffolds was mostly supported by both approaches, thus providing a high level of confidence in the inferred order and orientation of these scaffolds. A greater degree of uncertainty exists for the placement of the sequence in regions where only one of the approaches supports a particular arrangement, and this uncertainty is clearly demarcated. A set of “orphan” scaffolds was identified as being present on particular chromosomal elements based on their content of putative orthologs. However, these scaffolds were not placed within the polytene chromosome map with marker data. These orphan scaffolds potentially represent “islands” of assembled genome sequence localized within the pericentromeric heterochromatin of D. virilis. Heterochromatic regions of the Drosophila genome are known to be enriched with repetitive sequences (SMITH et al. 2007). To determine the proportion of each orphan scaffold that is represented by repetitive elements, each sequence was analyzed with Repeat Masker to identify interspersed repeats using the “Drosophila fruit fly genus” repeat library (http://www.repeatmasker.org). Three scaffolds of similar size (~1 Mb each) that mapped to medial positions of the X chromosome were used for comparison of element content. Physically mapped positions in the genome of D. virilis were localized within the assembled genome sequence and used to anchor the scaffolds to specific chromosomal positions. The orientation of these scaffolds was compared with the syntenic blocks identified through computational analysis to support scaffold joins based on both approaches, and to identify unique scaffold positions and orientations revealed by a single approach. D. mojavensis Chromosome Map Preparation. The D. mojavensis genome stock (15081-1352.22) was maintained at 17 C. Third instar larvae were dissected in a 45% acetic acid solution and their salivary glands removed. Salivary glands were then placed in fixative (1 volume lactic acid, 2 volumes water, 3 volumes acetic acid) for 5 minutes. A siliconized cover slip was placed on the salivary glands and compressed. Slides were then stored at -80 C for 24 hours. After freezing the cover slips were removed and slides were placed in a -80 C ethanol bath and allowed to come to room temperature. At this point slides were removed from the ethanol, air dried and stored at 4 C until hybridization. Immediately prior to hybridization, slides were incubated for 30 minutes at 65oC in 2X SSC, pH 7, cooled to room temperature, denatured with 0.14M Sodium Hydroxide in 2X SSC, washed multiple times in 2X SSC and dehydrated in successive washes of 70% and 95% ethanol, then air dried. Amplicons of 800-1100 bp in length were produced by standard PCR methods using appropriate primers (Supplemental Table 1). Amplified DNA was purified using Qiagen QIAquick PCR Purification kits (Qiagen Inc., Valencia CA), then biotinylated using Roche Biotin-Nick Translation Mix (Roche Applied Science, Mannheim, Germany). Briefly, 500 g of full-length amplicon was nick translated at 15oC for 30-40 minutes to produce biotinylated probes of 200-500 bp in length. Probes were precipitated in ethanol and sodium acetate in the presence of denatured salmon sperm DNA, washed once in 70% ethanol and dried. Probes were reconstituted in a hybridization buffer consisting of 2X SSC, 10% dextran sulfate, 50% formamide, pH 7, denatured at 65oC, and hybridized to slides containing prepared chromosomes for 15-20 hours at 37oC. Following a brief wash in 1X PBS, slides were incubated in 50mM Tris, pH 7.6 containing 4% BSA and a 1/100th volume of Vectastain (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Slides were washed 3 times with 1X PBS and developed in a solution of 50 mM Tris, pH 7.6 containing 0.15 mg/ml Diaminobenzidine and 0.08% Hydrogen Peroxide. The linkage map was determined from microsatellite markers identified by Ross and Markow (2006), Staten et al. (2004), and by Reed et al. (unpublished). Linkage relationships among 25 informative microsatellite markers were determined using 304 members of an F2 population were genotyped at 25 informative microsatellite markers (Reed et al. unpublished). Mapmaker 3.0 from the Whitehead Institute was used to calculate the linkage map (LANDER et al. 1987). We assigned markers to chromosome-based groups and then performed multipoint analysis on each chromosome group. Maximum likelihood marker order was calculated for each chromosome and the Haldane mapping function was used to assign linkage distances between markers in centiMorgans (cM). Markers were assigned to chromosomes using one or more of the following methods. 