Académique Documents
Professionnel Documents
Culture Documents
of Molecular Tools in
Aquaculture and Inland
Fisheries Management
MANUAL ON APPLICATION OF MOLECULAR TOOLS IN AQUACULTURE AND INLAND FISHERIES MANAGEMENT: PART 2
Part 2
Laboratory protocols
and data analysis
NACA Monograph 2
www.enaca.org
Manual on Application of
Molecular Tools in Aquaculture
and Inland Fisheries
Management
Part 2:
Laboratory protocols and data analysis
Contributors
Thuy Nguyen
Network of Aquaculture Centres in Asia-Pacific
Uthairat Na-Nakorn
Kasetsart University, Thailand
Wongpathom Kamonrat
Department of Fisheries, Thailand
Devin Bartley
Food and Agriculture Organization of the United Nations
Queensland University
of Technology
Brisbane, Australia
NACA MONOGRAPH SERIES
NACA is an intergovernmental organization that
promotes rural development through sustainable
aquaculture. NACA seeks to improve rural income,
increase food production and foreign exchange
earnings and to diversify farm production. The
ultimate beneficiaries of NACA activities are farmers
and rural communities.
ISBN 978-974-88246-2-8
List of tables....................................................................................................... 6
List of figures ..................................................................................................... 7
Acknowledgements........................................................................................... 9
Aims and scope .................................................................................................. 9
3
2.6. Single strand conformation polymorphism (SSCP) ................................ 45
2.6.1. Chemical preparation............................................................................ 45
2.6.2. Sample preparation............................................................................... 45
2.6.3. Gel running ............................................................................................ 46
2.6.4. Silver staining ........................................................................................ 46
2.7. Random amplified polymorphic DNA (RAPD) ........................................ 47
2.7.1. PCR preparation .................................................................................... 47
2.7.2. DNA amplification ................................................................................. 47
2.7.3. Electrophoresis ...................................................................................... 48
2.7.4. Staining and documentation ................................................................ 48
2.7.5. Scoring ................................................................................................... 49
2.7.6. Trouble shooting ................................................................................... 50
2.8. Amplified fragment length polymorphism (AFLP) ................................ 50
2.8.1. Restriction digestion of genomic DNA ................................................. 50
2.8.2. Adapter preparation ............................................................................. 51
2.8.3. Ligation of adapters .............................................................................. 51
2.8.4. Pre-amplification reactions ................................................................... 52
2.8.5. Selective amplification .......................................................................... 52
2.8.6. Gel electrophoresis ................................................................................ 52
2.8.7. Silver staining ........................................................................................ 54
2.8.8. Scoring ................................................................................................... 55
2.8.9. Gel preservation .................................................................................... 55
2.9. Microsatellites .......................................................................................... 55
2.9.1. Preparation of sequencing dye ............................................................ 56
2.9.2. Preparation of PCR cocktail .................................................................. 56
2.9.3. The PCR cycles ........................................................................................ 57
2.9.4. Check quality of PCR product using agarose gel ................................. 57
2.9.5. Protocol for polyacrylamide gel electrophoresis ................................. 58
2.9.6. Polyacrylamide gel preparation ........................................................... 58
2.9.7. Assembly of gel plate into the gel rig.................................................. 59
2.9.8. Electrophoresis ...................................................................................... 59
2.9.9. Chemical preparations .......................................................................... 60
2.9.10. Gel fixation and staining .................................................................... 61
2.9.11. Scoring gel ........................................................................................... 61
2.9.12. Trouble shooting ................................................................................. 62
2.10. Temperature gradient gel electrophoresis (TGGE) ............................... 62
4
3.1.4. Dominant markers................................................................................. 81
3.1.5. DNA sequences ...................................................................................... 84
3.2. Statistical tests ......................................................................................... 87
3.2.1. Neutrality tests ...................................................................................... 87
3.2.2. Linkage disquilibrium (LD) .................................................................... 88
3.2.3. Testing FST for significance ................................................................... 90
3.2.4. Estimating gene flow from mtDNA sequence data ............................ 91
3.2.5. The stream hierarchy model of gene flow .......................................... 93
3.2.6. Isolation by distance.............................................................................. 94
3.3. Presentation of data ................................................................................ 94
3.3.1. Minimum spanning networks............................................................... 95
3.3.2. Neighbour-joining trees ........................................................................ 96
3.3.3. Population trees .................................................................................... 97
3.3.4. Historical inference ............................................................................... 98
3.3.5. Statistical inference and interpreting the data ................................... 99
3.4. Commonly used software for data analysis......................................... 100
5
List of tables
6
List of figures
7
8
Acknowledgements
The senior author would like to thanks Mr. Pedro Bueno, former Director General
of NACA, without whose support the manual could not have become possible.
Encouragement from Prof. Sena De Silva, Deakin University (current Director
General of NACA) is very much appreciated. P. Mather and D. Hurwood would
like to acknowledge the Australian Centre for International Agricultural Research
(ACIAR) for funding support. We would like to thank Dr. Menchie Ablan from the
WorldFish Centre for her constructive comments.
This is the second part of the manual, “Application of molecular genetic techniques
in aquaculture and inland fisheries management”. The major aim of this part of
the manual is to provide step-by-step laboratory protocols and methodologies for
data analysis, and a guideline to design a population genetic study.
The scope covers most commonly used techniques for screening genetic variation,
general background on the methodologies for estimation of important parameters
in population genetic studies for different forms of molecular genetic markers.
▪ Section III - Data analysis and project design: will deal with aspects of data
management such as data analysis, interpretation and presentation, and a
guideline to design a population genetic studies.
9
10
Section 1
Molecular markers -
an overview
There are many levels at which we can 1.1 Nuclear markers
assess how different populations or
individuals may be, genetically. The The nuclear genome (nDNA) is very
traditional method and the approach large in most eukaryote species and
still employed in classical systematics recombines. Most species (although
has been to examine external morpho- not all) have a relatively large number
logical traits and infer divergence in of chromosomes with many DNA
the underlying genes that produce sequences available for analysis. Most
morphological phenotypes. There are eukaryote species are diploid (2N)
a number of problems that can be although some groups of fishes (e.g.
associated with this approach however, salmonids and catastomids) contain
including the fact that morphological polyploid lineages that can make
traits can often be highly conserved, genetic differentiation analyses more
convergent evolution can confuse complex. For most nuclear markers
relationships and associated with this, (unlike mitochondrial DNA (mtDNA)
environments can modify expression where offspring inherit from the
of underlying genes in diverse ways female parent only in most species),
hiding true patterns of relationship. male and female contribution to
Thus biologists have sought more offspring is nearly equal (the exception
fundamental markers with which to being where sex determination of the
assess genetic relationships and the individual is via sex chromosomes). For
advent of molecular genetics has nDNA both coding and non-coding
provided potential markers of genetic DNA sequences are present and
relationship that do not face the same individual sequences vary widely in
problems evident in comparisons their rates of evolution that are at
of morphology. This is because the least in part determined by functional
genotype is fixed at fertilisation and constraints on some coding sequences.
consequently cannot be influenced Thus there is a large amount of DNA
directly by the environment. Potential available for analysis and many ways
sources of direct genetic markers of of targeting individual sequences for
differentiation include; chromosome study.
morphology, protein variants, whole
DNA fragments and DNA sequences. To the uninitiated a diverse array of
Basically the simple linear arrangement molecular techniques and methodolo-
of four nucleotides (A, T, C, and G) gies are now available for analysing
contains a large amount of information DNA sequences and choosing appro-
on evolutionary rates, mutation rates, priate methods for the specific ques-
fixation rates and selection pressures. tions to be addressed can be a complex
There are also now a diverse array of and confusing process. When deciding
genetic markers to choose, both from on what type of genetic marker to use
the nuclear and cytoplasmic genomes. to characterise DNA genetic diversity in
a new study, a number of issues should
be considered: (1) choice of marker
13
to a large extent will depend on the because they can be very sensitive to
research question(s) to be addressed the PCR conditions used to generate
- choose a marker that can adequately them. This means that there is now
address the specific research questions, more pressure to demonstrate that
(2) relative costs, technical difficulty RAPDs are repeatable and inherited in
and necessary facilities/equipment may a Mendelian fashion before data are
require compromise – but choose the necessarily accepted.
most powerful marker available within
resource limitations, with the capacity The markers that led the revolution in
to address the questions and (3) some modern genetic diversity analyses were
questions are better addressed by Allozymes. First developed for analysis
synthesising data from different types of some human metabolic disorders in
of markers (e.g. combining data from the early 1960’s, evolutionary biologists
nDNA and mtDNA markers). Two broad quickly realised that they could be
classes of marker are available for used to investigate diversity in a wide
analysis of nDNA, so-called Dominant array of organisms at a much more
and Co-dominant markers. Dominant fundamental level than had been
markers (e.g. Random Amplified possible previously. While allozymes are
Polymorphic DNAs [RAPDs] and Ampli- not affected by environmental factors,
fied Fragment Length Polymorphisms unlike most DNA technologies, the
[AFLPs]) are assessed by presence variation observed is ‘one step’ away
/absence of a band on a gel and so we from variation at the most fundamental
are not able to directly assess if an indi- level because it is protein phenotypes
vidual with a band is homozygous (aa) that are compared rather than DNA
or heterozygous (ab) for the fragment. sequences directly. Studies commenced
So for genetic diversity studies with in the late 1960’s and early 1970’s and
dominant markers we can only estimate now a huge data set is available. While
expected heterozygosity assuming new technologies have taken over
H/W equilibrium and have no way of from allozymes to a large extent in
determining if the population conforms many labs, they still provide a relatively
or not. Thus, while dominant markers inexpensive, quick and sensitive tech-
have their uses (especially for example nique for screening genetic diversity.
AFLP’s in gene mapping studies), In some labs, researchers are returning
co-dominant markers (e.g. allozymes, to use this method because often the
microsatellites, SSCPs etc.) provide results achieved are comparable with
better analytical power because they that achieved from more sophisticated
allow direct determination of heterozy- but technically difficult and expensive
gosity in a sample. While many studies methodologies (e.g. microsatellites).
have used dominant RAPD markers to The main shortcomings of this tech-
document genetic diversity, in recent nique is the fact that tissue needs to
times questions have been raised about be stored at -80°C and that sampling
the reliability and repeatability of tissue is almost always invasive (i.e. the
RAPD’s particularly in animal species animal is killed).
14
One of the first technologies to directly not the larger intervening sequence of
target diversity at the DNA level DNA and (b) the relatively high cost of
directly was restriction fragment length the restriction enzymes.
polymorphism (RFLP). This relatively
simple approach uses commercially Development of the Polymerase Chain
available restriction enzymes (REs) that Reaction (PCR) in the late 1980’s revo-
are harvested from bacteria species lutionised the study of DNA sequences.
to cut DNA sequences at specific This is because the method in theory
sites (restriction sites). Each RE has a allows any DNA sequence to be ampli-
Section one
unique recognition site, so that when
a particular RE (e.g. EcoR-1) recognises
fied and millions of copies made of
just the targeted fragment that is then
its own specific restriction sequence available for analysis. Essentially PCR is
along a DNA molecule it cuts the in vitro DNA replication. Providing we
DNA at the site. This is known as a have some knowledge about the DNA
Title
restriction digest. The more restriction
sites along the sequence the more
sequence surrounding the sequence
of interest we can design Primers
places the RE will cut the DNA and (short DNA fragments usually (10 to
hence the greater will be the number 25bp in length - oligonucleotides)
of fragments produced. By exposing that bind to the DNA either side of a
the same piece of DNA from different target sequence and act as initiators
individuals to the same RE’s separately of replication for the target sequence.
we can detect any mutations in the A similar approach is used to obtain
recognition sites in individuals by the the complete sequence of a DNA
pattern of fragments produced on a gel fragment, because in sequencing
that separates DNA fragments by size. reactions the new nucleotides that are
For each individual, the combination added to a growing fragment have
of all size fragments after ‘cutting’ been prior labelled with fluorescent
with the RE should equal the total size dyes (a different colour each for A, T,
of the original DNA fragment. Thus G and C) and a laser reads the colour
we can estimate genetic divergence as they are incorporated individually
among individuals or populations in the growing strand and hence the
by comparing the number of DNA complete sequence of bp’s along a
fragments (or RFLPs) they share after fragment can be deciphered. The
restriction digest. Individuals with sequence data can then be stored
identical genotypes will have exactly on large databases (e.g. GenBank,
the same set of fragments for the same Australian National Genetic Informa-
piece of DNA. While RFLP analysis is still tion System – ANGIS) and accessed via
used for genetic diversity studies, the the net for comparative purposes.
approach is less popular now because
it is limited by (a) the ability of RFLP Microsatellites (or simple sequence
analysis to only provide information repeats – SSRs; or variable number
about the sites at which REs cut and tandem repeats VNTRs) currently are
the nDNA marker most favoured in
15
genetic diversity studies as they usually Once genetic data are available from
show very high levels of individual and individuals and or populations there
population variation. SSRs are either are a number of different genetic
simple (e.g. TGTGTGTGTG) or complex diversity indices that we can calculate
(e.g GAA(GA)17GAA) tandem repeats (depending on the data type). The
of short DNA sequences that are found ones most frequently used include;
at regular intervals right across the % polymorphic loci, relative allelic
genome of most eukaryote species. diversity, average heterozygosity,
While they are quite costly and time- Perhaps % polymorphic loci is the least
consuming to develop for a new species informative index (unless a relatively
they have great utility in intra-specific large number of independent loci are
studies. Most SSRs are embedded available) because the interpretation
in non-coding DNA sequences, are can be heavily biased by both the
noncoding themselves and conse- number of loci screened and their
quently accumulate mutations very function in the organism (if any).
rapidly. Thus individuals in outbred Relative allelic diversity and average
populations rarely share the same SSR heterozygosity are considered better
genotype across several loci. While measures of genetic diversity than %
this characteristic is advantageous in polymorphic loci but cannot always be
many intra-specific genetic diversity calculated e.g. heterozygosity cannot
applications it can be problematical in be calculated directly from markers
comparisons of more distantly related that show dominant inheritance. Other
individuals or populations because complications for specific types of
both Homoplasy and Null alleles can marker include the recognition that
be a problem. Where either or both phylogenetic inference from most
occur they will tend to result in under- nDNA is compromised potentially
estimates of true genetic divergence. by the fact that associations of
Another issue for use of microsatellite alleles/sequences along individual
data in phylogenetic studies is that the chromosomes in the nDNA have the
mode of evolution of microsatellite potential to be mixed up by chance
alleles has been debated. Unless it is it genetic recombination events and
has been determined for a particular confused by bi-parental inheritance (i.e.
species that mutations occur as an offspring can receive various combi-
accumulation or loss of single (stepwise nations of the parental chromosomes).
mutation model) or multiple repeats For fast evolving nDNA sequences like
then making the wrong assumption SSR’s we also need to consider that
can bias phylogenetic reconstructions homoplasy and null alleles could affect
of relationship. due to the problems our interpretations about genetic
outlined here, it is unwise to use SSRs diversity. Demographic events can
for phylogenetic reconstruction. also influence the patterns of genetic
diversity in some populations and
not in others, for example when one
population has been pushed through
16
a population bottleneck while another structure. Secondly, the lack of recombi-
has not. Population bottlenecks can nation means that mtDNA lineages will
result in loss of allelic diversity as evolve without the history of descent
population size declines. This loss is becoming jumbled over time as on
stochastic and essentially unrelated homologous chromosomes. This allows
to individual fitness. This means that us to differentiate between historical
current levels of allelic diversity may and contemporary processes that may
be related to past demographic events have influenced or determined the
(e.g. historical population bottlenecks) observed population genetic structure.
and hence may not accurately reflect Thirdly, mtDNA is generally considered
current relationships. selectively neutral (although this has
been challenged recently). Therefore
1.2. Mitochondrial DNA the effects of selection can largely be
markers removed as a confounding factor when
interpreting the data. In contrast, past
Mitochondrial DNA (mtDNA) differs or ongoing selection can be a signifi-
significantly from nuclear DNA in cant problem for some nuclear markers.
structure and mode of inheritance.
