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COMMUNICATIONS
Fingerprinting Birds' DNA with a Synthetic Polynucleotide Probe (TG)n
HANS
ELLEGREN
Departmentof AnimalBreeding
andGenetics,
SwedishUniversityof AgriculturalSciences,
Box7055, S-75007,Uppsala,Sweden
Unambiguous determination of genetic relationshipsis important to understandmating patternsand
population structures.For example, thorough social
studiesof lekking or cooperativelybreeding species
require that parentageand other closegenetic relationships be determined. Similarly, although extrapair copulations(EPC) have been documentedin several bird species(e.g. Birkhead 1987, Westneatet al.
1990), some details about the behavior cannot be eval-
uated unlessthe result of illegitimate matingscan be
quantified reliably. Until recently these have been
seriousobstacles,and although someattemptsto use
protein variantsas geneticmarkersto determine genetic relationships have been successful,many have
been inadequate. Basically this is a consequenceof
the low degree of polymorphism in protein systems
(Mumme et al. 1985, Romagnanoet al. 1989). Highly
polymorphic markersare required for effectivestudies of genetic relationships.
The discovery of families of hypervariable DNA
regionsin the human genome led to the development
of DNA fingerprinting. The techniquescreensseveral
hypervariable loci simultaneously to produce individual-specificfingerprints of the DNA (Jeffreyset
al. 1985a,b). The technique is alsoapplicable to birds
(Burke and Bruford 1987, Wetton et al. 1987), plants
(e.g. Dallas 1988), fungi (Braithwaite and Manners
1989), and nematodes (Uitterlinden
et al. 1991). This polynucleotide type of probe offers
several advantagescomparedwith oligonucleotides
and minisatellites (see below). I demonstrated that
the (TG), probe also detectsindividual-specifichybridization patterns in birds.
Five unrelated individuals of the Barn Swallow (Hi-
rundorustica)were analyzed.DNA waspreparedfrom
blood as describedby Gelter and Tegelstrfm (1990).
Ten microgramsof DNA from each individual was
digestedwith 50 U of restrictionendonucleaseHinfI
(Promega) according to the manufacturer's instruction. DNA was precipitated with ethanol, dissolved
in 1 x TE (10 mM Tris; 1 mM EDTA, pH 7.0) and
separatedin 20 cm 0.7% agarosegels at 40 V for 24
h. After electrophoresis,DNA was transferred to nylon filters (BiodyneB,Pall) under alkaline conditions.
The filters were prehybridized in 7% SDS; 0.263 M
Na2HPO4; 1 mM EDTA pH 8.0; 1% BSA for 2 h at
+65øC. The probe was a synthetic, double-stranded
polynucleotide with the simple repeat dT-dG x dAdC, "(TG),," (PharmaciaLKB Biotechnology).Agarose
electrophoresisindicatedan averageprobe length of
750 basepairs,but whether this was the actuallength
of the individual polymer or the length of multimer
annealedproductswasunknown. DNA (100-200 ng)
was radioactively labeled by standard nick translation. Hybridization was performed in the prehybridization solutionovernight at + 65øC.The filters were
et al. 1989). In
recent avian studies,DNA fingerprinting hasprovided a powerful method for analyzing parentage(Burke
et al. 1989, Gelter 1989, Birkhead et al. 1990, Gibbs et
washed twice in 2 x SSC (0.3 M NaC1; 0.03 M sodium
citrate); 0.1% SDS at +65øC and exposed to Kodak
X-AR film for 1-3 days at -70øC with intensifying
al. 1990,Gyllensten et al. 1990,Rabenoldet al. 1990,
screens.
Westneat 1990; see also Quinn et al. 1987).
