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STATE OF THE A R T I N THE MA N AGEMEN T OF A N I MAL GENE T I C RESOU RC ES
Section C
Molecular markers – a tool for

exploring genetic diversity
1 Introduction
DNA markers are useful in both basic (e.g. selection, paternity testing and food traceability).
phylogenetic analysis and search for useful This section focuses mainly on their application
genes) and applied research (e.g. marker assisted in characterization of AnGR diversity, and in the
Box 70
DNA, RNA and protein
DNA (deoxyribonucleic acid) is organized in pairs of polypeptide (an entire protein or one of the chains
chromosomes, each inherited from one of the parents. of a protein complex). The mRNA molecule is read
Each gene in an individual, therefore, has two copies, or translated three nucleotides (a codon) at a time.
called alleles, one on each chromosome of a pair. In Complementarity between the mRNA codon and the
mammals, genes are scattered along chromosomes, anti-codon of a transfer RNA (tRNA) molecule which
separated by long, mainly repetitive, DNA sequences. carries the corresponding amino acid to the ribosome
Genes are formed by coding sequences (exons) ensures that the newly formed polypeptide contains
separated by introns. The latter carry no protein- the specific sequence of amino acids required.
coding information, but sometimes play a role in the Not all genes are translated into proteins; some
regulation of gene expression. The instruction encoded express their function as RNA molecules (such as the
by genes is put into action through two processes. rRNA and tRNA involved in translation). Recently,
The first is transcription (copy) of genetic information new roles of RNA in the process of mRNA editing
into another type of nucleic acid, RNA (ribonucleic and in the regulation of gene expression have been
acid). Both exons and introns are transcribed into discovered (Storz et al., 2005; Aravin and Tuschl, 2005;
a primary messenger RNA (mRNA) molecule. This Wienholds and Plasterk 2005). Indeed, non-coding
molecule is then edited, a process which involves RNAs appear to be key players in various regulatory
removing the introns, joining the exons together, and processes (Bertone et al., 2004; Clop et al., 2006).
adding unique features to each end of the mRNA. A Thus, three types of molecules are available for
mature mRNA molecule is, thereby, created, which is investigating genetic characteristics at cellular, tissue
then transported to structures known as ribosomes and whole organism levels: the DNA which contains
located in the cell cytoplasm. Ribosomes are made of the encoded instruction; the RNA which transfers the
ribosomal RNA (rRNA) and proteins, and provide sites instructions to the cell “factory”; and the proteins
for the second process – translation of the genetic which are built according to the instructions, and
information, previously copied to the mRNA, into a make functioning cells and organisms.
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THE STATE OF THE WO RLD' S AN IMAL GENE T I C RESOURCES FOR FOOD AND AG RICULTURE
PART 4
search for functional variants of relevant genes. expressing proteins in specific tissues at different
It is important to note that RNA and proteins development or physiological stages.
also contain key information, and therefore Although analysis of single types of biomolecules
deserve parallel study; their role in the search for has proven extremely useful in understanding
functional variants is also explored below. biological phenomena, the parallel large-scale
Diversity among organisms is a result of investigation of DNA, RNA and proteins opens
variations in DNA sequences and of environmental up new perspectives in the interpretation and
effects. Genetic variation is substantial, and modelling of the complexity of living organisms.
each individual of a species, with the exception New scientific disciplines with the suffix “–omics”
of monozygotic twins, possesses a unique DNA are coming into existence. In these fields, recent
sequence. DNA variations are mutations resulting advances in the preparation, identification and
from substitution of single nucleotides (single sequencing of DNA, RNA and proteins, and in
nucleotide polymorphisms – SNPs), insertion or large-scale data storage and analysis, are bringing
deletion of DNA fragments of various lengths about a revolution in our understanding. A global,
(from a single to several thousand nucleotides), integrated view of an entire set of biological
or duplication or inversion of DNA fragments. molecules involved in complex biological processes
DNA variations are classified as “neutral” when
they cause no change in metabolic or phenotypic Box 72
traits, and hence are not subjected to positive, Recent developments in molecular
negative, or balancing selection; otherwise, they biology
are referred to as “functional”. Mutations in key
nucleotides of a coding sequence may change the Current revolutionary developments in molecular
amino acid composition of a protein, and lead to biological research relevant to livestock breeding
new functional variants. Such variants may have and genetic diversity conservation include:
an increased or decreased metabolic efficiency 1. establishment of the entire genome sequence
compared to the original “wild type”, may lose of the most important livestock species;
their functionality completely, or even gain a 2. development of technology to measure
novel function. Mutations in regulatory regions polymorphisms at loci spread all over the genome
may affect levels and patterns of gene expression; (e.g. methods to detect SNPs); and
for example, turning genes on/off or under/over- 3. development of microarray technology to
measure gene transcription at a large scale.
Information obtained through the sequencing
Box 71 of the entire genome (achieved for chickens and
The new “–omics” scientific disciplines almost complete for pigs and cattle), integrated
with SNP technology, will speed up the search
Genomics charts genes and the genetic variations for genes. Quantitative trait loci (QTL) mapping
among individuals and groups. It provides an to identify chromosome regions influencing a
insight into the translation of genetic information target trait, the presence of candidate genes
to metabolic functions and phenotypic traits. It located in the same region, and investigation of
unveils biological processes and their interactions their patterns of expression (e.g. by microarray
with environmental factors. Genomics involves and proteomic analyses) and their function across
the combination of a set of high-throughput species, will come together to identify key genes
technologies, such as proteomics and metabolomics, and to unravel the complexity of physiological
with the bioinformatic techniques that enable regulation for target traits.
the processing, analysis and integration of large
amounts of data. See below for further discussion of these developments.
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is emerging. Structural genomics, transcriptomics of functional variations. Molecular techniques