1) using BLAST (ALTSCHUL et al. 1994) of the flanking sequence against the D. melanogaster genome sequence to identify which Muller element and thereby to what chromosome in D. mojavensis it belonged (STATEN et al. 2004); 2) using allozyme markers assigned to chromosome by Zouros (1981; 1991) that differ between D. mojavensis and D. arizonae and looking for linkage between microsatellite and allozyme genotypes using non-recombinant male intermediaries; 3) looking for close linkage between assigned markers and unassigned makers. Chromosome assignments were then confirmed with BLAST against the D. mojavensis sequence. D. grimshawi Chromosome Map Preparation. Polytene chromosome maps (CARSON 1992) of the G1 line of Drosophila grimshawi served as the basis of our anchored scaffold figures. This strain was established in 1963 from an isofemale line and was recently sequenced (DROSOPHILA 12 GENOMES CONSORTIUM 2007). A total of 31 genes were localized via in situ hybridization to the polytene chromosomes of D. grimshawi, D. silvestris, D. nigribasis, or D. heteroneura using standard techniques (EDWARDS et al. 2001). Polytene band assignment (Supplemental Table 26) for non-homosequential species were made by using information of known inversion difference between species (see Results and CARSON et al. 1992). BLAST (ALTSCHUL et al. 1997) was used to determine genomic locations of each gene localized by in situ hybridization. Genome location was equated with polytene band assignments to place scaffolds on Muller elements by searching the D. grimshawi genome with the D. melanogaster homolog of each localized gene. Supplemental Results Unassigned or Orphan Scaffolds D. simulans. There are 659 unplaced scaffolds that contain called orthologs to euchromatic genes in D. melanogaster (Supplemental Tables 16 and 27). Some of these contain genes and sequences that are duplicates of sequence incorporated into the major aligned scaffolds. The total of unaligned sequence in these scaffolds is 3.9 Mb and the average size of the scaffolds is 5.9 Kb with the smallest being 0.5 Kb and the largest 178.3 Kb. A total of 82% of the scaffolds are <10 Kb; 15% are between 10 and 30Kb; 3% between 30 and 80 Kb and 1% >100 Kb. The remaining 20.9 Mb of sequence is in 9,280 scaffolds that have an average length of 2.2 Kb and are unassigned to a chromosome. Thus the majority of the unaligned scaffolds are quite small and many of the total have the potential to be incorporated into the large scaffolds that have been aligned to the chromosomes. D. sechellia. There are 58 unplaced scaffolds that contain called orthologs to euchromatic genes in D. melanogaster (Supplemental Table 17 and 27). As noted many of these contain genes and sequences that are duplicates of sequence incorporated into the major aligned scaffolds. The total of unaligned sequence in these scaffolds is 1.2 Mb and the average size of the scaffolds is 20.6 Kb with the smallest being 0.8 Kb and the largest 165.9 Kb. A total of 77% of the scaffolds are <10 Kb; 15% are between 10 and 30Kb; 4% between 30 and 100 Kb and 4% between 100 and 341 Kb. The remaining 46.1 Mb of sequence is in 14,555 scaffolds that have an average length of 3.2 Kb and are unassigned to a chromosome. The majority of the unaligned scaffolds are quite small and the 7 largest scaffolds either have only one or two called orthologs (5) or contain orthologs that are duplicated (2) in one of the aligned scaffolds and would thus appear not to provide much additional information to the aligned scaffolds reported here. D. erecta. There are 11 unplaced scaffolds that contain called orthologs to euchromatic genes in D. melanogaster (Supplemental Tables 18 and 27). As noted some of these contain genes and sequences that are duplicates of sequence incorporated into the major aligned scaffolds. The total of unaligned sequence in these scaffolds is 300 Kb and the average size of the scaffolds is 25.9 Kb with the smallest being 1.0 Kb and the largest 212.3 Kb. A total of 56% of the scaffolds are <10 Kb; 40% are between 11 and 90Kb; 4% >100 Kb. The remaining 25.3 Mb of sequence is in 5,091 scaffolds that have an average length of 5.0 Kb and are unassigned to a chromosome. Thus just more than half of the unaligned scaffolds are small and at least some of the larger ones have the potential to be incorporated into the scaffolds that have been aligned to the chromosomes. D. yakuba. There are 121 unplaced scaffolds that contain called orthologs to euchromatic genes in D. melanogaster (Supplemental Tables 19 and 27). Several of these contain genes and sequences that are duplicates of sequence incorporated into the major aligned scaffolds. The total of unaligned sequence in these scaffolds is 6.5 Mb and the average size of the scaffolds is 52.9 Kb with the smallest being 0.8 Kb and the largest 1.8 Mb. A total of 47% of the scaffolds are <10 Kb; 28% are between 10 and 30Kb; 15% between 30 and 80 Kb and 10% >100 Kb. The remaining 32.6 Mb of sequence is in 8,182 scaffolds that have an average length of 4.0 Kb and are unassigned to a chromosome. Thus a slight majority of the unaligned scaffolds are small and many of the total have the potential to be incorporated into the large scaffolds that have been aligned to the chromosomes. D. ananassae. There are 50 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 20 and 27). The average length of these scaffolds is 544 Kb. There