Single nucleotide polymorphisms (SNPs) are highly conserved in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques
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Macaca fascicularis (cynomolgus or longtail macaques) is the most commonly used non-human primate in biomedical research. Little is known about the genomic variation in cynomolgus macaques or how the sequence variants compare to those of the well-studied related species, Macaca mulatta (rhesus macaque). Previously we identified single nucleotide polymorphisms (SNPs) in portions of 94 rhesus macaque genes and reported that Indian and Chinese rhesus had largely different SNPs. Here we identify SNPs from some of the same genomic regions of cynomolgus macaques (from Indochina, Indonesia, Mauritius and the Philippines) and compare them to the SNPs found in rhesus.
We sequenced a portion of 10 genes in 20 cynomolgus macaques. We identified 69 SNPs in these regions, compared with 71 SNPs found in the same genomic regions of 20 Indian and Chinese rhesus macaques. Thirty six (52%) of the M. fascicularis SNPs were overlapping in both species. The majority (70%) of the SNPs found in both Chinese and Indian rhesus macaque populations were also present in M. fascicularis. Of the SNPs previously found in a single rhesus population, 38% (Indian) and 44% (Chinese) were also identified in cynomolgus macaques. In an alternative approach, we genotyped 100 cynomolgus DNAs using a rhesus macaque SNP array representing 53 genes and found that 51% (29/57) of the rhesus SNPs were present in M. fascicularis. Comparisons of SNP profiles from cynomolgus macaques imported from breeding centers in China (where M. fascicularis are not native) showed they were similar to those from Indochina.
This study demonstrates a surprisingly high conservation of SNPs between M. fascicularis and M. mulatta, suggesting that the relationship of these two species is closer than that suggested by morphological and mitochondrial DNA analysis alone. These findings indicate that SNP discovery efforts in either species will generate useful resources for both macaque species. Identification of SNPs that are unique to regional populations of cynomolgus macaques indicates that location-specific SNPs could be used to distinguish monkeys of uncertain origin. As an example, cynomolgus macaques obtained from 2 different breeding centers in China were shown to have Indochinese ancestry.
KeywordsMajor Histocompatibility Complex Class Rhesus Macaque Cynomolgus Macaque Macaque Species Breeding Center
The cynomolgus macaque (M. fascicularis) is used widely in biomedical research, advancing the study of simian immunodeficiency virus (SIV) pathogenesis [1, 2, 3], transplantation biology [4, 5], diabetes , and alcohol research , among others. Currently, more cynomolgus macaques are imported for use in biomedical research in the United States than are any other non-human primate species. While recent efforts have established many genetic tools for the study of the rhesus macaque, including the complete genome sequence , a microsatellite mapping set , and a collection of SNPs [10, 11], very few genetic resources are available for cynomolgus macaque genetic research . Specifically, the M. fascicularis genome sequence, gene expression arrays and a SNP map are not yet available to advance complex trait analysis in cynomolgus macaques.
The current range of the rhesus macaque extends from Eastern China through Western India and Pakistan. Within this territory, Indian and Chinese rhesus sub-populations have diverged into genetically distinct subpopulations, as assessed by both mtDNA analysis  and MHC allele distributions [25, 26, 27]. Recent genomic studies have shown that the vast majority (69%) of SNPs identified in Indian and Chinese rhesus macaque are private to one of the two subpopulations, and that the Chinese population has nearly twice as many SNPs as the Indian rhesus population [10, 11]. The dramatic difference in genetic heterogeneity between the Chinese and Indian rhesus macaques has been attributed to both a large expansion of the Chinese population and a contraction of the Indian rhesus population .
