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, 6 (6), e1000998

Copy Number Variation and Transposable Elements Feature in Recent, Ongoing Adaptation at the Cyp6g1 Locus

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Copy Number Variation and Transposable Elements Feature in Recent, Ongoing Adaptation at the Cyp6g1 Locus

Joshua M Schmidt et al. PLoS Genet.

Abstract

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for approximately 16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The molecular structure of the Cyp6g1 locus.
(A) The region encompassed by five overlapping PCR primer sets (E-H, A-B, A-D, C-D, E-G) that have been cloned are shown. Cyp6g1 exons are represented as black boxes above the line, Cyp6g2 exons as dark grey boxes below the line. The HMS-Beagle element and the Accord element sequences are represented as an empty box and as light grey boxes above the line respectively. (B) The complex structure of the locus is demonstrated by restriction digests (EcoRI; E, EcoRI-SacI; E/S and EcoRI-HindIII; E/H) of a plasmid containing the 12.5 kb A-D PCR product. The restriction sites for each digest are shown as lines drawn to scale with the locus structure. The 3.5 kb EcoRI-EcoR1 plasmid band is seen in all lanes. The sum of the molecular weights of the remaining fragments is shown at the bottom of each lane. The sums differ by intervals of 2.7 kb, which corresponds to the repeat unit represented in the top of the figure. * indicates 3.5 kb vector band. Sequences of E-H, A-B, C-F and E-G are lodged in Genbank with accessions HM214801, HM214799, HM214802 and HM214800 respectively.
Figure 2
Figure 2. The six alleles of Cyp6g1.
(A) The alleles are arranged from top to bottom in the order in which they arose. The M allele has a single copy of Cyp6g1 and lacks TE insertions. The A allele is single copy with an Accord insertion, the AA allele has two full length copies each with an Accord insertion. The BA allele has two full-length copies with the proximal copy containing HMS-Beagle. The BP allele has two full-length copies with the a copy containing HMS-Beagle and the b copy containing the P insertion. All of the lines assayed that have a P-element sequence in the Accord also have the HMS-Beagle insertion and so the BP class probably arose from the BA allele. The heterogeneous BPΔ class contains various low frequency variants that have scrambled P terminal repeats (Figure S3). PCR primers are shown as arrows and are named with a single letter. Note that primer L anneals to the HMS-Beagle sequence whereas primers H and I flank the transposable element insertion sites. (B) A gel demonstrating the diagnostic PCRs is shown on the right.
Figure 3
Figure 3. Temporal and geographic changes in Cyp6g1 allele frequencies.
(A) Lines established between the 1930s and the present. Flies were typed using the PCR assays described in Figure 2. (B) The frequency of the BP allele was scored in >40 flies from each of eight locations along the east coast of Australia. The populations are from Cape Tribulation (CT1, CT2), Gladstone/Alstonville (G/A), Maryborough (M), Rainbow Beach (RB), Coffs Harbour (CH), Wollongong (W), Bega (B). Error bars in both (A,B) represent the 95% binomial confidence interval.
Figure 4
Figure 4. DDT resistance correlates with Cyp6g1 allelic class.
The resistance of lines isochromosomal for their second chromosome are shown grouped on the x-axis by Cyp6g1 alleles (M, AA, BA, BP). The error bars represent the standard errors of the mean LC50 of the lines. Significant differences with preceding classes are indicated e.g. BA vs. BP females (One-tailed t-test: ***** p = 0.0005, **** p = 0.002, *** p = 0.003, ** p =  0.019, * p = 0.044). Shaded bars represent data from males, solid bars from females.
Figure 5
Figure 5. Cyp6g1 transcription in adult midgut and malphigian tubule.
Derived alleles exhibit higher gene expression than Cyp6g1[M]. (A) The allelic progression results in increased gene expression in the midgut with AA having a 2.6 fold increase (p = 0.036, one tailed t-test) over M and BA and BP both showing a 5 fold increase. There is also a significant 2 fold increase of BA over AA (p = 0.0006, one tailed t-test). In contrast, only the step between M and AA results in a significant increase in transcription (p = 0.04, one tailed t-test) in adult tubule (B), and all three derived alleles exhibited ∼3 fold increases in gene expression.
Figure 6
Figure 6. Population analysis of DDT resistance.
A DDT association study was conducted on 7500 females derived from a single population. (A) A dosage mortality analysis was undertaken to a identify dose that would kill the 5% of individual that were most susceptible to DDT and the dose that would kill all except the 5% of individuals that were most resistant to DDT. (B) The top three graphs show the allele frequencies of Cyp6g1-[BP] among the individuals that died on 2 ug of DDT (frequency of 0.15), among an unscreened field population sample (frequency of 0.31), and among the survivors of 120 ug of DDT (frequency of 0.58), respectively. Thus there is a strong association between DDT resistance and Cyp6g1-[BP]. The bottom three graphs show the frequency of a Cyp12d1 copy number variant among the same three groups (Cyp12d1 D/− = 0.81, 0.87 and 0.86) showing that it is not associated with field resistance. Note that flies carrying the Cyp12d1 duplication are denoted as D/−, whereas flies homozygous for a single copy are denoted as u/u. Error bars represent standard errors of the mean of biological replicates.

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