Basic Aquaculture Genetics PDF 2010

Southern Regional SRAC Publication No. 5001 Aquaculture Center Janu...

Southern Regional SRAC Publication No. 5001 Aquaculture Center January 2010 VI PR Basic Aquaculture Genetics Jason W. Abernathy,1 Eric Peatman 1 and Zhanjiang Liu* The inheritance of desirable traits in crops and gene. Most of the DNA forms a structure known as the livestock has been the foundation for selective breed- chromosome. Chromosomes are located in all nucleated ing for thousands of years. However, our understanding cells. The total number of chromosomes can differ among of genetics, which is the biological basis of heredity and species but will generally be constant in the same species. variation among organisms, has deepened dramatically Most aquatic species contain two sets of chromosomes, in the last several decades. This new knowledge expands one inherited from the father and one inherited from the the potential applications of genetics and genome tech- mother. Any organism with two sets of chromosomes is nologies in agricultural practices. In aquaculture settings, termed diploid, or 2N. We will focus on diploids in our current genetic improvement programs focus on select- explanations, unless otherwise noted. ing superior broodstock, using better breeding practices, The karyotype is the general appearance (size, increasing sustainability, and minimizing environmen- number, shape) of an organism’s chromosomes. There are tal problems. These programs have already led to more two types of chromosomes in eukaryotic species (species efficient, productive and profitable aquaculture systems, with nucleated cells): autosomes and sex chromosomes. but the genomics revolution promises to speed and Autosomes are all chromosomes that are not sex chromo- amplify genetic advances in the near future. This publica- somes. Sex chromosomes usually determine the fish’s sex, tion describes both basic genetic principles and applied genome technologies that are relevant to today’s aquacul- ture industry. Introduction to genetics Deoxyribonucleic Acid (DNA) T G A C G T C C A Genes and chromosomes A G T The basic unit of inheritance is the gene. Genes contain the biological code for the production of observ- able traits, or phenotypes, in an organism. Genes are arranged on a large molecule called deoxyribonucleic Chromosome acid (DNA). DNA consists of subunits called nucleo- tides (Fig. 1). The four nitrogenous bases that define the four different nucleotides are adenine (A), guanine (G), thymine (T) and cytosine (C). It is the combination of these subunits in a linear arrangement that codes for a Figure 1. The physical structure of deoxyribonucleic acid (DNA). DNA is characterized as having a double helix structure with a sugar-phosphate 1 The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries backbone. The nucleotides pair with each other to form the structure. and Allied Aquacultures, Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University Adenine pairs with thymine, while guanine pairs with cytosine. Courtesy: * Corresponding author National Human Genome Research Institute. either male or female. Autosomes are usually designated with a number, while the characteristic notation for sex Meiosis chromosomes is XX and XY in diploids, where XX are Chromosomes females and XY are males in the XY sex determination from parents Cell nucleus system. Many species of fish can have a different sex- determining system. Some may contain only autosomes. Some are hermaphroditic and either change sex as they Chromosomes grow or, in rare cases, possess male and female organs replicate. simultaneously. And in some fish species the sex chromo- somes have not yet been determined. Even though most eukaryotic species are diploid, Like chromosomes pair up. there are exceptions to the number of chromosome pairs (ploidy) in some aquatic species. Further, the number of chromosome sets can be manipulated in genetic improve- Chromosomes swap ment programs. Haploids (N) can be created, as well as sections of DNA. fish that contain chromosomes from the mother only Nucleus divides into Chromosome pairs divide. daughter nuclei. (gynogens) or from the father only (androgens). Triploids have three sets of chromosomes (3N) and tetraploids have Chromosomes divide. four sets of chromosomes (4N). In nature, tetraploids Daughter nuclei Daughter nuclei have single chromosomes divide again. are more common than triploids. The channel catfish is and a new mix of genetic material. diploid (2N) and contains 29 pairs of chromosomes (2N = 58). The salmonids (salmon and trout) and the common carp are widely thought to be tetraploid (4N). Since chromosomes occur in pairs in diploid organ- isms, each gene has at least two copies. Each copy of a gene, called an allele, is located at a specific location on Figure 2. The basic principle of meiosis. Courtesy: National Institute of each of the sister chromosomes. This location is called the General Medical Sciences. locus of the allele. Two alleles can be identical or can have variations in their DNA sequence. If alleles at a specific locus are identical, the organism is homozygous for that only one set of each chromosome pair. The process of cre- gene. Conversely, if alleles at a specific locus are different, ating haploid gametes is critical to reproduction in diploid then the organism is heterozygous for that gene. Differ- organisms. When an egg (N) is fertilized by the sperm ences in alleles within an individual can produce genetic (N), the correct number of chromosomes (2N) is recov- variance and thus different phenotypes in a population. ered in the zygote. The process of sperm or egg (gamete) The combination of alleles for a given trait is called the generation is called gametogenesis. More specifically, the genotype. production of sperm cells is referred to as spermatogen- esis, while the production of egg cells is called oogenesis. Chromosome replication and cell division In meiosis, at least three important processes occur to There are two forms of cell divisions—mitosis and produce genetic variability in the sperm and egg: crossing meiosis. As an organism grows, cells must divide and over, segregation, and independent assortment. First, the replicate to increase in number and to replace old or chromosomes in the gametocyte (precursor cell to an egg dying cells. Mitosis is the process by which a somatic cell or sperm) must undergo replication. After chromosome divides to produce two identical daughter cells. Somatic pairs replicate in diploids, the homologous chromosomes cells are all cells except egg and sperm and the cells that form pairs in bundles of four, called tetrads. At this point, produce them. During mitosis, the entire cell is dupli- the chromosomes that form tetrads can wrap around cated, including the chromosomes and all other cellular each other. The chromosomes may then break apart and material. Thus, each daughter cell contains a diploid set of pieces from different chromosomes can rejoin. Therefore, chromosomes. genetic material has been swapped from one chromosome This process differs from meiosis, which is associated to another. A portion of a chromosome that originated with gamete (egg and sperm) formation (Fig. 2). The eggs from the mother is exchanged between the homologous and sperm undergo cell division and replication, but pro- chromosome from the father, and vice versa. This process duce cells that contain a haploid (N) gamete, containing of recombination or physical exchange between homolo- 2 gous chromosomes is called crossing over. This occurs in Mendel’s law of independent assortment states that each the cell stage known as the primary gametocyte (primary gamete receives a random mixture of alleles, one at each spermatocyte in males, primary oocyte in females). locus, from each parent (Fig. 4). The processes of segrega- The other processes that increase genetic variation tion and independent assortment are random in that each include segregation and independent assortment. After secondary gametocyte or polar body receives a random chromosome duplication and crossing over in the pri- mixture of chromosomes from the mother and the father. mary gametocyte, the chromosome complement must The reduction of chromosomes from the primary game- be reduced from the diploid state (2N) to the haploid tocyte to the secondary gametocyte reduces the number state (N) in a cell division step for both the egg and of chromosomes from the diploid complement to the sperm. Each chromosome pair will separate, going from haploid complement. This process ensures that when