Australasian Science: Australia's authority on science since 1938

Decoding the Genome of the Tammar Wallaby

Wallaby neonate

The wallaby genome contains 1500 genes associated with smell, which the neonate uses to locate a teat in its mother’s pouch.

By Tony Papenfuss & Marilyn Renfree

The sequencing of the tammar wallaby genome has provided fascinating insights into its unique reproductive and immune systems.

The tammar wallaby (Macropus eugenii) is a small kangaroo. It is abundant on Kangaroo Island in South Australia, and is also found on the Abrolhos Islands, Garden Island and the Recherche Archipelago in Western Australia, as well as a few areas in the south-west corner of the mainland.

For more than 40 years, the tammar wallaby has been used as the model Australian marsupial for scientific research because, unlike most mammals, it has two types of control of its reproduction – a seasonal breeding pattern in the second half of the year controlled by day length, and a lactational breeding pattern in the first half of the year controlled by the sucking stimulus of the young in the pouch. The tammar was chosen for genetic sequencing because it is such a well-studied model with interesting biology.

The story of the tammar wallaby genome project begins between 130 and 160 million years ago during the

Cretaceous–Jurassic, when the tiny common ancestor of all mammals was scurrying around dodging dinosaurs. All mammals, including humans, mice and kangaroos, descend from this ancestral mammal, and there are many features in our genomes that date back to this time.

Eighty million years ago the supercontinent of Gondwana broke up to form the separate continents of Australia, Antarctica and South America. This isolated the marsupials in Australasia from those in South America. There is only one marsupial, the Virginia opossum, in North America.

Marsupial Sequencing
In 2003, the tammar wallaby and a South American marsupial, the grey short-tailed opossum, battled it out to see which would have their genome sequenced. The opossum is a pouchless marsupial from South America that had been more recently studied in some laboratories in the United States.

Initially, the National Human Genome Research Institute (NHGRI) in the US, which was responsible for funding the US program of genome sequencing, selected the opossum for sequencing, but they were impressed with our case for the wallaby and offered to help sequence it if we could find matching funds. Due to the efforts of Dr Sue Forrest from the Australian Genome Research Facility (AGRF) and others, it was decided that the tammar would be sequenced as well, albeit at a lower coverage, with costs split between the NHGRI in the USA and the State Government of Victoria, the AGRF, The Jack Brockhoff Foundation and AMRAD in Australia.

The building blocks of a genome are called nucleotides, which bond together to form the long molecules of DNA that carry genetic information in almost every cell in the body. The tammar genome contains about 2.9 billion nucleotides.

The job of sequencing the tammar genome was split between the AGRF, which has its headquarters in Melbourne, and the Human Genome Sequencing Center at the Baylor College of Medicine in Houston, Texas. To sequence the genome, it was smashed up into small fragments between 2000 and 6000 nucleotides long. Altogether about five million fragments or six billion nucleotides were sequenced, corresponding to about double the coverage of the entire tammar genome.

A sequenced genome is initially like a jigsaw puzzle with 10 million pieces. These pieces were then fitted back together by finding matches or overlaps between the 600-nucleotide-long strings of nucleotides.

Sequencing began in 2005 but it wasn’t until 2008, 6 long years since the initial proposal, that we finally received the first version of the assembled tammar genome. During this time new and cheaper sequencing technologies became available, so additional sequence was generated and three more versions of the assembly passed through our hands before the tammar genome paper was finally completed and published in August in Genome Biology.

But by this time the tammar wallaby was scooped of the honour of being the second published marsupial genome by the Tasmanian devil, which was sequenced using one of the next generation sequencing platforms by two different groups. While the Tasmanian devil will be the first marsupial to be sequenced twice, the tammar wallaby remains only the second marsupial to have its genome analysed in depth.

Making Sense of the Data
A sequenced genome is basically a big text file with about three billion nucleotide letters in it. So once you’ve got a genome sequence what do you do with it? How do you make sense of it?

The first job is to find all the genes. This, along with and many other analyses, is done by a bioinformatician. Bioinformatics is a relatively new area of science, and involves the application of mathematics, statistics and computer science to understanding biological data.

So bioinformaticians at the Walter and Eliza Hall Institute of Medical Research, the University of Sydney and the European Bioinformatics Institute in the United Kingdom started to search for all the genes. They used genes with the sequences known from other species to find similar genes in the tammar genome.

The next task was to try to see which genes were duplicated and which genes might have diverged from other species, and to start making sense and use of the data. Some of the results of these searches were very interesting indeed.

