In a landmark achievement that promises to accelerate comparative genomics efforts, and to serve as the basis for progress in numerous other areas of science research, including agriculture, human health and medicine, and complex disease genetics, scientists recently reported the sequencing and early analysis of the chicken genome, the first bird genome to be completely sequenced. The sequencing was carried out using state-of-the-art sequencers, reagents, and software from Applied Biosystems.

Elaine Mardis, Ph.D., co-director of the Genome Sequencing Center at Washington University School of Medicine in St. Louis, Missouri, and one of the three project managers for the chicken genome sequencing effort, emphasized the importance of Applied Biosystems sequencing technology to the chicken genome sequencing effort.

“We did, of course, use Applied Biosystems equipment and reagents to sequence this important genome. In particular, all reactions were performed with ABI Prism® BigDye® Terminator (v3.1) sequencing chemistry, and analyzed on the Applied Biosystems 3730xl DNA Analyzer platform.” Furthermore,” she said, “since early in 2003, we have used the KB™ Basecaller software from Applied Biosystems for all of our data analysis.”

“It's clear that having throughput capacity and sensitivity like that provided to us by the Applied Biosystems 3730xl DNA Analyzer, is key in producing genome sequence more cheaply and efficiently than ever before!”

Although there were numerous collaborating institutions involved in the genome analysis, all of the actual sequencing for the chicken genome project was carried out at Washington University.

Chicken Genome Augments Power of Comparative Genomics

As the first bird genome to be completely sequenced, the chicken genome fills in a large gap in the evolutionary tree of sequenced genomes and opens up new possibilities for insights and progress from comparative genomics studies.

“Located between mammals and fish on the tree of life, the chicken is well positioned to provide us with new insights into genome evolution and human biology,” said Francis S. Collins, M.D., Ph.D., director of the National Human Genome Research Institute (NHGRI) (1).

“By comparing the genomes of a wide range of animals, we can better understand the structure and function of human genes and, ultimately, develop new strategies to improve human health. With each genome that we sequence, this approach becomes more powerful.”

The NHGRI contributed $13 million to the chicken genome sequencing effort and is also supporting the sequencing of numerous other key genomes in order to facilitate comparative genomics and accelerate the advances that are predicted to come from these efforts (see reference 2).

Dennis Gilbert, Ph.D., the Chief Scientific Officer of Applied Biosystems, echoed Dr. Collins in emphasizing the importance the chicken genome sequence will have for the advancement of comparative genomics efforts.

“Comparative genomics is one of the most powerful techniques for unlocking the secrets of gene function. The chicken is at a particularly informative evolutionary distance from humans--neither too close nor too far--and the similarities and differences will be particularly important for understanding biology and the genetic causes of human disease.”

“The chicken genome can now be compared with the human genome--at the nucleotide level and researchers are already beginning to exploit this information,” Dr. Gilbert said.

Rick Wilson, Ph.D., co-director of the Genome Sequencing Center at Washington University School of Medicine in St. Louis. and leader of the chicken genome sequencing project, likewise emphasized the important position of the chicken genome.

"The chicken is really in an evolutionary sweet spot,” said Dr. Wilson (3). “It's at just the right evolutionary distance from all the other genomes we already have to provide us with a great deal of fresh insight into the human genome.”

“For nearly every aspect of biology, it allows us to distinguish features of mammalian biology that are derived or ancient, and it reveals examples of mammalian innovation and adaptation. The chicken genome is invaluable for shedding light on functional elements of the human genome and our unique evolutionary history.”

Professor Christopher Ponting, head of the Bioinformatics & Comparative Genomics group at the Medical Research Council in Oxford and leader of one of the chicken genome analysis teams, used the analogy of lenses to describe the power of comparative genomics (4).  

“Looking at mice, rats, and now the chicken, all of which are removed from us at different distances, is like peering through a collection of evolutionary lenses: each brings contrasting aspects of human biology into sharp focus. I‘m sure this research is going to make it much easier for medical researchers to pinpoint genetic changes that affect human heath and disease.”

Beyond Comparative genomics

In addition to its significance for comparative genomics study, the availability of the chicken genome sequence holds enormous promise for aiding advances and understanding in numerous other areas in which the chicken has historically played a major role—such as in studies of food production, nutrition, developmental biology, vaccine production, cancer research, and immunology—and also in the areas of infectious disease, particularly bird-borne diseases such as avian influenza (5, 6). The chicken genome should also prove useful in studies of complex diseases in humans. In addition, availability of the chicken genome sequence should be a boon for systematic studies of birds in general, as well as for the study of sex determination.

