Genomic sequence around butterfly wing development genes: annotation and comparative analysis

Abstract

Background: Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions.

Methodology/principal findings: We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes).

Conclusions: The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation.

Figure 1. Annotation of B. anynana genomic regions and fine-scale synteny with B. mori.

Each B. anynana BAC sequence is represented, with the corresponding scaffold in B. mori (including information on chromosomal location). Each putative gene is represented by a different color: B. anynana gene names in bold correspond to those on which BAC selection was based (Table 1), and B. mori gene names reflect SilkDB annotation (e.g., 010572 is SilkDB gene BGIBMGA010572). Exons are explicitly annotated for B. anynana as stripes of the same color (darker shade for duplicated exons). Arrows indicate the direction of transcription of each gene, and fine lines are used for highlighting chromosomal rearrangements. The figure contains a legend for the representation of sequence length, and for the protein-coding genes, repetitive sequence, transposable elements, and microRNA identified in this study. Details on all B. anynana predicted peptides can be found in Table S3.

Figure 2. Conservation of DNA sequence in relation to other lepidopterans.

VISTA plots of all BAC sequences against B. mori and, when available, other lepidopterans (moths Bombyx mori, Helicoverpa armigera, Spodoptera frugipera, and butterflies Papilio dardanus, Heliconius melpomene). Regions more than 70% conserved in a 100 bp window (VISTA default settings) appear as peaks with blue corresponding to annotated protein-coding regions and red to conserved non-coding sequence. Figure S2 shows close-up and extended analysis of regions around genes wingless and Ecdysone receptor.

Figure 3. Phylogenetic tree of Adh genes.

Neighbour-joining, unrooted tree reconstructed with MEGA 4 using the aminoacid sequence of the putative Adh genes in B. anynana (Bany, in green), together with the corresponding paralogs from chromosome 10 in B. mori (Bmori, in red, showing Silkdb gene accessions and BLAST results) and D. melanogaster (Dmel, in blue, showing FlyBase gene accessions). Numbers are bootstrap values for 1000 replicates.