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Genetic Diversity: Its Importance and Measurements.
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Pages: 251-295
CONSERVING BIOLOGICAL DIVERSITY: A MULTISCALED APPROACH

Edited by: Aabid Hussain Mir and Nazir Ahmad Bhat
ISBN: 978-93-86138-00-2
Edition: 2016
Published by: Research India Publications, New Delhi, India.
CHAPTER 17
Genetic Diversity: Importance and
Measurements
Tanmay Mukhopadhyay1 and Soumen Bhattacharjee1*
Abstract
Genes are the fundamental units of all biological variations and constitute the
raw material for evolution. It is the source of the enormous variety of life
forms, communities, and ecosystems. Genetic variation shapes and defines
individuals, populations, subspecies, species, and ultimately the kingdoms
of life on earth. Genetic diversity among individuals reflects the presence
of different alleles in the gene pool, and hence different genotypes within
populations. Genetic diversity has a great importance from the individualistic
and population point of view. All the phenotypic plasticity is dependent on the
genetic variability of any organisms which also helps it to adapt to and evolve
in different environmental pressures. Mainly three lines of evidences are there
1 Cell and Molecular Biology Laboratory, Department of Zoology, University of North Bengal,
Siliguri-734013, West Bengal.
* Corresponding author email: soumenb123@rediffmail.com; sbhnbu@gmail.com
252 Conserving Biological Diversity: A Multiscaled Approach
which support the ecological consequences on genetic diversity. Information

about genetic diversity is necessary for the development of appropriate
strategies in conservation biology as well as in many other applied fields. From
a basic evolutionary standpoint, genetic diversity is assumed to be crucial for
the evolutionary potential of a species. The most powerful catalyst in the field
of conservation has been the advances in genetic and molecular technologies,
leading to a wide variety of molecular methodologies for application in
conservation and population genetic studies. Recently, molecular methods have
been applied intensively in conservation biology and genetic diversity studies,
primarily as selective molecular tools, in resolving the empirical questions
of conservation and evolutionary concern. Several analytical statistical tools
and genetic diversity indices are now available to estimate or quantify genetic
diversity of an organism with more sophisticated way.
Keywords: Ecology and genetic diversity, Genetic diversity indices, Genetic

diversity, Importance of genetic diversity, Molecular markers.
Introduction
Genetic diversity is a study undertaken to classify an individual or population,
compared to other individuals or populations. It quantifies the magnitude of genetic
variability within a population which is a fundamental source of biodiversity. Genes
are the fundamental unit of biodiversity, the raw material for evolution, and the
source of the enormous variety of plants, animals, communities, and ecosystems
that we seek to protect, admire and use. Genetic variation shapes and defines
individuals, populations, subspecies, species, and ultimately the kingdoms of life
on earth. Genetic diversity among individuals reflects the presence of different
alleles in the gene pool, and hence different genotypes within populations. Genetic
diversity should be distinguished from genetic variability, which describes the
tendency of genetic traits to vary within populations (Laikre et al., 2010). Since
the beginning of the 20th century, the study of genetic diversity has been the major
focus of core evolutionary and conservation biology. The theoretical metrics
developed, such as genetic variance and heritability (Fisher, 1930; Wright, 1931),
provided the quantitative standards necessary for the evolutionary synthesis.
Further research has focused on the origin of genetic diversity, its maintenance and
its role in evolution. Simple questions such as “who breeds with whom” initiated
studies on the relatedness of populations. These investigations led to the formation
of ‘metapopulation’ theory, where a group of spatially separated populations of
the same species interact at some level and form a coherent larger group (Hanski,
Conserving Biological Diversity: A Multiscaled Approach 253
1998). The discovery of spatial structure in populations was a key element in the
early concepts and models of population ecology, genetics and adaptive evolution
(Wright, 1931; Andrewartha and Birch, 1954).
How different levels of genetic variation affect the rate of evolutionary change
within populations has also been intensively studied. Subsequently, the detection
of genetic variation has become more sensitive, firstly, through the utilization of
variations in enzymes (allozymes) and then through PCR-based marker systems,
allowing direct examination of DNA sequence variations. The precise detection of
genetic variation/diversity has greatly enhanced the studies of evolution. There is
no doubt that the genetic variation influences the fitness of individuals, and that
this is reflected in natural selection. In this regard, individual genotypes must vary
in ecologically important ways. Ecological adaptation is a significant factor for
example, in range expansion of different species. Species with different genotypes
conferring the highest levels of fitness are expected to survive and reproduce better,
shifting the gene pool over time towards higher frequencies of the alleles making
up the more successful genotypes (Ward et al., 2008). Fisher (1930) reported that
when an increase in fitness is allowed, genetic diversity can increase the population
growth rate, but only if the population is not regulated by other factors and if it
is experiencing directional selection. Despite the presence of genetic variation in
ecologically important traits, relatively little is known about the range of potential
ecological effects of genetic diversity on population dynamics, species interactions
and ecosystem processes (Hughes et al., 2008). This has led to the rise of the
field molecular ecology, which integrates the application of molecular population
genetics, phylogenetics and genomics to answer ecological questions.
Information about genetic diversity is necessary for the development of appropriate

