Download POLYMERASE-CHAIN-REACTION (PCR) ANALYSIS OF

The Auk 109(4):886-895, 1992
POLYMERASE-CHAIN-REACTION
MICROSATELLITES--A
GENETIC
NEW
RELATIONSHIPS
HANS
(PCR) ANALYSIS OF
APPROACH
TO STUDIES
OF
IN BIRDS
ELLEGREN
Departmentof AnimalBreedingandGenetics,
SwedishUniversityof AgriculturalSciences,
Box7055,S-75007 Uppsala,Sweden
A13STRACT.--Genomic
DNA libraries of the Barn Swallow (Hirundo rustica) and the Pied
Flycatcher(Ficedulahypoleuca)
were screenedfor the presenceof dinucleotidemicrosatellite
repeats.Two thymine-guanine repeats,"(TG),," and two thymine-cytosinerepeats,"(TC),,"
were isolatedand sequencedfrom the two species,respectively.Polymerase-chain-reaction
(PCR)analysisof 25 unrelatedBarn Swallowsand 10 unrelatedPied Flycatchersrevealed3
to 15 allelesper locusand heterozygosities
in the rangeof 0.46to 0.89.Mendelianinheritance
was confirmedfor all four loci in 10 Pied Flycatcherand 2 Barn Swallow families comprising
a total of 240 meioses.The occurrenceof nonparentalalleles in offspringfrom two Barn
Swallow families was consistentwith extrapair fertilization as revealed by a parallel DNAfingerprinting analysis.A DNA amount approximatelycorrespondingto 0.01/•1 blood was
used for PCR analysis.DNA was also prepared from feathersand approximately1% of a
preparation from a single remex or rectrix was sufficient for PCR amplification. One of the
primer pairsfrom the Barn Swallow alsoamplified a polymorphiclocusin the House Martin
(Delichonurbica)and in the Bank Swallow (R. riparia).Received
3 October1991,accepted
16 June
1992.
DURINGTHElast 10 to 15 years,moleculargenetictechniqueshaveopenednew perspectives
in biological research. Several ornithological
fields provide good illustrations.For example,
DNA-DNA hybridization has clearly contributed to avian systematics(Sibley and Ahlquist
1990), polymorphicpatternsof mitochondrial
DNA (mtDNA)
have aided in differentiation
studiesof speciesand populations(Kesslerand
Avise 1985),and polymorphismsof randomgenomic DNA segmentshave been usedto resolve
questionsabout parentage (Quinn et al. 1987).
Perhapsmost spectacularly,DNA fingerprinting, using minisatellite (Burkeand Bruford 1987,
ments in eukaryoticgenomes.The most abundant dinucleotide variant in mammals, thymine-guanine repeats "(TG),," shows copy
numbers in the order of 105 (Hamada et al.
1984a),on averageoccurringevery 30 kb (kilobasepairs) in the human genome(Stallingset
al. 1991).Consideringall possiblesimple motifs
there is probably one microsatelliteevery 6 kb
in man (Beckmannand Weber 1992).Although
their evolutionary conservation indicates a
functional or structuralsignificance,there is yet
no conclusiveevidencefor suggestedroles as
hot-spot regions for recombination (Pardue et
al. 1987),gene regulators(Hamada et al. 1984b),
Wetton et al. 1987)or syntheticsimple-repeat or sequencesstimulating packing and conprobes(Ellegren 1991a),has proved to be the densing of chromosomes(Stallingset al. 1991).
mostsensitivemethod for determiningthe geThe allelic variability at microsatelliteloci is
netic relationshipsof individuals in a popula-
usuallyhigh, with heterozygosities
approach-
tion (Burke 1989). Moreover, the isolation of
ing 90%. As for minisatellites, the polymorphismis due to varying numberof repeatunits
fied suchstudies(Gyllenstenet al. 1989,Hanot- (typically in range of 10-50), which can be rete et al. 1991).
