Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii

Abstract

Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.

Figure 1

Representative SNP–RFLP marker used in mapping analysis. Data for the marker cB21-4, indicating primers, PCR conditions, the sequence flanking the SNP and an example of the alleles detected (gel insert). The specific SNPs detected are indicated in parentheses within the sequence block using the following format (allele type I, allele type II and allele type III). While most markers define simple biallelic patterns, in some cases these differences occur closely spaced in the genome. This example illustrates two closely spaced SNPs, one unique to type II and one unique to type I, thus this marker is capable of distinguishing all three genotypes using a combination of restriction enzyme digestions. The slightly larger band in the undigested fragment of the type III strain is due to an 8 bp difference; however, this is not the basis for the SNP differences detected here. Data for the complete set of markers can be found at .

Figure 2

Genetic linkage maps for the 14 chromosomes of T.gondii. Individual markers are shown to the right of the vertical bar and chromosome numbers are given above each map. Markers that map to the same node are indicated to the right of a solid vertical bar. The corresponding genetic distances between each node are given to the left of each map and the total sizes in cM are shown at the bottom of each chromosome. Polymorphisms that are unique to type I are shown in red, those unique to type II are shown in green, those unique to type III are shown in blue and markers that contain multiple polymorphism as illustrated in Figure 1 are shown in yellow. Maps were constructed using MAPMAKER (22) from the analysis of 71 recombinant progeny using 250 genetic markers (Table 1). Markers that include data analyzed by Southern blot are followed by the suffix ‘.c’.

Figure 3

Mapping of drug resistance to SNF, FUDR and ARA-A across the genome of T.gondii. Plots indicate the log-likelihood association of resistance to each drug with markers aligned across the genome. In each case, a single locus was found to be statistically associated with resistance, and apparent secondary peaks were either non-significant or in non-informative regions of segregation distortion. Resistance to SNF was localized to chromosome IX, FUDR was localized to XI and ARA-A was mapped onto chromosome XII. Plots were generated from genome-wide scans using permutation analysis for marker associations from I × III crosses. Significance levels are given by dotted lines [lower line is significant (log likelihood of 1.7) while the upper line is highly significant (log likelihood of 3)].

Figure 4

Horizontal maps of the genome scaffolds comprising each of the T.gondii chromosomes. Chromosome numbers are indicated to the left as is the total size of scaffolds in base pairs. Scaffold numbers are given within or above the colored bars. Bars colored green indicated scaffolds that were mapped and oriented by linkage analysis. Bars indicated in light purple were linked genetically and oriented by BAC-end data. There are several remaining scaffolds that are linked by genetic data but where the orientation is still unknown (yellow bars). There are also five scaffolds that are linked only by BAC-end data (blue bars). Orientations that were supported by BAC-end data are indicated by a thin horizontal line connecting the bars.

Figure 5

Recombination frequency for T.gondii chromosomes and analysis of double-crossovers. (A) The genetic size of chromosomes was roughly correlated with their physical sizes as determined by summing the scaffolds for each chromosome shown in Figure 2. (B) Double-crossover analysis suggests that gene conversion processes in addition to double-crossovers contribute to recombination events spanning a genome segment containing only a single genetic marker. Vertical black bars show numbers, for all 71 progeny, of double-crossovers that are separated by two or more genetic markers, plotted versus estimated physical distance ranges separating the two crossovers (see Materials and Methods). Light gray sections of the four left bars represent additional recombination events spanning only one genetic marker (‘single-marker events’). As shown by the P-values and theoretical lines, the single-marker events in the two left bars are unlikely to occur by chance from the frequencies of true double-crossovers predicted by Poisson/uniform models (see Materials and Methods). The lines represent alternative models where dotted line indicates all cases, including all single-marker events, are assumed to be true double-crossovers and solid line indicates single-marker events in the two left bars are excluded from the numbers of double-crossovers.

Figure 6

Genetic and physical maps for chromosome III as drawn from CMap for T.gondii. CMap representation of the genetic map (left), scaffolds (center) and the assembled chromosome (right) for T.gondii chromosome III. Correspondences are shown in colored lines depicting the relationship between the data. Green lines indicate markers that were mapped and oriented genetically. Purple lines indicate markers that were mapped genetically and oriented by BAC-ends. As well, BAC-end linkages are indicated by blue horizontal dumbbell shaped bars. The scale for the left-hand map is based on genetic units, while the center and right-hand maps are based on physical size in base pairs. Differences in the map unit across the various nodes of the genetic map are indicated at the far left in kb/cM.