strategies in conservation biology as well as in many other applied fields. From
a basic evolutionary standpoint, genetic diversity is assumed to be crucial for the
evolutionary potential of a species. Research programs that aim to investigate
population structure provide evolutionary insights into the demographic patterns of
diverse organisms (Milligan et al., 1994). Furthermore, knowledge of population
structure of genetic resources is necessary for the development of strategies for
appropriate conservation of genetic diversity. Molecular phylogenetics and genetic
diversity analysis can help to clarify the taxonomic identity and evolutionary
relationships of the wild species. These methods can also help prevent miss-
identification and can carefully plan effective germplasm management strategies.
Variability and genetic diversity are important factors in evolution and also in
applied sciences because they determine the responses of a given organism to,
for example, environmental stress, natural selection and susceptibility to different
diseases.
Importance of Genetic Diversity

Genetic diversity is a trait for both for an individual or a population and is
characterized by the percentage of heterozygous alleles in diploid organisms (Nei,
1973). Genetic blueprint is the fundamental of all living organisms that carry a
specific genetic fingerprint. This is true irrespective of them being plants, animals,
or fungi, whether they are short or long-lived and whether they reproduce sexually
or asexually. Therefore, conservation, genetics and conservation genetics play a
role to a large extent for the restoration of living organisms. Although the basic
design underlying the conservation genetics may be very familiar, a very little
attention has been made to genetic considerations with respect to conservation
genetics.The genetic variability has an immense effect and has a great importance
on the survival and reproduction of any organism as well as populations, which
can be summarized as follows:
Effect on the individuals

Genetic variation or its deficiency within individuals can affect their survival and
reproducibility. In diploid organisms when both copies of a gene are identical (i.e.,
homozygous at that gene or locus), the expression of that gene may include traits
having less beneficial for survival or reproduction in particular circumstances.
This may cause several physiological or behavioral problems of genetic origin,
such as malformed physical structure, poor biochemical balance, improper organ
formation and function, altered social behavior, and susceptibility to different
diseases (Chai, 1976). Homozygosity or lack of heterozygosity is the common result
of inbreeding (sexual reproduction between closely related individuals) which
leads to greater chance of two alleles of a locus being identical by descent from
a common ancestor of both sides or by genetic drift. Small sized populations that
do not disperse or migrate well, or are constrained allopatrically from gene flow
with other populations, can be particularly susceptible to inbreeding depression,
which leads to reduced overall survival and reproduction of organisms with low
heterozygosity. Inbreeding depression arises from a variety of causes, including
expression of unfavorable or deleterious alleles, and often leads to lower survival
and birth or reproductive rates (Husband and Schemske, 1996; Meffe and Carroll,
1997). Inbreeding has a great impact on organism’s survivability and mortality. It
causes higher mortality, lower fecundity, reduced reproductive potentiality, reduced
growth rate, developmental instability, and reduces adaptive ability to withstand
environmental stress and lower competitive ability (Wright, 1977; Allendorf and
Leary, 1986; Ledig, 1986; Falconer, 1989).
Effect on the populations