M. fascicularis and M. mulatta co-exist in a limited geographic range in Mainland SE Asia. The precise evolutionary history of the macaques in the overlapping range is uncertain. For example, mtDNA sequence comparisons of both species cluster as discrete haplogroups [22, 28], consistent with complete lineage sorting and a lack of interspecies hybridization. However the limited genomic sequence comparisons to date, including portions of 4 autosomal loci and 2 Y chromosomal loci, suggest a closer relationship of the species than that predicted by mtDNA analysis [29, 30, 31], and suggests possible contemporary gene-flow between the macaques in Indochina . In addition, analysis of the MHC class II region has revealed a high level of allele sharing between rhesus and cynomolgus macaques, though no evidence of identical MHC class II haplotypes .
We recently reported the discovery and analysis of Chinese and Indian rhesus macaques SNPs in portions of 94 genes . Here we identified the composition and distribution of SNPs from cynomolgus macaques in 10 of those same gene regions. In addition, a SNP array of rhesus polymorphisms in 53 genes was used to genotype cynomolgus macaque DNAs from 5 different geographic regions (Indochina, Indonesia, Philippines, Mauritius and breeding centers in China). We found 51% of the polymorphisms were present in both cynomolgus and rhesus macaques. These findings suggests that the SNPs identified through genomic discovery efforts in rhesus macaque will have direct benefit to the development of cynomolgus macaque SNP research tools as well.
Distribution of SNPs identified by DNA sequencing 10 gene regions.
Total # SNPs
Private to M. f.
Shared with M. m. Chinese2
Shared with M. m. Indian2
Number of SNPs identified in M. fascicularis using an M. mulatta SNP array.
M. m. Indian SNPs (24 total)
M. m. Chinese SNPs (19 total)
M. m. Chn & Ind SNPs (21 total)
Chinese breeding centers
Total SNPs in M. fascicularis
The genotypes of two SNP loci, MAOA:116 and CCR1:641, are of particular interest. MAOA:116 alleles are largely fixed in the rhesus population, with the C-allele being found in Indian rhesus and the G-allele being found in Chinese rhesus . In the cynomolgus macaques 195/200 chromosomes carried the Indian rhesus C-allele. The rare G-allele was only observed in the Indochinese cynomolgus monkeys. At the CCR1:641 locus, the minor allele frequency was high (0.41) in the Indochinese and Chinese M. fascicularis, however no heterozygous genotypes were detected among the 41 macaques screened from these populations. The genotypes from both the SNP assay and direct sequencing were concordant at this locus.
We also used the SNP array to compare the genotypes of cynomolgus macaques from known geographic ancestry to those of animals imported from two breeding centers in China. Of the 12 SNPs in this assay that were only detected among the 25 Indochinese macaques, 11 were also found in the 16 animals obtained from China. The average minor allele frequency of these 12 specific SNPs was 0.19 in the Indochinese animals and 0.16 in the M. fascicularis from China.
We found that approximately half of the SNPs identified in M. fascicularis are also present in M. mulatta. The finding indicates that efforts to identify SNPs in either species will be beneficial in generating resources for both macaque species. The recent publication of the rhesus genome sequence has sparked interest in developing genome-wide SNP arrays for use in biomedical research . Such a rhesus macaque SNP array could be used to genotype M. fascicularis DNAs, which based upon this analysis, would capture about 50% of the genomic SNPs in cynomolgus macaques.
In this sampling, we identified 36 SNPs that appeared unique to a single geographic population of M. fascicularis, suggesting that genetic differences between the populations could underlie unrecognized phenotypic differences between these animals. Since genetic heterogeneity can complicate the reproducibility of results in biomedical studies, it would be prudent to use animals from only a single population of M. fascicularis in a single research study. Towards this goal, population-specific SNPs could be used to verify the ancestry of a research animal when it is uncertain. As example, the population-specific SNPs from this study identified cynomolgus macaques from two breeding centers in China as being of Indochinese descent. An expanded SNP discovery effort would readily identify more population markers, making it possible to identify hybrid M. fascicularis as well.
