The identification of markers segregating with resistance to Schistosoma mansoni infection in the snail Biomphalaria glabrata

Abstract

Both snail and parasite genes determine the susceptibility of the snail Biomphalaria glabrata to infection with the trematode Schistosoma mansoni. To identify molecular markers associated with resistance to the parasite in the snail host, we performed genetic crosses between parasite-resistant and -susceptible isogenic snails. Because resistance to infection in adult snails is controlled by a single locus, DNA samples from individual F2 and F1 backcross progeny, segregating for either the resistant or susceptible phenotypes, were pooled (bulked segregant). Genotypes for both parents were determined with 205 arbitrary decamer primers by random amplified polymorphic DNA-PCR. Of the 205 primers, 144 were informative, and the relative allele frequencies between the pools for these primers were determined. Two primers, OPM-04 and OPZ-11, produced fragments in the resistant parent of one cross that were inherited in a dominant fashion in the resistant F2 and backcross-bulked segregant progeny. Subsequent typing of DNA samples of individual progeny snails showed that the 1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced by primer OPZ-11 segregated in the same dominant fashion with the resistant phenotype. Sequence analysis of the 1.2-kb marker showed that it corresponds to a repetitive sequence in the snail genome with no homology to existing DNA sequences in the public databases. Analysis of the 1. 0-kb marker showed that it also corresponds to a repetitive sequence in the B. glabrata genome that contains an imperfect ORF, with homology to retrovirus-related group-specific antigens (gag) polyprotein.

Figure 1

(A) Primer OPM-04 RAPD products from genomic DNA of a resistant BS-90 parent (B), a susceptible M line parent (M), and four groups of resistant (–4) and susceptible (5) F₂ progeny. Arrow indicates the position of a 1.2-kb band in the resistant parent and offspring DNA. (B) Primer 0PZ-11 RAPD bands amplified from genomic DNA of resistant BS-90 (B), susceptible M line parents (M), and four groups of resistant (–4) and susceptible (5) F₂ progeny. Lane C shows amplification in the absence of template DNA. Arrow indicates position of 1.0-kb marker in resistant parent and progeny DNA. (C) Primer 0PM-04 amplification of resistant parents (B), susceptible parents (M), and F₁ backcross resistant (1) and susceptible (2) progeny. Lane C represents amplification in the absence of DNA. Note the position of a 1.2-kb band (arrow) amplified only in resistant parent and progeny. (D) Primer OPZ-11 amplification of resistant (B), susceptible (M) parents, and resistant (1) and susceptible (2) F₁ backcross progeny. Lane C shows RAPD–PCR without DNA template. Arrow shows position of 1.0-kb marker amplified only in the resistant parent and progeny.

Figure 2

(A) OPM-04 RAPD–PCR amplification of genomic DNA from resistant BS-90 (B), susceptible (M) parent snails, and 10 individual resistant progeny. Lane C represents amplification in the absence of template DNA. (B) OPM-04 amplification of DNA from resistant (B) and susceptible (M) parents and 10 individual susceptible progeny. Lane S represents amplification of S. mansoni DNA, and lane C shows amplification without DNA. (C) OPZ-11 amplification of DNA from resistant (B) and susceptible (M) parents and 10 individual resistant progeny. Lane C shows amplification in the absence of template DNA. (D) OPZ-11 amplification of resistant (B) and susceptible (M) parent snail DNA and DNA from 10 individual susceptible progeny. Lane S represents amplification of parasite DNA and lane C shows amplification without DNA.

Figure 3

Amplification of DNA from B. glabrata resistant strain BS-90 (B), susceptible strain M line (M), and resistant strain 10-R2 using primers OPM-04 (Fig. 3A) and OPZ-11 (Fig. 3B). Lane C in both cases represents the amplification in the absence of DNA template.

Figure 4

Cluster analysis of the RAPD data was performed as described in Materials and Methods. A total of 30 bands from the 2 RAPD primers (OPM-04 and 0PZ-11) were scored for 56 individuals, including the parent snails and offspring in a blind fashion. The hierarchical cluster analysis was performed with a binary distance matrix calculated from the data with a cluster analysis program that works with s-plus 4.0. Those individuals to the right are all resistant to the disease, and conversely, those to the left of the dashed line are susceptible.