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DNA fingerprinting in non-human populations
DNA fingerprinting in non-human populations Bill Amos and Josephine Pemberton University of Cambridge, Cambridge, UK DNA fingerprinting of non-human populations is beginning to fulfill its early promise, and in the past year there has been a flush of papers on mammalian breeding systems. However, many people, particularly field workers, believe that progress in this area has been slow. Attention is now focused on two amenable alternatives: microsatellite polymorphisms and randomly amplified polymorphic DNA. Of these, it is probable that microsatellites hold the key to rapid, efficient and highly informative screening of the genetic variability that exists within natural populations. Current Opinion in Genetics and Development 1992, 2:857-860
Introduction
Polygynous mating systems
In humans, DNA fingerprinting [1] visually identifies DNA polymorphism simultaneously at a large number of independently segregating, highly variable 'minisatellite' loci. The resulting genetic bar-codes can be used to determine individual identity and parentage and to estimate relatedness. Soon after the discovery of minisatellites, similar sequences were demonstrated in a wide variety of higher organisms and used to investigate a variety of issues in behavioural ecology [2]. Two emerging trends are discussed in this review. First, although the use of DNA fingerprinting in behavioural ecology has been dominated until recently by studies of bird populations, the past year has seen a surge of studies of mammalian social organization and reproductive behaviour. Second, increasing impatience with some aspects of the original multilocus technique has spawned a number of new approaches. Whether or not these methods justify the term DNA fingerprinting in its strictest sense, they provide interesting alternatives, each with its own pros and cons. Any new study should give careful consideration to which technique is most appropriate for the question being asked.
Mammalian social organization In mammals, gestation and lactation bias parental care of offspring more towards the female than in any other groups. For males, the principal way to increase reproductive success is to fertilize as many females as possible, although in a small proportion of species males take an alternative option and help to rear the offspring. In either case, paternity is critical to understanding the evolution of male behaviour.
Field studies on polygynous species have usually relied on observed matings, harem size and behavioural dominance as estimators of male reproductive success. With the advent of DNA fingerprinting it has become possible to explore the accuracy of these measures. In the seasonally breeding red deer (Cervua elaphus), males defend harems of females. Females enter a brief oestrus and usually mate only once with the harem male. In a long-term study on the Isle of Rum, Scotland, male reproductive success has been estimated from harem size and tile duration of harem holding. For a sample of 80 calves, Pemberton et al. [3] compared these observational measures with true paternity, as determined using DNA fingerprinting. Although the two methods gave almost identical rankings, molecular paternity revealed that the most successful males father more offspring than was previously estimated, and many less successful males were obtaining few or no paternities. Cercopithecine monkeys, such as macaques, typically live in mixed-sex groups. Oestrous females mate frequendy, often with many different males. Dominant males can monopolize matings, but previous studies of paternity, mostly using protein electrophoresis in captive groups, have failed to show a correlation between dominance and offspring number, or have given equivocal results. In a free-living population of long-tailed macaques (Macaca fascicularis) living in Sumatra, Indonesia, De Ruiter et al. [4], using protein electrophoresis and DNA fingerprinting to investigate the paternity of 45 offspring, clearly show that high-ranked males father most offspring. They attribute the contrasting results in captive and wild macaques to the artificial social conditions under which the former live. One way some mammalian males increase their mating success is to work together. In the Serengeti, Tanzania,
Abbreviation RAPD--random amplified polymorphic DNA.
© Current Biology Ud ISSN 0959-437X
857
858
Genomesand evolution male lions (Panthera leo) form coalitions which take over groups of cooperatively breeding, related females, kill existing cubs and father a new cohort. The larger the coalition the greater the chance of a takeover but the further any fitness spoils have to stretch. In one of the most impressive applications of DNA fingerprinting in any natural population to date, Packer et aZ [5 "°] and Gilbert el al. [6] examined relatedness between coalition members and the parentage of cubs. Whereas small coalitions (two males) may comprise unrelated males who both father cubs, larger coalitions (three to nine males) always contain some relatives, generally full or half brothers. Although junior members may not get the chance to father their own pups, they may still benefit through the inclusive fitness by helping their higher-ranking brothers to produce nephews and nieces. Like macaques and lions, pilot whales (Globicephala malaena) conmlonly live in mixed-sex groups or pods. Unlike these species, most pilot whale social behaviour goes unseen. Group structure can only be investigated by using molecular genetics to establish relatedness between members of stranded or hunted groups. DNA fingerprinting has shown that foetal cohorts are fathered eitiler by only a few unrelated males, or by a larger number of related males, and that the fathers are rarely present in the group [7]. This suggests that mating occurs during temporary meetings with other groups, whether mixedsex or all-male, raising the possibility that pilot whales display an entirely new mammalian mating system.
