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Genetic fingerprinting and giant panda paternity. Working in a laboratory may seem a far-removed pursuit from the more hands-on, active approaches to conservation, such as habitat management and making population surveys. Lab-based scientists are using molecular-genetic techniques, however, to help protect one of the world’s most threatened mammals from becoming extinct. The giant panda, Ailuropoda melanoleuca (Figure 1), is a secretive animal that lives in the mountainous bamboo-forests of China. After some scientific debate it has been agreed that the giant panda is a type of bear rather than a racoon, albeit a rather odd bear. Bears are carnivores, but giant pandas have adopted a diet of bamboo shoots; they have even evolved a ‘sixth-finger’ from an elongated wrist bone that helps them handle their leafy food. The evolution of the giant panda is of particular interest to scientists, but why is the general public so familiar with its distinctive black and white markings? After all, this animal was ‘discovered’ only relatively recently by the west in 1869 after an expedition to China by Pere David. Perhaps our interest is because pandas look cute or they have apparently rejected meat for a vegetarian diet? A drawback of having specialised ecological needs is that the giant panda particularly vulnerable to disturbance to its environment. A few hundred thousand years ago, giant pandas were found in Burma, northern Vietnam and much of eastern and southern China. Now, largely due to human settlement and deforestation, giant pandas are only found in a few reserves in south-west China (Figure 2). To make matters worse, the pandas’ staple diet, bamboo, suffered a severe population crash in the 1980s. While it is difficult to assess accurately the number of wild pandas (after all, they now only live in areas where humans cannot), there are probably less than 1,0001,500 individuals. The giant panda is one of the rarest mammals in the world and considered to be on the brink of extinction. The plight of the giant panda has sparked a high priority conservation effort. With the founding of the World Wildlife Fund, which adopted the giant panda for its logo, this species has established itself as the very symbol of nature conservation. Its iconic status probably explains why we are so aware of this shy creature. Parentage analysis Giant pandas have been kept and bred in zoos with the hope that they could be released into the wild. One particular worry is that small populations, such as in zoos or reserves, quickly become inbred and lose their genetic diversity. Inbreeding is known to be harmful, probably because genetic variation allows a species to adapt to changes in their environment. So it is not simply enough to breed more pandas, we must also make best use of the available pool of genetic diversity. We can do this, for example, by maximising the number of different males used for breeding, rather than let a single male dominate, and by maintaining corridors of habitat between reserves to allow animals to migrate and interbreed. Unfortunately, pandas find it difficult to reproduce in captivity. Because the population size is critically low, any failures to impregnate females could have serious implications for the conservation of this species. Since there is not enough time to experiment with the mating success of different male pandas, and thereby assess the quality of their sperm, fertilisation is guaranteed by artificial insemination with cryopreserved semen from several males in addition to natural copulation. With so many potential fathers, the true paternity of the cubs is not known! It is possible that one male fathers most or all of the cubs, and this would lead to inbred animals after a few generations. Identifying fathers is clearly an essential part of maintaining genetic variation in the giant panda. Wild populations present different problems for conservation biologists. Successful management plans for many species are based on knowledge of, for example, territorial behaviour, the size and overlap of animals’ home ranges and the breeding structure. Tracking individuals directly is the only way to be certain of movement throughout an area but this rarely provides information about successful mating. This information is important because if migrants fail to interbreed, then populations should be considered to be separate. Another drawback for direct tracking is that it is logistically difficult, time consuming and expensive even for large and conspicuous animals. Directly observing and following the movements the shy, mountain-dwelling giant panda is virtually impossible. For both wild and captive populations we need a way of specifically identifying parents and offspring; it would also be valuable to track the movements of wild animals. As explained below, genetic markers are ideal for both of these. Using genetic markers A genetic marker is simply a region of DNA that varies between individuals - it is polymorphic - each possible version of a region is called an allele. Because mammals have two complete copies of their genetic material in each cell - one from their mother and one from their father – the cells of each individual have two alleles for each marker region. We can look at a whole set of such regions, determine which alleles are present at each and we have a genetic ‘fingerprint’ which uniquely identifies that individual (see Box 1). Furthermore, if we also fingerprint possible parents, we can usually tell which is the offspring of which parent because each offspring inherits one allele at each region from each parent (see Box 2). The origin of this variation is mutation, but it may seem surprising that there can be so much variation in an animal’s DNA. After all, the DNA encodes the genes that contain the instructions for building and regulating an organism, so we might expect it to be very tightly regulated. However, only about 20% of a human’s (and similar for other animals) DNA does encode genes. The rest appears to be so-called ‘junk-DNA’ and mutations in this junk DNA usually have no effect. What’s more, because the genetic code is degenerate, even some mutations within genes, especially those at the third base of a codon, often have no effect on the gene’s product. Of course some mutations do alter genes and they provide the variation on which natural selection works. Some of these alterations are good, some bad and some have little or no effect on the fitness of the individual bearing this altered DNA sequence. For the most part, however, mutations in genes cause problems and are removed. Hence, there is typically not enough variation in genes themselves to provide unique fingerprints. Only about 20% of your DNA, however, encodes for genes; the rest appears to be so-called ‘junk DNA’ and mutations in this DNA usually have no effect. Within the junk DNA, where mutation is not constrained by natural selection, strange things can happen and odd strings of sequence can start to grow in length. Sometimes, the DNA copying machinery stutters and duplicates a particular sequence of nucleotides. Once this has happened it is more likely to happen again in the same place, so patches of repeating sequence can expand over many generations. The ‘junk’ DNA of most