Zoo Biology 9:91-98 (1990)

DNA “Fingerprints” and Paternity Ascertainment in Chimpanzees (Pan troglodytes) John Ely and Robert E. Ferrell

Deparfment of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.

Key words: genetic management, paternity exclusion, VNTR probes

INTRODUCTION

Although there is some evidence of a telomeric bias [Royle et al., 1987; Na- kamura et al., 19881, highly variable regions (HVRs) of DNA appear to be dispersed throughout the human genome and have also been detected in a broad array of species ranging from bacteria to non-human primates [Rogstad et al., 1988; Ryskov et al., 1988; Weiss et al., 19881. Alleles at hypervariable loci consist of a variable number of tandem repeats [VNTRs; Nakamura et al., 19871 of a short core sequence (1 1-60 bp), or mini-satellite [Jeffreys et al., 1985al. Allelic variation in the number of tandem repeats is detectable with any restriction endonuclease that cleaves DNA near but not within repeat units. Thus, the length of an observed restriction fragment is a function of the number of tandem repeat units within the fragment.

Individual VNTR loci have between 2 and 77 or more alleles [Nakamura et al.,

Received for publication August 23. 1989; revision accepted October 13, 1989.

Address reprint requests to Johii Ely, Department of Human Genetics, Crabtree Hall, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261.

0 1990 Wiley-Liss, Inc.

92 Ely and Ferrell

1987, 1988; Wong et al., 1987a,b]. Single VNTR loci with three or more alleles frequently have a mean heterozygosity of greater than 70% [Nakamura et a]., 19881. Under conditions of low stringency, hybridization of certain radiolabeled VNTR probes, including Jeffreys’ probes 33.6 and 33.15 [Jeffreys et al., 1985b], the alpha globin 3‘ HVR [Higgs et al., 1981; Fowler et al., 19881, and M13 bacteriophage [Vassart et al., 1987; Ryskov et al., 19881, to genomic DNA detects multiple hy- pervariable loci, producing a highly polymorphic pattern with a correspondingly high degree of individual uniqueness. The multiple bands detected in target DNA results from decreasing sequence homology and decreasing in-phase alignment of the core repeat units to the target DNA: reduced stringency allows sequences of reduced similarity to the labeled probe to hybridize [Jeffreys et al., 1985al. It has been estimated that Jeffreys’ probes detect on average about 15 scoreable loci [Jeffreys et al., 19861. Owing to the large number of bands detected per individual, and the relatively small probability of band-sharing between individuals, there is a very small overall probability that any two individuals picked at random will share all detected fragments. The polymorphic pattern detected on an autoradiograph has therefore been termed a DNA “fingerprint” [Jeffreys et al., 1985bl. DNA fingerprints have proven to be highly useful in resolving problems of individual identification in human affairs [Marx, 1988; Jeffreys et al., 1985~1.

One common situation involving problems of individual identification in animal breeding is paternity ascertainment. Knowledge of paternity is of great utility to the rational management of captive breeding colonies. Perhaps the primary benefit is the ability to avoid or at least reduce inbreeding, which is associated with reduced fertility [Wright, 19771, lower mean birthweight [Smith, 19861, increased neonatal mortality [Ralls et al., 1979; Ralls and Ballou, 19821, and reduced resistance to infectious disease [O’Brien and Evermann, 19881. Furthermore, accurate knowledge of pedi- grees so gained can then be used for the maximization of effective population size, the preservation of rare alleles, and the prevention of selectively disadvantageous geno- types. We report here on the application of DNA fingerprinting to paternity ascer- tainment among captive-born chimpanzees housed in multi-male groups as part of the National Chimpanzee Breeding and Research Program.