and proteomics are followed by metabolomics, have also proved useful in the investigation
and interactomics among others, and at a still of the origin and domestication of livestock
higher level of complexity, systems biology (Hood species, and their subsequent migrations, as
et al., 2004; Box 71). well as providing information on evolutionary
Investigation of biological complexity is a new relationships (phylogenetic trees), and
frontier which requires high-throughput molecular identifying geographical areas of admixture
technology, high computer speed and memory, among populations of different genetic origins.
new approaches to data analysis, and integration Subchapter 3.1 presents an outline of molecular
of interdisciplinary expertise (Box 72). techniques for the assessment of genetic diversity
within and between breeds.
Second role. Effective population size (Ne) is
2 The roles of molecular an index that estimates the effective number
technologies in characterization of animals in a population that reproduce and
contribute genes to the next generation. Ne is
Information on genetic diversity is essential in closely linked to the level of inbreeding and
optimizing both conservation and utilization genetic drift in a population, and therefore is
strategies for AnGR. As resources for conservation a critical indicator for assessing the degree of
are limited, prioritization is often necessary. New endangerment of populations (see Sections
molecular tools hold the promise of allowing the A and F). Traditional approaches to obtaining
identification of genes involved in a number of reliable estimates of Ne for breeding populations
traits, including adaptive traits, and polymorphisms are based on pedigree data or censuses. The
causing functional genetic variation (QTN necessary data on variability of reproductive
– Quantitative Trait Nucleotides). However, we success and generation intervals are often not
do not have sufficient knowledge to prioritize reliably available for populations in developing
conservation choices on the basis of functional countries. Molecular approaches may, therefore,
molecular diversity, and alternative measures be a promising alternative (see subchapter 3.2 for
are still needed. Phenotypic characterization further details).
provides a crude estimate of the average of the Third role. A top priority in the management
functional variants of genes carried by a given of AnGR is the conservation of breeds that have
individual or population. However, the majority unique traits. Among these, the ability to live and
of phenotypes of the majority of livestock species produce in challenging conditions, and to resist
are not recorded. infectious diseases are of major importance,
First role. In the absence of reliable phenotype particularly for developing countries. Complex
and QTN data, or to complement the existing traits, such as adaptation and disease resistance,
data, the most rapid and cost-effective measures are not visible or easily measurable. They can be
of genetic diversity are obtained from the assay investigated in experiments in which the animals
of polymorphisms using anonymous molecular are submitted to the specific environmental
genetic markers. Anonymous markers are likely conditions or are infected with the relevant
to provide indirect information on functional agent. However, such experiments are difficult
genes for important traits, assuming that and expensive to perform, and raise concerns
unique populations that have had a particular about animal welfare. This is the reason why
evolutionary history at the neutral markers (e.g. researchers are extremely interested in identifying
because of ancient isolation or independent genes controlling complex traits. Such genes can
domestication) are likely to carry unique variants be sought by a number of different approaches.
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PART 4
Tools being developed to target functional Box 73

variation are described in subchapter 3.3. Extraction and multiplication of DNA
and RNA
3 Overview of molecular The first step in DNA, RNA and protein analysis is
techniques extraction and purification from biological specimens.
Several protocols and commercial kits are available.
This section describes the most important The strategies applied depend on the source material
molecular techniques currently being utilized and and the target molecule. For example, DNA extraction
developed for the assessment of genetic diversity, from whole blood or white cells is relatively easy,
and for targeting functional variation. Box 73 while its extraction from processed food is rather
describes how DNA and RNA are extracted from difficult. RNA extraction from pancreatic tissue
biological material and prepared for analysis. The is difficult because of very rapid post-mortem
attributes of commonly used molecular markers degradation in this organ. Purity of DNA, RNA and
are outlined in Box 74, and sampling (a very proteins is often a key neglected factor in obtaining
important aspect of molecular studies) is discussed reliable results.
in Box 75. After isolating DNA (or RNA) from cells, the next
Protein polymorphisms were the first markers step is to obtain thousands or millions of copies of
used for genetic studies in livestock. However, the a particular gene or piece of DNA. DNA fragment
number of polymorphic loci that can be assayed, multiplication can be delegated to micro-organisms,
and the level of polymorphisms observed at typically E. coli, or accomplished in vitro using a
the loci are often low, which greatly limits their polymerase chain reaction (PCR). This technique,
application in genetic diversity studies. With which won the Nobel Prize for its inventor, Cary
the development of new technologies, DNA Mullis, exponentially amplifies any DNA segment
polymorphisms have become the markers of of known sequence. The key component in a PCR
choice for molecular-based surveys of genetic reaction is the DNA polymerase isolated from
variation (Box 74). Thermus aquaticus, a micro-organism adapted
to live and multiply at very high temperature.
3.1 Techniques using DNA markers to This thermostable Taq- (after Thermus aquaticus)
assess genetic diversity polymerase permits chain replication in cycles
and produces a geometric growth in the number
Nuclear DNA markers of copies of the target DNA. A PCR cycle includes
A number of markers are now available to detect three steps: i) DNA denaturation at 90–95 oC to
polymorphisms in nuclear DNA. In genetic diversity separate the DNA into two single strands to serve
studies, the most frequently used markers are as a template; ii) annealing of a pair of short single-
microsatellites. strand oligonucleotides (primers) complementary
to the target regions flanking the fragment of
Microsatellites interest, at 45–65 oC; iii) extension or elongation of
Currently, microsatellites (Box 74) are the newly synthesized DNA strands led by primers and
most popular markers in livestock genetic facilitated by the Taq-polymerase, at 72 oC. This cycle
characterization studies (Sunnucks, 2001). Their can be repeated, normally 25 to 45 times, to enable
high mutation rate and codominant nature amplification of enough amplicons (a fragment of a
permit the estimation of within and between- gene or DNA synthesized using PCR) to be detected.
breed genetic diversity, and genetic admixture
among breeds even if they are closely related.
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Box 74
Commonly used DNA markers
Restriction fragment length polymorphisms (RFLPs) recommendations for sets of microsatellite loci to be
are identified using restriction enzymes that cleave used for diversity studies for major livestock species,
the DNA only at precise “restriction sites” (e.g. which were developed by the ISAG–FAO Advisory
EcoRI cleaves at the site defined by the palindrome Group on Animal Genetic Diversity (see DAD-IS library
sequence GAATTC). At present, the most frequent use http://www.fao.org/dad-is/).
of RFLPs is downstream of PCR (PCR–RFLP), to detect Minisatellites share the same characteristics as
alleles that differ in sequence at a given restriction microsatellites, but the repeats are ten to a few
site. A gene fragment is first amplified using PCR, and hundreds bp long. Micro and minisatellites are
then exposed to a specific restriction enzyme that also known as VNTRs (Variable Number of Tandem
cleaves only one of the allelic forms. The digested Repeats) polymorphisms.
amplicons are generally resolved by electrophoresis. Amplified fragment length polymorphisms
Microsatellites or SSR (Simple Sequence Repeats) (AFLPs) are a DNA fingerprinting technique which
or STR (Simple Tandem Repeats) consist of a stretch detects DNA restriction fragments by means of PCR
of DNA a few nucleotides long – 2 to 6 base pairs amplification.
(bp) – repeated several times in tandem (e.g. STS (Sequence Tagged Site) are DNA sequences
CACACACACACACACA). They are spread over a that occur only once in a genome, in a known
eukaryote genome. Microsatellites are of relatively position. They needn’t be polymorphic and are used to
small size, and can, therefore, be easily amplified build physical maps.
using PCR from DNA extracted from a variety of SNPs are variations at single nucleotides which do
sources including blood, hair, skin or even faeces. not change the overall length of the DNA sequence in
Polymorphisms can be visualized on a sequencing the region. SNPs occur throughout the genome. They
gel, and the availability of automatic DNA sequencers are highly abundant and are present at one SNP in
allows high-throughput analysis of a large number of every 1000 bp in the human genome (Sachinandam
samples (Goldstein and Schlötterer, 1999; Jarne and et al., 2001). Most SNPs are located in non-coding
Lagoda, 1996). Microsatellites are hypervariable; they regions, and have no direct impact on the phenotype
often show tens of alleles at a locus that differ from of an individual. However, some introduce mutations
each other in the numbers of the repeats. They are in expressed sequences or regions influencing gene
still the markers of choice for diversity studies as well expression (promoters, enhancers), and may induce
as for parentage analysis and Quantitative Trait Loci changes in protein structure or regulation. These
(QTL) mapping, although this might be challenged SNPs have the potential to detect functional genetic
in the near future with the development of cheap variation.
methods for the assay of SNPs. FAO has published
Some controversy has surrounded the choice The mean number of alleles (MNA) per
of a mutation model – infinite allele or step- population, and observed and expected
wise mutation model (Goldstein et al, 1995) – for heterozygosity (Ho and He), are the most common
microsatellite data analysis. However, simulation parameters for assessing within-breed diversity.
studies have shown that the infinite allele mutation The simplest parameters for assessing diversity
model is generally valid for assessment of within- among breeds are the genetic differentiation
species diversity (Takezaki and Nei, 1996). or fixation indices. Several estimators have been
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Box 75 be calculated for the FST values between pairs

