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BIOTECHNOLOGY ANDGENETICS IN FISHERIES AND

AQUACULTURE

Second Edition

Andy BeaumontSchool of Ocean Sciences

Bangor UniversityWales, UK

Pierre BoudryIfremer

UMR M100 Physiologie et Ecophysiologie des Mollusques MarinsTechnopole de Brest-Iroise

Plouzane, France

Kathrin HoareSchool of Ocean Sciences


A John Wiley & Sons, Ltd., Publication

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AQUACULTURE

Second Edition

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AQUACULTURE

Second Edition

Andy BeaumontSchool of Ocean Sciences


Pierre BoudryIfremer

UMR M100 Physiologie et Ecophysiologie des Mollusques MarinsTechnopole de Brest-Iroise

Plouzane, France

Kathrin HoareSchool of Ocean Sciences


A John Wiley & Sons, Ltd., Publication

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This edition first published 2010C© 2010 by Andy R. Beaumont, Pierre Boudry and Kathrin HoareFirst edition published 2003

Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishingprogramme has been merged with Wiley’s global Scientific, Technical, and Medical business to formWiley-Blackwell.

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Library of Congress Cataloging-in-Publication Data

Beaumont, A. R. (Andy R.)Biotechnology and genetics in fisheries and aquaculture / Andy R.

Beaumont, Pierre Boudry, Kathrin Hoare. – 2nd ed.p. cm.

Includes bibliographical references and index.ISBN 978-1-4051-8857-9 (hardback : alk. paper)

1. Fishes–Molecular genetics. 2. Fishes–Breeding. I. Boudry, Pierre. II. Hoare, K.. III. Title.SH155.5.B43 2010639.3–dc22

2009031829

A catalogue record for this book is available from the British Library.

Set in 10/12 pt Times by Aptara R© Inc., New Delhi, IndiaPrinted in Singapore

1 2010

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http://www.wiley.com/wiley-blackwell



Contents

Preface to the second edition ixAcknowledgements xi

Chapter 1 What is genetic variation? 1

Deoxyribose nucleic acid 1Ribose nucleic acid 5What is the genetic code? 7Protein structure 7So what about chromosomes? 9How does sexual reproduction produce variation? 11Mitochondrial and chloroplast DNA 16

Chapter 2 How can genetic variation be measured? 19

DNA sequence variation 19DNA fragment size variation 36Protein variation 44Phenotypic variation 47

Chapter 3 Genetic structure in natural populations 49

What is a population? 49How are allele frequencies estimated? 51What is the relationship between alleles and genotypes? 51How do allele frequencies change over time? 53How does population structure arise? 54How are genetic markers used to study population structure? 55Levels of genetic differentiation in aquatic organisms 60Potential problems with allozymes and coding markers 61mtDNA variation 63Microsatellite variation 66Population structure in the flat oyster 68Mixed stock analysis (MSA) 73

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Chapter 4 Genetics of population size in conservationand aquaculture 77

Genetics of small population size in the wild 78Genetic markers in conservation 82Genetics of small population size in the hatchery 85Is there evidence of loss of genetic variation in the hatchery? 87How does hatchery propagation affect heterozygosity? 89Genetic markers for identification of hatchery product 93Genetic markers for pathogen identification 94

Chapter 5 Genetic variation of traits 99

Qualitative traits 99Quantitative traits 105What kinds of traits are important? 106Variation of a quantitative trait 106How can we estimate narrow-sense heritability? 109Realised heritability 115Correlated traits 118What types of artificial selections are there? 118Setting up a breeding programme 122Inbreeding, cross-breeding and hybridisation 123Current status of selective breeding programmes in aquaculture 126

Chapter 6 From genetics to genomics 129

What is the genome? 129Genome mapping 130Whole genome sequencing: the ‘big picture’ 137QTL mapping 137Application of QTLs in aquaculture and fisheries management 138Marker-assisted selection (MAS): from QTLs to genomic selection 139Transcriptomics 139

Chapter 7 Triploids and beyond: why manipulate ploidy? 145

How is it done? 146Production of gynogens and androgens 148Identification of ploidy change 149Value of Triploids 151Tetraploids 155Gynogens and androgens 157



Contents vii

Chapter 8 Genetic engineering in aquaculture 161

The DNA construct 162Transgene delivery 164Transgene integration 168Detecting integration and expression of the transgene 169Results of transgenesis efforts in fish 171So much for transgenics – what about cloning? 176Genethics 176

Glossary 181Index 193Colour plates appear between pages 52 and 53



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Preface to the second edition

The rate of change in the field of molecular genetics has been extremely fast since thedevelopment of DNA sequencing methodologies and the polymerase chain reaction.The new millennium has seen increasing miniaturisation and sophistication of thesetechniques, and this has led to second-generation sequencing methods that can producehundreds of thousands of DNA sequences from an organism in a very short space oftime. Because of the pace of these technological changes, some parts of the first editionof this book quickly became outdated.