Monogamy and helper systems In mammals, monogamy is rare (occurring in less than 5% of species) but widely scattered, and its evolutionary raison d'etre is still not known. Recently, Ribble [8] has used DNA fingerprinting to establish paternity for 82 infant Californian deermice (Peromyscus californi. cus), confirming their apparently monogamous mating system. Among related species, P. californicus is notable for the high survival of young to weaning [9] and it seems likely that paternal care is responsible, although further research, perhaps involving the removal of the males, is required to establish this point rigorously. In some monogamous carnivores, a reproductive pair may receive help in raising their offspring from other mature individuals, probably their previous offspring. In mammals, this strategy has several possible, but few as yet proved, advantages [10]. To understand such a system further, it is essential to know the precise family relationships between the principal pair, the helpers and the offspring being reared. For example, in one of many bird species with this rearing system, fingerprinting has shown that male helpers father some offspring, thus gaining direct benefits [ 11 ]. Sampling grey wolves (Canis lupus) is a considerable challenge to human ingenuity. However, Lehman et al. [12] have managed to sample 42 packs, from three regions in the United States, with varying completeness. Pack structure was investigated using a combination of mitochondrial- and minisatellite-DNA variation. While
most packs conformed to expectation, in that they consisted of an unrelated pair and their descendants, nine packs contained one or more individuals unrelated to the rest of the pack. Just what benefits accrue to an individual joining an unrelated pack remain obscure. Although some of these early mammalian fingerprinting studies confirm old ideas based on theory or observation, there are sufficient surprises to be wary of generalizations. After all, we must expect the earliest reports to come from studies where field workers guide their associated geneticists most accurately to the father. We have yet to see the mammalian equivalent to the case of the redwinged blackbird, in which Gibbs el aL [13] expected within-territory paternity, but instead uncovered a soap opera of between-territory paternities. However, we have no doubt that such a surprise will come in time.
A new generation of profiling techniques The vast potential of DNA fingerprinting in non-human populations, as indicated in the previous paragraphs, has never been in doubt. However, progress has been slower than man), expected. The reasons for this are easy to identify. First, multilocus fingerprinting requires Southern blotting of exceptional quality and, in the case of population studies that often involve hundreds of animals, consistency. Second, interpretation of DNA fingerprints, consisting of bands of widely varying intensities and infinitely variable position, has caused headaches. Accurate interpretation requires that adjacent, or nearby, tracks on the same gel are used to compare fingerprints from different individuals. In a paternity stud),, this means that a panel of males must be rerun near every single mother-offspring pair. This is wasteful of both samples and laboratory time. Several labs have explored computer storage and the comparison of band patterns, but computer matches still need experimental verification. Third, in many invertebrates, DNA content is limiting. For example, Blanchetot [14], using multilocus fingerprinting to examine relatedness in a honeybee (Apis mellifera) colony, obtained enough DNA for only one or two tests per insect and used these to look at relatedness in a colony. For many smaller species, DNA yield effectively precludes fingerprinting. A growing band of workers is thus abandoning multilocus fingerprinting in favour of techniques that overcome one or more of these difficulties. The new techniques that have been developed exploit either simpler methods for looking at minisatellite variation, or novel classes of highly variable DNA. No studies using these techniques in natural populations have yet been published, but many projects using them are in progress, and now is a good time to assess the alternatives.
Locus-specific minisatellites One way of simplifying a multilocus DNA fingerprint is to clone and analyze individual minisatellite loci. Many
DNA fingerprinting in non-human populations Amos, Pemberton minisatellite loci are truly hypervariable, with heterozygosities in excess of 95%, and genotypes can be scored, stored and used to accurately compare fingerprints from different individuals screened at widely different times. Furthermore, knowledge of which bands are allelic to which others greatly enhances the amount of genetic information derived per band; a handful of loci can be almost as informative as a multilocus fingerprint. Unfortunately, the very sequences that make minisatellites so variable are also, apparently, recognized by bacteria, making these loci extremely labile during cloning. In the absence of elaborate precautions [15], they seem to fall apart. Because of this, few non-human loci have been isolated so far (one exception being several probes cloned from various birds in the Burke laboratory in Leicester [16]), and single locus minisatellite analysis seems destined to be superceded by more accessible approaches.