are 12 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 653 Kb. The remaining 65.5 Mb of sequence is in 13,663 scaffolds that have an average length of 4.8 Kb and are unassigned to a chromosome. A total of 94.7 % of these scaffolds are less than 10 Kb, 3.0 % are between 10 and 20 Kb, 0.3 % are between 20 and 30 Kb, and 2.1 % are between 30 and 871 Kb. D. pseudoobscura. There are 158 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 21 and 27). The average length of these scaffolds is 26.4 Kb. There are 21 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 27.1 Kb. The remaining 20.3 Mb of sequence is in 4,692 scaffolds that have an average length of 4.3 Kb and are unassigned to a chromosome. A total of 88.7 % of the scaffolds are less than 10 Kb, 6.8 % are between 10 and 20 Kb, 2.1 % are between 20 and 30 Kb, and 2.0 % are between 30 and 150 Kb. D. persimilis. There are 124 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 22 and 27). The average length of these scaffolds is 73.0 Kb. There are 13 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 60.7 Kb. The remaining 48.2 Mb of sequence is in 12,600 scaffolds that have an average length of 3.8 Kb and are unassigned to a chromosome. A total of 95.9 % of the scaffolds are less than 10 kb, 3.1 % are between 10 and 20 Kb, 0.6 % are between 20 and 30 Kb, and 1.3 % are between 30 and 351 Kb. D. willistoni. There are 41 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 23 and 27). The average length of these scaffolds is 473.1 Kb. There are 10 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 73.0 Kb. The remaining 63.3 Mb of sequences is in 14,845 scaffolds that have an average length of 4.3 Kb and are unassigned to a chromosome. A total of 95.4 % of these scaffolds are less than 10 Kb, 2.2 % are between 10 and 20 Kb, 1.0 % are between 20 and 30 Kb, and 1.4 % are between 30 and 351 Kb. D. virilis. Four “orphan” scaffolds contain high scoring putative orthologs of genes located on the X chromosome of D. melanogaster, but they do not appear to be present in the euchromatic portion. Two scaffolds, 13036 and 12528, are organized as a conserved block based on syntenic groups identified in the other genome sequences. Supplemental Tables 24 and 27 lists these orphan scaffolds that likely represent “islands” of assembled sequence embedded within the pericentromeric heterochromatin. Consistent with the inference of a heterochromatic location, these four scaffolds contain approximately 35-45% interspersed repetitive sequence. In contrast, other similar sized scaffolds (A3, A4, and A5) located centrally within the polytenized region contain only 3-5% interspersed sequence. Therefore, these four orphan scaffolds are likely to represent the pericentric regions of the X and to comprise over 5 Mb of sequence that form initial building blocks from which more extensive analyses of the heterochromatic regions of D. virilis could proceed. Interestingly, these scaffolds do not contain any of the 24 annotated genes in the X heterochromatin of D. melanogaster. A conserved syntenic block comprised of three orphan scaffolds (12967, 10419 and 12728) that contain genes from element B was identified (Supplemental Table 24). Consistent with the orphan scaffolds from the X chromosome, each of these scaffolds contains a large amount (>28%) of interspersed repetitive sequence. A preliminary linkage analysis using markers developed within each of these scaffolds was performed to determine if these orphan scaffolds represent assembled regions of the pericentric heterochromatin of Chromosome 4. Using hybrids between D. virilis and D. americana (same arrangement of Chromosome 4) tight linkage was observed between single markers within each scaffold (Dvir\chico, Dvir\Ribonucleoside diphosphate reductase large subunit (RnrL), and Dvir\daughterless (da), respectively) and the Adh locus at the base of Chromosome 4 (4.7% recombinants, N = 275). Complete linkage was observed among the markers distributed among these three scaffolds and between this block and a molecular marker developed within the putative ortholog of the Dmel\nmd gene (zero recombinants, N = 275), which is located near the end of scaffold 12723 (B3) about 44 kb from the proximal end of the sequence organized on the map of Chromosome 4. Thus, these results demonstrate a centromeric location of these repeat enriched scaffolds containing over 1.7 Mb of the genome sequence and support the inference that each of these orphan scaffolds likely represents an assembled heterochromatic region. Again, annotated genes within the B•C heterochromatin of D. melanogaster are not present in these scaffolds. Two orphan scaffolds potentially map to the centromeric region of Element E, and an additional orphan scaffold (12736) has previously been shown by in situ hybridization of RpL15 to localize within the pericentromeric heterochromatin of this chromosome (SCHULZE et al. 2006). Each of these scaffolds contains a high proportion of their sequence as interspersed