MtDNA is a circular molecule that The most powerful method for deter-
undergoes no recombination and is mining variation is by direct sequencing
maternally inherited. The molecule (PCR) of the same DNA fragment for all
(generally 16-20kb in size) is made up individuals. This method is expensive,
of 13 protein coding genes, 2 ribosomal particularly for population studies
RNA genes (rRNA), 22 transfer RNA where sample sizes and number of
genes (tRNA) and section generally populations may be high. The previous
known as the D-loop or Control Region section has described several useful
(which is non-coding but is involved in techniques but not all of these are
the replication of the molecule). Unlike appropriate for mtDNA analysis. Here
the nuclear genome, the mitochondrial we will discuss two methods that
genome contains very little non-coding are particularly useful: Temperature
DNA. Gradient Gel Electrophoresis (TGGE)
and Single Strand Conformational
Several characteristics of mtDNA Polymorphism (SSCP). Both methods
make it a good choice of molecular have reasonable high throughput and
marker for population studies. Firstly, a high power to detect single base pair
its maternal inheritance and haploid mutations (but slightly less resolution
nature dictate that populations will than sequencing) therefore they can
exist at ¼ the effective population size identify unique haplotypes. Once
(Ne) as that seen with nuclear markers. all individuals have been assessed,
This characteristic will amplify the analysis of haplotypic frequency data
effects of drift (causing populations to is then possible. This method however,
differentiate), so mtDNA is therefore does not achieve the full potential of
sensitive for detecting population the data. Also, the power to isolate
17
contemporary from historical gene flow To improve the resolution of the
is significantly reduced. For a little more technique, TGGE is often conducted
financial outlay, one or two repre- in conjunction with Heteroduplex
sentatives of each unique haplotype Analysis (TGGE/HA). Nuclear (diploid)
detected in the screening process can DNA fragments can be heteroduplexed
be sequenced. This method can reduce to themselves but because mtDNA
the number of individuals requiring is haploid, an extra reference DNA
sequencing by an order of magnitude fragment (ideally from a moderately
(e.g. from 100’s to 10’s). divergent conspecific individual) needs
to be added. The heteroduplexing
As the name suggests, TGGE relies on process involves heating both the refer-
a temperature gradient to denature ence and sample DNA together in order
double-stranded DNA fragments that to reduce them to single strands. Upon
are differentially separated based cooling the strands reanneal. Apart
on their respective melting profiles. from the original double strands from
Another method, Denaturing Gradient the reference and sample fragments
Gel Electrophoresis (DGGE) is similar to recombining to themselves (homodu-
TGGE but uses a chemical gradient of plexes), mismatch pairings occurs with
increasing urea and formamide concen- one strand from the reference and one
trations to denature the DNA instead strand from the sample also recom-
of a temperature gradient. bining (heteroduplexes). Where heter-
oduplex fragments have nonperfect
The procedure involves elec- complementary matches, they tend to
trophoresing DNA through a have lower and more variable melting
polyacrylamide gel that is running points than homoduplexes resulting in
parallel to a temperature gradient. additional bands on the gel. TGGE is a
The double-stranded duplexes of DNA reliable method for DNA fragments up
migrate through the gel until they to ~700 base pairs in length.
reach their respective melting points
where progress is greatly reduced SSCP relies on electrophoresing
when the dsDNA begins to unwind. The single stranded DNA through a
melting point of a specific fragment of nondenaturing polyacrylamide gel.
DNA is a function of both the effect of Under appropriate conditions (i.e.
base sequence on the helix structure nondenaturing conditions) single
and the electrophoretic mobility of the stranded DNA folds into a particular
strand as it starts to unwind. Therefore shape (tertiary structure), with different
DNA fragments with different base sequences generally resulting in
pair sequences tend to display different different structure. The electrophoretic
melting points and hence stop at mobility through an acrylamide gel
different points on the gel. More is largely dependent on the shape of
details on TGGE techniques will be the fragment that is determined by
illustrated in Section 2.10. its unique DNA sequence, therefore
different haplotypes will provide
18
different banding patterns on the gel
when visualised. This method does not
require any special apparatus like TGGE
does and can detect nucleotide differ-
ences in fragments up to ~700 base
pairs in length. However, there tends to
be an inverse relationship between the
length of the fragment and the sensi-
tivity of the technique (i.e. resolution is
better with smaller fragments).
19
20
SECTION 2
Laboratory protocols
2.1. Allozyme electrophoresis suitable for detecting genetic diversity
of organisms showing weak popula-
The term “allozyme” refers to products tion differentiation such as marine
of different allelic forms of an enzyme organisms. Lastly, the technique only
coding gene. These products are detects a portion (usually <25%) of the
strands of amino acids called polypep- actual genetic variation because not
tides. Five of the 20 common amino all nucleotide changes lead to amino
acids that make up polypeptide chains acid substitutions. Additionally not
have weak electric charges (lysine, all amino acid substitutions result in
arginine and histidine are positively electrophoretically detectable mobility
charged with NH3+; aspartic acid and differences (Ryman & Utter 1987).
glutamic acid are negatively charged
with COO-). Thus, polypeptide chains Allozyme data are used for detecting
comprising different numbers of population structure, hybridisation,
these amino acids have different net species boundaries and for estimating
electrical charges so does the allozyme levels of gene flow (Hillis et al. 1996),
molecule which is made of one or more and investigating systematic relation-
than one type of polypeptide chains. ships (Richardson et al. 1986; Swofford
As such if crude extracts of enzymes et al. 1996).
are placed in an electric field, allozyme
molecules will be separated according There are four most commonly used
to their net-charge. methods of allozyme electrophoresis,
depending on types of medium used:
Allozyme electrophoresis involves the starch, polyacrylamide, agarose and
separation of products from isozyme cellulose acetate.
alleles on the basis of differential
migration due to varying surface 2.1.1. Gel preparation
charge when subjected to an electric
current. Thus different alleles are Buffer for electrophoresis
detected based on the mobility differ-
ences of their products at the end of Running buffer systems are chosen
the run, which are visualized via specific according to the types of tissue
histochemical staining procedures sampled and enzymes. Often stocks
(Richardson et al. 1986). of buffers are made for subsequent
dilution before use. The proper dilu-
The advantages of allozymes are their tions and running conditions are given
co-dominant nature, relatively low cost in Annex 1.
and availability of protocols common
for wide range of organisms. However The buffer used for electrophoresis is
the requirement for fresh tissues can called running or electrode buffer, and
be a major draw back. Moreover, that used for making gel is called gel
allozymes commonly have low levels of buffer. There are two different running
polymorphism hence they may not be systems based on the combination of
23
running and gel buffers - continuous 2. Gel mould(s) are often made from
and discontinuous electrophoresis Perspex (5mm thick should be fine).
systems. The running system that use There is no restriction on size of the
the same buffer for electrode and gel mould, however we recommend
is called continuous (e.g. the TC6, TC8, 200 mm x 200 mm x 12 mm for easy
TEB, TG and TM buffers; and when handling and suitable number of
the electrode buffer is different to samples that can be run (about 20
gel buffer it is called discontinuous samples could be stained for four
(e.g. the LiOH and Poulik buffers) (See enzymes per gel) (see Figure 1).
Annex 1).
3. Place the appropriate amount of
Making starch gel starch into the boiling flask. The
suggested amount is 55g of starch
1. Clean gel mould(s) using tissue per gel (200mm x 200mm x 12mm).
paper absorbed with ethanol (75- A one-litre boiling flask is sufficient
80%) to clean gel mould. Clean to make one gel; a 2-litre boiling
once again using distilled water. flask for making 2 gels.
The reason to clean the gel mould
properly is to avoid the gel sticking 4. Add 450 ml of buffer (exact amount
onto the plate and breaking. Set up varies with the buffer system; see
the plates on a sheet of newspaper Annex 1) to the starch, and swirl the
or paper towel, in case there is any mixture to suspend the starch. The
spillage. specific instructions for each buffer
are indicated on the stock bottles.
Each stock requires dilution for use.
12 mm
200 mm
24
5. Place flask with fully suspended 7. Pour the starch into the gel mould
starch on a hot plate with stirring with a continuous action; avoid
bar inside. Set heat on high and backtracking and zigzagging, as
stirrer on approximately 3/4. Pick up these can create discontinuities in
and swirl flask by hand every five the gel. Make certain that you use
minutes or so and check bottom all of the starch, equally distributed
of flask for signs of boiling. If between the plates.
indications of boiling keep swirling
vigorously until it abates. Return Figure 2. Connection to vacuum to
to hot plate and closely monitor. de-gas.
Localised boiling of starch while
T-bar
cooking is to be avoided at all costs
as it causes uneven cooking and
lumpy gels. Swirling need not be
violent, but just sufficient to prevent
over-cooking on the bottom of the To vacuum
flask. As the starch heats up, the
suspension becomes more viscous
and the stirring bar will stop;
remove flask from hot plate every
30 seconds or so and swirl for a few
seconds before returning it to the
hotplate for a total of a further 3
- 5 minutes until the starch mixture
begins to loosen up again and
becomes semi-translucent. At that
point, it is cooked.
25
10. Leave the covered gel at room 5. It is essential to keep the extract
temperature for at least 1 hour. cold to prevent deterioration of the
It is generally most convenient to enzymes (i.e. place grinding plate or
pour the gels one night before use. tubes on ice).
Depending on the weather, they
may be kept longer, but it is risky. It 6. Tough tissue may require powdered
is best to keep a consistent pattern, glass to improve homogenisation.
as the gels gradually lose moisture.
7. Soft, messy tissue works best if not
2.1.2. Sample preparation over-homogenised.
26
Figure 3. Example of a set of electrode boxes.
exact placement of this cut depends 6. Place the gel with samples onto
on the buffer and particular the buffer boxes, with the samples
enzymes (Discontinuous buffers: at the cathodal (black) end. Most
3 cm from end of gel; Continuous enzymes will migrate towards the
buffers: 3-6 cm, depending on anodal (red) end.
whether any of the enzymes
migrate cathodally (backwards, 7. Connect the gel to the buffer with
towards the black leads (–ve)). sponges (well rinsed in distilled
water).
4. Blot the excess liquid from the
inserts using tissue paper, and line 8. Cover the gel with a sheet of
up along the open cut (Figure plastic, to prevent dehydration and
4). Leave about 1.5mm between accidental electrocution.
samples; there is room for 25-30
samples on each gel. It is a good 9. Connect leads (red to red, black to
idea to have replicates of some black) to the buffer boxes, then to
samples on different gels, to be the power supply.
certain of electrophoretic resolution
on each gel. 10. Turn on power to desired current
or voltage. (Current varies with the
5. Push the gel together, making number of gels; voltage does not).
certain that there are no gaps.
27
11. Let it run until the bromphenol blue 15. Prepare the gel for slicing by cutting
migrates about 10cm, or until the off excess sections, and marking one
required separation has occurred corner for orientation.
(determined empirically for each
system). 16. Slice gels with fishing line. Flip
the top slice over for staining
12. As the run approaches completion, (remember to read it from right to
prepare staining solutions. left!)
These will keep for hours in the
refrigerator. Check to make certain 17. Place slices on perspex sheets (no air
that there is sufficient melted agar bubbles underneath) for stains with
on hand. an agar overlay; for liquid stains, use
a glass dish.
13. Turn off the power supply and
disconnect the leads at the power
supply, before disconnecting the
leads at the buffer boxes.
28
18. Melted agar is made by suspending A monomer protein/enzyme comprises
the flask in a beaker of boiling of single polypeptide chain which
water, until the agar is clear. Store is called a subunit. Therefore each
in 65ºC oven in flask. When using homozygote would produce a single
it, open the oven only as necessary, band of different mobility and the
and return the flask to the oven heterozygote would produce two
immediately. Make certain that bands of different mobility (Figure 5).
there are always two full 250 ml
flasks in the oven during busy times. A dimer protein/enzyme comprises two
polypeptide chains, either the same
2.1.4. Gel staining amino acid sequence or different.
Therefore homozygote individuals
See Annex 3 for a list of staining recipes produce only one type of polypeptide
for common enzymes. Staining can be chain and would show one band while
conducted in two ways, one is to mix a heterozygote would produce 3
the staining chemicals, then add agar bands, with a thick one equally distant
and poor over the gels, and another between the two outside bands. This
method involves dissolving staining is because there are twice as many
ingredients into staining buffer, which ways of producing a band comprised of
is used to soak the gel. Some enzymes two different peptides as there is one
may stain well in one method but not comprising two identical peptides.
the other.
A tetramer comprises 4 polypeptide
Note that most of staining ingredients chains thus a heterozygote produces 5
are relatively expensive, and either can bands. sometimes it is difficult to see
cause allergy or are poisons. Informa- the outer bands of a tetramer as they
tion on effects of chemicals is given on are approximately 1/6 of the intensity
the label or on a Materials Safety Data of the middle band.
Sheet (MSDS) provided by the chemical
supplier, so read carefully before Each staining may show products of
dealing with them. Care must be taken more than a single locus. This means
during handling, gloves and a mask that multiple enzymes catalyse the
must be worn at all times during the same reaction but are products of
staining process. different genes. They are termed
“isozymes”. While the products of
2.1.5. Gel scoring different alleles of a locus are termed
“allozymes”.
If the staining is successful bands of
enzyme/protein appear. The bands are 2.1.6. Trouble shooting
then scored for presumed genotypes
based on nature of each enzyme/ Allozyme electrophoresis needs experi-
protein. ence. It is quite difficult for beginners
to get good results. However, the
29
Figure 5. Electrophoretic phenotypes when one locus is expressed.
AAAA
AAAA’,AAA’A,AA’AA,A’AAA
Tetramer 4 AAA’A’,A’A’AA,A’AA’A,A’AAA’
AA’A’A’,A’A’A’A,A’AA’A’,A’A’AA’
A’A’A’A’
30
Table 1. Common problems in starch gel electrophoresis and solutions.
31
4. Add 4 µl Rnase-A (10 mg/ml) to 7. Depending on the size of the pellet,
each sample and mix by inverting add 30-200 µl TE buffer to the dried
tube 25 times. Incubate samples at DNA pellet. Some literature suggests
37°C for 1 hour then cool to room that TE may inhibit subsequent PCR
temperature. reactions. Although this does not
seem to be a major problem it may
5. Add 300 µl of Protein Precipitation be advisable to store DNA in 1/10
Solution to samples and vortex TE or deionised water. Allow the
vigorously for 20 seconds to mix DNA to rehydrate overnight at room
samples. Incubate on ice (or in temperature. Store at 2-8°C short
freezer) for 30 minutes. Centrifuge term or –20°C long term.
at high speed for 3-5 minutes. The
precipitated protein should form 8. DNA extraction of some tissues can
a tight pellet in the bottom of the be difficult by this method and if
tubes. If not repeat the vortex and this is the case there are several
ice incubation steps. other methods available. They are
generally more tedious and use
6. Pour off the supernatant some potentially nasty chemicals
containing DNA (leaving behind that need to be used in a fume
the precipitated protein) into a 1.7 hood.
ml centrifuge tube containing 720
µl of isopropanol. Mix by inverting 2.2.2. Phenol chloroform method
gently 25-50 times. Centrifuge at
high speed for 5 minutes to pellet Solutions required
the DNA (placing tubes at -20°C
for several hours or overnight aids ▪ TNES-urea: 10mM Tris-HCl pH 7.5,
in DNA precipitation). Pour off the 125mM NaCl, 10 mM EDTA pH 7.5,
supernatant and briefly drain tube 0.5% SDS, 4M Urea)
on a clean paper towel. Add 750 µl
70% ethanol and invert tube several ▪ TE: 10 mM Tris-HCl pH 7.5, 1mM
times to wash DNA. Centrifuge at EDTA
high speed for 1 minute. Carefully
pour off the ethanol. The DNA ▪ 2xCTAB: 100mM Tris pH 8.0, 1.4M
pellet may be loose so make certain NaCl, 20mM EDTA pH 8.0, 2% CTAB
that you don’t discard it. Drain the
tube on clean paper towel and ▪ 70% ethanol: 70ml of pure ethanol
allow DNA pellet to dry (room temp (molecular biological grade) with
or 37°C oven). It is essential that the 30ml autoclaved deionised water.
pellet is dry but if it is over dry it is
difficult to rehydrate. ▪ Proteinase K: 20mg/ml
▪ Phenol chloroform
32
▪ Isoamyl alcohol 11. Dry pellet in vacuum centrifuge
for 25 minutes or until ethanol is
Protocol evaporated.
1. Place tissue in 200µl TNES. 12. Add 100-200µl of TE and let sit for
several hours at room temperature.
2. Grind up tissue using plastic mortar
or tweezers. Add 500µl 2xCTAB to 2.2.3. DNA extraction from fish
tube. blood
9. Let it sit for at least 1 hour, then • High TE: 100 mM Tris-HCl, 40 mM
spin in cold room for at least 30 EDTA
minutes. • MGPL lysis buffer: 10 mM Tris-
HCl, 1 mM EDTA, 200 mM LiCl,
10. Pipette off supernatant, add 1 0.8% SDS
ml 70% cold (-20°C) ethanol. Mix • Proteinase K
gently, then spin in cold room for • TE: 10 mM Tris-HCl, 1 mM EDTA
5 minutes. Repeat once more to • Isopropanol
ensure that all salts are removed. • 70% ethanol
• NaCl
33
Procedures 6. The sample was air dried for 5 to
10 minutes and then resuspended
1. An aliquot of 20-100 µl* of the in 100 µl of TE. The samples were
blood/ethanol was transferred then adjusted to the appropriate
to a 1.9 ml microcentrifuge tube template concentration for use in
containing 1 ml of high TE (100 mM PCR.
Tris-HCI, 40 mM EDTA, pH 8.0). The
sample was vortexed and pulse spun 7. *The amount of blood/ethanol used
for 10-15 seconds to pellet the cells. in the extraction (step 1) must be
The supernatant was then removed. determined for each set of samples
and for each species since the initial
2. The cell pellet was resuspended in dilution factor of the blood may
250 µl MGPL lysis buffer (10 mM Tris- vary between collections and the
HCI, 1 mMEDTA, 200 mM LiCI, pH amount of DNA in the cells nay
8.0 and 0.8% SDS).To this was added vary from species to species. Both
Proteinase K to a final concentration of these factors will affect the yield
of 200 µg/ml. The sample was then from the extraction and must be
incubated at 45°C. After 15 min the taken into account.
sample was vortexed briefly and
further incubated until the cells 8. With the following modifications
were completely lysed (10-20 mm). this procedure has been used to
extract DNA, of sufficient quality
3. Following incubation, 500 µl of TE for PCR, from samples of skeletal
(10 mM Tris-HCI, 1 mM EDTA, pH muscle, tail fin (Ruzzante et al.
8.0) and 1996) and adipose fin (D. Cook,
unpublished results):
4. 750 µl of cold isopropanol were
added, and the sample mixed by 9. The time of digestion with
vortexing. Proteinase K (step 2 above) was
extended.
5. The DNA was then pelleted by a
1 minute spin at 14,000 rpm. The 10. Prior to the addition of isopropanol
supernatant was removed and the (step 3 above) the samples were
DNA pellet was washed with 70% spun, for one minute, to pellet
ethanol, pulse spun and the ethanol insoluble debris and the supernatant
removed. This was followed by a was transferred to a clean tube.
second pulse spin and removal of
the residual ethanol.