This second edition benefits from the addition of a new author (Dr Pierre Boudry)and in updating the book to take account of new technologies, we have taken theopportunity to correct errors, improve and reorganise the material from the first edition.Several chapters retain the same headings but with new material added to bring themright up to date. Using material from the first edition and adding fresh text and figures,we have introduced a new chapter based around the genetics of population size thatlinks directly to problems in aquaculture and in conservation. In addition, we haveprovided a new chapter called ‘Genetics to genomics’ that covers recent developmentsin genetic mapping, the search for quantitative trait loci and introduces the subject oftranscriptomics – the transcription of DNA sequence to RNA.

We have retained the introductory-level nature of the book but have increased thenumber of references at the end of each chapter for more advanced readers.

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Acknowledgements

The following have kindly given us permission to use some of their material in theillustrations for the book: Dr Ann Wood, Dr Karen Abey, Dr Shelagh Malham, DrCraig Wilding, Chris Beveridge, Alix Taylor and Natalie Horton. We thank the scientificpublishing companies that have allowed us to use figures from published sources and allsuch companies are acknowledged in figure legends.

We thank David Roberts from the School of Ocean Sciences, Bangor University, forphotography and assisting with preparation of figures.

Dr Pierre Boudry thanks the members of the Working Group on the Application ofGenetics in Fisheries and Mariculture (International Council for the Exploration of theSea) for sharing ideas and knowledge.

This second edition of the book would not have appeared without the support andencouragement of Nigel Balmforth and the keen editorial assistance of Kate Nuttall andCatriona Dixon of Wiley-Blackwell.

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Chapter 1

What is genetic variation?

Have you ever seen someone who looks and sounds exactly like you? Have you everseen your ‘spitting image’? Unless you are one of a pair of monozygotic twins (twinsproduced by the division of a single egg), you will not have done so. It is commonlyaccepted that all humans are different – indeed all humans there have ever been wereunique and were different from all humans living today. If this is true for Homo sapiens,is it also true for other sexually reproducing organisms? The answer is yes. Every salmon(Salmo salar) is different from every other salmon that has ever lived. Every mussel(Mytilus edulis) is different from every other mussel that has ever lived. This uniquenessof individuals within a species is the consequence of two factors: one is deoxyribosenucleic acid (DNA) and the other is sexual reproduction. These two factors produceand maintain the genetic diversity within a species, and an understanding of this isfundamental to our ability to sustainably exploit species of plants and animals. Withoutsex, the reproductive process can produce genetically identical individuals, as in plantswhere vegetative reproduction is common, and there are biotechnological methods thatcan produce clones (see Chapter 7).

Deoxyribose nucleic acid

The discovery by Watson and Crick of the structure of DNA in 1953 was a landmark inour understanding of how genetic information passes from generation to generation andhow it codes for the amino acids of proteins. In the half century since then, the fieldsof molecular biology and genetics have become inextricably linked and developments,particularly over the past 25 years, have opened up the potential of DNA biotechnologyand genomics.

The structure of DNA enables it to carry the information for a cell to reproduce itself.It is a polymeric molecule, that is, made up of a chain of subunits, consisting of chains ofnucleotide monomers. Each nucleotide contains a base, along with a sugar (deoxyribose)and a phosphate group (Figure 1.1). There are four individual bases: adenine, guanine,thymine and cytosine, and they are usually referred to by their first letter abbreviations,A, G, T and C. Two of the bases, A and G, have a double-ring structure and are knownas purines. The other two bases, T and C, are pyrimidines with a single carbon–nitrogenring.

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2 Biotechnology and Genetics in Fisheries and Aquaculture

Figure 1.1 The structure of DNA. Each nucleotide consists of a sugar, a phosphate and a base.Nucleotides are joined by a phosphodiester bond between the 5′ of one ribose sugar and the 3′ ofthe next. The chain of nucleotides therefore has a 3′ and a 5′ end.

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What is genetic variation? 3

Figure 1.2 The structure of DNA. Two polynucleotide strands are wrapped around each other inthe form of a double helix. Complementary base pairing occurs between the two strands such thatguanine (G) always bonds with cytosine (C), and adenine (A) always bonds with thymine (T).(Modified from Utter, F., Aebersold, P. & Winans, G. (1987) Interpreting genetic variation detectedby electrophoresis. In: Population Genetics and Fishery Management (eds N. Ryman & F. Utter),pp. 21–45. University of Washington, Washington, DC, USA, with permission from WashingtonSea Grant Program, University of Washington.)

