The sequence of a sheep

The nomadic pastoralists who overwintered at a semi-permanent settlement at Shanidar, in northern Iraq, some 9000 years ago, had similar tastes to modern consumers: they preferred tender young lamb to mutton.

The Shanidar site contained a high concentration of bones from one-year-old animals. Doubtless the sheep also contributed their warm fleeces to keep the farmers and their families warm against northern Iraq's bitterly cold winters.

Shanidar has provided some of the earliest evidence for the domestication of sheep, the first wild animals domesticated as livestock. Mitochondrial DNA analyses points to at least three independent domestication events across Eurasia that began as early as 11,000 years ago.

The people of Shanidar, and other unknown shepherds in Europe, the Middle East, and possibly in Asia, began a selection process that is recorded in the DNA of today's sheep breeds.

The international Sheep Genome Project will throw light on the origins and evolutionary relationships of modern breeds, and explore the full extent of the genetic diversity that ancient pastoralists captured during the domestication process.

The International Sheep Genomics Consortium (ISGC) completed a first pass sequence of the Ovis aries genome in January. The consortium, which includes researchers from across the world, has been taking the lead in developing genomic tools and reagents for use by the sheep genomics community.

Dr James Kijas, leader of CSIRO Livestock Industries sheep genome group, says the group was interested to see if the sheep genome could be sequenced using a new short-read technology like Roche's 454 FLX sequencers.

"The emergence of much higher throughput and cheaper sequencing methods has helped us tremendously," Kijas says.

The consortium decided to invest in using Roche 454's FLX automated sequencers in two locations, New Zealand's University of Otago and the Human Genome Sequencing Center at the Baylor College of Medicine in Houston Texas.

The 454 methodology generates reads which are shorter in length than traditional Sanger reads. However, the ability to generate 60 million base pairs of sequence and 250,000 reads in a single instrument run has opened the door to producing genome sequences much more quickly and cheaply.

Kijas says the approach was driven by the need to characterise the variable component of the sheep genome, so the project had sequenced DNA from six different sheep breeds.

Researchers in Texas sequenced the DNA of three of the animals, while the team in New Zealand sequenced the other three.

Dr John McEwan, a senior scientist from AgResearch who led the New Zealand component of the work, says the approach was different to that employed in other genome projects. "From the outset, we used next-generation sequencing technology, and we've done things in a slightly different order.

"The previous strategy was to do a reference sequence, and then use that as the basis for locating SNPs (single-nucleotide polymorphisms). In our case, we used the bovine genome as a template, because it's the nearest available neighbour to sheep among livestock breeds sequenced.

"We sequenced six individuals to identify SNPs, then roughly positioned them using the bovine genome as a reference.

"We intend in the longer term to proceed towards a sheep reference genome, but the primary driver is the need to identify SNPs for a SNP chip."

---PB--- Sheep SNP chip

The team produced 42 million reads and a total of nine gigabytes of DNA sequence. AgResearch and CSIRO researchers put it all together by jointly reconstructing a first pass of the sheep genome.

CSIRO bioinformation Dr Wes Barris says the approach involved using their previously-computed virtual sheep genome information to rearrange bovine chromosomes into virtual sheep chromosomes.

Sequence-comparison tools are then applied to map the millions of short sheep sequences onto the temporary virtual chromosomes, which provide a guide for locating and orienting the sheep sequences.

"We end up with sheep chromosomes made up of pure sheep sequence data," Barris says. "The procedure requires a lot of computing power. We have access to two computing clusters and have kept them busy around the clock doing sequence comparisons."

To increase the number of SNPs available to the international community, the consortium performed a second SNP discovery experiment. Kijas led a collaboration with Illumina, using its Solexa technology, which yielded nearly 50 million sheep sequence reads in a single machine run.

"We wanted to identify SNPs and obtain an estimate of their frequency within sheep populations at the same time," Kijas says. "To do this we collected a diverse set of 60 sheep, pooled their DNA into a single tube and then sequenced it with Solexa technology to search for additional polymorphic sites".

An immediate effect from the consortium's work will be the creation of a SNP chip carrying approximately 60,000 of the best SNPs. Kijas says researchers expect this new tool will dramatically accelerate the pace of gene discovery.

Australian researchers will be focusing on traits influencing disease resistance, meat quality, meat yield and wool fibre diameter in the Merino. Meat and Livestock Australia (MLA) and Australian Wool Innovation (AWI) have constructed large research flocks which will be used for the research.

"We're fortunate in having large flocks with highly accurate records for traits such as disease resistance, carcass parameters and even behavioural differences," Kijas says.

"The SNP chip will enable us to perform a genome-wide search for genes underpinning each of these traits."

Kijas says early work has shown that the level of genetic diversity present within sheep breeds is high compared to cattle.

"The ability to collect data from 60,000 SNPs across a large sample of breeds will give us detailed information about the origin and relationship between sheep populations." The consortium is now collecting 2000 sheep representing nearly 40 breeds for the experiment.

---PB--- Evolution of domestication

AgResearch's McEwen says two major centres of domestication have long been recognised, in the Middle East and Europe, and there is evidence of introgression between them in Iran and the Transcaucasus region.

Very recently geneticists have found evidence for a third centre of domestication in Asia, possibly in China.

"The new SNP chip should throw a lot of light on [the history of domestication]," McEwen says.

"For many years geneticists have used mitochondrial and Y-chromosome DNA to track how humans migrated around the world, but very recently SNP chips with variants of autosomal genes have been providing a more detailed history."

He says the contrasting selection pressures for sheep and cattle should make for interesting findings. Where cattle were originally domesticated as draught animals, to pull ploughs, and as a source of meat, sheep were initially bred as a food source, for meat and milk.

"People have been collecting wool from sheep for millennia, but strong selection pressure for traits like fleece quality began much more recently - perhaps only a few thousand years ago," he says.

That ancient division of interest is perpetuated on opposite sides of the Tasman Sea today. Most of Australia's wealth from sheep comes from wool; New Zealand's wealth rests on meat from fat lambs.

"We're very interested in speeding up the rate of genetic progress using whole-genome selection. We can genetically link elite animals at a younger age, which allows us to reduce the generation interval, and increase the rate of progress.

"We can also do whole-genome association studies to derive molecular breeding values, which is an improvement on doing measurements on individuals and using progeny testing."

McEwan says a number of traits the team is interested in are either sex-limited, or are not expressed until later in life.

"One is obviously reproductive ability, including the maternal component of lamb survival. We're interested in the ability to grow rapidly early in life, but without an equivalent increase in the mature size of the animal, so we finish up with a medium-sized animal that grows rapidly to maturity.

"A lot of New Zealand sheep have high mature weights, which is a problem for shearers.

"The other important traits are disease resistance - people are reluctant to challenge animals experimentally to assess disease resistance. Helminth parasites are the biggest problem, followed by facial eczema, viral pneumonia, footrot and flystrike.

"We also have a consortium in New Zealand studying susceptibility to Johne's disease in cattle, sheep and deer, and the sheep component of that hopes to make use of our SNP chip."