The finding that approximately half of the SNPs found in M. fascicularis overlap with those found in M. mulatta is remarkable, given that previous analysis showed that only 31% of SNPs in rhesus macaques are shared by both the Indian and Chinese subpopulations [10, 11]. The overlapping SNPs in M. fascicularis include ones that are private to Chinese or Indian rhesus, as well as those that are shared between Indian and Chinese populations. While there are more SNPs from the Chinese rhesus than Indian rhesus macaques present in cynomolgus macaques, there are about twice as many SNPs present in the Chinese rhesus population as whole. An evolutionary bottleneck that reduced the overall genetic diversity of the Indian rhesus macaque has been proposed , and such a contraction could also have reduced the representation of ancestral macaque SNPs in Indian rhesus monkeys.
There are several possible explanations for the high percentage of shared variants in these two macaque species. The SNPs identified in both M. fascicularis and M. mulatta could represent that the most ancient SNPs in the fascicularis group, those that predate the divergence of rhesus and cynomolgus macaques. Consistent with this idea, the majority of SNPs found in both Indian and Chinese rhesus macaques were also found in M. fascicularis. Interestingly, although mtDNA analysis supports the divergence of these two species 1.8 MYA [32, 33], the few studies to date of nuclear DNA loci have suggested a closer relationship [29, 30, 31]. Our findings also suggest a more complex evolutionary history than that suggested by mtDNA alone.
It is possible that relatively recent gene flow between these two macaque species has contributed to the high rate of overlap between M. mulatta and M. fascicularis SNPs. The Y-chromosome sequence studies of Tosi et al. suggest interspecies hybridization, though only within the current overlapping range of Chinese rhesus macaques and Indochinese cynomolgus macaques [30, 31]. However, in this study we found that Indonesian cynomolgus macaques also share a high percentage of SNPs with the rhesus macaques. Thus if interspecies hybridization contributed to the shared sequences, it likely would have occurred before or during periods of glaciation, when land bridges could have permitted the migration of macaques as far South as Indonesia. Nonetheless, gene flow between Chinese rhesus and M. fascicularis does not explain all of the overlapping SNPs, since there is also evidence of Indian-specific rhesus variants in M. fascicularis. Due to the geographic barriers that separate India and Indonesia, SNPs common to both of these populations are more likely a consequence of either sequence conservation or convergent evolution.
Selective pressure to maintain some of the sequence variants could have contributed to the retention of some SNPs in both M. fascicularis and M. mulatta. Possible evidence of selective pressure can be found within this study. By both direct sequence comparisons and SNP array genotyping, we found that one SNP locus (CCR1:641) had a high minor allele frequency (0.41) in the macaques derived from Indochina and Chinese breeding center, and yet no heterozygous individuals were detected, a striking departure from Hardy-Weinberg equilibrium. This finding could be the consequence of inadequate sample size, or a technical issue that was resolved neither by direct sequencing nor by the SNP array. However it is also possible that a heterozygous genotype at CCR1:641, or at alleles tightly linked to this locus, is associated with decreased survival. This is not implausible, since CCR1 encodes the chemokine receptor 1 protein, which is involved in leukocyte recruitment in response to pathogenic infections .
The MAOA:116 locus has fixed alleles in the Indian and Chinese rhesus macaque populations and was included in this study of M. fascicularis. The allele present in > 99% of Indian rhesus (C) was also found in almost all of the M. fascicularis, with the only exceptions being a few individuals from the Indochinese population. This skewed presence of the Indian rhesus allele in the M. fascicularis animals is striking. The MAOA gene encodes monoamine oxidase A, a protein that is involved in the breakdown of neurotransmitters, including norepinephrine and serotonin. Some alleles of MAOA are thought to influence aggressive and impulsive behaviors in primates . Perhaps selective pressure favors different MAOA alleles in varying macaque populations or environments.
There were no fixed alleles detected in this study that distinguish M. fascicularis and M. mulatta, and thus there is no direct evidence of gene replacement between species. Based upon the morphological and anatomical differences between the macaques, one would expect some gene replacement to be present. Additional sequencing of larger regions of genomic DNA will be needed to resolve the rate of allele fixation between these two macaque species.