Sampling genetic material of populations (Weir and Cockerham, 1984)
to test the null hypothesis of a lack of genetic
Sample collection is the first and the most important differentiation between populations and,
step in any diversity study. Ideally, samples should therefore, the partitioning of genetic diversity
be unrelated and representative of the populations (e.g. Mburu et al., 2003). Hierarchical analysis
under investigation. Generally, the sampling of of molecular variance (AMOVA) (Excoffier
30 to 50 well-chosen individuals per breed is et al., 1992) can be performed to assess the
considered sufficient to provide a first clue as to distribution of diversity within and among groups
breed distinctiveness and within-breed diversity, if a of breeds.
sufficient number of independent markers is assayed Microsatellite data are also commonly used to
(e.g. 20–30 microsatellites; Nei and Roychoudhury, assess genetic relationships between populations
1974; Nei, 1978). However, the actual numbers and individuals through the estimation of genetic
required may vary from case to case, and may be distances (e.g. Beja-Pereira et al., 2003; Ibeagha-
even lower in the case of a highly inbred local Awemu et al., 2004; Joshi et al., 2004; Sodhi et
population, and higher in a widely spread population al., 2005; Tapio et al., 2005). The most commonly
divided into different ecotypes. used measure of genetic distances is Nei’s standard
The choice of unrelated samples is quite genetic distance (DS) (Nei, 1972). However, for
straightforward in a well-defined breed, where it closely related populations, where genetic drift
can be based on the herd book or pedigree record. is the main factor of genetic differentiation, as
Conversely, it can be rather difficult in a semi-feral is often the case in livestock breeds, particularly
population for which no written record is available. in the developing world, the modified Cavalli-
In this case, the use of a geographic criterion is highly Sforza distance (DA) is recommended (Nei et
recommendable, i.e. to collect a single or very few al., 1983). Genetic relationship between breeds
(unrelated) animals per flock from a number of flocks is often visualized through the reconstruction of
spread over a wide geographic area. The record of a phylogeny, most often using the neighbour-
geographical coordinates, and photo-documentation joining (N-J) method (Saitou and Nei, 1987).
of sampling sites, animals and flocks is extremely However, a major drawback of phylogenetic tree
valuable – to check for cross-breeding in the case reconstruction is that the evolution of lineages
of unexpected outliers, or for identifying interesting is assumed to be non-reticulate, i.e. lineages can
geographic patterns of genetic diversity. A well-chosen diverge, but can never result from crosses between
set of samples is a long-lasting valuable resource, lineages. This assumption will rarely hold for
which can be used to produce meaningful results even livestock, where new breeds often originate from
with poor technology. Conversely, a biased sample cross-breeding between two or more ancestral
will produce results that are distorted or difficult to breeds. The visualization of the evolution of
understand even if the most advanced molecular tools breeds provided by phylogenetic reconstruction
are applied. must, therefore, be interpreted cautiously.
Multivariate analysis, and more recently
Bayesian clustering approaches, have been
proposed (e.g. FST and GST), the most widely suggested for admixture analysis of microsatellite
used being FST (Weir and Basten, 1990), which data from different populations (Pritchard et
measure the degree of genetic differentiation al., 2000). Probably the most comprehensive study
of subpopulations through calculation of the of this type in livestock is a continent-wide study
standardized variances in allele frequencies of African cattle (Hanotte et al., 2002), which
among populations. Statistical significance can reveals the genetic signatures of the origins,
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secondary movements, and differentiation of (2006a) investigated, respectively, chicken and