a a primary gametocyte to a secondary gametocyte. Each sperm (N) fertilizes an egg (N), the correct complement of secondary gametocyte will contain a single replicated chromosomes (2N) will be created in the zygote (fertilized chromosome from each homologous pair. In spermato- egg). The exception to independent assortment of chro- genesis, all chromosome pairs separate and one of the chromosomes of each pair goes to a secondary spermato- cyte. In oogenesis, all chromosome pairs separate and AABB aabb P generation one of the chromosomes of each pair goes to a secondary oocyte or a polar body. The process by which the dupli- AB ab Gametes cated chromosomes separate and form secondary game- tocytes is the basis for Mendel’s law of segregation (Fig. 3). AaBb F1 generation AB AB AA aa P generation AABB Ab Ab AABb AABb A a Gametes aB aB AaBB AAbb AaBB ab ab Aa F generation 1 AaBb AaBb AaBb AaBb Aabb aaBB Aabb A a aaBb aaBb A AA Aa aabb F generation 2 a Aa aa F2 generation Figure 4. Mendel’s law of independent assortment of alleles of different Figure 3. Mendel’s law of segregation. This is the classic example of genes. Gregor Mendel also observed the trait producing seed shape, either independent segregation of alleles, using a single observable trait. smooth or rough seeds, along with seed color. In this example, the dominant Gregor Mendel used the observable trait of seed coloration in his plant alleles are yellow (A) and smooth (B) seeds, whereas the recessive alleles experiments. In this example, the dominant allele is yellow seed color are green (a) and rough (b) seeds. The parental cross produces F1 progeny (A), whereas the recessive allele is green seed color (a). One homozygous that are all heterozygous dominant (AbBb) for the traits, and thus yellow dominant (AA) and one homozygous recessive (aa) parent were crossed to smooth seeds. The F2 generation, produced by a cross of F1 generation produce F1 progeny that are all heterozygous dominant (Aa) for the trait (AaBb x AaBb), yielded progeny with various genotypes and phenotypes. (and all yellow seeds). A cross between two F1 progeny (Aa x Aa) produces F2 Two new phenotypes appear in the F2 generation: yellow rough seeds and progeny with three genotypes (AA, Aa and aa), and two phenotypes (yellow green smooth seeds. Since the two genes were segregating and assorting or green seeds). Seed coloration is characterized as either homozygous independently of each other during meiosis, multiple combinations of alleles dominant (AA) yellow, heterozygous dominant (Aa) yellow, or homozygous (and traits) were produced. Note that independent assortment of genes may recessive (aa) green. not hold true for all combinations of alleles (such as with linked genes). 3 mosomes (and alleles) is linkage. If the genes that control not be morphologically distinct. Many sex chromosomes a trait are located on the same chromosome and are close resemble autosomes, and/or some sex-determining genes together, they are said to be physically linked. In this case, may be located on autosomes. In these cases, sex-linked these genes may be inherited together. traits must be studied to determine the sex chromosomes The secondary gametocytes consist of either two sec- or sex genes that are located on autosomes. ondary spermatocytes (males) or a secondary oocyte and The most common sex-determining system is the a polar body (females). Each secondary oocyte normally XY diploid system. This system is the most common in produces only one haploid egg; the rest are polar bodies. known fish species as well as in humans. Channel catfish In females, after segregation and independent assortment, have the XY system. This system was so named because chromosomes at one end are pinched off with a little sur- the sex chromosomes in humans resemble a Y or an X. rounding cytoplasm (yolk). This forms the first polar body. In the XY system, the sex chromosomes in females are Polar body cells are non-functional and are a byproduct identical (XX), while those in males are a mix (XY). The of oogenesis. Since the egg is the major site of nourish- Y chromosome is found only in males. Since the pairs ment to the embryo, a high concentration of cytoplasm is in females are the same, these chromosomes are termed necessary and hence the unequal division of cytoplasm homogametic, while in the male they are called heteroga- in the oocyte versus the polar body. The first polar body metic. may or may not divide again to produce two small haploid All eggs contain only a single X chromosome. In cells. The other daughter cell is the secondary oocyte. The males, half the sperm population contains a single X secondary oocyte and first polar body produce one mature chromosome and half contains a single Y chromosome. egg and one secondary polar body. Secondary oocytes are In the XY system, the heterogametic sex (male) is the one not stimulated to egg production until after they have con- responsible for sex determination in the population. If an tacted the sperm. In males, the two secondary spermato- egg (X) is fertilized with a sperm carrying the X chromo- cytes produce four sperm cells. All sperm cells have the some, the sex of the offspring will be female (XX). If an haploid number of chromosomes that have been indepen- egg (X) is fertilized with a sperm carrying the Y chromo- dently assorted and contain equal amounts of cytoplasm. some, the offspring will be male (XY). Again, the result- ing offspring will be diploid. It is likely that only a portion Development of each sex chromosome is ultimately responsible for sex When an egg and sperm are fused through fertiliza- determination. Other traits (genes) may be located on the tion, a diploid zygote is produced. This is a single cell; XY chromosomes in many different aquaculture species. mitosis of the zygote and subsequent daughter cells is There are several other sex-determining systems in responsible for the growth and development of the fish. fish besides the XY system. One is the WZ system. It Fish eggs are mostly composed of yolky cytoplasm. In works exactly the opposite to the XY system; a homoga- general, after fertilization cell division begins in a thin, metic fish is a male while a heterogametic fish is a female. yolk-free region of the egg called the blastodisc. This Blue tilapia is thought to have the WZ sex-determining initial cell division is known as the cleavage phase and the system. The female blue tilapia contains the different set initial cells are termed blastomeres. Blastomere divisions of sex chromosomes (WZ), while the male blue tilapia are rapid and cells build upon one another to constitute contains the same set of sex chromosomes (ZZ). Hence, the blastoderm. These early cells mix and can give rise to the W chromosome is the sex-determining chromosome. a variety of cell and tissue types. After a period of rapid The sperm (Z) that fertilizes an egg of one chromosome cell cleavage, cell divisions begin to slow and cell move- type (Z) will produce males (ZZ), while a sperm (Z) that ment begins. Blastomeres begin to cluster and segregate fertilizes an egg of another chromosome type (W) will throughout the embryo. After the blastoderm has filled produce females (WZ). In other words, sex is controlled about half the yolk, or sooner depending on the species, by different types of eggs, not different types of sperms as germ layers begin to form through the process of gastru- in the XY sex-determination system. lation, or cell restructuring. These layers will develop into Another system is the ZO sex-determining system. the tissue and organ systems of the fish. From a genetic The dwarf gourami fish and a sole fish have been identi- standpoint, zygotic gene expression generally begins dur- fied as having the ZO system. Here, the females are ZO ing the cell division stages. and the males are ZZ. In these species, the females are the sex-determining species and are the heterogametic sex. Sex determination The female produces haploid eggs with either the Z chro- Sex genes are the main determinants of gender. The mosome or no sex chromosome at all. If an egg with the sex chromosomes of different fish species may or may Z chromosome (Z) is fertilized by a sperm (Z), then the 4 offspring will be male (ZZ). If an egg has no sex chromo- Qualitative genetic traits some (O), the resultant fertilization will produce a female Phenotypes can fall under the category of qualitative offspring (ZO). traits, or traits that can be simply described as one or the The XO system also exists in some species of fish, other. A trait is qualitative if it can be sorted into one