Plasmodium falciparum Recombinants
The human malaria parasite Plasmodium falciparum recombinants survives pressures from the host’s immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two main mechanisms used by the parasite to generate genomic diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion, and drug resistance.
Materials and methods
- Parasites and cultivation of parasites.
Thirty-two independent recombinant progeny of P. falciparum from the 7G8 × GB4 cross and the two parental lines have been previously described. Parasites were maintained in RPMI 1640 medium containing 5% human erythrocytes O+ (5% hematocrit), 0.5% Albumax (GIBCO, Life Technologies, Grand Island, NY, USA), sodium bicarbonate 24 mM and gentamicin 10 μg/ml at 37 °C. C under an atmosphere of 5% CO2, 5% O2 and 90% N2.
- Genechip® microarray, DNA hybridization and data normalization
The PFSANGER Genechip® was purchased from Affymetrix, Inc. (Santa Clara, CA, USA), and array hybridization was performed at the National Cancer Institute Microarray Facility (Frederick, MD, USA). . The probes on the array were designed based on the P. falciparum genome v2.1.1 sequence (3D7) which covers genomic regions where unique probes with a reasonably wide thermal range can be designed.
Due to recent updates to genome databases, all probe sequences were reassigned to new coordinates along each chromosome according to the 3D7 genome sequence in PlasmoDB v6.0. DNA extraction, labelling, microarray hybridization, data collection and normalization have been described. Hybridized chips were washed and stained following the Affymetrix EukGE-WS2v5 protocol and scanned at 570nm emission wavelength with the Affymetrix 3000 scanner. CEL files of scanned images were processed with the R/Bioconductor package and the robust multichip analysis method.
The programs retrieved the individual probe hybridization signal, subtracted background noise, quantile-normalized signals across all chips, and log2 transformed the data into a final data matrix. The raw and normalized data obtained for this publication have been deposited in NCBI’s Gene Expression Omnibus and can be accessed through the GEO series accession number [GEO: GSE25656].
- Single trait polymorphism and parental genotype assignment
SFP calls were recorded and validated using an internal Perl script as previously described and validated. An SFP was defined as a three-fold or greater reduction in signal intensity than that of the reference 3D7 genome, regardless of the number or type of substitutions covered by a probe. A probe was assigned as SFP(‘1’) if the signal reduction was at least three times (conservative to reduce false positives) that of 3D7 and was not named SFP(‘0’) if the signal change was less than 3.0.
For each progeny, there were generally four different possible genotypes: both 7G8 and GB4 are the same as 3D7, designated ‘0_0’; both 7G8 and GB4 are the same but different from 3D7(‘1_1’); 7G8 is different from 3D7 and GB4 is not (‘1_0’); and GB4 is different from 3D7, and 7G8 is not (‘0_1’).
From these SFP calls, we selected probes that have differential SFP calls between the two parents (i.e., one parent was ‘1’ and the other was ‘0’), then the signals of each progeny’s probes were assigned individually based on comparisons with the signals of the two parents. Because single-probe calls were shown to be error-prone, and SFP was called only if at least two continuous probes indicated a polymorphism. To avoid overlapping redundant probe calls, we collapsed all overlapping probes within 25 bp into one SFP (SFP).
Here, we use a high-density mosaic matrix to estimate the rate of genetic recombination among 32 offspring of a P. falciparum genetic cross (7G8 × GB4). We detected 638 recombination events and built a high-resolution genetic map. By comparing the genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots.
Like centromeres in other organisms, P. falciparum centromere sequences are found in chromosomal regions largely devoid of recombination activity. Hotspot-enriched motifs were also identified, including a 12-bp G/C-rich motif with a 3-bp periodicity that can interact with a protein containing 11 arrays.
These results show that the P. falciparum genome has a high rate of recombination, although it also follows the general rule of meiosis in eukaryotes with an average of about one crossover per chromosome per meiosis. The GC-rich repetitive motifs identified in the hot spot sequences may play a role in the observed high recombination rate. The lack of recombination activity in the centromeric regions is consistent with observations of reduced recombination near the centromeres in other organisms.