Genetic characterization of Indian-origin and Chinese-origin rhesus macaques (Macaca mulatta)

Genetic differences between Indian-origin and Chinese-origin rhesus macaques are as great as those between some primate species and can influence the results of experiments in which both are used as animal models for the study of the same human diseases. Unfortunately, many breeding facilities do not know with certainty the origin of the founders of their rhesus breeding colonies. Here I summarize the most definitive of the genetic traits among the microsatellite (STR) loci and mitochondrial DNA sequences that my laboratory previously reported to characterize Indian-origin and Chinese-origin rhesus macaques and then estimate the frequencies of these traits and their reliability as indicators of country of origin. The expression of diagnostic traits at two or more of four different unlinked loci provides a nearly 100% reliability in distinguishing rhesus macaques of Indian and Chinese origin.     

Population genetics of short tandem repeat (STR) loci

To investigate the population genetics of short tandem repeat (STR) polymorphisms in human populations, we have studied the allele frequency distributions of four STR loci (HUMTH01, HUMVWA31, HUMF13A1 and HUMFES) in 16 different population surveys which can be categorised within three broadly defined ethnic groups: Caucasian, Asian (Indian subcontinent), and African (Afro-Caribbean and US black). We have observed that allele frequency distributions of populations within ethnic groups are similar; consequently, genetic distances are an order of magnitude lower than between ethnic groups. Inbreeding coefficients (F-statistics) and calculations of the number of mean heterozygous loci per individual, along with estimates of variance, did not suggest that the populations were substructured. This included a study of an immigrant Asian population known to comprise at least three different sub-groups. Finally, an indication of the discriminating power is given by calculation of likelihood ratios (LR) of each individual tested across all four loci. Approximately 70% of Caucasians give an LR of greater than 10,000; the test is even more discriminating in Afro-Caribbeans-approximately 90% of tests are greater than 10,000.Editor's commentsThe authors present data generated by the move from VNTR to STR loci for human identification. The data they present for samples within major racial groupings address some of the concerns about population substructuring discussed by Balding and Nichols in this volume.     

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.     

Assessment of genetic management at three specific-pathogen-free rhesus macaque (Macaca mulatta) colonies

Genetic management is required to maintain genetic diversity by minimizing inbreeding and genetic subdivision in colonies of animals bred for biomedical research. Polymorphic short tandem repeat (STR) loci are useful for genetic management because they facilitate parentage assignments, genetic characterization of individuals, and estimates of baseline population genetic parameters. Using highly informative STR loci, we estimated gene diversity and F-statistics to determine the level of genetic heterogeneity and genetic structure of three specific-pathogen-free (SPF) rhesus macaque (Macaca mulatta) colonies. Effective population sizes, variance in male reproductive success, and rate of decrease in genetic variability also were estimated for two of the three colonies. We documented the overall success of genetic management in maintaining genetic diversity in captive colonies. We report that even genetically managed SPF colonies, despite maintaining high and stable levels of gene diversity (over 0.75), are prone to genetic subdivision due to different management strategies, founder effects, genetic isolation, and drift. These processes are accelerated by the high variances in male reproductive success and low adult sex ratios that are typical of captive rhesus macaque breeding groups, both of which reduce the effective population sizes of these groups.     

Simple sequence repeat (SSR) polymorphisms for colony management and population genetics in rhesus macaques (Macaca mulatta)

Cross-species amplification of 72 SSR (predominantly tetranucleotide) loci from the DNA of six rhesus macaques of diverse regional origins was conducted using human primers for the polymerase chain reaction (PCR). Thirteen of these primer pairs, which consistently and unambiguously amplified polymorphic fragments from these six samples and which exhibited Mendelian properties, were also used to amplify SSR loci for 176 male rhesus macaques that are founders of six different captive breeding colonies. These include four groups of macaques originating in India and one group of macaques each that originated in China and Thailand. Gene diversity based on the SSR loci provided a reliable estimate of average heterozygosity but was between two and four times higher than that for 9 protein coding loci. Based on the SSR loci, Chinese rhesus were more genetically diverse and unique than were rhesus from India or Thailand, a conclusion not consistent with data based on protein coding loci. The six most informative SSR loci are unlinked and provide probabilities of single parent exclusion and genetic identity exceeding 0.99 and one in one million, respectively, both of which are reasonable standards for colony management purposes. Am. J. Primatol. 42:199–213, 1997. © 1997 Wiley-Liss, Inc.     