of at including a sunfish species. In this system, the males least two categories. These traits are not measured over a are heterogametic (XO) and determine the sex of the range, as is the case with quantitative traits. An example offspring. In a way, the XO sex-determination system is of a qualitative trait would be fish pigmentation; the fish is similar to the XY sex-determination system except that either normally pigmented or albino. Qualitative traits are in the XO system the males contain just one set of sex often the simplest to characterize because they are likely chromosomes (X). to be controlled by only one or a few genes, unlike quan- Several other sex-determining systems have been dis- titative traits. Since an aquaculture species can be catego- covered in fish. Many of these systems are more complex rized by these traits, the population can be described by than the XY system and its variants. These systems have the ratios of its members with these traits. The number multiple sex chromosomes. One example of a multiplex of fish with each trait are simply added up and described, system is the WXY sex-determining system, common such as 3:1, 1:1, and so on. For example, a population of 20 to the platyfish used in aquaria. Here, both males and fish has either a fan tail or a round tail, a qualitative trait. females can be either homogametic or heterogametic If 15 fish have fan tails and five fish have round tails, the and determine the sex of the offspring. In WXY, the W ratio of fan tails to round tails is 3:1. Described another chromosome acts as a modifying chromosome that can way, one-quarter of the fish population has round tails block the male-determining ability of the Y chromosome. and three-quarters of the population has fan tails. Quali- Therefore, the XY and YY offspring are males and the tative phenotypes are the same in different environments. XX, WX and WY offspring are females. Other multiplex A number of factors influence these ratios, including systems include the X1X1X 2X 2/X1X 2Y, ZZ/ZW1W2, and the number of genes needed to produce the phenotype XY1Y2/XX systems. Finally, sex-determining genes also and gene action. The genetic mechanisms described here can be located on autosomes. Some aquaculture species are typically referred to as classical genetics or Mendelian do not have any morphologically distinct sex chromo- genetics. Gene action can be characterized as single or somes. In those species, sex is determined by a number of multiple genes producing a qualitative phenotype. Clas- female or male genes located on specific autosomes. sic qualitative traits are dominant or recessive. If a single One important concept to introduce is genetic sex allele is expressed over the other at the same locus, then the versus phenotypic sex. Fish, being lower vertebrates, have mode of action is termed dominance. Complete dominance much flexibility in sex organ development, so a pheno- mode of action describes the expression of alleles at the typic sex may not reflect the chromosome composition of same locus where one copy (the dominant allele) masks the the fish. A number of factors can influence expression of effect of the other copy (the recessive allele). The phenotype the sex phenotype. Some of the most common factors are expressed is termed the dominant phenotype, and the other temperature, salinity, population density in tanks, pres- is the recessive phenotype. When dominant alleles occur, sure, hormone treatments, radiation and photoperiods. there are three genotypes possible, while only two pheno- Sex modifications may be induced or may occur naturally types can be produced (Fig. 3). A classic example of com- in some populations. For instance, hormone treatment plete dominance of a single locus is the gene for albinism in (estrogen) of developing males in some fish species can the channel catfish. The dominant genotype that produces produce phenotypic adult females even though the fish normally pigmented catfish can be labeled as AA or Aa, contain the XY chromosomes. Conversely, hormonal where the capital (A) is the dominant allele and the lower treatment can lead to the development of all males even case (a) is the recessive allele. Both AA and Aa fish produce though the fish have female genetic makeup (XX chromo- normal pigmentation. Only fish with the complete recessive somes). For instance, a common practice in aquaculture pigment gene (aa) will be albino. Hence, with dominance is treating tilapia with 17α-methyltestosterone (MT) to there are three combinations of genotypes (AA, Aa, aa), induce all-male populations. As male tilapia grow much while only two phenotypes are produced—normal (AA faster than females, such hormonal treatment leads to or Aa) or albino (aa) coloration. Fish that are homozygous increased yields. In such cases, however, the fish may dominant (AA) will obviously be normally pigmented, but harbor XY chromosomes (genetic males and phenotypic so will fish with one dominant (A) and one recessive (a) males as well), or they may harbor XX chromosomes allele (heterozygous dominant, Aa). With complete domi- (genetic females, but phenotypic males). nance, only parents that carry recessive alleles (either Aa or aa) can produce offspring with a recessive trait (Fig. 3). 5  There are two other possibilities concerning qualita- Quantitative genetic traits tive traits—multiple alleles and sex-linked alleles. When Quantitative traits are phenotypes that have a range of alleles at more than one locus control a qualitative pheno- expression. These are traits that can be measured, rather type, there are two possibilities of gene action. There are than either/or traits such as normal pigmentation versus either epistatic effects or non-epistatic effects. Epistatic albinism. Phenotypes that can be observed and measured effects are interactions between genes that can cause mod- may vary in a population. Therefore, genetic programs ifications or suppressions of phenotypes. Thus, combina- must be able to properly analyze and understand the traits tions of multiple alleles at different loci can cause different of interest. Commercially important traits in aquacul- phenotypes than the simple case of either/or patterns. ture include length, weight, growth rate, feed conversion, A classic example of epistasis for a qualitative trait in oxygen tolerance, percentage of body fat, meat production, aquaculture is the scale patterns in the common carp (Fig. disease resistance, and stress resistance, just to name a 5). Common carp scaling includes wild-scaled, mirror, few. These traits are said to be quantitative because they linear or leather types. Wild-scaled carp have scales all typically vary among individuals within a population. about the fish; mirror carp have scales scattered around Quantitative traits are measured using a continuous distri- the fish; linear carp have scales arranged in a linear array; bution system and statistics. Such traits are described and leather carp have very few scales. These patterns are reported around their central tendencies, such as average controlled by genes (S and N) from two loci. One loci (S) (mean), variance, standard deviation and range. These determines the degree of scales, either wild-scaled (SS or traits can be controlled by a single gene, but are usually Ss) or mirror-scaled (ss). The other loci (N) modify these controlled by several to many genes. These traits are also phenotypes in the following manner: influenced by the environment. Gene expression levels, a. (SS nn or Ss nn); wild-scaled carp the environment, and the interaction of the two can play a b. (SS Nn or Ss Nn); linear carp significant role in the variation of quantitative traits. c. (ss nn); mirror carp Quantitative traits are under a constant variance. d. (ss Nn); leather carp Each gene (except linked genes) is segregated and inde- There is another combination of alleles possible for pendently assorted. As many quantitative traits are scale patterns: the homozygous dominant (NN) form of controlled by multiple genes, each of those genes will be the locus (N). This inheritance is lethal to embryos in the segregating and sorting independently as well. Since so common carp. many genes are involved and each locus is independently Note that the discussions on qualitative traits have assorted, the potential for a variety of genetic combina- concentrated on genes on autosomal chromosomes. How- tions in the offspring is large. Because quantitative traits ever, some phenotypes can be controlled by genes on sex are also influenced by the environment, the action of both chromosomes