34
11. NaCl to 50 mM was added after 2.3.2. Methods
the TE (step 3 above). This was
necessary, to ensure efficient There are two methods to quantify
recovery of DNA, because of DNA sample. The conventional method
the lower concentration of DNA of electrophoresis of DNA sample of
obtained from tissue samples unknown concentration with a known
compared to that obtained from standard is applied in most labs where
blood. a spectrophometer is not available.
Another method is using a spectropho-
2.3. DNA quality and meter that directly quantifies the DNA
quantification concentration in the resuspended DNA
extraction.
35
6. Load 2 of DNA marker (HindIII 11. Compare the intensity of the DNA
digested λ DNA for example) into bands of the samples with the
one of the wells. intensity of the λ bands. As the
amount of DNA present in each
7. Run the gel at 70-100V until the dye λ band is known (information is
is about 2.5 cm from the origin. often provided by the supplier),
the amount of DNA of each sample
8. Move the gel to a tray with can be estimated by comparing the
ethidium bromide (1µl ethidium fluorescent yield of the sample with
bromide in 100 µl ddH20) those of the λ bands.
(HAZARD!!!). Let the gel stain for 5-
10 min, and then de-stain for about 12. Quality of extracted DNA can also
2 min in another container with be assessed by looking at the gel.
ddH2O only. Good quality DNA will show as a
sharp intense band. Degraded DNA
9. Illuminate the gel with UV extracts will show various degree of
light (CAUTION – UV LIGHT IS smearing. See Figure 7 for example.
HAZARDOUS!!! – WEAR MASK
OR UV PROTECTION GLASSES IF
EXPOSED TO UV LIGHT).
Figure 6. Agarose gel analysis of genomic DNA isolated from Tor tambroides: 1, 2:
good DNA quality; 3-4: DNA with RNA; 5-6: degraded DNA; 7-8; DNA with salts.
36
Spectrophometric determination 2.3.3. Trouble Shooting
of DNA concentration
Common problems and solutions are
Dilute 1.5 µl of DNA to 1500 with summarised in Table 2.
deionised water and read at A230, A260
and A280. The A260/A280 ratio provides 2.4. Polymerase chain
an estimate of the purity of the DNA. reaction (PCR)
In a pure sample, this ratio is approxi-
mately 1.8. Lower values indicate The advent of PCR has greatly acceler-
protein or phenol contamination. A230 ated the progress of studies on the
should be less than A260 and may be genomic structure and processes of
the same as A280. High A230 reading various organisms. Any gene region,
indicates that residual phenol remains even in highly complex genomes, can
in the preparation. An A260 of 1 corre- be specifically amplified using this
sponds to approximately 50 µl/ml of technique if the flanking nucleotide
double-stranded DNA in a 1 cm quartz sequences are known (Saiki et al. 1988).
cuvette. Nucleic acid concentration is The PCR procedure involves replicating
calculated as follows: target regions of DNA, which are
flanked by regions of known sequences
A260 * 50 mg/µl * 0.001 µl/ml * dilution (Erlich 1989). Synthetic nucleotide
factor (1500 µl/1.5 µl) (µg/µl) primers (usually 18-30 bp long) that
are complementary to each of the
37
flanking regions are needed. These The following basic PCR protocol is
are combined with a small amount of from The Simple Fools Guide to PCR
DNA (at nanogram levels), plus free by Palumbi et al. (1991). The amounts
deoxynucleotides (dNTPs), a reaction listed below are for 25 µl reaction
buffer, and Taq DNA polymerase volumes so if you use larger or smaller
(isolated from the hot spring bacteria volumes, adjust each component
Thermus aquatica). During a series of accordingly. Again, it is advisable to
up to 30 heating and cooling cycles, the include a negative control in each
DNA is denatured into single-stranded round of PCR to ensure solutions are
molecules, the two primers anneal to not contaminated. Similarly, the inclu-
their complementary sequences on sion of a positive control (i.e. a sample
either side of the target region, and the has been amplified successfully
the DNA polymerase replicates the previously) can help isolate problems
region downstream from each primer. should amplification fail.
The amount of target DNA will double
with each temperature cycle, so that 2.4.1. Chemicals required
even low starting copy numbers of the • 10X PCR buffer (supplied with
target sequence will generate trillions Taq)
of copies by the end of the last cycle. • 50mM MgCl2 (supplied with Taq)
• Taq DNA polymerase
PCR can significantly decrease the • 2.5 mM of each dNTPs, mix
amount of time required to isolate together
a desired segment of the genome. • 10 µM each primer
Also, PCR allows DNA analysis to be • Extracted genomic DNA
performed from small and sometimes • Autoclaved deionised H2O
minute tissue sample. However, for
most uses of PCR, one must determine 2.4.2. PCR mixture
the sequences of regions flanking a
given locus, and this can entail consid- Table 3 represent the volume and
erable effort when working with a new concentration of each component in
species. The use of arbitrary primers (in one PCR reaction of 25 µL total volume.
random amplified polymorphism DNA
- RAPD), does allow one to identify To minimise pipetting errors, add the
genetic markers relatively quickly in a components together in a master
species for which extensive sequence mix except for sample DNA and Taq.
information may not be available. Simply multiply the amounts per
Furthermore, a significant number of reaction by the number of reactions
universal primers have been developed to be performed plus two, one for the
and used to amplify a number of gene blank and one as a spare (in case of
regions in different species. pipetting error). The Taq is added at
the last moment. Just before adding
the DNA template, as it degrades very
38
Table 3. Basic PCR mixture.
39
▪ Annealing at (Tm-10°C) for 15- ▪ A further extension step of 2
30 seconds. Tm is provided with minutes at 72°C following the last
primers. The standard estimate of cycle.
annealing temperature is:
This basic protocol can be adjusted to
Tm (°C)= [(G+C)x4]+[(A+T)x2]-5 maximise PCR efficiency if required.
Generally annealing temp may be
▪ Always use the lower Tm of the two lowered if the primers were not
primers “perfect” or increased for increased
specificity. Also, concentration of MgCl2
▪ Extension at 72°C for 15-90 seconds can also be changed; it is advised that a
(depending on the length of the series of MgCl2 concentration should be
gene fragment to be PCR) tried and a general observation is that
high concentration of MgCl2 provides
higher yield, however it may also
produce non-specific PCR products.
Figure 7. Verification of the PCR product on gel. (Lane M1: 100bp DNA ladder;
lane 1-4: PCR products of ATPase6-8 fragment amplified from Tor species DNA,
fragment is approximately 1000 bases long; lane 5: PCR products of cytochrome
b fragment amplified from Tor species DNA, the fragment is approximately 450
bases long; the last two lanes are negative control.
40
2.4.4. Visualising PCR products 3. Only one band is formed.
As in the description above, it is
PCR products are assessed in the same possible that the primers fit to the
way as DNA extracts (see Protocol 4). desired locations, and also at other
However, rather than a high molecular locations. In this case, different
weight DNA band close to the sample bands may be present in one lane
origin the PCR product should migrate on a gel.
at the rate appropriate to its predicted
size. This is verified by comparison with 2.4.5. Trouble shooting
the DNA marker (which also assesses
quantity). If the predicted size of the Common PCR problems are summarised
PCR product is small, be careful not to in Table 5.
run the gel too long so as samples do
not run off the gel. 2.5. Restriction fragment
length polymorphism (RFLP)
Before the PCR product is used in
further applications, it has to be RFLPs were the first DNA markers to be
checked if: used by population biologists (Parker
et al. 1998). The technique involves
1. There is product formed. cutting a DNA strand at specific
Not every PCR is successful. There nucleotide sequences using a restriction
is for example a possibility that the endonuclease and thereby producing a
quality of the DNA is poor, that one pool of different sized DNA fragments.
of the primers doesn’t fit, or that RFLP variation can be visualised directly
there is too much starting template, by staining with ethidium bromide
or even at least one ingredient was following electrophoresis of the DNA
left out! in an agarose gel. This can be done
for small molecules, such as the entire
2. The product is of the right size. mitochondrial DNA, which produce a
It is possible that there is a product, manageable number of fragments with
for example a band of 500 bases, many restriction enzymes (Landsmann
but the expected gene should be et al. 1981; Tegelström 1992). The most
1800 bases long. In this case, one appropriate method of analysis involves
of the primers probably fits on a restriction sites, whereby actual sites
part of the gene closer to the other are mapped to specific positions on
primer. It is also possible that both the strand of DNA of interest. Scoring
primers bind to a totally different is based on the loss or gain of a site,
gene (non-specific priming). this giving an accurate resolution of
relationships. An alternative method
is fragment analysis which scores the
different fragments as either present or
absent. However, this method assumes
that fragments of similar length on a
41
Table 5. Common PCR problems.
42
gel are homologous. Unfortunately, Incubate for at least one hour at
this assumption can be misleading required temperature (enzyme manu-
since multiple fragments may make facturer’s instruction). Once incubation
up a single band on a gel. In addition, time is up, place the tube on ice to
different cleavage sites may produce bring down the condensation, then
similar banding patterns, thus giving freeze until required.
erroneous relationship among samples
(Fetzner & Crandall 2001). Similar to setting up PCR reaction, it is
advisable to make a restriction digest
A more efficient approach which is mix sheet for multiple samples. Make
now used relatively commonly is to a master mix and then aliquot the
amplify the DNA region of interest appropriate volume to each tube. PCR
then conduct restriction analysis on the product is added last.
amplified fragment. PCR products are
treated with restriction enzymes and 2.5.2. Running buffer
the fragments separated on an agarose
gel and visualised by ethidium bromide 10x TBE (dilute to 1x for use):
staining to identify RFLP profiles (Karl • 10.8 g Tris
& Avise 1992). An advantage of this • 5.5 g Boric acid
approach is that the PCR fragment • 0.744 g EDTA
can first be sequenced from a number • make up to 1 litre with dd H2O
of individuals in the first instance, to
allow detection of polymorphic restric- 2.5.3. Running a gel
tion sites. Mitochondrial DNA is most
commonly used for this method of 1. Gels used for RFLP analyses are
genetic analysis as it has been proven often made of 2% Agarose in 1x
to allow easy detection of genetic TBE buffer.
differences at population levels (Hillis
et al. 1996). 2. Place TBE gel into running chamber,
pour 1x TBE buffer over the gel.
2.5.1. Restriction digest mix
3. Load all (10 µL) digested PCR
For one reaction: product into a well (mix with
• Restriction enzyme buffer 1.0 µl loading dye). Load marker for
• Restriction enzyme 0.2 µl justifying band size.
• PCR product 8.8 µl
4. Run the gel at 150V, 60 mA.
43
5. Once the run is finished, place gel in Figure 8 shows that individuals
the gel in a water bath with 1% 1 and 2 have the same haplotype A,
ethidium bromide (HAZARD!!!) for individuals 3, 4, 7 have haplotype B and
10 min, then destain in distilled H2O individuals 5, 6 and 8 have haplotype C.
for 5 min.
When more than one restriction
6. View the gel under UV light. enzyme is used, the final haplotype is
the combination of all haplotypes that
2.5.4. Documentation of results resulted from single restriction digest
(see Table 7).
Restriction digest of PCR product results
in banding patterns as shown in Figure 2.5.5. Trouble shooting
8 when running on an agarose gel.
Each pattern is a haplotype and often Common problems with RFLP are
assigned a letter (e.g. A, B, C…) or a summarised in Table 8.
number (1, 2, 3…). Example from the
44
Table 8. Common problems with RFLP.
45
2. Heat mixture to 95°C for 5 minutes ▪ All solutions should be made fresh
and immediately place on ice (keep just before use. All staining steps
on ice until you load sample). are performed with gentle shaking
of the gel at room temperature in
2.6.3. Gel running a fume hood. Use enough of each
solution to cover the gel.
1. Running Buffer is 0.5X TBE.
1. Remove the gel from electrophoresis
2. Cool gel in refrigerator for 1 hour apparatus, separate the glass plates
and then pre-run for 15 minutes at and remove the spacers. Transfer
5W constant power prior to loading. the gel on the bottom glass plate
to a clean staining tray. The gel may
3. Flush wells with buffer and load float off the plate during staining
samples. and can be removed.
4. Run at 5W for 2-10 hrs (you 2. Wash gel twice for 3 minutes in
must determine this yourself but wash solution. Take care to remove
remember that single strand DNA the last of the wash solution before
migrates slower than double adding the silver nitrate solution.
stranded DNA).
3. Incubate the gel in silver nitrate
5. It is important power be kept solution for 10 minutes (remove
constant throughout the run. solution).
46
2.7. Random amplified markers, as compared to codominant
polymorphic DNA (RAPD) nuclear markers revealed by RFLPs,
allozymes or microsatellites (Lewis
RAPD markers are produced by PCR & Snow 1992) although it should be
using short oligonucleotide primers noted occasionally RAPD markers have
of random sequences. Different RAPD been detected that show patterns
patterns arise when genomic regions consistent with codominant markers
vary according to the presence/absence which contain microsatellite loci (Garcia
of complementary primer annealing & Benzie 1995). Because of their short
sites. The primers are typically 10 bp length, RAPD markers may produce
long (Williams et al. 1990) and no some artifactual amplification products,
specific knowledge of a particular DNA and careful control of DNA quality and
sequence is required. Allelic variation amplification conditions is necessary to
usually consists of the presence or ensure reproducible banding patterns
absence of particular amplification (Carlson et al. 1991; Riedy et al. 1992;
products, which can be separated on Scott et al. 1993). Ideally and where
agarose gels stained with ethidium possible, undertake crosses of known
bromide. The RAPD process typically genotypes to demonstrate Mendelian
reveals several polymorphic genetic inheritance of bands, and repeat
segments per primer within popula- experiments to confirm banding patters
tions; other segments may appear as before scoring.
monomorphic bands within or across
populations (Hadrys et al. 1992). The 2.7.1. PCR preparation
degree of variability observed for many
primers suggests that the technique Stock and final concentrations per 25
will be useful for a variety of questions, µl of reaction mixture is presented in
including individual identification, Table 9.
pedigree analysis, strain identification,
and phylogenetic analysis (Parker et al. 2.7.2. DNA amplification
1998).
Place PCR tubes in a thermal cycler.
RAPD markers are rarely inherited Amplify using the most commonly used
as codominant alleles (Parker et al. temperature profile as shown in Table
1998). Losses of a priming site results 10.
in complete absence of the enclosed
amplified segment, not simply a shift in After amplification remove the PCR
mobility on the gel. In heterozygotes, tubes from the thermal cycler. Add 3 µl
therefore, differences may appear only of 10x loading buffer to each tube. Mix
as differences in band intensity, which by flicking the bottom of the tube and
is not usually a reliable phenotype spin to collect the mixture. The mixture
for PCR analysis. As a consequence, is now ready for loading in the agarose
information on the parental origin of gel. It is may be advisable to run and
alleles may be inaccessible for RAPD
47
Table 9. Stock and final concentration of RAPD-PCR.
Get a gel mould and seal both edges 8. Close tank and attach electrode
with 1” masking tape. Place in a level wires to the power supply. Run for 3
platform and attach combs. Some gel h at 150 V (running time depending
making design may not need this step. on the buffer used and length of
the gel/ length need to run).
1. Prepare 1.4% agarose by weighing
3.5 g agarose. Transfer this to a 500 2.7.4. Staining and documentation
ml flask and add 250 ml 0.5x TBE
buffer. 1. After electrophoresis, switch off the
power supply and remove the tank
2. Boil for 6 min in a microwave. Allow cover.
the solution to cool to 60°C.
4. Fill the electrode tank with 0.5x TBE Table 10. Example temperature profile
buffer. for RAPD-PCR.
48
2. Remove the gel from the moulder 2.7.5. Scoring
and transfer in a tray with ethidium
bromide (5 µl) staining solution in ▪ Designate a name or a number for
a 500 ml H2O. Stain for 10 min. EtBr each RAPD marker based on the
staining solution can be reused. molecular size and primer used.
Please take care when working with
ethidium bromide, it is a mutagen, ▪ It is a good idea to keep the gel
hence may need to wear double images for checking purposes. An
gloves when handling gels with example of a RAPD gel is shown in
ethidium bromide. Figure 9.
3. After staining, destain by rinsing Score RAPD bands using a binary system
with ddH2O in a different container. of 0 (in the absence of the band) and 1
(if the band is present). Remember that
4. Photograph/score the gel under UV only sharp and clear bands are scored.
light. Again, some laboratories are Microsoft Excel would be useful for this
well equipped with gel imaging purpose, because from this software
system, but some other laboratories we can export data to other genetic
may have to wear UV protection software easily.
glasses while viewing gels under UV
light.
Figure 9. An example of a RAPD gel. Bands are PCR products of two fish species
(samples 1-4 are from the first species and samples 5-8 are from the second
species), amplified using primer OPA20. There are many bands but only sharp
bright bands should be scored (OPA20-1 to OPA 20-7).
49
2.7.6. Trouble shooting The key feature of AFLP is the capacity
for screening of many different
Common problems with RAPD are DNA regions distributed randomly
summarised in Table 11. throughout the genome simultane-
ously. The high reliability of the
2.8. Amplified fragment technique is the result of combining
length polymorphism (AFLP) the strengths of two methods, the
replicability of restriction fragment
AFLP was introduced by (Vos et al. analysis and the power of PCR (Vos
1995). The AFLP protocol involves the et al. 1995). Thus, AFLP allows the
following steps: (1) DNA digestion detection of polymorphisms of genomic
with two different restriction enzymes restriction fragments by PCR amplifica-
(typically EcoR I and Mse I), (2) ligation tion. AFLP markers have proven useful
of double-stranded adapters to the for assessing genetic differences among
ends of the restriction fragments, (3) individuals, populations and independ-
optional DNA pre-amplification of ently evolving lineages, such as species
ligated product directed by primers (Mueller & Wolfenbarger 1999).
complementary to adapter and
restriction site sequences, (4) DNA 2.8.1. Restriction digestion of
amplification of subsets of restriction genomic DNA
fragments using selective AFLP primers
and labelling of amplified products, 1. Prepare digestion mixture as per
(5) separation of fragments via elec- Table 12.
trophoresis, and (6) scoring fragments
as either presence or absence among 2. Heat one oven to 70°C and another
samples. to 37°C
50
Table 12. Preparation of digestion mixture.