Food Production. The chicken is one of the most important sources of high-quality protein (meat and eggs) for much of the world, and an increased demand for such protein in developing countries is expected for the years ahead. Chicken meat and eggs are likely to be a major contributor to meeting this demand, as the establishment of poultry production is less cost-intensive than for other meat animals and cattle and/or swine consumption is not compatible with religious practices in some developing nations. Research that can lead to improved productivity in this area can have enormous benefits, and it is likely that the knowledge of the chicken genome sequence will greatly aid such studies.

Nutrition. The chicken genome sequence is likely to serve as a resource for researchers seeking to enhance the nutritional value of poultry and egg products. Because of the huge economic importance of chickens and because feed is the #1 expense for chicken farmers and because of the vast numbers of chickens produced, more is known about chicken nutrition than about the nutrition of almost any other animal, including humans.

Cancer, Vaccine Production, and Immunology. The first tumor virus (Rous sarcoma virus) and the first oncogene (src) were identified in the chicken. More doses of Marek’s disease vaccine (targeted against a cancer-causing herpes virus of chickens) are made than any other vaccine—human or animal. And, of course, the viruses used in the production of many human vaccines are grown in chicken eggs. And it was the chicken immune system that provided the first indications of distinctions between T-cells and B-cells, with the B-cell being named originally from the “b” in “bursa” of Fabricius, an organ in chickens and other birds, that produces the antibody-producing cells. In the chicken, immunoglobulin diversity is generated by somatic mutation via gene conversion using sequences from flanking pseudogenes rather than by the VDJ recombination process that predominates in human and mouse. Availability of the chicken genome sequence is likely to aid work to understand the processes by which immunoglobulin diversity is generated.

Developmental Biology and Gene Regulation. The chicken has long been one of the primary models for developmental biology as its embryonic development occurs in an egg (in ovo) rather than inside the body (in utero). “Because it’s so easy to look at and manipulate gene activity as an embryo develops in the egg, scientists often use the chicken in studies of development,” said LaDeana Hillier, one of the leaders of chicken genome sequencing project (3). “Much of what we know about human limb formation has been uncovered through studies of chickens,” added Dr. Jeremy Schmutz and Dr. Jane Grimwood in a commentary (7). In addition, the chicken has provided an important model system for studies of gene regulation. Many of the pioneering studies of steroid hormone regulation involved the chicken oviduct (ovalbumin gene regulation). And, because chicken red blood cells retain their nucleus, they have been the classical model system in which chromatin structure has been studied.

Bird-Borne Diseases of Humans. Analysis of the chicken sequence may also provide insights that may prove useful in combating bird-borne diseases such as salmonella, campylobacter, and avian flu. The possible spread to humans of avian flu has recently become a particularly serious concern and knowledge gained from studying the chicken genome, as representative of all birds, may be very helpful in work to better understand and combat this disease threat.

Complex Diseases in Humans. The chicken genome sequence may also prove helpful in the elucidation of complex disease genetics in humans. While much progress has been made in identifying genes underlying relatively rare monogenic diseases, much less progress has been made in the area of the far more common complex diseases (diabetes, cancer, hypertension, etc.) that are believed to be caused by combinations of multiples genes that are individually of relatively small effect. The chicken has long been the subject of study of multigenic inheritance of quantitative traits (e.g., size, weight, fertility, egg production, etc.), and insights from these studies may be directly applicable to the study of the multigenic inheritance of complex diseases in humans. Identification of the multiple relevant genes in complex diseases in humans has been very difficult. The process typically requires large, well-defined families and careful measurement of trait values. With the availability of the chicken genome sequence, and the corresponding maps and markers, the component genes can first be identified in chickens—and then the corresponding human genes can hopefully be found and tested for their roles in human inherited traits such as high blood pressure, obesity, etc. [see also reference (8)].

Chicken Models for Human Diseases. The chicken has also provided models for the study of a number of human diseases, including scleroderma, vitelligo, muscular dystrophy, and autoimmune thyroiditis. Another bird, the Japanese quail, is the only non-primate model for the study of age-related macular degeneration (AMD) and an inbred chicken line has served as a model for cleft palate.

Systematic Studies of Birds. The chicken genome sequence will likely provide the molecular underpinnings for systematic studies of birds in general (a class/order that has over 9,600 species)--for the study of bird behavior, sexual selection and courtship, vocal communication, rearing of young, migration, and the evolution of color patterns and other morphological features, for example.