as well. The mode of inheritance of a trait the environment and the multiplex of genes involved pro- may be different when the allele is linked to one of the sex duces the distribution of these traits in a population. chromosomes. Components of phenotypic expression In an individual, a quantitative trait is determined by its genes, the environment, and the interactions of the (a) two. This relationship is the sum of any phenotype: P = G + E + GE (b) Where: P is the phenotypic value for an expressed phenotype G is the effect of genetics due to a particular pheno- (c) type E is the value for environmental influences (d) GE is the combined effect of the environment on the genotype of the individual Genetics and environment interact differently in each Figure 5. Different phenotypes caused by epistatic effects in common carp: individual. An individual may be influenced differently (a) wild-scaled carp, (b) linear carp, (c) mirror carp, and (d) leather carp. 6 by different environments. Different individuals may The relationships and differences among these genetic respond differently to the same environment. variance values, the way they are inherited, and their Importantly, the individual genetic value can be bro- proportional amounts are important in any breeding ken down into its principal components. The genetic value program. (G) can be described as the sum of additive effects (A), dominance effects (D), and epistatic or interaction effects Genetic variance and breeding (I) such that: Genetic variance is a key component in exploiting the traits of interest in a breeding program. From the G=A+D+I equation above, components of genetic variance include Additive effects are due to the cumulative effects of epistatic effects, additive effects, and dominance effects. alleles at all loci for a given trait. This value is independent Epistatic effects are rarely used in a breeding program of other interactions among alleles or other combinations because there so many possible combinations of alleles of alleles, so additive effects are preserved through meiosis in this measurement of genetic variance. Most programs and these values are predictably passed from parents to focus on additive and dominance genetic variance. The offspring. relative contribution of either of these effects on the phe- Dominance effects are due to the interaction of alleles at notype of interest determines the best approach to use in each locus. During meiosis, homologous chromosomes are a breeding program. separated and reduced from the diploid to the haploid state. Additive effects are passed down from parent to Since gametes are haploid, they can contain no dominance offspring and dominance effects are created with each effect; new pairs of alleles form after fertilization to create generation. If additive effects are large, and thus the varia- new and random diploid states. Thus, dominance effects are tion within a population is large, fish with a desirable trait created in different combinations with each generation. can be selected and bred. If the additive genetic variance Epistatic effects are also important in the genetic is small, then selecting fish for a specific trait within a equation. These effects are caused by the interaction of population may not produce progeny that express the alleles at different loci. Simply stated, this is the effect trait better than the parents. In this case, a recombination of interactions between genes. During meiosis, epistatic of alleles would be the expected approach in the breed- effects for most genes are disrupted by segregation and ing program. More alleles are introduced through an independent assortment (Fig. 4). Therefore, most epistatic influx of new genetic material, based on trial and error. effects are recreated with each generation. New strains of fish can be introduced into the breeding Since quantitative traits have a distribution and vari- program (intraspecific hybridization) or different species ance in a population, one way to understand these traits is bred (interspecific hybridization). A new combination to analyze their variance and effects. Breeding programs of alleles may produce offspring that harbor the desired can exploit the genetic variance in a population in order combination of phenotypes. to minimize its effect. The equation for phenotypic effects For a selection program to be effective, the amount can be modified from the individual to be relevant to the of additive variance should be determined. The measure population. The way to study quantitative traits in a popu- of heritability (h2) describes the proportion that additive lation is to study variance for a trait or traits. Variance genetic variance contributes to a phenotype in a popula- is introduced from the environment (VE), from genetics tion. Heritability describes the percentage of a phenotype (VG), and from the interaction between genetics and the that can be inherited in a predicted manner. This value environment (VGE). Thus, the sum of the phenotypic should be reliable since, for a quantitative phenotype, the variance (VP) for any quantitative trait is: genotype is not affected by meiosis. These values range from 0 to 1 and are a percentage. A heritability value of VP = VG + VE + VGE 1 suggests that the phenotype observed is 100 percent Genetic variance is used in breeding programs to explained by the additive genetic variance. A heritability assist in the selection of a stock of fish. To study genetic value of 0 suggests that additive genetic effects do not con- variance, the value must be broken down into its princi- tribute to the phenotype. In general, a heritability value pal components. Genetic variance is the sum of additive greater than 0.2 (h2 = 0.2) suggests that a trait may be reli- genetic variance (VA), dominance genetic variance (VD), ably exploited in a selection program. If a trait’s heritabil- and epistatic or interaction genetic variance (VI) such ity is lower than h2 = 0.15, a selection-based program may that: not be the answer because dominance genetic variance is more important in this case. Just remember that we VG = VA + VD + VI are dealing with quantitative traits here. These traits may 7 be affected by the environment and the population, and Improving a trait using hybridization is done through can vary between generations. Direct selection of traits trial and error. Some of the hybrids may have superior with low inheritance may be difficult; in such situations, traits and some may not, but hybridization is the best way genome-based technologies such as marker-assisted selec- to increase performance when the heritability for a trait tion may be more applicable. is very low because hybridization creates new combina- tions of alleles through dominance genetic variance. This Genetic improvement programs process is independent of heritability, so hybridization programs can be used even when heritability is high. Selection Selection and hybridization can both be used to increase Selection is a process in which individuals with fish performance. desired phenotypes for a particular trait are identified and used as future broodstock to produce progeny that Intraspecific hybridization are also superior for the trait. Selection programs include Intraspecific hybridization can be used to improve mass selection and family selection. In mass selection, the fish performance in one of two ways: It can be used to performance of all individuals is compared and selection produce a new strain that can undergo selection for a is based on the performance of each, disregarding the trait, and it can also be terminal with the hybrids being parentage. In family selection, the average performance of the end product. In general, hybrids have better fitness families is compared and whole families are selected. because of greater genetic variability. This is known as Selection programs are reliable only if the genes hybrid vigor or enhancement through outbreeding. responsible for the genetic variation are passed to the To create a new breed, a cross must be made between offspring. Additive genetic variance is transmitted to individuals of two strains with different genetic back- offspring in a calculated and reliable manner. Heritabil- grounds, followed by a selection program to improve ity (h2) values must be taken into account. These values performance. A selection program using hybrids usually are a direct measurement of genetic variance explained cannot begin until the second generation of fish (F2) is by additive genetic effects. The heritability estimates for a spawned. This is due