51
2.8.4. Pre-amplification reactions • Eco RI + ANN oligo (50 ng/µl)
0.50 µl
1. Make reaction mixture as follows • Mse I + CNN oligo (50 ng/µl)
(ingredients are per reaction): 0.60 µl
• Eco RI + A oligo (50 ng/ µl) 0.5 µl • DNTPs (5mM) 0.80 µl
• Mse I + C oligo (50 µl) 0.5 µl • 10X PCR buffer 2.00 µl
• DNTPs (5mM) 2.0 µl • Taq polymerase (5U/µl) 0.08 µl
• 10X PCR buffer 2.0 µl • MgCl2 (50mM) 1.20 µl
• Taq polymerase (5U/ µl) 0.1 µl • ddH2O 13.82 µl
• MgCl2 (50mM) 1.2 µl • Template DNA from pre-selective
• ddH2O 11.9 µl PCR 1.00 µl
• Template DNA from restriction/
ligation 2.0 µl 1. Run under following thermal cycle:
4. Blot testing can be used to test 3. Test product using dot blot if
reaction success. Dot 2 µl of necessary
ethidium bromide (HARZARD!!!)
and 3 µl of product on plexi-glass. 4. Combine 8 µl formamide-loading
Use 3 µl of cocktail as control. buffer and PCR product.
Visualise dots using UV box.
2.8.6. Gel electrophoresis
2.8.5. Selective amplification
1. Acrylamide gel solution:
1. Make reaction mixture as follows • 42g urea
(ingredients are per reaction): • 10ml 10x TBE
• 15ml 40% acrylamide
52
• ddH20 up to 100ml 10. In an Eppendorf tube combine 1ml
of 95% EtOH 0.05% acetic acid and
1. Combine urea, TBE, and 2µl of bind silane.
approximately 25ml water in a
beaker. Stir with heat until urea 11. Treat glass same as above
dissolves. description of Sigmacote. Use a
great deal of pressure when wiping
2. Transfer solution to 100ml- with EtOH. Change gloves.
graduated cylinder and add water
up to 85ml. Transfer to vacuum 12. In the event of a contamination
flask. of either Sigmacote or Bind silane
on the respective glass, soak in
3. Add acrylamide to flask and degas 10%NaOH.
for approximately 10min.
13. While horizontal, place spacers on
4. Transfer solution to beaker and add IPC and long glass on top.
100µl TEMED and 500µl 10% fresh
APS. Draw solution into syringe. 14. Erect vertically and clamp side
Keep tip submerged at all times. braces.
5. Place tube on syringe and turn it 15. Attach caster base insert pegs and
upward. Push air out of tube and turn. Be sure to do this while vertical
pinch end of tube. Insert into Caster and that you can see the space in
base. between glass plates through caster
base hole.
6. Glass preparation (all glass must be
scrupulously clean!) 16. Check to see if comb will easily
insert between glass. If not, adjust.
7. Wipe integrated buffer chamber
(IPC) unit with chem-wipe and 17. Lean clamps on top of tube racks.
ethanol.
18. Inject gel solution.
8. Glass should be treated with
Sigmacote about every 5 gels run 19. Insert combs and adjust unit to
or until top of gel sticks to long horizontal position.
glass. Saturate a chem-wipe with
Sigmacote and wipe vertically and 20. Allow gel to polymerise for at least
horizontally. Wait five minutes and 1 hour.
wipe glass three times with ethanol.
Change gloves. 21. Gel loading
53
22. Fill bottom tray with 1X TBE so 4. Stain the gel: Transfer the gel to
about ½ inch of the bottom of gel staining solution and agitate well
unit is submerged. Fill IPC until ½ for 30 minutes.
inch above short glass. Use needle
and flush out well 5. Pour 1L of the developing solution
into a tray. Transfer staining solution
23. Run gel at 75W for 1 hour. to beaker. Rinse tray and fill with
ddH2O.
24. Flush well again and insert comb
without piercing gel. 6. Rinse gel for 5-10 seconds ONLY.
Transfer to developing solution.
25. Load 4.5 µl sample.
7. Agitate in developing solution until
26. Run gel for 10min and then remove bands begin to appear. Transfer gel
comb (good time to make fix/stop to remaining chilled developing
and developing solution). solution for 2-3 minutes.
27. Run gel for total of 2 hours and 8. Fix the gel: add 1L of Fix/stop
50minutes. (Light blue dye should solution directly to developing
migrate 1 inch below bottom rib of solution and agitate for 2-3 minutes
IPC.
9. Rinse gel twice for two minutes each
28. Insert tube in IPC and drain buffer. in ddH2O.
29. Pull glass apart and wash IPC. 10. Dry gel on glass
54
**chill to 10°C. Place in freezer for 2. Carefully transfer gel to 3.5% acetic
approx 4 hours and stir to break up ice acid and soak for 3 min without
prior to use. agitation. Rinse in ddH2O for 2
minutes without agitation.
Immediately before use add:
• 3 ml of 37% formaldehyde 3. Drain excess water from gel and
• 400 µl aliquot sodium thiosulfate smooth a sheet of chromatography
(discard remaining) paper over gel.
55
polyacrylamide gel to allow resolution for different species, care should be
of alleles that may differ in size by as exercised. Ideally target products
few as two base pairs. should be sequenced to verify that they
are ‘real’ microsatellites.
A major disadvantage of microsatel-
lites is that identifying appropriate It is also common in the region that the
regions from a genomic library for a size of gels used to screen variation are
new species can be time-consuming often too small and much of the ‘real’
and expensive. Known primers are variation is not detected, leading to
not usually useful for amplifying the potential problems with H/W conforma-
same locus across related taxa unless tion. Labs should therefore consider
the microsatellite region is flanked applying a more rigorous approach to
by highly conserved sequences where silver staining microsatellite analysis, or
priming site are located (FitzSimmons use less technically demanding nuclear
et al. 1995). In addition, the presence markers such as allozymes.
of null alleles (alleles that do not
amplify due to mutational changes in The protocol presented here can be
the priming site) can complicate the applied in a small laboratory with
analysis as well. However, microsatel- minimum equipment.
lites have been extremely useful in
fish and crustacean population studies 2.9.1. Preparation of sequencing
and are quickly becoming the marker Dye
of choice for a variety of applications
(Wright & Bentzen 1994; Xu et al. Mix 10 mg of bromophenol blue, 10
1999). mg xylene, 200 µl 0.5 M EDTA and add
formamide until a final concentration
In the last few years microsatellites of 10 ml is reached. Store at 4°C.
have become one of the most popular
molecular markers with applications in 2.9.2. Preparation of PCR cocktail
many different fields. High polymor-
phism and the relative ease of scoring Each PCR reaction should contains the
represent the two major features that following ingredients which can be
make microsatellites of large interest added directly to each tube, or mixed
for many genetic studies. The major all ingredients except DNA template as
drawback of microsatellites is that a PCR cocktail. The ingredients for each
they need to be isolated de novo from reaction of 10 µl containing:
species that are being examined for the • 1 x PCR buffer (available with
first time. High output microsatellite Taq polymerase)
library screening requires an automatic • 1.0-2.0 mM MgCl2
sequencer which is only available in a • 100 µM of mixed dNTPs
few labs in Asian countries. However, • 0.5 µM of reverse and forward
in the case that primers used for primer
microsatellites have been developed • 0.2 unit of Taq polymerase
56
• Add autoclaved ultra pure water 2.9.3. The PCR cycles
up to the desired volume
• 5-10 ng of DNA template The PCR cycles are summarised in Table
14 below.
In each PCR tube containing 1 µl DNA
template, 9µl cocktail is added. 2.9.4. Check quality of PCR
product using agarose gel
Note:
Before the PCR products are separated
▪ Add 1 µl DNA template (diluted on acrylamide gel it is recommended to
DNA) in all PCR microtubes with perform a preliminary check on agarose
labels (use different pipette tips gel as follows.
to prevent contamination). While
preparing cocktail, the microtube 1. Load 5 µl of each PCR product mixed
should be kept on ice to prevent the with the stop dye in a well of the
reaction to start. agarose gel. Same pipette tip can be
use by washing it in 0.5 x or 1 x TBE
▪ Taq DNA polymerase should be buffer.
added last to prevent reaction. It
should be taken out from the deep 2. Perform electrophoresis using a
freeze at the time of addition to the voltage of 100 volt for around 30
cocktail and returned to the freezer minutes in small electrophoresis and
immediately after use. 40 minutes in big electrophoresis.
57
2.9.5. Protocol for polyacrylamide 2.9.6. Polyacrylamide gel
gel electrophoresis preparation
58
6. Then clip the open side with clips to 4. Fill the upper chamber with 1 x TBE
prevent the gel from leaking. Clamp buffer up to the margin.
the long sides with the binding
clips, 2 clips/side. Make sure that the 5. Pour 1 x TBE buffer in the lower
clips clamp on middle of the spacer buffer chamber until the gel bottom
width. is properly submerge.
7. Leave it to polymerize for 2 – 3 6. Insert the comb until the tip of the
hours. teeth just contact the gel surface.
Check for bubbles, if present remove
8. After polymerization, it by blowing buffer with a pipette
polyacrylamide gel can be used into the groove.
immediately.
7. Close the upper and lower chamber
2.9.7. Assembly of gel plate into lids. Plug in the power lead assembly
the gel rig and start prerun by setting the
power supply to 100 W and operate
1. Check whether the drain bottle at for 30 min to warm the gel.
the back of the gel rig is empty and
properly attached to the drain tube. 2.9.8. Electrophoresis
Prepare running equipment set
according to the instruction for each 1. About ten minutes prior the
model. completion of the prerun, prepare
samples for loading.
2. Remove the clips and pieces of tape,
gently rinse the sandwiched gel 2. Flash centrifuge the samples and
plate under running tapped water. then denature at 94ºC for 3 min
Remove the excess polyacrylamide and then immediately place the
gel sticking outside the glass plates. denatured samples on ice.
Gently remove the comb, and rinse
until all debris is washed out from 3. Load 2-3.5 µl of the sample into
the gel space. Then dry all the each sample well. Load size markers
surfaces with paper towel. to a well ahead of the first sample
and a well after last sample.
3. Place the gel plates on the supporter
in the lower buffer chamber with 4. Plug in the HV lead assembly, adjust
the short gel plate facing the the power supply to 50 W and run
aluminum plate of the sequencer. for 1.30 hours.
Close the sequencer properly
according to the manual for each 5. When the electrophoresis is
model. completed, turn off the power
supply and disconnect the HV
assembly.
59
6. Open the upper and lower buffer • Denature at 95ºC for 30 sec
chambers and drain. Then uninstall • Annealing and extension at 70ºC
the gel according to the manual for for 2 min
each sequencer model.
▪ Hold at 4ºC.
2.9.9. Chemical preparations
▪ After the reaction is completed
Preparation of M13 ladder add to each tube 5 µl of a stop dye
follwed by a flash spin. Prior to
M13 ladder solution (10 µl) is prepared loading to electrophoresis gel the
according to a protocol modified from solutions must be denatured.
Promega Corporation (undated) as
follows: Developing solution
Place the tubes in a PCR machine ▪ Add 100 ml of glacial acetic acid to
programmed as follows: 900 ml of autoclaved distilled water
and mix.
▪ One cycle of denature at 95ºC for 2
min ▪ Pour it in a plastic bottle and close
properly.
▪ 60 cycles of:
60
Staining solution 7. Then stain the gel by pouring silver
nitrate solution into the tray and
▪ Weigh 1 gram of silver nitrate and shake for 30 minutes.
put in a plastic jug.
8. Remove silver nitrate by pouring
▪ Add 1 liter of autoclaved distilled into a designated bottle. Do
water. not throw it elsewhere. Before
discarding, the silver nitrate should
▪ Add 1.5 ml of 37 % formaldehyde to be kept under sunlight.
the solution.
9. Wash the stained gel with
▪ The preparation should be done autoclaved distilled water 1 time (no
quickly to protect from light and longer than 5-10 seconds).
keep in the dark place before use.
10. Pour developing solution over it,
2.9.10. Gel fixation and staining and shake until the bands appear
and the color of the gel turns to
1. Prepare two plastic trays and two light brown.
plastic buckets.
11. Add fix/stop solution and shake for
2. Place the sandwiched plates on a 3 minutes.
laboratory bench. Then carefully
separate the plates by inserting a 12. Wash the plate twice with distillated
small metal rod and lift. The gel water for 2 minutes each.
would tightly adhere to the short
plate. 13. Keep the plate to dry over night at
room temperature.
3. Put the short plate with the gel on
top in the tray. Then pour fix/stop 2.9.11. Scoring gel
solution to the tray, and shake for
20 minutes. Scoring of microsatellite genotypes
is straightforward. The homozygotes
4. After 20 minutes remove the fix/stop produce a single band whereas the
solution and keep in a bucket for heterzygotes produce two bands.
further use. However, some problems may emerge
for example dinucleotide microsatel-
5. Pour 1 liter autoclaved distilled lite almost always produces stutter
water into the tray (with the short bands which may lead to miscoring of
plate) and shake for 2 minutes then homozygotes. Some loci comprise null
remove. alleles which refer to alleles that do not
give bands. Therefore heterozygotes
6. Repeat it twice (all together 3 are mis-scored as homozygotes.
times).
61
2.9.12. Trouble shooting The procedure involves the elec-
trophoresing of DNA through a
Common problems with microsatellites polyacrylamide gel that is running
are summarised in Table 15. parallel to a temperature gradient.
The double-stranded duplexes of DNA
2.10. Temperature gradient migrate along the gel until they reach
gel electrophoresis (TGGE) their respective melting points where
the progress is greatly reduced when
As the name suggests, this method the dsDNA begins to unwind. The
relies on a temperature gradient to melting point of a specific fragment of
denature double-stranded DNA frag- DNA is a function of both the effect of
ments that are differentially separated base sequence on the helix structure
based on their respective melting and the electrophoretic mobility of the
profiles. Another method, Denaturing strand as it starts to unwind. Therefore
Gradient Gel Electrophoresis (DGGE) DNA fragments with different base
is similar to TGGE but uses a chemical pair sequences tend to display different
gradient of increasing urea and forma- melting points and hence stop at
mide concentrations to denature the differing points on the gel.
DNA instead of a temperature gradient.
Figure 10. TGGE apparatus. Both heated (eg. 60ºC) and cooled (eg. 20ºC) water is
pumped through opposite ends of the heating block creating a linear temperature
gradient. Samples are loaded at the cool end of the gel and electrophoresed to a
point in the gel where the temperature denatures the double-stranded fragments.
The gel is then silver stained to visualise the bands.
Power Pack +
Polyacryamide Gel
Temperature
Buffer Buffer
Gradient Block
62
Table 15. Common problems with microsatellite.
Dark, swirling patterns on the gel • Inadequate agitation during the staining steps
surface • Inadequate rinsing before the development step
Yellow gel • Improper fixing of the gel
• Poor quality sodium carbonate was used
Gray gel • The sodium thiosulfate was not added to the
developing solution
Band stain yellowish-brown with poor • Dirty template DNA
contrast, as opposed to dark gray
63
To improve the resolution of the
technique, TGGE is usually conducted
in conjunction with Heteroduplex
Analysis (TGGE/HA). Nuclear (diploid)
DNA fragments can be heteroduplexed
to themselves but because mtDNA
is haploid, an extra reference DNA
fragment (ideally from a moderately
divergent conspecific individual) needs
to be added. The heteroduplexing
process involves heating both the refer-
ence and sample DNA together in order
to reduce them to single strands. Upon
cooling the strands recombine. Apart
from the original double strands from
the reference and sample fragments
recombining to themselves (homodu-
plexes), mismatch pairings occurs with
one strand from the reference and one
strand from the sample also recom-
bining (heteroduplexes). Where heter-
oduplex fragments have nonperfect
complementary matches, they tend to
have lower and more variable melting
points than homoduplexes resulting in
additional bands on the gel. TGGE is a
reliable method for DNA fragments up
to ~700 base pairs in length.
64
SECTION 3
Data analysis
3.1. Analysis of molecular information provided - DNA sequences.
data Once the direct analysis of nucleotide
sequences has been developed, we
One of the main goals in biodiversity will consider other markers which
conservation is the preservation of provide indirect estimates of nucleotide
genetic diversity. Traditionally, the divergence between alternative alleles
study of genetic diversity has fallen - haplotypic markers, such as RFLPs
within the realm of population and SSCPs. Additionally, we consider
genetics, particularly in regard to dominant markers, such as RAPDs and
comparing levels of genetic diversity AFLPs, for which the “presence” allele
within and among populations and in is dominant over the “absence” allele.
making inferences on the nature and Furthermore, wherever appropriate,
intensity of evolutionary processes some computer programs available for
from the observed patterns of genetic use in population genetics and analysis
diversity. Hence, there is a long tradi- of molecular variation are introduced
tion as well as a wealth of conceptual and discussed.
tools in population genetics for
analysing, measuring and partitioning 3.1.1. Co-dominant Markers
genetic diversity.