Comparative study of genetic variation at 15 STR loci in three isolated populations of the Bosnian mountain area

Fifteen autosomal STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX, and FGA) were studied in three geographically close but isolated populations from the Bosnian mountain area. The three villages are Bobovica, Dejcici, and Lukomir. DNA was obtained from 83 individuals, and the allele frequencies and genetic diversity among the three sample groups were compared. In addition, seven of the STR loci (CSF1PO, D13S317, D3S1358, D5S818, D7S820, FGA, TH01) were used in a comparative population analysis of the Bjelasnica-Treskavica region and the Adriatic islands of Brac, Hvar, and Korcula. Although the sample sizes are relatively small, the observed variation within any of the small isolated populations is high and comparable to less isolated groups. In addition, even though the populations are geographically isolated, the STR data are similar among the populations. The most significant frequency differences were observed at the TH01 locus. Although the specific allele distributions in any untyped population cannot be determined a priori, we find support for a high degree of diversity for the STR loci in most populations. In addition, the multiple locus profile is highly informative not only for various population studies but also for forensic studies, even when specific population data are not available.     

中国五个民族STR位点遗传多态性（2）

通过对我国汉回蒙藏维５个民族的５０个家系和５００份样本的ＳＴＲ基因扫描、基因分型和遗传结构分析,获得了ＳＴＲ基因传递方式及遗传特征的大量科学数据。研究结果表明在９个ＳＴＲ位点上汉族有６０种ＳＴＲ等位基因,１４９种基因型;回族有６３种ＳＴＲ等位基因,１４４种基因型;蒙古族有６９种ＳＴＲ等位基因,１７３种基因型;藏族有７７种等位基因,１６８种基因型;维吾尔族有７０种ＳＴＲ等位基因,１４８种基因型。中国     

Genetic characterization of rhesus macaques (Macaca mulatta) in Nepal

Indian-origin rhesus macaques (Macaca mulatta) have long served as an animal model for the study of human disease and behavior. Given the current shortage of Indian-origin rhesus, many researchers have turned to rhesus macaques from China as a substitute. However, a number of studies have identified marked genetic differences between the Chinese and Indian animals. We investigated the genetic characteristics of a third rhesus population, the rhesus macaques of Nepal. Twenty-one rhesus macaques at the Swoyambhu Temple in Kathmandu, Nepal, were compared with more than 300 Indian- and Chinese-origin rhesus macaques. The sequence analyses of two mitochondrial DNA (mtDNA) loci, from the HVS I and 12 S rRNA regions, showed that the Nepali animals were more similar to Indian-origin than to Chinese-origin animals. The distribution of alleles at 24 short tandem repeat (STR) loci distributed across 17 chromosomes also showed greater similarity between the Nepali and Indian-origin animals. Finally, an analysis of seven major histocompatibility complex (MHC) alleles showed that the Nepali animals expressed Class I alleles that are common to Indian-origin animals, including Mamu-A*01. All of these analyses also revealed a low level of genetic diversity within this Nepali rhesus sample. We conclude that the rhesus macaques of Nepal more closely resemble rhesus macaques of Indian origin than those of Chinese origin. As such, the Nepali rhesus may offer an additional resource option for researchers who wish to maintain research protocols with animals that possess key genetic features characteristic of Indian-origin rhesus macaques.     