African cattle pastoralism. pig diversity throughout Europe; Hanotte et al.
Molecular genetic data, in conjunction with, (2002) obtained data on cattle at the scale of
and complemented by, other sources such as almost the entire African continent; Tapio et al.
archaeological evidence and written records, (2005) assessed sheep diversity at a large regional
provide useful information on the origins and scale in northern European countries; and Cañon
subsequent movements and developments of et al. (2006) studied goat diversity in Europe and
genetic diversity in livestock species. Mapping the Near and Middle East. However, for most
the origin of current genetic diversity potentially species, a comprehensive review is still lacking.
allows inferences to be made about where Ongoing close coordination between large-scale
functional genetic variation might be found projects promises the delivery of a global estimate
within a species for which only limited data on of genetic diversity in the near future for some
phenotypic variation exist. species such as sheep and goats. In the meantime,
Combined analysis of microsatellite data new methods of data analysis are being developed
obtained in separate studies is highly desirable, to permit the meta-analysis of datasets that have
but has rarely been possible. This is because most only a few breeds and no, or only a few, markers
population genetic studies using DNA markers in common (Freeman et al., 2006). This global
are limited to small numbers of breeds, often perspective on livestock diversity will be extremely
from a single country (Baumung et al., 2004). valuable to reconstruct the origin and history of
Often, different subsets of the FAO-recommended domestic animal populations and, indirectly, of
markers are used, and no standard samples are human populations. It will also highlight regional
genotyped across projects. The application of and local hotspots of genetic diversity which may
different microsatellite genotyping systems causes be targeted by conservation efforts.
variation between studies in the estimated size
of alleles at the same loci. To promote the use SNPs
of common markers, FAO is now proposing an SNPs (Box 74) are used as an alternative to
updated, ranked list of microsatellite loci for the microsatellites in genetic diversity studies. Several
major livestock species3. FAO recommends the technologies are available to detect and type
use of the markers in the order of ranking, to SNP markers (see Syvänen, 2001, for a review).
maximize the number of markers overlapping Being biallelic markers, SNPs have rather low
among independent investigations. For some information content, and larger numbers have to
species, DNA from standard animals is available. be used to reach the level of information obtained
For example, aliquots of sheep and goat standard from a standard panel of 30 microsatellite loci.
DNA used in the European Union (EU) Econogene However, ever-evolving molecular technologies
project have been distributed to other large-scale are increasing automation and decreasing the
projects in Asia and Africa, and can be requested cost of SNP typing. This is likely, in the near
through the Econogene Website (http://www. future, to permit the parallel analysis of a large
econogene.eu). number of markers at a lower cost. With this
There are only a few examples of large-scale perspective, large-scale projects are ongoing in
analyses of the genetic diversity of livestock several livestock species to identify millions (e.g.
species. Hillel et al. (2003) and SanCristobal et al. Wong et al., 2004) and validate several thousands
of SNPs, and identify haplotype blocks in the
3
Lists and guidelines can be found in the DAD-IS library at genome. Like sequence information, SNPs permit
http://www.fao.org/dad-is.
a direct comparison and joint analysis of different
experiments.
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PART 4
SNPs seem to be appealing markers to apply in Mitochondrial DNA markers

the future for genetic diversity studies because they Mitochondrial DNA (mtDNA) polymorphisms
can easily be used in assessing either functional or have been extensively used in phylogenetic and
neutral variation. However, the preliminary phase genetic diversity analyses. The haploid mtDNA,
of SNP discovery or SNP selection from databases carried by the mitochondria in the cell cytoplasm,
is critical. SNPs can be generated through various has a maternal mode of inheritance (individuals
experimental protocols, such as sequencing, inherit the mtDNA from their dams and not from
single-stranded conformational polymorphism their sires) and a high mutation rate; it does not
(SSCP) or denaturing high-performance liquid recombine. These characteristics enable biologists
chromatography (DHPLC), or in silico, aligning to reconstruct evolutionary relationships between
and comparing multiple sequences of the same and within species by assessing the patterns of
region from public genome and expressed mutations in mtDNA. MtDNA markers may also
sequence (EST) databases. When data have not provide a rapid way of detecting hybridization
been obtained randomly, standard estimators between livestock species or subspecies (e.g.
of population genetic parameters cannot be Nijman et al., 2003).
applied. A frequent example is when SNPs The polymorphisms in the sequence of the
initially identified in a small sample (panel) of hypervariable region of the D-loop or control
individuals are then typed in a larger sample of region of mtDNA have contributed greatly to the
chromosomes. By preferentially sampling SNPs at identification of the wild progenitors of domestic
intermediate frequencies, such a protocol will bias species, the establishment of geographic patterns
the distribution of allelic frequencies compared of genetic diversity, and the understanding of
to the expectation for a random sample. SNPs do livestock domestication (see Bruford et al., 2003,
hold promise for future application in population for a review). For example, the Middle Eastern
genetic analyses; however, statistical methods origin of modern European cattle was recently
that can explicitly take into account each method demonstrated by Troy et al. (2001). The study
of SNP discovery have to be developed (Nielsen identified four maternal lineages in Bos taurus
and Signorovitch, 2003; Clark et al., 2005). and also demonstrated the loss of bovine genetic
variability during the human Neolithic migration
AFLPs out of the Fertile Crescent. In the same way,
AFLPs are dominant biallelic markers (Vos et multiple maternal origins with three mtDNA
al., 1995). Variations at many loci can be arrayed lineages were highlighted in goats (Luikart et
simultaneously to detect single nucleotide al., 2001), with Asia and the Fertile Crescent as
variations of unknown genomic regions, in which possible centres of origin. Recently, a third mtDNA
a given mutation may be frequently present lineage was discovered in native Chinese sheep
in undetermined functional genes. However, (Guo et al., 2005), a fourth in native Chinese goats
a disadvantage is that they show a dominant (Chen et al., 2005), and a fifth in Chinese cattle
mode of inheritance; this reduces their power (Lai et al., 2006). In Asian chickens, nine different
in population genetic analyses of within-breed mtDNA clades have been found (Liu et al., 2006),
diversity and inbreeding. Nevertheless, AFLP suggesting multiple origins in South and Southeast
profiles are highly informative in assessing the Asia. All these results indicate that our current
relationship between breeds (Ajmone-Marsan knowledge of livestock domestication and genetic
et al., 2002; Negrini et al., 2006; De Marchi et diversity remains far from complete. For further
al., 2006; SanCristobal et al., 2006b) and related discussion of the origins of domestic livestock
species (Buntjer et al., 2002). species see Part 1 – Section A.
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3.2 Using markers to estimate Mapping exercises are generally accomplished

