Fig 1.
Map of the study area and the location of sampled communities in Ecuador.
Purple circles indicate the location of Coamine (CE), La Extensa (EX) and Chaquizhca (CQ) in Loja Province, and El Bejuco (BJ) in Manabí.
Fig 2.
Step-by-step of 2b-RAD library and genomic data preparation for triatomine genomic population analysis.
(1) gDNA is extracted from heads, legs and thorax of triatomine bugs. (2) After that, gDNA is processed using the 2b-RAD protocol [38] and (3) libraries are sequenced on Illumina instruments. (4) Once the data is delivered, it is trimmed and filtered before (5) used in genotyping software such as STACKS [58]. (6) Then, genotypes are exported from the cloud (MySQL repository) and filtered if large amount of missing data is present. (7) Finally, the polymorphic loci of interest are exported in conventional file formats for population genomic analysis. See Table 1 for an overview of the technique and for particular recommendations.
Table 1.
Overview of 2b-RAD genotyping: key considerations and further reading.
Fig 3.
Example of two samples processed with 2b-RAD protocol.
1.8% agarose gel electrophoresis showing gDNA (a), digested DNA (b) and PCR product (c) in 2 samples for each of the 3 IIB-REases (AlfI, CspCI and BcgI).
Fig 4.
Comparison of read depth and marker identification in R. ecuadoriensis using 3 different Type IIB restriction enzymes.
In line with in silico predictions, AlfI, an abundant in silico cutter did not produce enough molecular markers as compared to BcgI and CspCI, less abundant in silico cutters. In the diagram, enzymes with abundant in silico restriction sites (dark gray rectangles) within the genome (dark blue solid line with yellow squares or SNPs) are more likely to produce fragments (light blue, green and orange rectangles) at different locations among samples during a random experiment. This may yield insufficient read depth and thus compromise polymorphic marker discovery (dark blue rectangles with a yellow square).
Table 2.
Relationship between number of reads and polymorphic loci obtained from STACKS analysis.
Fig 5.
Relationship between the number of reads and polymorphic loci obtained by each Type IIB restriction enzyme.
Lines show the comparison of the relationship between the increased number of reads obtained by AlfI (Magenta square), BcgI (Dark blue point) and CspCI (Light blue triangle) IIB-REases, and increasing numbers of polymorphic loci discovered after STACKS analysis. Different read abundances were obtained by randomly subsampling the dataset of each enzyme, and analyzing these in STACKS separately as independent datasets. In the figure, A) shows polymorphic loci with up to 2 SNPs shared by at least 90% of samples and best fit logarithmic (Magenta), geometric (Dark blue) and exponential (Light blue) growth curves. B) shows polymorphic loci with up to 2 SNPs shared by at least 80% of samples and best fit geometric (Magenta and Dark blue) and Power-law (Light blue) growth curves.
Fig 6.
Genetic clusters (K = 2) assigned by STRUCTURE.
The blue columns (1—CE, 2—EX and 3—CQ) indicate the samples from Loja, and the purple column (4—BJ) indicate the samples from Manabí. A) BcgI and B) CspCI datasets, respectively.