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Apanel of 20 highly variable microsatellite polymorphisms in rhesus macaques (Macaca mulatta) selected for pedigree or population genetic analysis.

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American Journal of Primatology 67:377–383 (2005)
BRIEF REPORT
A Panel of 20 Highly Variable Microsatellite
Polymorphisms in Rhesus Macaques (Macaca mulatta)
Selected for Pedigree or Population Genetic Analysis
JEFFREY ROGERS1,2n, MACKENZIE BERGSTROM1, ROY GARCIA IV1,
JORDAN KAPLAN1, ANN ARYA1, LEILAH NOVAKOWSKI1, ZACH JOHNSON1,
AMANDA VINSON1, and WENDY SHELLEDY1
1
Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio,
Texas
2
Southwest National Primate Research Center, San Antonio, Texas
This paper reports 20 new microsatellite loci that are highly polymorphic
in rhesus macaques (Macaca mulatta). We screened known human
microsatellite loci to identify markers that are polymorphic in rhesus
macaques, and then selected specific loci that show substantial levels of
heterozygosity and robust, reliable amplification. The 20 loci reported
here were chosen to include one highly informative microsatellite from
each rhesus monkey autosomal chromosome. Fourteen of the 20
polymorphisms are tetranucleotide repeats, and all can be analyzed
using standard PCR and electrophoresis procedures. These new rhesus
markers have an average of 15.5 alleles per locus and average
heterozygosity of 0.83. This panel of DNA polymorphisms will be useful
for a variety of different genetic analyses, including pedigree testing,
paternity analysis, and population genetic studies. Many of these loci are
also likely to be informative in other closely related Old World monkey
species. Am. J. Primatol. 67:377–383, 2005.
r 2005 Wiley-Liss, Inc.
Key words: rhesus macaque; microsatellite; polymorphism; STR; tetranucleotide
INTRODUCTION
Rhesus macaques are among the most widely investigated nonhuman
primates. A recent report by the National Institutes of Health indicated that
rhesus monkeys are the most commonly used nonhuman primate in biomedical
research (www.ncrr.nih.gov/compmed/rhesusworkshopreport.pdf). This species is
widely used in studies of reproductive biology, behavior, developmental
Contract grant sponsor: NIH; Contract grant numbers: RR08781; RR15383.
n
Correspondence to: Jeffrey Rogers, Department of Genetics, Southwest Foundation for Biomedical
Research, 7620 N.W. Loop 410, San Antonio, TX 78227. E-mail: jrogers@darwin.sfbr.org
Received 26 October 2004; revised 21 February 2005; revision accepted 13 March 2005
DOI 10.1002/ajp.20192
Published online in Wiley InterScience (www.interscience.wiley.com).
r
2005 Wiley-Liss, Inc.
378 / Rogers et al.
psychology, aging, immunology, infectious disease, and other aspects of
biomedicine [e.g., Barr et al., 2004; Golos, 2004; Lane et al., 2002; Moore et al.,
2003; Sauermann, 2001; Wolf, 2004]. In addition, the behavior, ecology, and
population genetics of wild or semi-free-ranging populations have received
intensive study for many years [e.g., Melnick, 1988; Richard et al., 1989; Sade,
1972; Southwick et al., 1965].
Over the past 20 years, genetics has become a central element in primate
research. In many circumstances, the genetic characteristics of a species are the
primary focus of investigation, such as when DNA sequences or patterns of gene
expression are compared within or among species. Increasingly, data concerning
genetic differences among individuals within a species are used to investigate
other aspects of primate biology [DiFiore, 2003; de Ruiter, 2004]. For example,
recent studies have used molecular data to identify the fathers of specific infants
born in wild primate groups [Buchan et al., 2004], determine paternity in captive
primate colonies [Newman et al., 2002], and even infer kinship relationships
among wild, unhabituated primates [Bradley et al., 2004].