effective population size following the co-segregation of polymorphic
Hill (1981) suggested using gametic phase markers in structured experimental populations
disequilibrium of DNA polymorphisms to estimate (e.g. F2 or backcross) or existing populations
effective population size (Ne). This estimation
can be based on genotypes for linked markers Box 76
(microsatellites or SNPs). The expected correlation QTL mapping
of allele frequencies at linked loci is a function of
Ne and the recombination rate. Ne can, therefore, If a QTL for a target trait exists, the plus- and minus-
be estimated from the observed disequilibrium. variant allele of the unknown responsible gene (Q and
Hayes et al. (2003) suggested a similar approach q) will co-segregate with the alleles at a nearby M1
based on chromosome segment homozygosity, marker (M1 and m1) that we are able to genotype
which, in addition, has the potential to estimate in the laboratory. Let us hypothesize that M1 co-
Ne for earlier generations, and therefore allows segregates with Q and m1 with q, that is M1 and Q
a judgement of whether an existing population are nearby on a same chromosome and m1 and q on
was of increasing or decreasing size in the past. the homologous chromosome (M1Q and m1q).
The study demonstrated, with example data sets, Let us also assume that an F2 population derived
that the Holstein-Friesian cattle breed underwent by the mating of heterozygous F1 individuals is
a substantial reduction of Ne in the past, while genotyped. Following the genotyping, F2 progenies are
the effective population size of the human grouped on the base of their marker genotype (M1M1
population is increasing, which is in agreement and m1m1; M2M2 and m2m2; ... MnMn and mnmn),
with both census and pedigree studies. and afterwards the average phenotype of the groups
is compared. If no QTL is linked to a given marker (e.g.
3.3 Molecular tools for targeting M2), then no significant difference will be detected
functional variation between the average phenotypic value of the M2M2
and m2m2 progenies for the target trait. Conversely,
Approaches based on map position: when progenies are grouped by their genotype at the
quantitative trait loci (QTL) mapping marker M1, then the group M1M1 will mostly be QQ
Genetic markers behave as Mendelian traits; in at the QTL, and the group m1m1 will mostly be qq. In
other words, they follow the laws of segregation this case, a significant difference is observed between
and independent assortment first described by progeny averages, and therefore the presence of a
Mendel. Two genes that are located on the same QTL is detected. In species, such as poultry and pigs,
chromosome are physically linked and tend to be where lines and breeds are commonly interbred
inherited together. During meiosis, recombination commercially, this exercise can be accomplished in
between homologous chromosomes may break experimental populations (F2, BC) while in ruminants
this linkage. The frequency of recombination two (daughter design – DD) or three (grand-daughter
between two genes located on the same design – GDD) generation pedigrees are generally
chromosome depends of the distance between used. In DD the segregation of markers heterozygous
them. Recombination rate between markers in a sire (generation I) is followed in the daughters
is, therefore, an indication of their degree of (generation II) on which phenotypic data are collected.
linkage: the lower the recombination rate, the In GDD, the segregation of markers heterozygous in
closer the markers. The construction of genetic a grand-sire (generation I) are followed in his half-sib
maps exploits this characteristic to infer the sons (generation II), whose phenotype is inferred from
likely order of markers and the distance between those of the grand-daughters (generation III).
them.
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PART 4
under selection programmes (families of full regions of a model organism for which complete
siblings or half siblings). Medium to high density genome information is available.
genetic maps of a few hundred to a few thousand Occasionally, key information on gene function
markers are available for most livestock species. arrives from an unexpected source. This was the
To identify a QTL for a given trait, a family case with the myostatin gene, the function of
segregating for the trait is genotyped with which was first discovered in mice and then found
a set of mapped molecular markers evenly to be located in cattle in the chromosomal region
spread over the genome (Box 76). A number of where the double-muscling gene had previously
statistical methods exist to infer the presence of a been mapped (McPherron and Lee, 1997).
significant QTL at a given marker interval, but all It is clear that identifying the responsible
rely on the fact that families possess a high level gene (quantitative trait genes – QTG) and the
of linkage disequilibrium, i.e. large segments functional mutation (QTN) of a complex trait is
of chromosomes are transmitted without still a substantial task, and several approaches
recombination from parents to progeny. are needed to decrease the number of positional
The result of a QTL mapping experiment is the candidate genes. Information on gene function
identification of a chromosome region, often is fundamental in this respect. However, we are
spanning half of a chromosome, in which a still ignorant about the possible function(s) of the
significant effect is detected for the target trait. majority of genes identified by genome and cDNA
Modern research is actively using mapping to (complementary DNA) sequencing. This is why the
identify QTL influencing adaptive traits. Examples investigation of patterns of gene expression may
of such traits include, in chickens, increased provide useful information, in combination with
resistance to Salmonella colonization and the positional approach previously described, to
excretion (Tilquin et al., 2005), and susceptibility identify candidate genes for complex traits. This
to develop pulmonary hypertension syndrome combined approach is referred to as genetical
(Rabie et al., 2005); and in cattle, trypanotolerance genomics (Haley and de Koning, 2006). New
(Hanotte et al., 2002). advances in the investigation of patterns of gene
The QTL mapping phase is generally followed by expression are described in the next section.
the refinement of the map position of the QTL (QTL Alternative approaches are presently being
fine mapping). To accomplish this task, additional investigated to detect adaptive genes using
markers, and above all additional recombination genetic markers (Box 77). They are now at the
events in the target area, are analysed. A clever experimental stage, and only further research will
approach has recently been designed and applied permit an evaluation of their efficacy.
to the fine mapping of a chromosome region The ultimate goal of QTL mapping is to identify
on BTA14 carrying a significant QTL for milk fat the QTG, and eventually the QTN. Although only
percentage and other traits (Farnir et al., 2002). a few examples exist to date in livestock, these
This approach exploits historical recombination in are the kind of mutations that could have a
past generations to restrict the map position to direct impact on marker assisted breeding and on
a relatively small 3.8 cM (centimorgan) region, a conservation decision-making. Conservation models
size that has permitted the positional cloning of considering functional traits and mutation need to
the gene (DGAT1) (Grisart et al., 2002). be developed, as an increasing number of QTG and
Following fine mapping, the genes determining QTN will be uncovered in the near future.
the performance trait can be sought among the
genes that are located in the regions identified. Investigating patterns of gene expression
Candidate genes may be sought in the same species In the past, the expression of specific traits,
(e.g. when a rich EST map is available or when such as adaptation and resistance, could only be
the genome is fully sequenced) or in orthologous measured at the phenotypic level. Nowadays, the
368
Box 77
The population genomics approach
An alternative approach to the identification of particular, genes involved in adaptation to extreme

