A new multiplex SNP genotyping assay for detecting hybridization and introgression between the M and S molecular forms of Anopheles gambiae

Abstract

The M and S forms of A. gambiae have been the subject of intense study, but are morphologically indistinguishable and can only be identified using molecular techniques. PCR-based assays to distinguish the two forms have been designed and applied widely. However, the application of these assays towards identifying hybrids between the two forms, and backcrossed hybrids in particular, has been problematic as the currently available diagnostic assays are based on single loci, and/or are located within a multi-copy gene. Here we present an alternative genotyping method for detecting hybridization and introgression between M and S molecular forms based on a multi-locus panel of single nucleotide polymorphisms (SNPs) fixed between the M and S forms. The panel of SNPs employed are located in so called “islands of divergence” leading us to describe this method as the “Divergence Island SNP” (DIS) assay. We show this multi-locus SNP genotyping approach can robustly and accurately detect F1 hybrids as well as backcrossed individuals.

Spatiotemporal dynamics of gene flow and hybrid fitness between the M and S forms of the malaria mosquito, Anopheles gambiae

“…A few studies have used multiple restriction fragment length polymorphism assays and/or mismatch primer-based SNP assays to genotype one SNP in each of the three (X, 2L, and 3L) islands of divergence (26,36). We extended this approach with the development of the DIS assay (35) to describe A. gambiae populations throughout West Africa ( Fig. 1 and SI Appendix, Table S3).…”

“…1 and SI Appendix, Table S3). The DIS assay includes 7 SNPs spanning 4.4 Mbp on the X chromosome, 5 spanning 2.2 Mbp on chromosome 2L, and 3 spanning 117 kb on chromosome 3L (35). Genotypes at these 15 SNPs were used to designate individuals as M form, S form (homozygous for form-specific alleles at a minimum of 13 of the 15 SNPs), F 1 hybrids (heterozygous at all 15 SNPs), and backcross (individuals with mixed M, S, and heterozygous genotypes at 3 or more SNPs).…”

“…We used the method described in ref. 35 to illustrate the distribution of DIS genotypes among each study population, using what we refer to as "DIS maps" (Figs. 2 and 3 and SI Appendix, Figs.…”

“…In this study, we used a simple multilocus SNP genotype approach to distinguish M, S, F 1 hybrids, and backcross individuals (35), which we call the "divergence island SNP" (DIS) assay (SI Appendix). We describe the spatial and temporal distribution of hybridization frequencies, the extent to which hybridization results in introgression, and the fitness of hybrid genotypes in nature.…”

Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa

“…Genomic DNA was extracted using a DNeasy extraction kit (Qiagen). Species determination of mosquitoes from the A. gambiae complex were made with the combination of species diagnostic assays ( Fanello et al , 2002; Favia et al , 2001; Santolamazza et al , 2008; Scott et al , 1993) and a divergence island SNP (DIS) genotyping assay ( Lee et al , 2014) while other species were identified by morphology.…”