repeats (>30%), but also contains single-copy genes located within element E of D. melanogaster. The 632-kb scaffold 12936 contains eight such genes, and conservation of syntenic groups indicates this scaffold forms a block with the 3,548-kb scaffold 12958. However, this later scaffold contains an almost even mixture of genes from elements E, D, and the centromeric heterochromatin of these fused elements within the genome of D. melanogaster (Supplemental Table 24). Mosaic gene composition from elements D and E was previously recognized around the RpL15 gene of D. virilis (SCHULZE et al. 2006). This gene is present within orphan scaffold 12736, which is also a mosaic of element D and E genes (Supplemental Table 24). The RpL15 gene has been localized by in situ hybridization to the pericentromeric heterochromatin of Chromosome 2 (element E) of D. virilis (SCHULZE et al. 2006), whereas it is located in the heterochromatin of Chromosome 3L (element D) of D. melanogaster (SCHULZE et al. 2005). Appearance of frequent movement of genes between elements within these pericentric scaffolds of D. virilis may derive from a single exchange between arms of Chromosome 3 (i.e., via pericentric inversion) within the D. melanogaster lineage. The remaining scaffolds in the genome assembly have not been placed in relation to the chromosomes of D. virilis. Although these unplaced scaffolds are numerous, in total they only represent 43.7 Mb of sequence, or about 21% of the assembly. The largest of these unplaced scaffolds is 2.2 Mb in size (13045) that appears to be devoid of any putative orthologs of genes currently identified in D. melanogaster. This large scaffold is exceptional, because only three others are greater than 500 kb and the remaining scaffolds are less than 500 kb in size. Putative orthologs of D. melanogaster genes were identified on 54 of these smallest scaffolds that collectively represent 4.6 Mb of the unplaced scaffolds. D. mojavensis. There are 37 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 25 and 27). The average length of these scaffolds is 175.8 Kb. There are 9 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 5.3 Kb. The remaining 34.1 Mb of sequence is in 6,785 scaffolds that have an average length of 5.0 Kb and are unassigned to a chromosome. A total of 94.4 % of these scaffolds are less than 10 Kb, 2.4 % are between 10 and 20 Kb, 0.2 % are between 20 and 30 Kb, and 3.0 % are between 30 and 626 Kb. D. grimshawi. There are 152 unplaced scaffolds with genes that are orthologous to D. melanogaster euchromatic genes (Supplemental Tables 26 and 27). The average length of these scaffolds is 29.9 Kb. There are 5 unplaced scaffolds with genes that are orthologous to D. melanogaster heterochromatic genes. The average length of these scaffolds is 6.0 Kb. The remaining 60.4 Mb of sequence is in 17,267 scaffolds that have an average length of 3.5 Kb are unassigned to a chromosome. A total of 96.8 % of these scaffolds are less than 10 Kb, 1.9 % are between 10 and 20 Kb, 0.1 % are between 20 and 30 Kb, and 1.2 % are between 30 and 309 Kb. General Observations. The majority of unplaced scaffolds are relatively small fragments relative to all scaffolds in the assemblies. These scaffolds are not gene rich. The largest scaffolds that are unaccounted for in the mapping tend to be on Muller F where few markers exist and will require additional efforts to orient the DNA to the polytene chromosomes. 3. Notes on Chimeric Assembly Scaffolds Determined via synteny processing using Synpipe (BHUTKAR et al. 2006). D. sechellia misjoins confirmed by sequencing center. Species Muller CAF1 Scf. gi gb D. sechellia A/E 4 80980796 CH480819.1 Scf_4:Coordinates(1..3,309,224) maps to Muller_A Scf_4:Coordinates(3,343,645..End) maps to Muller_E Species Muller CAF1 Scf. gi gb D. sechellia B/C/E 5 80980795 CH480820.1 Scf_5:Coordinates(1..356,260) maps to Muller_C Scf_5:Coordinates(369,296..5,091,023) maps to Muller_B Scf_5:Coordinates(5,120,468..End) maps to Muller_E Species Muller CAF1 Scf. gi gb D. sechellia D/E 0 80980800 CH480815.1 Scf_0:Coordinates(1..9,924,332) maps to Muller_D Scf_0:Coordinates(9,934,039..End) maps to Muller_E Species Muller CAF1 Scf. gi gb D. willistoni D/C 181009 111135209 CH964154.1 Scf_181009:Coordinates(1..2,748,112) maps to Muller D Scf_181009:Coordinates(2,841,197..End) maps to Muller C 4. File Formats for Supplemental Tables a. Scaffold joining based on syntenic evidence Each species has a corresponding MS-EXCEL file called: Supplemental Table x <species>_scf_joins.xls for Supplemental Tables 4 to 14. Each file has separate worksheets corresponding to individual Muller element. Scaffolds joined together are placed in blocks identified by the marker "Blocks". If additional joins can be made with weaker evidence, the corresponding blocks are repeated after a RED separator marked "Weak Scf. Joins" and these blocks now include additional joins inferred with weaker evidence. In a number of cases, assembly errors have been identified and highlighted