The (TG)nprobe revealed highly polymorphic hybridization patterns on Barn Swallow DNA digested
with HinfI (Fig. 1). Basedon the limited sampleof 5
unrelated birds, the average probability that 2 random individuals would displayidentical fingerprints
was estimatedto be 8.1 x 10-•2 (calculatedaccording
sart et al. 1987). Minisatellites derived from other
to Jeffreys198•b). This estimate is derived from a
species,including the Willow Warbler (Phylloscopus mean band frequencyof 0.36 and an averageof 24.8
trochilus),have been used (e.g. Gyllensten et al. 1989). scoreablebands(larger than a fragment size of 2 kilo
However, syntheticoligonucleotides(15-25 basepairs) base pairs) per individual. In total, I scored 66 rewith simple repeat motifs (e.g. di- or trinucleotide solvablebandsamong the • individuals. Distinct and
repeats)alsoproduce individual-specific fingerprints variable hybridization patternswere alsoobtained for
and chickens (Gallus
in a variety of species(Epplen 1988, Sch•fer et al. Canada Geese(Brantacanadensis)
1988, Kashi et al. 1990). Simple repeat motifs are probgallus)(datanot shown).The probabilitythat two Barn
ably presentin all eukaryoticgenomesand their poly- Swallowswould haveidenticalDNA fingerprintswith
morphism is due to a varying number of repeated the (TG)• probe (8 x 10-22)is somewhat higher than
units (Tautz 1989).
that describedfor other avian speciesand fingerprintThe synthetic polynucleotide probe (TG). detects ing probes(e.g. Burke and Bruford 1987, Gyllensten
highly specificDNA fingerprints in horses(Ellegren et al. 1990, Meng et al. 1990). Becauseof the small
DNA fingerprinting has traditionally been performedby usinggeneticallyclonedhuman minisatellite probes(e.g. Jeffreys'clones33.6 and 33.15) or by
a tandemly repeated DNA segment present in the
wild-type genomeof the bacteriophage
M13 (seeVas-
956
The Auk
108: 956-981.
October
1991
October
1991]
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Communications
957
sample size (i.e. band frequencies could not be less
than 0.2), however, the probability estimateis likely
to be an underestimate.
I believe that the polynucleotideprobe(TG)• would
be suitablefor parentagetesting in birds. But for a
correct assessment of the amount
of information
that
can be receivedby this fingerprinting probe,family
studiesmustdemonstrateto which degreethe restriction fragmentsare independently inherited. However, little or no allelism or linkage hasbeen found
in the species/fingerprintingprobecombinationsso
far tested(seeBurke 1989).A rough estimateof the
usefulness
of the (TG)• probefor paternitytestingin
Barn Swallowscan be obtainedas follows. The probability that an autosomalband from an offspring's
fingerprint is presentin the mother equals
(1 + q - q2)/(2 - q),
where q is the meanallelic frequencyderived from
q= 1 - (I -x)%
and x is the mean band frequency(Georgeset al.
1988). From this probability the proportionof an
offspring'sfragmentsexpectedto be presentin the
mothercanbe determined.Theremainingfragments
in the offspringmustbe of a paternalorigin.Forthe
BarnSwallow,q = 0.20ßand the probabilityto make
14.1
7.2
6.4
5.7
4.8
4.3
3.7
an incorrectpaternity determination can thus be approximately estimatedto 1.1 x 10-4 (0.368-8;
8.8 is the
average number of paternal bands). It must be em-
phasized,however,thatthenumberof paternalbands
islikely to vary andthe estimateisjustfor an average
offspring.
Foroffspringcarryingfewerpaternalbands,
the confidencein excludingandassigningparentage
will
2.3
be lower.
Syntheticpolynucleotides
are easyto obtain and
1.9
are convenientas DNA probesbecausethey are in a
ready-to-useform for hybridization. Minisatellite
probesare usually cloned into plasmid vectorsand
must regularly be preparedon a large scale.This is
not difficult,but it is time-consuming
and requires
basicbiochemicalequipment,often not easilyavailable. Another attractivefeature of polynucleotides
with simplerepeatmotifsis that they, in contrastto
somecommonminisatelliteprobes,are not protected
by patents.Moreover,in comparisonwith oligonucleotides,polynucleotideprobesoffer several technical advantagesduring the labeling and hybridization procedures.
