VARlATION AND CONGRUENCE OF MICROSATELLITE MARKERS
FOR PEROMYSCUS LEUCOPUS
CHERYL A. SCHMIDT
Department of Biological Sciences,
Texas Tech University, Lubbock, TX 7940983131
Present address: Department of Biology, Central Missouri State University, Warrensburg, MO 64093
A frequent challenge to ecologists and evolutionary biologists is the need to determine the
genetic relationships among individuals, demes, and metapopulations. Genetic markers that
provide sufficient resolution could be used to elucidate mating systems, determine parentage, and assess gene flow among populations. I developed a set of (CT)n. (GT)n' and
(GATA)n microsatellite markers for the white-footed mouse, Peromyscus leucopus. Variation at six microsatellite loci was assessed for populations ranging from Maine to New
Mexico (transect populations) and for a second set of populations in South Dakota. Alleles
per locus ranged from II to 29. Alleles per locus per population ranged from 6.5 to 8.8,
with an average of 7.3. Microsatellite data were congruent with allozymic. chromosomal,
and other data in depicting asymmetrical gene flow between two chromosomal cytotypes.
Discriminant function- analysis of microsatellite data provided ~92% correct classification
to cytotype. 63% correct classification to populations within the transect from Maine to
New Mexico, and 100% correct classification to populations in South Dakota. The ecological distribution of Peromyscus leucopus and the extensive ecological and genetic knowledge already established for this species, combined with the highly polymorphic microsatellite markers developed in this study, make this an excellent system for addressing questions
relevant to ecology, behavior, evolution, and conservation biology.
Key words: Peromyscus leucopus, white-footed mouse, microsatellites, conservation biology. population genetics, molecular markers
A plethora of topics could be addressed
if genetic markers could assign individuals
to family lineages, populations, metapopulations, and subspecies (Baker, 1994). Recent developments in molecular biology,
particularly characterization of highly variable loci such as microsatellites, have the
potential to yield genetic markers with a
level of resolution adequate to address these
issues.
Peromyscus leucopus is perhaps the most
studied nongame mammal in North America (Lackey et aI., 1985). The white-footed
mouse ranges across eastern North America
south of Canada (Hall, 1981). This species
occupies wooded and brushy areas and usually avoids grasslands and coniferous forest
with little understory (Lackey et aI., 1985).
This habitat requirement results in a variety
Journal of Mammalogy. 80(2):522-529. [999
of local distributional patterns ranging from
large continuously distributed populations
to small isolated populations.
An extensive genetic data base already
exists for this species. A contact zone in
central Oklahoma between what are described as northeastern and southwestern
chromosomal cytotypes has been extensively studied (Baker et aI., 1983; Nelson et aI.,
1987; Stangl, 1986; Van Den Bussche et aI.,
1993). Investigators have used a variety of
character sets, including morphology (Simmons et aI., 1992), allozymes (Krohne and
Baccus, 1985; Nelson et aI., 1987; Robbins
et aI., 1985; Schnake-Greene et aI., 1990;
Tolliver et aI., 1987), and restriction-site
analysis of mitochondrial DNA (Nelson et
aI., 1987) in search of markers to distinguish either between the two cytotypes or
522
May 1999
SCHMIDT-MICROSATELLI1E MARKERS FOR PEROMYSCUS
among local populations of P. leucopus
within the northeastern part of its range. Although those studies have not yielded markers at the population level, all provide important background information.
Microsatellites are tandem repeats of
simple (one to six nucleotides) motifs with
overall lengths usually of :=:;100 base pairs
(Litt and Luty, 1989) and are major sources
of genetic variation (Tautz et aI., 1986). Microsatellite polymorphisms are present
among individuals (Litt and Luty, 1989;
Tautz, 1989; Weber and May, 1989), inbred
strains of rats (Serikawa et al., 1992), and
populations of invertebrates (Choudhary et
aI., 1993) and vertebrates (Ellegren et aI.,
1992). Microsatellites are ubiquitous in eukaryotes, with some motifs occurring in
higher copy number per haploid genome
than others (Moran, 1993; Tautz and Renz,
1984). In particular, the GT/CA tandem repeat has been shown to be common in
many mammal species (Hamada et aI.,
1982; Tautz and Renz, 1984; Van Den Bussche et aI., 1995). Janecek et aI. (1993) examined abundance of all possible dinucleotide motifs in P. leucopus and concluded
that GT/CA was the most common dinucleotide repeat, followed by CTIGA.