with comments. In some cases, scaffolds have been broken up and parts have been assigned to different Muller elements. Approximate coordinate ranges have been provided. Columns in each worksheet: Flag: 0=Normal/Default, 1=Further joins with weak evidence possible for this scaffold, 2=Comparative evidence exists but is weaker (typically from a single far-off species) Species: Name of species Muller: Name of Muller element this scaffold was mapped to CAF1_Scf: CAF1 name of the scaffold gi: Genbank scaffold id gb: Genbank scaffold accession Scf_Size: Scaffold size in base pairs Orientation: +/- orientation of this scaffold in the corresponding block of joined scaffolds. Orientation is relevant within a block only (does not indicate how the whole block is oriented on the chromosome) Evidence_Code: S: Syntenic information from one or more species used to infer join SG: Syntenic support with some intermediate markers within a scaffold gap B: Joining evidence based on a species-specific breakpoint (weaker than above two) LS: Localized scrambling with larger syntenic context preserved (weaker than first two) Marker_Pair: Internal ids of the pair of markers (homologous genes) used to infer this join D. mel through D. grim columns: x=No information 0=This species does NOT support this scaffold join (with the preceeding scaffold) 1=This species supports this scaffold join (with the preceeding scaffold) Additional column for any comments. b. Scaffold Order: File Format This file presents the summary of the scaffold order based on computational and mapping analysis. Each species is presented under a different tab in the spreadsheet. Information for the six Muller elements is listed within the single sheet providing the orientation of the scaffolds on each chromosomal arm. CAF1_Scf: CAF1 name of the scaffold gi: Genbank scaffold id gb: Genbank scaffold accession Scf_Size: Scaffold size in base pairs Orientation: +/- orientation of this scaffold c. Marker Data: File Format for the species in the melanogaster group Each species has a corresponding MS-EXCEL file called: Supplemental Table x <species>_marker.xls for Supplemental Tables 16 to 19. Each file has eight tabs. Tab 1, Scaffold Summary provides the following information in the columns: Scaffold: name of the scaffold Size: Length of the scaffold in nucleotides Genes: Number of genes within the scaffold Total: Total number of nucleotides or number of genes Tabs 2 to 7, Chromosome N (Muller element) correspond to the Nth chromosome of D. melanogaster or equivalent Muller element and have the following information in the columns, Scaffold: name of the scaffold Start: beginning coordinate for the gene marker End: end coordinate for the gene marker Dmel FBgn: Dmel gene identifier number Dmel Gene Symbol: symbol for the gene name Dmel Cytology: location of the gene marker on the D. melanogaster polytene map D<species> FBgn: <species> gene identifier number D<species> Gene Symbol: D<species> Cytology: Tab 8, Small Scaffolds summarizes the information about unplaced scaffolds and has the following information in the columns, Scaffold: name of the scaffold Start Coordinate: beginning coordinate for the gene marker End Coordinate: end coordinate for the gene marker Dmel FBgn: Dmel gene identifier number Dmel Gene Symbol: symbol for the gene name Dmel Cytology: location of the gene marker on the D. melanogaster polytene map d. Marker Data: File Format for the species not in the melanogaster group Each species has a corresponding MS-EXCEL file called: Supplemental Table x <species>_marker.xls for Supplemental Tables 20 to 26. Each file has separate worksheets corresponding to each of the six Muller elements. Columns in each worksheet: Symbol: symbol for the gene name Gene: gene name Gen_Map: location of the gene marker on the genetic map Cyto_Map: location of the gene marker on the polytene map GenBank_Acc: GenBank accession number of the CAF1 scaffold that includes the gene marker CAF1_Scf: name of the CAF1 scaffold that includes the gene marker Start: beginning coordinate for the gene marker End: end coordinate for the gene marker BLAST: FlyBase: mel_Orth: the orthologous gene name of the D. melanogasterortholog. Reference: reference for the genetic or physical map data. The full reference is located at the bottom of each spreadsheet. 