largely to the principle of dominance trait should be as large as possible to ensure the efficacy of genetic variation; first-generation hybrids (F1) generally a selection program. do not pass on the hybrid vigor (and superior traits) to Many traits may be correlated, positively or nega- all offspring. The hybrids are created using two distinct tively. For instance, fast growth rate is often correlated strains and, therefore, additive genetic effects should be with a more efficient feed conversion rate in some species increased. This means that subsequent generations are (and the opposite can be true in other cases). Therefore, suitable for use in a selection program. selection for fast-growing fish also selects for fish with Another approach is to conduct a selection pro- a better feed conversion rate. However, if the traits are gram within a strain and then use hybridization to try to negatively correlated, the breeder must be careful because improve performance. However, not all hybrids will show selection for one trait may negatively affect the other. For improved performance. Success depends not only on the instance, fast growth may be negatively correlated with strains selected but also on the reciprocal hybridization. reproduction capacity so that selecting for fast-growing This means that a male of one strain crossed with a female fish could reduce reproductive capacity. Therefore, in of a different strain may produce progeny with different selection programs for certain traits other important traits, and vice versa. The hybrids that perform the best traits must be carefully monitored to determine if there is will be determined by experimentation. any correlation among the traits. Interspecific hybridization Hybridization Fish of different species may be crossed to produce If heritability is very low, selection methods will not more productive progenies. Interspecific hybridization is be the best way to increase performance. To improve a usually used to exploit hybrid vigor, or the tendency of a trait in this instance, greater genetic variation for new crossbred organism to have qualities superior to those of combinations of alleles is needed. New combinations can either parent. However, if progenies are fertile, interspe- be created by mating fish with different genetic histories, cific hybridization has also been used to improve genet- a process called hybridization. All offspring are called ics through introgression, a process by which the genes hybrids. The parents can be of the same species, but dif- of one species flow into the gene pool of another. This is ferent strains (intraspecific hybridization). Or, the parents achieved by backcrossing an interspecific hybrid with one can be of different species (interspecific hybridization). of its parents. The same principles apply as with intraspe- 8 cific hybridization: Genetic improvement is based on new reproduction difficult. combinations of alleles. In aquaculture, triploid grass carp are often grown Interspecific hybrids must be able to produce prog- in ponds along with other species to control grasses and eny. Once this is established, a breeding program can be weeds. While there is limited use for triploids in large attempted. But many between-species hybrids are sterile, scale aquaculture, using the process for species with do not reproduce as readily as the parents, or produce extremely high fecundity, such as oysters, has shown a progenies that are non-viable or abnormal. Spawning does good level of success. Farmers need large numbers of eggs not occur naturally between many species that can be since many eggs do not survive handling and shocking. hybridized, so these species must be artificially spawned. Another way to produce triploids is to mate a dip- The process of producing a superior fish involves loid with a created tetraploid. This method may increase experimenting with combinations of strains, species and viability, but requires a tetraploid population. reciprocal crosses. Tetraploids have four sets of chromosomes (4N). They An example of successful interspecific hybridiza- can be created by shocking a zygote when it is undergoing tion is crossing channel catfish and blue catfish. Each of mitosis. Shock should be applied after the chromosomes these species has several different superior traits. Channel have replicated and as the nucleus is about to divide into catfish is best for commercial production because of its two. The shock prevents the nucleus and cell from divid- growth rate. Blue catfish has a more uniform body shape, ing so that it retains four chromosomes. yields more fillet, is easier to seine, and is more resistant One reason to produce tetraploids is to create trip- to certain diseases. A cross between female channel cat- loids, as mentioned above. Triploids can be created more fish and male blue catfish produces viable offspring. The efficiently by mating diploids with tetraploids than they hybrids have a faster growth rate and are more disease can with shock treatments, because shocking causes resistant than the parental species. With this knowledge, significant losses. Many tetraploids can produce viable hybridization programs can produce fish that can be offspring, so that once a population of tetraploids is cre- selected for further genetic improvements. ated, they could be propagated without creating a new population every time. Polyploidization Most fish species are diploid; they contain two sets Production of gynogens or androgens of chromosome pairs (2N), one set inherited through the Gynogens are fish that contain chromosomes only mother and the other set inherited through the father. from the mother. They are produced by activating oocyte Polyploids have more than the diploid number of chro- division with irradiated sperm and then restoring dip- mosomes. Polyploidy can be induced in fish by using loidy to the developing zygote. Irradiation destroys the techniques such as temperature variation or pressure DNA in the sperm, but the sperm still can penetrate the applied to the eggs to create triploids (3N) or tetraploids egg and induce cell division. After activation of the egg (4N). with irradiated sperm, the second polar body normally is Triploids are created by shocking newly fertilized extruded, resulting in haploid embryos that eventually die eggs. The egg does not expel the second polar body when if no additional treatments are given. One way to restore shocked. This creates a fertilized egg with one nuclei from diploidy is to block the extrusion of the second polar body the egg (N), one from the sperm (N), and one from the by temperature or pressure shocks. Gynogens so created second polar body (N). During development, the three are called meiogens or meiotic gynogens. Meiotic gyno- haploid nuclei will fuse and create a triploid. Triploids gens are not completely homozygous, even though they also can be created by temperature shocks. contain genetic material only from the mother, because Triploids are created to increase fish growth and to of recombinations between chromosomes in the ovum control populations by inducing sterility. Triploids should and in the second polar body. Another way to recover grow larger because the cells are larger (containing more diploidy is to block the first cell cleavage after doubling of genetic material and larger nuclei). And since triploids are the chromosomes. This is done with chemical treatment, usually sterile, less energy is needed to produce gametes temperature shocks, or hydrostatic pressure. Gynogens and this energy may be diverted to growth. Triploids are created this way are called mitogens or mitotic gynogens. sterile because the normal 2N number of chromosomes Mitotic gynogens also contain genetic material only from is disrupted, so that segregation and independent assort- the mother and are 100 percent homozygous. Gynogens ment are disrupted. This makes gametogenesis difficult can be very useful for genetic studies. They are used because the chromosomes cannot be divided equally. to reduce genetic variations and to produce all-female Many triploids also have abnormal gonads, which makes populations in the XY sex-determination system. In 9 certain cases, homozygous genetic material can reduce leading to constitutive production of elevated growth the complexity of a study. For instance, for whole genome hormone, which in turn induces fast growth. An induc- sequencing, a completely homozygous DNA template can ible promoter can be used to detect specific contaminants reduce the complexities caused by DNA sequence varia- for environmental monitoring. For