vealedby in vitroamplificationof the DNA segRecently,a new type of genetic marker, mi- ment of interest using the polymerasechain
crosatellites, has received considerable attenreaction(PCR;Saiki et al. 1988)and subsequent
tion in genome analyses(Litt and Luty 1989, gel electrophoresis(reviewedby Beckmannand
Tautz 1989, Weber and May 1989). Microsatel- Soller 1990, Weber 1990, Litt 1991). In humans,
locus-specific
minisatelliteprobeshas simpli-
lites are tandem stretches of mono-, di-, tri- or
microsatellites
tetranucleotidesequencemotifsthat frequently
occur as randomly dispersed repetitive ele-
serving as genetic markers in the human genome (HUGO) project,for instancefacilitating
886
have become
invaluable
tools
October1992]
AvianMicrosatellites
the localizationof geneswith impactson genetic diseases(Froguel et al. 1992, Goto et al.
1992).The hypervariablenature of thesemarkersalsomakesthem highly suitablefor identity
or parentagetesting (Edwardset al. 1991, 1992,
Ellegren et al. 1992).
In addition to work on humans, microsatel-
887
servedas a positive control. A negative control involving PCR mastermix without added DNA was
alwaysincluded.
DNA preparation.--DNAwas either preparedfrom
venousbloodor from singlefeathers.In the former
case,1 #1blood (frozen in EDTA capillarsand newly
thawed) was diluted to 1 ml with water and incubated
at room temperaturefor 15 min. After centrifugation
at 10,000x g for 2 min, the supernatantwascarefully
lites have been analyzed in the mouse (Mus
musculus;
e.g. Love et al. 1990),somedomestic removed(leavingabout25 #1)and discarded.A 5%
animals(e.g. Fries et al. 1990, Crawford et al. Chelex (Biorad) solution was added to a final volume
1990, Ellegren et al. 1992, Wintere et al. 1992), of 200 #1 and incubated at 56øCfor 15 min. The mixand whales (Tautz 1989, Sch16tterer et al. 1991).
ture was vortexed for 10 s, boiled for 8 rain and then
In this study,I report the molecularcloning of
avian microsatellites.Four dinucleotiderepeats
vortexedagain.Followingcentrifugationasabove,1
were isolated from the Barn Swallow (Hirundo
rustica)and the Pied Flycatcher(Ficedulahypoleuca),and the polymorphismwas investigated
by PCR amplificationof DNA from unrelated
individuals
and families.
to 5 #1of the supernatantwere taken to PCR and the
remainderfrozen. Beforebeing usedagain,the sam-
ple wasvortexedand centrifugated.Chelexis a chelatingstyrenedivinylbenzeneresinthatformsmulticoordinatedcomplexeswith bivalent metal ions that
otherwisemay catalyzethe breakdownof DNA during boiling (cf. Ellegren 1992).
Various types of feathers (primaries,secondaries
and remiges)were usedfor DNA preparation.Feath-
METHODS
ers were collected from adult birds one to three weeks
Cloneisolation.--The procedure followed Ellegren
et al. (1992). Shortly, size-fractionated(200-500 base
pairs)genomiclibrarieswere constructedfrom Barn
Swallow DNA digestedwith AluI, HaeIII and HinfI
in the plasmid vector pBluescriptSKII+, and from
Pied FlycatcherDNA digestedwith the same enzymes in the phage vector M13mp18. The libraries
were transferredto nylon membranesand screened
with radioactivelylabelledpolynucleotide(TG),and
poly(thymine-cytosine),
(TC),, probesfor the presence of microsatellite-containingclones. Positive
beforeanalysisand were storedat room temperature
betweenwhen they were collectedandanalyzed.Approximately5 mm of the root part of a single shaft
was cut into a few pieceswith a surgicalblade and
transferred to 200 #1 5% Chelex and 2.5 #1 proteinase
K (8 mg/ml). The mixturewasincubatedat 56øCfor
at least4 h, and an additionalportion of proteinase
K was addedone-halfway through the incubation
step.After this treatmentthe samplewashandledas
above(vortexing,boiling, vortexingand centrifugation) and 1 to 5 #1was taken to PCR. When handling
and preparingthe feathersthe possiblerisk of concloneswere isolated and sequenced.