Each nucleotide is a single unit that joins with neighbouring nucleotides in a linearfashion to make up a polynucleotide chain. Particular carbon atoms in the 5-carbonstructure of deoxyribose are referred to by numbers, 1′ (pronounced prime) to 5′. Thelink between nucleotides is formed when the 5′ of one bonds to the 3′ of the next via aphosphodiester bond (Figure 1.1). It is the sequence of the four bases in a polynucleotidechain, which acts as the code for genetic information.

The complete DNA molecule actually consists of two polynucleotide chains, orstrands, wrapped around each other in the form of a double helix. The sugar + phosphatebackbones are at the outside of the molecule while the bases point towards the middleof the structure; the two strands of the molecule run in opposite directions (Figure 1.2).

The functional beauty of the DNA molecule is a result of complementary base pairingwhere G can only bond with C, and A can only bond with T, at the middle of themolecule. It means that the two strands are complementary such that the base sequenceof one strand predicts and determines the base sequence of the other strand. Becauseone strand predicts the other, it can be used to replicate the sequence.

The replication process produces daughter molecules, each of which has one parentalstrand and one copied strand. This is called semi-conservative replication. Replicationof DNA takes place every time a cell divides. The cell’s entire DNA is progressivelyunwound, revealing short single-stranded regions which can be copied by DNA poly-merase enzymes. Unwinding does not begin at the ends of the molecule, but at pointscalled replication origins, and it then proceeds from these points along the DNA. Thenew strands of DNA being synthesised during replication are always synthesised in the5′ to 3′ direction. This means that as the original strands separate, one new strand canbe continuously synthesised against its copy strand (the leading strand), while the otherhas to be synthesised intermittently in short lengths as enough copy strand (the laggingstrand) becomes available (Figure 1.3).

Considering the enormous numbers of bases and coded information in the DNAof a cell, replication needs to be extremely accurate. Even a very small incidence ofmistakes in copying would result in the loss of important genetic information within

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Figure 1.3 Replication of DNA. As the DNA double helix unwinds, new DNA is synthesisedcontinuously in a 5′ to 3′ direction on the leading strand and in 5′ to 3′ directed segments (Okazakifragments) on the lagging strand.

a few cell divisions. However, during the replication process, various proofreadingactivities take place and almost all errors are corrected by removing the incorrect baseand inserting the correct one. In spite of proofreading, a few errors are inevitable whensuch high numbers of bases are to be copied and it is estimated that about one in every3 billion bases is incorrectly inserted. Such errors are called point mutations, and they canalso be induced at much higher rates by certain chemicals and radioactivity. Althoughthere are very few of them at each genome replication event, they are nevertheless thefundamental source of variation which fuels the process of evolution. Without sucherrors, no genetic change at the DNA level would take place, preventing any adaptationof organisms in a changing environment, but with too many errors, daughter cells wouldtoo often be non-viable and the organism carrying that DNA would soon become extinct.

Functional sequences, i.e. coding DNA, represent only a small fraction of the totalgenome, for example around 3% in humans. The rest is made up of what has been called‘junk DNA’. Whether all of it is really ‘junk’ is not known, and several hypotheseshave been proposed to explain how junk DNA might have a function, notably in termsof regulation of gene expression. Some of this junk DNA consists of pseudogenes,genes that for some reason or another have become non-functional. Yet other parts ofnon-coding DNA consist of dispersed or clustered repeated sequences of varying length,from 1 base pair (bp) to thousands of bases (kilobases, kb) in length, known as ‘repetitiveelements’. Junk DNA also includes transposons that encode proteins with no clear value

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Figure 1.4 Generalised structure of a gene. The open reading frame for a gene begins with anupstream initiation codon and ends with a downstream termination codon. Many genes have aregion, or regions, of non-coding DNA within them. These introns are spliced out of the mRNAduring transcription so that only the codons within the exons are translated into amino acids.

to their host genome. The dispersed repeated sequences occur as copies spread acrossthe genome and can be categorised as long or short interspersed nuclear elements, longterminal repeats and DNA transposons. The clustered repeated sequences, where therepeated sequence occurs in tandem copies, are classed as satellites, minisatellites ormicrosatellites, depending on the length of the repeat unit, and these have turned out tobe useful genetic markers, as will be explained in later chapters. Between them, theserepeated elements can constitute up to 40% of the genome.