MATERIALS AND METHODS

Chimpanzee blood samples were provided by Patricia Alford, D.V.M., Uni- versity of Texas System Cancer Center, Science Park, Bastrop, Texas; Jo Fritz, Primate Foundation of Arizona, Tempe, Arizona; and R. Brent Swenson, D.V.M., Yerkes Regional Primate Research Center, Atlanta, Georgia. In most cases, subspe- cific designations are unknown (Nathan Flesness, personal communication). The paternity cases discussed herein refer only to the Bastrop colony. Blood specimens were collected into dextrose-citric acid-containing vacutainers and shipped to Pitts- burgh by overnight courier. High molecular weight genomic DNA was isolated from peripheral white cells harvested from 20 ml of anti-coagulated whole blood by the method of Miller et al. [ 19881. Ethanol-precipitated DNA was spooled out with a 1 pl inoculating loop and transferred to a microcentrifuge tube containing 1 ml of 10 mM Tris-HC1, 0.2 mM EDTA, pH 7.5. Six micrograms of DNA was digested to completion with 4 ~1 of the restriction endonuclease, Hae I11 (10 Units/pl). Samples from the offspring, the dam, and all putative sires were electrophoresed through 1%

DNA Fingerprints in Chimpanzees 93

agarose gels until all fragments less than 0.5 Kb had run off the gel, then transferred to nylon filters (Zetabind) overnight, using a 1 M ammonium acetate, 0.04 M NaOH transfer solution [Westneat et al., 19881. Whole, wild-type MI3 phage was radiola- beled with 32P-CTP by random oligonucleotide priming [Feinberg and Volgelstein, 19831. After overnight pre-hybridization, the filters were hybridized in a solution consisting of 7% SDS, 1 mM EDTA (pH 8.0), 0.263 M Na,HPO,, 1% BSA, and no competitor DNA, for 36-48 hours at 60"C, then washed [Westneat et al., 19881. Autoradiography was performed with intensifying screens at - 80°C between 12 and 120 hours. Fragment sizes were estimated by comparison to size standards run on each gel.

RESULTS

The 15 bp core unit repeated in tandem at two locations within the protein I11 gene of M13 bacteriophage [Vassart et al., 19871 is complementary to variable sequences in the chimpanzee. Preliminary hybridizations involving known family groups demonstrate that the detected fragments segregate in families according to the rules of Mendelian inheritance, as illustrated in Figure 1. M13 can therefore be used as a DNA probe for paternity ascertainment. Barring possible mutations, fragments present in an offspring but absent in its mother are paternal bands and are used to identify the true sire. Figures 2 and 3 illustrate the assignment of paternity in several multimale cases.

A band of approximately 4.4 Kb in the male offspring depicted in Figure 3 cannot be attributed either to the dam or to the putative sire (who possesses all other non-maternal bands) and evidently represents a mutation to a new fragment length. The mutation rate among fragments detected by the myoglobin-derived core sequence is estimated at 0.00417 per generation [Jeffreys et al., 1985a; see also Jeffreys et al., 19881, and the evident mutation is consistent with the expected 1.75 new mutations expected under a Poisson process with parameter 0.00417 for the 21 offspring re- solved with this method. A more extensive study including estimation of the mutation rate for MI 3-detected fragments is in preparation. In addition, we are currently typing a series of polymorphic protein-coding loci to confirm the results of the fingerprint studies.

Where reliable information on potential sires was available, paternity could be accurately assigned, and in known cases of paired breeding the biological father was never excluded by this procedure. Electrophoresis time and length of autoradio- graphic exposure were varied to obtain optimum resolution and visualization of both small and large DNA fragments. Many of the paternal alleles present in an offspring are shared by several potential sires. However, to date we have been able to identify at least two exclusionary bands per putative sire prior to calling an exclusion. Al- though cases may involve up to six potential sires [Alford and Bloomsmith, 19891, exclusionary power seems unrelated to the number of males and appears to be more dependent on the number of non-maternal fragments identified in an offspring.