Microsatellites The potential of microsatellites as locus-specific polymorphic markers was recognized simultaneously by three groups [17-19]. Microsatellites are short tracts of repeating di- or trinucleotide motifs, within which molecular 'slippage' (DNA-strand misalignment) leads to high rates of gain and loss of repeat units. Screening relies on PCR, which uses two oligonucleotide primers, complementary to nearby opposing sites, to initiate repeated rounds of DNA synthesis across the intervening region. The DNA between, and including, the primer sites is thus amplified exponentially. If PCR primers are designed to bind to unique DNA sequences either side of a microsatellite region, the length of the amplified product will reflect the number of repeat units in the array. Most workers aim for fragments about 100-250bp in length, which can be unambiguously scored to a precise number of nucleotide bases using polyacrylamide sequencing gels. Finally, for economy of effort, careful primer design can allow several loci to be amplified and electrophoresed simultaneously. To us, microsatellites appear to offer the best blend of characteristics cu[rently available. They occur in all higher organisms tested so far, and are abundant, widely distributed in the genome, and easy to clone. Through PCR, they can be screened effciently from little, poor quality starting material. Variability is allelic and at perhaps 2-10 alleles per locus, generally lower than for minisatellite loci, although it can be very high; one pilot whale locus has 54 alleles scored in 200 related animals (W Amos, C Schloetterer, D Tautz, unpublished data). One further merit of using microsatellites is that primers
often work well on related species. For example, Schloetterer et al. [20 °-] have shown that four sets of primers derived for pilot whales will all amplify products in a wide range of other species, covering virtually all extant cetacean groups. In the majority of cases, variability was observed.
Random amplified polymorphic DNAs At present, there is much interest in the use of random amplified polymorphic DNAs (RAPDs) as markers, both for quantitative traits in applied studies, and for individuals and populations in the wild. A recent review [21] outlines the uses to which RAPDs may be put in natural populations, including parentage testing and population differentiation. As with microsatellite analysis, the RAPD technique relies on PCR. Normally, PCR is directed towards a specific DNA region, high stringency ensuring that the primers bind to unique sites and only one product results. However, at lower stringency, primers can bind less precisely to many more sites. Whenever two such sites lie suffciently close to one another, and in the correct orientation, a PCR product will result. RAPD analysis involves using almost any short primer sequence, usually about 10 nucleotides in length, at a stringency such that a number of these anonymous PCR products are generated. Individual variability is screened on agarose gels and, just as with multilocus fingerprinting, relatedness is deduced from the number of bands that two individuals have in common. Importantly, RAPD bands are usually non-allelic, being scored simply as present or absent. Just as many multilocus fingerprinting probes can be applied to most organisms, so can RAPDs; indeed, the), may be more applicable because there are no a p r i o r i sequence requirements. Furthermore, RAPDs are technically much simpler and faster, and more 'off-the-shelf'. Finally, being PCR-based, the technique can be applied to extremely small samples of DNA, such as those available from insects, where DNA yield is often insufficient for multilocus DNA fingerprinting. As promising as RAPD technology appears to be, its application in population analysis is not straightforward. First, although it is easier technically to use than multilocus fingerprinting, there still remains the problem of interpreting complex banding patterns in agarose gels. Second, because the bands produced in RAPD analysis are anonymous, there is frightening scope for artifacts. Two scenarios spring to mind. Many natural populations harbour internal parasites whose DNA will co-purify and co-PCR with that of the host, and will be indistinguishable from the host's DNA bands on the final gel. Furthermore, because many field workers obtain samples from study organisms as and when the), can, inevitably some sampies will contain more degraded DNA than others and different tissues will give rise to DNA containing different impurities. Either reason could alter a RAPD band profile; without the framework of an allelic system for reference, such changes could be very misleading. We can see two areas where RAPDs may be the technique of choice. First, as a source of anonymous genetic markers for analyzing quantitative traits and genome mapping. Second, for paternity analysis in cases w h e r e DNA is limited and where speed and convenience are more important factors than genetic resolving power. A prime example of this is the analysis of parentage in large, captive-reared broods, particularly insects, as in the dragonfly study mentioned in [21].
859
860
Genomes and evolution
Conclusions DNA fingerprinting of non-human populations has proved somewhat slow, but where analysis is complete, the results have justified the wait. Fingerprinting, in its original form, will probably remain the method of choice for straightforward paternity testing where the father is one of only a few candidates. In most other instances, new techniques can lend greater speed and yield more information, suggesting that molecular analysis will become an essential component of any field study. Among the alternatives that are currently available, we predict that the future lies increasingly with microsatellites.
7.
A~IOS W, BARRETtJA, DOVER GA: Breeding Behaviour of Pilot Whales Revealed by DNA Fingerprinting. Heredi O, 1991, 67:49-55.
8.
RIBBLEDO: The Monogamous Mating System of Peromyscus californicus as Revealed by DNA Fingerprinting. Bebat, Ecol Sociobiol 1991, 29:161-166.
9.
RIBBLEDO: Lifetime Reproductive Success and its Correlates in the Monogamous Rodent, Peromyscus californicus. J Anita Ecol 1992, 61:457-468.
10.
E,Xlt.ENST (EDS): Evolution of Cooperative Breeding in Birds and Mammals. In Bebat,ioural Ecology. an Et'ohttiona O , / ~ proach. Edited by Krebs JR, Davies NB. Oxford: Blackwells; 1991.