instance, P450 oxidase tions between the two sets of chromosomes in regular promoters are inducible upon exposure to certain con- diploid individuals. taminants. Transgenic fish with these promoters, along Androgens contain chromosomes only from the with a marker gene, would express the marker gene when father and are produced by fertilizing irradiated eggs the transgenic fish is exposed to the contaminants. with regular sperm, followed by doubling of the paternal In spite of these advantages, genetic engineering is an genome. Fertilizing an irradiated egg with normal sperm unconventional approach and the uncertainty of its effect produces a haploid zygote. The zygote is shocked after on both food safety and ecological safety has generated replication, during cleavage, to prevent cell division. The much public resistance to its application in aquaculture. two haploid nuclei fuse together to create a diploid zygote To date, transgenic fish have had very limited use and with all-male chromosomal material. This produces two limited economic impact. The well-documented com- identical copies of haploid male chromosomes. Androgens mercial application of transgenic fish is the ornamental also can be created by using tetraploid males to fertilize glowing zebrafish, the GloFishTM, created in Singapore and irradiated eggs, which produces diploid offspring. commercially available in the United States. Androgens are completely homozygous and are often Although the technology for producing transgenic referred to as doubled haploid. Androgens are useful fish is mature, the production and verification process is for many genetic studies, including reduction of genetic long and the cost is still high. Major obstacles for using variation and production of all-male or all-female popula- transgenic fish are social resistance and regulations. tions. For instance, in the XY sex-determination system, Therefore, recent studies have focused on assessing trans- YY males can be produced by androgenesis. Mating YY genic fish in terms of food and ecosystem safety, and on males with XX females produces an all-male (XY) popu- ways to contain transgenic populations. lation. In some cases, as in tilapia production, all-male populations are desirable because of their higher growth Molecular genetics and genomics rate. Molecular genetics is an emerging field in fish breed- ing programs. It is the study of genetic material (geno- Genetic engineering types) to help determine if fish possess certain traits of Genetic engineering is the process by which a gene(s) interest (phenotypes). One such method of genetic testing or a functional part of a gene is transferred into an organ- that will soon become reality for the aquaculturist is DNA ism. The gene may come from the same species as the marker-assisted selection. When a certain trait of inter- recipient or a different species. A transgenic fish is pro- est is studied, and a genetic marker found for this trait, a duced upon successful gene transfer. The desirable gene is DNA test can determine which fish in the population will then propagated in the offspring. A number of processes be the best to use in a breeding program. Some agricul- are involved in genetic engineering. First, a gene of inter- tural programs such as beef and poultry have imple- est is cloned and inserted into a vector, such as a bacterial mented these technologies in their selection and breeding plasmid DNA. The plasmid is then isolated from the bac- programs already. As our knowledge of the genetics of teria in large quantities. The gene of interest, or the DNA aquaculture species increases, genetic testing will become inserted into the plasmid, is removed from the vector and a reality for this industry also. injected into the fish zygote, where it is expected that the The entire DNA composition of an organism is called new gene will become part of the host fish DNA. Once the its genome. Genomics is the study of the entire genome or transferred DNA is incorporated into the germ cells, it DNA of a species and how genes interact within the whole is inherited as a part of the genome; fish so produced are organism. Whole genome sequences for many important transgenic fish. aquaculture species should be known in the very near Genetic engineering has the advantage of break- future because of advances in sequencing technologies, but ing the species barrier. It avoids epistasis by transferring the complete DNA sequence of most aquaculture species is specific gene(s). Genetic control elements also can be presently unknown. Genomic programs may still be use- manipulated to allow a gene to be controlled by a different ful for any breeding program. Maps of useful traits (their promoter, as desired. Such promoters can be constitutive position along the chromosomes) will be valuable in the or inducible. For instance, a growth hormone gene can integration of genomic data with traditional selection pro- be placed under the control of a constitutive promoter, grams. Long-term goals of a genomics program would be to 10 identify sets of genes and be able to map multiple produc- tion traits to their chromosomes to assist in selection. Original double-stranded DNA Genomics concepts and examples Separate strands of aquaculture genomics research and anneal primers 5’ 3’ Genomics is a very active research field. Rather than Primers 3’ 5’ attempting a thorough review of all the knowledge and progress made, we will discuss some basic concepts of genomics to help readers get to the genomics literature. 5’ 3’ In aquaculture, major progress has been made in genom- ics research on many finfish and shellfish species such as 5’ Atlantic salmon, rainbow trout, tilapia, carp, striped bass, shrimp, oyster and scallop. We will use research in catfish as examples for convenience. 3’ 5’ New primers Molecular markers 5’ 3’ A first step toward improving aquaculture programs through molecular genetics is to identify molecular markers. DNA sequences vary within a population; that New strands is, alleles at a given locus may be different within popu- lations. Such differences are termed polymorphisms. Identifying polymorphic markers within a species can have commercial importance. For example, a molecular marker can be identified that differentiates a population of slow-growing fish from a population of fast-growing fish based on allele usage. To identify molecular mark- Desired ers, blood or tissue samples may be collected in the field, fragment while DNA isolation and the molecular techniques must strands be performed in a laboratory. There are many techniques for identifying molecular Figure 6. The polymerase chain reaction (PCR). The DNA strands are markers. When no genetic information is available for the separated by heating. Primers anneal to the target (complementary) aquaculture species of interest, random amplified poly- sequence and the DNA sequence between the primers is replicated. The morphic DNA (RAPD) and amplified fragment length process is repeated and the number of DNA molecules is doubled with each polymorphism (AFLP) markers can be used. DNA of cycle of PCR to produce many copies of the desired fragment. Courtesy: the aquatic species is isolated and RAPD or AFLP tech- National Human Genome Research Institute. niques are used to try to find polymorphisms in the DNA. RAPD and AFLP markers are identified with the help of polymerase chain reactions (PCR). PCR uses primers observed. The AFLP technique also can identify polymor- (short sequences of synthesized DNA) that bind to DNA phic DNA by using PCR. Of the two techniques used to and amplify a stretch of DNA between the primer bind- identify molecular markers, AFLP is highly robust and ing sites (Fig. 6). In RAPD, short random primers are more reliable, but requires more steps than RAPD, as well synthesized and PCR with low annealing temperature is as some specialized equipment and training. The basis of performed on the DNA. The reaction is visualized on a gel polymorphism in AFLP is also caused by differences in and polymorphisms may be identified between DNA sam- DNA sequences between samples, observed as the pres- ples; they are seen as a presence or absence of an amplified ence or absence of amplified product on a gel. While product. The basis of polymorphism in RAPD markers RAPD and AFLP markers are a quick and economical is the differences in DNA sequences between samples. If way to identify polymorphic DNA between samples, they the DNA sequence at the primer binding site(s) and/or the are inherited as dominant markers. Dominant markers, length of the DNA sequence between