Variability of fifteen autosomal microsatellite DNA loci in five populations of aboriginal South Siberians

The allele distributions for 15 STR loci included in the AmpFISTR SGM Plus and AmpFISTR Profiler Plus kits ("Applied Biosystems", USA) were determined in 261 healthy unrelated individuals belonging to five indigenous populations of South Siberia: in Buryats, Altaians, Tofalars, Sojots and Khakassians. No significant differences in allele frequencies were found between populations studied. Combined power of discrimination (PD) for the STR loci investigated were estimated for the populations under study.     

Assessing natural introgression in 2 biomedical model species, the rhesus macaque (Macaca mulatta) and the long-tailed macaque (Macaca fascicularis)

Rhesus macaque (Macaca mulatta) and long-tailed macaque (Macaca fascicularis) are the 2 most commonly used primate model species in biomedical sciences. Although morphological studies have revealed a weak hybridization at the interspecific contact zone, in the north of Indochina, a molecular study has suggested an ancient introgression from rhesus to long-tailed macaque into the Indo-Chinese peninsula. However, the gene flow between these 2 taxa has never been quantified using genetic data and theoretical models. In this study, we have examined genetic variation within and between the parapatric Chinese rhesus macaque and Indo-Chinese long-tailed macaque populations, using 13 autosomal, 5 sex-linked microsatellite loci and mitochondrial DNA sequence data. From these data, we assessed genetic structure and estimated gene flow using a Bayesian clustering approach and the "Isolation with Migration" model. Our results reveal a weak interspecific genetic differentiation at both autosomal and sex-linked loci, suggesting large population sizes and/or gene flow between populations. According to the Bayesian clustering, Chinese rhesus macaque is a highly homogeneous gene pool that contributes strongly to the current Indo-Chinese long-tailed macaque genetic makeup, whether or not current admixture is assumed. Coalescent simulations, which integrated the characteristics of the loci, pointed out 1) a higher effective population size in rhesus macaque, 2) no mitochondrial gene flow, and 3) unilateral and male-mediated nuclear gene flow of approximately 10 migrants per generation from rhesus to long-tailed macaque. These patterns of genetic structure and gene flow suggest extensive ancient introgression from Chinese rhesus macaque into the Indo-Chinese long-tailed macaque population.     

Paternity assessment in rhesus macaques (Macaca mulatta): Multilocus DNA fingerprinting and PCR marker typing

Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)₄, (CA)₈, and (CAC)₅. Paternity assessment by multilocus DNA analysis with probe (CAC)₅ alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair. Am. J. Primatol. 44:1–18, 1998. © 1998 Wiley-Liss, Inc.     

Allelic polymorphism of six microsatellite DNA Loci in populations of Sakha (Yakutia)

The allele frequency distributions of six STR loci (D3S1358, D16S539, THOI, D8S1179, LPL, and HUMvWFII) used in forensic practice were analyzed in populations of Sakha (Yakutia): three ethnogeographical groups of Sakha (Yakuts), Evenks, Yukagirs, Dolgans, and Russians. There were significant differences between Russians and all other populations by five markers. The total discriminating power (PD) of the locus system studied was assessed for each population. The interpopulation genetic difference (F _ST) was 0.005. Data on the allelic polymorphism of the above STR loci were used to analyze phylogenetic relationships among the populations of Sakha and those of other regions: East Europe, South Siberia, Chukotka, and Kamchatka.     

An initial genetic linkage map of the rhesus macaque (Macaca mulatta) genome using human microsatellite loci

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate species in biomedical research. To create new opportunities for genetic and genomic studies using rhesus monkeys, we constructed a genetic linkage map of the rhesus genome. This map consists of 241 microsatellite loci, all previously mapped in the human genome. These polymorphisms were genotyped in five pedigrees of rhesus monkeys totaling 865 animals. The resulting linkage map covers 2048 cM including all 20 rhesus autosomes, with average spacing between markers of 9.3 cM. Average heterozygosity among those markers is 0.73. This linkage map provides new comparative information concerning locus order and interlocus distances in humans and rhesus monkeys. The map will facilitate whole-genome linkage screens to locate quantitative trait loci (QTLs) that influence individual variation in phenotypic traits related to basic primate anatomy, physiology, and behavior, as well as QTLs relevant to risk factors for human disease.     

