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Genome Res. Jun 2005; 15(6): 885–889.
PMCID: PMC1142479

A BAC-based physical map of the Drosophila buzzatii genome

Abstract

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps, and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation, and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and an ~18× expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be ~23×. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9555 clones, and assembled them using FingerPrint Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

A variety of genomic resources have been developed as part of the Drosophila Genome Project, including the high-quality sequence and annotation of the Drosophila melanogaster genome (Adams et al. 2000; Celniker and Rubin 2003). Comparatively few genomic resources have been available for other species within the genus Drosophila. Phylogenetic analyses indicate that two main lineages exist within the Drosophila genus, which diverged ~60 million years ago (Powell 1997; Tamura et al. 2004). One lineage leads to the Sophophora subgenus with ~300 species (including D. melanogaster and D. pseudoobscura), whereas the other one leads to the subgenera Drosophila (including Drosophila virilis and Drosophila buzzatii) and Idiomyia (Hawaiian species), with ~700 and 375 described species, respectively (Powell 1997; http://taxodros.unizh.ch/). Thus, many Drosophila species are relatively distantly related to D. melanogaster, and genomic resources developed for this species therefore have a somewhat limited applicability to them (Segarra et al. 1995; Podemski et al. 2001; Ranz et al. 2001; González et al. 2002). Fosmid and BAC libraries for some Drosophila species have been produced or are currently in production (http://www.genome.gov/; http://tdgc.arl.arizona.edu/baclibraries.htm). Recently, the genome sequence of Drosophila pseudoobscura became available (Richards et al. 2005), and whole-genome shotgun sequences of 10 other Drosophila species are available or in progress (http://rana.lbl.gov/drosophila/multipleflies.html).

Here, we describe the construction of a BAC library and a BAC-based physical map of the D. buzzatii genome. D. buzzatii belongs to the repleta species group of the Drosophila subgenus (Wasserman 1992), a group comprising ~100 species that has been used for studies of ecological adaptation and speciation for more than 60 years (Spencer 1941; Crow 1942; Wharton 1942; Barker and Starmer 1982; Barker et al. 1990). Efforts to map the genome of D. buzzatii began 50 years ago with the comparative analysis of its salivary gland chromosomes to establish the phylogenetic relationships between repleta group species (Wasserman 1954, 1962; Ruiz et al. 1982; Ruiz and Wasserman 1993). This was followed by the linkage mapping of a small number of visible mutants (Schafer et al. 1993). In the last 10 years, ~400 DNA markers have been mapped by in situ hybridization to the D. buzzatii chromosomes (Ranz et al. 1997, 2003; Laayouni et al. 2000; Casals et al. 2003). No large-insert genomic libraries or clone-based physical maps were previously available for this species.

Results and Discussion

Construction of D. buzzatii BAC library

We constructed a BAC library from the D. buzzatii st-1 strain. High molecular-weight (HMW) DNA was prepared from adults, partially digested with EcoRI and EcoRI methylase, size fractioned, and cloned into the pTARBAC2.1 shuttle vector (Hoskins et al. 2000; Osoegawa et al. 2004). The D. buzzatii BAC library comprises 18,353 clones arrayed in 48 microtiter plates (see Methods). We determined the average insert size to be 152 kb, by EcoRI restriction fingerprinting of 9555 clones (Fig. 1A). The size distribution is somewhat skewed to the right, which results in a very high proportion (98.6%) of cloned inserts larger than 100 kb. The size of the genomes of the repleta group species is ~220 Mb, with ~70% in the euchromatin (Ranz et al. 2001), thus the expected redundancy of the library is ~18×. We hybridized two gridded filters containing the entire library with six euchromatic gene probes. The average number of positive clones per probe was 23, which provides an estimate of the actual representation of the euchromatin in the library (see Supplemental material).

Figure 1.
(A) Size distribution of the 9555 Drosophila buzzatii BAC clones analyzed by fingerprinting. (B) Distribution of clones in contigs and (C) contig sizes for the 345 contigs in the fingerprint map.

Fingerprinting and automatic contig assembly

To build a physical map of the D. buzzatii genome, we first fingerprinted and assembled into contigs 9555 BAC clones using high-throughput methods (Marra et al. 1997; Schein et al. 2004). The fingerprint data were automatically assembled using FingerPrint Contigs (FPC) software (Soderlund et al. 1997, 2000) with a cut-off score of 10-11. This threshold value represents the maximum allowable probability of a chance match between any two clones. The automated assembly produced 634 contigs and 516 unmatched clones (i.e., singletons, see Supplemental material for further details).

Hybridization of BAC clones to salivary gland chromosomes

We hybridized to the D. buzzatii chromosomes 552 clones representing 443 contigs. The information from 427 clones giving one primary hybridization signal was used for map construction. We also hybridized a subset of 163 BAC clones to the chromosomes of Drosophila repleta, another species of the repleta group whose cytological maps (Wharton 1942) have been used as the standard reference for all species in this group (Wasserman 1992). The results allowed us to revise the homology between chromosomes and chromosomal segments of D. buzzatii and D. repleta (Ruiz and Wasserman 1993) and to reconstruct the D. buzzatii chromosomes using the D. repleta cytological maps (Wharton 1942).