Preliminary work for this study included
screening the same genomic library of P.
leucopus used by Janecek et al. (1993) with
(GATA),. In that screening, 16% of the
clones hybridized with the (GATA), probe,
yielding an estimate of > 13,000 copies in
the haploid genome (Baker, 1994). Microsatellite loci (GT)" (CT)" and (GATA),
have great potential for yielding markers
able to distinguish among groups of whitefooted mice from subpopulations and cytotypes.
My objective was to develop a set of rnicrosatellite markers for Peromyscus leucopus and assess geographic variation at those
loci to determine 1) if the microsatellite
data were congruent with chromosomal, allozymic, and mtDNA data relative to introgression of the northeastern cytotype into
the southwestern form, and 2) the level of
523
discrimination among popUlations provided
by that set of microsatellite markers.
MATERIALS AND METHODS
A sublibrary specific for microsatellites (CT)n'
(GT)n and (GATA)n was constructed from a cosmid genomic library (Janacek et al., 1993) of a
wild-caught male P. Jeucopus (TK 27500) from
Garza Co., Texas. Genomic clones containing
(CT)n, (GT)n or (GATA)n were selected following hybridization methods described by Sambrook et a1. (1989) and Van Den Bussche et al.
(1995). Selected clones were digested with Sau
3Al and ligated into the phagemid Bluescript
(BSKS--Stratagene, La Jolla, CA), followed
by transfonnation using Stratagene XLI-Blue
Escherichia coli competent cells (Stratagene
Catalog number 200236).
Clones from the microsatellite sublibrary
were selected for sequencing following hybridization as previously described. Selected clones
were sequenced by Sanger-dideoxy chain termination (Sanger et al., 1977) using Sequenase
II and the T3 and T7 primers for pBluescript-.
Primers were developed complementary to
the sequence of bases upstream and downstream
of the repetitive microsatellite region for each
locus. Three criteria were used in selecting
regions to be used for complementary primers.
The first was relatively even distribution of the
four nucleotides. That criterion was used to
avoid developing primers complementary to
base sequences inherently variable due to tandem repeats. The second desirable condition was
that the sequence to which the complementary
primer was to be developed should contain
2::40% cytosine-guanine. Cytosine and guanine,
with their triple hydrogen bonds, enhance primer
annealing and stability on the priming site during amplification. The third criterion was to
make the distance between primers of a pair
range between 100 and 300 base pairs. Primer
pairs were developed only for microsatellite
clusters containing 2:: I 0 tandem repeats. Whenever possible, primers were 20 base pairs in
length. Primers (Table 1) were synthesized on
the 0.2 IJ-M scale, cleaved, deprotected. desalted,
and nonphosphorylated. Each primer pair initially was put through a survey of PCR conditions to detennine optimal annealing temperatures and concentrations of MgCl 2 and genomic
DNA
(Table 2).
Individuals from populations ranging from
524
JOURNAL OF MAMMALOGY
Vol. 80, No.2
TABLE 1.-Primer sequences for microsatellite loci ofPeromyscus leucopus. PL indicates species;
the next 2-4 letters indicate the type of repeat; the number, followed by A or B identifies the specific
primer pair.