Genetic variability/diversity can influence any population in several ways. The
growth rate of the population is lowered due to reduced fecundity and survival
of the inbred individuals, which in turn increases the risk of extinction of the
population concerned due to stochastic phenomena in near future (Goodman,
1987). Lower capability for population growth also reduces the potentiality of a
population to recover from population crisis due to several environmental stress
and genetic bottlenecks which cause further inbreeding depression within the
population (Schmitt and Ehrhardt, 1990; Miller, 1994; Keller et al., 1994).There
are several factors that affect the population fitness. Therefore, if the population
growth is not adequate to compensate the loss in fitness, a decline in genetic
variation will eventually decrease the potentiality of the population to evolve in
the changing environment. Fisher (1930) reported that the heritability of a trait is
directly proportional to the evolutionary response to the selection, which in turn
is proportional to the expected heterozygosity of gene diversity of that particular
trait (Falconer, 1989). James (1971) has reported that populations having genetic
polymorphism in several alleles or having multiple alleles for a specific trait
show great evolutionary adaptability. Thus, population with lower heritability,
reduced heterozygosity, and fewer polymorphic loci will probably adapt more
slowly and attain lesser adaptation than the population which is more diverse
and having greater genetic diversity. Therefore, a sudden fluctuation in genetic
variability in small populations can reduce the rate of adaptability sufficiently
to cause small populations to go extinct in the face of environmental changes to
which large populations would likely be able to adapt (Burger and Lynch, 1995).
Every population should have an effective size to withstand genetic erosion and
the loss of genetic variation due to genetic drift/genetic bottlenecks to preserve
long-term survival potentiality. Mutations can basically influence the feasibility of
small populations by bringing in inbreeding depression, by maintaining adaptive
genetic variation in quantitative characters, and through the disintegration of
fitness by the accumulation of mildly detrimental mutations. Lande (1995) has
suggested that highly detrimental mutations have a low effect to promote genetic
variation because incorporation of new genetic variation is much slower than
the overall mutation rate in populations, therefore, considering the mutations are
not detrimental. The effective population size needs to be in an order of 5000 to
ensure long-term viability of the population (Lande, 1995). Random genetic drift
is the major driving force that effects to change the allele frequencies in small
populations. Therefore, deleterious mutations occasionally get fixed in small
populations replacing the more adaptable alleles. As the deleterious mutations
accumulate in the populations, the population size may decrease and genetic drift
becomes even more pronounced, commonly called as Genetic Meltdown (Kimura,
1983; Lacy, 1987).
Phenotypes and functions are dependent on genetic variability

Biologists refer to two basic expressions of variability; the genotype and the
phenotype. The genotype (genetic makeup of an organism) of organisms with
nucleated cells (multi-cellular plants, fungi and animals), is represented by the
nucleus (chromosomal DNA) of each cell. Additional genetic content resides in
other organelles within the cell, such as in mitochondria (mitochondrial DNA
in animals) and chloroplasts (chloroplast DNA in plants). A large number of
genes constitute the multi-cellular organism’s genotype, and genes can be found
at multiple sites (loci) on chromosomes. In higher plants and animals (but not
fungi, bryophytes, and many marine invertebrates), the adult organism has two
copies of each gene, one of each derived from each single parent. When the two
copies are the same, the individual is homozygous for that gene; if the two copies
are different, the individual is said to be heterozygous for that gene. The various
forms of a gene are referred to as alleles; when these forms are identical across
a population, the gene is considered as monomorphic in nature, and if more than
one allele of that particular gene exists,, the gene is considered to be polymorphic
in nature. Consequently, phenotype is the expression of these genes in a particular
environment and is influenced by the environmental context at every level, from
the cell to the whole organism. It is very hard to separate the phenotypic variation
in an organism’s trait with a genetic basis. Sometimes, mistakes commonly occur
in assuming a genetic basis of a trait, when the observed variation is solely due
to phenotypic plasticity. Genetic variation does not solely affect the fitness of
individuals or populations, rather both the genes and the environment ultimately
contribute to the fitness and restoration success.
A number of studies have been carried out that suggested how the genetic
composition and genetic variability affect the form and function of organisms
(Hamrick et al., 1979; Hedrick, 1985; Primack and Kang, 1989; Rehfeldt, 1990;
Allen et al., 1996; Hartl and Clark, 1997). In fact, the recognition of genetic
variation among individuals was a primary insight that led to the formulation
of evolutionary theory as we know it today (Freeman and Herron, 1998). Genes
regulate body size, shape, physiological processes, behavioral traits, reproductive
characteristics, tolerance of environmental extremes, dispersal and colonizing
ability, the timing of seasonal and annual cycles, disease resistance, and many
other traits (Raven et al.,1986). Thus, genetic variation in ecology is one of the
fundamental forces that shape the biology and the survival of living organisms.
Genetic diversity helps to adapt to environmental variability