genome regions carrying relevant genes has recently environments, disease resistance, etc. Many of
been proposed. It consists of the detection of these traits, which are of great importance to the
“selection signatures” via a “population genomics” sustainability of animal breeding, are difficult or
approach (Black et al., 2001; Luikart et al., 2003). impossible to investigate by classic QTL mapping
Three main principles of the population genomics or association study approaches. The potential of
approach to QTL mapping are that: population genomics has recently been investigated
1. neutral loci across the genome will be similarly from a theoretical point of view (Beaumont and
affected by genetic drift, demography, and Balding, 2004; Bamshad and Wooding, 2003), and
evolutionary history of populations; through experimental work with different types of
2. loci under selection will often behave differently markers in natural populations (AFLPs: Campbell
and, therefore, reveal “outlier” patterns of and Bernatchez, 2004; microsatellites: Kayser et al.,
variation, loss of diversity (increase of diversity 2003; SNPs: Akey et al., 2002). The approach has
if the loci were under a balanced selection), recently been applied within the Econogene project
linkage disequilibrium, and increased/decreased (http://lasig.epfl.ch/projets/econogene). In preliminary
Gst/Fst indices; and analyses, three SNPs in MYH1 (myosin 1), MEG3
3. through hitchhiking effects, selection will also (callypige), and CTSB (cathepsin B) genes in sheep
influence linked markers, allowing the detection have shown significant outlier behaviour (Pariset
of a “selection signature” (outlier effects), et al., 2006).
which can often be detected by genotyping a Within the same project, a novel approach based
large number of markers along a chromosome on Spatial Analysis Method (SAM) has been designed
and identifying clusters of outliers. This to detect signatures of natural selection within the
approach utilizes phenotypic data at the breed genome of domestic and wild animals (Joost, 2006).
level (or subpopulations within a breed), rather Preliminary results obtained with this method are in
than at the individual level, and thereby nicely agreement with those obtained by the application
complements classical QTL mapping approaches of theoretical models in population genetics, such as
within pedigrees. those developed by Beaumont and Balding (2004).
The population genomics approach can also SAM goes a step further compared to classical
identify genes subjected to strong selection approaches, as it is designed to identify environmental
pressure and eventually fixed within breeds, and in parameters associated with selected markers.
transcriptome (the ensemble of all transcripts in genes expressed in a tissue at a given time. Thus,
a cell or tissue), and the proteome (the ensemble the techniques contribute to the decoding of
of all proteins) can be directly investigated by the networks that are likely to underlie many
high-throughput techniques, such as differential complex traits.
display (DD) (Liang and Pardee, 1992), cDNA- -Omics technologies are often compared to
AFLP (Bachem et al., 1996), serial analysis of gene turning on the light in front of a Michelangelo
expression (SAGE) (Velculescu et al., 1995; 2000), fresco rather than using a torch that permits a
mass spectrometry, and protein and DNA view only of parts of the whole. The overall view
microarrays. These techniques represent a allows the meaning of the representation to be
breakthrough in RNA and protein analysis, understood and its beauty to be appreciated. In
permitting the parallel analysis of virtually all reality, the power of these techniques is paralleled
369
PART 4
at present by the difficulty and cost involved in and sequenced – sequencing of the concatemer
applying them and in analyzing the data produced. clones results in the quick identification of
The isolation of homogeneous cell samples is numerous individual tags; (iii) the expression level
rather difficult, and is an important prerequisite in of the transcript is quantified by the number of
many gene expression profiling studies. The large times a particular tag is observed.
number of parallel assays results in low cost per Microarrays can be used to compare, in a single
assay, but at a high cost per experiment. Equipment experiment, the mRNA expression levels of several
is expensive, and high technical skill is needed in thousands of genes between two biological
all experimental phases. This is in addition to the systems, for example, between animals in a
general difficulty in analysing RNA compared to normal environment and animals in a challenging
DNA. RNA is very sensitive to degradation, and environment. Microarray technology can also
particular care has to be taken while extracting it provide an understanding of the temporal and
from tissues that have a very active metabolism. spatial patterns of expression of genes in response
Indeed, sample conservation and manipulation to a vast range of factors to which the organism
is one of the keys to success in RNA analysis is exposed.
experiments. The application of nanotechnologies Very small volumes of DNA solution are printed
to the analysis of biological molecules is opening on a slide made of a non-porous material such
up very promising perspectives in solving these as glass, creating spots that range from 100 to
problems (Sauer et al., 2005). 150 μm in diameter. Currently, about 50 000
Data handling is a further problem. Molecular complementary DNAs (cDNAs) can be robotically
datasets such as gene expression profiles spotted onto a microscope slide. DNA microarrays
can be produced in a relatively short time. contain several hundreds of known genes, and a
However, the standardization of data between few thousands of unknown genes. The microarray
laboratories is needed for consistent analysis is spotted with cDNA fragments or with
of different biological datasets. Agreements prefabricated oligonucleotides. The latter option
on standardization, as well as the creation of has the advantage of a higher specificity and
interconnected databases, are essential for the reproducibility, but can be designed only when
efficient analysis of molecular networks. the sequence is known. Microarray use is based on
the principle of “hybridization”, i.e. the exposure
Transcript profiling of two single-stranded DNA, or one DNA and one
This section briefly describes SAGE and microarray RNA, sequences to each other, followed by the
techniques. Descriptions of other techniques measurement of the amount of double-stranded
may be found in a number of recent reviews molecule formed. The expression of mRNA can
(e.g. Donson et al., 2002). SAGE generates be measured qualitatively and quantitatively. It
complete expression profiles of tissues or cell indicates gene activity in a tissue, and is usually
lines. It involves the construction of total mRNA directly related to the protein production induced
libraries which enable a quantitative analysis of by this mRNA.
the whole transcripts expressed or inactivated at Gene expression profiling contributes to the
particular steps of a cellular activation. It is based understanding of biological mechanisms, and
on three principles: (i) a short sequence tag (9–14 hence facilitates the identification of candidate
bp) obtained from a defined region within each genes. The pool of genes involved in the expression
mRNA transcript contains sufficient information of trypanotolerance in cattle, for example, has
to uniquely identify one specific transcript; (ii) been characterized by SAGE (Berthier et al., 2003),
sequence tags can be linked together to form long and by cDNA microarray analysis (Hill et al., 2005).
DNA molecules (concatemers) which can be cloned The parallel investigation of the expression of
370
many genes may permit the identification of Mass spectrometry (an analytical technique
master genes responsible for phenotypic traits for the determination of molecular mass)
that remain undetected by differential expression in combination with chromatographic or
analysis. These master genes may, for instance, electrophoretic separation techniques, is
possess different alleles all expressed at the same currently the method of choice for identifying
level, which promote the expression of downstream endogenous proteins in cells, characterizing
genes with different efficiency. In this case, the post-translational modifications and determining
master gene can be sought either by exploiting protein abundance (Zhu et al., 2003). Two-
current knowledge of metabolic pathways, or dimensional gel electrophoresis is unique
via an expression QTL (eQTL) approach (Lan et with respect to the large number of proteins
al., 2006). In this approach, the level of expression (>10 000) that can be separated and visualized
of the downstream genes is measured in a in a single experiment. Protein spots are cut
segregating population. The amount of transcript from the gel, followed by proteolytic digestion,
of each gene is treated as a phenotypic trait, and and proteins are then identified using mass
QTL that influence the gene expression can be spectrometry (Aebersold and Mann, 2003).
sought using methodologies described above. It is However, standardization and automation of
worth noting that data analysis for the detection two-dimensional gel electrophoresis has proved
of QTL is still quite difficult to master. This is also difficult, and the use of the resulting protein
true for transcript profiling techniques because of patterns as proteomic reference maps has only
the many false signals that occur. been successful in a few cases. A complementary
technique, liquid chromatography, is easier to
Protein profiling automate, and it can be directly coupled to mass
The systematic study of protein structures, post- spectrometry. Affinity-based proteomic methods
translational modifications, protein profiles, that are based on microarrays are an alternative
protein–protein, protein–nucleic acid, and approach to protein profiling (Lueking et al.,
protein–small molecule interactions, and the 2003), and can also be used to detect protein–
spatial and temporal expression of proteins in protein interactions. Such information is essential
eukaryotic cells, are crucial to understanding for algorithmic modelling of biological pathways.
complex biological phenomena. Proteins are However, binding specificity remains a problem
essential to the structure of living cells and their in the application of protein microarrays,
functions. because cross-reactivity cannot accurately be
The structure of a protein can be revealed by predicted. Alternative approaches exist for
the diffraction of x-rays or by nuclear magnetic detecting protein–protein interactions such as
resonance spectroscopy. The first requires a the two hybrid system (Fields and Song, 1989).
large amount of crystalline protein, and this is However, none of the currently used methods
often restrictive. In order to understand protein allow the quantitative detection of binding
function and protein–protein interactions at the proteins, and it remains unclear to what extent
molecular level, it would be useful to determine the observed interactions are likely to represent
the structure of all the proteins in a cell or the physiological protein–protein interactions.
organism. At present, however, this has not been Array-based methods have also been developed
achieved. Interestingly, the number of different for detecting DNA–protein interaction in vitro
protein variants arising from protein synthesis and in vivo (see Sauer et al., 2005, for a review),
(alternative splicing and/or post-translational and identifying unknown proteins binding to
modifications) is significantly greater than the gene regulatory sequences. DNA microarrays
number of genes in a genome. are employed effectively for screening nuclear
extracts for DNA-binding complexes, whereas
371
PART 4
protein microarrays are mainly used for identifying Box 78