Detecting signatures of inter‐regional and inter‐specific hybridization among the Chinese rhesus macaque specific pathogen‐free (SPF) population using single nucleotide polymorphic (SNP) markers

Background While rates of gene flow between rhesus and longtail macaque populations near their hybrid zone in Indochina have been quantified elsewhere, this study demonstrates that the inter‐specific introgression is not limited to the Indochinese hybrid zone but is more geographically widespread. Methods Twelve rhesus and longtail macaque populations were analyzed using single nucleotide polymorphic (SNP) loci. Results There is evidence for inter‐specific admixture between Chinese rhesus and mainland longtails, with implications for genetic diversity both in the Chinese super‐SPF population at the California National Primate Research Center and in other primate facilities. Eastern Chinese rhesus appeared more highly derived than western Chinese rhesus, and allele sharing between longtails and Chinese rhesus was not random with regard to geographic distance, but no significant nuclear genetic differences between eastern and western Chinese rhesus were detected among the 245 genic SNPs assayed. Conclusion The implications of this inter‐specific admixture for the use of Chinese rhesus and mainland longtail in biomedical research should be considered.     

Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil

Allozyme variation in New World screwworm,Cochliomyia hominivorax (Coquerel), populations from Brazil was examined. Variability was observed in 8 of 13 enzyme loci and the frequency of the most common allele was <0.95 for seven loci. Observed and expected heterozygosities were 0.159 and 0.165, respectively. Comparisons of the Brazilian populations with previously studied populations from Costa Rica resulted in Nei's genetic distances of between 0.000 and 0.006, with the greatest distance being between populations within Brazil. Comparisons with Mexican populations using only three loci resulted in genetic distances ≤0.031. Goodness-of-fit statistics for Hardy-Weinberg equilibrium and Wright'sF statistics indicated small deviations from expected equilibrium genotype frequencies and low levels of differentiation between populations within Brazil. Differentiation among screwworm populations from Brazil, Costa Rica, and Mexico was minimal.     

Improved haplotype analysis of human myelin basic protein short tandem repeat loci

We report an improved haplotype analysis of the human myelin basic protein gene (MBP) short tandem repeat (STR) polymorphism. The polymorphic G-->A transition and 2 conventional STR polymorphisms, MBPA and MBPB, were simultaneously determined by an amplified product length polymorphism technique. After the MBPC fragments containing MBPA and MBPB were amplified, the linkage of these 2 STR loci was determined by a second amplification, using polymerase chain reaction (PCR) technique, of the isolated MBPC fragments. The present haplotype analysis dispensed with family studies for the haplotyping of MBPA and MBPB. Polymorphisms of the MBP loci studied in German and Japanese populations showed a high genomic variation. Haplotype analysis of the MBP loci showed distinct differences between the German and the Japanese populations. Consequently, haplotype analysis of the MBP loci promises to be useful in forensic identification and paternity testing.     

GENETIC DIVERSITY AND POPULATION STRUCTURE IN CAMELLIA JAPONICA L. (THEACEAE)

Camellia japonica is a widespread and morphologically diverse tree native to parts of Japan and adjacent islands. Starch gel electrophoresis was used to score allelic variation at 20 loci in seeds collected from 60 populations distributed throughout the species range. In comparison with other plant species, the level of genetic diversity within C. japonica populations is very high: 66.2% of loci were polymorphic on average per population, with a mean number of 2.16 alleles per locus; the mean observed and panmictic heterozygosities were 0.230 and 0.265, respectively. Genotypic proportions at most loci in most populations fit Hardy-Weinberg expectations. However, small heterozygote deficiencies were commonly observed (mean population fixation index = 0.129). It is suggested that the most likely cause of the observed deficiencies is population subdivision into genetically divergent subpopulations. The overall level of population differentiation is greater than is typically observed in out-breeders: The mean genetic distance and identity (Nei's D and I) between pairs of populations were 0.073 and 0.930, respectively, and Wright's Fst was 0.144. Differences among populations appeared to be manifested as variation in gene frequencies at many loci rather than variation in allelic composition per se. However, the patterns of variation were not random. Reciprocal clinal variation of gene frequencies was observed for allele pairs at six loci. In addition, principal components analysis revealed that populations tended to genetically cluster into four regions representing the geographic areas Kyushu, Shikoku, western Honshu, and eastern Honshu. There was a significant relationship between genetic and geographic distance (r = 0.61; P < 0.01). Analysis of variance on allozyme frequencies showed that there was approximately four times as much differentiation among populations within regions, as among regions. It is likely that the observed patterns of population relationships result from the balance between genetic drift in small subpopulations and gene flow between them.     