Organisms live in complex environment that vary in spatial and temporal scale and
is characterized by several factors such as weather, disturbance events, resource
availability, population sizes of competitors, etc. (White and Walker, 1997). If a
group of organisms were to live in a completely stable physical and biological
environment, then a relatively narrow range of phenotypes might be optimally

adapted to those conditions. Under these circumstances, organisms would benefit
more by maintaining a narrow range of genotypes adapted to prevailing conditions,
and allele frequencies might eventually attain equilibrium. By contrast, if the
environment is patchy, heterogeneous, unpredictable over time, or includes a wide
and changing variety of diseases, predators, and parasites, then subtle differences
among individuals increase the probability that some individuals against others,
will survive to reproduce i.e., the traits of the organisms are exposed to natural
selection. Since differences among individuals are determined at least partly
by genotype, population genetic theory predicts that in variable environments a
broader range of genetic variation or higher heterozygosity will persist (Cohen,
1966; Chesson, 1985; Tuljapurkar, 1989; Tilman, 1999). Any population can
tolerate the stochastic environmental variations through genetically controlled
traits. These traits are important from the stand point of resistance and resilience
ability of the population to tolerate freezing conditions, drought or inundation,
high or low light availability, salinity, heavy metals, soil nutrient deficiencies,
extreme soil pH values, fluctuating temperature, dissolved oxygen, novel diseases
in all groups of organisms (Huenneke, 1991).
Plant populations often include individuals with a range of phenological calendar.

For instance, Great Basin shrub populations include individuals that leaf out and
flower over a period of weeks, increasing the likelihood of persistence of the
population through periods of unusually early or late growing conditions (Falk et
al., 2001). Knapp et al. (2001) documented flowering periods in a population of
individual blue oak trees and found that trees initiated flowering over a period of a
month in the spring. A diverse array of genotypes appears to be especially important
in disease resistance (Schoen and Brown, 1993; McArdle, 1996). Genetically
uniform populations are occasionally vulnerable to diseases and pathogens and
such uniformity also predisposes a population to transmit disease from one
individual to another by direct contact or proximity. More diverse populations are
more likely to include individuals resistant to specific diseases. Moreover, infected
individuals occur at lower density, and thus diseases or pathogens may move more
slowly through the population. Finally, genetic variation is a factor in competition
among individuals in real ecological communities. Among animals, behavioral
traits may regulate inter-specific competition. Since organisms make energetic or
life history trade-offs among traits (for example, allocating energy between growth
and reproduction), genetic variability is an important factor with regard to how
populations function (Thompson and Plowright, 1980; Fowler, 1981; Gurevitch,
1986; Goldberg, 1987; Manning and Barbour, 1988; Welden, Slauson and Ward,
1988; Grace and Tilman, 1990; Tilman and Wedin, 1991; Wilson and Tilman,
1993; Delph et al., 1998).
Ecology and Genetic diversity

Genetic diversity is a measure that quantifies the magnitude of genetic variability
in the natural populations and is the fundamental source of biological diversity
in nature. Over the nine decades the study of genetic diversity is considered as
a principal domain for evolutionary and conservation biologists (Wright, 1920
and Fisher, 1930). The genetic diversity provides the raw material for evolution
by natural selection, influences the fitness of individual genotypes and vary in
ecologically important ways (Fisher, 1930; Ford, 1964; Endler, 1986). However,
the simple presence of heritable trait variation does not mean that different levels
of genetic diversity will have predictable ecological consequences. For example,
by increase in fitness, genetic diversity can increase in population but only if the
population is not regulated by other evolutionary factors (Fisher, 1930). Thus,
despite the obvious presence of genetic variation for ecologically important traits,
we know relatively little about the range of potential ecological effects of genetic
diversity for population dynamics, species interactions and ecosystem processes
(Figure 1). Frankham et al. (2002) have reported the short term ecological effects of
genetic diversity in small or endangered populations. Several agricultural practices
has been carried out where genetically modified crops have been harvested for
better yield and production, as well as decreased risk of herbivores and pathogens
(Zhu et al., 2000). Mainly three lines of evidences lend foundation of the study of
the ecological effects on genetic diversity. First, the ecological consequences of
genetic diversity has focused on how the number of species and functional groups
e.g. trophic structure within communities affects the stability and functioning of
the ecosystem (Hooper et al., 2005).
Figure 1: Processes underlying potential direct and indirect impacts of genetic

