Ancestry, Plasmodium cynomolgi prevalence and rhesus macaque admixture in cynomolgus macaques (Macaca fascicularis) bred for export in Chinese breeding farms.

BACKGROUND: Most cynomolgus macaques (Macaca fascicularis) used in the United States as animal models are imported from Chinese breeding farms without documented ancestry. Cynomolgus macaques with varying rhesus macaque ancestry proportions may exhibit differences, such as susceptibility to malaria, that affect their suitability as a research model. METHODS: DNA of 400 cynomolgus macaques from 10 Chinese breeding farms was genotyped to characterize their regional origin and rhesus ancestry proportion. A nested PCR assay was used to detect Plasmodium cynomolgi infection in sampled individuals. RESULTS: All populations exhibited high levels of genetic heterogeneity and low levels of inbreeding and genetic subdivision. Almost all individuals exhibited an Indochinese origin and a rhesus ancestry proportion of 5%-48%. The incidence of P. cynomolgi infection in cynomolgus macaques is strongly associated with proportion of rhesus ancestry. CONCLUSIONS: The varying amount of rhesus ancestry in cynomolgus macaques underscores the importance of monitoring their genetic similarity in malaria research.

Heterospecific SNP diversity in humans and rhesus macaque (Macaca mulatta).

BACKGROUND: Conservation of single nucleotide polymorphisms (SNPs) between human and other primates (i.e., heterospecific SNPs) in candidate genes can be used to assess the utility of those organisms as models for human biomedical research. METHODS: A total of 59,691 heterospecific SNPs in 22 rhesus macaques and 20 humans were analyzed for human trait associations and 4207 heterospecific SNPs biallelic in both taxa were compared for genetic variation. RESULTS: Variation comparisons at the 4207 SNPs showed that humans were more genetically diverse than rhesus macaques with observed and expected heterozygosities of 0.337 and 0.323 vs. 0.119 and 0.102, and minor allele frequencies of 0.239 and 0.063, respectively. In total, 431 of the 59,691 heterospecific SNPs are reportedly associated with human-specific traits. CONCLUSION: While comparisons between human and rhesus macaque genomes are plausible, functional studies of heterospecific SNPs are necessary to determine whether rhesus macaque alleles are associated with the same phenotypes as their corresponding human alleles.

Use of genome-wide heterospecific single-nucleotide polymorphisms to estimate linkage disequilibrium in rhesus and cynomolgus macaques.

Rhesus and cynomolgus macaques are frequently used in biomedical research, and the availability of their reference genomes now provides for their use in genome-wide association studies. However, little is known about linkage disequilibrium (LD) in their genomes, which can affect the design and success of such studies. Here we studied LD by using 1781 conserved single-nucleotide polymorphisms (SNPs) in 183 rhesus macaques (Macaca mulatta), including 97 purebred Chinese and 86 purebred Indian animals, and 96 cynomolgus macaques (M. fascicularis fascicularis). Correlation between loci pairs decayed to 0.02 at 1146.83, 2197.92, and 3955.83 kb for Chinese rhesus, Indian rhesus, and cynomolgus macaques, respectively. Differences between the observed heterozygosity and minor allele frequency (MAF) of pairs of these 3 taxa were highly statistically significant. These 3 nonhuman primate taxa have significantly different genetic diversities (heterozygosity and MAF) and rates of LD decay. Our study confirms a much lower rate of LD decay in Indian than in Chinese rhesus macaques relative to that previously reported. In contrast, the especially low rate of LD decay in cynomolgus macaques suggests the particular usefulness of this species in genome-wide association studies. Although conserved markers, such as those used here, are required for valid LD comparisons among taxa, LD can be assessed with less bias by using species-specific markers, because conserved SNPs may be ancestral and therefore not informative for LD.

A genetic comparison of two alleged subspecies of Philippine cynomolgus macaques.

Two subspecies of cynomolgus macaques (Macaca fascicularis) are alleged to co-exist in the Philippines, M. f. philippensis in the north and M. f. fascicularis in the south. However, genetic differences between the cynomolgus macaques in the two regions have never been studied to document the propriety of their subspecies status. We genotyped samples of cynomolgus macaques from Batangas in southwestern Luzon and Zamboanga in southwestern Mindanao for 15 short tandem repeat (STR) loci and sequenced an 835 bp fragment of the mtDNA of these animals. The STR genotypes were compared with those of cynomolgus macaques from southern Sumatra, Singapore, Mauritius and Cambodia, and the mtDNA sequences of both Philippine populations were compared with those of cynomolgus macaques from southern Sumatra, Indonesia and Sarawak, Malaysia. We conducted STRUCTURE and PCA analyses based on the STRs and constructed a median joining network based on the mtDNA sequences. The Philippine population from Batangas exhibited much less genetic diversity and greater genetic divergence from all other populations, including the Philippine population from Zamboanga. Sequences from both Batangas and Zamboanga were most closely related to two different mtDNA haplotypes from Sarawak from which they are apparently derived. Those from Zamboanga were more recently derived than those from Batangas, consistent with their later arrival in the Philippines. However, clustering analyses do not support a sufficient genetic distinction of cynomolgus macaques from Batangas from other regional populations assigned to subspecies M. f. fascicularis to warrant the subspecies distinction M. f. philippensis.