An integrated physical map of the D. buzzatii genome

Information from the fingerprint assembly, the cytological localization of BACs, and the library screening with gene probes was merged to produce an integrated physical map (see Methods). Manual editing and merging allowed us to reduce the number of contigs from the initial set of 634 to a final set of 345. Figure 1B shows the distribution of clones within contigs. The mean number of clones per contig is 26, and the largest number of clones in a contig is 351. The fingerprints of a subset of overlapping clones within each contig were compared, and the size of the genomic region covered by each contig was estimated. The average contig size is estimated to be 338 kb (Fig. 1C). Some of the contigs are quite large (30 contigs are larger than 800 kb), although many (216) are relatively small (100-300 kb). The largest contig is ~1.9 Mb.

Using the cytological data, we anchored 181 contigs to the D. buzzatii chromosomes. These contigs contain 7788 (81.5%) of the fingerprinted clones (Supplemental Table S1). Maps of the D. buzzatii chromosomes with the cytological localization of the 427 markers and the 181 contigs they represent are shown in Figure 2. A complete list of clones and in situ hybridization results is given in Supplemental Table S2.

Figure 2.
Integrated BAC-based physical map of the Drosophila buzzatii genome. (We consider the cytological map to be a kind of physical map.) Vertical lines indicate the relative position of the 427 BAC clones that produced a primary hybridization signal and represent ...

The size and cytological span of 15 of the largest contigs were used to estimate the DNA content per cytological band in the salivary gland chromosome map. Taking into account the total number of bands and the total size of the contigs included in our integrated map (Fig. 2), we estimate that the physical map covers ~89% of the euchromatic portion of the D. buzzatii chromosomes. The cytological data indicate that BAC coverage extends nearly to the telomeres, while pericentric regions are less well represented, probably because of the high content of repetitive DNA in these regions (Fig. 2).

Unrestricted access to the resources described in this paper is provided. A database containing all of the fingerprint images and analyses, clone sizes, contig composition, library screenings, and in situ hybridization images can be accessed using iCE (Fjell et al. 2003) at http://www.bcgsc.ca/ice. The D. buzzatii BAC library (CHORI-225) is available from BACPAC Resources (http://bacpac.chori.org/). We expect that the BAC library and high-coverage BAC-based physical map will be highly useful resources not only for those working in D. buzzatii as a model system but also to all those interested in the comparative analysis of genomes. The usefulness of this BAC-based physical map extends to many repleta group species, because their cytological relationships have been determined using D. repleta chromosomes as a reference (Wasserman 1992). In addition, the library has already been used to successfully sequence part of the Hox gene complex of D. buzzatii (Negre et al. 2005). Finally, the D. buzzatii map may help in the assembly of some of the Drosophila genomes currently being sequenced, particularly that of Drosophila mojavensis, which also belongs to the repleta species group.

Methods

Flies

The D. buzzatii strain used to construct the BAC library and to map BACs by in situ hybridization (st-1) is fixed for the standard arrangement in all chromosomes and was produced by Betrán et al. (1998). The D. repleta stock used for in situ hybridization was no. 1611.6 from the National Drosophila Species Resource Center (Bowling Green, OH).

BAC library construction

The library was constructed according to the improved methods described in detail in Frengen et al. (1999) and Osoegawa et al. (1999, 2004). HMW DNA was prepared from 3 g of adult flies, including equal numbers of females and males, as described in Hoskins et al. (2000). The partially digested HMW DNA was size-fractionated by Pulse Field Gel Electrophoresis, and fractions corresponding to 150-250-kb DNA fragments were recovered by electroelution and cloned in pTARBAR2.1. See Supplemental material for further details.

In situ hybridization of BAC clones

In situ hybridizations were carried out as in González et al. (2002). Probes were labeled with biotin-16-dUTP. The hybridization temperatures were 37° for D. buzzatii chromosomes and 25° for D. repleta chromosomes. We hybridized 552 BAC clones to D. buzzatii salivary gland chromosomes; 506 gave positive results, and 427 producing a single primary hybridization signal were used in physical map construction (Supplemental Tables S1 and S2). Nine clones gave two signals; these may represent chimeric clones or mixtures of two clones caused by cross-well contamination. This low rate (1.6%) is in agreement with the low level of chimerism observed in other BAC libraries (Osoegawa et al. 2001). In total, 70 clones gave more than two hybridization signals and/or hybridized to the pericentromeric regions and the microchromosome, probably because of repetitive DNA content. The density of transposable elements increases near Drosophila centric heterochromatin (Kaminker et al. 2002). Clones from such repeat-rich regions cannot be assigned to a particular chromosomal site. Thus, there is a relative scarcity of markers in our physical map near the centric heterochromatin, especially on the X-chromosome (Fig. 2). Examples of the different types of hybridization results are shown in Supplemental Figure S1.

Acknowledgments

We thank Susan Celniker, Bàrbara Negre, Barret Pfeiffer, Theresa Ren, and Chung Li Shu for help. This work was supported by grant BMC2002-01708 from the Dirección General de Enseñanza Superior e Investigación Científica (MEC, Spain) awarded to A.R.

Footnotes

[Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: S. Celniker, B. Negre, and B. Pfeiffer.]

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.3263105.

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