Assembly of the Genome of the Disease Vector <i>Aedes aegypti</i> onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

<div><p>The mosquito <i>Aedes aegypti</i> transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of <i>Ae. aegypti</i>, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes <i>Ae. aegypti</i> resistant to the human parasite <i>Brugia malayi</i>, and generated a list of candidate genes that could be affecting the trait.</p></div

Examples of two misassembled scaffolds.

<p>Individual contigs are shown as gray rectangles. Contigs with markers in this study are indicated with an * above the scaffold and are labeled with their position on the genetic map. The new scaffolds created by splitting misassemblies (identified in this study only) are shown with solid lines. New scaffolds with suffixes ‘a,’ ‘b,’or ‘c’ contain markers that allow placement on the genetic map. New scaffolds with suffixes ‘m’ or ‘n’ fall between conflicting markers and therefore contain a misassembly and cannot be placed on the genetic map. A) Supercontig 1.1 with markers from the previously published integrated map <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652-Timoshevskiy1" target="_blank">[20]</a> shown below. The mapping of markers to two different chromosomes indicates a misassembly within the scaffold, which is supported by both markers sets. B) Supercontig 1.48 with synteny with <i>An. gambiae</i> shown below the scaffold and the different colors indicating different chromosome arms. Both our markers and syntenic breaks with <i>An. gambiae</i> indicate that this scaffold is misassembled in at least two instances.</p

Summary of our assembly of the genome onto a genetic map.

1<p>scaffolds which have been ordered along the chromosome (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd-0002652-g002" target="_blank">Figure 2</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a>).</p>2<p>number of mapped scaffolds after splitting misassemblies.</p>3<p>scaffolds assigned to the chromosome only (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a>).</p

A linkage map of <i>Ae. aegypti</i>.

<p>The map was constructed using SNPs identified in RAD tag sequences. Positions in cM are indicated to the left of each linkage group. Scaffolds mapping to each position on the map are shown to the right of each linkage group and are named by the last 2–4 digits of their supercontig ID number. The region linked with resistance to infection by <i>B. malayi</i> is highlighted in red. Scaffolds with suffixes a–d were previously misassembled and have been split and assigned new scaffold names (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd-0002652-g001" target="_blank">Figure 1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a> for additional information).</p

The correlation between the cM position of a scaffold on our genetic map and its position on the previously published integrated map [20].

<p>The positions of scaffolds on chromosomes 1 (A), 2 (B), and 3 (C) are significantly correlated between the map presented here and the previously published map order (Spearman correlation, A: ρ = 0.803, <i>P</i> = 0.0017; B: ρ = 0.899, <i>P</i> = 0<0.0001; C: ρ = 0.857, <i>P</i> = 0.0004). Outliers are potentially caused by unidentified scaffold misassemblies. In cases where we found a scaffold to be misassembled, the correlation was performed by using the genetic map position of the contig closest to the one used in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652-Timoshevskiy1" target="_blank">[20]</a>. In one instance on Chromosome 2 and two instances on Chromosome 3, scaffolds mapped to a different chromosome in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652-Timoshevskiy1" target="_blank">[20]</a> than on our genetic map and these are not included in this analysis.</p

The relationship between genetic (cM) and physical map (Mb) positions and estimated local recombination rates across the three chromosomes of <i>Ae. aegypti</i>.

<p>The physical length was measured as the number of base pairs mapped to a particular genetic position for chromosomes A) 1, B) 2, and C) 3. Local recombination rates for chromosomes D) 1, E) 2, and F) 3 show depressed recombination in the centromeric regions of each chromosome.</p

Resistance to <i>B. malayi</i> is determined by a single locus that is dominant in action and maps to the first chromosome.

<p>A) Average frequencies of the reference allele (the SNP allele in the published genome) in the pools of refractory versus susceptible mosquitoes. A cluster of markers with ∼100% frequency in susceptible pools (vertical dotted lines) and ∼50% frequency in refractory pools (horizontal dotted line) is consistent with resistance being determined by a single locus that is dominant in action. Red points indicate statistically significant differences in allele frequencies between refractory and susceptible pools at a genome-wide significance of <i>P</i><0.01. B) Manhattan plot of the minimum <i>P</i>-values for each scaffold, with the x-axis representing a physical map created from the linkage map. Only markers from contigs used to assemble the linkage map are shown here (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a>). Dotted line indicates significance at a genome-wide significant of <i>P</i><0.01.</p