unknown DNA-binding proteins at proteome- Databases of biological molecules
wide level. In the future, these two techniques
will reveal detailed insights into transcriptional A number of databases exist which collect information
regulatory networks. on biological molecules:
Many methods of predicting the function of DNA sequence databases:
a protein are based on its homology to other • European Molecular Biology Lab (EMBL): http://
proteins and its location inside the cell. Predictions www.ebi.ac.uk/embl/index.html
of protein functions are rather complicated, and • GenBank: http://www.ncbi.nlm.nih.gov/
also require techniques to detect protein–protein • DNA Data Bank of Japan (DDBJ): http://www.
interactions, and to detect the binding of proteins ddbj.nig.ac.jp
to other molecules, because proteins fulfil their Protein databases:
functions in these binding processes. • SWISS-PROT: http://www.expasy.ch/sprot/sprot-
top.html
• Protein Information Resource (PIR): http://pir.
4 The role of bioinformatics georgetown.edu/pirwww/
• Protein Data Bank (PDB): http://www.rcsb.org/
Developing high-throughput technologies would pdb/
be useless without the capacity to analyse the
Gene identification utility sites Bio-Portal
exponentially growing amount of biological data. • GenomeWeb: http://www.hgmp.mrc.ac.uk/
These need to be stored in electronic databases GenomeWeb/nuc-geneid.html
(Box 78) associated with specific software • BCM Search Launcher: http://searchlauncher.
designed to permit data update, interrogation bcm.tmc.edu/
and retrieval. Information must be easily accessible • MOLBIOL: http://www.molbiol.net/
and interrogation-flexible, to allow the retrieval • Pedro’s BioMolecular Research tools: http://
of information, that can be analysed to unravel www.biophys.uni-duesseldorf.de/BioNet/Pedro/
metabolic pathways and the role of the proteins research_tools.html
and genes involved. • ExPASy Molecular Biology Server: http://www.
Bioinformatics is crucial to combine information expasy.ch/
from different sources and generate new Databases of particular interest for
knowledge from existing data. It also has the domestic animals:
potential to simulate the structure, function and http://locus.jouy.inra.fr/cgi-bin/bovmap/intro.pl
dynamics of molecular systems, and is therefore http://www.cgd.csiro.au/cgd.html
helpful in formulating hypotheses and driving http://www.ri.bbsrc.ac.uk/cgi-bin/arkdb/browsers/
experimental work. http://www.marc.usda.gov/genome/genome.html
http://www.ncbi.nlm.nih.gov/genome/guide/pig/
http://www.ensembl.org/index.html
5 Conclusions http://www.tigr.org/
http://omia.angis.org.au/
Molecular characterization can play a role in http://www.livestockgenomics.csiro.au/ibiss/
uncovering the history, and estimating the http://www.thearkdb.org/
diversity, distinctiveness and population structure http://www.hgsc.bcm.tmc.edu/projects/bovine/
of AnGR. It can also serve as an aid in the genetic
management of small populations, to avoid
excessive inbreeding. A number of investigations
372
have described within and between-population high-throughput –omics technologies are