Sex Determination. In addition, the availability of the chicken genome sequence may foster advances in the understanding of sex determination, which is handled differently in birds and humans [in birds, the female bears the sex-determining chromosome (W) whereas in humans, the male bears the sex determining chromosome (Y)].

A Platform for a Research Revolution

"The draft sequence of the chicken genome and the findings provided in this first-level analysis truly revolutionize what research can be accomplished with this agriculturally and biomedically important species," said Mary Delany, Ph.D., a geneticist in the University of California at Davis (UC-Davis) Department of Animal Science and a coordinator of the department’s participation in the chicken genome analysis (9). Dr. Delany is a world-recognized authority on the biology and genetics of the chicken.

“Before the genome was sequenced, we, as researchers, were essentially ‘blind,’ but now we are able to ‘see’ the genome and more easily explore the mechanisms by which it operates,” Dr. Delany said.

Concern Over Loss of Chicken Lines. The great opportunities offered by the availability of the chicken genome sequence highlight a concern that Dr. Delany has written of in the past (10)--namely, that avian lines are dwindling in number worldwide, and are being lost in the United States due to budget cuts. The availability of such lines is believed to be crucial to much of the work that can now proceed because the sequence is available. A letter (11) describing this concern was published in Nature the week following the report on the chicken genome sequence and analysis.

The Publications

The report describing the chicken genome sequence and its preliminary analysis by the International Chicken Genome Sequencing Consortium was published in Nature on December 9, 2004 (12). The members of the consortium subjected the chicken genome sequence to rigorous analysis from numerous different perspectives in order to provide their comprehensive report.

Two related papers were also published in the same issue of Nature. One described a genetic variation map of 2.8 million SNPs generated on the basis of comparative sequence analysis for three domestic chickens and the wild chicken (red jungle fowl) whose DNA was used for chicken genome sequencing project (13). The other reported on physical mapping of the chicken genome sequence (14).

As noted earlier, all of the sequencing of the chicken genome was done at the Genome Sequencing Center of the Washington University School of Medicine, by scientists using Applied Biosystems sequencing technology.

Highlights from Early Analysis

Already the early analysis of the chicken genome sequence has resulted in a number of significant findings—some expected and some surprising. [see also references (7, 15)]

Little Repetitive DNA and Much “Dark Matter”

First of all, the chicken was found to have almost as many genes as the human (20,000 to 23,000 genes in the chicken versus an estimated 25,000 to 30,000 genes in the human), despite having only one-third the amount of DNA (1.l billion base pairs versus 3 billion base pairs).

The relatively greater gene density of the chicken genome was attributed in large part to chicken having much less repetitive DNA than the human [see also reference (16)]. “The recognizable repetitive content (sometimes referred to as “junk DNA”) of the chicken genome is only about 10% as compared to about 50% for humans,” said LaDeana Hillier (3), who was the first author on the genome analysis article. One hypothesis the researchers mentioned for this was that the chicken genome might have evolved to optimize metabolism and to minimize the amount of repetitive DNA.

Analysis indicated that the reduced repetitive DNA in the chicken genome is associated with reduced numbers of interspersed repeats, processed pseudogenes, and segmental duplications relative to the corresponding numbers typically seen in mammalian genomes. Interspersed repeats are remnants of insertion events mediated by active or extinct transposons. Processed pseudogenes and segmental duplications are often flanked by direct repeats that are considered to be a hallmark of a DNA insertion.

As much as 85% of the chicken genome was found to include neither genes nor repetitive DNA, and has no known function. The authors suggested that this genetic "dark matter" may contain previously unrecognized regulatory elements, but also may include ancient DNA repetitive elements that have mutated beyond recognition.

Many Orthologues and a Conserved Core of Vertebrate Genes

Roughly 60% of the protein-coding genes in the chicken correspond to a single matching gene (orthologue) in the human, the authors reported. The average sequence similarity of these matching genes is 75.3 %; while the corresponding figure for matching genes between rodents and humans is higher (88%), as would be expected given the respective evolutionary distances. Comparative analysis of human, chicken, and fish indicated that there is a conserved core of genes that is likely to be present in most vertebrates.

The degree of sequence similarity between matching human and chicken genes was found to be different for different groups of genes—with the similarity being higher than  average for genes involved in cytoplasmic and nuclear functions, and lower than the average for genes involved in reproduction, host defense, and adaptation to the environment.