Microsatellite loci (sometimes called simple sequence repeats (SSRs) or short
tandem repeats (STRs)) are short segments of DNA that accumulate high levels of
variation within populations or species [Ellergren, 2004; Litt & Luty, 1989; Weber
& May, 1989]. These loci consist of tandemly repeated nucleotide sequences or
motifs that are two to six basepairs in length. Their value for population genetics
and other analyses derives from a high frequency of mutations that increase or
decrease the number of repeat units in a given locus. Over time, these changes in
the number of repeats present in a locus generate a range of allele lengths within
a given population of animals. Some microsatellite loci exhibit more than 20
different alleles in a primate population (see below). Alleles are distinguished by
comparing the total length of the repeated segments. Most (but not all)
microsatellite polymorphisms appear to have no functional consequences, and
thus they are assumed to evolve as neutral genetic markers. Researchers have
taken advantage of these informative loci to examine genetic variation within
natural populations, infer kinship relationships among animals, and reconstruct
the history of differentiation among populations. Microsatellite markers are also
useful for the genetic management and analysis of captive colonies of primates,
since they are well suited for paternity testing and monitoring of population
variability, as well as other genetic analyses such as linkage mapping and
investigation of chromosomal rearrangements [e.g., Moore et al., 1998].
Given the value of microsatellites as informative genetic markers, and the
intense research interest in rhesus macaques, it is no surprise that various
researchers have worked to identify this type of polymorphism in this species
[Hadfield et al., 2001; Kayser et al., 1996; Morin et al., 1997; Smith et al., 2000].
However, no one genetic locus is guaranteed to be highly informative in all rhesus
monkey populations. In addition, there are advantages to using panels of
microsatellites that show no pairwise genetic linkage between loci, so that
independent inheritance of alleles across loci can be assumed. Moreover,
experience in a number of laboratories has shown that allelic variation in
tetranucleotide repeat markers (those microsatellites in which the repeat motif is
four basepairs in length) is more reliably scored than in dinucleotide repeat loci,
primarily because it is easier to distinguish a set of alleles that all differ by four
basepairs in length than it is to distinguish alleles that differ by only two
basepairs. For these reasons, we report here a new panel of 20 highly polymorphic
microsatellite polymorphisms for rhesus macaques, one for each of the 20 rhesus
autosomal chromosomes. Fourteen of the loci are tetranucleotide repeats, and all
Am. J. Primatol. DOI 10.1002/ajp
New Microsatellites in Rhesus Monkeys / 379
produce readily scored microsatellite genotypes when standard laboratory
procedures are used. We anticipate that these loci will be useful in a variety of
research applications.
METHODS AND MATERIALS
DNA Sampling
Whole blood or tissue was obtained from pedigreed rhesus macaques housed
at the Oregon National Primate Research Center in Beaverton, Oregon, and from
the Southwest National Primate Research Center in San Antonio, Texas.
Genomic DNA was isolated from white blood cells or tissue using standard
phenol/chloroform extraction methods. This search for new rhesus DNA
polymorphisms was conducted as part of a larger program intended to construct
a complete genetic linkage map of the rhesus genome. Consequently, we were able
to confirm the Mendelian pattern of inheritance for potential polymorphisms by
genotyping the loci in five three-generation families of rhesus macaques,
consisting of a total of 865 animals from the Oregon and Southwest National
Primate Research Centers.
Initial Identification and Genotyping of Polymorphic Microsatellites
To identify informative microsatellites in rhesus macaques, we first tested
300 human microsatellite loci that were known to be polymorphic in baboons
(Papio hamadryas) [Rogers et al., 2000]. Initial screening of these potential
polymorphisms employed DNA samples from eight breeding males that had sired
substantial numbers of offspring in the rhesus pedigrees. We tested each
fluorescently-labeled primer pair using standard PCR conditions (50 ng genomic
DNA, 1 buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2+),
additional MgCl2+ to a final concentration of 2.0 mM, 0.80 mM each primer,
200 mM each dNTPs, and 0.25 U Taq polymerase (TaKaRa, Otsu, Japan)) in a
final volume of 6 ml. The thermal cycling method followed a ‘‘touchdown’’
procedure that consisted of an initial denaturing step at 951C for 5 min, followed
by 10 cycles of denaturing at 941C for 40 sec, annealing starting 101C above the
final annealing temperature for 30 sec and decreasing 11C per cycle, and
extension at 721C for 30 sec. Following the initial 10 cycles, the method included
25 cycles of standard amplification at the final annealing temperature, followed by
extension at 721C for 7 min, and holding at 41C. The final annealing temperature
for these ‘‘touchdown’’ PCR protocols ranged from 481 to 521C. The resulting
PCR products were analyzed on ABI377 automated DNA sequencers using ABI
GeneScan Analysis (v.3.1.2) and Genotyper (v.2.5) software (Applied Biosystems
Inc., Foster City, CA). The microsatellite markers that reliably produced
genotypes that could be scored with standard methods (see below), produced
three or more alleles among the eight male rhesus tested, and did not produce
spurious amplification products that interfered with effective genotyping were
selected for genotyping in the full pedigrees.