Primer code
Approximate product size
in base pairs
Primer sequence 5'-3'
PLCT5A
PLeT5B
PLGTl5A
PLGTl5B
PLGTl6A
PLGTl6B
PLGT22A
PLGT22B
PLGT48A
PLGT48B
PLGT50A
PLGT50B
PLGT55A
PLGT55B
PLGT56A
PLGT56B
PLGT58A
PLGT58B
PLGT62A
PLGT62B
PLGT66A
PLGT66B
PLGT67A
PLGT67B
PLGATA29A
PLGATA29B
PLGATA68A
PLGATA68B
PLGATA70A
PLGATA70B
CCTCCTAGTGTCTGAGGTG
CATAGTCATAGCTTTAGAA
172
GATCAAGTCTCACTATGTAG
256
GACCTCCACAAATACACTGT
GACAGACACCAGAGGTCACG
220
TCATAGTAACATATGCTCAT
GATCTCTAGTCTGTACACA
198
ATTACTATTTTCTCTTATG
CAGAGATAACATGCATGCCA
264
TGACTGAAAGAGCCAGTCCT
GCATCACAGATTCGAATCTG
180
CTAATGCTAATATCTAGAAG
GCTCAGTGGGTCAAGGCACT
GACTAAGTCTCACTATGTAG
GATCAAGTCACGCGC
AGCTGATACATCCAC
GATCTTGTGAACACGCTTCT
TTGATGGCTCTGGAGAGGCT
AGAGCAGTGACTAGAAATAG
GTTCATCAACTGCATTCAGT
CTCTGTCTGCCACACATGCT
GTGCCATCACAGATGTGACA
GCACTTGCTGCATCACTGAA
TCACTACAGAGCCTGGGCTG
GCAGCAGCAAGCGACTCTAT
GCCTGGTCTACAGAGTGAGT
GATCGTAGATAATAGGTAGA
TCTGAAAATGCAGTGTTCAT
CTTGGTATGCATCGCCATCT
TAATCTCTGTAGCTTCATGT
Maine to New Mexico were selected for this
study (Table 3). That set of populations was referred to as "transect populations." Transect
populations were selected to detenrune if micro-
333
154
164
161
147
283
126
183
224
satellite data were congruent with other data in
depicting asymmetrical gene flow between the
two cytotypes and evaluate effectiveness of microsatellite data in assigning individuals to cy-
TABLE 2.-Amplification conditions for microsatellites of Peromyscus leucopus used in assessing
variation at regional and local scales.
Primer pair
PLGT15
PLGT16
PLGT48
PLGT58
PLGT66
PLGATA70
MgCl 2
(jL1I25 J.Ll rxn)
1.5
1.5
3.0
2.5
1.5
2.5
Annealing
temperature
(0C)
58
55
58
58
55
58
Percent
polyacrylamide gel
4
6
4
10
10
4
Time at
65 mAmps
5
5
5
7
7
5
h
h
h
h
h
h
May 1999
SCHMIDT-MICROSATELLITE MARKERS FOR PEROMYSCUS
525
TABLE 3.-Localities o/populations ofPeromyscus leucopus screened/or variation at six microsatellite loci.
State
Kansas
Maine
New Mexico
New York
Oklahoma
Oklahoma
Oklahoma
Oklahoma
South Dakota
South Dakota
South Dakota
South Dakota
Texas
County
Riley
Penobscott
DeBaca
Rockland
Hughes
Kiowa
McClain
McIntosh
Beadle
Clay
Clay
Yankton
Dickens
Locality (n)
Not specified (10)
0.6 mi W on Gardiner RD off Hwy 2 (9)
16.5 mi S, 2 mi E Taiban, Ben Hall Ranch (10)
Bear Mountain Trailside Zoo & Museum (6)
4.5 mi E Wetumkah (10)
1.5 mi S, 0.8 mi W Mountain View (2)
2 mi E Blanchard (10)
2.2 mi E Raiford (10)
0.2 mi S, 1 mi E Huron, on James River (9)
Game Production Area 1 mi S of Vennillion Airport, on Missouri River (10)
Highline Game Production Area, 7.6 mi W Vennillion, on Missouri River (8)
Woods in NW quadrant of intersection of Hwy 50 and James River (10)
I mi E Afton Springs, Robert Baker Ranch (9)
totypes and populations within the transect.