11.
RABENOLDPP, RABENOI.DKN, PIPER WH, I-bvtT3OCKJ, ZACK SW: Shared Paternity Revealed by Genetic Analysis in Cooperatively Breeding Tropical Wrens. Nature 1990, 348:538-540.
Unlike the original multilocus technique, none of the new methods discussed here provides an individual-specific DNA pattern, and so the implication of the term fingerprinting is no longer valid. Tl'ms there is a case to be made for a less explicit generic temL such as DNA profiling.
12.
LEHMANN, CLM~.KSON P, MECH LD, MEIER TJ, WAYNE RK: A Study of the Genetic Relationships within and Among Wolf Packs Using DNA Fingerprinting and Mitochondrial DNA. Behat, Ecol Sociobiol 1992, 30:83-94.
13.
GIBBSI-IL, WEATHERHEADPJ, BOAG PT. WHITE BN, TABAK LM, HOYSAK DJ: Realized Reproductive Success of Polygynous Red-winged Blackbirds Revealed by DNA Markers. Science 1990, 250:1394-1397.
References and recommended reading
14.
BIANCHETOTA: Genetic Relatedness in Honeybees as Established by DNA Fingerprinting. Heredi O, 1991, 82:391-396.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of speckfl interest •, of outsumding interest
t5.
ARMOURJAL, POVEY S, JEREMIAH S, JEFFREYS AJ: Systematic Cloning of H u m a n MinisateUites from Ordered Charomid Libraries. Genomics 1990, 8:501-502.
16.
lt-x-aOrTl..O, BURKET, ARMOURJAI., JEFFREYS AJ: Hypervariable Minisatellite DNA Sequences in the Indian Peafowl P a v o CtYstatus. Genomic.v 1991, 9:587-597.
17.
TAUTZ D: Hypervariable Simple Sequences as a General Source of Polymorphic DNA Markers. Nucleic .-Icids Res 1989, 17:6463-6471.
1.
JEFFREYSAJ, Wll£ON V, THI.:IN SL: Hypervariable Minisatellite Regions in H u m a n DNA. Nalttre 1985, 314:67-73.
2.
BURKET: DNA Fingerprinting and O t h e r Methods for the
Study of Mating Success. Trend~ Ecol Et,ol 1989, 4:139-144. 3.
PEMBERTONJM, AH~ON SD, GUINNESS FE, CLU'I'rON-BROCKTII, DOVER GA: Behavioural Estimates of Male Mating Success Tested by DNA Fingerprinting in a Polygynous Mammal. Behav Ecol 1992, 3:66-75.
18.
IJ'rr M, Lul~' JA: A Hypervariable Microsatellite Revealed by in-Vitro Amplification of a Dinucleotide Repeat within the Cardiac Muscle Actin Gene. Am .I Httm Genel 1989, 44:397-401.
4.
DE RUITERJR, SCHEFFRAHNW. TROMNIELENGJJM, UITTERI.INDEN AG, MARTIN RD, V,'a'~ HOOFF JARAM: Male Social Rank and Reproductive Success in Wild Long-tailed Macaques. Paternit}, Exclusions by Blood Analysis and DNA Fingerprinting. In Paterni O, in Primates: Genetic Tests a n d ?beoriex hnplications of H u m a n DNA Fingelprinting. Edited by Martin RD, Dixson AF, Wickings EJ. B:LSeI: S Kargel AG; 1992.
19.
WEBI-:RJL, ~'bw PE: Abundant Class of H u m a n DNA Polymorphisms w h i c h Can be Typed Using the Polymerase Chain Reaction. Am .I Httm Genet 1989, 44:388-396.
5. °o
PACKERC, GIt.BERT DA, PUSEY AE, O'BmEN ~I: A Molecular Genetic Analysis of Kinship and Cooperation in African Lions. Nature 1991, 351:562-565. This paper describes how subordinate male lions help their brothers to take over a pride, even if they themselves do not obtain paternities. Both parentage testing and estimates of relatedness were used to unravel the lion's complex breeding system, establishing a good example of inclusive fitness benefits in a mammal. 6.
GILBERTDA, PACKER C, PUSlP," AE, STEPHENS JC, O'BRIEN SJ: Analytical DNA Fingerprinting in Lions: Parentage, Genetic Diversity and Kinship. J ltered 1991, 82:378-386.
20. •.
SCHI.OE'rW_RF_RC, AMOS W, TAUT/. D: Conservation of Polym o r p h i c Simple Sequence loci in Cetacean Species. Nature 1991, 354:63-65. This paper is the first to make a rigorous comparison of homologous microsatellite loci in related species. Four loci, cloned from the longfinned pilot whale, are compared across 11 species, representing most major cetacean radiations. The microsatellite flanking sequences are found to be conserved and fnost loci are variable in all species tested. 21.
l-l*d)ttYs H, BAtJCK M, SCHIERWATERB: Applications of Amplified Polymorphic DNA (RAPD) in Molecular Ecology. Mol Ecol 1992, h55-63.
