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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