They canbe labeledby standardnick
translation, unincorporatedlabeled nucleotidescan
easilybe separated,and hybridizationand washing
conditionsaresimilarto that usedfor genomicclones.
The syntheticpolynucleotide(TG)• detectedindividual-specificfingerprints in Barn Swallow DNA,
andis likely to be suitablefor parenthoodtestingin
otherbirds.This type of probeoffersseveraladvantages compared with cloned minisatellites and oligonucleotides, and would thus facilitate the use of
DNA fingerprintingin birds.
Fig. I.
DNA fingerprints of five unrelated indi-
viduals of the Barn Swallow (Hirundo rustica). The
probewas the syntheticpolyunucleotide(TG)•. Fragment sizes(in kilo basepairs)are indicatedto the left.
I thank H,•ikan Tegelstrfm, who provided avian
DNA samples. Leif Andersson, H•kan Tegelstrfm,
David Westneat, and an anonymous referee made
valuable commentson the manuscript.
LITERATURE CITED
BmKHEAD,
T. R. 1987. Sperm competitionin birds.
Trends
Ecol. Evol. 2: 268-272.
ß T. BURKEßR. ZANN, F. M. HUNTER, & A. P.
KRUPA. 1990. Extra-pair paternity and intraspecificbrood parasitismin wild Zebra FinchesTae-
958
Short
Communications
niopygia
guttata,revealedby DNA fingerprinting.
Behav.
Ecol. Sociobiol.
27: 315-324.
hypervariable minisatellite probes detect DNA
polymorphism in the fungus Colletotrichum
gloeosporioides.
Curt. Genet. 16: 473-475.
BURKE,
T. 1989. DNA fingerprinting and other methodsfor the studyof matingsuccess.
TrendsEcol.
Evol. 4: 139-144.
--,
Nature
327: 149-152.
N. B. DAVIES,M. W. BRUFORD,& B. J. HATCH-
WELL.1989. Parentalcareandmatingbehaviour
of polyandrousDunnocks Prunellamodularisrelatedto paternityby DNA fingerprinting.Nature
338: 249-251.
85: 6831-6835.
SANDBERG.
1991. DNA fingerprintingin horses
using a simple (TG), probe and its applicationto
population comparisons.Anim. Genet. In press.
EPPLEN,
J.T. 1988. On simple repeated GAT/CA sequencesin animal genomes:a criticalreappraisal.
J. Heredity 79: 409-417.
--,
1132.
variability of DNA fingerprintsin three species
of swan.Heredity 64: 73-80.
MUMME,R. L., W. D. KOENIG,R. M. ZINK, & J. A.
MARTEN.1985. Geneticvariationandparentage
in a Californiapopulationof AcornWoodpeckers. Auk
102: 305-312.
1987. DNA markeranalysisdetectsmultiple maternity and paternity in singlebroodsof the Lesser Snow Goose. Nature
326: 392-394.
1989. Genetic and behavioural
differ-
HAYDOCH,
& S.W. ZACH.1990. Sharedpaternity
revealedby genetic analysisin cooperatively
breeding tropical wrens. Nature 348: 538-540.
ROMAGNANO,L., T. R. McGUIRE, & H. W. POWER. 1989.
Pitfallsand improvedtechniquesin avian parentage studies.Auk 106: 129-136.
$CH•FER,
R., H. ZISCHLER,
U. BIRSNER,
A. BECKER,
& J.
T. EPPLEN.1988. Optimized oligonucleotide
entiation associatedwith speciationin the flyprobesfor DNA fingerprinting.Electrophoresis
9: 369-374.
catchersFicedulahypoleuca
and F. albicollis.
Ph.D.
dissertation,Uppsala, Uppsala Univ.