B ?m~os and J Pemberton, Department of Genetics, UniversiW of Cambridge, Downing Street, Cambridge CB2 3EH, UK,
Introduction
Polygynous mating systems
In humans, DNA fingerprinting [1] visually identifies DNA polymorphism simultaneously at a large number of independently segregating, highly variable 'minisatellite' loci. The resulting genetic bar-codes can be used to determine individual identity and parentage and to estimate relatedness. Soon after the discovery of minisatellites, similar sequences were demonstrated in a wide variety of higher organisms and used to investigate a variety of issues in behavioural ecology [2]. Two emerging trends are discussed in this review. First, although the use of DNA fingerprinting in behavioural ecology has been dominated until recently by studies of bird populations, the past year has seen a surge of studies of mammalian social organization and reproductive behaviour. Second, increasing impatience with some aspects of the original multilocus technique has spawned a number of new approaches. Whether or not these methods justify the term DNA fingerprinting in its strictest sense, they provide interesting alternatives, each with its own pros and cons. Any new study should give careful consideration to which technique is most appropriate for the question being asked.
Mammalian social organization In mammals, gestation and lactation bias parental care of offspring more towards the female than in any other groups. For males, the principal way to increase reproductive success is to fertilize as many females as possible, although in a small proportion of species males take an alternative option and help to rear the offspring. In either case, paternity is critical to understanding the evolution of male behaviour.
Field studies on polygynous species have usually relied on observed matings, harem size and behavioural dominance as estimators of male reproductive success. With the advent of DNA fingerprinting it has become possible to explore the accuracy of these measures. In the seasonally breeding red deer (Cervua elaphus), males defend harems of females. Females enter a brief oestrus and usually mate only once with the harem male. In a long-term study on the Isle of Rum, Scotland, male reproductive success has been estimated from harem size and tile duration of harem holding. For a sample of 80 calves, Pemberton et al. [3] compared these observational measures with true paternity, as determined using DNA fingerprinting. Although the two methods gave almost identical rankings, molecular paternity revealed that the most successful males father more offspring than was previously estimated, and many less successful males were obtaining few or no paternities. Cercopithecine monkeys, such as macaques, typically live in mixed-sex groups. Oestrous females mate frequendy, often with many different males. Dominant males can monopolize matings, but previous studies of paternity, mostly using protein electrophoresis in captive groups, have failed to show a correlation between dominance and offspring number, or have given equivocal results. In a free-living population of long-tailed macaques (Macaca fascicularis) living in Sumatra, Indonesia, De Ruiter et al. [4], using protein electrophoresis and DNA fingerprinting to investigate the paternity of 45 offspring, clearly show that high-ranked males father most offspring. They attribute the contrasting results in captive and wild macaques to the artificial social conditions under which the former live. One way some mammalian males increase their mating success is to work together. In the Serengeti, Tanzania,
Abbreviation RAPD--random amplified polymorphic DNA.
© Current Biology Ud ISSN 0959-437X
857
858
Genomesand evolution male lions (Panthera leo) form coalitions which take over groups of cooperatively breeding, related females, kill existing cubs and father a new cohort. The larger the coalition the greater the chance of a takeover but the further any fitness spoils have to stretch. In one of the most impressive applications of DNA fingerprinting in any natural population to date, Packer et aZ [5 "°] and Gilbert el al. [6] examined relatedness between coalition members and the parentage of cubs. Whereas small coalitions (two males) may comprise unrelated males who both father cubs, larger coalitions (three to nine males) always contain some relatives, generally full or half brothers. Although junior members may not get the chance to father their own pups, they may still benefit through the inclusive fitness by helping their higher-ranking brothers to produce nephews and nieces. Like macaques and lions, pilot whales (Globicephala malaena) conmlonly live in mixed-sex groups or pods. Unlike these species, most pilot whale social behaviour goes unseen. Group structure can only be investigated by using molecular genetics to establish relatedness between members of stranded or hunted groups. DNA fingerprinting has shown that foetal cohorts are fathered eitiler by only a few unrelated males, or by a larger number of related males, and that the fathers are rarely present in the group [7]. This suggests that mating occurs during temporary meetings with other groups, whether mixedsex or all-male, raising the possibility that pilot whales display an entirely new mammalian mating system.