Polymorphic loci identified in the initial screening were then genotyped in
the large rhesus families with the use of human PCR primers fluorescently
labeled with TET, FAM, or HEX (primer information available at http://
www.ncbi.nih.gov). PCR amplification reactions were optimized using a range
of magnesium concentrations (1.5–3.0 mM) and annealing temperatures
(46–541C). We employed the following amplification protocol in a 6-ml total
volume in 96-well PCR plates (all reagents from TaKaRa): 50 ng genomic DNA,
Am. J. Primatol. DOI 10.1002/ajp
380 / Rogers et al.
1 buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2+), 0.80 mM each
primer, 200 mM each dNTPs, 0.25 U Taq polymerase, and additional magnesium
as required for optimized protocols requiring 41.5 mM MgCl2+. The thermal
cycling method again used ‘‘touchdown’’ procedures. All PCR procedures
were performed on ABI 9600 and 9700 thermal cyclers, and the PCR products
were run in panels of four to six markers with TAMRA-labeled MapMarker
Low (BioVenture, Inc., Murfreesboro, TN) size standard on ABI 377 sequencers.
Fragment size analysis was performed with GeneScan Analysis (v.3.1.2) and
Genotyper (v.2.5) software.
Screening and Genotyping of Additional Loci
After we tested the first 300 primer pairs, we selected 1,150 microsatellite
primer pairs from published human linkage maps [Kong et al., 2002]. To increase
efficiency, we analyzed these human loci (not previously known to be polymorphic
in rhesus or any other Old World monkey) using slightly different procedures.
Screening in this second phase again used DNA from eight rhesus monkey males,
and was done in 96-well plates. For both screening and genotyping of families
during this second stage, we amplified the genomic DNA and visualized the PCR
products using proprietary ABI 5-dye technology and reagents. The thermal
cycling method consisted of an initial denaturing step at 951C for 12 min followed
by 10 cycles of denaturing at 941C for 15 sec, annealing at 48–541C for 15 sec, and
extension at 721C for 30 sec. The remaining 20 thermal cycles used denaturing
temperature of 891C, annealing at the same temperature as the first 10 cycles,
and extension at 721C, all for the same time periods. The amplification reactions
concluded with a final extension at 721C for 10 min and a final hold at 41C.
Genotypes for the full pedigrees in this second stage were again determined using
ABI377 automated sequencers and ABI GeneScan Analysis (v.3.1.2) and
Genotyper (v.2.5) software, but the size standard used in this phase was ABI
GeneScan 500 LIZ.
RESULTS
We identified a total of 382 previously unknown microsatellite polymorphisms in rhesus macaques. Of the 300 human loci originally studied in baboons,
only 154 (51.3%) showed at least three alleles in our screen of eight rhesus
monkeys. The 1,150 human loci tested later produced another 228 markers
(19.8%) with three or more alleles, although 96 additional human loci exhibited
two alleles in the screening test, and may also be useful in rhesus. The 382
markers are being used to construct a genetic linkage map of the rhesus
genome, and further information about many of those loci can be found at
http://www.snprc.org. Table I lists a subset of these polymorphisms, including
one highly informative locus for each rhesus autosomal chromosome. Fourteen
of these markers exhibit allelic differences consistent with tetranucleotide repeats, while the other six loci are dinucleotide repeats. The sequences
of the repeat units are not known, as the PCR products have not been
sequenced.
The 20 loci in this panel are highly polymorphic. The average number of
alleles per locus is 15.5, with a range of 7–31. The average heterozygosity is 0.831,
with a range of 0.75–0.91. The specific allele sizes and frequencies, and other
details for each locus (including the PCR primer sequences) are available at
http://www.snprc.org.