TAUTZ,
D. 1989. Hypervariabilityofsimplesequenc& H. TEGELSTR•M.1990. Restriction enzyme
es as a general source for polymorphic DNA
markers. Nucl. Acids Res. 17: 6463-6471.
cleavageof nuclear DNA revealsdifferencein
repetitive sequencesin two speciespairs (Ficedula UITTLERLINDEN,
A. G., P. E. SLAGBOOM,
T. E. JOHNSON,
and Parus). Auk 107: 429-431.
GEORGES,
M., g. S. LEQUARRE,M. CASTELLI,R. HANSET,
& G. VASSART.
1988. DNA fingerprintingin domesticanimalsusing four different minisatellite
probes.Cytogenet.Cell Genet.47: 127-131.
GIBBS,H. L., P. J. WEATHERHEAD,
P. T. BOAG,B. N.
WHITE, L. M. TABAK,& D. J. HOYSAK. 1990. Re-
alized reproductivesuccess
of polygynousRedwinged Blackbirds revealed by DNA markers.
Science 250: 1394-1397.
GYLLENSTEN,
U. B., S. JAKOBSSON,
& H. TEMRIN. 1990.
NO evidencefor illegitimate young in monogamousand polygynouswarblers.Nature 343:168170.
,
--,
& A. C. WILSON.
1989.
Nu-
cleotidesequenceand genomicorganizationof
bird
minisatellites.
Nucl.
Acids
Res. 17: 2203-
2214.
pervariable "minisatellite" regions in human
DNA.
& J. VIJG. 1989. The Caenorhabditis
elegans
genome containsmonomorphicminisatellitesand
simplesequences.
Nucl. AcidsRes.17:9527-9530.
VASSART,
G., M. GEORGES,
R. MONSIEUR,H. BROCAS,
A.
$. LEQUARRE,& D. CHRISTOPHE. 1987.
A se-
quencein M13 phagedetectshypervariableminisatellites in human and animal DNA. Science 235:
683-684.
WESTNEAT,
D. F. 1990. Geneticparentagein the IndigoBunting:a studyusingDNA fingerprinting.
Behav. Ecol. Sociobiol. 27: 67-76.
, P. W. SHERMAN, & M. L. MORTON. 1990. The
ecologyand evolutionof extra-paircopulations
in birds.Pp. 331-370in Currentornithology(D.
M. Power, Ed.). New York and London, Plenum
Press.
WETTON,J. H., R. E. CARTER,D. T. PARKIN,& D. WAL-
TERS.1987. Demographicstudyof a wild House
JEFFREYS,
A. J., V. WILSON,& $. L. THEIN. 1985a. Hy-
--,
riety of vertebrates. Nucl. Acids Res. 18: 1129-
RABENOLD,
P. P., K. N. RABENOLD,
W. H. PIPER,J.
ELLEGREN,H., L. ANDERSSON,M. JOHANSSON,& K.
GELTER,H.P.
SOLLER.
1990. Largerestrictionfragmentscontaining poly-TG are highly polymorphicin a va-
QUINN, T. W., J. S. QUINN, F. COOKE,& g. N. WHITE.
DALLAS,
J.F. 1988. Detectionof DNA "fingerprints"
of cultivatedrice by hybridization with a human
minisatellite DNA probe. Proc. Natl. Acad. Sci.
USA
KASHI, Y., Y. TIKOCHINSKY,E. GENISLAV,F. IRAQI, g.
NAVE, J. $. BECKMANN,Y. GRUENBAUM,& M.
MENG, A., R. E. CARTER,& D. T. PARKIN. 1990. The
& M. W. BRUFORD.
1987. DNA fingerprinting
in birds.
"fingerprints" of human DNA. Nature 316: 7679.
BRAITHWAITE,
K. S., & J. M. MANNERS. 1989. Human
--,
[Auk,Vol. 108
Nature
--,&
Sparrowpopulationby DNA fingerprinting.Nature 327: 147-149.
314: 67-73.
. 1985b. Individual-specific
Received
13 December
1990,accepted
30 April 1991.