genomes contains tens of thousands of regions like this where short sequences, such as CA, GTT or GATA, repeat dozens or hundreds of times (see Figure 3). These regions are called microsatellites and the number of repeats at each microsatellite is often immensely variable between individuals. To put it another way, they are polymorphic with many alleles at each region. A genetic fingerprint is a representation of the alleles present at a number of different microsatellites. The probability of individuals having the same alleles at several loci becomes very remote. Forensic scientists typically use a standard set of human microsatellites for identifying individuals from DNA extracted from material, such as blood, hair or saliva, that contains nucleated cells. For giant pandas we can generate a fingerprint in exactly the same way, once we have identified a panel of panda microsatellites. Paternity and indirect tracking In giant pandas, genetic fingerprinting has identified the fathers of many captive-bred cubs. It was found that in almost all cases the male involved with the natural mating sired the cub. The reasons for this are unclear at present, although the sperm used in artificial insemination may lose some quality when frozen for preservation. So we can identify parents in captivity, but what about tracking pandas in the wild? The answer lies in panda dung. As food passes through the digestive system it scrapes the lining of the gut and picks up epithelial cells which of course contain DNA. When we extract DNA from faeces we get a mixture of panda, bacteria and bamboo genetic material. However, the PCR amplifies only the panda DNA because we design PCR primers to match particular panda microsatellites. DNA can also be obtained from hair and saliva. This is important for conservation biology because we do not have to see or interfere with our organism. The potential for this used of extraneous material is huge. The genetic structure of brown bears in North America has already been studied using microsatellite fingerprints from faecal samples. By collecting faeces it is therefore possible to count the number of individuals, identify breeding pairs and their offspring and also track the movements of these individuals; indeed the bears are never seen or disturbed. DNA is also taken from museum specimens, such as bone and skin, which allows present-day diversity to be compared with that prior to industrialisation and habitat loss from human populations expansion. Thanks to junk-DNA, the pedigrees of many captive panda cubs have now been established. The need to prevent an erosion of genetic diversity within the captive panda populations has lead to exchanges of animals between different zoos. This increased collaboration should help achieve the ultimate goal of augmenting the wild population with healthy individuals that were bred in captivity. Terms explained Polymorphic: Having variation in the sequence of DNA among individuals. Degenerate code: A genetic code in which some amino acids may each be encoded by more than one codon. The genetic code is degenerate because there are many instances in which different codons specify the same amino acid. Codon: Three bases in a DNA or RNA sequence which specify a single amino acid. Key Points Loss of genetic diversity is believed to be detrimental to species’ survival prospects; more genetic diversity can be maintained in larger populations. Much DNA does not code for proteins – it is apparently junk-DNA. Junk DNA can withstand high rates of mutation and offer highly polymorphic regions of DNA to use as genetic markers. The breeding and family structure of both captive and wild populations is usually difficult to determine without using genetic markers. As DNA is inherited from each parent, the paternity of captive-bred offspring can be assigned without breeding information. Because many junk-DNA sequences are unique to individuals, specific genetic profiles recovered DNA extracted from extraneous material, such as hair or faeces, permits the natural movements of animals to be tracked without direct observation. Key Words conservation, DNA, genetic diversity, genetic fingerprint, giant panda, microsatellite Acknowledgments We wish to thank The Wellcome Trust for funding the travel fellowship to Shen Fujun and Prof S.J. Kemp that allowed us to develop a panel of giant panda microsatellites. The North of England Zoological Society generously financed the travel and accommodation of P.C. Watts to conferences held by the Chengdu Research Base for Giant Panda Breeding in China. Box 1 How do we determine the genetic fingerprint? Microsatellites usually vary between individuals because of loss or gain of a repeat and so their alleles vary in length. We can amplify a particular microsatellite by PCR and then accurately measure its length by gel electrophoresis. PCR (Polymerase Chain Reaction) is a very convenient way of copying specific regions of DNA. It is incredibly sensitive so we need only tiny quantities of starting DNA. We can specifically target panda DNA because we know the sequences surrounding our microsatellites. We design short pieces of DNA – primers – to match these sites and thus define the region between which the PCR will amplify. Electrophoresis Since DNA is negatively charged, we can separate different sized alleles by sieving the DNA fragments through a gel matrix under the influence of an electric current. Smaller fragments of DNA migrate faster than larger fragments, a process called gel electrophoresis. By running DNA fragments of known size alongside our samples, we can accurately measure the length of each microsatellite allele (see Figure 4). Box 2 Analysis of parentage and populations Parentage analysis is easy with microsatellites provided they have enough alleles and you have samples from enough possible parents. Each allele at each microsatelite in a panda cub DNA must have come from one of its parents. So for each microsatellite, we can eliminate as possible parents any animals not having at least one allele the same size as the cub. We don’t have to check many microsatellites before we have eliminated all but the actual parents. Population assignment Because microsatellite alleles have no function, they are not selected for or against and so are usually in Hardy-Weinberg equilibrium (see Biological Sciences Review, Vol. 15, No. 4, pp. 7-9). This means that for each microsatellite we can predict the expected number of pandas that will have any combination of alleles. So when we fingerprint a panda we can ask ‘what is the probability of this particular fingerprint occurring in each population?’. Some alleles are more common in one panda population than in another, so a particular microsatellite fingerprint is more likely to arise in one than the other. That panda is therefore probably a member of the population in which its fingerprint is most likely to occur. Figure 1. Giant pandas eating bamboo at Chengdu Research Base for Giant Panda Breeding, Chengdu, P.R. China. Figure 2. Sketch map of the distribution of giant panda fossils from the Pleistocene (light blue area) and present day geographic range of the giant panda (dark blue). Figure 3. A microsatellite sequence Figure 4. A genetic fingerprint. Figure 1. Figure 2. Russia Mongolia Beijing Chengdu Figure 3 Figure 4