Regional Origin
Rhesus macaques of Indian and Chinese origins and cynomolgus macaques of different regional origins (Indochina, Indonesia, Mauritius, Philippines and Malaysia) have been found to respond differently to experimental treatment effects, including experimental infection with certain pathogens. Examples are the differences between Indian and Chinese rhesus macaques in susceptibility to SIV infection and the difference between Indonesian and Philippine cynomolgus macaques in their susceptibility to infection with Plasmodium knowlesi. These differences have been assumed to result from genetic differences between these animals.
The UC Davis Molecular Anthropology Laboratory [MAL] identified and estimated the frequencies of more than 4,000 rhesus macaque SNPs in Indian and Chinese rhesus macaques using Roche's 454 pyrosequencing technology (Malhi et al., 2007; Satkoski et al., 2008).
Shown below are PCA plots illustrating the ability of 23 STRs and 92 of these 4,000 SNPs that the MAL randomly selected to differentiated Indian rhesus macaques from Chinese rhesus macaques. The acronyms in the boxes represent the mitochondrial DNA (mtDNA) haplogroups of the rhesus macaques. The image reflects the greater power of SNPs than STRs to identify the region of origin of rhesus macaques. The MAL then selected the 96 of these 4,000 SNPs that maximally differentiated between Indian and Chinese rhesus macaques for the regional origin test. These SNPs identify the region of origin of rhesus macaques with a high degree of reliability.
A program called STRUCTURE (Pritchard et al., 2000) is then used to estimate the proportions of Indian and Chinese ancestry of each animal, which the MAL reports to you.
Many SNPs are highly conserved (hence, shared) between rhesus and cynomolgus macaques (Street et al., 2008). The image below represents the effectiveness of the same 92 rhesus SNPs in the image above for differentiating cynomolgus macaques from the five different geographic regions of their range. As these 92 SNPs were randomly selected among the 4,000 SNPs identified, they do not represent SNPs that maximally differentiate the two regional populations that are included in the test. As illustrated below, the country of origin of different Indochinese cynomolgus macaques cannot be distinguished from each other due to their close genetic similarity but are easily distinguished from all insular populations of that species.
The MAL genotyped all 4,000 of the rhesus SNPs identified in each of the five regional populations of cynomolgus macaques and identified all of the shared SNPs between rhesus and cynomolgus macaques. The MAL found 800 SNPs that were shared between rhesus macaques and only one of the five regional populations of cynomolgus macaques; these SNPs are shown below.
The MAL selected 96 of these SNPs, as evenly divided among the five regional populations of cynomolgus macaques as possible and that maximally differentiate among the five populations for inclusion on their regional origin panel of SNPs for cynomolgus macaques. The MAL's regional origin service will provide you with the regional of origin of your cynomolgus macaque based on the 96 most informative of the SNPs in the above image that have been selected because their frequencies in the population that uniquely shares that SNP with rhesus macaques is high (over 40%).
Note that approximately 75% of all SNPs shared between rhesus macaques and only one of the regional populations of cynomolgus macaques are shared between rhesus macaques and Indochinese cynomolgus macaques. This is because the two species have experience a long history of admixture on the Southeast Asian mainland. The MAL selects up toed the 96 highest frequency rhesus SNPs uniquely shared with Indochinese cynomolgus macaques as a panel of SNPs to estimate the proportion of rhesus macaque ancestry in Indochinese cynomolgus macaques.
The MAL usually genotypes SNPs using the SNaPshot assay which is based on fluorescent labeled single nucleotide base extension. The test resembles the methodology of Sanger (di-deoxy base termination) sequencing. A multiplex reaction to identify 10-12 different SNPs is set up using di-deoxy NTPs (ddNTPs) and primers specific to each SNP that are of differing lengths and that end at the nucleotide just before the SNP being assayed. Each ddNTP is labeled with a different fluorescent dye.
Because the addition of the ddNTP to the primer sequence will not allow any additional nucleotide to be added its identity (A,T,C or G) can be determined by its characteristic dye and the specific length of the amplicon (the length of the primer sequence plus 1 base pair). The length of each amplicon and its dye identifies both the SNP being assayed in the multiplex reaction and the nucleotide at the SNP position, respectively.
The MAL runs 5 or 6 different multiplex reactions sufficient to genotype all the SNPs that they MAL finds most informative for the tests. The fragment lengths and their diagnostic dyes, excited by a laser beam, are identified using the MAL's ABI 3130xl Genetic Analyzer (sequencer).
For genotyping larger numbers of animals (orders >200 animals) the MAL uses an Illumina Bead Array assay on a Golden Gate platform at the UC Davis Genome Center. As construction of these assays and waiting to gain access to the platform may require delay in processing your order, please allow up to 6 weeks to obtain the MAL's report.