Development and validation of a SNP-based assay for inferring the genetic ancestry of rhesus macaques (Macaca mulatta).

Rhesus macaques (Macaca mulatta) are an important primate model species in several areas of biomedical research. The wide geographic distribution of this species has led to significant genetic differentiation among local and regional populations. These regional differences can be important factors in the selection of the most appropriate subjects for particular research studies, as animals from different populations can respond differently to the same experimental treatment. Consequently, it is valuable to confirm the ancestry of individual rhesus monkeys from geographically distinct populations. Using DNA samples obtained from rhesus macaques from six National Primate Research Centers, we tested a set of 384 potential ancestry informative single nucleotide polymorphisms (SNPs) and identified a final panel of 91 SNPs that can reliably distinguish Indian-origin from Chinese-origin rhesus monkeys. This genetic test can be used to determine the ancestral origin of animals and to detect individuals that are hybrids between these two regional populations. To demonstrate use of the SNP panel, we investigated the ancestry of 480 animals from the Yerkes NPRC (YNPRC) for which the colony records were insufficient to clearly establish ancestry. Three of the YNPRC animals tested were determined to be hybrids. This SNP ancestry tool will be useful to researchers, colony managers, and others who wish to evaluate the ancestral origin of individual rhesus macaques, and therefore will facilitate more effective and efficient use of these animals in biomedical research.

Expression levels of 10 candidate genes in lung tissue of vaccinated and TB-infected cynomolgus macaques.

The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomolgus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1ß, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non-vaccinated animals.

Identifying human-rhesus macaque gene orthologs using heterospecific SNP probes.

We genotyped a Chinese and an Indian-origin rhesus macaque using the Affymetrix Genome-Wide Human SNP Array 6.0 and cataloged 85,473 uniquely mapping heterospecific SNPs. These SNPs were assigned to rhesus chromosomes according to their probe sequence alignments as displayed in the human and rhesus reference sequences. The conserved gene order (synteny) revealed by heterospecific SNP maps is in concordance with that of the published human and rhesus macaque genomes. Using these SNPs' original human rs numbers, we identified 12,328 genes annotated in humans that are associated with these SNPs, 3674 of which were found in at least one of the two rhesus macaques studied. Due to their density, the heterospecific SNPs allow fine-grained comparisons, including approximate boundaries of intra- and extra-chromosomal rearrangements involving gene orthologs, which can be used to distinguish rhesus macaque chromosomes from human chromosomes.

Genetic diversity and population structure in wild Sichuan rhesus macaques.

Because wild rhesus macaque (Macaca mulatta) populations have suffered major declines, there is a growing need to characterize their genetic and population structure in order to protect the genetic integrity of this species. In this study, we genotyped a sample comprising 120 wild rhesus macaques from six sites in Sichuan Province for 30 nuclear microsatellite (STR) loci using an ABI 3130xl genetic analyzer. Bayesian analyses and PCA clearly differentiated monkeys from Heishui from those at other sites. The samples from all six sites exhibited high gene diversity suggesting that the Sichuan wild rhesus macaque populations are not threatened by a lack of genetic diversity. Deviation from Hardy-Weinberg equilibrium was more frequent in the Danba and Heishui populations. This may be due to the more fragmented habitat and less disturbance by humans in this area that foster greater subpopulation structuring than occurs in eastern China. We suggest that this population subdivision is the result of both long-term geographic barriers and human activity.

A large-scale SNP-based genomic admixture analysis of the captive rhesus macaque colony at the California National Primate Research Center.

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Center's (CNPRC) super-Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian-origin animals. The results also reveal no evidence of recent gene flow from long-tailed and pig-tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long-tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long-tailed, and pig-tailed macaques likely predate the divergence among these groups.

Genotyping single nucleotide polymorphisms (SNPs) across species in Old World Monkeys.