A gene is a unit of information which is held as a code in a discreet segment ofDNA. This code specifies the amino acid sequence of a protein or the sequence of anRNA molecule. Scientists were surprised to discover quite early on that the sequenceinformation for a single gene was often not continuous along the DNA, but wasinterspersed with pieces of non-coding sequence. The coding parts of a gene sequenceare exons, and the non-coding parts are introns (Figure 1.4). Before a gene can beexpressed, the DNA that encodes it has to be transcribed into ribose nucleic acid (RNA).

Ribose nucleic acid

The structure of RNA is similar to that of DNA except that (a) the sugar is ribose insteadof deoxyribose, (b) in the place of thymine, a similarly structured base called uracil (U)is present and (c) the molecule consists of only a single polynucleotide strand. RNAmolecules are produced by the process of transcription of the linear sequence of basesin DNA and are then used in the translation of that sequence into a chain of amino acidsthat go to make up a protein. The type of RNA transcribed from the sequence is calledmessenger RNA (mRNA), and translation of this sequence into a string of amino acidsis undertaken by ribosomal RNA (rRNA) and transfer RNA (tRNA) molecules.

During transcription of the DNA, an RNA copy is made of one of the strands ofDNA (Figure 1.5). The two strands of DNA are called the template strand and thenon-template strand. Other names for the non-template strand are the sense (+) strandor the coding strand. The RNA is synthesised by RNA polymerase enzymes using the

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Figure 1.5 Transcription and translation of DNA. Introns are removed from pre-messenger RNAbefore translation takes place. The polypeptide chain is formed from amino acids coded for by themRNA and brought together by tRNA. (Modified from Utter, F., Aebersold, P. & Winans, G. (1987)Interpreting genetic variation detected by electrophoresis. In: Population Genetics and FisheryManagement (eds N. Ryman & F. Utter), pp. 21–45. University of Washington, Washington, DC,USA, with permission from Washington Sea Grant Program, University of Washington.)

template strand and is therefore a copy of the non-template (sense or coding) strand ofDNA. Because this RNA is a direct copy of the DNA, it contains both the coding (exons)and the non-coding sequences (introns) of the gene. Introns are removed from this pre-messenger RNA, and the subsequent molecule is the final mRNA. The mRNA moleculesare transported from the nucleus into the cytoplasm where the message is translated intoa sequence of amino acids by rRNA in bodies known as ribosomes. Amino acids arebrought to the ribosomes by tRNA molecules, each specifying a particular amino acid(Figure 1.5), and synthesised, in the presence of rRNA, into a linear sequence.

The detailed mechanics and biochemistry of the processes of transcription and trans-lation are outside the scope of this book, but can be found in most standard genetictexts. The term ‘gene expression’ encompasses both of these processes and their variouscontrolling steps. ‘Transcriptomics’ is the study of the transcriptome, the set of RNA

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transcripts produced by the genome of a cell, an organ or a whole organism at any onetime. For the purposes of this book, the reader need only appreciate the key concept thata coding sequence of bases in DNA leads, by a direct copying process involving RNA,to the production of a sequence of amino acids, the building blocks of proteins. This iswhat has been called the ‘central dogma’: information is transferred from DNA to RNAto protein. Some exceptions to the central dogma occur, such as retroviruses and prions,but they are outside the scope of this book.

What is the genetic code?

How are the four bases (A, C, G and T) in DNA organised to provide an unambiguouscode for the 20 amino acids present in proteins? The ‘words’ of the code consist ofthree bases. There are 43 = 64 possible combinations of the four bases into a tripletcode, and it is these 64 triplet codons which define the 20 amino acids. Because thereare more than 20 codons, the genetic code has some redundancy – most amino acidsare coded for by more than one codon. The codons are written using the symbol U, foruracil (in mRNA), rather than T, for thymine (in DNA). Three codons (UAA, UAG andUGA) do not encode amino acids but act as signals for protein synthesis to stop and arecalled termination codons or stop codons. The triplet AUG codes for methionine (formylmethionine in bacteria and mitochondria) and is the signal for protein synthesis to start.It is thus the initiation codon which sets the reading frame. The amino acid sequence ofall proteins therefore starts with methionine, but this is sometimes removed later. Detailsof the amino acids encoded by the various codons are given in Table 1.1. Note that theredundancy of the code is not random. In particular, the first two bases of the codonsfor an amino acid are usually the same. It is generally only the third base which varies.Different organisms often show particular preferences for one of the several codons thatencode the same given amino acid and this is called codon bias. How these preferencesarise within a genome is a debated question.

