Marker-assisted mapping enables effective forward genetic analysis in the arboviral vector Aedes aegypti, a species 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. This study provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods by improving upon the current re-based genetic sexing strains. MAM does not require densely populated markers and can be readily applied throughout the genome to facilitate the mapping of genes responsible for insecticide- and viral-resistance. By enabling effective forward genetic analysis, MAM bridges a significant gap in establishing Ae. aegypti as a model system for research in vector biology. As large regions of suppressed recombination are also common in other plant and animal species including those of economic significance, MAM will have broad applications beyond vector biology.

Introgression of the Aedes aegypti Red-Eye Genetic Sexing Strains Into Different Genomic Backgrounds for Sterile Insect Technique Applications

“…The recent discovery of the gene responsible for the red eye phenotype in Aedes aegypti, namely cardinal, opens the way for a faster and easier transfer of the sexing characters of the red eye GSS in local genomic background and will thus avoid the long (10-11 generations) and tedious genetic crosses described in this manuscript (Chen et al, 2021). This can be achieved by using CRISPR/Cas9 targeted mutagenesis of the cardinal gene of the local population to de novo develop a red eye mutant line.…”

“…Females of the Red Eye strain and males from IDN, SGP and LKA populations were used to initiate the three introgression crosses while the respective crosses with males from BRA, MEX and THA populations are reported in the study of Chen et al (2021) (Figure 1A).…”

Genome-wide Association Study Reveals New Loci Associated With Pyrethroid Resistance in Aedes aegypti

“…The Ae. aegypti genome has a low per mega base recombination rate (approximately 0.3 cM/Mb), with long recombination deserts ( Juneja et al, 2014 ; Dudchenko et al, 2017 ; Chen et al, 2021 ), and our markers density after QC is on average about 12 SNPs/Mb, indicating good genomic coverage. Of course, recombination is not uniform across genomes, nevertheless, the number of markers we use have reasonably good power to detect significant associations.…”

Selective targeting of biting females to control mosquito-borne infectious diseases

“…Although no female contamination was observed in this system, the cost of flowsorting systems and their relatively low throughput are substantial barriers to their usage. Most recently, a sex-linked spontaneous red-eye mutation is being explored for sex separation by eye color [51], and the determination that cardinal is the causal gene for this mutant [52] provides an opportunity to expand its use in other mosquito species.…”