Discovery of novel genic-SSR markers from transcriptome dataset of an important non-human primate, Macaca fascicularis

Macaca fascicularis, also known as the cynomolgus macaque, is an important non-human primate animal model used in biomedical research. It is an Old-World primate widely distributed in Southeast Asia and is one of the most abundant macaque species in Malaysia. However, the genetic structure of wild cynomolgus macaque populations in Malaysia has not been thoroughly elucidated. In this study, we developed genic-simple sequence repeat (genic-SSR) markers from an in-house transcriptome dataset generated from the Malaysian cynomolgus macaque via RNA sequencing, and applied these markers on 26 cynomolgus macaque individuals. A collection of 14,751 genic-SSRs were identified, where 13,709 were perfect SSRs. Dinucleotide repeats were the most common repeat motifs with a frequency of 65.05%, followed by trinucleotide repeats (20.55%). Subsequently, we designed 300 pairs of primers based on perfect di- and trinucleotide SSRs, in which 105 SSRs were associated with functional genes. A subset of 30 SSR markers were randomly selected and validated, yielding 19 polymorphic markers with an average polymorphism information content value of 0.431. The development of genic-SSR markers in this study is indeed timely to provide useful markers for functional and population genetic studies of the cynomolgus macaque and other related non-human primate species

Expediting the sampling, decalcification, and forensic DNA analysis of large elephant ivory seizures to aid investigations and prosecutions

The illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/ optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action

DNA forensic case study: species identification from suspected crocodile penis

Department of Wildlife and National Parks (PERHILITAN) began developing the capacity on wildlife DNA forensic since 2009 to assist in law enforcement activities. Most of the forensic cases require DNA species identification of animal parts where key morphological characters are missing. Among the cases frequently confiscated are from traditional Chinese medicine (TCM), which often claim to use animal parts such as reproductive organs. Dried crocodile penises, in particular, are believed to have medicinal benefits and are highly demanded in TCM industries since millennials ago. In this case study, we analysed four enforcement cases comprising of 44 exhibits which resemble crocodile penis using the partial cytochrome b gene of the mitochondrial DNA. Sequence similarity searches were conducted using both the BLAST search engines of GenBank and also PERHILITAN’s MyWILDNA database to identify the species. Out of 44 exhibits, 22 items produced DNA sequences in which three were found to be derived from Crocodylus porosus while the remaining was identified as Bos taurus, Bos javanicus, and Bos indicus. This case study showed that most of TCM which claimed to be derived from crocodile penis turned out to be counterfeit products

Genetic diversity and population structure of long-tailed macaque (Macaca fascicularis) populations in Peninsular Malaysia

Background: The genetic diversity and structure of long-tailed macaques (Macaca fascicularis) in Peninsular Malaysia, a widely used non-human primate species in biomedical research, have not been thoroughly characterized. Methods: Thirteen sites of wild populations of long-tailed macaques representing six states were sampled and analyzed with 18 STR markers. Results: The Sunggala and Penang Island populations showed the highest genetic diversity estimates, while the Jerejak Island population was the most genetically discrete due to isolation from the mainland shelf. Concordant with pairwise Fst estimates, STRUCTURE analyses of the seven PCA-correlated clusters revealed low to moderate differentiation among the sampling sites. No association between geographic and genetic distances exists, suggesting that the study sites, including island study sites, are genetically if not geographically contiguous. Conclusions: The status of the genetic structure and composition of long-tailed macaque populations require further scrutiny to develop this species as an important animal model in biomedical research

Rediscovery of nycticebus coucang insularis Robinson, 1917 (Primates: Lorisidae) at Tioman Island and its mitochondrial genetic assessment

Slow lorises (Nycticebus) consist of eight species native to Southeast Asia while three species are recognised in Malaysia - N. coucang, N. menagensis and N. kayan. This study reports on the rediscovery of the subspecies N. coucang insularis Robinson, 1917 in Tioman Island and the genetic assessment of its mitochondrial DNA variation. Morphological measurements conform the specimen as the putative N. coucang but with distinct colour and markings. Two mitochondrial DNA segments (cytochrome b and control region) were produced from the subspecies representing their first registered sequences in GenBank. Genetically, the subspecies showed 99% of nucleotide similarity to N. coucang species type for both the DNA segments and constitute its own unique haplotype. Phylogenetic trees constructed using three methods (neighbour joining, maximum likelihood and Bayesian inference) showed two major groups within Nycticebus; the basal group was formed by N. pygmaeus while the second group consisted of the remaining Nycticebus species. The phylogenetic position of the subspecies, however, remains unresolved due to the observed mixing between N. coucang and N. bengalensis. Several reasons could lead to this condition including the lack of well documented voucher specimens and the short DNA fragments used. In addition, the possibility of hybridisation event between N. coucang and N. bengalensis could not be excluded as a possible explanation since both species occur sympatrically at the Isthmus of Kra region until the Thailand-Malaysia border. The rediscovery of this subspecies displays the unique faunal diversity that justifies the importance of Tioman Island as a protected area