Polymerase-chain-reaction
analysis.--Microsatellite- taminationwas carefully avoidedby using a separate
flanking PCR primers were synthesizedon the basis surgicalbladefor eachfeather.Unlessotherwisestated, datawill refer to analysisof DNA preparedfrom
of the derived sequenceinformation.Primer labelling
blood.
(end-labelling with gamma-32p-dATP)
and PCR reagentswere as previouslydescribed(Ellegrenet al.
RESULTS
1992).PCR (10 #1)wascarriedout with approximately
50 ng templateDNA and 2 pmol primer (one primer
labelled) in small Eppendorf tubes or in microtiter
plate wells using a thermal cycler (Coy Laboratory
Products,Inc., or Techne) in the following way: 1
cycle of 95øCfor 2 min, 55øCfor 30 s and 72øCfor 1
min; 25 to 29 cyclesof 94øCfor 1 min, 55øCfor 30 s
and 72øCfor ! min. After the final cycle,a prolonged
extensionstep of 5 min was added.
I mixed 1 #1 of the product from the amplification
reactionwith 95%formamideand dye, heatedto 75øC
and separatedin 6% denaturingpolyacrylamidegels
(standard"sequencing"gels). After electrophoresis,
gelswere dried and amplificationproductsvisualized
by autoradiographyfor 2 to 24 h. The sizesof alleles
were determined by electrophoresisof an accompanying sequence reaction. Amplification of the
cloned alleles also aided in size determination
and
Isolationof microsatellites.--Screening
of about
2,000recombinantplasmidclonesfrom the Barn
Swallow library with (TG), and (TC), probes
yielded two TG-positive clones (STG1 and
STG4). Sequenceanalysis of these clones revealed one perfect stretchof 12 TG repeatsand
one compound structure of GA-GT repeats.
Screeningof approximately the same number
of phageclonesfrom the PiedFlycatcherlibrary
yielded two TC-positive clones (PTC2 and
PTC3).Theseconsistedof eight and 12 perfect
TC-repeats,respectively,and were derivedfrom
different loci. Primers (21 or 24 bp) for PCR
amplificationwere synthesizedaccordingto the
DNA sequencesflanking the repeat regions,
888
HANSELLEGREN
[Auk,Vol. 109
TABLE1. Repeat structure and primer sequencesat two Barn Swallow (STG! and STG4) and two Pied
Flycatcher (PTC2 and PTC3) microsatellite loci.
Locus
STG1
STG4
PTC2
PTC5
Primer A (5'-3')
Repeatstructure
TGAACAATGAGAGAACTGATGCAA
( GT) •
CATCAAGAGAGGGATGGAAAGAGG
( GA) • ( GT)?A( TG) •
TGATCGAAAGACCTGTAAGAT ( TC) •
GTGTTCTTAAAACATGCCTGGAGG
( CT) •
Primer B (5'-3')
CATCACTCAGATGAGAAACTGGAA
GAAAAGATTATTTTTCTTTCTCCC
ATCAGCGTTAGACCAATACTCTTA
GCACAGGTAAATATTTGCTGGGCC
following recommendationsgiven by Luty et
al. (1990). Repeat structuresand primer com-
size differencescorrespondingto one and two
repeat units, respectively).This situation did
positions are indicated in Table 1.
not prevent scoring since the occurrence of an
PCR amplification
andvariability.--PCRanaly- adjacentand slightly shorter allele was recogsis of Barn Swallow and Pied FlycatcherDNA nized asa strongsignal causedby the combined
using primers designedto flank the microsatel- radiation from an extra band and a main band.