We found that 52% of the SNPs identified in M. fascicularis are also found in M. mulatta. The high rate of overlap suggests that the evolutionary relationship of rhesus and cynomolgus macaques may be closer than that suggested by previous morphological and mtDNA analysis. It also indicates that future SNP discovery efforts in either macaque species will generate information that will be useful for both species. Future efforts to identify cynomolgus SNPs would not only advance genetic research in this widely used animal model, but would also generate tools for verifying the origins of animals for studies where ancestry is important.
M. fascicularis DNAs used in this study were obtained from at least 2 sources for each geographic region: 25 from Indochina (21 from Cambodia and 4 from Vietnam; SNBL USA, Everett, WA; Alpha Genesis, Yemassee, SC); 20 from Indonesia (Tinjil Island, founded by animals native to Western Java and Southern Sumatra; Java via SNBL USA, Everett, WA; Worldwide Primates, Miami, Florida); 23 from Mauritius (Charles River Laboratories, Wilmington, MA, via the University of Washington and the University of Wisconsin); 16 from the Philippines (SICONBREC, Tanay, Rizal Province, Luzon Island; Del Mundo Trading, Mandaluyong, (central) Manila, Luzon island; Jan Vacek, Located in ILOILO, on the Island of Panay.); 16 from China (SNBL USA, Everett, WA; Alpha Genesis, Yemassee, SC).
The 10 gene regions chosen for direct DNA sequence analysis were selected from those previously analyzed in rhesus macaques . In each case, PCR amplification was achieved using Taq Polymerase (Fermentas, Inc., Hanover, MD) in accordance with the manufacturers protocol, along with primers designed from the rhesus macaque genomic sequence (see Additional file 4). Amplification products were separated by agarose gel electrophoresis and isolated using Montage Gel Extraction Kit (Millipore, Inc., Bedford, MA). The DNA fragments were sequenced using the PCR amplification primers and Big Dye Chemistry; the products were separated on a Genetic Analyzer 3130 (Applied Biosystems, Inc., Foster City, CA). Sequence electropherograms were visually inspected and compared using Sequencher 4.7 (GeneCodes, Inc., Ann Arbor, MI).
A custom SNP array was used to genotype 64 previously identified rhesus SNPs, using iPLEX reagents and protocols for multiplex PCR, single base primer extension and generation of mass spectra in accordance with the manufacturer's instructions (Sequenom, Inc., San Diego, CA). The multiplex reactions included 28, 17, 12 and 7 primer sets.
This work was supported by grants RR00163 and RR00166, from the National Center for Research Resources, National Institutes of Health. It was also supported in part by SNBL USA, Ltd. (Everett, WA). We thank both the reviewers and other individuals who provided insightful comments on the manuscript, including Carlos Bustamante, Joe Felsenstein, Samone Khouangsathiene, David O'Connor, Eliot Spindel and Roger Wiseman. We also thank Jennifer Dunfield, Christine Howard, Samone Khouangsathiene and Erik McArthur for their assistance in preparing the manuscript. We are grateful for the generous contributions of M. fascicularis DNAs from our colleagues: Roger Wiseman and David O'Connor (Wisconsin National Primate Research Center, Madison, WI) contributed Mauritian cynomolgus DNAs. Nickolas Lerche (California National Primate Research Center, Davis, CA) provided DNAs from Philippine M. fascicularis and Kathy Grant (Oregon National Primate Research Center, Beaverton, OR) provided DNAs from Indonesian animals. Travis Rogers at the Vollum DNA Sequencing Core, Oregon Health Sciences University, preformed the DNA sequencing. Kenneth Beckman, Director of the Functional Genomics Laboratory at Children's Hospital in Oakland Research Institute in Oakland, CA provided assistance with the Sequenom iPLEX SNP assay.
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