Allele / genotype nomenclature
This summary on methods for analysing
variation using molecular markers will The first assignment before analysing
start with a brief outline of the main data is that a genotype should be
population genetic concepts involved. interpreted from observed phenotype.
These ideas were developed for simple
situations, such as the one-locus It may be appropriate here to provide
two-alleles case, and were refined and some variation of allele nomenclature.
generalised later. However, the main Allozyme alleles are often named
features are best understood by taking using alphabetical characters following
the simplest case, which in terms of a alphabetical order with A being the
molecular marker, can be understood as slowest moving allele as shown in
an allozyme locus with only two alleles. Figure 11. This is the simplest case, i.e.
In this situation, we are dealing with a monomeric enzyme with two alleles,
co-dominant markers, such as those which give phenotypes.
generated by allozyme electrophoresis
and microsatellite techniques, for However, sometimes alleles can
which all possible genotypes (both be named based on the distance,
homozygotes and the heterozygote) measured in mm, the protein produced
can be easily ascertained. from them migrates in the gel relative
to the distance the protein produced
The manual will then move to the from the most common allele migrates
case of the richest possible markers in in the gel.
terms of the amount and quality of the
67
Figure 11. Hypothetical electrophopherograms of a monomeric enzyme.
1 2 3 4 5 6
Allele A
Gel A
Allele B
AA AB BB AB AA
168 bp 1 2 3 4 5 6 7 8 9 10
160 bp
156 bp
168/168 160/160 160/160 168/156 156/156
160/160 160/156 168/160 156/156 156/156
68
Some enzymes can be dimeric or
tetrameric where product of different The frequency of an allele is
alleles interact with each other as given by:
shown in Figure 12.
2H0 + He (1)
Proportion of polymorphic loci (P) 2N
where:
Proportion of polymorphic loci (P) is
estimated by using number of loci H0 = number of homozygotes for
polymorphic divided by the total that allele.
number loci examined.=
He = number of heterozygotes for
Example: Assuming the individuals that allele.
depicted in Figure 11 and Figure 12
were also analysed for seven other loci N = number of individuals
all of which were monomorphic (not examined.
variable).
1 2 3 4 5 6
Allele A
Gel B
Allele B
1 2 3 4 5 6
Allele A
Gel C
Allele B
69
if frequency of the common allele does He is mainly determined by loci with
not exceed 0.95 (P95). P99 is used when two or more alleles at an appreciable
the sample size is larger than 100. frequency (0.1 to 0.9). It is not sensitive
to the loss of low frequency alleles.
Average Expected Heterozygosity
(He) Average number of alleles per
locus (An)
Average expected heterozygosity (He)
is average proportion of loci at which Examples from Gel A, B and C plus
an individual is expected to be hetero- seven monomorphic loci:
zygous based on HWE.
An = (7+6)/10 = 1.30
Expected heterozygosity at a locus (h)
is one minus the sum of the squared Testing for Hardy-Weinberg
allele frequencies: equilibrium
70
Average number of alleles per locus (An) is only calculated for co-dominant
markers, and is estimated as the total number of alleles detected summed
over all loci divided by the total number of loci examined.
hopefully can determine what those ratio of p2, 2pq and q2, for a two
forces are in order to manage the allele polymorphism, where p is the
population. frequency of allele A and q if the
frequency of allele B.
The Hardy-Weinberg principle serves
as a kind of a null hypothesis, i.e. there We use Gel A as an example (please
is random mating, no selection, no note that this is only an example with
mutation and no migration occuring a small sample size, in practice we will
in the population studied. It tells us have to generally deal with much larger
what to expect if all of the seven stated number of samples).
conditions are true in the population. If
we sample a population and find that Expected proportion of AA:
the genotype frequencies are different p2 = 0.6662 = 0.44
from Hardy-Weinberg expectations,
then we can conclude that one or Expected proportion of BB:
more of these assumptions is violated, q2 = 0.3342 = 0.11
or at least one evolutionary process is
operating, or sometimes variations are Expected proportion of AB:
not properly scored. This motivates us 2pq = 2 x 0.666 x 0.334 = 0.45
to study the population in more detail.
Expected number of AA:
The usual way to compare a set of 0.44 x 6 = 2.66
observed values to a set of expected
values (based on some null hypothesis) Expected proportion of BB:
is to use a goodness of fit test. The 0.11 x 6 = 0.67
most commonly used goodness of fit
test for Hardy-Weinberg conditions is Expected proportion of AB:
the χ2 test. 0.45 x 6 = 2.67
71
Observed distribution and expected It is important to note that interpreting
Hardy-Weinberg equilibrium distribu- goodness of fit tests for Hardy-
tion of genotypes can be summarised in Weinberg equilibrium is not always
the table below: straightforward. Detecting significant
deviations requires large sample sizes
Table 16. Observed and expected HWE. and strong disturbing forces. Lack of
significance cannot be interpreted to
Genotypes mean that evolutionary processes are
AA AB BB not operating. Different processes may
Observed 3 2 1 be acting in ways that are often not
Expected 2.66 2.67 0.67 detectable with a goodness of fit test,
(O - E)2 0.12 0.45 0.11 or they may be too weak to be detect-
(O - E)2/E 0.04 0.17 0.16 able with a given sample size.
χ2 = 0.04 + 0.17 + 0.16 = 0.37 For formal testing try one of the online
tools (Online HWE and Association
The degrees of freedom (df) in a test Testing; Online; HWE Test (Multi-
involving n classes are usually equal allelic Markers), Genetic Calculation
to n-1. That is, if the total number of Applets (up to four allele), or freeware
individual (6 in this example) is divided (Arlequin v3.01; PopGene; GDA;
among n classes (3 genotypic classes in TFPGA). One important point is to
the example), then once the expected choose an exact test for multiallelic
numbers have been computed for n-1 markers because χ2 tests are inappro-
classes (2 in the example), the expected priate when there are multiple alleles
number of the last class is set. Thus in (Guo & Thompson 1992). It has been
the above example there are only two argued that even for large samples, a
degrees of freedom in the analysis. χ2 test is inappropriate and exact tests
should be used in assessment of HWE
Check the χ2 value of 0.37 at df = 2 (Wigginton et al. 2005).
in Table 17 we will have P value >
0.05 and therefore we accept the null The Hardy-Weinberg Principle suggests
hypothesis of Hardy-Weinberg equilib- that as long as the assumptions are
rium in the population in our example. valid, allele and genotype frequencies
The difference between the observed and expected values can be tested for
statistical significance using a χ2 test for goodness of fit.
χ2 = (Observed − Expected )2
∑ (3)
Expected
72
will not change in a population in whole genome without changing
successive generations. Thus, any the allele frequencies. Rare-male
deviation from HWE may indicate: mating advantage also tends to
increase the frequency of the rare
1. Small population size results allele and heterozygosity (in reality,
in random sampling errors and random mating does not occur
unpredictable genotype frequencies all the time). Cryptic population
(a real population’s size is always stratification is another reason for
finite and the frequency of an allele departure from HWE.
may fluctuate from generation to
generation due to chance events). 3. A very high mutation rate in the
population (typical mutation rates
2. Assortative mating which may be are < 10-5 per generation) or massive
positive (increases homozygosity; gene flow from a genotypically
self-fertilization is an extreme different population interfering
example) or negative (increases with the allele frequencies.
heterozygosity), or inbreeding
which increases homozygosity in the
df Probabilities
0.95 0.90 0.70 0.50 0.30 0.20 0.10 0.05 0.01 0.001
1 0.004 0.016 0.15 0.46 1.07 1.64 2.71 3.84 6.64 10.83
2 0.10 0.21 0.71 1.39 2.41 3.22 4.61 5.99 9.21 13.82
3 0.35 0.58 1.42 2.37 3.67 4.64 6.25 7.82 11.35 16.27
4 0.71 1.06 2.20 3.36 4.88 5.99 7.78 9.49 13.28 18.47
5 0.15 1.61 3.00 4.35 6.06 7.29 9.24 11.07 15.09 20.52
6 1.64 2.20 3.83 5.35 7.23 8.56 10.65 12.59 16.81 22.46
7 2.17 2.83 4.67 6.35 8.38 9.80 12.02 14.07 18.48 24.32
8 2.73 3.49 5.53 7.34 9.52 11.03 13.36 15.51 20.09 26.13
9 3.33 4.17 6.39 8.34 10.66 12.24 14.68 16.92 21.67 27.88
10 3.94 4.87 7.27 9.34 11.78 13.44 15.99 18.31 23.21 29.59
11 4.58 5.58 8.15 10.34 12.90 14.63 17.28 19.68 24.73 31.26
12 5.23 6.30 9.03 11.34 14.01 15.81 18.55 21.03 26.22 32.91
13 5.89 7.04 9.93 12.34 15.12 16.99 19.81 22.36 27.69 34.53
14 6.57 7.79 10.82 13.34 16.22 18.15 21.06 23.69 29.14 36.12
15 7.26 8.55 11.72 14.34 17.32 19.31 22.31 25.00 30.58 37.70
20 10.85 12.44 16.27 19.34 22.78 25.04 28.41 31.41 37.57 45.32
25 14.61 16.47 20.87 24.34 28.17 30.68 34.38 37.65 44.31 52.62
30 18.49 20.60 25.51 29.34 33.53 36.25 40.26 43.77 50.89 59.70
50 34.76 37.69 44.31 49.34 54.72 58.16 63.17 67.51 76.15 86.66
Accept Reject
73
4. Selection of one or a combination It has to be remembered that when
of genotypes (selection may be HWE is tested, mathematical thinking
negative or positive). Selective is necessary. When the population
elimination of homozygotes as is found in equilibrium, it does not
in some autosomal dominant necessarily mean that all assump-
diseases, where homozygotes for tions are valid since there may be
the mutation may die in utero, is an counterbalancing forces. Similarly, a
example (in a very large sample, this significant deviation may be due to
could violate HWE). Similar to this sampling errors (including Wahlund
selection, sampling error (selection effect, see below and Glossary), misclas-
bias) may also affect HWE if bias sification of genotypes, measuring two
concerned ethnicity. or more systems as a single system,
failure to detect rare alleles and the
5. Unequal transmission ratio inclusion of non-existent alleles. The
(transmission ratio distortion Hardy-Weinberg laws rarely holds true
or segregation distortion) of in nature (otherwise evolution would
alternative alleles from parents to not occur). Organisms are subject to
offspring. mutations, selective forces and they
move about, or the allele frequencies
6. Different gene frequencies in males may be different in males and females.
and females. The gene frequencies are constantly
changing in a population, but the
7. Gels have not been read correctly. effects of these processes can be
assessed by using the Hardy-Weinberg
In most population genetic estimations law as the starting point.
(like linkage disequilibrium calcula-
tions), HWE is assumed. This means that Wahlund effect: Reduction in observed
genotype probabilities are determined heterozygosity (increased homozy-
by allele frequencies and nothing gosity) because of pooling discrete
interferes with this. If this assumption subpopulations with different allele
is not met, the estimations will not be frequencies that do not interbreed as
accurate. When HWE is assumed, this a single randomly mating unit. When
means that genotype probabilities are all subpopulations have the same
determined by allele frequencies, i.e., gene frequencies, no variance among
there is no transmission ratio distortion, subpopulations exists, and no Wahlund
selection against a genotype (lethality) effect occurs (FST = 0). Isolate breaking
etc. If HWE is violated, statistical is the phenomenon that average
methods using allele frequencies heterozygosity temporarily increases
may not be valid and methods that when discrete subpopulations make
use genotype frequencies should be contact and interbreed (this is due to
preferred (Xu et al. 2002) a decrease in homozygotes). It is the
opposite of Wahlund effect.
74
To calculate D, first one needs to calculate genetic identity (I). Nei’s coef-
ficient of genetic identity (I) between two taxa is given by:
∑ xi y i
I= (4)
( ∑ xi2 y i2
Where xi and yi are the frequencies of the ith allele in population X and Y
respectively.
The mean genetic identity (I) is the mean over all loci studied (including
monomorphic ones) and is most conveniently calculated as:
I xy
I= (5)
Ix Iy
Where Ixy, Iyand Iy are the means, over all loci of ∑ xi y i , ∑ xi2 and ∑ y i2
respectively.
75
there is population subdivision, there is was formalized in the first instance by
almost inevitably some genetic differ- Wright (1951) in a series of hierarchical
entiation among the subpopulations, F-statistics.
e.g. difference in allele frequencies
among the subpopulations. Genetic Assuming that there are three levels
differentiation may result from natural of population structure: Individuals
selection favouring different genotypes (I), Subpopulations (S) and the Total
in different subpopulations, but may population (T). F-statistics can be
also result from random processes in illustrated as:
the transmissions of alleles from one
generation to the next or from chance ▪ FIS is inbreeding coefficient
differences in allele frequencies among (formula 6): is the mean reduction
the initial founders of the subpopula- in heterozygosity of an individual
tions. due to non-random mating within a
subpopulation, i.e. a measure of the
One of the main effects that popula- extent of genetic inbreeding within
tion subdivision has on genetic subpopulations. FIS ranges from -1.0
diversity, is the reduction in observed (all individuals are heterozygous) to
heterozygosity compared with +1.0 (no observed heterozygotes).
expected heterozygosity. The extent of
reduction in observed heterozygosity ▪ FST is fixation index (formula
can be used to quantify the level of 7): is the mean reduction in
genetic of differentiation between the heterozygosity of a subpopulation
subpopulations. This quantification (relative to total population) due to
genetic drift among subpopulations,
i.e. a measure of the extent of
HS − HI
FIS = (6)
HS
H T − HS
FST = (7)
HT
H T − HI
FIT (8)
HT
Where:
• HI is the observed heterozygosity averaged across all subpopulations.
• HS is the expected heterozygosity across all subpopulations.
• HT is the expected hterozygosity for the total population.
76
genetic differentiation among ▪ FST = 0.05-0.15 indicates moderate
subpopulations. FST ranges from 0.0 genetic differentiation
(no differentiation) to 1.0 (complete
differentiation - subpopulations are ▪ FST = 0.15-0.25 indicates very large
fixed for different alleles). genetic differentiation
▪ FIT is overall fixation index (formula ▪ FST = > 0.25 indicates extensive
8): is the mean reduction of genetic differentiation
heterozygosity of an individual
relative to total population. The three F-statistics described above
can be extended to include higher
Examples: The value FST = 1 was levels of hierarchy. For example, if we
estimated for two subpopulations of have a series of subpopulations of a
a fish species. This means that there is fish species which naturally occur in
absolute differentiation between the three separate river basins, then the
two subpopulations, with each fixed following FST related statistics could be
for a different allele. Simply speaking, estimated:
this can be interpreted as 100% of
the total genetic variation is between ▪ FST: the variance among
subpopulations, with zero variation subpopulation relative to the total
present within subpopulations. variance
The value FST = 0.47 was estimated for ▪ FSC: the variance among
two subpopulations of another fish subpopulations within groups
species. That is, there is a substantial (denoted as C)
differentiation among subpopulations,
and 47% of total genetic variation is ▪ FCT: the variance among groups
distributed among subpopulations, relative to the total variance.
with 53% of the variation within
subpopulations. What FST may tell us about gene
flow
Although FST has a theoretical range of
0 to 1, the observed maximum is usually A single population may split into two
less than 1. Wright (1978) suggests the subpopulations at some point, which
following general guidelines for the then each diverge randomly over time.
interpretation of FST based on allozyme However, some subpopulations may
loci: share some migrants (for example fish
migrate during flooding seasons from
▪ FST = 0.00-0.05 may be considered one pool to another). If all subpopula-
as indicating little genetic tions share migrants with all other
differentiation subpopulations with equal chance, then
77
there is a simple relationship between those generated from techniques
FST and migration (m): such as Restriction Fragment Length
Polymorphism (RFLP) and Single
1 Strand Conformation Polymorphism
FST = (SSCP). However, in the case of RLFP,
4Nm + 1
it is important to distinguish between
restriction fragment data and restric-
where Nm is the actual number of tion site data.
individuals that migrate. However, it
is only a relative measure of migration ▪ Restriction fragment data are
between populations. Sometimes Nm those generated from RFLP using
is referred to as such as in Arlequin randomly chosen restriction
software. enzymes to digest the whole
genome, such as the mitochondrial
3.1.2. Haplotypic markers (RFLPs, genome without knowledge of the
SSCPs, TGGEs) actual sequences, and for which only
the size of the generated fragments
In order to analyse genetic diversity are available.
using haplotypic markers, it is necessary
to provide clear classification of the
different kinds of haplotypic markers.
Haplotypic genetic markers include
2mXY
S= (10)
mX mY
Where:
• mX and mY are the number of restriction sites in sequence X and Y,
respectively
• mXY is the number of restriction sites shared by both sequences
• Proportion of nucleotide differences is estimated as:
p = 1− r S (11)
π = [− ln S ]/r (12)
3 3
d=− ln(1 − p) (13)
4 4
78
▪ Restriction site data are those ments are due to different mutations
generated by RFLP technique, for along the sequences. However, the
which the precise location of a recognition site are mostly single
recognised sequence for a restriction nucleotide. Therefore the method of
enzyme is known. It is common analysing data is slightly different to
nowadays that the actual sequences restriction site data.
of a gene are firstly generated
for a number of representative Diversity within population
individuals of several populations,
and restriction enzymes are For restriction site data, Levels of
designed on the basis of sequence diversity within population is expressed
differences. by estimates of proportion of shared
restriction sites, proportion of nucle-
Data generated from SSCP technique, otide differences, nucleotide diversity
can be converted into haplotypes. and number of substitutions per site.