Monogamy and helper systems In mammals, monogamy is rare (occurring in less than 5% of species) but widely scattered, and its evolutionary raison d'etre is still not known. Recently, Ribble [8] has used DNA fingerprinting to establish paternity for 82 infant Californian deermice (Peromyscus californi. cus), confirming their apparently monogamous mating system. Among related species, P. californicus is notable for the high survival of young to weaning [9] and it seems likely that paternal care is responsible, although further research, perhaps involving the removal of the males, is required to establish this point rigorously. In some monogamous carnivores, a reproductive pair may receive help in raising their offspring from other mature individuals, probably their previous offspring. In mammals, this strategy has several possible, but few as yet proved, advantages [10]. To understand such a system further, it is essential to know the precise family relationships between the principal pair, the helpers and the offspring being reared. For example, in one of many bird species with this rearing system, fingerprinting has shown that male helpers father some offspring, thus gaining direct benefits [ 11 ]. Sampling grey wolves (Canis lupus) is a considerable challenge to human ingenuity. However, Lehman et al. [12] have managed to sample 42 packs, from three regions in the United States, with varying completeness. Pack structure was investigated using a combination of mitochondrial- and minisatellite-DNA variation. While
most packs conformed to expectation, in that they consisted of an unrelated pair and their descendants, nine packs contained one or more individuals unrelated to the rest of the pack. Just what benefits accrue to an individual joining an unrelated pack remain obscure. Although some of these early mammalian fingerprinting studies confirm old ideas based on theory or observation, there are sufficient surprises to be wary of generalizations. After all, we must expect the earliest reports to come from studies where field workers guide their associated geneticists most accurately to the father. We have yet to see the mammalian equivalent to the case of the redwinged blackbird, in which Gibbs el aL [13] expected within-territory paternity, but instead uncovered a soap opera of between-territory paternities. However, we have no doubt that such a surprise will come in time.
A new generation of profiling techniques The vast potential of DNA fingerprinting in non-human populations, as indicated in the previous paragraphs, has never been in doubt. However, progress has been slower than man), expected. The reasons for this are easy to identify. First, multilocus fingerprinting requires Southern blotting of exceptional quality and, in the case of population studies that often involve hundreds of animals, consistency. Second, interpretation of DNA fingerprints, consisting of bands of widely varying intensities and infinitely variable position, has caused headaches. Accurate interpretation requires that adjacent, or nearby, tracks on the same gel are used to compare fingerprints from different individuals. In a paternity stud),, this means that a panel of males must be rerun near every single mother-offspring pair. This is wasteful of both samples and laboratory time. Several labs have explored computer storage and the comparison of band patterns, but computer matches still need experimental verification. Third, in many invertebrates, DNA content is limiting. For example, Blanchetot [14], using multilocus fingerprinting to examine relatedness in a honeybee (Apis mellifera) colony, obtained enough DNA for only one or two tests per insect and used these to look at relatedness in a colony. For many smaller species, DNA yield effectively precludes fingerprinting. A growing band of workers is thus abandoning multilocus fingerprinting in favour of techniques that overcome one or more of these difficulties. The new techniques that have been developed exploit either simpler methods for looking at minisatellite variation, or novel classes of highly variable DNA. No studies using these techniques in natural populations have yet been published, but many projects using them are in progress, and now is a good time to assess the alternatives.
Locus-specific minisatellites One way of simplifying a multilocus DNA fingerprint is to clone and analyze individual minisatellite loci. Many
DNA fingerprinting in non-human populations Amos, Pemberton minisatellite loci are truly hypervariable, with heterozygosities in excess of 95%, and genotypes can be scored, stored and used to accurately compare fingerprints from different individuals screened at widely different times. Furthermore, knowledge of which bands are allelic to which others greatly enhances the amount of genetic information derived per band; a handful of loci can be almost as informative as a multilocus fingerprint. Unfortunately, the very sequences that make minisatellites so variable are also, apparently, recognized by bacteria, making these loci extremely labile during cloning. In the absence of elaborate precautions [15], they seem to fall apart. Because of this, few non-human loci have been isolated so far (one exception being several probes cloned from various birds in the Burke laboratory in Leicester [16]), and single locus minisatellite analysis seems destined to be superceded by more accessible approaches.
Microsatellites The potential of microsatellites as locus-specific polymorphic markers was recognized simultaneously by three groups [17-19]. Microsatellites are short tracts of repeating di- or trinucleotide motifs, within which molecular 'slippage' (DNA-strand misalignment) leads to high rates of gain and loss of repeat units. Screening relies on PCR, which uses two oligonucleotide primers, complementary to nearby opposing sites, to initiate repeated rounds of DNA synthesis across the intervening region. The DNA between, and including, the primer sites is thus amplified exponentially. If PCR primers are designed to bind to unique DNA sequences either side of a microsatellite region, the length of the amplified product will reflect the number of repeat units in the array. Most workers aim for fragments about 100-250bp in length, which can be unambiguously scored to a precise number of nucleotide bases using polyacrylamide sequencing gels. Finally, for economy of effort, careful primer design can allow several loci to be amplified and electrophoresed simultaneously. To us, microsatellites appear to offer the best blend of characteristics cu[rently available. They occur in all higher organisms tested so far, and are abundant, widely distributed in the genome, and easy to clone. Through PCR, they can be screened effciently from little, poor quality starting material. Variability is allelic and at perhaps 2-10 alleles per locus, generally lower than for minisatellite loci, although it can be very high; one pilot whale locus has 54 alleles scored in 200 related animals (W Amos, C Schloetterer, D Tautz, unpublished data). One further merit of using microsatellites is that primers
often work well on related species. For example, Schloetterer et al. [20 °-] have shown that four sets of primers derived for pilot whales will all amplify products in a wide range of other species, covering virtually all extant cetacean groups. In the majority of cases, variability was observed.
