DNA Technology

Dorian J. Garrick
Institute of Veterinary, Animal and Biomedical Sciences, Massey University

Introduction

Genetic differences between individuals reflects differences in their DNA sequences. Genetic changes in a population come about by changes in DNA sequences over time. This involves the creation of new sequences by mutations and other accidents of nature, and subsequent selection of favourable DNA sequences. Natural selection is achieved by survival of the fittest. To the extent that survivors are genetically different from non-survivors, the favourable genes for survival will be the only genes passed on to the next generation. Farmers do not see the DNA in their plants and animals, rather they see the performance that results from the joint effects of DNA and the environment. Farmers have been manipulating DNA for thousands of years simply by preferentially breeding from individual plants and animals that have favourable characteristics and by culling individuals with undesirable features.

Recent advances in DNA technologies include the development of methods to compare DNA strands at regions of interest in different individuals and to sequence DNA. This paper describes some uses of these technologies to identify and improve the characteristics of plants and animals.

DNA Structure and Function

Deoxyribose Nucleic Acid (DNA) is a chemical that stores instructions, known as genes, that enable the cells that constitute an organism to manufacture substances and to control these processes. The structure of DNA is described as a double-helix, represented by two strands, each strand consisting of a sequence of four compounds (Adenine, Cytosine, Guanine, and Thymine), known as bases. The strands are complementary, in that the base sequence on one strand determines the sequence on the other strand. When Adenine appears in any position on one strand, the corresponding position on the other strand will contain Thymine. Cytosine and Guanine are similarly paired. This unique structure allows the strands to separate and perfectly reform in two exact copies, as occurs during growth. A paired strand of DNA is known as a chromosome. In most species, the chromosomes exist in pairs, one member of the pair being inherited from the sire, the other member of the pair inherited from the dam.

A strand of DNA involves important sequences of base pairs (known as exons) that code for certain compounds, and supposedly unimportant sequences (known as introns) that separate the exons. A mutation or new change to the sequence of base pairs in an exon will usually affect a cell process whereas changes in intron sequences are believed to be of little or no functional consequence. Accordingly, when DNA is compared among individuals in a population, far more variation is usually found in intron sequences than in exon sequences. One form of variation in introns involves monotonous replication of small units of DNA, such as Adenine-Cytosine repeats, known as micro satellites. The number of such repeats can vary widely among individuals in the population, allowing this information to be used as a so-called marker. For example with respect to one such microsatellite marker, a particular individual may have 5 repeats on one chromosome and 7 repeats on the other chromosome in the pair. Any one individual carries at most two variants (one on each chromosome in a pair) but tens or hundreds of variants (polymorphisms) may be present over a population.

The development of a technique known as PCR (the Polymerase Chain Reaction) has revolutionised the assessment of marker genotypes. This technique involves multiplication of the DNA sequences from a specific chromosome region. The lengths of the multiplied sequences are then compared between DNA samples from different individuals. The lengths of the sequences directly reflect the number of repeat sequences. This knowledge can be used in a variety of applications.

DNA Tests to Find Carriers of Some Known Recessive Genes

Most productive traits are controlled by a large number of genes, each gene having a relatively small effect. In contrast, many congenital disease conditions result from a single genetic defect. True breeding normal individuals would carry two copies of the operational gene, and are said to be homozygous, implying that all their zygotes (sperm or eggs) carry the same genetic information with respect to that particular gene. On rare occasions, a defect known as a mutation may occur, whereby the sequence of base pairs is altered, such that the gene passed on to offspring is no longer operational. Provided the resultant offspring has inherited a normal copy of the gene from the other parent, the process for which the gene is responsible will continue. Most individuals carry a number of such defects, described as being recessive as these are masked by the dominant action of the normal gene. These individuals would be described as heterozygous for this condition, as they produce zygotes that differ in their genetic information. Exactly half of the resultant sperm or eggs would carry the defect. Occasionally, an offspring will be produced that has inherited the same defective gene from both parents, and the process for which the gene is responsible will no longer occur normally. Depending on the condition, these individuals may not survive until birth, may die at a young age, or perhaps be infertile.

In traditional breeding programmes, it has been very difficult, or expensive, to identify the heterozygotes or apparently normal individuals that carry one copy of the defective gene. Progeny testing suspected carriers could sometimes be used to identify carrier males, provided known carrier females or daughters were available. The advent of DNA tests allows for heterozygous animals to be so identified, directly from DNA obtained from cells contained in a sample of blood, semen, milk, fibre or other tissues. Such tests can be applied to young animals (often while in utero), and in future will perhaps be applied to embryos prior to their transfer to recipients.

DNA tests for a number of such conditions (BLAD, citrullinemia, Factor XI, DUMPS) are now routinely applied to dairy bulls prior to widespread use through artificial insemination. Similar tests have been developed to identify black bulls carrying the gene for red coat colour and to test for particular milk protein variants. Research is ongoing to identify tests for presence of horned/polled genes, spider syndrome, and within AgResearch, for the presence of the Booroola and Inverdale genes.

DNA Tests to Identify or Confirm Parentage

Breeders are often interested in confirming the parentage of individual plants or animals. Suppose, as in the earlier example, an individual carries a marker with 5 and 7 repeats. This individual must have received the 5 repeat variant from one parent and the 7 repeat variant from the other parent. Any parent that contains neither the 5 or 7 repeat variant can immediately be excluded from having been a parent of this individual. If, as is often the case, one parent is known, deduction of the identity of the other parent is straightforward. Through a process of elimination, and the use of say five different microsatellite markers, parentage can usually be established. This technique can be used in multiple sire joinings, or could be applied to identify parentage of animals as their carcasses are being processed through meat plants. Commercial parentage services for deer and cattle using DNA tests are available in New Zealand through GenomNZ, a company formed by AgResearch.