Different conformation of DNA frag-
Example:
2mXY 2*2
S= = = 0 .8
mX + mY 3 + 2
The length of the recognition sequence r = 6. Therefore proportion of
nucleotide differences:
p = 1 − r S = 1 − 6 0 .8
79
∑ mk rk dk
d= k
(14)
∑ mk rk
k
Where:
• mk = average number of bands for the restriction enzyme k
• rk = length of the sequence recognised by the enzyme k
• dk = estimated nucleotide divergence
∑ mk rk dk
d AB = k
(15)
∑ mk rk
k
Where:
• mk = average number of bands for the restriction enzyme k
• rk = length of the sequence recognised by the enzyme k
80
focus on inter-population divergence,
then it can be estimated as formula Haplotype diversity:
(16). j
H = 1 − ∑ pi2 (16)
3.1.3. Population structure i
81
into account, data are treated in a very same position in the gel, and that the
similar way to that already described researcher is capable of matching bands
for restriction fragment data. In the from different lanes within and among
case of RAPD data, r corresponds to gels, and that each locus can be treated
the primer length used for random as a two-allele system, with a presence
amplification (usually r=10). However, and an absence allele. Lynch and
there are a number of assumptions that Milligan (1994) adopt the following
have to be made as outlined by Clark estimate for the gene frequency, q, of
and Lanigan (1993): the null allele at one locus (formula 17).
x
q= (17)
var( x )
1−
8x2
82
If loci have been sampled in population A, the average gene diversity in this
population is:
1 X
hA = ∑ hA (18)
X i =1 i
1 n
h= ∑ hA (19)
n A=1
The heterozygosity between populations A and B at the ith locus can be
estimated by:
2∑ H AB
H= (23)
n(n − 1)
83
treats RAPDs as restriction data, and Number of segregating sites is the
therefore F-statistics can be estimated number of variable nucleotide sites in a
as mention in Section 3.1.1 If the sample of sequences. The disadvantage
data showed evidence of Mendelian of this measure is that it does not
inheritance, then the principle used for incorporate the length of the sequence
co-dominant marker can be applied. analysed and hence is not comparable
It is suggested that when dealing with across the data set.
dominant markers such as RAPDs and
AFLPs, it is ideal if an inheritance study Proportion of segregating sites is the
can be conducted to test for Mendelian number of segregating sites divided by
inheritance patterns of the alleles. the length of the sequences.
2∑ d j
i< j
d= (23)
n(n − 1)
84
Seq1 Seq2 Seq3 average nucleotide diversity within
Seq1 * subpopulations, then we can derive
Seq2 2 * a familiar expression for an FST, like
Seq3 1 1 * nucleotide measure of subpopulation
differentiation:
The average number of pairwise
πT − π S π B
nucleotide differences between the = (25)
three sequences will be: πT πT
85
allelic calculations (FST), it is assumed As a result, the two values are
that all alleles are equidistant from different. This is because the two forms
each other, while in the nucleotide are really measuring different proper-
diversity calculations (ΦST), there are ties of the data.
different distances between different
alleles. (This can indeed be applied to One is not necessarily any ‘better’
any other type of distance calculation than the other. Even in terms of simply
you might require, such as between detecting whether or not there is
microsatellite alleles). In fact, now we significant differentiation between
can actually calculate the allelic FST subpopulations, it depends entirely
in exactly the same way we calculate on the data set as to which form of
ΦST (in AMOVA – see below) by simply FST - allelic or nucleotide distance
making all the distances between – is the more powerful statistically.
alleles equal one (i.e., replace the So the bottom line is that it is useful
pairwise distance matrix by a unity to calculate both types of FST for one
matrix). This is shown below. FST and given data set.
ΦST values then can be estimated as
shown in the example that follows.
π T − π S 0.48 − 0.16
Φ ST = = = 0.67
πT 0.48
86
In population genetics studies, DNA zygote advantage. A classic example
sequences can be analysed using the of this is the mammalian major histo-
ARLEQUIN software package, where compatibility complex (MHC) system
important factors such as number of genes and other compatibility system
variable sites, haplotype frequency, in other organisms: the self-incompat-
pairwise number of nucleotide ibility system of plants, fungal mating
differences etc. can be obtained. types and invertebrate allorecognition
Furthermore, analysis of population systems. In all these genes, a very high
differentiation using F-statistics is also number of alleles are present. This
implemented, or population genetic can be interpreted as an indicator of
structure using Analysis of Molecular some form of balancing (diversifying)
Variance (AMOVA) can also be applied selection. In the case of neutral
using this software. polymorphisms, a single common allele
and a few rare alleles are expected. The
3.2. Statistical tests frequency distribution of alleles is also
informative. A large number of alleles
The rapid rate of increase of technical showing a relatively even distribution
applications to population genetic is against neutrality expectations and
studies has been paralleled by the suggestive of diversifying selection.
number of statistical tests developed to
analyse them and probably exceeded Most tests detect selection by rejecting
by the number of computer software neutrality (observed data deviate
programs available to perform the significantly from what is expected
analyses. We certainly do not have under neutrality). This deviation,
the time or scope to investigate them however, may also be due to other
all. Here, and in the next section, we factors such as changes in population
only look at a few basic analyses (both size or genetic drift. The original
quantitative and qualitative) that are neutrality test was Ewens-Watterson
useful for understanding population homogeneity of neutrality based on a
structure. comparison of observed and predicted
homozygosity calculated by Ewens’s
3.2.1. Neutrality tests sampling formula which uses the
number of alleles and sample size. This
Several methods have been designed test is not very powerful.
to use DNA polymorphism data
(sequences and allele frequencies) to Other commonly used statistical tests
obtain information on past selection of neutrality include: Tajima’s D (theta,
events. Most commonly, the ratio of θ), Fu and Li’s D, D* and F. Tajima’s test
non-synonymous (replacement) to (Tajima 1989) is based on the fact that
synonymous (silent) substitutions (dN/dS under the neutral model estimates of
ratio; see below) is used as evidence the number of segregating/polymor-
for overdominant selection (balancing phic sites and the average number of
selection) of which one form is hetero- nucleotide differences are correlated.
87
If the value of D is very large or very For other tests and software to perform
small, the neutral ‘null’ hypothesis is these statistics, see websites in DnaSP.
rejected. DnaSP calculates D and its
confidence limits (two-tailed test). 3.2.2. Linkage disequilibrium (LD)
Tajima did not base this test on the
coalescent but Fu and Li’s tests (Fu & Li Two ‘alleles’ can be present on the
1993) are directly based on the coales- same chromosome (positive LD), or not
cent. The tests statistics D and F require segregate together (negative LD). As a
data from intraspecific polymorphism result, specific alleles at two different
and from an outgroup (a sequence loci are found together more or less
from a related species), while D* and than expected by chance. LD is the
F* only require intraspecific data. non-independence, at a population
DnaSP uses the critical values obtained level, of alleles carried at different
by Fu and Li (1993) to determine the positions in the genome. In this case,
statistical significance of D, F, D* and F* the expected frequency of a two-locus
test statistics. DnaSP can also conduct haplotype can be calculated as the
the Fs test statistic (Fu 1996). The probability of the occurrence of two
results of this group of tests (Tajima’s independent (or joint) events simply by
D and Fu & Li’s tests) based on allelic multiplying their gene frequencies. The
variation and/or level of variability same situation may exist for more than
may not clearly distinguish between two alleles. Its magnitude is expressed
selection and demographic alternatives as the delta (Δ) value and corresponds
(bottleneck, population subdivision) to the difference between the expected
but this problem only applies to the and the observed haplotype frequency.
analysis of a single locus (demographic If there is no LD, Δ will be zero (or not
changes affect all loci whereas selection significantly different from zero), if
is expected to be locus-specific which there is positive LD it will be a positive
are distinguishable if multiple loci are value. It can also be negative if the
analysed). Tests for multiple loci include two alleles tend not to occur together.
the HKA test described by Hudson et The statistical significance of LD,
al. (1997). This test is based on the which depends on the sample size,
idea that in the absence of selection, and the magnitude of LD are separate
the expected number of polymorphic issues. The statistical significance is
(segregating) sites within species and determined usually by Fisher test and
the expected number of ‘fixed’ differ- the magnitude is determined by either
ences between species (divergence) are Δ value or alternative measures. The
both proportional to the mutation rate, magnitude can be normalised (for allele
and the ratio of them should be the frequencies) to have the same range of
same for all loci. Variation in the ratio values for any frequency.
of divergence to polymorphism among
loci suggests selection. Ideally, haplotype frequencies should
be calculated from family data. Obvi-
ously, this gives the most accurate
88
results. In practice, however, when that there is little or no advantage to
family data are not available, Δ and constructing haplotypes from family
two-locus haplotype frequencies data rather than unrelated individuals.
are calculated from a sample of the The major point is that when using
population data by constructing 2x2 population data, genotyping errors
contingency tables for each allele pair. become an issue. When genotyping a
A contingency table for this purpose large number of markers, an error rate
contains the individual (observed) of only 1% will produce a large number
values cross-classified by levels for two of inaccurate haplotypes. Genotyping
different attributes. A common 2x2 errors are not the only possible sources
table constructed in genetic studies is of accuracy problems. Other factors
as follows in the table below. include sample size, allele frequency
distributions and departures from HWE.
Counts for each combination of levels
(presence or absence) of the two There are other measures of LD.
factors (alleles) are placed in each cell. Because the value of Δ depends on
The corresponding Δij is estimated by allele frequencies a normalisation of
the formula (usually in HLA studies): Δ is needed. This is achieved by taking
into account the allele frequencies:
Δij = (d/N)1/2 – [((b+d)/N)((c+d)/N)]1/2 (26) normalised delta value (D’) = ΔAB /
Δmax. Δmax is the lesser of pApb or
The haplotype frequency (HFij) equals papB if Δ is positive or pApB or papa
GFi x GFj + Δij, where GF is the gene if Δ is negative. Because the sign is
frequency (the proportion of the arbitrary, |D’| is often used rather than
chromosomes carrying a particular Δ’. Therefore, D’ (normalised LD) is
allele). The haplotype frequency scaled to remove allele frequency
calculated with this formula from the effects. In a large enough sample, D’ =
population data compares reasonably 1 that indicates complete LD and D’ =
well with estimates obtained directly 0 corresponds to no LD. |D’| is directly
from counting haplotypes constructed related to recombination fraction and
from family segregation data. This its generalization to more than two loci
method generates a reliable estimate is the only measure of LD not sensitive
of a haplotype frequency with the to allele frequencies. ASSOCIATE and
exception of very small haplotype HAPLOVIEW are some of the software
frequencies. Also for other parts of that calculate D’ values.
the genome, it has been reported
Allele i
Present (+) Absent (-) Row totals
Present (+) a (+/+) b (+/-) a+b
Allele j
Absent (-) c (-/+) d (-/-) c+d
Column totals a+c b+d N=a+b+c+d
89
Another linkage disequilibrium statistic LD include changes in population
is the square of the correlation coef- demographics (such as population
ficient (r2) between the alleles at locus growth, bottlenecks, geographical
A and B: r2 = Δ2/ (pA pa pB pb) which subdivision, admixture and migra-
can also be expressed as r2 = Δ2 / (pA tion) and selective forces. Admixture
(1-pA) pB (1-pB)) (for two loci with two (intermixture of populations) would
alleles each). The measure r2 has several cause LD if the mixing populations
properties that make it more useful. In have different allele frequencies.
brief, for low allele frequencies r2 has LD will also be erased faster in large
more reliable sample properties than populations than in small ones (chance
|D’|. in small populations maintain LD).
Permanent LD may result from natural
LD estimates can be calculated using selection if some gametic combinations
various software such as ARLEQUIN, confer higher fitness than alternative
GDA, PopGene amongst others. combinations. Regional LD may also
be variable according to haplotype. An
Interpretation of LD Data: The patterns example has been presented for HLA
of LD observed in natural populations haplotypes. Haplotype-specific patterns
are the result of a complex interplay of LD may reflect haplotype-specific
between genetic factors and the recombination hotspots as has been
population’s demographic history. LD is shown for mouse MHC.
usually a function of distance between
the two loci. This is mainly because Note that LD has nothing to do with
recombination acts to break down HWE and should not be confused with
LD in successive generations. When a it.
mutation first occurs it is in complete
LD with the nearest marker (D’ = 1.0). 3.2.3. Testing FST for significance
Given enough time and as a function
of the distance between the mutation Once we have calculated our FST values,
and the marker, LD tends to decay we need to know whether they repre-
and in complete equilibrium reaches sent significant population structure or
a D’ = 0 value. Thus, it decreases every not. Theoretically FST ranges from zero
generation of random mating unless (no structure) to one (total differentia-
some process opposes the approach tion). Any values that fall in between
to linkage ‘equilibrium’. However, these extremes need to be statistically
physical distance could account for less determined for significance. That is we
than 50% of the observed variation are testing the null hypothesis that
in LD. One genetic phenomenon that Ho: FST = 0 is not significantly different
affects LD is gene conversion. Gene from 0.
conversion is an important mechanism
in the breaking down of allelic associa-
tions over short distances, i.e., decay
of LD. Other factors that influence
90
There are several methods for significantly different from zero (i.e.
determining the significance of FST reject the null hypothesis at the α=0.05
values. Firstly, the estimated FST can be level).
compared to a χ2 distribution with the
relationship between FST and the χ2 Another powerful test to determine
critical value as: whether significant differentiation
exists among sample sites is the Exact
χ2(crit) = 2N*FST(k-1) (27) Test of Raymond and Rousset (1995)
which is analogous to Fisher’s exact test
with degrees of freedom: and is based on a ‘number of popula-
tions’ x ‘number of haplotypes’ contin-
df = (k-1)(s-1) gency table. This test employs a Markov
chain random walk method that
As can be seen, this method is affected provides an unbiased estimate of the
by sample size (N), the number of exact probability of incorrectly rejecting
alleles present in the sample (k) and the null hypothesis (null hypothesis =
the number of population samples (s). there is no differentiation among sites)
Therefore the greater the number of which is committing a Type I error. It is
samples and/or a more variable marker a particularly powerful test with small
will increase the power of this test. sample sizes and rare alleles. This is a
Although this is a simple test (it can ‘whole of table’ test that estimates the
be calculated by hand), it is however, probability of observing a table less
a relatively conservative test with a likely than the observed configuration
moderate chance of committing a Type under the null hypothesis of panmixia.
II error (i.e. incorrectly accepting that
there is no structure). 3.2.4. Estimating gene flow from
mtDNA sequence data
A more powerful method for
determining the significance of FST Directly quantifying gene flow is
is the Permutation Test. This method extremely difficult (if not impossible)
permutes (randomises) haplotypes in natural systems because observing
among populations and calculates an all migrants (dispersers) is often not
FST. This process is repeated many times possible and even if you can observe
(e.g. 1000) resulting in a distribution all dispersers you have no idea as to
of FST values. We can then place the FST how many of them actually contribute
observed from the original data into their genes to future generations in
the distribution and make a judgement the receiving population and in what
as to the level of significance. For proportion.
example if 95% of the simulated FST
values fall below our true FST value,
then we can say that we are 95%
confident that the estimated value is
91
We can however, estimate gene flow There are several limitations however,
(Nem) indirectly from inference gained to this method of estimating gene
from the relationship between genetic flow. 1) the gene flow model used
drift and gene flow at equilibrium (i.e. (usually the island model) may not be
FST) where: appropriate for the system that you
• Nem = number of migrants are working with; 2) the assumption of
• Ne = effective population size drift/gene flow equilibrium is usually
• m = proportion of receiving violated; 3) it does not account for sex
population that is made up of biased dispersal (i.e. for mtDNA we are
migrants only estimating female gene flow and
dispersal may be male mediated; 4) it
The relationship between gene flow has been argued that the relationship
and population structure is: between Nem and FST only holds true
for global FST estimates and therefore
FST = 1/(1+2Nemf) (for mtDNA) (28) is not relevant to pairwise comparisons
(which is what we are really interested
Notice that here we are estimating in); 5) it assumes dispersal is equal in
effective female gene flow as only both directions which is highly unlikely
females can contribute their mtDNA in freshwater systems.
genes to subsequent generations. As
we can estimate FST from the data, we Another potential problem with this
are able to estimate effective dispersal. method relates to the genetic marker
Note that as Nem increases, FST chosen for the study. Any FST value less
decreases, as would be expected. This than 1 (absolute differentiation) will
equation is specifically the relationship result in a positive estimate of gene
between gene flow and structure under flow. We know that FST is the parti-
the assumptions of the island model tioning of variation within and among
of gene flow. The relationship for the populations so that a hyper-variable
stepping-stone model is: marker in a very large population may
result in a surprisingly low level of
FST = 1/(1+2Nemf)( 2Neµ / Nemf)½ (29) differentiation. The lower the FST, the
higher the estimate of Nem, even if two
The first thing to notice is that this populations do not share any haplo-
model incorporates the effects of the types in common. Furthermore, two
mutation rate (µ). As population size populations that are totally isolated
(Ne) increases, the stepping-stone from each other today, may have
model approaches the island model. haplotypes in common from a time
It has been argued that the difference when there was connectivity (ancestral
between these two models in esti- retention). In this case, the methods
mating gene flow is negligible. Most for estimating gene flow above will
computer programs that calculate Nem be measuring historical rather than
in this way, do so under the assump-
tions of the island model.