The MAL offers a second test for region of origin that is based on mitochondrial DNA (mtDNA) sequencing. MtDNA is extra-nuclear and, because it is inherited exclusively through the matriline, does not experience recombination. MtDNA of different animals come to differ due to mutations and occurred in their unique maternal ancestral lineage. This allows the MAL to arrange different animals' mtDNA sequences in an ancestor/descendant relationship to one another.
In addition, the control region of mtDNA evolves approximately two orders of magnitude more rapidly than nuclear DNA allowing differences between remotely related individuals to be identified. A characteristic series of mutations (SNPs) in the mtDNA of an animal defines its mtDNA haplotype that it shares with its mother and all his mother's offspring and her matrilineal ancestors excepting mutations that occurred in the evolutionary history of that matriline.
Due to ongoing mutations, many matrilines differ by only one or a few mutation and comprise a haplogroup, a group of closely related haplotypes. All full-blood Indian rhesus macaques share one of two major haplogroups (Ind1 and Ind2) while all full-blood Chinese rhesus macaques have one of three major haplogroups, ChiE, ChiS, and ChiW, that are found predominately in eastern, southern and western China, respectively. ChiW can be further subdivided into three subhaplogroups, ChiW1, ChiW2, and ChiW3 (Smith and McDonough 2005).
Similarly, all full-blood cynomolgus macaques belong to one of two major haplogroups Fas1 and Fas2, each of which is further subdivided into several subhaplogroups that are strictly limited to specific geographic regions as illustrated by the neighbor-joining tree below (Smith et al., 2007).
As illustrated by the tree below, full-blood Indian and Chinese rhesus macaques can easily be distinguished from each other, as can full-blood cynomolgus macaques from Mauritius, Philippines, Indochina, and Indonesia/Malaysia, but their distinctive mtDNA haplogroups or subhaplogroups.
The tree above is based on an 835 base pair sequence that encompasses the mtDNA control region. Note that Indian and Chinese rhesus macaques and Philippine, Mauritian and Indochinese cynomolgus macaques are easily identified by their characteristic mtDNA haplogroups or subhaplogroups. Both Malaysian and Indonesian cynomolgus macaques are interspersed among the remaining subhaplogroups and cannot be distinguished from each other by their mtDNA haplotypes.
Moreover, animals whose ancestry traces to more than a single geographic region could have a predominant origin in one geographic region based on nuclear DNA markers yet have mtDNA indicative of another country. Thus, this test is appropriate only when there is confidence that animals tested are have not experienced hybridization with animals from a different geographic region.
The MAL provides sequencing of this 835 base pair sequence of the mtDNA control region using the Sanger (di-deoxy termination method) on the MAL's ABI 3130xl Genetic Analyzer (sequencer) as a service. The Sanger method incorporates some di-deoxy NTP (ddNTPs), each differentially fluorescently labeled, in the sequencing reaction. Unlike dNTPs, ddNTPs are chemically modified to prevent the addition of an additional dNTP (or ddNTP) to the sequence.
As the template DNA is first "read" by the Taq polymerase, the appropriate dNTP, as well as some fluorescent labeled ddNTP, is added to the primer sequence. This is the shortest amplicon that will be amplified and its fluorescent dye (red, green, blue or yellow) identifies the first nucleotide in sequence of interest. Similarly, the next smallest amplicon generated (one base pair larger than the first one) is end-labeled with the dye that defines the second nucleodide in the sequence of interest.
This process continues until the largest amplicon is amplified that is labeled with the dye designating the last nucleotide in the sequence of interest. When all amplicons are ordered by their size, the order of the fluorescent dyes identifies the order of nucleotides in the sequence of interest.
The MAL reports to you the sequence, subhaplogroup/haplogroup and geographic region of origin of each animal implied by their mtDNA.
Malhi, R.S., B. Sickler, D. Lin, J. Satkoski, D. George, S. Kanthaswamy, D. G. Smith. 2007.MamuSNP. A SNP resource for rhesus macaques (Macaca mulatta). PLoSONE 2(5):e438.
Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155, 945-959.
Satkoski, JA, RS Malhi, S Kanthaswamy, RY Tito, VS Malladi and DG Smith. 2008. Pyrosequencing as a method for SNP identification in the rhesus macaque (Macaca mulatta). BMC Genomics 9:256.
Smith, D.G. and J.W. McDonough. 2005. Mitochondrial DNA variation in Chinese and Indian rhesus macaques (Macaca mulatta). American Journal of Primatology 65:1-25.
Smith, D.G., J.W. McDonough and D.A. George. 2007. Mitochondrial DNA variation within and among regional populations of longtail macaques (Macaca fascicularis) in relation to other species of the fascicularis group of macaques. American Journal of Primatology 69:182-198.
Street, S. L., Kyes, R. C., Grant, R., & Ferguson, B. (2007). Single nucleotide polymorphisms (SNPs) are highly conserved in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. BMC Genomics, 8, 480. doi:10.1186/1471-2164-8-480.
