G C A T T A C G G C A T genes Review Malaria in the ‘Omics Era’ Mirko Pegoraro and Gareth D. Weedall * School of Biological and Environmental Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK; [email protected] * Correspondence: [email protected] Abstract: Genomics has revolutionised the study of the biology of parasitic diseases. The first Eukaryotic parasite to have its genome sequenced was the malaria parasite Plasmodium falciparum. Since then, Plasmodium genomics has continued to lead the way in the study of the genome biology of parasites, both in breadth—the number of Plasmodium species’ genomes sequenced—and in depth— massive-scale genome re-sequencing of several key species. Here, we review some of the insights into the biology, evolution and population genetics of Plasmodium gained from genome sequencing, and look at potential new avenues in the future genome-scale study of its biology. Keywords: Plasmodium; malaria; genomics; methylomics; methylation 1. Introduction Since the genome of the malaria parasite Plasmodium falciparum was published in 2002 [1], alongside that of its mosquito vector [2] and its human host (Consortium and International Human Genome Sequencing Consortium, 2001) [3], malaria genomics has led the way in the study of eukaryotic pathogens. Since then, a growing number of Plasmodium species’ genomes have been sequenced and large-scale population resequencing Citation: Pegoraro, M.; Weedall, G.D. studies have been carried out in P. falciparum and several other species. These efforts have Malaria in the ‘Omics Era’. Genes allowed the evolutionary and population genomics of malaria parasites to be studied in 2021, 12, 843. https://doi.org/ unprecedented detail and provide a model for the application of genomics to the study 10.3390/genes12060843 of other parasite species. The application of other genome-wide sequencing analyses (e.g., transcriptomics and methylomics) broadens our knowledge of Plasmodium biology Academic Editor: Asis Khan and presents new frontiers to study. Here we consider some of these and how they can be studied at scale. This review presents some of the key advances in the study of the Received: 28 April 2021 evolutionary genomics of malaria parasites made possible by the application of genomic Accepted: 27 May 2021 sequencing technologies. Published: 30 May 2021 2. Expanding Horizons: Sequencing Across the Genus Plasmodium Publisher’s Note: MDPI stays neutral The genus Plasmodium contains a huge number of species that parasitise a wide range with regard to jurisdictional claims in of vertebrate hosts (Figure1). For a broad view of the phylogenetic relationships among published maps and institutional affil- the malaria parasites, see [4]. At the time of writing, 5-6 Plasmodium species are known iations. to infect humans. Four of these have been known for many years: Plasmodium falciparum and Plasmodium vivax are the most widespread and clinically important species, with Plasmodium malariae and Plasmodium ovale both less common and less well studied. More recently, these have been joined by Plasmodium knowlesi, a zoonotic malaria that primarily Copyright: © 2021 by the authors. infects macaques, and P. ovale has been recognised as not one but two (probable) species: Licensee MDPI, Basel, Switzerland. P. ovale curtisi and P. ovale wallikeri [5]. This article is an open access article Plasmodium genomes are, in some ways, rather attractive genomes to sequence: hap- distributed under the terms and loid in the vertebrate host (and in vitro culture), not too large (around 23 Mb, encoding conditions of the Creative Commons around 5500 genes for P. falciparum) and with 14 well-defined chromosomes. In other Attribution (CC BY) license (https:// ways they present technical challenges: they are extremely AT-rich, the resulting lowered creativecommons.org/licenses/by/ complexity of parts of the genome increasing the challenge in sequencing and assembly. 4.0/). Genes 2021, 12, 843. https://doi.org/10.3390/genes12060843 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 19 Genes 2021, 12, 843 2 of 18 (http://creativecommons.org/licenses complexity of parts of the genome increasing the challenge in sequencing and assembly. /by/4.0/). However, these challenges have been overcome and as a result, a large and growing num- However, these challenges have been overcome and as a result, a large and growing num- ber of Plasmodium species’ genomes have been fully sequenced and assembled, to varying ber of Plasmodium species’ genomes have been fully sequenced and assembled, to varying levels of completeness. For some reference genomes, initial sequencing and assembly ef- levels of completeness. For some reference genomes, initial sequencing and assembly forts using first- and second-generation sequencing technologies have subsequently been efforts using first- and second-generation sequencing technologies have subsequently been improved using third-generation, long read sequencing technology. Additional strains improved using third-generation, long read sequencing technology. Additional strains havehave also also been been independently independently sequenced sequenced and and assembled assembled for for several several species species [6 [6–9–].9]. FigureFigure 1. 