92
contemporary dispersal patterns. Being be partitioned at different hierarchical
able to distinguish between these two levels within the system (Meffe &
scenarios is dealt with in section 3.3.1. Vrijenhoek 1988). In this model the
total genetic diversity (HT) is parti-
Because of the limitations of this tioned into several components:
method, it is strongly suggested that
estimates of the number of migrants HT = HC + DCR + DRS + DST (30)
never be treated as absolute values • HC = variation within populations
(although this is tempting for manage- • DCR = differences among
ment purposes). At best, one could populations in a river
infer differences in dispersal rates by • DRS = differences among rivers in
orders of magnitude (e.g. Nem between a drainage
populations A and B is 10 times greater • DST = divergence among
than between populations A and C). drainages
• Under the SHM it is expected
3.2.5. The Stream hierarchy model that DCR < DRS < DST
of gene flow
Analysis of Molecular Variance
The island and stepping-stone models (AMOVA) (Excoffier et al., 1992) is a
are not really appropriate for FST and method of analysis that is ideal for
gene flow analyses in freshwater investigating population structure and
systems for several reasons, including gene flow in a hierarchical fashion in
the dendritic nature of river systems freshwater systems. It is analogous
and the unequal bi-directional gene to the standard Analysis of Variance
flow which is idiosyncratic to individual (ANOVA), a standard parametric
species. For this reason there has not statistical procedure for partitioning
been a simple equation that describes error (variation about the estimate of
the relationship between FST and gene the mean) within and among treat-
flow in these systems. However a gene ments. Similarly, AMOVA can partition
flow model has been proposed (Stream genetic variation within and among
Hierarchy Model (SHM)) to explain populations. Although this is essentially
the distribution of genetic variation in what normal FST analysis does, AMOVA
freshwater systems were variation can can partition genetic variation at
Table 18. AMOVA table showing the partitioning of genetic variation at different
hierarchical levels.
93
spatially different hierarchical levels we cannot use a standard correlation
(i.e. hierarchical FST). Hence, genetic is that the basic assumption of the
variation can be partitioned into test of independence of the samples is
• FIS = among individuals within violated. This is because all estimates of
populations both genetic and geographic distance
• FSC = among populations within are pairwise measures. Therefore the
region (rivers) same population will be included in
• FCT = among regions within total many of the data points in the analysis.
(among river drainages)
To test for IBD we use a permutation
Due to the restricted nature of dispersal procedure similar to that described
within and among river drainages, the above. Firstly we calculate our pairwise
Stream Hierarchy Model predicts that FST matrix (other measures of genetic
FCT >> FSC. The significance of these distance may be used) and a pairwise
parameter estimates can be determined geographic distance matrix and
using a permutations test. Of course perform a normal correlation test to
this method relies on a hierarchical get our correlation coefficient (r). The
sampling strategy as described later. geographic matrix is then randomised
and another correlation coefficient
3.2.6. Isolation by distance is calculated. This process is repeated
many times until we have a distribu-
Sometimes in river systems the only tion of simulated r values. We then
barrier to dispersal is distance. In vast place the observed r value into the
drainages such as the Mekong River, distribution and determine the level of
it may be physically impossible for an significance as described previously.
individual to traverse the geographic
distance between two populations 3.3. Presentation of data
within a single lifetime. If this is the
case, then a signature of isolation by An important component of population
distance (IBD – not to be confused with data analysis is to be able to display
‘identical by descent’) may result. To the data is a way that makes it easy
test for this pattern, we generally use to understand and to assist in commu-
a Mantel’s test that is an extension nicating the results to managers. The
of a standard parametric correlation methods can be categorised as either
analysis. Here we are looking for a qualitative or quantitative or some-
correlation between levels of genetic times a combination of both. The most
differentiation (FST – the dependent common way of displaying mtDNA
variable) and geographical (or stream) data is by gene trees of which there are
distance (the independent variable). several types.
A positive relationship will indicate
IBD (i.e. the greater the geographic
distance separating two populations,
the higher the FST). The reason that
94
3.3.1. Minimum spanning geographically widespread. On the
networks other hand, tip haplotypes are gener-
ally more recently evolved (derived)
A minimum spanning network (or and are therefore younger and usually
cladogram) displays the relationship represented in fewer individuals.
among unique haplotypes in the
sample (see Figure 13), relying on the If we relate an individual haplotype’s
number of base pair differences. This position in the network to its
method is particularly appropriate geographic distribution we have some
for intraspecific studies using mtDNA power (albeit qualitative) to be able
analysis and can provide significant to differentiate between historical
qualitative insight into both population gene flow or ancestral retention and
and evolutionary processes that may contemporary gene flow. For instance,
have influenced observed patterns. if internal haplotypes are widespread
but tip haplotypes are restricted
Figure 13. A minimum spanning then historical gene flow is a more
network of four mtDNA 16S rRNA likely scenario. On the other hand,
haplotypes obtained from 16 indi- widespread internal and tip haplotypes
viduals of the critically endangered indicate contemporary gene flow. The
Mekong giant catfish, Pangasianodon study of the relationship between
gigas (Na-Nakorn et al. 2006). population genealogies and their
geographical distributions is referred to
Pg02 as phylogeography.
95
a strong statistical framework, it still require considerable computational
relies on a largely qualitative interpre- power. However, one of the most
tation of the results. A few limitations appropriate for intraspecific studies
exist with network analysis. The most is also one of the simplest. The
obvious is that the data are not used neighbour-joining method clusters
to their full potential. Simply using haplotypes based on the level of
the number of base pair differences distance/similarity that allows for
to determine the relationship among unequal rates of molecular change
haplotypes excludes the opportunity among haplotypes resulting in a tree
to incorporate more complex mutation with varying branch lengths. The basic
models that may better reflect the method is to calculate the pairwise
evolutionary history of the population. distances among all haplotypes. These
Secondly, homoplasy caused by multiple distances are then scaled based on the
substitutions at a single nucleotide site distances among all other pairwise
can mask the true relationship among comparisons. The haplotypes that share
haplotypes. For example the following the lowest scaled value are joined first
DNA sequences produce the network through node 1 (a node is the internal
below. point in a tree where two or more
branches converge). The next haplotype
ATCAG is then joined to the tree through node
2 with the distance between node 1
ACCAG and node 2 calculated and so on until
all haplotypes are joined. Although this
AGCAG is a simple interpretation of the algo-
rithm for building a neighbour-joining
In this case it is impossible to discern tree, we will not go into further detail
which haplotypes are tip and which here but the method is readily available
are internal. Also homoplasy can in many computer programs.
make it difficult to group closely
related haplotypes with any statistical Node
probability. Furthermore, incomplete
sampling may result in a haplotype
being labelled as a tip when in fact it is
internal (and possibly ancestral to the
whole cladogram).
96
An advantage that the neighbour- distances. Once again there are several
joining method has over networks population distance methods available
is that the appropriate distance of which we will look at two here.
method can be applied and that the
confidence of haplotype groupings can The first is Nei’s DA which is a net
be determined statistically. The most genetic divergence among populations
common form of testing the haplotype where:
groupings (clades) statistically is boot-
π1 + π2
strapping. Bootstrap values provide an DA = π12 - (31)
2
estimate of how well a particular node
in the tree is supported. Bootstrapping
has become a general term for a This method is sensitive to differential
different permutation tests relating to drift pressure between populations
gene trees. For DNA data, bootstrap- and is therefore a good measure when
ping involves randomly removing a there is little variation in population
nucleotide base or a number of bases sample size.
from every haplotype sequence and
generating a neighbour-joining tree. Another measure is the Carvalli-Sforza
The base positions are then replaced chord distance DCS which standardises
and others removed to build another distances with respect to drift and
tree. This process is repeated (usually therefore is more appropriate when
>500 times). Finally the tree from the drift is the main process causing
total data set is calculated. The boot- differentiation:
strap values are the percentage of time
that a particular node was supported
2 2
during the permutations. Although DCS = 1 − cos θ (32)
π
neighbour-joining trees are more
statistically rigorous, one disadvantage
is that it is not always easy to differen- where:
tiate between ancestral and recently
derived haplotypes. θ = ∑ xi y i
97
Figure 14. An example result of MDS analysis.
Stress: 0.01
MS
TC KU
BeR RC GoC
BC
EC
LC
DiC
Tully KC
TRH C
Herbert CaC
Nth Johnstone ChC TGL
UT NC
Barron
DC
98
population structure. Historical Based on the mode of this distribution
demographic fluctuations (expansions and the underlying mutation rate
or bottlenecks) can influence the of the marker used in the study, the
‘neutrality’ of the observed data and timing of the expansion event can
confound interpretations (especially be estimated. A population that has
from tests that assume neutrality). remained large and stable over time
would result in a multimodal (ragged)
Many populations have undergone distribution due to the process of
significant size changes in their past. random lineage sorting. Lineage
One way to test for a historical expan- sorting refers to the long term effect of
sion is by using the mismatch distribu- drift within a population where many
tion. A mismatch analysis is simply the internal haplotypes have gone extinct
frequency distribution of all nucleotide (see below) resulting in clade structure
pairwise differences between all within a single population.
individuals in the sample. It has been
shown that particular historical demo-
graphic events will leave a signature in
the distribution. For example, a popula-
tion that has undergone an expansion
will tend to retain more newly
evolved haplotypes than under stable
conditions because as the population This is just one of many examples
expands, the effects of genetic drift are of how the analysis of mtDNA in
reduced. This will provide a MSN with association with the concept of a
one or a few internal tips (usually with molecular clock can elucidate important
a relative high frequency) and many historical events influencing current
low frequency tip haplotypes resulting population structure. In the example
in what is commonly referred to as a of the mismatch distribution above,
‘star phylogeny’. Under the expansion tests for neutrality would reject the
model, the mismatch distribution from null hypothesis of neutral evolution.
this star phylogeny should approximate Instead of inferring that our marker
a smooth Poisson curve. is under selection, we can propose
that it is actually a neutral marker but
that demographic influences have
influenced the observed structure.
99
final content of this section concerns All estimated parameters that contain
the pitfalls of over-interpreting our variation are just that – an estimate.
results. As such, there will always be a certain
degree of error around that estimate.
Firstly, we will quickly revisit the Secondly, we will never be 100% sure
probability of committing a Type I that our sample actually represents the
error. In many ecological analyses, there population truthfully. For example, the
is a need to make multiple comparisons absence of a haplotype from a sample
from the data set. It has been argued does not mean that it is absent from
that this will result in an overall reduc- the population, but simply we may
tion in the significance level of the not have sampled intensely enough
test (i.e. α increases). The reasoning is to capture it. Section 12 highlighted
that some tests will return a significant certain aspects of gene flow estima-
result by chance that equates to tion that places limitations on the
committing a Type I error where the interpretation of the estimated values
null hypothesis is rejected incorrectly (e.g. non-equilibrium, inappropriate
(e.g. out of 100 tests, 5 may be found models). Many genetic statistical tests
significant by chance). To overcome are conservative in nature and there-
this problem, a Bonferroni Correction fore Type II errors are always possible.
for multiple comparisons is usually An acceptance of the null hypothesis
implemented. The rule of thumb for does not mean it is true, but only that
the correction is to divide the apriori there was insufficient power to reject
significance level by the number of it. Additionally, a rejection of the null
tests performed (e.g. α=0.05 and 20 hypothesis does not necessarily mean
tests, then α=0.05/20 = 0.0025). This that the alternative hypothesis is true.
correction is usually invoked when we Finally mitochondrial DNA, although
are estimating values within a popula- an ideal marker in many respects, is
tion (e.g. HWE tests or neutrality tests). a single locus and as such, inherently
Some maintain that it is also applicable decreases the power of any test.
to pairwise FST although an exact test
of differentiation (whole of table Taking the factors listed above into
test) largely removes the necessity. account, there is always a risk of over-
Currently however, the application of interpreting data. In a good population
the Bonferroni test in ecological studies study, the genetic data should comple-
is contentious. ment the ecological data rather than
dictating the inference made from it,
Whether to correct for multiple tests especially where the data is used for
or not raises an important point. Even making management decisions.
though we may be confident that we
have correctly accepted or rejected the
hypothesis under investigation, how
does this relate to the true biological
processes underlying our analyses?
100
Table 19. Commonly used programs for population genetic analysis.
101
3.4. Commonly used
software for data analysis
Many software programs have been
developed to analyze data in relation
to molecular population genetic
analysis. Their easy access,
implementation of sophisticated,
powerful statistical techniques and
user-friendly interface make them an
attractive alternative to performing
calculation on spreadsheets or writing
a program by oneself. Although there
are a number of programs available
for cost-free downloading from
the internet, only some of the most
commonly used ones are mentioned
here.
102
Section 4
Project design
In population genetic studies, project lations?’ we already have some sense of
design is probably the most critical the spatial sampling strategy required.
step for several reasons. Generating The more concise the question or
molecular genetic data requires questions being asked, the greater the
expensive chemicals and facilities, it chance that an appropriate design will
may also involve destructive sampling be formulated, and consequently we
of animals and therefore careful will have a greater confidence in the
planning is recommended in order to ecological interpretations from the
obtain the best information at the least resulting data.
cost if possible. A project often goes
through several steps of planning such Problems associated with biodiversity
as identification of problems to be in relation to aquaculture and fisheries
addressed, conducting a pilot study to often can be addressed by studying
identify suitable markers, followed by genetic variation within and/or among
the development of sampling strate- populations. These populations under
gies, collecting and preserving samples, study could be either wild or farmed
generating and analysing data. stocks. Population genetics can be
useful in:
105
▪ Understanding species biology These factors need to be taken into
(mating patterns, dispersal and account to reduce error in the statistical
migration). results. The errors that can arise fall
into two categories: Type I and Type
II errors. Type I or α error arises if the
4.2. Statistical inference null hypothesis is rejected when it is
actually true (e.g. inferring structure
Once we have our concise questions, when there is none). The level of Type
we need to be able to test them in a I error that we are willing to accept in
statistical manner. That is, we need an many biological/ecological analyses is
experimental design that will allow us commonly 5% (i.e. α = 0.05). That is,
to either accept or reject our hypoth- we are willing to accept that the null
eses with a certain level of probability hypothesis will be incorrectly rejected
that minimizes errors in interpreting 5% of the time.
the data. Using the question above,
‘does migration between points A and Type II or β error occurs when we fail
B result in introgression of genetic to reject the null hypothesis when it is
material between the respective actually false. Although the probability
populations?’ we can formulate specific of committing a Type I error is the
testable hypotheses: pre-specified significance level (α), the
probability value of committing a Type
Null Hypothesis N0: There is no genetic II error is unspecified and generally
differentiation among populations A unknown. However the power of the
and B (i.e. panmixia) test can be defined as 1 – β (the prob-
ability of rejecting the null hypothesis
or when it is truly false). Even though it is
difficult to quantify β, the relationship
N0 : FST between A and B is not signifi- between the two error types is known.
cantly different from zero For a given sample size the value of α
is inversely related to β. The lower the
The goal of the experimental design chance of committing a Type I error, the
is to provide sufficient power to higher the chance of committing a Type
confidently accept or reject the null II error. The only way to reduce these
hypothesis. The statistical power of the error rates simultaneously is to increase
analyses will depend on quantitative the sample size. In other words,
factors such as how many sample sites, the greater the sample size the less
how many individuals per site, how likelihood there is of making incorrect
many genetic loci were assayed and conclusions. This fact needs to be taken
even how many base pairs of DNA were into account when designing the study.
included per locus. As will be seen in later sections, the
capacity for making errors (especially
Type II) in population genetic studies is
considerable.
106
4.3. Pilot study allozymes can be screened using body
tissues such as body slime or finclips
Population genetic studies often (Mather and Ruscoe, 1992). The second
involve large sample sizes and are factor is the availability of funding;
usually expensive; as such a small-scale sequencing is rather expensive and
pilot study is desirable to identify therefore should be used when the
suitable markers before large-scale budget allows, otherwise only small
screening of genetic variation. number of individuals are sequenced,
Although there is no exact rule of then the results can be used to develop
thumb as to how many individuals or new primers for SSCP or restriction
populations that should be used in the enzyme for RFLP.
pilot study, it is recommended that
about 10-20 individuals of at least two There are no clear answers of how
populations (originally from different many loci should be screened. The
geographic areas) be used to screen for rule of thumb is that more the better.
polymorphisms. A reasonable sample However, more often than not that
size of 10-20 individuals for each research budgets are limited and
population will increase the possibility therefore number of loci employed
of detecting intra-population variation, also limited. Again, one should consult
while small sample sizes (2-5 individuals colleagues and literature on relevant
per population) may be enough to species to understand which loci are
provide an idea on inter-population likely to be variable and the level of
variation, depending on the variability that variation. This and the question
of the markers. being asked will help determine an
approximate number of loci. For
It is recommended that one should example, the question, “Is there
consult colleagues and the relevant population sub-structuring in this
literature before conducting a pilot sample?” may require analyses of many
study. There may be some established loci, whereas the question, “Is there
markers and methods that are available more than one species in this sample?”
which should be adopted rather than would require analysis of only one or a
repeating the same development few diagnostic loci.
process, when lots of loci need to be
screened, and much more resources are 4.4. How many samples?
needed.