Inheritancepattern.--To investigatethe inherlites gaveamplificationproductsof varying but
approximately expectedsize. No individual ex- itancepattern of the avian microsatellites,
they
hibited more than two intense bands consistent
were applied to families previously analyzed
with a locus-specificamplification. Each"main" by DNA fingerprinting (data and DNA kindly
band
was associated
with
one or a few
fainter
bandsappearingimmediately below (i.e. being
shorter than the main product; Fig. 1). These
"extra"
bands seem to be inevitable
artifacts in
provided by H•kan Tegelstr•Sm,Department of
Genetics,Uppsala University). In 10 Pied Flycatcher (n = 52 offspring) and 2 Barn Swallow
(n = 8 offspring) families, DNA fingerprinting
PCR amplification of dinucleotide microsatellites, and are presumed to be the result of slippage events as the Taqpolymerasesynthesizes
the complementarystrand of the repeat region
(e.g. Litt and Luty 1989,Weber and May 1989,
Luty et al. 1990). Cloning experiments have
using the Jeffreys33.15 probe (Jeffreyset al.
1985)had failed to detectany illegitimateoffspring (the probe revealshighly variable DNA
demonstrated
allele at the PTC2, PTC3, STG1 and STG4 loci
that extra bands lack one or a few
fingerprints in the two species;Gelther et al.
1992, Smith et al. 1991). In microsatellite am-
plifications of these families, every offspring
repeat units (Luty et al. 1990, Riesset al. 1990), was also present in either of the parents; for
a situationsupportedby the factthat extrabands homozygousoffspring,the allele was found in
occur with multiples of the length of the repeat both parents (Fig. 2). Data were consistentwith
unit. The possibilitythat the extrabandswould every parent transmitting one allele to eachoffreflecta geneticheterogeneityat the genomic spring and, thus, autosomalinheritance was inlevel can be ruled out by noting that amplifi- dicated in a total of 240 meioses (208 for Pied
cationsof cloned microsatellitealleles display Flycatcherand 32 for BarnSwallow).However,
extra band patterns identical to genomic am- sex linkage could not be completely excluded
for STG1 since offspring were unsexedand the
plifications (e.g. Ellegren et al. 1992).
Analysis of unrelated individuals revealed femaleappearedto be homozygousin both famextensivepolymorphismat all four avian micro- ilies.
satellite loci (e.g. Fig. 1). Ten Pied Flycatchers
Detectionof extrapairfertilization.--Given the
showed four different alleles at the PTC2 locus
assumption that microsatellites are stably in(expectedheterozygosity[Hexp]
= 0.49)and eight herited, the hypervariable nature of these
allelesat the PTC3 locus(H•xp= 0.76). A more markerssuggests
that they couldbe usefulfor
extensivescreeningof 25 unrelated Barn Swal- detecting extrapair paternity or intraspecific
lows revealed heterozygositiesof 0.89 and 0.46 brood parasitismin birds. In two Barn Swallow
at the STG1and STG4loci,respectively.As many families, DNA fingerprinting had demonstratas 15 alleles were found at STG1. The observed
ed two (of three) and four (all) illegitimate offallele frequenciesat the Barn Swallow loci are spring, respectively(Tegelstr•Smunpubl. data;
summarized
in Table 2.
extrapairpaternityis known from previousDNA
The extra bands of one allele sometimes were
studiesof the BarnSwallow [Smith et al. 1991]).
in juxtapositionto the other allele (e.g. hetero- An analysis of the segregationpattern at the
zygousindividuals 2 and 6 in Fig. 1 show allele STG1 and STG4 microsatellite loci in these two
October1992]
AvianMicrosatellites
889
TABLE2. Estimatedallele frequenciesat the Barn
Swallow STG1 and STG4 microsatellite loci (n = 25
individuals).
Allele size (bp)
Frequency
STG1
200
228
230
232
234
236
238
240
242
244
252
256
260
262
268
0.02
0.05
0.16
0.11
0.05
0.04
0.07
0.11
0.20
0.02
0.02
0.05
0.05
0.07
0.02
STG4
135
137
139
Fig. 1. PCR amplificationof the STG1 microsatellite locus in eight unrelated Barn Swallows. Alleles
0.14
0.70
0.16
indicatedby arrows.
obtainedafter overnight exposure(Fig. 4). The
strengthof the signalsvaried between individuals suggestingdifferent DNA yields from the
families revealednonparentalalleles in all six
illegitimate offspring.One of the female alleles
was present in all offspring, whereas the two
malesapparentlydid not transmitallelesto any
offspring (Fig. 3).