Individual chimpanzees have an average of about 20 resolvable Hae I11 bands in the length interval between approximately 1.6 and 12 or more Kb (Figs. 1-3). By comparison with studies performed using fingerprinting probes on other species, the estimated incidence of band sharing among unrelated chimpanzees (P = .487) is higher than that estimated for feral bird populations [Burke and Bruford, 19871, about

94 Ely and Ferrell

Fig. 1. DNA “fingerprints” of two known chimpanzee families involving a single male demonstrate that the detected fragments segregate in families. Fragments marked in each of the offspring that do not appear in the dam are paternal fragments. All fragments in an offspring can be traced to at least one of the parents. Fragment sizes (in Kb) are estimated by comparison to a size standard run on each gel, as indicated by the scale on the left.

equal to domesticated animals, including dogs, cats, and horses [Jeffreys and Morton, 1987; Georges et al., 19881, and lower than inbred strains of mice [Jeffreys et al., 19871. A similar high degree of band-sharing has also been observed in captive marmosets [Dixson et al., 19881.

DNA Fingerprints in Chimpanzees 95

Fig. 2. DNA “fingerprints” used to identify the true sire of an animal born in a multi-male housing situation. A paternal fragment around 2.75 Kb excludes the male on the left. A pair of paternal fragments just below 5 Kb excludes the male on the right. Owing both to inclusion and exclusion, the sire is identified as the middle male.

DISCUSSION

Among captive-born chimpanzees, the use of DNA fingerprints revealed by hybridization of genomic DNA digests to bacteriophage MI3 DNA has proved to be an efficient procedure for the assignment of paternity. Because of the high degree of variability revealed by this probe, all 21 cases to date involving multiple potential sires result in compatibility with only a single sire and the direct exclusion of paternity

96 Ely and Ferrell

Fig. 3. DNA “fingerprints” used to identify the true sire of an animal born in a multi-male housing situation. In this instance, the true sire is identified as the first male by a trio of fragments near 10 Kb, all of which are absent in the second male. A fragment of approximately 4.4 Kb in the offspring cannot be attributed to either the dam or the putative sire and apparently represents a novel mutation. See text for discussion.

for the non-included males. The frequency of band sharing among randomly selected chimpanzees seems higher than among individuals from other outcrossing vertebrate species. However, historical information on the geographical origin of wild-caught chimpanzees is so poor that we cannot precisely estimate the degree of relatedness among the individuals studied here.

Even with a high probability of band-sharing, none of the 21 paternity cases

DNA Fingerprints in Chimpanzees 97

tested so far has failed to be resolved with M13. Using a genera1 formula for the simple case of k alleles at n unlinked loci [Chakravarti and Li, 19831, where n = 15 [Jeffreys et al., 19861, k = l /q = 4.1, and q = p/2 = 0.487/2 [q2 being approx- imately zero for small q, see Jeffreys et al. [1985b]), the mean probability of exclu- sion using MI3 is estimated at greater than P = ,999. By comparison, paternity exclusion among captive rhesus macaques ( M . mulatta) using a combination of 35 electrophoretic markers, leucocyte antigens, and red cell antigens resulted in a com- bined probability of exclusion of P = .995 [Smith et al., 19841.

In order to gain some insight into the frequency and distribution of hypervari- able loci in the chimpanzee, we are extending our studies to a number of locus- specific VNTRs of the type described by Nakamura et al. [1987, 19881. The results obtained with these probes should corroborate the reliability of the M13 results reported here.

CONCLUSIONS

1. The use of molecular genetic techniques based on the availability of DNA probes that detect hypervariable sequences in the genomic DNA of common chim- panzees is a powerful technique for the assignment of parentage.

2. In the 21 cases analyzed here, this assignment includes both the unique assignment of biological fathers in multi-male situations and the simultaneous exclu- sion of all other potential fathers.

3. These techniques are applicable to the resolution of paternity questions and the unique identification of individual animals in any management situation.

ACKNOWLEDGMENTS

This work was supported by the National Chimpanzee Breeding and Research Program, Grant R24 RR03577. We acknowledge the enthusiastic cooperation of Pat Alford, Mike Keeling, Jo Fritz, Brent Swenson, and their staffs. We thank three anonymous reviewers for their perceptive and helpful comments.

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