The development of DNA markers is becoming increasingly useful in the field of primatology for studies on paternity, population history, and biomedical research. In this study, we determine the efficacy of using cross-species amplification to identify single nucleotide polymorphisms (SNPs) in closely related species. The DNA of 93 individuals representing seven Old World Monkey species was analyzed to identify SNPs using cross-species amplification and genotyping. The loci genotyped were 653 SNPs identified and validated in rhesus macaques. Of the 653 loci analyzed, 27% were estimated to be polymorphic in the samples studied. SNPs identified at the same locus among different species (coincident SNPs) were found in six of the seven species studied with longtail macaques exhibiting the highest number of coincident SNPs (84). The distribution of coincident SNPs among species is not biased based on proximity to genes in the samples studied. In addition, the frequency of coincident SNPs is not consistent with expectations based on their phylogenetic relationships. This study demonstrates that cross-species amplification and genotyping using the Illumina Golden Gate Array is a useful method to identify a large number of SNPs in closely related species, although issues with ascertainment bias may limit the type of studies where this method can be applied.

Phylogenetic analysis of chinese rhesus macaques (Macaca mulatta) based on mitochondrial control region sequences.

Between one and six subspecies of Chinese rhesus macaques (Macaca mulatta) have been proposed based on morphological differences and/or their geographic distribution. In this study, a 489 base pair fragment of the mitochondrial control region was amplified from 230 DNA samples collected from rhesus macaques in the Sichuan province in Western China. The fragment was then sequenced and aligned with 208 sequences from wild rhesus macaques, sampled throughout the species' geographic range in China downloaded from GenBank. Phylogenetic analysis of the 182 unique sequences identified among these samples divided Chinese rhesus macaques into two western haplogroups (haplogroups A and B) and three older eastern haplogroups (haplogroups C, D, and E), whose differentiation probably occurred during the penultimate glacial event. During the warming after the penultimate glacial event, haplogroups A, B, and E rapidly expanded and a relatively young subhaplogroup of haplogroup E, E', limited to Southern China but shared with Vietnamese rhesus macaques, was reintroduced from Indochina during the last glacial event. One haplotype most closely related to subhaplogroup E' probably represents the isolation of Hainan Island, to where it is restricted, from the mainland by the formation of the Qiongzhou Strait approximately 8,500 years ago. The distribution of haplogroups both informs the phylogeographic history of dispersal of Chinese rhesus macaques and has implications for their suitability as animal models in biomedical research.

Association between hybrid status and reproductive success of captive male and female rhesus macaques (Macaca mulatta) at the California National Primate Research Center (CNPRC).

The California National Primate Research Center (CNPRC) houses more than 1,000 rhesus macaques (Macaca mulatta) of mixed Chinese-Indian ancestry. Most of these animals are kept in outdoor field cages, the colony's long term breeding resource. Since 2001, hybrids comprised between 4 and 49% of the field cage populations, but in most cases have represented a maximum of 10% of those populations. The increasing prevalence of hybrids is partly due to management efforts to distribute genetic diversity effectively and minimize genetic subdivisions. However, other factors may also contribute to the spread of hybrids within the colony, most notably variance in socio-sexual behaviors and physical attributes. It is known that hybrids of some species exhibit heterosis, such as early maturation, that can enhance reproductive success, and anecdotal observations of mixed groups of hybrid, Indian and Chinese animals at the CNPRC suggest that hybrids are more sexually active. To determine whether hybrids experienced a reproductive advantage, a study was conducted using birth records of 5,611 offspring born in the CNPRC colony between 2003 and 2009. We found that while the degree of Chinese ancestry (DCA) appeared to influence the maturational schedule of both males and females (maturation was inversely related to proportion of Chinese ancestry), DCA had no independent effect on either male or female RS or rank. Therefore, we have found no evidence that a hybrid phenotype confers an absolute reproductive advantage in our colony.

The complete genome and genetic characteristics of SRV-4 isolated from cynomolgus monkeys (Macaca fascicularis).

At least 5 serotypes of exogenous simian retrovirus type D (SRV/D) have been found in nonhuman primates, but only SRV-1, 2 and 3 have been completely sequenced. SRV-4 was recovered once from cynomolgus macaques in California in 1984, but its genome sequences are unknown. Here we report the second identification of SRV-4 and its complete genome from infected cynomolgus macaques with Indochinese and Indonesian/Indochinese mixed ancestry. Phylogenetic analysis demonstrated that SRV-4 was distantly related to SRV-1, 2, 3, 5, 6 and 7. SRV/D-T, a new SRV/D recovered in 2005 from cynomolgus monkeys at Tsukuba Primate Center in Japan, clustered with the SRV-4 isolates from California and Texas and was shown to be another occurrence of SRV-4 infection. The repeated occurrence of SRV-4 in cynomolgus monkeys in different areas of the world and across 25years suggests that this species is the natural host of SRV-4.