5. List of Supplemental Tables 1. Supplemental Table 1. D. mojavensis oligonucleotide primers 2. Supplemental Table 2. List of genome assembly scaffolds assigned to each Muller element (euchromatic sequence) in each of the 11 species. The first sheet (README) includes notes on format. The data are found in the Excel spreadsheet, supp_file_all_species_euchr_scaffolds.xls 3. Supplemental Table 3. List of genome assembly scaffolds assigned to heterochromatic sequence in each of the 11 species. The first sheet (README) includes notes on format. The data are in the Excel spreadsheet, supp_file_all_species_het_scaffolds.xls 4. Supplemental Table 4. Scaffold-join predictions for D. simulans. The data are in the Excel spreadsheet, Dsim_scf_joins.xls. Notes on content and format of scaffold-join predictions for each species are found in README_File Formats.pdf 5. Supplemental Table 5. Scaffold-join predictions for D. sechellia. The data are in the Excel spreadsheet, Dsec_scf_joins.xls. 6. Supplemental Table 6. Scaffold-join predictions for D. erecta. The data are in the Excel spreadsheet, Dere_scf_joins.xls. 7. Supplemental Table 7. Scaffold-join predictions for D. yakuba. The data are in the Excel spreadsheet, Dyak_scf_joins.xls. 8. Supplemental Table 8. Scaffold-join predictions for D. ananassae. The data are in the Excel spreadsheet, Dana_scf_joins.xls. 9. Supplemental Table 9. Scaffold-join predictions for D. pseudoobscura. The data are in the Excel spreadsheet, Dpse_scf_joins.xls. 10. Supplemental Table 10. Scaffold-join predictions for D. persimilis. The data are in the Excel spreadsheet, Dper_scf_joins.xls. 11. Supplemental Table 11. Scaffold-join predictions for D. willistoni. The data are in the Excel spreadsheet, Dwil_scf_joins.xls. 12. Supplemental Table 12. Scaffold-join predictions for D. virilis. The data are in the Excel spreadsheet, Dvir_scf_joins.xls. 13. Supplemental Table 13. Scaffold-join predictions for D. mojavensis. The data are in the Excel spreadsheet, Dmoj_scf_joins.xls. 14. Supplemental Table 14. Scaffold-join predictions for D. grimshawi. The data are in the Excel spreadsheet, Dgri_scf_joins.xls. 15. Supplemental Table 15 Scaffold_Order.xls. Summary of scaffold order and orientation for the 11 Drosophila species. 16. Supplemental Table 16 Dsim_Marker.xls. Genetic and physical marker data for D. simulans. Notes on content and format of marker file is found in README_File_Formats.pdf 17. Supplemental Table 17 Dsec_Marker.xls. Genetic and physical marker data for D. sechellia. Notes on content and format of marker file is found in README_File_Formats.pdf 18. Supplemental Table 18 Dere_Marker.xls. Genetic and physical marker data for D. erecta. Notes on content and format of marker file is found in README_File_Formats.pdf 19. Supplemental Table 19 Dyak_Marker.xls. Genetic and physical marker data for D. yakuba. Notes on content and format of marker file is found in README_File_Formats.pdf 20. Supplemental Table 20 Dana_Marker.xls. Genetic and physical marker data for D. ananassae. Notes on content and format of marker file is found in README_File_Formats.pdf 21. Supplemental Table 21 Dpse_Marker.xls. Genetic and physical marker data for D. pseudoobscura. Notes on content and format of marker file is found in README_File_Formats.pdf 22. Supplemental Table 22 Dper_Marker.xls. Genetic and physical marker data for D. persimilis. Notes on content and format of marker file is found in README_File_Formats.pdf 23. Supplemental Table 23 Dwil_Marker.xls. Genetic and physical marker data for D. willistoni. Notes on content and format of marker file is found in README_File_Formats.pdf 24. Supplemental Table 24 Dvir_Marker.xls. Genetic and physical marker data for D. virilis. Notes on content and format of marker file is found in README_File_Formats.pdf 25. Supplemental Table 25 Dmoj_Marker.xls. Genetic and physical marker data for D. mojavensis. Notes on content and format of marker file is found in README_File_Formats.pdf 26. Supplemental Table 26 Dgri_Marker.xls. Genetic and physical marker data for D. grimshawi. Notes on content and format of marker file is found in README_File_Formats.pdf 27. Supplemental Table 27 Assembly Summary Data.xls. Information on assembly scaffolds for the 11 Drosophila species. This includes classification of each scaffold as: (1) mapped to a chromosome; (2) assigned to a chromosome based on Synpipe, but not mapped; (3) assigned to heterochromatin; (4) unassigned to a chromosome. The spreadsheet has summary information for all species on a single page as well as a detail spreadsheet for each species. 6. Supplemental Literature Cited ALEXANDER, M. L., 1976 The genetics of Drosophila virilis, pp. 1365-1427 in The Genetics and Biology of Drosophila, edited by M. ASHBURNER and E. NOVITSKI. Academic Press, New York. ALTSCHUL, S. F., M. S. BOGUSKI, W. GISH and J. C. WOOTTON, 1994 Issues in searching molecular sequence databases. Nat. Genet. 6: 119-129. ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHAFFER, J. ZHANG, Z. ZHANG et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402. ANDERSON, W. W., 1993 Linkage map of Drosophila pseudoobscura, pp. 3.252-253.253 in Genetic Maps: Locus Maps of Complex Genomes, edited by S. J. O'BRIEN. