Marker-assisted mapping enables forward genetic analysis inAedes aegypti, an arboviral vector with vast recombination deserts

Abstract

Aedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up-to-47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here we report the development of a marker-assisted-mapping (MAM) strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using MAM, we mapped a transgene that was inserted in a > 100 Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. The identification of the causal gene of the sex-linked re mutation provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods. To facilitate genome-wide forward genetics in Ae. aegypti, we generated and compiled a number of lines with markers throughout the genome. Thus, by overcoming the challenges presented by the vast recombination deserts and the scarcity of markers, we have shown that effective forward genetic analysis is increasingly feasible in this important arboviral vector species.

Atypical landscape of transposable elements in the large genome ofAedes aegypti

“…For Ae. aegypti , the meiotic recombination rate in cM/Mb is shown using data from Chen et al ., 2022. The overlaid light grey shading indicates the 123-Mb region of reduced male recombination identified by Fontaine et al ., 2017 around the male-determining (M) locus on chromosome 1.…”

“…Increased accumulation of retrotransposons is particularly noticeable within the 123-Mb region of chromosome 1 that contains the sex-determining locus and displays reduced male recombination in Ae. aegypti (Fontaine et al ., 2017; Chen et al ., 2022). Remarkably, a localized inversion of TE accumulation between LTR and LINE retrotransposons occurs at the precise genomic location of the male-determining (M) locus on chromosome 1.…”

Recovery of Metagenomic Data from theAedes aegyptiMicrobiome using a Reproducible Snakemake Pipeline: MINUUR

“…We selected the data sets to cover a range of sequencing depths, DNA extraction method and sequencing platform (Figure 2C). All raw FASTQ files of published sequencing data were retrieved from the European Nucleotide Archive (ENA) under the project accession numbers PRJEB33044 (37), PRJNA255893 (50), PRJNA385349 (51), PRJNA718905 (35), PRJNA776956 (52) and PRJNA992905 (53).…”

“…For this study, we used five published genome datasets of Aedes aegypti publicly available on the European Nucleotide Archive (35,37,(50)(51)(52)(53). We selected the data sets to cover a range of sequencing depths, DNA extraction method and sequencing platform (Figure 2C).…”

“…Whole genome shotgun sequencing is commonly used to study mosquito genomics at the individual (35,36) and population (37,38) level; meaning non-mosquito sequence data (we refer to these as non-reference reads, since they do not map to the reference genome of interest) are a source to identify mosquito microbiome members using metagenomics. Genomic surveillance programs such as the Anopheles gambiae 1000 Genomes Project contain a large number of genomic samples with each release (39) and, at time of writing, currently 100,514 Aedes aegypti whole-genome sequencing runs are deposited on the European Nucleotide Archive.…”