diversity on the ecological functioning. Solid black lines indicate direct ecological
consequences of genetic diversity; dotted lines indicate effects of natural selection,
which depend on genetic diversity.
Secondly, there is a growing interest on the ecological effects of the variance

component around the mean within the experimental or observable units of a
particular describable variable (Inouye, 2005). Several lines of research provide
detailed information regarding how the genetic differences between individuals
have influenced the species interaction and the interplay between the genetic
and ecological dynamics (Turkington and Harper, 1979). Finally, the community
genetics has bridged the fields like evolutionary biology, population genetics and
community ecology (Whitham et al., 2003). Community genetics focuses the
genetic diversity as a hierarchical concept and is not limited to single taxonomic
and genetic level. Therefore, the variation in ecologically important traits such as
growth rate, competitive ability, immune function, virulence etc., the amount of
genetic diversity at any level of population can have important ecological effects.
Genetic diversity can affect the community at the same trophic level (Figure 1).
For example, multi-species communities of grassland plants with higher genetic
diversity per species maintained higher species diversity over time than the
communities with lower genetic diversity (Booth and Grime, 2003). In this system,
genotypic interactions between species contribute to the effects of overall genetic
diversity (Whitlock et al., 2007). Mechanisms underlying the effects of genetic
diversity on community may apply equally to the effects of species diversity on
community, but mechanisms specific to genetic diversity can also contribute to
community-level impacts. The effect of genetic diversity on kin recognition has
been implicated in the successful invasion of the Argentine ant (Linepithema
humile), where the low allelic diversity of the invader decreased the precision of
the recognition system, allowing this species to form large, competitively dominant
super-colonies (Tsutsui et al., 2003). Genetic diversity also appears to influence
behavior in this species, with ants from low diversity colonies showed greater
aggression and lower mortality in encounters with ants from high-diversity colonies
(Tsutsui, 2004). Several studies have been carried out how the genetic diversity
affects the higher trophic level in an ecosystem. The number of plant genotypes
affects the arthropod community translating into positive effects on total arthropod
species richness but not on total abundance (Johnson et al., 2006). The mechanisms
underlying these effects differed depending on arthropod trophic level, increases
with predator species richness and abundance could be attributed to spatial and
temporal niche, complementarity among genotypes contributed to the increase in
abundance of arthropod species. In the study mentioned above, genotypic richness
increased the abundance of plant-associated species by increasing plant abundance
(Reusch et al., 2005), while genotypic richness positively affected arthropod
species richness by increasing both the total abundance and the diversity of plant
resource availability (Crutsinger et al., 2006). Another study also suggested the
effects of plant genetic diversity on the abundance and/or diversity of invertebrate
communities (Tovar-Sanchez and Oyama, 2006). Effects of genetic diversity on

community dynamics across trophic levels can also occur due to rapid evolution,
as illustrated by modified predator–prey and host–pathogen dynamics (Pimentel,
1968). Genetic diversity in dominant plant species can also influence fluxes of
nutrients and energy, i.e., ecosystem-level processes (Madritch et al., 2006).
Moreover, genetic diversity is not the sole driving force to affect the ecological
processes. Genetic diversity influences the ecological processes only when the
four conditions are met. The conditions are as follows: first, when a community or
ecosystem is dominated by one or a few primary habitat-forming species, genetic
diversity can play a role similar to species diversity in an ecosystem (Crutsinger
et al., 2006). Interestingly, relatively few studies focused on the genetic diversity
of foundation species. However, the habitat-forming species have widest ranging
effects at the ecosystem level, but greater focus should be placed on the genetic
diversity of the dominant species. Secondly, when genetic diversity in one species
affects the abundance or distribution of a keystone species (i.e. a species with an
effect disproportionate to its biomass in the community) it can have large indirect
ecological impacts (Crawford et al., 2007). Thirdly, an important prediction
proposes that genetic diversity will only have prominent ecological effects for
species that exhibit measurable genetic diversity within populations for relevant
traits, and thus these effects cannot be assumed without the genetic diversity data.
For example, populations those are highly inbred or have experienced a recent
selective force for genes controlling ecologically important traits will likely exhibit
low genetic diversity. Finally, given the documented importance of genetic diversity
and species diversity for disturbance response and stability, genetic diversity
will be most relevant in highly variable environments or those subject to rapid
anthropogenic change (Reusch et al., 2005; Hooper et al., 2005). Therefore, species
diversity and genetic diversity can be both a cause and consequence of ecological
processes (Vellend and Geber, 2005). For example, genetic diversity enables prey
populations to evolve, which can affect predator population dynamics and in turn
drive further ecological and evolutionary changes within the prey population,
leading to predictable predator–prey eco-evolutionary dynamics (Yoshida et al.,
2003). Moreover, genetic variation in one species can allow for coexistence with
its competitors, while at the same time, competitor species diversity maintains this
genetic variation (Lankau and Strauss, 2007). There are undoubtedly numerous
additional reciprocal effects between genetic diversity and ecological factors, as
genetic diversity and evolutionary processes can influence a range of population,
community and ecosystem responses (Fussmann et al., 2007). It is clear that the
level of genetic diversity within a population can affect the productivity, growth
and stability of that focal population, as well as inter-specific interactions within
communities, and ecosystem-level processes.
Molecular tools to assess Genetic Diversity