the primer sites is as the name implies, will generate a marker with a single different between samples, polymorphisms will likely be dose of alleles. Thus, with dominant markers, dominant 11 homozygous and heterozygous genotypes are not dis- identify important genes from each species. Molecular tinguishable on the basis of the presence or absence of markers can also help identify strain and parentage, and amplified products on a gel. Dominant markers are gener- thus confer lineage-specific information. ally less informative than co-dominant markers. Commu- nications of dominant markers across laboratories can be Genomic mapping difficult as well. Genome mapping techniques include linkage map- In aquaculture species where some genetic informa- ping, physical mapping, molecular cytogenetic mapping tion exists, more molecular markers can be identified. by fluorescent in situ hybridizations, and radiation hybrid Highly robust and informative markers include microsat- mapping. We will not cover molecular cytogenetic map- ellite markers and single nucleotide polymorphism (SNP) ping and radiation hybrid mapping in this publication. markers. Microsatellites are stretches of DNA within To use linkage mapping, multiple molecular mark- a genome that contain simple sequence repeats. As we ers must be identified and a resource family defined. A know, DNA sequences are composed of four nucleotides: resource family is a population of individuals (parents A, T, C and G. When the combination of nucleotides at a and progeny) whose DNA is used for genotyping. Some locus is repeated, such as CACACACACACA, a microsat- planning must be done when choosing a resource fam- ellite exists. Microsatellites are generally highly polymor- ily for genetic studies. If the resource family is highly phic, abundant, distributed throughout the genome, and informative, a monohybrid cross can be used if parents inherited as co-dominant markers. This makes microsat- are true-breeding, or homozygous, for alternate forms of ellites very useful in developing polymorphic markers. a trait (Fig. 4). True-breeding parents produce offspring In some cases, the DNA sequences differ at the primer with a phenotype of interest, say disease resistance. When binding site(s) used to amplify the locus by PCR. This a species is mated from true-breeding parents (P genera- would lead to the non-amplification of the allele (so-called tion), the offspring are termed the first filial generation, null allele). The basis of polymorphism between samples or F1 generation. These F1 progeny are used to create F2 for microsatellites is the number of sequence repeats at a progeny by self-mating, back-crossing, or hybridiza- locus such as (CA)8 versus (CA)10. Use of microsatellites as tion. For linkage mapping studies, the F2 generation (and molecular markers requires prior DNA sequence infor- beyond) are chosen as resource families. If the F1 genera- mation to identify these repeats, as well as some extra cost tion is chosen as a resource family, only heterozygous loci and training. of the parents are segregating; the vast majority of homo- SNP markers are co-dominant markers and using zygous loci of the parents are not segregating. As a result, them requires some prior DNA sequence information. the F1 resource family may not be fully informative, as all SNP markers are also very useful and provide allele- heterozygous siblings will have genotypes with no allele specific information. SNPs are defined as a base change segregation. For instance, if homozygous P fish (AA) at any given position along the DNA chain (e.g., A to and (aa) are mated all progeny will have the Aa genotype G, or C to T). Theoretically, SNPs should have a total of and no allele segregation will be observed. F2 progeny four alleles (A, T, G and C at any position); observations produced by a backcross of F1 fish will produce offspring suggest, however, that they most often exist as bi-allelic with the genetic diversity to use as a resource family, as markers (e.g., the two alleles can be A or G). SNPs can Aa x Aa produces progeny with different genotypes (AA, be identified either within an individual (between sister Aa, and aa alleles are possible). As a practical example, F1 chromosomes in diploid organisms) or between indi- interspecific hybrid catfish (channel catfish x blue catfish) viduals. For example, one allele has the DNA sequence have been created for their superior performance in AATAGCTG and another allele has the sequence several commercially important traits. An F2 generation AATACCTG. In this case, an SNP marker has been iden- was created by mating F1 generation hybrids with channel tified at that locus. catfish and is currently being used as a highly informative Identified molecular markers can have several uses in resource family. genetic analysis. When a polymorphic marker has been Linkage maps are created using multiple polymorphic identified between populations selected for important molecular markers within a resource family. Remember, traits, individuals can be selected for likely trait perfor- recombination of alleles occurs by crossover of homolo- mance based upon their genotype (marker-assisted selec- gous chromosomes. However, recombination frequency is tion). Further, molecular markers can help identify DNA not consistent throughout a genome. Genes on different variation useful for inducing new and favorable traits chromosomes segregate completely independently. When in a selection program where needed. When using an genes are close together on the same chromosome, they interspecific hybrid system, molecular markers can help are physically linked. These genes are expected to have a 12 lower recombination frequency than genes on the same ful in locating specific regions along the chromosome for chromosome but far away from each other. In a resource further study. QTL analysis forms the basis for marker- family, when multiple loci are screened by using multiple assisted selection, where loci that correspond to candidate molecular markers, linkage maps can be created, usually genes or traits can be used to assist classical breeding with the aid of software programs. While the creation of programs. linkage maps is time-consuming and can be complex, the Ultimately, the best genome map can be obtained by process simply involves the reconstruction of chromo- sequencing the whole genome for the species of interest. somes (by creating linkage groups) using the recombina- Sequencing a whole genome is costly and requires time, tion differences of molecular markers. When recombina- effort and specialized equipment. Even so, the whole tion frequency between markers is low (near 0 percent), genome sequence of a species can provide a wealth of it is expected that the markers are linked and little or no information. Most whole genome sequencing projects to recombination has occurred. Conversely, when recombi- date are performed by shotgun sequencing and/or mini- nation frequency between markers is high (approaching mal tiling path sequencing. Shotgun sequencing is done 50 percent), it is expected that the markers are indepen- by sequencing many random, short (~500 nucleotides) dently assorted during meiosis. The creation of linkage segments of DNA and assembling the sequence with the groups is very useful in determining the position and help of computers to re-construct the chromosomes. order of markers/alleles along chromosomes. With minimal tiling path sequencing, DNA is sequenced Another useful genomic mapping strategy is physi- in an orderly manner and guides are used to help in the cal mapping. Physical maps are created with the help of reconstruction process. An example of the minimal tiling a DNA library. A library, in terms of genetics, is any col- path method would be using a BAC library to sequence lection of DNA fragments that have been inserted into a individual BACs and assembling each BAC one by one. cloning vector for propagation. For physical mapping, the Both strategies are effective, and many projects use a whole genome of a species can be fragmented and used combination of the methods to provide a highly accu- to construct large-insert DNA libraries. One example of a rate genome sequence. Recent progress in whole genome large-insert DNA library is a bacterial artificial chromo- sequencing includes the use of next-generation (second some (BAC) library. A BAC library contains many long generation and third generation of sequencers) sequenc- pieces (~200,000 nucleotides) of genomic DNA of the ing technologies. Currently, next-generation sequencing species of interest. BAC