Genetic structure and expansion of golden jackals (Canis aureus) in the north-western distribution range (Croatia and eastern Italian Alps)

The golden jackal, widely distributed in Europe, Asia and Africa, is one of the less studied carnivores in the world and the genetic structure of the European populations is unknown. In the last century jackals strongly declined mainly due to human persecution, but recently they expanded again in eastern Europe. With the aim to determine the genetic structure and the origin of expanding jackals, we analyzed population samples obtained from Bulgaria, Serbia, Croatia (Dalmatia and Slavonia) and individuals sampled in north-eastern Italy. Samples were typed at the hypervariable part of the mitochondrial DNA control-region (mtDNA CR1) and at 15 canine autosomal microsatellite loci (STR), and analyzed using multivariate, Bayesian and landscape genetic methods. The mtDNA CR1 was monomorphic, showing a single haplotype shared among all the populations. The STR loci were variable, with 2–14 alleles and intermediate values of heterozygosity (Ho = 0.47; He = 0.51). Genetic diversity was significantly partitioned (θ_ST = 0.07; P < 0.001) and the populations were partially distinct, perhaps in consequence of recent fragmentations. Jackals from Dalmatia were the most genetically differentiated. Assignment testing and gene flow analyses suggested that jackals colonizing Italy have admixed origins from Dalmatian and Slavonian populations. They are not first generation migrants, suggesting that dispersal towards north-eastern Italy is a stepping-stone process. Golden jackal and wolf colonization patterns might be different, with prevalent short-distance dispersal in jackals versus prevalent long distance dispersal in wolves. The admixed origin of jackals in the Alps ensures abundant genetic variability, which may enhance adaptive fitness and expectancy of population growth. The intersections between Dinaric–Balkan and Eastern Alps are areas of population expansion and admixture, highlighting their conservation, ecological and evolutionary values.     

Identification of Country of Origin and Admixture Between Indian and Chinese Rhesus Macaques

We describe a restriction analysis that distinguishes between rhesus macaques of unmixed Indian and Chinese ancestry and between western and eastern Chinese ancestry. We amplified a 254-bp fragment of mitochondrial DNA (mtDNA) that contains restriction sites hypothesized to be diagnostic of country of origin for samples from 534 and 567 individuals alleged to be of solely Indian or solely Chinese origin, respectively. After digestion with the MaeIII, SmlI, and BccI restriction enzymes, the polymerase chain reaction (PCR) products of only 3 of the 1101 samples exhibited restriction patterns uncharacteristic of their alleged country of origin. A sample comprising 392 of these rhesus macaques was genotyped for 24 nuclear microsatellite (STR) loci. Principal coordinates analysis confirmed marked genetic similarity of regional populations within each country but a substantial difference between Indian and Chinese rhesus macaques. Using STRUCTURE (Pritchard and Wen, 2003),we assigned probabilities of Chinese and Indian ancestry to each sample based on its STR genotypes. We assigned all the unmixed rhesus macaques to their correct countries of origin with probabilities >0.95. We constructed an artificial sample of 1st-generation hybrid Indian/Chinese rhesus macaques by randomly sampling from the genotypes of Indian and Chinese individuals. STRUCTURE assigned robabilities of Chinese and Indian ancestry to hybrids that closely corresponded with the proportions of alleles in that sample drawn from unmixed Chinese and Indian rhesus macaques.