The most powerful catalyst in the field of conservation has been the advances
in genetic and molecular technologies, leading to a wide variety of molecular
methodologies for application in conservation and population genetic studies.
To date, molecular methods have been applied vastly in conservation biology
primarily as selectively neutral molecular tools for resolving the empirical
questions of conservation and evolutionary relevance (Primmer, 2009). The first
step of molecular biological technologies in the field of conservation genetics
was taken up in 1960 with the introduction of protein polymorphism analysis
(Ridgeway, 1962), followed by the mitochondrial DNA (mtDNA), Restriction
Fragment Length Polymorphism (RFLP)-based methodologies (Bowen and
Avise, 1990), Randomly Amplified Polymorphic DNA (Williams et al., 1990),
and more recently by microsatellite marker-based technologies (Taylor et
al., 1994), Inter Simple Sequence Repeat (ISSR) markers (Zietkiewicz et al.,
1994), Diversity Array Technology (DArT) (Kilian et al., 2005) and other high
throughput platforms. Therefore, the applications of particular types of genetic
markers are becoming more and more specialized to achieve a particular goal
to solve the specific questions of conservation concern (Schlotterer, 2004). The
‘genomic era’ was started when the genome of the evolutionary, ecologically and
commercially important model organisms were successfully sequenced (Kohn et
al., 2006). There are several benefits like larger number of molecular markers of
a wide variety of organisms, SNP and microsatellite marker’s high sophistication
and additional use of neutral markers that enhance the fidelity of conservation
study, enabling researchers to look deep into the problems regarding conservation
concern (Sanchez and Endicott, 2006; Kohn et al.,2006). Expressed Sequence
Tag (EST) libraries are the valuable resource for the study of conservation and
evolutionary genetics using bioinformatic tools by CASCADE databases, called
in silico SNP mining pipeline (Guryev et al., 2005). More recently, genomic
technological advances like Next Generation Sequencing (NGS) technologies and
“deep sequencing” or “ultra-high throughput sequencing” technologies (Holt and
Jones, 2008) provide a wider path to understand the empirical questions regarding
the conservation genetics of model as well as non-model organisms.
Restriction Fragment Length Polymorphism (RFLP)

Restriction fragment length polymorphism (RFLP) has much greater power
and was originally developed for mapping human genes (Botstein et al., 1980).
This technique quickly proved its utility in virtually all species. O’Brien (1991)
grouped genetic markers into two types: Type I markers, are associated with a gene
of known functions, while Type II markers are associated with anonymous gene
segments of one sort or another. Variations in the characteristic pattern of a RFLP
digest can be caused by base pair deletions, mutations, inversions, translocations
and transpositions which result in the loss or gain of a restriction endonuclease

recognition site, resulting in a fragment of different length and polymorphism. Only
a single base pair difference in the recognition site cause the restriction enzyme
not to cut the DNA. If the base pair mutation is present in one chromosome but
not the other, both fragments will be present on the gel, and the sample is said to
heterozygous for the marker. Only co-dominant markers exhibit this behaviour
which is highly desirable, while dominant markers exhibit a present/absent
behaviour which can limit data available for analysis. RFLP has some limitations
since it is time consuming.
Randomly Amplified Polymorphic DNA (RAPD or AP-PCR)