The presence of nonparental microsatellite
allelesmay either be due to falseparentageand/
or to mutation.Here, falseparentagewasknown
from previous experimentsand, thus, it is reasonable that the observed microsatellite patternsresultedfrom it. The lack of male-specific
alleles suggeststhat all offspring in these families were the outcomeof cuckoldry.Sincethe
preparations.
mutation
rate at microsatellite
loci seem
to be
Cross-species
analysis.--Primersflanking the
BarnSwallow microsatellites
were alsoapplied
to DNA preparedfrom the House Martin (Delichonurbica)and from the Bank Swallow (R. ri-
paria;in both cases,feather preparationsused).
The annealing temperaturein the PCR reaction
was decreased
to 45øC to allow
a certain
mis-
matchbetween the primers and the heterogenous sequences.Whereas no specificampliftcation product was obtained at STG1, the STG4
locus yielded a distinct two-allele polymorphism in six unrelated individuals of both species (data not shown). The House Martin and
Bank Swallow
alleles were
identical
to two of
significantly lower than at minisatellite loci
(Kwiatkowskiet al. 1992),the presenceof a non~
parental microsatelliteallele may in practicebe
used as a marker of false parentage (see Dis-
the BarnSwallow alleles,but the 135 bp allele
that showeda frequencyof just 0.14 in the Barn
Swallow was presentin 10 of 12 chromosomes
cussion).
in the other species.Whether these shared al-
Analysisoffeathers.--Asan alternativeto blood
sampling,featherswere evaluatedfor their suitability in serving asa DNA sourcefor PCR analysis. By a simple protease/boiling procedure,
DNA was preparedfrom about5 mm of the root
part of single remex and rectrix from the Barn
Swallow. Using approximately 1% of a preparation asstartingmaterial in PCR reactions,autoradiographysignalsof suitablestrengthwere
lelesreflecta commonancestryof allelesor are
due to a denovogeneration of alleles of the same
size cannot
be resolved.
DISCUSSION
Previous hybridization studies have indicated the frequent occurrenceof dinucleotide repeats in avian genomes (Hamada et al. 1982),
890
HANS ELLEGREN
[Auk, VoL 109
b
F
M
F
½
M
F
M
Fig. 2. PCR amplification of avian microsatelliteloci in families with parentageconfirmedby DNA fingerprinting.F is female,M ismale,arrowson eachsideindicateallelesof femaleandmale origin,respectively.
Analysisof: (a) a Pied Flycatcherfamily at PTC3 locus,(b) a different Pied Flycatcherfamily at PTC2 locus,
and (c) a Barn Swallow family at STG1 locus.
be useful for monitoring the geneticvariability
in populations aswell asfor investigating close
show that microsatellites
in the Barn Swallow
geneticrelationships(e.g. parentage).
In the latter casean important prerequisite is
and the Pied Flycatcherdisplay a considerable
degreeof polymorphism(i.e. hypervariability), that alleles are stably inherited without a siga factsuggestingthat this type of markercould nificant elementof mutation to new length albut this constitutesthe first report of the molecularcloning of suchsequences.
Presentdata
a
M
b
F
M
F
Fig.3. Detectionof extrapairfertilizationin two BarnSwallowfamilieswith aid of PCR-analyzed
microsatellitepolymorphisms.
M (male)is putativefather,F (female)is putativemother,solidarrowson eachside
refer to maleand femalealleles,respectively,and openarrowsindicatenonparentalalleles.(a) Analysisof
a BarnSwallowfamilyat STG1locus,and (b) analysisof a differentfamilyat STG4locus.DNA fingerprinting
had revealedthat all offspringin family a and offspring1 and 3 in familyb were illegitimate.