Protein structure

Proteins have many tasks. Some form the structure of tissues, others – the enzymes –act as specific catalysts of biochemical reactions, and yet other proteins, such as hor-mones, have a regulatory function. By their very nature, proteins are bound to be highlycomplex molecules, but it is possible to categorise their structure into four basic levels.The primary structure of a protein is the linear sequence of the chain of amino acids (thepolypeptide chain) and this, as we have seen already, is directly related to the sequenceof bases in the DNA which codes for it. Although most amino acids are pH neutral, twoare negatively charged and two positively charged. In addition, some are hydrophilic (at-tracted to water) and others hydrophobic (repelled by water). Thus, secondary structureof a protein is based on characteristic patterns produced by the properties and interac-tions of particular types of amino acids within the chain. One such secondary structure

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Table 1.1 The genetic code showing the amino acids coded by the 64 triplet combinations of thefour bases. The bases down the left-hand side represent the first position in the reading frame, thebases along the top indicate the second position and the bases down the right-hand side showthe third position.

Second base

First base U C A G Third base

U Phe Ser Tyr Cys UPhe Ser Tyr Cys CLeu Ser Stop Stop ALeu Ser Stop Trp G

C Leu Pro His Arg ULeu Pro His Arg CLeu Pro Gln Arg ALeu Pro Gln Arg G

A Ile Thr Asn Ser UIle Thr Asn Ser CIle Thr Lys Arg AMet Thr Lys Arg G

G Val Ala Asp Gly UVal Ala Asp Gly CVal Ala Glu Gly AVal Ala Glu Gly G

Ala, Alanine; Arg, Arginine; Asn, Asparagine; Asp, Aspartic acid; Cys, Cysteine; Glu, Glutamic acid; Gln, Glutamine;Gly, Glycine; His, Histidine; Ile, Isoleucine; Leu, Leucine; Lys, Lysine; Met, Methionine; Phe, Phenylanaline; Pro,Proline; Ser, Serine; Thr, Threonine; Trp, Tryptophan; Tyr, Tyrosine; Val, Valine.

is an alpha-helix, another is a pleated sheet. The tertiary structure is dependent on howthese secondary structures become folded in three dimensions. Therefore the DNA code,through the linear relationship of the various amino acids, dictates both the secondaryand tertiary structures. This is an important point because it reveals that point mutationsin the DNA coding for a particular protein can have far-reaching consequences on thefinal size, shape and overall charge of that protein.

Many proteins are composed of two or more polypeptide chains (subunits), and thesubunits making up a protein may be identical or they may be different. The genericname for proteins with more than a single subunit is oligomers. This is the level ofquaternary structure of proteins and it enables larger proteins to be produced withoutrequiring a very long gene sequence in the DNA. It also allows greater functionalityin proteins by combining different activities within a single molecule. Proteins with asingle subunit are called monomers, those with two subunits are dimers and those withfour are tetramers. For example, glucose phosphate isomerase, an enzyme involved inthe production of energy from the breakdown of carbohydrates, is a dimer, with twosubunits coded by the same gene, while haemoglobin, which carries oxygen around inthe blood, is a tetrameric molecule consisting of two alpha-globin and two beta-globinchains, each coded by different genes.

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So what about chromosomes?

In fish and shellfish, as with all other eukaryote organisms, the DNA molecules in thenucleus are combined with proteins, mainly histones, to make chromosomes. Each chro-mosome represents a single DNA molecule. Chromosomes are usually only clearly visi-ble and identifiable when cells are dividing, at which time the chromosomes have alreadydivided into daughter chromatids. However, the daughter chromatids retain connectionto each other at a position called the centromere, or primary constriction, and this is thelast part of the chromosome to divide. The position of the centromere on the chromosomecan be central (metacentric), between the centre and one end (submetacentric), veryclose to one end (acrocentric), or terminal (telocentric). The number of chromosomes,their lengths and the positions of their centromeres are unique to each species and thesecharacters are used as descriptors for the species karyotype (Figure 1.6). Chromosomesthemselves mutate and evolve (Box 1.1), and before the advent of allozyme markers,some geneticists spent much of their time squinting down microscopes following the

Figure 1.6 Metaphase chromosome spread and karyotype of Mytilus edulis. There are sixmetacentric and eight submetacentric pairs of chromosomes. Haploid number N = 14, diploidnumber 2N = 28. Scale bar = 5 μm. (Reproduced with permission from Dixon, D.R. & Flavell, N.(1986) A comparative study of the chromosomes of Mytilus edulis and Mytilus galloprovincialis.Journal of the Marine Biological Association, UK, 66, 219–228, Cambridge University Press.)

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