May 14, 1987 - quail DNA that no individual bands could be readily distin- guished and ... were.no instances of a scored (parent-specific) band occurring.
Reprinted
from Nature,
Vol. 327, No. 6118, pp. 149-152, 14 May 1987
@ Macmillan
Journals
DNA fingerprinting in birds T. Burke & M. w. Bruford Department of Zoology, University of Leicester, Leicester LEl 7RH, UK
Several regions of the human genome are highly variable in populations because the number of repeats in these regions of a short 'minisatellite' sequence varies at high frequency. Different minisatellites have a core sequencel,2 in common, however, and probes made up of tandem repeats of this core sequence detect many highly variable DNA fragments in several species including humansl,3, cats4, dogs4 and mice!. The hypervariable sequences detected in this way are dispersed in the genome and their variability means that they can be used as a DNA 'fingerprint', providing a novel method fQrthe identification of individuals2,6,confirmation of biological relationships7,8 and human genetic analysis9,1O.We show here that human minisatellite-derived probes also detect highly variable regions in bird DNAs. Segregation analysis in a house sparrow family confirms that these regions comprise many mostly heterozygous dispersed loci and we conclude that house sparrow DNA fingerprints are analagous to those of humans. Fingerprint analysis identified one nestling, with fingerprint bands not present in the parent pair's fingerprints, which we conclude resulted from an extrapair copulation. Extrabond copulations have been described in many wild bird speciesll-13, but their success and hence adaptive significance have rarely been quantifiablel4-2O. DNA fingerprinting will be of great significance to studies of the sociobiology, demography and ecology of wild birds. Hybridization patterns obtained from conspecifics of 16 species indicate that bird DNAs have many different and very variable regions with similarity to both of the human minisatellite poly-(core) probes 33.6 and 33.15. Figure 1 shows examples from six species (representing four oscine families and two non-passerine orders). In most cases the band patterns have a similar complexity to human DNA fingerprints (shown for comparison in Fig. 1). There was, however, one striking exception: probe 33.15 hybridized so intensively to Hinfl-digested Japanese quail DNA that no individual bands could be readily distinguished and, unusually, hybridization was more complex for larger fragment sizes. Thus an abundant class of Japanese quail DNA, probably comprising a tandem-repetitive satellite DNA containing the occasional Hinfl restriction site, has sequence
Table 1 Intraspecific similarities of band patterns produced by human minisatellite probes Species Enzyme Human HinfI HS A/uI PF A/uI CB A/uI HinfI JQ BE A/uI R A/uI
Probe 33.6 x nb 13.8 0.21 6.0 0.28 8.0 0.13 5.0 0.42 17.0 0.47 23.0 0.30
-
-
Probe 33.15 x nb 14.7 0.21 15.0 0.17 22.0 0.27 15.5 0.20
25.5
Combined x nb 28.5 0.21 27.0 0.22 30.0 0.23 20.5 0.26
0.28
Probabilities of band sharing in six bird species (refer to Fig. 1 for names), with the human data (from ref. 2) for comparison. Band comparisons were made for the better resolved and less complex larger fragments only, to 4 kb for the human, PF and JQ digests, and to 2 kb for the others where the fragments are found to be of smaller average size. Here, nb refers to the mean number of bands in the compared size range; x was found as the mean of each pairwise comparison of the proportion of individual A's bands that were matched by a band of similar intensity and electrophoretic mobility in B. A small number of weakly hybridized bands, that although apparent in A would have been obscured, if present, by a stronger band in B, were excluded. As only two individuals have been compared in each case here, confidence limits have not been estimated.
