Table 1.
Primers used to validate the linear configuration and 4 structural types of the mitochondrial genome.
Figure 1.
1D-PFGE NotI Restriction Fragment Length Polymorphism (RFLP) analyses of B. microti R1 and Gray strains (left) and their corresponding Southern hybridizations (right) using a radiolabeled telomeric probe.
PFGE conditions used are: 1% agarose gel in 1X TAE using a pulse condition of 1 min for 10h at 6 V/cm (10.5°C). Arrows indicate the location of the 8 telomeric regions in the B. microti genome. Fragments showing restriction polymorphism are indicated with white arrows and stars. *: Undigested genomic DNA. 1: Chromosome I, 2: Chromosome II, 3: Chromosome III, 4: Chromosome IV. a and b represented the two telomeric regions of each chromosome.
Figure 2.
Whole Genome Map Assemblies of chromosomes I, II, III and IV of the B. microti R1 and Gray strains.
Each multicolored horizontal line represents a single molecular map. Colors are used to distinguish KpnI restriction fragments. Blunt ends indicate true ends of chromosomes. The horizontal gray line on top of the assemblies represents the assembled chromosome from each map contig.
Table 2.
Whole Genome Mapping assembly statistics for Babesia microti Gray and R1 strains.
Figure 3.
Comparison of the middle region of the KpnI restriction map of contig KIII_ctg350 with the 3′ end and 5′ end of chromosomes III and IV, respectively.
The 3′ end of chromosome III (IIIb) and 5′ end of chromosome IV (IVa) share a common region, which includes two KpnI fragments of 3.8 and 5.6 kb. This region is in one orientation on chromosome III and the opposite orientation on chromosome IV. The regions of the chromosomes missing from the original assembly are shown in white (sizes are in kb).
Figure 4.
Schematic representation of the extremities of the four chromosomes of the R1 and Gray strains based on WGM analysis.
Gray boxes represent the terminal ends of each chromosome as revealed by Whole Genome Mapping. Chromosome sizes are based on WGM calculations.
Figure 5.
Comparison of the Whole Genome maps (WGM) of each chromosome of the R1 and Gray strains.
Restriction sites are shown as vertical lines within each WGM. Differences between maps are indicated by the alignment connectors. The regions of the chromosomes that are different between the two WGMs are shown in white. The NotI restricted site and predicted size of the restriction fragments are represented for each extremity. Underlined values are calculated according to the predicted position of the NotI site.
Figure 6.
Evidence for a linear configuration and 4 structural types of the mitochondrial genome in B. microti by PCR analysis.
A. Schematic representation of the linear configuration of the mitochondrial genome of B. microti and the 4 structural types. The mitochondrial chromosome carries three protein encoding genes: cob (cytochrome b), cox1 (cytochrome c oxidase sub-unit 1) and cox3 (cytochrome oxidase sub-unit 3). The ribosomal RNA genes are fragmented in apicomplexa (43, 44). Two genes encoding rRNA of the small ribosomal (ssu6 and ssu7) and six genes encoding rRNA of the large ribosomal sub-unit have been characterized in B. microti (lsu1, lsu2, lsu3, lsu4, lsu5 and lsu6). IR-A and IR-B represent the two independent inverted repeats. Arrows represent primers 1 to 9 (Table 1) used in PCR reactions to demonstrate the linear configuration and the 4 structural types of the mitochondrial genome of B. microti. B. Fragment sizes expected from PCR analyses for the 4 linear types I, II, III and IV. C. Agarose gel analysis of the different PCR products. The PCR fragments amplified are in agreement with the prediction.
Figure 7.
Evidence for a linear configuration and 4 structural types of the mitochondrial genome in B. microti by Southern analysis.
A. Schematic representation of the linear configuration and the 4 structural types of the mitochondrial genome of B. microti. The protein encoding genes were used as genetic markers and target with specific probes (dark lines). Gene names are as described in Figure 6. IR-A and IR-B represent the two independent inverted repeats. DraI and EcoRI sites used to digest the genomic DNA and their positions on the molecules are represented. B. Southern blot analysis preformed with cox1, cob or cox3 probes (black bars) on of the R1 strain's mitochondrial DNA either undigested (U) or digested with DraI (D) or EcoRI (E). The theoretical sizes and associated types of mtDNA molecules are represented. Eth. Br.: Ethium bromide-stained agarose gel.
Figure 8.
Phylogenetic analysis on the left panel is based on a combination of three protein sequences encoded by the genome of different apicomplexan mitochondria (cox1+cob+cox3).
About 63% of the sites were conserved for the phylogenetic analysis. The scale indicates the inferred number of substitutions. Boostrap values are associated to each branch. The unrooted tree was calculated using the Phygeny.fr web site and default options of the one-click procedure. The schematic structures of mitochondrial genomes in apicomplexa (right panel) are based on species marked by a “+” in (A). Inverted repeats are represented by arrows. T. parva is presenting short inveted repeats that are not described in other Theileria and true-Babesia species. Dashed lines figure out the concatenated form of the molecule. * T. orientalis is presenting an inversion of the cox3 gene. The gene order is highly conserved among Plasmodium species [47]. Variations are associated to rRNA genes and resolution site. The P. falciparum genome organization is also present in other species such as P. floridensis, P. mexicanum or P. reichenovi. A slight variation in the molecule structure of P. berghei and P. gallineum does not change the gene order (Fig. S3 in Supporting Information S1). This later organization is present in other species such as P. fragile, P. knwolesi, P. sinium, P. vivax or P. yoelii.
Figure 9.
Genome size and chromosome number in apicomplexan species.
Number of nuclear chromosomes and genome size are shown in panel on the right. Piroplasma taxa shown in dark color; B. microti stripped. Schematic of phylogenetic relationships among species shown on the left. Resolution of the interior node in the piroplasma clade marked with ‘*’ resolved according to [34], based on study with 150 nuclear genes. Resolution differs from that obtained with mitochondrial sequences (Fig. 8) and the 18S rDNA gene [6].