Genome-wide discovery and development of polymorphic microsatellites from Leishmania panamensis parasites circulating in central Panama

Abstract

BackgroundThe parasite Leishmania panamensis is the main cause of leishmaniasis in Panama. The disease is largely uncontrolled, with a rising incidence and no appropriate control measures. While microsatellites are considered some of the best genetic markers to study population genetics and molecular epidemiology in these and other parasites, none has been developed for L. panamensis.FindingsHere we have developed and tested a new panel of microsatellites for this species, based on high-throughput genome-wide screening. The new set of microsatellites is composed of seventeen loci, mainly spanning trinucleotide or longer motifs. We have evaluated the sensitivity and specificity of the panel based on a sample of 27 isolates obtained from cutaneous leishmaniasis patients from central Panama and also several reference species from both L. (Leishmania) and L. (Viannia) subgenera. The genetic equilibrium was assessed both intra- and inter-loci, while the reproductive mode was evaluated using several tests. The new SSR panel shows high polymorphism and sensitivity, as well as good specificity. The preliminary data described here for L. panamensis suggest extensive departure from Hardy-Weinberg proportions, significant linkage disequilibrium and strong deficit of heterozygotes. Several recombination tests involving multilocus linkage disequilibrium and a phylogenetic approach allowed rejection of frequent recombination in our dataset.ConclusionsThe genome-wide strategy described here proved to be useful to identify and test new polymorphic SSR loci in Leishmania. The new panel of polymorphic microsatellites is a valuable contribution to the existing molecular markers for the study of genetic structure and other aspects of this important species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1153-2) contains supplementary material, which is available to authorized users.

Molecular Identification of Parasites Causing Cutaneous Leishmaniasis in Panama

“…48,76 Probably, the analyzed region of the hsp70 gene (1,245 pb) presents a low polymorphism to readily distinguish between closely related Leishmania species. In this regard, complementary studies using more powerful molecular analysis tools, like multi-locus sequence typing and complete genome sequencing, [22][23][24]77,78 are necessary to clearly define the phylogenetic position of the Leishmania isolates (here referred as Leishmania (V.) sp. 1, Leishmania (V.) sp.…”

Genomic Diversification, Structural Plasticity, and Hybridization in Leishmania (Viannia) braziliensis

“…Several nuclear and mitochondrial DNA markers as well as different molecular techniques have been used with the purpose to evaluate inter-and intra-specific genetic variability of New/ Old world Leishmania species, such as L. major, L.infantum, L. donovani, L. tropica, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis (Cupolillo et al, 2003;Nolder et al, 2007;Kuhls et al, 2013;Restrepo et al, 2013;Marco et al, 2015;Restrepo et al, 2015;Fotouhi-Ardakani et al, 2016;Cysne-Finkelstein et al, 2018;Quaresma et al, 2018;Banu et al, 2019). This has suggested high intra-specific variability, especially for L. braziliensis from Brazil (Marlow et al, 2014), Argentina (Marco et al, 2015) and Colombia (Herrera et al, 2017).…”

Intraspecific Genomic Divergence and Minor Structural Variations in Leishmania (Viannia) panamensis

“…The reported studies so far have focused on L. braziliensis, where the majority report consistent findings in describing its high intraspecific genetic diversity [16,26,43] even in cell populations isolated from a single L. braziliensis strain ("sub-strain") [44], while in L. panamensis the results are not yet concordant, probably for the molecular approaches used. While studies using MLST reveal extreme genetic homogeneity within L. panamensis [10], others using MLEE [5], sequencing of specific genes (Cytb/HSP70) [6], AFLP [13], or microsatellites [14], have reported a high intraspecific genetic diversity in this species. Considering that DNA high-throughput sequencing has the potential to identify changes in the whole genome structure, we used this approach to reveal the intraspecific genomic variability of L. panamensis from Colombian clinical isolates and Panamanian genomes retrieved from databases.…”

“…Several nuclear and mitochondrial DNA markers (cytochrome b, COII, gp63, 18S-rRNA, mini-exon, HSP-70, and ribosomal internal transcribed spacer regions (ITS-rDNA)) as well as different molecular techniques such as polymerase chain reaction (PCR) [9], multilocus sequence typing (MLST) [10], multilocus enzyme electrophoresis (MLEE) [5,11], random amplified polymorphism (RAPD) [12], amplified fragment length polymorphisms (AFLP) [13] and microsatellites [14], have been used to evaluate the inter and intraspecific genetic variability of different Leishmania species (L. tropica, L. major, L. donovani, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis) [13][14][15][16][17][18][19]. However, the identification of such genetic variants across the whole genome has become possible only with the arrival of DNA-seq.…”