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doi: 10.1111/j.1461-0248.2006.00889.x
REVIEWS AND
SYNTHESES
Department of Ecology,
Abstract
Recent improvements in genetic analysis and genotyping methods have resulted in a
rapid expansion of the power of molecular markers to address ecological questions.
Microsatellites have emerged as the most popular and versatile marker type for ecological
applications. The rise of commercial services that can isolate microsatellites for new
study species and genotype samples at reasonable prices presents ecologists with the
unprecedented ability to employ genetic approaches without heavy investment in
specialized equipment. Nevertheless, the lack of accessible, synthesized information on
the practicalities and pitfalls of using genetic tools impedes ecologists! ability to make
informed decisions on using molecular approaches and creates the risk that some will use
microsatellites without understanding the steps needed to evaluate the quality of a
genetic data set. The first goal of this synthesis is to provide an overview of the strengths
and limitations of microsatellite markers and the risks, cost and time requirements of
isolating and using microsatellites with the aid of commercial services. The second goal is
to encourage the use and consistent reporting of thorough marker screening to ensure
high quality data. To that end, we present a multistep screening process to evaluate
candidate loci for inclusion in a genetic study that is broadly targeted to both novice and
experienced geneticists alike.
Keywords
Homoplasy, linkage, marker isolation, Mendelian inheritance, microsatellites, molecular
ecology, neutrality, null alleles, population genetics, simple sequence repeats.
Ecology Letters (2006) 9: 615629
INTRODUCTION
Table 1 Brief summary of some ecological questions that can be addressed using neutral genetic markers, sorted by the type of data required
In the past decade, the process of isolating new microsatellites has been streamlined with technological advances and
protocol optimization to make the process cheaper, more
efficient and more successful (Zane et al. 2002; Glenn &
Schable 2005). See Appendix S1 for a conceptual schematic
of a microsatellite locus isolation process. A quick search on
the web using appropriate search terms, such as $microsatellite isolation service!, will produce a long list of providers
in locations across the globe. Some services specialize in
certain taxa, such as plants or mammals, and will generally
work with you to tailor the product to your needs. These
laboratories typically require 26 months to develop markers, and most cost less than USD 1500 per locus, or 1015
loci for c. USD 10 000. Although this expense is not trivial,
it is roughly the cost of a PCR machine, and is far less
expensive than equipping a full molecular laboratory if you
do not already have access to one. The cost and time to
delivery also depend on whether the loci are tested for
quality and amplification protocols are optimized as part of
the isolation service. Some services will even write up a
primer note publication with shared authorship. Many of the
laboratories that offer microsatellite isolation also offer
genotyping services, and can take you from start to finish
for your entire study. As an alternative to sending out
samples, it is also possible to establish collaboration with an
academic laboratory with the necessary technical expertise
and equipment, or at many universities, work closely with a
central sequencing facility.
Table 2 Summary of the quality control screening protocol with checklist of suggested tests
Assumption
3. Linkage equilibrium
4. Selective neutrality
5. Mendelian inheritance
6. Every allele differs in length
*These tests are currently beyond the standards required of most ecological uses of microsatellites and should be viewed as optional (see text
for more detail).
about microsatellite mutation behaviour, inheritance mechanisms and distribution across chromosomes are uncovered,
the list of suggested tests will likely evolve.
While we cannot know whether or not most studies
employ such quality control measures, the majority of
publications fail to report the results for most of these tests
(Table 3). In our survey of 50 recent microsatellite studies,
28% of studies mention the importance of quality control
screening but fail to report any results of statistical tests.
Moreover, most testing was carried out post hoc and relied
heavily on testing for HardyWeinberg Equilibrium (HWE).
Table 3 shows that roughly twice the failure rate is detected
with explicit tests for null alleles, inheritance and neutrality
compared with indirect inferences based on testing for
HWE. While the continuing trend of shortening manuscripts makes thorough reporting of quality control testing
more challenging, the use of web-based data repositories
which are commonly linked to printed manuscripts would
be an easy solution to this problem, and would facilitate the
comparison and meta-analysis of data sets.