1.Phylogeny Phylogeny of of malaria malaria parasites, parasites, showing showing the the relationships relationships among among human-infecting human-infecting species. spe- Thecies. tree The is atree schematic is a schematic diagram diagram indicating indicating topology topology (branch lengths(branch are lengths not to are scale). not Theto scale). phylogenetic The positionsphylogenetic of human-infecting positions of humanPlasmodium-infectingspecies Plasmodium are indicated. species More are detailedindicated. subtrees More detailed are shown sub- for majortrees lineagesare shown of for simian major malaria lineages parasites of simian and malaria for the parasites Laverania and subgenus for the Laverania of ape parasites. subgenus Host of speciesape parasites. are indicated Host species in brackets are indicated (hu = human; in brackets ma = macaque; (hu = human; ch = chimpanzee;ma = macaque; go ch = gorilla).= chimpan- Tree topologieszee; go = gorilla). are after Tree [4], andtopologies after [10 are] for after the [4] Laverania., and after [10] for the Laverania. AlongsideAlongsidePlasmodium Plasmodium falciparum falciparum[1], [1] the, otherthe other human human malaria malaria parasites parasitesP. vivax P.[6 ,vivax11], P.[6,11] malariae, P. ,malariaeP. ovale,curtisi P. ovaleand curtisiP. ovale and wallikeriP. ovale wallikeri[12–14] have[12–14] been have sequenced. been sequenced. Other mem- Other bersmembers of the of subgenus the subgenus Laverania, Laverania, parasites parasites of African of African great great apes, apes, have have been been sequenced sequenced includingincluding the the chimpanzee chimpanzee parasites parasitesP. reichenowiP. reichenowiand andP. adleri P. adleriand and gorilla gorilla parasites parasitesP. gaboni P. ga-, P.boni blacklocki, P. blacklockiand P. billcollinsiand P. billcollinsi[10,15]. [10 A ,P.15 vivax]. A P.-like vivax parasite-like parasite of great of apes great has apes also has been also sequencedbeen sequenced [13]. The [13] simian. The parasitesimian parasite of macaques of macaques and zoonotic and zoonotic human pathogen human pathogenP. knowlesi P. hasknowlesi been sequencedhas been sequenced [16,17], as [16,17] well,as as otherwell as simian other simian parasites parasites including includingP. coatneyi P. coatneyi[18] and[18]P. and cynomolgi P. cynomolgi[8,9]. [8,9] Outside. Outside of the of primatethe primate malarias, malarias, the the genomes genomes of of rodent rodent malaria malaria parasitesparasitesP. P. berghei berghei, ,P. P. chabaudi chabaudi chabaudichabaudi and P. yoelii yoelii yoelii,, important important model model species species for for the thestudy study of ofmalaria malaria biology, biology, have have been been sequenced sequenced [7,19 [7,19–22]–22. ].Two Two avian avian malaria malaria species, species, P. P.gal gallinaceumlinaceum andand P.P. relictum relictum, have, have been been sequenced sequenced [23] [23. Additional]. Additional strains strains have have also also been beenindependently independently sequenced sequenced and assembled and assembled for P. forfalciparumP. falciparum [24], P.[24 vivax], P. [6,11] vivax and[6,11 P.] andcyno- P.molgi cynomolgi [8,9]. [Together,8,9]. Together, this represents this represents an enviably an enviably richly richly sequenced sequenced genus. genus. HavingHaving so so many many species’ species’ genomesgenomes providesprovides aa rich resource for for studying studying evolution evolution in inthe the genus. genus. For For example, example, an an early early study study of of comparative comparative structural structural genomics genomics between between P. P. falciparum and rodent malaria parasites showed extensive chromosome structural rear- rangement and that lineage-specific genes could often be found at the breakpoints of these translocations [25]. More recently, third-generation long read sequencing has provided Genes 2021, 12, 843 3 of 18 further insights into the genomic architecture of Plasmodium. The central region of each chromosome, around the centromere, comprises a relatively stable ‘core genome’ charac- terised by broad co-linearity and one-to-one homology of the genes among genomes. An estimated 90% [26] or more [24,26] of the genome has been suggested