The sequence analysis revealed that orthologues of certain immune-related genes previously thought to be specific only to mammals are, in fact, present in the chicken. These include orthologues for cathelicidin (an antimicrobial protein), colony stimulation factor (CSF), leukemia inhibitory factor (LIF), certain interleukins (IL-3, IL-4, IL-9, IL-13, and IL-26), and three secretoglobins.

The IL-26 gene was previously known only in humans. The discovery of an IL-26 orthologue in chickens means that the chicken may now serve as a model organism in which researchers can investigate the function of IL-26. It is the only animal model now available for such studies.

Expansions/Contractions of Multi-Gene Families

The scientists noted that many of the differences between chickens and humans can be associated with expansions and contractions in multigene families, as well as with innovations within gene families in different lineages.

Keratins. Chickens were found to have a much-expanded gene family for the type of keratin protein used in scales, claws, and feathers. Mammals, on the other hand, have an expanded gene family of the type of keratin used in hair formation.

Milk, Teeth, and Pheromones. Chickens were found to be missing the genes involved in the production of milk proteins, tooth enamel, and the detection of pheromones. The researchers said this might mirror the evolution of the mammary glands and the nose in mammals, and the loss of teeth in birds.

Olfaction. The chicken genome showed evidence for a recent and rapid expansion of certain olfactory receptors and was found to have total number of olfactory genes approximately equal to the total found in humans. [see also references 17-19]. This finding was somewhat surprising and suggested that the long-held belief that chickens have a poor sense of smell might have to be reconsidered. On five chicken chromosomes, the olfactory receptor genes were found to be located in clusters close to the telomere, and this positioning, by analogy with the situation with human olfactory receptor gene clusters, may be associated with their rapid evolution.

Taste. Analysis revealed that the chicken, overall, has fewer genes for G-coupled taste receptors than humans do. While similar numbers of genes for type I receptors (sweet and unami taste) were found in chickens and mammals, only three genes for type II receptors (bitter taste) were found in chickens compared to the approximately 30 genes for type II receptors typically found in mammals. Birds may have a limited capacity for bitter taste, or they may have recruited other G-protein-coupled receptor subtypes for sensing bitter compounds, the authors suggested.

Interferon. Humans have an extensive family of alpha-interferon genes, but analysis showed that these genes are absent from the chicken. The expansion and diversification of interferon genes in mammals is thought to be an innovation that came in response to challenges posed by different pathogens, the researchers said.

Light-Dependent Enzymes. Chickens were shown to possess genes coding for certain light-dependent enzymes that mammals do not produce. It is believed that mammals once had genes for such enzymes, but lost them in the course of evolution. It is thought that these losses reflect a period in early mammalian history in which mammals were active mainly at night, the authors said.

Expansions/Contractions of Domain Elements

In addition to the expansion and contractions of protein-coding gene families, there have also been expansions and contractions in DNA segments coding for domain elements of proteins. For example, the largest domain-level expansion seen in humans versus chickens is found in the Kruppel-associated box (KRAB) domain whose presence coincides with C2H2 zinc fingers in transcription factors. KRAB domains are found in more than 400 human genes, but in fewer that 140 genes in the chicken. These domains are completely absent in the Fugu fish genome.

Overall Loss of Genes in Chicken Is Surprising

Very surprisingly, the analysis has indicated that, among all the vertebrate genomes that have been sequenced to date, the chicken genome is the only one to have apparently lost more genes that it has gained over an extended period of time. Furthermore, the chicken appears to have many fewer of the set of genes that are widely conserved in metazoans, and were presumably present in the common ancestor of mammals and birds.

New Start Sites for 2,000 Human Genes

Alignment of chicken and human genes indicates that approximately 2,000 human genes may actually start at different sites than previously thought. The discovery of these “true” start sites, which appear to lie inside the previously hypothesized boundaries of the genes, may have implications for the understanding of human disease and the design of new therapies.

Fewer Gene Duplication Events in Chicken

Analysis indicated there have been significantly fewer gene duplication events in the chicken lineage than in the human lineage. The two genomes show similar numbers for older duplications that may have taken place before the ancestral lineages diverged, the authors said.