RAPD was the first PCR-based molecular marker technique developed and
it is by far the simplest method for genetic diversity analyses (Williams et al.,
1990), especially when other sophisticated markers are unavailable. Short PCR
primers (approximately 10 bases in length) are randomly and arbitrarily selected
to amplify random DNA segments throughout the genome, hence the “Randomly”
or “Arbitrarily Primed PCR (AP-PCR)”. The resulting amplification product is
generated at the region flanking a part of the 10 base pair priming sites in the
appropriate orientation. RAPD products are usually visualized on agarose gels,
after staining with ethidium bromide in a particular concentration. RAPD markers
are easily developed and because they are based on PCR amplification followed
by agarose gel electrophoresis, they are quickly and readily detected in very short
time. RAPD technique was used extensively in studying genetic diversity within/
between plant species and animal species. Most RAPD markers are dominant and
therefore, heterozygous individuals cannot be distinguished from the homozygotes.
This contrasts with RFLP markers which are co-dominant and therefore, can
distinguish among the heterozygote and homozygotes. Thus, in contrast to
standard RFLP markers and especially VNTR loci, RAPD markers generate less
information per locus examined. One disadvantage of using RAPD technique is the
reproducibility between different gel runs which is due to the short primer length
and low annealing temperature. However, if carefully chosen and PCR conditions
standardized, RAPD gels can be of much value in certain situations.
Inter-Simple Sequence Repeat (ISSR)

The Inter-Simple Sequence Repeat (ISSR) is semi-arbitrary marker amplified by
polymerase chain reaction (PCR) in the presence of one primer complementary
to a target microsatellite. Each band corresponds to a DNA sequence delimited
by two inverted microsatellites (Zietkiewicz et al., 1994; Tsumara et al., 1995;
Nagaoka and Ogihara, 1997). It does not require genome sequence information,
leads to multi-locus, highly polymorphic patterns and produces dominant markers
(Mishra et al., 2003). ISSR-PCR is a fast, inexpensive genotyping technique
based on length variation in the regions between microsatellites. This method has
a wide range of uses, including the characterization of genetic relatedness among
populations, genetic fingerprinting, gene tagging, detection of clonal variation,
cultivar identification, phylogenetic analyses, detection of genomic instability
(Brenner, 2011), and assessment of hybridization. ISSR markers are also suitable
for the identification and DNA fingerprinting (Gupta and Varshney, 2000; Gupta
et al., 2002). ISSRs are like RAPDs markers in that they are quick and easy to
handle, but they seem to have better reproducibility of SSR markers because of the
longer length of their primers.
Simple Sequence Repeats (SSR) or Microsatellites

Simple Sequence Repeat (SSR) (also called microsatellites) markers are repeats of
short nucleotide sequences, usually equal to or less than six bases in length, that
vary in number of repeats (Rafalski et al., 1996; Reddy et al., 2002). SSRs are
becoming the most important molecular markers in both animal and plant studies.
SSR are stretches of 1 to 6 nucleotide units repeated in tandem and spread randomly
in eukaryotic genomes. SSRs are very polymorphic due to the high mutation rate
affecting the number of repeat units. Such length-polymorphisms can be easily
detected on high resolution gels (e.g. sequencing gels). It is suggested that the
variation or polymorphism of SSRs are a result of polymerase slippage during
DNA replication or unequal crossing overs (Levinson and Gutman, 1987). SSRs
are not only very common but are also hypervariable for numbers of repetitive
DNA motifs in the genomes of eukaryotes (Rallo et al., 2000; van der Schoot
et al., 2000). SSRs have several advantages over other molecular markers. For
example (i) microsatellites allow the identification of many alleles at a single
locus, (ii) they are evenly distributed all over the genome, (iii) microsatellites
can offer more detailed population genetic insights, (iv) they are co-dominant,
highly polymorphic and specific (Jones et al., 1997), (v) very repeatable, little
DNA is required and so cheap and easy to run, (vi) need a small amount of medium
quality DNA and (vii) the analysis can be semi-automated and performed without
the need of radioactivity (Guilford et al., 1997), (vii) with the advance of DNA
isolation technology, it is possible to identify loci in highly degraded ancient
DNAs (aDNAs), where traditional enrichment procedures have been unsuccessful
(Allentoft et al., 2009) (ix) with the development of high-throughput sequencing
platforms, SSR has recently become faster and efficient (Santana et al., 2009).
However, since genomic sequencing is needed to design specific primers, it is not
very cost effective and also requires much background knowledge and optimization
for each species before use.
Amplified Fragment Length Polymorphisms (AFLP)