Random amplified polymorphic DNAs At present, there is much interest in the use of random amplified polymorphic DNAs (RAPDs) as markers, both for quantitative traits in applied studies, and for individuals and populations in the wild. A recent review [21] outlines the uses to which RAPDs may be put in natural populations, including parentage testing and population differentiation. As with microsatellite analysis, the RAPD technique relies on PCR. Normally, PCR is directed towards a specific DNA region, high stringency ensuring that the primers bind to unique sites and only one product results. However, at lower stringency, primers can bind less precisely to many more sites. Whenever two such sites lie suffciently close to one another, and in the correct orientation, a PCR product will result. RAPD analysis involves using almost any short primer sequence, usually about 10 nucleotides in length, at a stringency such that a number of these anonymous PCR products are generated. Individual variability is screened on agarose gels and, just as with multilocus fingerprinting, relatedness is deduced from the number of bands that two individuals have in common. Importantly, RAPD bands are usually non-allelic, being scored simply as present or absent. Just as many multilocus fingerprinting probes can be applied to most organisms, so can RAPDs; indeed, the), may be more applicable because there are no a p r i o r i sequence requirements. Furthermore, RAPDs are technically much simpler and faster, and more 'off-the-shelf'. Finally, being PCR-based, the technique can be applied to extremely small samples of DNA, such as those available from insects, where DNA yield is often insufficient for multilocus DNA fingerprinting. As promising as RAPD technology appears to be, its application in population analysis is not straightforward. First, although it is easier technically to use than multilocus fingerprinting, there still remains the problem of interpreting complex banding patterns in agarose gels. Second, because the bands produced in RAPD analysis are anonymous, there is frightening scope for artifacts. Two scenarios spring to mind. Many natural populations harbour internal parasites whose DNA will co-purify and co-PCR with that of the host, and will be indistinguishable from the host's DNA bands on the final gel. Furthermore, because many field workers obtain samples from study organisms as and when the), can, inevitably some sampies will contain more degraded DNA than others and different tissues will give rise to DNA containing different impurities. Either reason could alter a RAPD band profile; without the framework of an allelic system for reference, such changes could be very misleading. We can see two areas where RAPDs may be the technique of choice. First, as a source of anonymous genetic markers for analyzing quantitative traits and genome mapping. Second, for paternity analysis in cases w h e r e DNA is limited and where speed and convenience are more important factors than genetic resolving power. A prime example of this is the analysis of parentage in large, captive-reared broods, particularly insects, as in the dragonfly study mentioned in [21].
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Genomes and evolution
Conclusions DNA fingerprinting of non-human populations has proved somewhat slow, but where analysis is complete, the results have justified the wait. Fingerprinting, in its original form, will probably remain the method of choice for straightforward paternity testing where the father is one of only a few candidates. In most other instances, new techniques can lend greater speed and yield more information, suggesting that molecular analysis will become an essential component of any field study. Among the alternatives that are currently available, we predict that the future lies increasingly with microsatellites.
7.
A~IOS W, BARRETtJA, DOVER GA: Breeding Behaviour of Pilot Whales Revealed by DNA Fingerprinting. Heredi O, 1991, 67:49-55.
8.
RIBBLEDO: The Monogamous Mating System of Peromyscus californicus as Revealed by DNA Fingerprinting. Bebat, Ecol Sociobiol 1991, 29:161-166.
9.
RIBBLEDO: Lifetime Reproductive Success and its Correlates in the Monogamous Rodent, Peromyscus californicus. J Anita Ecol 1992, 61:457-468.
10.
E,Xlt.ENST (EDS): Evolution of Cooperative Breeding in Birds and Mammals. In Bebat,ioural Ecology. an Et'ohttiona O , / ~ proach. Edited by Krebs JR, Davies NB. Oxford: Blackwells; 1991.
11.
RABENOLDPP, RABENOI.DKN, PIPER WH, I-bvtT3OCKJ, ZACK SW: Shared Paternity Revealed by Genetic Analysis in Cooperatively Breeding Tropical Wrens. Nature 1990, 348:538-540.