Transect populations were either NE or SW of
hybrid populations in Oklahoma described by
Baker et aI. (1983) and Stangl (1986). Chromosomal cytotype was known for individuals
from popUlations in Oklahoma and assumed for
the other populations based on position NE or
SW of the Oklahoma contact zone. In addition
to the transect populations, four populations
from southeastern South Dakota were selected
to evaluate perfonnance of microsatellite loci in
assigning individuals to populations at a more
local level relative to transect populations. Each
population was represented by individuals
caught on the same night from a small area (single locality given) to increase probability of
sampling a single subpopulation at each locality.
Primer pairs used to detennine variation in
number of tandem repeats of selected microsatellite loci within and among populations of P.
leucopus were GT15, GTI6, GT48, GT58,
GT66, and GATA70 (Table I). One primer of
each pair was end-labeled by a standard g35S_
AT?, T4 polynucleotide kinase reaction (Sambrook et al., 1989) prior to PCR amplification of
homologous loci from genomic DNA of individuals from each subpopulation. Optimal parameters established for each locus were used during
amplification (Table 2). End-labeled amplification products were denatured at 80°C for ;;:::3 min
before loading onto a denaturing polyacrylamide
gel. The percentage of polyacrylamide was
based on the predicted size range of amplification products (Table 2). Sequenced M13 was
electrophoresed on each side of the population
samples as a size standard. The gel system used
had a continuous buffer welI from top to bottom
that provided a constant temperature across the
gel, minimizing bowing of the buffer front and
allowing lines to be drawn between identical
base positions of the two MI3 sequences. That
provided an efficient means for determining
sizes of alleles in population samples.
Variation at each microsatellite locus was
characterized based on number of alleles, range
of allele sizes, and average observed heterozygosity across all populations. Mean number of
alleles per locus per population of 9-10 individuals also was calculated as an indicator of variability provided by the entire set of microsatellite loci
Discriminant function analysis was used to assess effectiveness of microsatellite variation in
assigning individuals to cytotype, populations
along the transect, and populations in South Dakota. Lengths of alleles in base pairs served as
predictors. Resubstitution and cross-validation
options of the DISCRIM procedure (SAS Institute Inc., 1985) were initially applied, using genotypes from all six loci. The cross-validation
option was applied by using about one-half of
the individuals from each sample to construct
the discriminant function equation, and then
testing the equation on the remaining individuals. Subsequently, a stepwise discriminant function analysis (STEPDISC procedure; SAS Institute Inc., 1985) was conducted to determine if
sufficient classification could be achieved with a
subset of loci. Loci indicated by the stepwise
discriminant function procedure were reevalu-
526
JOURNAL OF MAMMALOGY
ated using Proc DrSCRIM with resubstitution
and cross-validation for comparison to the perfonnance of all six loci.
RESULTS
Microsatellite sublibrary.-The microsatellite sublibrary was comprised of 14
(CT)n-specific microtiter plates, 7 (GT)nspecific microtiter plates, and 13 (GATA)nspecific microtiter plates, totaling 34 plates
(3,264 potential microsatellite-bearing
clones). That sublibrary yielded 104 clones
that hybridized strongly with respective microsatellite probes.
Primer development.-Sixty-eight of the
strongly hybridizing clones ultimately were
selected for sequencing. Although the first
nticrosatellite successfully sequenced was a
(GATA)n repeat, (GT)n microsatellite sequences accumulated faster than the other
two, as might have been expected due to
the relative abundance of the three motifs
in P. leucopus. Hence, the final set of microsatellite sequences used to develop primer pairs comprised 1 (CT)", II (GT)", and
3 (GATA)" loci (Table I).
Of the IS primer pairs that ultimately
were designed and synthesized, six (GT15,
GTl6, GT48, GT58, GT66, and GATA70)
were optimized efficiently and used in the
subsequent population study. The remainder (CT5, GT22, GT50, GT55, GT56.
GT62, GT67, GATA29, and GATA68) did
not produce amplification products during
trial surveys and were not considered.
Application of microsatellite primers.As predicted, amplified microsatellite loci
were variable, with all six loci polymorphic
for all populations examined (Table 4).