libraries can be used to create can produce hundreds of thousands to tens of millions physical maps by a technique called DNA fingerprinting. of DNA sequences from a sequencing reaction. As more The genomic DNA contained in a BAC library is isolated sequencing technologies are developed and they become and fragmented to create “fingerprints,” or highly spe- less costly, whole genome sequencing projects for many cific DNA patterns based on the nucleotide composition aquaculture species are expected. of the DNA sequence. Overlapping fingerprints are then used to reconstruct the DNA, with the goal of creating a Gene expression studies map spanning the entire genome of the species of inter- Other important research is the study of gene expres- est. Physical maps are used in whole genome sequencing sion. The central dogma in molecular biology is the flow projects and are a useful resource for many other genomic of genetic information—DNA to RNA to proteins. A gene projects, including further marker and gene identification, (DNA) is transcribed into RNA. There are several types whole genome comparisons to other highly characterized of RNA; messenger RNA (mRNA) translates the genetic species (map-rich or model species) such as zebrafish and information to proteins, the biologically active endpoint Tetraodon, and in map integration projects. of many genes. Many gene expression studies involve the When linkage and physical maps are created, the study of mRNA. The level of expression of a given gene maps can be integrated (aligned) together, which is par- has a direct relation to the amount of mRNA present for ticularly useful in analyzing quantitative trait loci (QTL). that gene. When candidate genes have been identified, QTLs are regions of DNA where a correlation has been gene expression studies can help determine where (what identified with a trait(s) of interest. QTLs can be added cell or tissue) and how much (quantity of mRNA present) to genomic maps. By integrating the linkage and physi- of the gene has been expressed. There are many reasons cal maps, a QTL identified within a linkage map can be to perform gene expression studies. An example would be located along the physical map. If the marker corresponds determining gene expression in a control versus a treat- to a known gene, the function of this gene may be deter- ment group, such as in healthy fish versus diseased fish. mined. If the QTL occurs along a region of DNA where Several genomic tools can help in gene expression no function can yet be assigned, maps are especially use- studies. The most common technique for studying the 13 expression of individual genes is reverse transcription- to the genes that are expressed in one group but not the PCR (RT-PCR). PCR requires DNA templates, so for gene other(s). The traditional Sanger sequencing method has expression studies mRNA must be converted to comple- limitations. The highly efficient next generation sequenc- mentary DNA, or cDNA, using reverse transcriptase, ers have the ability to discover the entire mRNA compo- thus the name RT-PCR. Once the mRNA is converted to sition of an organism without any subtraction or nor- cDNA, the PCR process works the same way as regular malization, which greatly facilitates genome-scale gene PCR. The idea is that if the starting material contains expression studies. more of a specific mRNA, more PCR products will be Once cDNA libraries have been made and ESTs generated using specific PCR primers than when the generated, these resources can be used to develop micro- starting material contains less of the mRNA. This type arrays. In the absence of a whole genome sequence in of RT-PCR is sometimes referred to as semi-quantitative most of the aquaculture species today, microarrays are PCR because the quantification is not always perfect. often created using all available EST sequences for a spe- Quantification of starting mRNA relies on the PCR to be cies of interest. Common ESTs are combined (clustered) conducted under identical conditions and stays within to create a unique set of sequences. These sequences are the log phase of PCR amplification. A better approach used to synthesize DNA “features.” Features are applied is to use real time RT-PCR. It provides a highly accurate to a media to create an array of sequences. These features assessment of gene expression levels but is costly and can detect the presence and level of expressed genes in requires specific equipment. a population of cDNA through hybridization. Microar- Gene expression profiling is used to assess gene expres- rays are very powerful tools, limited only by the num- sion at the genome scale. When working with a species for ber and quality of EST resources available with which which little or no genetic data is available, cDNA libraries to design features. Many studies involving microarray are useful in gene discovery projects. Not all genomic DNA technology have been and are currently being performed corresponds to the gene coding (protein coding) sequence. in aquaculture species, and there can be many applica- Therefore, by using a cDNA library for sequencing (instead tions. One general application would be to determine of genomic DNA), the sequences correspond to gene coding which genes are differentially expressed in a control group products. Single-run sequencing does not guarantee that versus a treatment group. This is generally performed the complete mRNA (cDNA) will be sequenced. Usually, using a specific tissue or cell type. An example would be only a short fragment of the complete cDNA is sequenced. to study the expression profile of healthy fish liver tissue Single-run sequences of cDNA are called expressed (control) versus liver from a diseased fish (treatment) to sequence tags (ESTs). Many gene products in an organism attempt to discover genes involved in disease resistance can be discovered by generating a large set of ESTs. The or disease susceptibility. In this example, when cDNA is frequency of ESTs in a large-scale EST sequencing project created from both tissues, the microarray can be used to is a rough reflection of the gene expression patterns of the detect the genes that are up-regulated or down-regulated organism. However, repeated sequencing of the most abun- between the tissues. These data are useful in determin- dantly expressed transcripts prevents the rarely expressed ing a global gene expression profile between treatment genes from being sequenced at all. groups, and also in further research to produce species To create a highly efficient cDNA library for gene with superior performance traits. discovery projects, multiple sources (cells and tissues) can be used. To circumvent the problem of repeated sequenc- ing of the most highly expressed genes, various types of Afterword DNA libraries can be created, including normalized and An understanding of the principles of genetics is subtracted libraries. Normalized and subtracted cDNA useful in any aquaculture program. Genetics programs libraries are often created in the effort to sequence the can help increase productivity in aquaculture systems, as greatest number of genes. Since cDNAs in a library are in using hybridization and selection to produce strains sequenced at random, gene products will be sequenced at with superior performance traits. Genetics research has a frequency relative to their expression levels represented improved the quality and production of aquatic species in the library. A normalized library maximizes gene throughout the years, but there is a need for further and discovery because all genes represented in the library are faster genetic gains. Much of the progress to date has been at a more equal abundance (in theory, at least). Subtracted made using traditional selection. As molecular genetics libraries are useful when comparing two or more expres- and genomics tools and technologies are implemented in sion groups. Subtracted libraries eliminate the cDNA aquaculture systems, they will complement and extend shared by the groups. The remaining cDNA corresponds classical genetic improvement programs. 14  SRAC fact sheets are reviewed annually by the Publications, Videos and Computer Software Steering Committee. Fact sheets are revised as new knowledge becomes available. Fact sheets that have not been revised are considered to reflect the current state of knowledge. The work reported in this publication was supported in part by the Southern Regional Aquaculture Center through Grant No. 2007-38500-18470 from the United States Department of Agriculture, National Institute of Food and Agriculture. 16