Cross Species use of Human Microarray Genotyping Technology for Bornean Orangutan (Pongo pygmaeus) SNP Discovery

Previous genetic studies of orangutans (Pongo spp.) have relied mainly upon mitochondrial DNA or microsatellite short tandem repeats (STR) for genomic genotyping analysis. Scientists have yet to take advantage of the genetic closeness of the great apes to humans for genomic analysis by using advanced techniques available for human genotyping. To genotype orangutans at Tanjung Puting National Park, we developed a novel combination of a methyl-based magnetic enrichment capture of genomic fecal DNA with genotyping on a human targeted single nucleotide polymorphism (SNP) microarray, and compared this to additional microsatellite (STR) micro-capillary genotyping. We successfully isolated 125 known human genomic SNP loci (0.08% of those targeted) which hybridized orangutan DNA on the human targeted Illumina Infinium QC array. We estimated genetic diversity and relatedness (r) using three estimators for a total of 32 (21 female and 9 male) wild orangutans at the Camp Leakey study site. Average TrioML relatedness within the sample, estimated from our combo SNP/STR dataset, was at a range consistent with half and first cousins (r = .082). All sampled males and females had relatives within the study site indicating we have verified a local, closely related community of wild orangutans at Camp Leakey

The Origin of Malarial Parasites in Orangutans

Background Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans. Methodology/Principal Findings We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-142) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-142 and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-142 in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites. Conclusion The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates

Segments of the left domain of the orang-utan mitochondrial DNA control region, of varying lengths and positions, as published and analysed in prior studies.

<p>Many were cut by the original authors from longer sequences (<i>i</i>.<i>e</i>. larger PCR amplicons, containing flanking regions) prior to analysis and/or publication; only those bases derived from the control region are specified here. Nucleotide positions correspond to those of the complete Sumatran orang-utan mitochondrial DNA genome (GenBank accession code X97707; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168715#pone.0168715.ref050" target="_blank">50</a>]) and are estimated for unpublished sequences based on the lengths noted in each manuscript. The control region spans positions 15484–16499 of the reference genome.</p

Relative position of primers used in DNA amplification and sequencing of the complete orang-utan mitochondrial DNA control region.

<p>The sizes of each region in this schematic are proportional to their sizes in the orang-utan mitochondrial genome.</p

Nucleotide diversity throughout the complete orang-utan mitochondrial DNA control region.

<p>Diversity was computed for alignments comprising, a) all 38 sequences (1022 sites), b) 13 sequences of Sumatran maternal ancestry (1018 sites), and c) 25 sequences of Bornean maternal ancestry (1002 sites). Data were computed under the sliding window method, using a window length of 100 sites and a step size of 25 sites, excluding sites with gaps.</p

Diversity indices computed from three alignments of 35 novel and unique complete control-region sequences generated in this study, plus three published complete control-region sequences, for all sequences and for those of Bornean or Sumatran orang-utans only.

<p>Alignments were trimmed to the lengths of various shorter segments sequenced in prior studies.</p