Number of alleles per locus ranged from II
(GT48) to 29 (GATA70). Mean number of
alleles per locus per population (9-10 individuals) ranged from 6.5 to 8.8, with an
average of 7.3. Average heterozygosity per
locus ranged from 0.39 (GT58) to 0.75
(GT66), with an overall mean of 0.58.
Distribution of unique alleles among
transect populations followed a pattern indicative of introgression of the northeastern
Vol. 80, No.2
cytotype into the southwestern form. Of 27
alleles unique to populations within the
range of the southwestern cytotype, two
(GT58-16 and GATA70-6) were common
to all four southwestern populations but
were not found in northeastern popUlations.
Although 53 unique alleles were observed
in one or more populations from the northeastern portion of the transect, none of
these 53 alleles were common to all northeastern popUlations. Two non-unique alleles
(GTl5-12 and GT48-11) were found in all
six northeastern populations and in one and
two southwestern popUlations. Three alleles
were found in five of the six northeastern
populations (missing from either the populations in Maine or New York) and in one
or two of the southwestern populations.
Discriminant function analysis based on
microsatelIite variation at all six loci correctly assigned 92% of the individuals to
their respective cytotype (known or presumed) via the resubstitution procedure.
Cross-validation yielded 88% correct assignment of individuals to cytotypes. Four
of the seven misclassified individuals were
from northeastern populations and were assigned to the southwestern cytotype; three
were from southwestern popUlations (all
three from New Mexico) and assigned to
the northeastern cytotype. Stepwise discriminant function selected two loci, GT16
and GATA70, using a 0.15 retention criterion. Subsequent direct discriminant function analysis using only those two loci
yielded 91 % correct assignment by both resubstitution and cross-validation.
Assignment of individuals to popUlations
within the transect by direct discriminant
function analysis using data for all six microsatellite loci provided 63% correct classification by resubstitution and 42% by
cross-validation. Stepwise discriminant
function analysis using a 0.15 retention criterion, followed by iterations of direct discriminant function analyses with subsets of
the loci retained by stepwise discrimination,
produced a subset of four loci that performed favorably well relative to the entire
SCHMIDT-MICROSATELLITE MARKERS FOR PEROMYSCUS
May 1999
TABLE 4.-Summary of variation at six microsatellite loci in Peromyscus leucopus.
Parameter
Locus
OTIS
OT16
OT48
GT58
GT66
GATA70
No. of Range (bp)
allelesa.b of alleles
22
24
11
28
27
29
197~227
158-221
242-269
98-140
85-120
216-278
Range
ofHb
X
H"
0.20-0.80
0.50-1.00
0.20-0.89
0.00-0.80
0040-1.00
0040-1.00
0.50
0.66
0.47
0.39
0.75
0.70
• Mean number of alleles across all loci = 23.5; mean num·
ber of alleles per locus per populations = 7.3 (range of 6.58.8).
b Mean heterozygosity per population and mean number of
alleles per locus per population calculated for populations rep·
resented by nine or more individuals.
set. The subset comprising GT16, GT48,
GT58, and GATA70 yielded 60% correct
classification by resubstitution and 45% by
cross-validation.
By resubstitution, the discriminant function equation based on data from all six microsatellite loci correctly assigned 100% of
the individuals from South Dakota to the
locality from which they were captured.
Cross-validation yielded 91 % correct assignment. Stepwise discriminant function
analysis with significance level for retention
set at 0.15, followed by iterations of direct
discriminant function analysis using stepwise-selected subsets, yielded optimal assignment with five of the six loci (GTI5,
GT16, GT48, GT66, and GATA70; resubstitution = 100%, cross-validation = 94%).
However, with only four of the loci (GTI5,
GT16, GT66, and GATA70), resubstitution
correctly assigned 94%, and 91 % were correctly assigned by cross-validation.