Once working primers are developed (see Appendix S1),
2030 individuals from each of 35 broadly distributed
populations can be genotyped (see Appendix S2) for the
preliminary screening outlined below. When the entire
collection of samples in the study is genotyped, the analyses
of the tests in the screening process should be repeated and
the results reported in any subsequent publication. The
recommended steps in the screening process are outlined
below (with references that provide more extensive overviews of these topics), and a checklist of necessary tests is
presented in Table 2. We note here that some specialized
statistical analyses make other critical assumptions, such as
conformity to a specific mutational model, constant
population size, or migration-drift equilibrium that are
considered beyond the scope of this review.
Frequency of
reporting (%)
Survey
result (%)
10
84
36
78
12
26
8
34
2.1
13.6
34.6
10.8
5.3
1.6
5.3
1.8
2.4
25.2
33.5
24.2
7.7
5.8
10.5
7.8
16
4.7 11.2
1.4 1.9
Frequency of Reporting indicates the percentage of the 50 studies that performed each test.
The Survey Result values are the mean percentage of loci that failed the test 1 SD. We
present both the values for those studies that tested explicitly for each quality control step,
and those that inferred a violation post hoc after detecting an unexpected deviation from
HardyWeinberg Equilibrium (HWE; noted by asterisk). We excluded studies that used
microsatellite markers taken from previously published research studies to minimize the
chance that tests of assumptions were carrried out previously and therefore not reported in
the current study. A list of the references for the 50 studies is included in Appendix S3 in
Supplementary Material.
*Includes tests of deviation from HWE.
Mendelian inheritance
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Appendix S1. Conceptual flowchart for developing new microsatellite markers base d on the enrichment
technique (one of many methods that are in use see Zane et al. 2004 and Glenn 2005), and primer
optimization steps.
A. Extract DNA from a single tissue sample.
B. Create a DNA library:
1. Cut the genome into 500 bp fragments pieces with a restriction enzyme digest.
2. Attach linker DNA to the ends of each fragment linker DNA has a known sequence so that
primers can be designed to bind to them.
3. Amplify the DNA fragments using primers for the linker ends with PCR.
C. Separate out fragments with repeat sequences:
1. Mix the DNA fragments with a microsatellite probe (an oligonucleotide made of a repeat
sequence of your choice) that can be recovered magnetically.
2. Promote the hybridization of probes to any complementary repeat sequences in the DNA
fragments by heating to denature the DNA and cooling slowly.
3. Hold a magnet to the tube to attract the probes (now bound to the DNA), and wash away the
rest of the unbound DNA with a series of rinses.
D. Sequence the fragments to find microsatellite loci:
1. Using primers for the linker DNA, amplify DNA with PCR to concentrate it.
2. Clone the DNA to prepare it for sequencing - insert it into a plasmid, inoculat e bacteria with the
plasmid, grow the bacteria to replicate the DNA.
3. Isolate the DNA from the bacteria.
4. Sequence microsatellite DNA in the plasmid with primers targeted to the insertion points on the
plasmid.
E. Examine the sequences to find microsatellite repeats.
F. Design primers for the flanking region of the microsatellites (with help from a primer selection software
program such as Primer3 which selects optimal primer sites) and have them made.
G. Attempt amplification of loci with the new primers. Use a gradient of PCR conditions in which the
temperatures, times, magnesium and primer concentrations vary to find optimal conditions.
H. Use gel electrophoresis to confirm the presence of PCR products. Discard primer pairs that fail to
amplify after several attempts.
I. Check for polymorphism by running the successful primer pairs on 10-20 individuals. Estimate allelic
diversity and heterozygosity levels. Discard invariant loci.
J. Check for reliability. Rerun the successful primer pairs on the same individuals twice more to ensure
that genotype scoring is consistently reproducible. Discard loci with unreliable amplification.