Functional Urea Cycle in Chickens

In contrast to what has been the prevailing belief for the last 40 years, analysis of the chicken genome sequence indicated that genes involved in the urea cycle do exist intact in that genome. Because of the reported absence in the chicken liver of the enzymatic activity that initiates the urea cycle [i.e., the activity of carbamyl phosphate synthetase 1 (CPS1)], it has long been thought that the urea cycle did not function in the chicken. It had been suggested that this might be related to birds excreting waste in the form of uric acid rather than urea, perhaps for the purpose of avoiding the buildup of soluble urea in the developing egg. The sequence analysis, however, indicated that there is a full-length, apparently functional, gene for CPS1 in the chicken genome. It will be interesting to determine the expression profile of this gene to understand its in the urea cycle or an alternative pathway, Dr. Delany said.

Domestic Chickens Retain Surprising Amount of Genetic Diversity

In what the researchers termed a “big surprise,” domestic chickens were found to have retained a remarkable and unexpected level of genetic diversity despite the intense selection they have experienced during commercial breeding. This was a conclusion of the Nature article that specifically compared SNPs in three lines of domestic chicken--a broiler (meat producer), a layer (egg producer), and a silkie (an ornamental)—against those in the sequenced chicken genome sequence (from a red jungle fowl, the wild ancestor of these domestic chickens).

Chicken Genome Sequence Will Help Narrow Search for Functional Elements

Researchers said that about 2.5% of human DNA could be aligned with sequences in the chicken genome, and this is deemed very important as it means that scientists can focus on this small subset of the human genome as the most likely regions in which to find functional elements that have been conserved between chicken and human.

Functional elements include not only protein-coding genes, but also non-coding RNAs, regulatory elements, structural elements, and perhaps additional elements whose function is not yet known.

“We believe that the bits of DNA that are most resistant to change are those that have been most crucial to our survival throughout evolutionary history,” said the MRC’s Professor Ponting (4). “The 2.5% corresponds to 70 million letters of DNA and among these is where we can look first for mutations linked to human disease. In effect, the chicken genome has helped us condense the human genome to something more manageable.”

Evolutionary distance is critical to the ability to detect functional elements in genome comparisons. For instance, while human-fish comparisons can reveal conserved protein-coding sequences, the evolutionary distance between them is too great to allow other conserved functional elements to be revealed.

The evolutionary distance between chickens and humans, on the other hand, is such that it will permit the identification, not only of conserved protein-coding sequences, but also of sequences for conserved functional elements that do not code for protein. And the ability to learn more about these non-protein-coding functional elements holds great promise for aiding the elucidation of important biological mechanisms that are poorly understood, or even unknown, at this time.

The evolutionary distance between humans and rodents is too small to allow for the identification of non-coding functional elements as it is much harder to distinguish between random matches and truly conserved sequence at these shorter evolutionary distances.

With regard to the detection of conserved sequence regions, it is important that genomes are sufficiently diverged so that non-conserved bases are rarely the same by chance, but, at the same time, are close enough to allow reliable alignment across conserved bases.

The evolutionary distance between the chicken and the human is such that any preserved sequence similarity is likely to be functional—i.e., it has been preserved because it has a vital function. For closer species, such as the mouse and rat, not enough time has elapsed for the separation of chance sequence similarities and functional sequence similarities.

For more distant species, such as the pufferfish (Fugu rubripes), the additional time has allowed for increased shuffling of the genome that reduces the ability to make the extended sequence alignments that can reveal the key sequence preservations that reveal conserved non-coding functional elements.

Interestingly, researchers have found that non-coding sequences that align between chickens and mammals are often located far from genes and are frequently organized in clusters, and appear to be under selection for functions that are not yet understood.

Functional Non-Coding RNA in the Chicken

The researchers said that they have identified at least 571 non-coding RNA (ncRNA) “genes” from over 20 distinct gene families in the chicken genome. The researchers believe that a large percentage of these ncRNAs will prove to be functional. This belief is based, in part, on the fact that the number of predicted pseudogenes for these ncRNAs in the chicken is much smaller than the number predicted to exist in humans. The set of high-percentage-functional ncRNA genes in the chicken can be used to help identify functional human ncRNA genes, the researchers suggested.

Paucity of Processed Pseudogenes in the Chicken

The chicken genome was found to contain only 51 processed pseudogenes, far fewer than the approximately 15,000 typically found in mammalian genomes, the authors said. This large difference is attributed, they suggest, to the chicken having a particular dominant reverse transcriptase enzyme that is highly specific and that does not recognize poly-adenylated mRNA. Mammalian reverse transcriptases tend to be relatively non-specific (promiscuous) and can recognize processed mRNA (i.e., mRNA with introns removed and poly-A tail attached) and use it as template to produce DNA that is then reinserted into genome (to create a processed pseudogene).