DISCUSSION
Variation provided by the six microsatellite loci in my study indicates that rnicrosatellites hold great potential as markers for
population studies of small mammals. The
high levels of polymorphism at these loci
in P. leucopus (number of alleles per locus
ranging from 11 to 29; mean number of alleles was 23.5; Table 4) exceed the varia-
527
tion at rnicrosateIIite loci reported for large
mammals such as North American wolflike canids (Roy et al., 1994; range = 1020 alleles per locus, X ~ 13.6) and black
bears (Ursus americanus-Paetkau and
Strobeck, 1994; range = 9-19 alleles per
locus, X = 11.5), although heterozygosity
values are generally comparable.
Loci for which the primers for P. leucopus were developed are variable enough
to address many questions in behavioral
ecology, such as the elucidation of mating
systems and parentage. These markers have
been successfully used in taxa ranging from
other species of Peromyscus to species of
Microtus and Clethrionomys. For P. leucopus, these markers provide adequate variation to examine patterns of gene flow
among local popUlations and, therefore, can
be used to determine impacts of habitat
variation and other ecological parameters
on genetic structure of metapopulations.
This information would not only enhance
conservation decisions but also lend insight
into the impact of habitat distribution on local extinction, loss of genetic variability,
popUlation differentiation, and speciation.
Detailed studies of carefully selected model
species and the synthesis of these data into
strategies to wisely manage populations of
endangered taxa and their habitats are needed.
My study was not designed to intensively
examine patterns of gene flow across the
hybrid zone in central Oklahoma, but there
is evidence for asymmetrical gene flow
across that hybrid zone. Two alleles
(GT58-16 and GATA70-6) that were present in all four southwestern populations did
not occur in any of the popUlations within
the range of the northeastern cytotype. This
indicates a lack of gene flow from SW to
the NE. Conversely, no alleles were found
in all northeastern cytotype popUlations
without also being found in one or more
southwestern popUlations. This indicates
that there is gene flow from the NE to the
SW. These results demonstrate congruence
of these data with earlier chromosomal, al-
JOURNAL OF MAMMALOGY
528
lozymic and mtDNA data (Baker et aI.,
1983; Nelson et aI., 1987; Stangl, 1986;
Stangl and Baker, 1984), which also indicated that gene flow across the hybrid zone
is asymmetrical with introgression of the
northeastern alleles into the southwestern
genome.
The ability to assign individuals to populations based on microsatellite genotypes
could contribute significantly to studies of
natural populations ranging from behavior
to conservation genetics. The set of microsatellite markers developed in this study
provides such assignment capabilities.
These markers can correctly assign 92% of
the individuals to the correct cytotype, 63%
of the individuals to populations within the
transect, and 100% of the individuals within
South Dakota to their respective populations.
Misclassification of individuals to presumed cytotypes may be a reflection of either limitations or strengths of this set of
markers. All misclassified individuals were
from populations for which the cytotype
was assumed. Therefore. it is possible that
misclassified individuals were actually of
the cytotype to which they were assigned
based on discriminant function analysis.
Anthropogenic movement of small rodents
is common and could have resulted in mice
with the southwestern cytotype being present in northeastern populations, and vice
versa. Such movement presents an increasing challenge to studies of natural populations.
ACKNOWLEDGMENTS
I thank R. J. Baker for providing the opportunity, substantial funding, and unending encouragement to use the genomic library of P.
Jeucopus and the facilities at Texas Tech University to conduct this research. This research
was facilitated by numerous field collectors over
many years, particularly R. J. Bradley, R. V. D.
Bussche. M. J. Hamilton. and C. A. Porter,
among others. J. L. Longmire provided significant technical advice. R. Van Den Bussche provided invaluable advice and moral support. J.
Cathey. M. Maltbie. E. Reat. J. Peppers, and oth-
Vol. 80, No.2
er members of R. J. Baker laboratory also provided laboratory assistance and camaraderie.
This work was funded in part by a graduate fellowship from the Institute of Biotechnology at
Texas Tech University, two grants-in-aid of research from Sigma Xi. one grant-in-aid of research from the American Society of MammaIogists, and a Helen Hodges Charitable Trust Fellowship.
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Submitted 20 January 1998. Accepted 1 September
1998.
Associate Editor was Robert K. Rose.