Tammar Immune Genes
The major histocompatibility complex (MHC) is a large cluster of genes found in the genomes of all jawed vertebrates. The genes found in the MHC play key roles in the immune system.

In eutherian mammals like humans and mice, the MHC is split into three regions. The opossum was the first marsupial to have its MHC studied, with a group of Australians and Americans finding that the opossum’s MHC contained the same kinds of genes as the human MHC but with the regions in a different order that was actually closer to the order seen in the chicken.

When the tammar genome was examined it turned out that only two out of these three regions were present. Remarkably, one family of genes was spread to locations over most of the chromosomes. This explosion of the tammar MHC seems to have had no ill effects on the animal. In fact, it disproves the old theory that the genes in the MHC complex must be clustered on the same chromosome.

Kangaroos give birth to highly underdeveloped young that lack a mature, adaptive immune system. After birth, the young climbs into the mother’s pouch where it attaches to a teat and remains there as it continues to develop. The pouch can be a dirty place that is rich in bacteria, yet the young flourish there. The pouch’s secret is a cocktail of antimicrobial proteins in the milk and in the pouch young itself. We now know that these antimicrobials can even kill drug-resistant pathogens, so they have potential for the development of new antibiotics for the human medical armoury.

The Tammar’s Amazing Smell
There are about 25,000 genes in the human genome. About 400 of these are olfactory receptor (OR) genes responsible for our sense of smell. We also have another 600 OR genes that don’t actually work.

The tammar genome revealed a remarkable 1500 OR genes. This is one of the largest olfactory receptor gene families in a mammal.

Why does the tammar have so many genes for smelling? The answer is probably twofold. First, its ancestors had to evade the now-extinct marsupial predators like the 150 kg marsupial lion (Thylacoleo) in Australia and the marsupial sabre-toothed tigers (Thylacosmilus) in South America, and even Australian mainland populations of the Tasmanian tiger (Thylacinus cynocephalus).

But there is another important reason for the tammar’s sophisticated sense of smell. The tiny neonate has to crawl up (against gravity) to reach the opening of the pouch and, once there, needs smells to direct it down into the pouch to locate one of the four available teats. These odours are almost certainly pheromones produced by the mammary glands of the mother.

Pheromones are also essential for the detection of females that are ready to mate. The male tammar spends a great deal of time sniffing at the pouch and genital openings of his female companions.

Development from Top to Tail
A family of genes called HOX genes controls the development of the body plan in early life across the entire animal kingdom. HOX genes are arranged in four clusters in mammalian genomes. The genes switch on like traffic lights over time from the head to the toe. The genes are very similar between mammals, but differences in where and when they are switched on can change the body plan.

Since marsupials have a number of differences from other mammals (again, in their reproductive tract and in the development of the distinctive body plan of kangaroos) we were led to study the HOX genes in the tammar. The genes themselves are remarkably similar to human HOX genes, but we found two tiny genes called micro-RNAs that were specific to marsupials. Comparing the tammar and human HOX clusters turned up a brand new human (and tetrapod) micro-RNA gene.

Looking at where the HOX genes were actually turned on in the limbs, we found that the timing of gene expression that directs the differentiation of the limb and digit structures differed a little from those described in humans, mice, bats and chickens. It appears that differences in the elements that control HOX expression may be responsible for the special anatomy of the kangaroo hind limb, such as its fused and reduced toes and its hopping locomotion.

Reproduction and Lactation
Marsupials are mammals, but their reproductive strategy differs from those of eutherian mammals. They have a fully functional placenta but their pregnancies are very short – a month or less in all marsupials. The newborn is effectively an exteriorised fetus and is very small, weighing only 400 mg in the tammar.

Instead of lengthening their pregnancies, they have expanded lactation and now have the most sophisticated form of lactation of any mammal. Their milk changes in composition throughout the whole of lactation – which extends for 9 months in the tammar – and it is tailor-made for every stage of the post-natal development of the young. The genes that control some of these changes are now known for the tammar.

The young itself undergoes most of its development during this lengthy period of pouch life. Most of their organ systems develop and grow after birth, including the testes and ovaries. Thus the tammar, and other marsupials, provide unique access for the study of organs that are normally inaccessible during their development in utero in other mammals, making them invaluable biomedical models for understanding mammalian development.

Conclusion
There were many unexpected findings from our study. Having the genome of the tammar has given us new information about many facets of kangaroo biology and will continue to provide a resource to aid biologists making exciting new discoveries about marsupials – and mammals like humans – for many years to come.

Tony Papenfuss and Marilyn Renfree are with the Bioinformatic Division of the Walter and Eliza Hall Institute of Medical Research and the University of Melbourne’s Department of Zoology.