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. AQUADRO, C. F., A. L. WEAVER, S. W. SCHAEFFER and W. W. ANDERSON, 1991 Molecular evolution of inversions in Drosophila pseudoobscura: The amylase gene region. Proc. Natl. Acad. Sci. USA 88: 305-309. ASHBURNER, M., 1967 Patterns of puffing activity in the salivary gland chromosomes of Drosophila. Chromosoma 21: 398-428. ASHBURNER, M., and F. LEMEUNIER, 1976 Relationships within the melanogaster species subgroup of the genus Drosophila (Sophophora). I. Inversion polymorphisms in Drosophila melanogaster and Drosophila simulans. Proc. R. Soc. Lond. B. 193: 137-157. BABCOCK, C. S., and W. W. ANDERSON, 1996 Molecular evolution of the sex-ratio inversion complex in Drosophila pseudoobscura: Analysis of the Esterase-5 gene region. Mol. Biol. Evol. 13: 297-308. BALLARD, J. W. O., and D. G. BEDO, 1991 Population cytogenetics of Austrosimulium bancrofti (Diptera: Simulidae) in eastern Australia. Genome 34: 338-353. BECKENBACH, A. T., 1981 Map position of the esterase-5 locus of D. pseudoobscura: usable marker for "sex-ratio". D. I. S. 56: 23-24. BHUTKAR, A., S. RUSSO, T. F. SMITH and W. M. GELBART, 2006 Techniques for multi-genome synteny analysis to overcome assembly limitations. Genome Informatics 17: 152-161. BIEMONT, C., L. MONTI-DEDIEU and F. LEMEUNIER, 2004 Detection of transposable elements in Drosophila salivary gland polytene chromosomes by in situ hybridization, pp. 21-28 in Mobile Genetic Elements: Protocols and Genomic Applications, edited by W. J. MILLER and P. CAPY. Humana Press, Totowa, NJ. BONDINAS, G. P., M. G. LOUKAS, G. N. GOULIELMOS and D. SPERLICH, 2002 The actin loci in the genus Drosophila: establishment of chromosomal homologies among five nearctic species of the Drosophila obscura group by in situ hybridization. Chromosoma 111: 256266. BRIDGES, C. B., 1935 Salivary chromosome maps with a key to the banding of the chromosomes of Drosophila melanogaster. J. Heredity 26: 60-64. CARSON, H. L., 1992 Inversions in Hawaiian Drosophila, pp. 407-439 in Drosophila Inversion Polymorphism, edited by C. KRIMBAS and J. R. POWELL. CRC Press, Boca Raton, FL. CARSON, H. L., J. TONZETICH and L. T. DOESCHER, 1992 Polytene chromosome maps for Hawaiian Drosophila, pp. 441-453 in Drosophila Inversion Polymorphism, edited by C. KRIMBAS and J. R. POWELL. CRC Press, Boca Raton, FL. DAVIS, T., K. EDWARDS, L. DOESCHER, J. KURIHARA, A. LACRE et al., 1998 Physical mapping of Hawaiian Drosophila chromosomes using homologs of Drosophila melanogaster genes. A. Dros. Res. Conf. 39: 825B. DOBZHANSKY, T., 1950 The chromosomes of Drosophila willistoni. J. Heredity 41: 156-158. DOBZHANSKY, T., and A. H. STURTEVANT, 1938 Inversions in the chromosomes of Drosophila pseudoobscura. Genetics 23: 28-64. DONALD, H. P., 1936 On the genetical constitution of Drosophila pseudo-obscura, Race A. J. Genet. 33: 103-122. DROSOPHILA 12 GENOMES CONSORTIUM, 2007 Evolution of genes and genomes on the Drosophila phylogeny. Nature 450: 203-218. EDWARDS, K. A., T. DAVIS, D. MARCEY, J. KURIHARA and D. YAMAMOTO, 2001 Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function. Gene 275: 195-205. ENGELS, W. R., C. R. PRESTON, P. THOMPSON and W. B. EGGLESTON, 1986 In situ hybridization to Drosophila salivary chromosomes with biotinylated DNA probes and alkaline phosphatase. Bethesda Research Laboratory Focus 8: 6-8. GUBENKO, I. S., and M. B. EVGEN'EV, 1984 Cytological and linkage maps of Drosophila virilis chromosomes. Genetica 65: 127-139. HAMBLIN, M. T., and C. F. AQUADRO, 1999 DNA sequence variation and the recombinational landscape in Drosophila pseudoobscura: a study of the second chromosome. Genetics 153: 859-869. HARSHMAN, L. G., 1977 A technique for the preparation of Drosophila salivary gland chromosomes. D. I. S. 52: 164. KOVACEVIC, M., and S. W. SCHAEFFER, 2000 Molecular population genetics of X-linked genes in Drosophila pseudoobscura. Genetics 156: 155-172. KRESS, H., 1993 The salivary gland chromosomes of Drosophila virilis: a cytological map, pattern of transcription and aspects of chromosome evolution. Chromosoma 102: 734742. LAKOVAARA, S., and A. SAURA, 1972 Location of enzyme loci in chromosomes of Drosophila willistoni. Experientia 28: 355-356. LANCEFIELD, D. E., 1922 Linkage relations of the sex-linked characters in Drosophila obscura. Genetics 7: 335-384. LANDER, E. S., P. GREEN, J. ABRAHAMSON, A. BARLOW, M. J. DALY et al., 1987 MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. LEMEUNIER, F., and M. ASHBURNER, 1976 Relationships within the melanogaster species subgroup of the genus Drosophila (Sophophora). II. Phylogenetic relationships between six species based upon polytene chromosome banding sequences. Proc. R. Soc. Lond. B. 193: 275-294. LEMEUNIER, F., B. DUTRILLAUX and M. ASHBURNER, 1978 Relationships within the melanogaster subgroup species of the genus Drosophila (Sophophora). III. The mitotic chromosomes and quinacrine fluorescent patterns of the polytene chromosomes. Chromosoma 69: 349-361. LEVINE, R. P., and E. E. LEVINE, 1955 Variable crossing over arising in different strains of Drosophila pseudoobscura. Genetics 40: 399-405. LOZOVSKAYA, E. R., D. A. PETROV and D. L. HARTL, 1993 A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning. Chromosoma 102: 253-266. MACHADO, C. A., R. M. KLIMAN, J. A. MARKERT and J. HEY, 2002 Inferring the history of speciation from multilocus DNA sequence data: The case of Drosophila pseudoobscura and close relatives. Mol. Biol. Evol. 19: 