October
1992]
AvianMicrosatellites
891
leles. I found no mutation among 240 meioses
(with parentage confirmed by DNA fingerprinting), indicating that mutation events
should be rare at these dinucleotide repeats.In
humans,an extensivebody of literatureon microsatellitestabilityhasemergedin recentyears.
Thus far, only a single report describeslength
mutations (two) at dinucleotide microsatellite
loci (Kwiatkowski et al. 1992). These mutations
were found in a sampleof 4,480meiosesleading
to an estimated mutation rate of 4.5 x 10-4 per
meiosis.This figure should however be related
to numerousreportswhich have failed to detect
any spontaneousmutation to new length alleles. For example, completely stable inheritancehasbeen found in samplesof a few hundred to thousands of meioses,as investigated
by Dracopoliand Meisler (1990), Love et al.
(1990; mouse data), Kwiatkowski et al. (1991),
Morral et al. (1991), Oudet et al. (1991), Petersen
et al. (1990), Richards et al. (1991), Small et al.
(1991), Todd et al. (1991; mousedata), Weber et
al. (1991), Decker et al. (1992), Edwards et al.
(1992),Fornageet al. (1992),Froguelet al. (1992),
Fig. 4. PCR amplificationof STG1 microsatellite
locusin six unrelated BarnSwallowsusingDNA pre-
pared from single feathers.N is a negativecontrol
with PCR master mix, but without added DNA. Ar-
rows refer to interpreted alleles.
Goto et al. (1992), Hazan et al. (1992), and Wilkie
et al. (1992). Moreover, documented data sets
with 20 to 100 stable meioses exceed 100 (for
hybridization with one multilocus DNA fingerprinting probe. However, severalmicrosatinstance, see recent issues of Nucleic Acids Reellite loci can be analyzed simultaneouslyin
search).Altogether, there may be in the order multiplex PCR reactionsand also separatedin
of 50,000 human microsatellite meioses pub- the same gel (Luty et al. 1990, Edwards et al.
lished and still only two mutationshave been 1991, Ellegren et al. 1992, Hazan et al. 1992).
found (i.e. mutation rate of about 10-4 to 10-5). ApplyingJamieson's
(1965)formulaefor excluClearly, this givesevidencefor a significantly sionpower,a combinationof five microsatellite
more stable situation than the high mutation loci each having a heterozygosityof about 0.8
rate characterizingminisatellite loci (Jeffreyset yieldsan efficiencyof 0.98 to 0.99for detection
al. 1988;reportedratesof 1.1 x 10-2 to 2 x 10-3 of falsepaternity (given known maternity). The
in birds, Burke and Bruford 1987, Westneat
samepoweris reachedwith only threeloci hav1990).Furthermore, observationsof very strong ing a polymorphismcorrespondingto the Barn
linkage disequilibrium between microsatellite Swallow STG1 locus. The efficiency given by
loci and adjacentpolymorphicmarkersare like- this combination of markers would imply that,
ly to reflect stability even on an evolutionary if there is a match between the alleles of an
time scale(Chehab et al. 1991, Oudet et al. 1991). offspringand its putative father, paternity will
Although it remainsto be shown,using ped- be incorrectlyassignedin 1 or 2 casesof 100.
igree material with parentage confirmed by
However, as soon as the alleles of an offspring
DNA fingerprinting, that avian microsatellites and either of its putative parentsdo not match,
are as stable as in humans, it is difficult from a an illegitimate mating can almost certainly be
molecularpoint of view to seewhy they would concluded due to the apparent stability of the
behave in a different manner. Thus, it is likely systems.To properly distinguish between exthat avian microsatellites
will serve as effective
trapair fertilization and intraspecificbrood parasitismin caseswhen offspringlackallelesfrom
tools for the detection of extrapair parentage.
Practicalaspectsof microsatellites
in parentage their putative father but display allelespresent
testing.--With regard to parentage testing, an
in their putative mother, probability estimates
analysisof the polymorphismat onemicrosatel- establishedon the basisof allele frequenciesin
lite locus will
not of course be as effective
as
the population need to be considered.
