Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.

Significance: Rhodnius prolixus is a major vector of Chagas disease, an illness caused by Trypanosoma cruzi which affects approximately 7 million people worldwide. This report describes the first genome sequence of a nondipteran insect vector of an important human parasitic disease. This insect has a gene repertoire substantially distinct from dipteran disease vectors, including immune signaling pathways that display major departures from the canonical network. Large gene expansions related to chemoreception, feeding, and digestion have facilitated triatomine adaptation to a blood-feeding lifestyle. This study provides information about the physiology and evolution of an important disease vector that will boost understanding of transmission of a life-threatening parasite and may lead to the development of innovative control methods.

The GC content of human DNA varies widely across the genome, ranging from 30% to 60%, and regions of hundreds of kilobases (often referred to as isochores [Bernardi 2000]) may have relatively homogeneous base compositions. This compositional heterogeneity appears to be very widespread in eukaryotes (Nekrutenko and Li 2000) and may represent an important level of genome organization, insofar as gene density (Zouback, Clay, and Bernardi 1996 ), gene length (Duret, Mouchiroud, and Gautier 1995 ), and patterns of codon usage (Sharp et al. 1995 ), as well as the distribution of different classes of repetitive elements (Soriano, Meunier-Rotival, and Bernardi 1983 ; Duret, Mouchiroud, and Gautier 1995 ), are all correlated with GC content. Despite intensive investigation, the underlying cause(s) of the observed heterogeneity remains contested, with two major hypotheses competing: Bernardi has suggested that selection is primarily responsible for maintaining the observed patterns (Bernardi and Bernardi 1986 ; Bernardi 2000 ), a view supported by recent analysis of polymorphism in the human MHC cluster (Eyre-Walker 1999 ), whereas the balance of opinion has favored systematic mutational bias as the ultimate cause (Filipski 1987 ; Suoeka 1988; Wolfe, Sharp, and Li 1989 ; Francino and Ochman 1999 ). Ultimately, however, it is not clear why either selection for increased GC content or mutation bias should promote such marked local variation in genomic nucleotide content.

Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly. WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm. Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.

We report a genome-wide search of Y-linked genes in Drosophila pseudoobscura . All six identifiable orthologs of the D. melanogaster Y-linked genes have autosomal inheritance in D. pseudoobscura . Four orthologs were investigated in detail and proved to be Y-linked in D. guanche and D. bifasciata , which shows that less than 18 million years ago the ancestral Drosophila Y chromosome was translocated to an autosome in the D. pseudoobscura lineage. We found 15 genes and pseudogenes in the current Y of D. pseudoobscura , and none are shared with the D. melanogaster Y. Hence, the Y chromosome in the D. pseudoobscura lineage appears to have arisen de novo and is not homologous to the D. melanogaster Y.

The heterochromatic state of the Drosophila Y chromosome has made the cloning and identification of Y-linked genes a challenging process. Here, we report application of a procedure to identify Y-linked gene fragments from the unmapped residue of the whole genome sequencing effort. Previously identified Y-linked genes appear in sequenced scaffolds as individual exons, apparently because many introns have become heterochromatic, growing to enormous size and becoming virtually unclonable. A TBLASTN search using all known proteins as query sequences, tested against a blastable database of the unmapped fragments, produced a number of matches consistent with this scenario. Reverse transcription–PCR and genetic methods were used to confirm those that are expressed, Y-linked genes. The five genes reported here include three protein phosphatases ( Pp1-Y1 , Pp1-Y2 , and PPr-Y ), an occludin-related gene ( ORY ), and a coiled-coils gene ( CCY ). This brings the total to nine protein-coding genes identified on the Drosophila Y chromosome. ORY and CCY may correspond, respectively, to the fertility factors ks-1 and ks-2 , whereas the three protein phosphatases represent novel genes. There remains a strong functional coherence to male function among the genes on the Drosophila Y chromosome.