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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
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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
Supplemental Table 15. 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.
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.
17. Supplemental Table 17 Dsec_Marker.xls. Genetic and physical marker data for D.
sechellia.
18. Supplemental Table 18 Dere_Marker.xls. Genetic and physical marker data for D. erecta.
19. Supplemental Table 19 Dyak_Marker.xls. Genetic and physical marker data for D.
yakuba.
20. Supplemental Table 20 Dana_Marker.xls. Genetic and physical marker data for D.
ananassae.
21. Supplemental Table 21 Dpse_Marker.xls. Genetic and physical marker data for D.
pseudoobscura.
22. Supplemental Table 22 Dper_Marker.xls. Genetic and physical marker data for D.
persimilis.
23. Supplemental Table 23 Dwil_Marker.xls. Genetic and physical marker data for D.
willistoni.
24. Supplemental Table 24 Dvir_Marker.xls. Genetic and physical marker data for D. virilis.
25. Supplemental Table 25 Dmoj_Marker.xls. Genetic and physical marker data for D.
mojavensis.
26. Supplemental Table 26 Dgri_Marker.xls. Genetic and physical marker data for D.
grimshawi.
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.
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