892
HANSEL•.EGREN
[Auk,Vol. 109
If the extremevariability producedby DNA
ysisallows samplesto be collectedeven when
fingerprinting can be dispensedwith, locus- birdsare absent.For example,featherscollected
specificsystemsoffer the generaladvantageof at nests or from areas where birds have been
a simplergeneticinterpretation.This is further moltingare likely to give necessary
DNA yields.
accentuatedin microsatellite analysis since:(a) Sinceabout 1%of the preparationsfrom single
alleles can be distinguished(which may not be remiges and rectrices was sufficient for PCR
the case in agarose electrophoresisof large analysis,smaller feathers (e.g. covertsor body
minisatellite alleles of similar size; Devlin et al.
feathers)also would probably be suitable for
1991)and definedby size, thusallowing com- analysis.
puterization and the exchange of data or colOne promisingpossibilitywith feathersas a
laborative studiesbetween laboratories;(b) re- DNA sourceand PCR as a hypersensitivedecent advanceswith "sequencinginstruments" tection method is to DNA type specimensof
and softwaredesignedto registratemicrosatel- birds in museums.Although old DNA generlite polymorphismspermit an automatedand ally is degradedto someextent,relatively short
nonradioactive assay for these markers (Ed- microsatelliteregionsmay be intact (cf. Hagelwards et al. 1991); and (c) even with manual berg et al. 1991). Using the microsatelliteloci
handling the time schedulefrom DNA prepa- describedin this study,I have typed more than
ration to result need not be longer than 24 h.
100-year-oldBarnSwallowsand Pied FlycatchAn obviouslimitation,however,to the prac- ers stored as museum skins (Ellegren 1991b,
tical value of microsatellitepolymorphismsis 1992).The obviousbenefit of suchan approach
that primersdesignedfor one specieswill only is that the geneticvariability may be monitored
amplify a correspondinglocusin closelyrelated overtime. Thiscanhavemajorsignificancewhen
species. This has been shown for some mamdesigningconservation
strategiesfollowing,for
mals(Moore et al. 1991),including somewhales example, an extensive population bottleneck.
(SchliSttereret al. 1991). In the latter case, miFurthermore,thisapproachwould allow the gecrosatellite-flanking
primerscouldamplifyhet- netic analysisof extinct species(cf. Thomaset
erologoussequencesin speciesthought to have al. 1989).
been separatedfor 20 million years. The Barn
Swallow STG4 primers also amplified a polyACKNOWLEDGMENTS
morphic locusin the HouseMartin and the Bank
Swallow (estimatedsplit 5-10 million yearsago;
Sibley and Ahlquist 1990), showing that amplification of microsatellitesin closelyrelated
speciesholds true for birds as well.
Generalconsiderations
of PCRanalysis.--PCRis
most efficiently performed with sequences
shorter than 1 to 2 kb. Thus, microsatellites are
more adaptableto PCR than minisatellitesdue
to their smallersize.Regardingthe minute template-DNA requirements,I useda DNA amount
approximatelycorrespondingto a blood vol-
ume of 0.01 /•1 for each PCR reaction.Using
conventionalblood-samplingtechniques,this
would imply that the DNA amount will not
limit the possibility of performing repeated
DNA analyses. Moreover, the demonstration
that feathersmaybe usedfor DNA typing adds
a new perspectiveto the sampling routines in
genetic studies of birds. First, collecting feathers is obviously easier than collecting blood.
Second,sincefeathersmaybe storedat ambient
temperaturebefore DNA preparation,there is
I am grateful to: Hfikan Tegestr/Sm
for generously
providing DNA, blood samplesand unpublished
DNA-fingerprinting datanecessaryfor investigating
the inheritance of the microsatellites; Rauno Alatalo,
Arne Lundberg,Anders Pape Moller and Jan Petterssonfor collectingsamples;and Leif Andersson
for valuablecommentson the manuscript.This study
was given financialsupportby the Uddenberg-Nordingskaand Nilsson-Ehlefoundations.
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