Am. J. Primatol. DOI 10.1002/ajp
a
1
3
7/21
6
4
5
14/15
8
10
20/22
12
2q
2p
11
9
17
13
18
19
16
Human
chromosome
D1S1594
D3S3045
D21S1246
D6S2419
D4S1645
D5S1466
D15S823
D8S1466
D10S179
D20S171
D12S372
D2S1333
D2S146
D11S1352
D9S934
D17S791
D13S797
D18S869
D19S559
D16S403
Marker
16
18
19
25
13
13
20
11
18
15
15
13
14
11
7
16
9
31
7
18
Number
of
allelesa
0.81
0.84
0.90
0.89
0.82
0.75
0.89
0.79
0.84
0.80
0.89
0.86
0.82
0.85
0.76
0.88
0.75
0.91
0.76
0.81
Heterozygosity
in
rhesus
Tetra
Tetra
Tetra
Tetra
Tetra
Tetra
Tetra
Tetra
Di
Di
Tetra
Tetra
Di
Di
Tetra
Di
Tetra
Tetra
Tetra
Di
Repeat
type
119–200
176–248
380–450
145–250
240–314
279–327
320–385
121–170
115–160
108–134
150–200
266–334
193–219
228–248
189–213
159–193
180–214
175–347
110–130
150–175
Range
(bp)
5
4
5
5
4
4
4
5
4
4
4
4
4
4
4
4
5
5
5
4
Dye
type
Allele numbers are based on genotypes from 865 pedigreed rhesus monkeys from Oregon and Southwest primate centers
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Rhesus
chromosome
TABLE I. Polymorphic Microsatellite Loci in Rhesus Macaques
5Dye50
TD50
5Dye48
5Dye48
TD50
TD50
TD50
5Dye48
TD48
TD52
TD46
TD48
TD50
TD50
TD50
TD48
5Dye48
5Dye48
5Dye50
TD48
Annealing
temperature
2
2
2
2
2
2
2
2
2
2
2
2
1.5
2
2
2
2
3
2
2
MgCl2
0.67
0.82
0.78
0.85
0.80
0.86
0.73
0.68
0.71
0.89
0.84
0.88
0.85
0.79
Heterozygosity
in
baboons
New Microsatellites in Rhesus Monkeys / 381
Am. J. Primatol. DOI 10.1002/ajp
382 / Rogers et al.
DISCUSSION
Highly variable nuclear DNA polymorphisms in nonhuman primates,
especially polymorphic loci that are unlinked and therefore inherited independently of each other, are useful for a variety of purposes. The rhesus loci reported
here are known to be unlinked, because all have been used for genome-wide
linkage analyses in this species (see http://www.snprc.org). The microsatellites
reported here could be used for paternity testing or other aspects of pedigree
analysis. These loci will also be valuable for studies of population genetic
variation, including assessments of variability within or differentiation among
natural populations. In addition, it is likely that many of the 20 loci described
here will also be polymorphic in other species of macaques, or in more distantly
related Old World monkey species [Nair et al., 2000].
A number of microsatellites have already been described in rhesus monkeys
[Hadfield et al., 2001; Kayser et al., 1996; Morin et al., 1997; Smith et al., 2000].
The additional polymorphisms reported here add to this literature. One
advantage of the novel loci presented here is their substantial level of
heterozygosity. Less than half of the microsatellite loci previously published for
rhesus macaques have heterozygosity values greater than 0.70, while all of these
new loci exhibit values greater than 0.75. A second advantage is that most of our
new loci are tetranucleotide repeat polymorphisms, which are often more easily
analyzed and more reliably compared across laboratories and populations.
Previous studies [e.g., Hadfield et al., 2000; Morin et al., 1997] also identified
variable tetranucleotide repeats, but the ability to perform effective genetic
analyses of rhesus monkeys and other macaques is enhanced when researchers
are provided with the widest possible array of polymorphisms from which to
choose.
ACKNOWLEDGMENTS
We thank Dr. Judy Cameron, Gary Heckman, and the animal-care staff of the
Oregon NPRC for their assistance regarding the rhesus monkeys from that
center. We also thank members of the Dept. of Comparative Medicine, SFBR, for
their help in studies of monkeys at the Southwest NPRC. This work was
supported by NIH grants RR08781 and RR15383 to J. Rogers.
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