H. Order fluorescently labeled primers for the remaining loci. Complete the full screening process detailed
in the text. Discard problematic loci.
K. Streamline the genotyping of the full dataset with the remaining loci by establishing a multiplex PCR
protocol primers for multiple loci (labeled with different dyes) are amplifi ed in a single PCR reaction.
100
125
150
175
200
A An ideal output: The two alleles of this heterozygote are even in height and easy to distinguish from
the stutter peaks adjacent to them during PCR some products are 1, 2 or 3 repeats short due to errors
in replication (similar to step-wise mutation) and show up as evenly spaced peaks with decreasing height
to the left of the true peak. Some loci show extensive stuttering and others show virtually none. The
stutter bands are useful for distinguishing microsatellite products from non-specific or non-target products.
Notice that there are pull-up peaks in the green section of the spectrum. Pull-up is due primarily to
spectral overlap in the emission spectra of the dyes which the sequencer records as a false peak in a
different color. This is a common artifact of the DNA sequencers analysis process that can create
confusion in some situations.
B Two examples of trickier outputs: The blue genotype is a heterozygote but the 2 alleles are only 1
repeat different in size. This creates a characteristic pattern for loci with stutter in which the second peak
is higher than the first because of the additive intensity of the larger alleles first stutter peak and the
smaller alleles true peak. If the first two peaks were equal in height it would be difficult to determine if the
genotype has one or two alleles. One clue is that there are 4 stutter peaks to the right of the largest peak
instead of 3 (assuming the pattern from plot A is characteristic of the blue locus). The second locus in
this plot is shown with black dye and has larger alleles. This locus has no stutter perhaps because there
are few repeats so that the Taq polymerase doesnt make stepwise errors. This genotype is also a
heterozygote, but the larger allele is faint. Larger alleles usually show at least slightly shorter peaks
because PCR is less efficient for longer products. Here, the larger allele is so small that it could be easily
overlooked or mistaken for noise. If any less PCR product were loaded onto the gel it might not show up
at all in which case it would be an example of large allele drop-out, another common source of inflated
homozygosity counts.
C More examples: When multiple loci are loaded in the same gel lane for efficiency, or amplified together
in one PCR multiplex reaction, allele peaks can overlap and be sometimes easy to miss. Here the
green and blue loci share an allele size. Distinguishing the true alleles is made even more difficult due to
the occurrence of green pull-up peaks. If the green allele product were less intense than the blue allele
product (instead of equal as shown here), it might be mistaken for a pull up peak. Re-running the green
locus separately in such cases will minimize scoring error. Here again, the blue locus shows two alleles
that differ by one base pair. The heights are the same because this locus does not show strong stutter.
But the blue locus does show small flanking peaks the left side is a very faint stutter so only the largest
stutter peak is visible, and the right side might be an A-Tail, when the Taq adds an extra adenine
nucleotide onto some copies of the product, increasing it by 1 bp. It will not be mistaken for a
microsatellite allele because an extreme height difference would not occur for 2 alleles so close in size,
as their amplification efficiencies should be similar. The black locus is a homozygote. Even though there
is a small black peak on the left side of the plot, a smaller allele is almost never shorter than a larger
allele. An additional clue is that the larger black peak is quite fat and tall -- PCR produces approximately
double the amount of product when an allele is homozygous because it does not compete for the Taq
with a second allele. The purple locus represents a split peak problem that occurs from high rates of ATailing by the Taq. The +A peaks occur for all of the stutter peaks, making the scoring difficult,
especially for heterozygotes with closely sized alleles. Although the true allele is denoted here, a locus
with alleles that look like the purple one would be too difficult to score reliably. Usually the problem can be
corrected by adding an extra extension step to the PCR program that gives the Taq time to add an A-Tail
to all copies of the product consistently (bumping all alleles and stutter peaks up in size by 1 bp from their
true length).
D Unscorable loci: The blue locus is a stegosaur with unacceptably high stutter. The black locus has
too many non-specific artifact peaks to reliably choose the microsatellite alleles. The green locus has
been overloaded on the gel or has unusually high PCR product concentration and is smearing in the lane.