The discussion above clearly demon-
Choice of markers depends on several strates the need for maximising
factors. The first factor is the status numbers to gain credible results. A
of species under study. For example, perfect experimental design would
allozyme electrophoresis may not require that every individual was
be suitable for studying species that sampled and that every bit of DNA
are rare or endangered if destructive in each individual was sequenced.
sampling is required, although many Although this would totally eliminate
107
error (at least that generated from the general rule of thumb for required
experimental design), it is clearly not sample size where α = 0.05 and β = 0.50
possible. Constraints (financial, time, (i.e. a power of 0.5) is
resource, logistical) exist that prevent a
perfect design. Therefore, it is desirable 2n = 1/FST
to design a study that fits within these
constraints but still provides statistically so that to detect an FST value of 0.01
powerful results. you would require only 50 individuals
(Slatkin and Barton 1989). Once again
Unfortunately, there are few absolute this is a posthoc test and is not really
benchmarks with which to decide on usable in the design stage (unless
sample size. The number of sample sites you can establish the desired level of
required is reflected by the question differentiation prior to sampling) but
that you are asking. Ultimately, we rather a test to determine whether
assume that our spatial sampling is sampling was adequately undertaken.
representative of the entire system
under investigation and that the scale The number of diploid individuals in
is fine enough to detect the effects of each of two samples required to detect
population processes. a given difference in allele frequency
(Δp) given the actual frequencies of
The number of individuals sampled per the allele in the population (p) and the
site should be representative of the power desired (1-β).
greater population or deme from which
the sample is taken. That is, the allelic When using mtDNA a good rule of
frequencies in the sample should be the thumb to use is a sample size of 30
allelic frequencies in the real popula- individuals. It has been shown that a
tion. There are specific calculations sample size of 30 will provide a 95%
to determine relevant samples sizes chance of detecting all haplotypes that
needed to obtain a certain statistical exist in the population at a frequency
power (see table opposite) but this of 0.10 or more.
is dependent on the relative allelic
frequencies in the population. But it is The current philosophy of achieving
clear that high power to detect small high statistical power in population
differences in allele frequency requires genetic studies is through increasing
large sample sizes. As allele frequencies the number of loci assayed rather than
in the study populations are generally increasing the number of individuals
unknown prior to the sampling, these per sampled population. However a
values cannot be implemented in balance is required here as lab work
the experimental design. Therefore, tends to be more costly and labour
maximising the number of individuals intensive. It should be noted that
within constraints is advisable. A mtDNA is a single circular molecule
with no recombination (i.e. a single
locus), so that increasing the number
108
p
Power Δp 0.55 0.70 0.80 0.90 0.95
50% 0.05 760 645 492 276 146
0.10 190 162 123 69 50
0.20 48 40 31 25 50
0.50 6 9 13 25 50
109
variation. Continue in this way until the that there is no significance, is there
whole catchment is sampled (see Figure any point in continuing on to smaller
15 below). This design can further spatial scales? It is strongly suggested
be expanded to investigate genetic that if this is the case however, that a
differentiation among river drainages. reduced sample from finer spatial scales
The same design in each drainage is assayed. This is because factors other
would be ideal, but if we already have than population processes (i.e. gene
an estimate of finer scale structuring flow and drift) may have influenced
from one drainage, then only a few population structure (e.g. historical
samples spread around the second drainage rearrangement).
drainage are necessary to estimate
the ‘among drainage’ component of
genetic differentiation. 4.6. Temporal sampling
Figure 15. A stylised depiction of a For specific questions, sampling
hierarchical sampling scheme. schemes may be required to be more
rigorous over a temporal scale rather
than a spatial scale. For example, the
4 question may be ‘is the exploitation of
a fishery reducing genetic variation?’
In this case we are interested in the
partitioning of genetic variation among
sampling times rather than among
2°
sites. In these instances, it is important
to maintain the same experimental
design among sampling times (i.e.
sample size, genetic markers used,
1°
statistical analyses employed) to be able
to make valid conclusions concerning
temporal fluctuations in genetic
variation.
110
4.7. Which DNA Marker? 4.8. Field trip planning,
tissue collection and storage
Intuitively we may expect that the
more alleles (haplotypes) present for Preparation for collecting tissues in
a particular marker, the greater the the field is a very important step.
statistical power to detect genetic Often field trips are expensive and
differentiation among populations. labour intensive, and as such careful
However, this is not always the case. planning would help to reduce cost,
Remember that FST analysis partitions time and energy. Field trip preparation
genetic variation within and among should take into account characteristics
populations. The more variation of species to be collected, and the
partitioned within populations, the less environment where they live. Required
there is remaining to be partitioned equipments and facilities such as boat,
among populations (i.e. reducing nets, waders, dissecting kits, liquid
FST). So a highly variable marker may nitrogen containers etc. should be
indicate panmixia when in fact gene well planned for. Also, if there is more
flow is highly restricted (Type II error). than one on going project at a time,
consider the possibility of combining
There are no right or wrong markers the sampling for more than one species
to use in population studies, but some in one trip to reduce travel cost.
general rules can be applied. The level
of genetic variation within a popula- It is also important to plan for storage
tion is a function of both population packages. Field trips often conducted
size (large population - low effect of in the remote areas where a market
genetic drift) and the mutation rate is not available to purchase plastic
(generating variability). So for large bags, aluminum foil or tubes. If it is
populations, it would be wise to choose planned to collect whole specimens of
a more slowly evolving marker (e.g. fish, plastic bags may be convenient.
an rRNA gene). Conversely, in small This is mostly related to protein-based
populations the fast evolving control techniques in which tissues from
region may be more appropriate. If we different organs are required. In case
are unsure of the possible size of the of DNA-related work, small amount of
population, then a protein coding gene tissue is sufficient and therefore small
with a moderate mutation rate would vials and good sealing cap will be fine.
be a good place to start. Although a Sample labeling is extremely important,
certain degree of trial and error will as samples will become useless without
be required, many studies already exist correct labels. It is advisable to use
that may provide insight into the best waterproof and non-smearing ink
marker to use. permanent marker pens to label the
samples. Also, try to test the stability
of ink in different environment such
as freezing in ultracold freezers, liquid
nitrogen etc. before taking to the field.
111
Care should be taken when dissecting 70% or higher concentration at room
tissues from animals. It may be good to temperature. Ethanol may evaporate
apply anesthetic drugs to immobilize overtime, and therefore care should
the animal before dissection. It is also be taken to ensure there is sufficient
important to note that in some coun- ethanol to cover the tissue. Fish scales
tries, a permit is needed before the can be kept dry at room temperature;
field sampling. Handling animal should this is perhaps most convenient in case
follow international and national samples are requested from elsewhere,
regulations. Destructive sampling where ethanol may be a constraint as
should be avoided as much as possible. most courier companies do not accept
consignments with ethanol.
We may repeat here that for projects
involving allozyme electrophoresis,
whole specimens may be needed to
obtain tissues from different organs
such as liver, muscle, eyes, heart are
required. However, for DNA-related
work then only small amounts of tissue
are required, for fish for example, a
small piece (one square centimeter) of
finclip or a few scales should be suffi-
cient, or for crustacean species, a leg
may be enough. In the latter case, the
animal can be released back into their
original place (rivers, ponds, tanks). It
is also noted that an extra amount of
tissue may be useful, as some projects
may require more than one marker and
therefore more tissue will be required.
112
Conclusion
113
114
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Annex 1: List of commonly used buffers in
allozyme electrophoresis
* See Annex 2.
** The current for discountinuous buffers will drop during the run; the voltage may
be increased to compensate.
119
120
Annex 2: Stock solutions of buffers used in
allozyme electrophoresis
TC6 TC8
Per 1 litre final volume: Per 1 litre final volume:
• 66.6g Tris (Sigma 7-9) • 104g Tris
• 39.5g Citric acid (monohydrate) • 39.5g Citric acid
• pH6; Store in refrigerator • pH8
• Store in refrigerator
TEB
Per 1 litre final volume: TM
• 109g Tris Per 1 litre final volume:
• 30.9g Boric acid • 60.5g Tris
• 7.4g Disodium EDTA • 58g Maleic acid
• Store at room temperature • 18.6g EDTA
• 10g MgCl2
TG • 25g NaOH
• 30g Tris • Adjust to pH7.4 with NaOH Store
• 144g Glycine at room temperature
• Make up to 1 litre.
• Store at room temperature LiOH electrode buffer
Per 1 litre final volume:
LiOH gel buffer • 6.3g LiOH*
Per 1 litre final volume: • 59.4g Boric acid
• 27.2g Tris • Store at room temperature
• 7.5g Citric acid
• 200 ml Electrode stock buffer *Carefully - the dust is irritating to
• Store in refrigerator breath
121
122
Annex 3: Staining recipes for common enzymes
Each of the assay recipes which follow Add 25 ml of the melted agar, mix and
are in amounts suitable for staining pour onto gel slices.
two gels; the width of the gel is about
10mm, and 16mm long (i.e. 2 x 8cm).
Standard procedures for all assays are Standard recipe amounts
given below. • MTT: 4 mg
• PMS: About 0.2 mg - tiny amount
on tip of spatula.
Assay buffers: • Add PMS last, just before agar.
All assays with PMS must be
Tris-HCl pH 8 incubated in the dark.
• 70 ml 1M Tris • NAD: 3 mg
• 30 ml 1M HCl • NADP: 2.5 mg
• make to 1000 ml with H2O • G6PD: 10 µl of a 1000 units/ml
solution.
Phosphate pH 7 • Add G6PD just before adding
• 100 ml 1M Na2HPO4 (14.2 g) PMS.
• 98 ml 1M NaH2PO4 (14.3 g)
• make to 1000 ml with H2O Acid phosphatase (Acph)
• α-napthyl acid phosphate 50 mg
• 0.1 M Na acetate buffer pH5 100
Agar overlay recipes ml
• Incubate gel in substrate solution
Heat 2% agar suspension in water in a 30 minutes at 37˚ C then add 25
flask in beaker of boiling water until mg
agar solution is clear. This solution can • Fast black K in 20 ml H2O.
be prepared in advance, and kept in a
stoppered flask in the 60˚C oven. Adenosine deaminase (Ada)
• Adenosine 30 mg
Mix contents of enzyme assay recipe in • MTT
25 ml buffer (including any substrate • Xanthine oxidase 1 unit
solutions).
123
• Nucleoside phosphorylase 5 units Alkaline phosphatase (Alph)
add just before PMS • β-napthyl acid phosphate 50 mg
• PMS • TEB electrode buffer 100 ml
• Phosphate buffer 25 ml • Incubate gel in substrate solution
• Agar overlay 30 minutes at 37˚C, then add 25
mg fast black K in 20 ml H2O.
Adenylate kinase (Ak)
• ADP 10 mg ARGININE PHOSPHOKINASE (Apk)
• Glucose 20 mg • Phosphoarginine 10 mg
• MTT • ADP 10 mg
• PMS • Glucose 100 mg
• NAD • MTT
• MgCl2 • PMS
• G6PD • NAD
• Hexokinase 10 mg • MgCl2
• Tris-HCl buffer 25 ml • G6PD
• Agar overlay • Hexokinase 10 mg
• Tris-HCl buffer 25 ml
Alcohol dehydrogenase (Adh) • Agar overlay
• Ethanol 5 ml
• MTT Catalase (Cat)
• PMS ▪ Solution A:
• NAD • H2O2 1 ml of 28% solution
• Tris-HCl buffer 20 ml • Phosphate buffer 15 ml
• Agar overlay • H2O 100 ml
124
Creatine kinsase (Ck) • Na2HPO4 14.2 g
• Phosphocreatine 50 mg (try more • H2O to 500 ml (should be pH 7.4)
if that doesn’t work) • H2O 25 ml
• ADP 10 mg • Incubate gells in substrate
• Glucose 20 mg solution for 30 minutes at 37˚C,
• MTT then add 50 mg fast blue BB in
• PMS 20 ml H2O.
• NAD
• MgCl2 Glutamate dehydrogenase (Gdh)
• G6PD • 0.1 M sodium glutamate 10 ml
• Hexokinase 10 mg • MTT
• Tris-HCl buffer 25 ml • PMS
• Agar Overlay • NAD (use NADP in some species)
• Tris-HCl buffer 15 ml
Esterase (Est) • Agar overlay
• Substrate solution 2 ml (substrate
solution contains : β-napthyl α-glycerophosphate
acetate 1 g & acetone 100 ml) dehydrogenase (α-Gpd)
• Phosphate buffer 100 ml • 0.1 M α-glycerophosphate 10 ml
• Incubate gels in substrate • MTT
solution for 20 minutes at 37˚C, • PMS
then add 50 mg fast blue BB • NAD
in 20 ml H2O. If enzyme is very • Phosphate buffer 15 ml
active, add the dye directly to the • Agar overlay
substrate solution.
Guanine deaminase (Gda)
Glucose-6-phosphate • Guanine 30 mg
dehydrogenase (G6pd) • MTT
• Glucose-6-phosphate 40 mg • PMS
• MTT • Xanthine oxidase 20 µl (1 unit)
• PMS • Nucleoside phosphorylase 10 µl
• NADP (5 min.)
• MgCl • Phosphate buffer 25 ml
• Tris-HCl buffer 25 ml • Agar overlay
• Agar Overlay
Hexokinase (Hk)
Glutamate-oxaloacetate • Glucose 2 g
transaminase (Got or AAT) • ATP 25 mg
• MTT
▪ Substrate solution 25 ml: • PMS
• α-ketoglutaric acid 365 mg • NAD
• L-aspartic acid 1331 mg • MgCl2
• EDTA 0.5 g • G6PD
125
• Tris-HCl buffer 25 ml Malate dehydrogenase (Mdh)
• Agar overlay
▪ Substrate solution 10 ml:
Isocitrate dehydrogenase (Idh) • L-malic acid 13.4 g
• Na3 isocitrate 40 mg • 2 M Na2CO3 49 ml
• MTT • Water to 100 ml
• PMS • Adjust to pH 7 with Na2CO3
• NADP • MTT
• MgCl • PMS
• Tris-HCl buffer 25 ml • NAD
• Agar overlay • Tris-HCl buffer 15 ml
• Agar overlay
Lactate dehydrogenase (Ldh)
▪ Substrate solution 10 ml (5 ml for Malic enzyme (Me)
vertebrates): • As for Mdh, but substitute NADP
• DL lactic acid (85% solu.) 10.6 ml for NAD
• 1 M Na2CO3/water 49 ml
• Water to 100 ml Mannose-6-phosphate isomerase
• Adjust to pH 7 with 0.5 M (Mpi)
Na2CO3 • Mannose-6-Phosphate 20 mg
• MTT • MTT
• PMS • PMS
• NAD • NAD
• Tris-HCl buffer 15 ml (20 ml for • G6PD
vertebrates) • Phosphoglucose isomerase 20 µl
• Agar overlay (20 units) add just before PMS
• Tris-HCl buffer 25 ml
Leucine aminopeptidase (Lap) • Agar overlay
• L-leucyl-β-napthylamide 40 mg
• dissolved in 1 ml dimethyl Nucleoside phosphorylase (Np)
formamide (or 1 or 2 drops of • Inosine 30 mg
acetone) • MTT
• Phosphate buffer 100 ml • PMS
• Incubate gel in substrate solution • Xanthine oxidase 20 µl (1 unit)
30 minutes at 37˚C, then add 25 add just before PMS
mg fast black K in 20 ml H2O • Phosphate buffer 25 ml
• Agar overlay
126
Peptidase (Pep) • Tris-HCl buffer 25 ml
▪ Peptide 20 mg. Peptides used: • Agar overlay
• L-leucyl-glycylglycine (LGG)
• L-leucyl-proline (LP) Sorbitol dehydrogenase (Sdh)
• L-leucyl-l-tyrosine (LT) • Sorbitol 200 mg (or more if
needed up to 1 gm)
▪ Dissolve LT and o-Dianisidine in • MTT
• 2 drops of 0.1 M HCl • PMS
• o-dianisidine 5 mg • NAD
• L-amino acid oxidase 5 mg • Tris-HCl buffer 25 ml
• Horseradish peroxidase 5 mg • Agar overlay
• Phosphate buffer 25 ml
• Agar overlay Superoxide dismutase (Sod)
• MTT
Phosphoglucomutase (Pgm) • PMS
• Glucose-1-phosphate 60 mg (try • Tris-HCl buffer 25 ml
more if needed) • Agar overlay
• MTT
• PMS
• NAD
• MgCl2
• G6PD
• Tris-HCl buffer 25 ml
• Agar overlay
6-phosphogluconate
dehydrogenase (6pgd)
• Na3 6-phosphogluconic acid 20
mg
• NADP
• MTT
• PMS
• Tris-HCl buffer 25 ml
• Agar overlay
127
128
Annex 4: Practical exercises
There is a fish species that have been 4. Fifty individuals from each of the
used for aquaculture in the last 20 years three populations of fish were
or so. A national broodstock center sampled and scored for five enzyme
was established and the first batch of loci as represented below. The three
broodstock were domesticated from populations sampled were from
the wild population of a river nearby. (1) the national broodstock centre,
The center artificially breeds the fish (2) the river nearly, and (3) a farm
and distributes fingerlings to farmers which had originally obtained only a
for grow-out. Some farmers have also small number of brood fish from the
successfully bred the fish, recruiting national broodstock centre 20 years
broodstock from what they breed and ago. How do you use the data to
recently observe slow growth, loss of answer the above questions?
productivity as well as high level of
deformity. The environmental authority
also claims there is evidence that
farmed escapees have interbreed with
wild fish.
129
Population 1: River.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
B
Enzymes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
B
Enzymes
130
Population 3: National Broodstock Centre.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
B
Enzymes
Population 1: River.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
A1
A2
Primer
A3
A4
131
Population 2: Farmer’s boodstock.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
A1
A2
Primer
A3
A4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
A1
A2
Primer
A3
A4
132
Haplotypic markers Ava II annd Hinf I. After digestion,
the restriction digested products were
The same individuals of fish above run on agarose gels and stained with
were used for RFLP analysis. The D-loop ethidium bromide. The gel images were
region of the mtDNA was amplified taken as below. Do these data show
for all 150 individuals. PCR products similar results found using allozymes
were subjected to digestion using 3 and RAPDs?
restriction enzymes, namely EcoR I,
Population 1: River.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Eco RI
Restriction
Ava II
Hinf I
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Eco RI
Restriction
Ava II
Hinf I
133
Population 3: National broodstock center.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Eco RI
Restriction
Ava II
Hinf I
134
Manual on Application
of Molecular Tools in
Aquaculture and Inland
Fisheries Management
www.enaca.org