Mitochondrial DNA diversity of the long-tailed macaque (Macaca fascicularis) from the northern region of Peninsular Malaysia

We examined the genetic diversity of 64 long-tailed macaques from the northern states of Peninsular Malaysia covering the states of Perlis and Kedah including the Langkawi Island using the complete control region (CR) segment of the mitochondrial DNA. Standard genetic diversity including nucleotide diversity, haplotype diversity and genetic divergence were calculated. Moderate nucleotide diversity (π = 0.021) was observed which is higher than a previous study on the Penang M. fascicularis population. Twenty-three haplotypes were detected with haplotype diversity, h of 0.936. Haplotype sharing was observed among Langkawi and Perlis macaques indicating historical connection between the island and the mainland. Phylogenetic trees constructed grouped the samples into 4 groups without any obvious populations structuring

Three divergent subpopulations of the malaria parasite plasmodium knowlesi

Multilocus microsatellite genotyping of Plasmodium knowlesi isolates previously indicated 2 divergent parasite subpopulations in humans on the island of Borneo, each associated with a different macaque reservoir host species. Geographic divergence was also apparent, and independent sequence data have indicated particularly deep divergence between parasites from mainland Southeast Asia and Borneo. To resolve the overall population structure, multilocus microsatellite genotyping was conducted on a new sample of 182 P. knowlesi infections (obtained from 134 humans and 48 wild macaques) from diverse areas of Malaysia, first analyzed separately and then in combination with previous data. All analyses confirmed 2 divergent clusters of human cases in Malaysian Borneo, associated with long-tailed macaques and pig-tailed macaques, and a third cluster in humans and most macaques in peninsular Malaysia. High levels of pairwise divergence between each of these sympatric and allopatric subpopulations have implications for the epidemiology and control of this zoonotic species

A revisit to a low-cost method for the isolation of microsatellite markers: the case of the endangered Malayan tapir (Tapirus indicus)

There are many approaches to develop microsatellite markers. Despite the availability of the more advanced technology in the market, due to budget constraints, we revisited an easy and rapid polymerase chain reaction (PCR) cloning-sequencing method to design microsatellite markers for Tapirus indicus. Using six random amplified microsatellite markers, this study had rapidly generated 45 unique genomic sequences containing microsatellites. After screening 15 terminal and seven intermediate microsatellite loci, we shortlisted five and seven which were amplified either by single- or multiplex PCR using the economical three-primer PCR method. Genotyping attempts were made with ten T. indicus individuals using three of the terminal microsatellite loci and all seven intermediate loci. However, none of the terminal microsatellite loci were considered useful for population genotyping studies, while the seven intermediate loci showed good amplification but were monomorphic in the ten samples and the subsequent 51 tapir samples. Despite successful detection of amplified loci, we would like to highlight that, researchers who are interested in this alternative method for isolation of microsatellite loci to be cautious and be aware of the limitations and downfalls reported herein that could render these loci unsuitable for population genotyping

Using Y-Chromosome to elucidate the evolution and dispersal pattern of the long-tailed macaques (Macaca fascicularis) in Southeast Asia

We employed a combined segment of the testis-specific protein (TSPY) and the sex determining region (SRY) of the Y-chromosome gene to elucidate the evolutionary pattern of the long-tailed macaques (Macaca fascicularis) in Southeast Asia. A maximum-likelihood (ML) tree and a phylogenetic network were constructed using 147 sequences of M. fascicularis from the Peninsular Malaysia and Sarawak including sequences from the other regions of the species range taken from the other previous studies. Measurements of standard genetic diversity indices were calculated. Our findings revealed that the M. fascicularis are separated into two major groups of the continental and the insular lineages. Furthermore, the continental lineage is separated into two faunal regions demarcated at the Isthmus of Kra. The Y-chromosome dataset revealed a dominant haplotype emerging at around 0.25 (±0.1) million years ago (mya) which was shared by 82 samples from the southern region of the Isthmus of Kra which ranges from Songkhla, Thailand, the Malay Peninsula and downwards to Sumatra, Indonesia. The insular lineage emerged at around 0.61 (±0.4) mya which occupied the island of Borneo and the Philippines. We also confirmed that the introgression of the M. mulatta Ychromosome into the Indochinese M. fascicularis (Vietnam and Cambodia) are absent in the M. fascicularis haplotypes from the southern region of the Isthmus of Kra. Keywords: Macaca fascicularis, Y-chromosome, phylogenetic tree and network, dispersal route, time estimates

RNA sequencing (RNA-Seq) of lymph node, spleen, and thymus transcriptome from wild Peninsular Malaysian cynomolgus macaque (Macaca fascicularis)

The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53-63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development