Appendix S3. Citations for the papers used to generate Table 3. We examined the most recent
papers in the journals Molecular Ecology and Evolution and chose the first 25 from each journal,
in reverse chronological order from July 2005 issues, that used microsatellites to assess
ecological and genetic traits of single or multiple species. We excluded studies that used
microsatellite markers taken from previously published research studies to minimize the chance
that some tests were done previously and therefore not reported in the current study. We then
surveyed these papers to estimate the frequency and results of reported marker screening as
explained in Part III of the text and Table 2.
Molecular Ecology
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Bowen, B.W., Bass, A.L., Soares, L. & Toonen, R.J. (2005). Conservation implications of
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Charmantier, A. & Reale, D. (2005). How do misassigned paternities affect the estimation of
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Fredsted, T., Pertoldi, C., Schierup, M.H. & Kappeler, P.M. (2005). Microsatellite analyses
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Molecular Ecology, 14, 2363-2372.
Funk, W.C., Blouin, M.S., Corn, P.S., Maxell, B.A., Pilliod, D.S., Amish, S. & Allendorf, F.W.
(2005). Population structure of Columbia spotted frogs (Rana luteiventris) is strongly
affected by the landscape. Molecular Ecology, 14, 483-496.
Goossens, B., Chikhi, L., Jalil, M.F., Ancrenaz, M., Lackman-Ancrenaz, I., Mohamed, M.,
Andau, P. & Bruford, M.W. (2005). Patterns of genetic diversity and migration in
increasingly fragmented and declining orang- utan (Pongo pygmaeus) populations from
Sabah, Malaysia. Molecular Ecology, 14, 441-456.
Hauswaldt, J.S. & Glenn, T.C. (2005). Population genetics of the diamondback terrapin
(Malaclemys terrapin). Molecular Ecology, 14, 723-732.
Jones, K.L., Krapu, G.L., Brandt, D.A. & Ashley, M.V. (2005). Population genetic structure in
migratory sandhill cranes and the role of Pleistocene glaciations. Molecular Ecology, 14,
2645-2657.
Keeney, D.B., Heupel, M.R., Hueter, R.E. & He ist, E.J. (2005). Microsatellite and mitochondrial
DNA analyses of the genetic structure of blacktip shark (Carcharhinus limbatus)
nurseries in the northwestern Atlantic, Gulf of Mexico, and Caribbean Sea. Molecular
Ecology, 14, 1911-1923.
Magalon, H., Adjeroud, M. & Veuille, M. (2005). Patterns of genetic variation do not correlate
with geographical distance in the reef-building coral Pocillopora meandrina in the South
Pacific. Molecular Ecology, 14, 1861-1868.
Maki-Petays, H., Zakharov, A., Viljakainen, L., Corander, J. & Pamilo, P. (2005). Genetic
changes associated to declining populations of Formica ants in fragmented forest
landscape. Molecular Ecology, 14, 733-742.
McRae, B.H., Beier, P., Dewald, L.E., Huynh, L.Y. & Keim, P. (2005). Habitat barriers limit
gene flow and illuminate historical events in a wide-ranging carnivore, the American
puma. Molecular Ecology, 14, 1965-1977.
Mesquita, N., Hanfling, B., Carvalho, G.R. & Coelho, M.M. (2005). Phylogeography of the
cyprinid Squalius aradensis and implications for conservation of the endemic freshwater
fauna of southern Portugal. Molecular Ecology, 14, 1939-1954.
Michaux, J.R., Hardy, O.J., Justy, F., Fournier, P., Kranz, A., Cabria, M., Davison, A., Rosoux,
R. & Libois, R. (2005). Conservation genetics and population history of the threatened
European mink Mustela lutreola, with an emphasis on the west European population.
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Otero-Arnaiz, A., Casas, A., Hamrick, J.L. & Cruse-Sanders, J. (2005). Genetic variation and
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