Ltd.,
1987
similarity to probe 33.15. The probability of band sharing between unrelated individuals within each species sampled from the wild is similar to that found in humans (Table 1). Greater similarity was observed in the Japanese quail, as expected from the fact that they originated from a relatively inbred laboratory population. The genetics and germline stability of the detected DNA fragments were investigated in a house sparrow family, consisting of 2 adults and the 11 offspring raised by them in four successive broods, belonging to a closely monitored wild population17. DNA from each individual was hybridized under two different stringencies to probes 33.6 and 33.15 (Fig. 2). In nine offspring all DNA fragments were also present in either or both parents, but two nestlings were found to contain mismatched bands not present in either parent. These novel bands may have arisen by mutation, or may indicate incorrect parentage. As nestling 'X' contains many mismatched bands we conclude that X cannot be the progeny of this parental combination (Fig. 2 legend). We cannot, however, reject the mutation hypothesis for the nestling containing a single mismatching band; many such mutant bands have been observed at mammalian minisatellite loci, and their mutation rate is known to vary significantll'lO. The observed rate in house sparrows of 1/288 = 0.0035 (see Fig. 2 legend) is close to that of -1/240 for humans I, and mutation is therefore a more likely explanation for a single mismatch than is incorrect parentage, even if an actual parent were a close relative of the assigned one. The segregation of parental bands in the 10 nestlings excluding X was investigated. As with humans9, probes 33.6 and 33.15 detected completely different sets of bands. Some bands, idt;ntified from their electrophoretic mobility and their pattern of distribution. among nestlings were detected in more than one hybridization experiment (identified by letters in Fig. 2). There were. no instances of a scored (parent-specific) band occurring in all 10 offspring; thus all the bands scored were heterozygous. Although we consider it likely that some bands will be homozygous, we note that bands specific to one parent will tend to include those at the more polymorphic loci. The mean transmission frequency for 61 different parental bands was 0.52, consistent with the 1: 1 mendelian segregation expected for heterozygous loci. The cosegregation of bands in a parent was investigated by counting the number of gametic recombination events recorded in the sibship (Table 2 legend). Closely linked b.ands are expected to show no recombination, and allelic bands are expected to show no co-inheritance. From binomial expectati~n, the total number of pairwise comparisons among the 29 maternal and 32 paternal fragments that are expected to show complete linkage or allelism by chance is only 0.80 and 0.96 comparisons respectively. As many more linked and allelic combinations were actually found (Fig. 2), we conclude that most cosegregating bands represent DNA fragments in close linkage, possibly single mini satellites that have been cleaved into two or more fragments, and that most apparently allelic bands represent true alleles. The number of recombination events occurring in the sibship between each pair of loci was compared with that expected assuming independent assortment (Table 2). As the observed distributions are generated from pairwise and hence nonindependent comparisons the usual goodness-of-fit tests are inappropriate, but inspection reveals no suggestion of clumping. We conclude that the house sparrow loci scored here are dispersed throughout the genome, as are similar loci in the four mammalian species that have so far been investigated4.5.9. The mean number of loci scored in a human DNA fingerprint (29.5, using both probes)9 is two to three times that for cats (10.0) and dogs (13.0) (ref. 4). The mean number scored in house sparrows at the lower stringency (21.5) is intermediate, whereas few (7.5) are detected at the higher stringency (Fig. 2). Alleles in the higher stringency fingerprints are represented by an average 1.20 fragments, whereas the corresponding value for the lower stringency is 1.07 and that for humans is only 1.01.
.
