Analysis of the Genetic Variation of the Fruitless Gene within the Anopheles gambiae (Diptera: Culicidae) Complex Populations in Africa

Abstract

Targeting genes involved in sexual determinism, for vector or pest control purposes, requires a better understanding of their polymorphism in natural populations in order to ensure a rapid spread of the construct. By using genomic data from An. gambiae s.l., we analyzed the genetic variation and the conservation score of the fru gene in 18 natural populations across Africa. A total of 34,339 SNPs were identified, including 3.11% non-synonymous segregating sites. Overall, the nucleotide diversity was low, and the Tajima’s D neutrality test was negative, indicating an excess of low frequency SNPs in the fru gene. The allelic frequencies of the non-synonymous SNPs were low (freq < 0.26), except for two SNPs identified at high frequencies (freq > 0.8) in the zinc-finger A and B protein domains. The conservation score was variable throughout the fru gene, with maximum values in the exonic regions compared to the intronic regions. These results showed a low genetic variation overall in the exonic regions, especially the male sex-specific exon and the BTB-exon 1 of the fru gene. These findings will facilitate the development of an effective gene drive construct targeting the fru gene that can rapidly spread without encountering resistance in wild populations.

Keywords: Africa; An. gambiae s.l; Fruitless; genomics; vector control.

Figure A1

Variant density (bp⁻¹) in the genomic region of the Fruitless gene; upper figure is the genomic region of the Fruitless gene (rectangles correspond to the exonic regions, simple lines are the intronic regions, and dark-red end is the 3 prime UTR); lower figure is the variant density plot.

Figure A2

Median of nucleotide diversity in a window of 500 bp across the genomic region of the Fruitless gene; red points indicate the mean of the nucleotide diversity; AGO: Angola; BFA: Burkina Faso; CAF: Central African Republic; CIV: Côte d’Ivoire; CMR: Cameroon; COD: Democratic Republic of Congo; GAB: Gabon; GHA: Ghana; GIN: Guinea; GMB: Gambia; MLI: Mali; MOZ: Mozambique; MWI: Malawi; TZA: Tanzania; UGA: Uganda.

Figure A3

Median of Tajima’s D in a window of 500 bp across the genomic region of the Fruitless gene; red points indicate the mean of the Tajima’s D. Horizontal line indicates the null value of the Tajima’s D test. AGO: Angola; BFA: Burkina Faso; CAF: Central African Republic; CIV: Côte d’Ivoire; CMR: Cameroon; COD: Democratic Republic of Congo; GAB: Gabon; GHA: Ghana; GIN: Guinea; GMB: Gambia; MLI: Mali; MOZ: Mozambique; MWI: Malawi; TZA: Tanzania; UGA: Uganda.

Figure A4

Haplotype diversity in a window of 500 bp across the genomic region of the Fruitless gene; red points indicate the mean of the haplotype diversity. AGO: Angola; BFA: Burkina Faso; CAF: Central African Republic; CIV: Côte d’Ivoire; CMR: Cameroon; COD: Democratic Republic of Congo; GAB: Gabon; GHA: Ghana; GIN: Guinea; GMB: Gambia; MLI: Mali; MOZ: Mozambique; MWI: Malawi; TZA: Tanzania; UGA: Uganda.

Figure A5

H12 statistic in chromosome X (window size = 4 kb) of An. gambiae s.l population. High values of H12 in a given genomic region indicates a signal of positive selection in this genomic region within the population [36]. Magenta bands indicate the fru region (X: 1283016-1373662) and the Cyp9k1 region (X:15240572-15242864). The H12 values were low in the fru region, but higher in the Cyp9k1 region shown to be involved in the pyrethroid resistance in An. gambiae s.l populations [42]. AGO: Angola; BFA: Burkina Faso; CAF: Central African Republic; CIV: Côte d’Ivoire; CMR: Cameroon; COD: Democratic Republic of Congo; GHA: Ghana; GIN: Guinea; GMB: Gambia; MLI: Mali; MWI: Malawi; TZA: Tanzania; UGA: Uganda.