Unlike the original multilocus technique, none of the new methods discussed here provides an individual-specific DNA pattern, and so the implication of the term fingerprinting is no longer valid. Tl'ms there is a case to be made for a less explicit generic temL such as DNA profiling.
12.
LEHMANN, CLM~.KSON P, MECH LD, MEIER TJ, WAYNE RK: A Study of the Genetic Relationships within and Among Wolf Packs Using DNA Fingerprinting and Mitochondrial DNA. Behat, Ecol Sociobiol 1992, 30:83-94.
13.
GIBBSI-IL, WEATHERHEADPJ, BOAG PT. WHITE BN, TABAK LM, HOYSAK DJ: Realized Reproductive Success of Polygynous Red-winged Blackbirds Revealed by DNA Markers. Science 1990, 250:1394-1397.
References and recommended reading
14.
BIANCHETOTA: Genetic Relatedness in Honeybees as Established by DNA Fingerprinting. Heredi O, 1991, 82:391-396.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of speckfl interest •, of outsumding interest
t5.
ARMOURJAL, POVEY S, JEREMIAH S, JEFFREYS AJ: Systematic Cloning of H u m a n MinisateUites from Ordered Charomid Libraries. Genomics 1990, 8:501-502.
16.
lt-x-aOrTl..O, BURKET, ARMOURJAI., JEFFREYS AJ: Hypervariable Minisatellite DNA Sequences in the Indian Peafowl P a v o CtYstatus. Genomic.v 1991, 9:587-597.
17.
TAUTZ D: Hypervariable Simple Sequences as a General Source of Polymorphic DNA Markers. Nucleic .-Icids Res 1989, 17:6463-6471.
1.
JEFFREYSAJ, Wll£ON V, THI.:IN SL: Hypervariable Minisatellite Regions in H u m a n DNA. Nalttre 1985, 314:67-73.
2.
BURKET: DNA Fingerprinting and O t h e r Methods for the
Study of Mating Success. Trend~ Ecol Et,ol 1989, 4:139-144. 3.
PEMBERTONJM, AH~ON SD, GUINNESS FE, CLU'I'rON-BROCKTII, DOVER GA: Behavioural Estimates of Male Mating Success Tested by DNA Fingerprinting in a Polygynous Mammal. Behav Ecol 1992, 3:66-75.
18.
IJ'rr M, Lul~' JA: A Hypervariable Microsatellite Revealed by in-Vitro Amplification of a Dinucleotide Repeat within the Cardiac Muscle Actin Gene. Am .I Httm Genel 1989, 44:397-401.
4.
DE RUITERJR, SCHEFFRAHNW. TROMNIELENGJJM, UITTERI.INDEN AG, MARTIN RD, V,'a'~ HOOFF JARAM: Male Social Rank and Reproductive Success in Wild Long-tailed Macaques. Paternit}, Exclusions by Blood Analysis and DNA Fingerprinting. In Paterni O, in Primates: Genetic Tests a n d ?beoriex hnplications of H u m a n DNA Fingelprinting. Edited by Martin RD, Dixson AF, Wickings EJ. B:LSeI: S Kargel AG; 1992.
19.
WEBI-:RJL, ~'bw PE: Abundant Class of H u m a n DNA Polymorphisms w h i c h Can be Typed Using the Polymerase Chain Reaction. Am .I Httm Genet 1989, 44:388-396.
5. °o
PACKERC, GIt.BERT DA, PUSEY AE, O'BmEN ~I: A Molecular Genetic Analysis of Kinship and Cooperation in African Lions. Nature 1991, 351:562-565. This paper describes how subordinate male lions help their brothers to take over a pride, even if they themselves do not obtain paternities. Both parentage testing and estimates of relatedness were used to unravel the lion's complex breeding system, establishing a good example of inclusive fitness benefits in a mammal. 6.
GILBERTDA, PACKER C, PUSlP," AE, STEPHENS JC, O'BRIEN SJ: Analytical DNA Fingerprinting in Lions: Parentage, Genetic Diversity and Kinship. J ltered 1991, 82:378-386.
20. •.
SCHI.OE'rW_RF_RC, AMOS W, TAUT/. D: Conservation of Polym o r p h i c Simple Sequence loci in Cetacean Species. Nature 1991, 354:63-65. This paper is the first to make a rigorous comparison of homologous microsatellite loci in related species. Four loci, cloned from the longfinned pilot whale, are compared across 11 species, representing most major cetacean radiations. The microsatellite flanking sequences are found to be conserved and fnost loci are variable in all species tested. 21.
l-l*d)ttYs H, BAtJCK M, SCHIERWATERB: Applications of Amplified Polymorphic DNA (RAPD) in Molecular Ecology. Mol Ecol 1992, h55-63.
B ?m~os and J Pemberton, Department of Genetics, UniversiW of Cambridge, Downing Street, Cambridge CB2 3EH, UK,