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

Resistance to <i>B</i>. <i>malayi</i> is dominant and sex-linked, and it impairs early stages of <i>B</i>. <i>malayi</i> development.

<p>Female progeny from a backcross inherited the resistance phenotype of their maternal grandparent, whether resistance was measured as A) the proportion of mosquitoes infected with L3 stage parasites or B) the number of L3 stage parasites at 10 days after infection. S♀ x R♂ (referred to as susceptible from this point forward) were created by crossing an LVP^S female with an LVP^R male, followed by backcrossing the F₁ progeny to LVP^S. R♀ x S♂ (referred to as resistant) were created in the same way except an LVP^R female was crossed with an LVP^S male in the parental generation. By 24 hours after infection, many <i>B</i>. <i>malayi</i> microfilariae have migrated from the midgut to the thoracic tissues in both C) susceptible and D) resistant hosts. At 48 hours after infection, microfilariae are molting into the non-feeding L1 developmental stage in E) susceptible hosts, whereas growth is arrested in F) resistant hosts. By 72 hours after infection, nearly all surviving <i>B</i>. <i>malayi</i> are in the L1 stage in G) susceptible hosts, whereas they are dead or dying in H) resistant hosts. I) Genome-wide gene expression levels of <i>B</i>. <i>malayi</i> are comparable in resistant and susceptible hosts at 12 hours after infection, whereas by 48 hours, gene expression, and presumably growth, of <i>B</i>. <i>malayi</i> is higher in susceptible mosquitoes. <i>B</i>. <i>malayi</i> gene expression is estimated by dividing the total number of RNA-seq reads mapping to the <i>B</i>. <i>malayi</i> genome by the total number of reads mapping to the <i>Ae</i>. <i>aegypti</i> genome.</p

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

Association mapping demonstrates that a single dominant locus controls resistance to <i>B</i>. <i>malayi</i> in a Kenyan population of <i>Ae</i>. <i>aegypti</i>.

<p>A) Mapping with an additive model of inheritance produces a small number of significant associations on chromosome 1 and in regions unassigned to chromosomes. B) Including the most significant marker in panel A as a covariate in the analysis results in no significant associations, suggesting that a single locus controls infection. C) A dominance model of inheritance produces highly significant associations, whereas D) a recessive model does not. Red-dotted line shows genome-wide significance at <i>P</i><0.01; black-dotted line shows significance at <i>P</i><0.05. The x-axis represents a physical map (bp) made by arranging scaffolds along the genetic map [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004765#ppat.1004765.ref019" target="_blank">19</a>], with scaffolds mapping to the same genetic map position being ordered randomly. To correct genome misassemblies, scaffolds that contain any gene with at least 10 segregating sites and an <i>F</i>_ST greater than 0.1 in the RNA-seq data were moved to the unassembled region. In the dominance model, 13 sites on 4 scaffolds map to 0 cM and a single site maps to 12 cM.</p

Immune-related genes that are differentially expressed in response to <i>B</i>. <i>malayi</i> infection.

<p>Immune-related genes are from the ImmunoDB database or manual curation, and only those that are significantly differentially expressed for either or both genotypes (or have a significant interaction term) are shown. Expression is log₂ fold-change in response to <i>B</i>. <i>malayi</i>, with genes in blue being upregulated and genes in red being downregulated. Grey means insufficient coverage to estimate expression. (A) At 12 hours post infection, almost all genes are being expressed in the same direction and with similar magnitudes. (B) At 48 hours, a subset of genes are being expressed differently in response to infection. The green boxes indicate statistical significance in susceptible mosquitoes (left, FDR<0.2), resistant mosquitoes (middle, FDR<0.2) or a significant interaction (dark green right, <i>P</i><0.01).</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

Differentially expressed genes in the genomic region controlling resistance.

<p>Genes shown either map to the genetic map position associated with resistance (chromosome 1, 0 cM) or are on an unassembled scaffold with a significant association at a genome-wide significance of <i>P</i><0.01. They are significantly differentially expressed either 1) between resistant and susceptible genotypes prior to infection (FDR<0.20; black border) or 2) in response to infection in either genotype at 12 or 48 hours after infection (FDR<0.20; black border), or 3) respond to infection differently between the genotypes (* indicates a significant interaction between genotype and infection at <i>P</i><0.01). Several genes have been assigned to chromosome 1 but have an unknown genetic map position. ^AOn a misassembled scaffold, ^BMapped to chromosome 1 previously without positional information [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004765#ppat.1004765.ref019" target="_blank">19</a>], ^CNewly mapped to chromosome 1 using <i>F</i>_ST estimates from RNA-seq data. NA indicates no genetic mapping information.</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