2
Fig. 1 Hybridization patterns obtained from avian DNAs probed with the human minisatellite probes 33.6 and 33.15, with the DNA fingerprints produced in a single human DNA for comparison. Arrowheads, estimated positions of 20, 10, 8, 6, 4 and 2 kb fragments for tracks to the right. The avian DNAs were obtained from apparently unrelated mated pairs of five species sampled in the wild (HS, house sparrow, Passer domesticus; PF, pied flycatcher, Ficedula hypoleuca; CB, corn bunting, Miliaria calandra; BE:,European oX
R
BE
,---,
kb 20
10 8 6
4
bee-eater ,I Apiasterrme;:;;-p.sl; R, rook,
Corvus fru'giregUsf and 'one laboratory population (JQ, Japanese quail, Coturnix coturnix japonica). DNAs were digested with restriction enzy-
mes having a four-base recognition 2 L I L I L---J L I L J L--J L ' L I '---' L--J L...J '---' ' ' sequence: HinfI (Human, JQ) or Probe:33.1533.6 33.15 33.6 33.15 33.6 33.15 33.6 33.15 33.6 33.15 33.6 33.15 Alul (HS, PF, JQ, BE, R). Methods. Human DNA was prepared from blood2. Bird tissues variously consisted of washed and lysed red blood cell fractions densely pelleted at 15,000g during an earlier isozyme surveyl7 and since stored for up to 6 years at -70°C (HS), frozen heparinized whole blood (BE, JQ), whole blood collected into at least one volume of 1x SSC (0.15 M NaCI, 15 mM trisodium citrate, pH 7.0, autoclaved) and kept at ambient temperatures for up to 3 days before freezing at -20°C (CB, R), or pectoral muscle freshly frozen at -20°C (PF). All blood was originally collected by venipuncture. For DNA isolation, muscle tissue was .first finely chopped at O°C, cooled in liquid nitrogen and ground until particulate. About 0.5 g muscle or the equivalent of 50 fLlwhole blood were well suspended in 4 ml 0.1 M Tris-CI pH 8.0,0.1 M NaCI, 1 mM EDTA before incubation with 2.5 units ml-I proteinase K (Sigma) and 0.5% SDS for at least 1.5 h at 55°C. This was followed by two phenol/chloroform and one chloroform extraction, then ethanol precipitation with 0.2 M sodium acetate. Typically, 50 fLlavian blood yields > tOOfLg DNA. For gels and hybridization, 5 fLgsamples of DNA were digested with 15 units of HinfI or Alul in the presence of 4 mM spermidine trihydrochloride (Sigma1 at 37°C for 16 h, and recovered after phenol extraction by ethanol precipitation, followed by washing in 80% ethanol. Digested DNAs were resuspended in a loading mix and electrophoresed in 0.7% or 1.0% (for PF only) agarose gels and blotted onto nitrocellulose filters (Schleisher and Schull, 0.45 fLm pore size) as described elsewhere2. The 32P-labelled probe DNAs were prepared from the human mini satellite M13 recombinants 33.6 and 33.15 by primer extensionl.2. Southern blots were hybridized overnight at 62°C in the presence of 50fLgml-I denatured salmon DNA (SigmaType Ill, sheared in 0.2M NaOH,20 mM EDTA at 100°C for 20min, neutralized with HCl and recovered after phenol/chloroform extraction by ethanol precipitation), 2xDenhardt's, 0.1% SDS, 6% (w/v) polyethylene glycol 6,000 (BDH) and 1 x SSC, and autoradiographed for 1-7 days at -70°C using Kodak X-Omat or Amersham MP film and a single Cawo intensifying screen. The auto radiographs for human and house sparrow were obtained from a single filter; hybridization to probe 33.6 was carried out after the removal, confirmed by autoradiography, of all previously hybridized 33.15 by immersing the filter in water at 85°C and allowing it to cool to 20 °C. Thus there is greater independence among bands in the more complex of the sparrow fingerprints, possibly because many linked fragments occur in the unresolved region, but house sparrow bands are less independent than those of humans. This may suggest that the sparrow fragments are more often clustered into tight linkage groups than in humans, or that Alul (as used for sparrow fingerprints) cleaves fragments internally more frequently than HinfI (as used for humans). Having demonstrated the independence of most bands in the lower stringency fingerprints, we can estimate the theoretical probability that two individuals will share the same pattern from a knowledge of the frequency with which bands of similar mobility are shared. For probe 33.15, this probability is -2 x 10-20 for two random unrelated individuals and -9 x 10-10 for two sibs; for probe 33.6 the respective probabilities are -3 x 10-14 and -5 x 10-7 (Fig. 2 legend). Knowledge of the independence of bands is also required in the analysis of relatedness. We wish to know if either adult is a parent of nestling X (see Fig. 2). As we have identified linked and allelic bands we can consider the alleles, or 'haplotypes' comprising closely linked bands, at the independent loci detected at either or both stringencies. As all the scored haplotypes are heterozygous, we expect half to occur in an offspring. As X has fewer than expected of the haplotypes of the male member of the pair (M) (P