Figure A6

Conservation score and nucleotide diversity in a window of 12 bp within the BTB and connector region of the Fruitless gene; upper figure shows the BTB and connector region (rectangles correspond to the exonic regions, and the simple lines are the intronic regions); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot.

Figure A7

Conservation score and nucleotide diversity in a window of 12 bp within the zinc-finger A (ZnFA) region of the Fruitless gene; upper figure shows the ZnFA region (the rectangle corresponds to the exonic region, and the simple line is the intronic region); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot.

Figure A8

Conservation score and nucleotide diversity in a window of 12 bp within the zinc-finger B (ZnFB) region of the Fruitless gene; upper figure shows the ZnFB region (the rectangle corresponds to the exonic region, and the simple line is the intronic region); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot.

Figure A9

Conservation score and nucleotide diversity in a window of 12 bp within the zinc-finger C (ZnFC) region of the Fruitless gene; upper figure shows the ZnFC region (the rectangle corresponds to the exonic region, the dark red end is the 3 prime UTR region, and the simple line is the intronic region); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot.

Figure A10

Conservation score and nucleotide diversity in a window of 12 bp within the Cyp9k1 (X:15240572-15242864), a gene shown to be under selection pressure [36]; upper figure shows the genomic region of the Cyp9k1 (the rectangle corresponds to the exonic region, the dark red ends are the 3′ and 5′ UTR regions, and the simple line is the intronic region); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot. The conservation score remains high in the exonic regions compared to the intronic ones, as it is found in the fru gene.

Figure 1

Heat map showing the allelic frequencies of the non-synonymous mutations with maximum allelic frequencies that are > 5% in at least one population. The vertical axis of the heat map shows the non-synonymous variant positions in the X chromosome, and the horizontal axis shows the populations of An. gambiae s.l. The gradient color bar shows the distribution of the allelic frequencies. AGO: Angola; BFA: Burkina Faso; CAF: Central African Republic; CIV: Côte d’Ivoire; CMR: Cameroon; COD: Democratic Republic of Congo; FRA: Mayotte; GAB: Gabon; GHA: Ghana; GIN: Guinea; GMB: Gambia; GNB: Guinea-Bissau; MLI: Mali; MOZ: Mozambique; MWI: Malawi; TZA: Tanzania; UGA: Uganda.

Figure 2

Variation of the heterozygosity within the non-synonymous SNPs with maximum allelic frequencies that are superior to 5% in at least one population of An. gambiae complex from West, Central, and East Africa. The vertical axis shows the difference between the observed and the expected heterozygosity; the horizontal axis shows the non-synonymous SNPs positions. Positive values (Obs. het > Exp. het) mean an excess of heterozygotes at this position; negative values (Obs. het < Exp. het) mean a deficit of heterozygotes at this position; null values (Obs. het = Exp. het) mean no deviation from the Hardy–Weinberg equilibrium at this position. Obs. het.: observed heterozygosity, Exp. het.: expected heterozygosity, CAR: Central African Region, EAR: Eastern African Region, WAR: Western African Region.

Figure 3

Linkage disequilibrium between the non-synonymous SNPs with maximum allelic frequencies that are greater than 5% in at least one population; upper figure shows allelic frequencies; lower figure shows linkage disequilibrium value (−1 indicates no LD, and +1 indicates perfect LD).

Figure 4

Conservation score and nucleotide diversity in a window of 12 bp within the sex-specific region of the Fruitless gene. The upper figure shows the five transcripts of the Fruitless gene (rectangles correspond to the exonic regions, simple lines are the intronic regions, and dark-red end is the 3 prime UTR within the AGP000080-RC transcript); lower figure shows the conservation score (blue line) and the nucleotide diversity (dark fill) plot.

Figure 5

Conservation score and nucleotide diversity in a window of 12 bp within the sex-specific region of the Fruitless gene. The upper figure is the sex-specific region of the Fruitless gene (rectangle corresponds to the male-specific region, rectangle and simple line correspond to the female-specific region, and red dashes are the probable stop codon within the female-specific region); lower figure is the conservation score (blue line) and the nucleotide diversity (dark fill) plot.