472-488. MARQUES, E. K., M. NAPP, H. WINGE and A. R. CORDEIRO, 1966 A corn meal, soybean flour, wheat germ medium for Drosophila. D. I. S. 41: 187. MATSUBAYASHI, H., M. MATSUDA, Y. TOMIMURA, M. SHIBATA and Y. N. TOBARI, 1992 Cytological mapping of Om mutants of Drosophila ananassae. Japanese Journal of Genetics 67: 259-264. MOORE, B. C., and C. E. TAYLOR, 1986 Drosophila of southern California. III Gene arrangements of Drosophila persimilis. J. Heredity 77: 313-323. NOOR, M. A., M. D. SCHUG and C. F. AQUADRO, 2000 Microsatellite variation in populations of Drosophila pseudoobscura and Drosophila persimilis. Genet. Res., Camb. 75: 25-35. ORR, H. A., 1995 A new linkage map of the D. pseudoobscura X chromosome. D. I. S. 76: 127128. ORTIZ-BARRIENTOS, D., A. S. CHANG and M. A. NOOR, 2006 A recombinational portrait of the Drosophila pseudoobscura genome. Genet. Res., Camb. 87: 23-31. PAPACEIT, M., M. AGUADE and C. SEGARRA, 2006 Chromosomal evolution of elements B and C in the Sophophora subgenus of Drosophila: evolutionary rate and polymorphism. Evolution 60: 768-781. PAPACEIT, M., and E. JUAN, 1998 Fate of dot chromosome genes in Drosophila willistoni and Scaptodrosophila lebanonensis determined by in situ hybridization. Chromosome Research 6: 49-54. POWELL, J. R., 1997 Progress and Prospects in Evolutionary Biology: The Drosophila Model. Oxford University Press, New York, NY. PRAKASH, S., 1974 Gene differences between the sex-ratio and standard gene arrangements of the X chromosome and linkage disequilibrium between loci in the standard gene arrangement of the X chromosome in Drosophila pseudoobscura. Genetics 77: 795-804. ROHDE, C., 2000 Polimorfismo cromossômico e elementos transponíveis em Drosophila willistoni. Ph. D. Thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil ROSS, C. L., and T. A. MARKOW, 2006 Microsatellite variation among diverging populations of Drosophila mojavensis. J. Evol. Biol. 19: 1691-1700. SCHAEFFER, S. W., and C. F. AQUADRO, 1987 Nucleotide sequence of the Adh gene region of Drosophila pseudoobscura: Evolutionary change and evidence for an ancient gene duplication. Genetics 117: 61-73. SCHAEFFER, S. W., P. GOETTING-MINESKY, M. KOVACEVIC, J. PEOPLES, J. L. GRAYBILL et al., 2003 Evolutionary genomics of inversions in Drosophila pseudoobscura: Evidence for epistasis. Proc. Natl. Acad. Sci. USA 100: 8319-8324. SCHULZE, S. R., B. F. MCALLISTER, D. A. R. SINCLAIR, K. A. FITZPATRICK, M. MARCHETTI et al., 2006 Heterochromatic genes in Drosophila: A comparative analysis of two genes. Genetics 173: 1433-1445. SCHULZE, S. R., D. A. R. SINCLAIR, K. A. FITZPATRICK and B. M. HONDA, 2005 A genetic and molecular characterization of two proximal heterochromatic genes on chromosome 3 of Drosophila melanogaster. Genetics 169: 2165-2177. SEGARRA, C., and M. AGUADE, 1992 Molecular organization of the X chromosome in different species of the obscura group of Drosophila. Genetics 130: 513-521. SEGARRA, C., G. RIBÓ and M. AGUADÉ, 1996 Differentiation of Muller's chromosomal elements D and E in the obscura group of Drosophila. Genetics 144: 139-146. SMITH, C. D., S. SHU, C. J. MUNGALL and G. H. KARPEN, 2007 The Release 5.1 annotation of Drosophila melanogaster heterochromatin. Science 316: 1586-1591. SPASSKY, B., and T. DOBZHANSKY, 1950 Comparative genetics of Drosophila willistoni. Heredity 4: 201-215. STATEN, R., S. D. SCHULLY and M. A. NOOR, 2004 A microsatellite linkage map of Drosophila mojavensis. BMC Genetics 5: 1-8. STURTEVANT, A. H., and E. NOVITSKI, 1941 The homologies of the chromosome elements in the genus Drosophila. Genetics 26: 517-541. STURTEVANT, A. H., and C. C. TAN, 1937 The comparative genetics of Drosophila pseudoobscura and D. melanogaster. J. Genet. 34: 415-432. TAN, C. C., 1936 Genetic maps of the autosomes in Drosophila pseudoobscura. Genetics 21: 796-807. TAN, C. C., 1937 The cytological maps of the autosomes in Drosophila pseudoobscura. Zeitschrift fur Zellforschung u. mikr. Anatomie 26: 439-462. TOBARI, Y. N., 1993 Linkage maps, pp. 49-51 in Drosophila ananassae. Genetical and Biological Aspects, edited by Y. N. TOBARI. Japan Scientific Society Press, Tokyo. TOBARI, Y. N., B. GONI, Y. TOMIMURA and M. MATSUDA, 1993 Chromosomes, pp. 23-48 in Drosophila ananassae. Genetical and Biological Aspects, edited by Y. N. TOBARI. Japan Scientific Society Press, Tokyo. VIEIRA, J., C. P. VIEIRA, D. L. HARTL and E. R. LOZOVSKAYA, 1997 A framework physical map of Drosophila virilis based on P1 clones: applications in genome evolution. Chromosoma 106: 99-107. YARDLEY, D. G., 1974 Linkage analysis of the -amylase locus in pseudoobscura. D. I. S. 51: 25. ZOUROS, E., 1981 An autosome-Y chromosome combination that causes sterility in Drosophila mojavensis x Drosophila arizonensis hybrids. D. I. S. 56: 167-168. ZOUROS, E., 1991 Searching for speciation genes in the species pair Drosophila mojavensis and D. arizonae, pp. 233-247 in Molecular Techniques in Taxonomy, edited by G. M. HEWITT, A. W. B. JOHNSTON and J. P. W. YOUNG. Springer-Verlag, Berlin.