DNA Testing for Marker-assisted Selection

Most productive traits are polygenic, so-called because they result from the actions of large numbers of genes. Selection is aimed at identifying individuals with superior sets of these genes. In such cases, traditional improvement programmes for plants and animals have relied on phenotypic measurements to identify superior individuals. Individuals that have proven to be superior through the measured performance of progeny have been widely used with no knowledge as to exactly the number, size or location of genes that have contributed to this superiority. The application of DNA markers provides an opportunity to identify any genes of moderate effect (known as quantitative trait loci or QTLs), such that selection might be effective without the need to wait and measure actual performance.

Two approaches are being used to ascertain the existence, size and nature of these QTLs. The first approach involves crossing between genetically distant strains, preferably inbred lines. Examples of such crosses that are currently being researched or proposed for research in New Zealand are between Merinos and Romneys, Jerseys and Limousins, lean and fat sheep selection lines, nematode parasite resistant by susceptible sheep lines, facial eczema resistant by susceptible sheep selection lines, and bloat susceptible by resistant lines of dairy cattle. The aim of these experiments is best explained by use of one of these examples, say the Jersey and Limousin. The fact that Jerseys breed true in the sense of producing Jersey offspring, implies that this breed tends to be homozygous for genes that make Jerseys what they are. Limousin cattle are quite different in appearance and performance and as these differences between the breeds are genetically-based, will tend to be homozygous for different forms of the genes. When a first-cross (or F1) Jersey-Limousin animal is produced, it will be heterozygous for all these genes that make the two breeds different. That is, the F1 will inherit one copy of the Jersey form of the gene from the Jersey parent and one copy of the Limousin form of the gene from the Limousin parent. When an F1 bull is used as a sire, half of its offspring will inherit the Jersey form of the gene and the other half will inherit the Limousin form.

Consider one section of DNA in the F1 animal, for which there is a marker. The F1 animal will have two variants of the marker, one corresponding to the Jersey parent, the other to the Limousin parent. The offspring of this F1 parent can be distinguished according to the origin of the marker, to produce two groups of offspring. If these two groups of offspring differ in any physical or performance characteristics, then this implies that the gene responsible may be located close to the region where the marker is located. A systematic search using 200 or more markers, spaced all along every chromosome can enable discovery of any large or moderate-sized genes that make Jerseys and Limousins different. Such a finding would be of enormous scientific interest, but may be of little benefit from a selection viewpoint with regard to the base parent lines, in this case the Jersey and Limousin. This is because the Jersey will already be fixed with the Jersey form of the gene and the Limousin will be fixed with the Limousin form of the gene. The finding could be useful if a new breed was to be created, making use of the F1 animals.

An alternative approach to find QTLs involves study within sire families. Suppose a sire is heterozygous for a marker. The offspring can be categorised according to the marker variant and their performance compared. If their performance is different, this indicates that the sire is also heterozygous for a production gene located near to the marker. If there is no difference between groups of progeny according to their marker variant, there may still be a QTL near the marker but this would not be segregating if this sire is homozygous for the QTL. Such a study would require all offspring to be genotyped for a range of markers, which can be an expensive exercise. A slight variation of this approach involves assessing only the progeny-tested sons of a sire and determining if the merits of the sons are different according to their marker variants. This is known as a grandsire design and is currently being used by Livestock Improvement Corporation to investigate QTLs for dairy production traits. This design will not detect as many genes and is not as powerful as crosses between inbred lines because every sire family will be homozygous for some QTLs. However, it has the advantage that any genes that are detected could be used for selection. For example, the sons of a bull could be selected soon after birth, with only those sons retained that carry the marker variant associated with the higher level of performance.

Any application of marker-assisted selection, whether between strains or within families will not result in the creation of animals that could not have occurred naturally. The technology simply offers the option of identifying the genotypes of some individuals without the need to wait for and measure actual performance.

Use of DNA Technology to Create New Gene Combinations (Transgenics)

Applications of techniques including those described in the previous section have allowed some specific genes to be identified. The base sequences of some genes have been derived allowing the physiological and biochemical pathways for gene expression to be determined. Specific genes can then be recreated and inserted into DNA sections in other individuals of a different species. New organisms created by artificially moving genes are known as transgenics.

Transgenic laboratory animals are extremely valuable in studies of basic science, and have proven invaluable in gaining understanding of BSE (mad cow) and related diseases. In other studies, transgenic laboratory mice have been created by copying the testes determining region (or TDR) from the Y chromosome onto another chromosome. The TDR gene is responsible for the formation of testes and thereby the creation of male animals. The transgenic mice produce offspring with a sex ratio of 3 males to one female.

Some transgenic animals and plants are of interest from a farming viewpoint. In plant breeding, transgenic tomatoes have been created such that the tomato fruit will not over-ripen as readily, prolonging the shelf life of the product. A gene for resistance to the herbicide Roundup has been transferred into Soyabeans allowing this crop to be sprayed with Roundup to kill weeds without affecting the bean crop. In the animal area, a human gene has been transferred to sheep such that the gene is expressed in the mammary of the ewe and resulting in the production of a pharmaceutical product in the milk. A small flock of sheep would then be milked to capture the pharmaceutical product. The creation and use of transgenic plants and animals are, and will continue to be, the subjects of considerable moral and ethical debate.

Summary

DNA is an essential building block responsible for animals and plants being what they are. Preferential selection of certain individuals, whether by natural or artificial means has resulted in changed performance in later generations only as a consequence of DNA changes. Recent improvements in knowledge of the structure and function of DNA has led to development of some DNA technologies that have been of enormous benefit to some human endeavours, notably basic science and medicine. New applications of DNA technologies will increasingly impact livestock farming in future decades.