diversity – some at quite a large scale. However, used to this end. The identification of QTN
these studies are fragmented and difficult offers new opportunities and challenges for
to compare and integrate. Moreover, a AnGR management. Information on adaptive
comprehensive worldwide survey of relevant diversity complements that on phenotypic and
species has not been carried out. As such, it is neutral genetic diversity, and can be integrated
of strategic importance to develop methods into AnGR management and conservation
for combining existing, partially overlapping decision-tools. The identification of unique
datasets, and to ensure the provision of standard alleles or combinations of alleles for adaptive
samples and markers for future use as worldwide traits in specific populations may reinforce the
references. A network of facilities collecting justification for their conservation and targeted
samples of autochthonous germplasm, to be utilization. Gene assisted selection also has the
made available to the scientific community under potential to decrease the selection efficiency gap
appropriate regulation, would facilitate the currently existing between large populations
implementation of a global survey. raised in industrial production systems, and small
Marker technologies are evolving, and it is local populations, where population genetic
likely that microsatellites will increasingly be evaluation systems and breeding schemes cannot
complemented by SNPs. These markers hold great be effectively applied. Marker and gene assisted
promise because of their large numbers in the selection may not, however, always represent
genome, and their suitability for automation in the best solution. These options need to be
production and scoring. However, the efficiency evaluated and optimized on a case-by-case basis,
of SNPs for the investigation of diversity in animal taking into account short and long-term effects
species remains to be thoroughly explored. The on population structure and rates of inbreeding,
subject should be approached with sufficient and cost and benefits in environmental and
critical detachment to avoid the production of socio-economic terms – in particular impacts on
biased results. people’s livelihoods.
Methods of data analysis are also evolving. New As in the case of other advanced technologies,
methods allow the study of diversity without a it is highly desirable that benefits of scientific
priori assumptions regarding the structure of the advances in the field of molecular characterization
populations under investigation; the exploration are shared across the globe, thereby contributing
of diversity to identify adaptive genes (e.g. to an improved understanding, utilization and
using population genomics, see Box 77); and the conservation of the world’s AnGR for the good of
integration of information from different sources, present and future human generations.
including socio-economic and environmental
parameters, for setting conservation priorities
(see Section F). The adoption of a correct
sampling strategy and the systematic collection
of phenotypic and environmental data, remain
key requirements for exploiting the full potential
of new technologies and approaches.
In addition to neutral variation, research is
actively seeking genes that influence key traits.
Disease resistance, production efficiency, and
product quality are among the traits having
high priority. A number of strategies and new
373
PART 4
Box 79
Glossary: molecular markers
For the purpose of this section the following With this knowledge, it is thought that the
definitions are used: identification of a few alleles of a haplotype block can
Candidate gene: any gene that could plausibly unambiguously identify all other polymorphic sites
cause differences in the observable characteristics in this region. Such information is very valuable for
of an animal (e.g. in disease resistance, milk protein investigating the genetics behind complex traits.
production or growth). The gene may be a candidate Linkage: The association of genes and/or markers
because it is located in a particular chromosome that lie near each other on a chromosome. Linked
region suspected of being involved in the control of genes and markers tend to be inherited together.
the trait, or its protein product may suggest that it Linkage disequilibrium (LD): is a term used
could be involved in controlling the trait (e.g. milk in the study of population genetics for the non-
protein genes in milk protein production). random association of alleles at two or more loci,
DNA: the genetic information in a genome is not necessarily on the same chromosome. It is not
encoded in deoxyribonucleic acid (DNA), which is the same as linkage, which describes the association
stored in the nucleus of a cell. DNA has two strands of two or more loci on a chromosome with limited
structured in a double helix, which is made of a sugar recombination between them. LD describes a situation
(deoxiribose), phosphate, and four chemical bases in which some combinations of alleles or genetic
– the nucleotides: adenine (A), guanine (G), cytosine markers occur more or less frequently in a population
(C) and thymine (T). An A on one strand always than would be expected from a random formation of
pairs with a T on the other through two hydrogen haplotypes from alleles based on their frequencies.
bonds, while a C always pairs with a G through three Linkage disequilibrium is caused by fitness
hydrogen bonds. The two strands are, therefore, interactions between genes or by such non-adaptive
complementary to each other. processes as population structure, inbreeding, and
Complementary DNA (cDNA): DNA sequences stochastic effects. In population genetics, linkage
generated from the reverse transcription of mRNA disequilibrium is said to characterize the haplotype
sequences. This type of DNA includes exons and distribution at two or more loci.
untranslated regions at the 5’ and 3’ ends of genes, Microarray technology: a new way of studying
but does not include intron DNA. how large numbers of genes interact with each other
Genetic marker: a DNA polymorphism that can be and how a cell’s regulatory networks control vast
easily detected by molecular or phenotypic analysis. The batteries of genes simultaneously. The method uses
marker can be within a gene or in DNA with no known a robot to precisely apply tiny droplets containing
function. Because DNA segments that lie near each functional DNA to glass slides. Researchers then
other on a chromosome tend to be inherited together, attach fluorescent labels to mRNA or cDNA from the
markers are often used as indirect ways of tracking the cell they are studying. The labelled probes are allowed
inheritance pattern of a gene that has not yet been to bind to cDNA strands on the slides. The slides are
identified, but whose approximate location is known. put into a scanning microscope that can measure the
brightness of each fluorescent dot; brightness reveals
Haplotype: a contraction of the phrase “haploid
how much of a specific mRNA is present, an indicator
genotype”, is the genetic constitution of an individual
of how active it is.
chromosome. In the case of diploid organisms, the
haplotype will contain one member of the pair of Primer: a short (single strand) oligonucleotide
alleles for each site. It may refer to a set of markers sequence used in a polymerase chain reaction (PCR)
(e.g. single nucleotide polymorphisms – SNPs) found RNA: Ribonucleic acid is a single stranded nucleic
to be statistically associated on a single chromosome. acid consisting of three of the four bases present in
DNA (A, C and G). T is, however, replaced by uracil (U).
374
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STATE OF THE A R T I N THE MA N AGEMEN T OF A N I MAL GENE T I C RESOU RC ES

Molecular markers – a tool for

is emerging. Structural genomics, transcriptomics of functional variations. Molecular techniques

Tools being developed to target functional Box 73

Box 75 be calculated for the FST values between pairs

secondary movements, and differentiation of (2006a) investigated, respectively, chicken and

SNPs seem to be appealing markers to apply in Mitochondrial DNA markers

3.2 Using markers to estimate Mapping exercises are generally accomplished

An alternative approach to the identiﬁcation of particular, genes involved in adaptation to extreme

protein microarrays are mainly used for identifying Box 78

have described within and between-population high-throughput –omics technologies are

Nei, M. & Roychoudhury, A.K. 1974. Sampling

Weir, B.S. & Basten, C.J. 1990. Sampling strategies for

Weir, B.S. & Cockerham, C.C. 1984. Estimating F-

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