Genetic Crosses and Linkage Mapping in Schistosome Parasites

Abstract

Linkage mapping - utilizing experimental genetic crosses to examine cosegregation of phenotypic traits with genetic markers - is now 100 years old. Schistosome parasites are exquisitely well suited to linkage mapping approaches because genetic crosses can be conducted in the laboratory, thousands of progeny are produced, and elegant experimental work over the last 75 years has revealed heritable genetic variation in multiple biomedically important traits such as drug resistance, host specificity, and virulence. Application of this approach is timely because the improved genome assembly for Schistosoma mansoni and developing molecular toolkit for schistosomes increase our ability to link phenotype with genotype. We describe current progress and potential future directions of linkage mapping in schistosomes.

Keywords: Schistosoma; fine mapping; functional analysis; heritability; linkage; phenotype.

Figure 1.. Heritable phenotypes in schistosomes.

(A and B) Host specificity of S. mansoni to the snail intermediate host (redrawn from [6]). (A) Infection rates of a Brazilian snail (BgBRE) challenged with miracidia from two different S. mansoni lab populations (SmBRE and SmGUA), with doses ranging from 1–50 miracidia/snail. The sympatric Brazilian S. mansoni (SmBRE) infection rate reaches 100% with 10 miracidia, but parasites from Guadalupe (SmGUA) are unable to infect BgBRE. Similarly, (B) shows infection rates of another Brazilian snail (BgBS90) challenged with miracidia from two different S. mansoni lab populations (SmBRE and SmLE). Only SmLE, from Puerto Rico) is able to infect BgBS90. Note that infection rates of SmLE plateau at ~50% indicating that this snail population is polymorphic for susceptibility to SmLE. (C) Larval response to snail odors (data from [7]). The behavior of miracidia to host snail species varies between S. mansoni populations. Egyptian S. mansoni (SmEG) respond strongly (showing rapid change in swimming direction) only to their sympatric host snail (B. alexandrina), while Brazilian S. mansoni are indiscriminate and respond to Biomphalaria spp. from both the New and Old World and even to unrelated species (Lymnea). Genetic crosses [7] suggest recessive inheritance of snail specific behavior in SmEG. (D) Cercarial shedding intensity. S. mansoni (SmPR1) show rapid change in numbers of cercariae shed in response to laboratory selection for high or low shedding (redran from [1]). Surprisingly, high levels of virulence to the snails is associated with low rather than high cercarial shedding. The rapid response to selection of both cercarial shedding intensity and virulence strongly suggest that these traits are heritable. (E) Cercarial shedding time in omani S. mansoni (redrawn from [55]). A parasite population primarily recovered from rodents (grey shading) shows a nocturnal shedding of cercaria larvae from the snail (B. pfeifferi), while populations infecting humans (no shading) show a normal diurnal shedding of cercaria larvae. Genetic crosses indicate that this trait is heritable [56] in other population studied.

Figure 2.. Genetic crosses and linkage information aid genome assembly.

A S. mansoni genetic map [64] generated using a genetic cross allows unordered scaffolds assembled from short-read genome sequence to be anchored and ordered to chromosomes (only chr. 6 is shown here). Neighboring scaffolds can then be stitched together using additional sequence data (adapted from [31]).

Figure 3.. Classical linkage mapping of oxamniquine resistance.

(A) Linkage analysis. Measurement of OXA resistance and genotyping of individual worms from a two generation cross (insert) resulted in unambiguous mapping (LOD = 31, dotted line shows genome wide threshold for significance) of a single genome region underlying OXA resistance (red line). When the marker showing the highest LOD is used as a cofactor in the analysis (blue line) no additional peaks are observed, suggesting that OXA-R is a monogenic trait. (B). This genome region contains 17 genes (Red and blue genes are transcribed in opposite directions) (C). We narrowed down this list to one gene (Smp_089320, red outline; Y: Yes, N: No) by searching for genes that show amino acid differences between the parents, are expressed in adult worms, and encode for proteins of a size consistent with previous biochemical work [62]. (D). Functional analysis: RNAi knockdown (left panel: control RNAi treatment; right panel: RNAi targeted against Smp_089320) of OXA-sensitive parasite demonstrate involvement of Smp_089320, a sulfotransferase. Figure adapted from [13].

Figure 4.. Schistosome linkage analysis using Bulk Segregant analysis (BSA).

(A). BSA using pooled F2 progeny – in this case one pool is selected while a second pool is left unselected. Pools of surviving parasites are then quantitatively genotyped across the genome. This can be done by genome or exome sequencing of pools, and estimation allele frequencies from read depth of variable sites. (B). Validation of BSA methods for detecting OXA resistance. Plot reveals genome regions that show significant (-log₁₀ p-value) differences in alleles frequencies between pools of parasites that were either treated with OXA or treated with drug diluent only [68]. The peak observed in chr. 6 is within 1 cM of the locus mapped using classical QTL methods [13]. Figure redrawn from [68] using version 7 of the S. mansoni genome. Alternating gray and white shading marks chromosome boundaries, while unassembled genome regions are shown in black.

Figure I

(in Box 1). Key lifecycle features relevant to linkage mapping.