Amplified Fragment Length Polymorphisms (AFLP) based genomic DNA
fingerprinting is a technique used to detect DNA polymorphism. AFLP is a PCR-
based technique (Vos et al., 1995), and has been reliably used for determining
genetic diversity and phylogenetic relationship between closely related genotypes.
AFLP analysis combines both the reliability of restriction fragment length
polymorphism (RFLP) and the convenience of PCR-based fingerprinting methods.
AFLP markers are generally dominant and do not require prior knowledge of the
genomic composition. AFLPs are produced in great numbers and are reproducible.
The AFLP is applicable to all species giving very reproducible results and it has
the advantage of the extensive coverage of the genome under study. In addition,
the complexity of the bands can be reduced by adding selective bases to the
primers during PCR amplification. Disadvantages of this technique are that alleles
are not easily recognized, have medium reproducibility, labour intensive and have
high operational and development costs (Karp et al., 1997). Moreover, AFLP
requires knowledge of the genomic sequence to design primers with specific
selective bases. There are several advantage of this technique (i) no need for
prior knowledge of any sequence information, (ii) multiple bands are produced
per each experiment, (iii) these bands are produced from all over the genome,
(iv) the technique is reproducible (Vos and Kuiper, 1997; Blears et al., 1998), (v)
have highly discriminatory power, and (vi) the data can be stored in database like
AmpliBASE MT (Majeed et al., 2004) for comparison purposes.
Single Nucleotide Polymorphism (SNP’s)

Single Nucleotide Polymorphism (SNP) represents DNA sequences that differ by
a single base when two or more individuals are compared. They are responsible
for specific traits or phenotypes, or may represent neutral variation that is useful
for evaluating diversity in the context of evolution. SNPs are the most widespread
type of sequence variation in genomes discovered so far. About 90% of sequence
variants in humans are different in single bases of DNA (Collins, 1998). Several
disciplines such as population ecology and conservation and evolutionary genetics
are benefitting from SNPs as genetic markers. There is a widespread interest in
finding SNP’s because they are numerous, more stable and potentially easier to
score. Within coding regions there are on average, four SNPs per gene with a
frequency >1%. About half of these cause amino acid substitutions: termed non-
synonymous SNPs (nsSNPs) (Cargill et al., 1999). SNPs are rapidly replacing
simple sequence repeats (SSRs) as the DNA marker of choice for applications
in conservation and population genetics because they are more abundant, stable,
amenable to automation, efficient, and increasingly cost-effective (Edwards and
Batley, 2010). There are limitations to the discovery of SNP’s in the non-model
organism due to the expenses and technical difficulties involved in the currently
available SNP isolation strategies (Brumfield, 2003; Seddon et al., 2005). Typical
direct SNP discovery strategies involve sequencing of locus specific amplification
(LSA) products from multiple individuals or sequence determination of expressed
sequence tags (EST-sequencing) (Twyman, 2004; Suh and Vijg, 2005). Other direct
strategies include Whole Genome Shotgun Sequencing (WGSS) and Reduced
Representation Shotgun Sequencing (RRSS) approaches. If comparative sequence
data are available in public or other databases, various sequence comparison
algorithms that identify nucleotide differences provide an alternative means
to empirically discover SNPs (Guryev et al., 2005). The software applications
PyroBayes and Mosaik are being used to differentiate between true polymorphisms
and sequence errors (Hillier et al., 2008). The new development in technologies
that collect high-throughput data contributes substantially in the progress in
evolutionary genomics (Gilad et al., 2009). The next generation sequencing
(NGS) technologies have the potential to revolutionize genomic research and
enable to focus on a large number of outstanding questions more easily (Rafalski,
2002). The NGS provides the capacity for high-throughput sequencing of whole
genomes at low cost. They have advantage of improving the capacity to finding
novel variations that are not covered by genotyping arrays (Imelfort et al., 2009).
Diversity Array Technology (DArT)

Diversity array technology (DArT) is a microarray hybridization-based technique
that can simultaneously screen thousands of polymorphic loci without any prior
sequence information. The DArT methodology works in a high multiplexing
level and is able to simultaneously type several thousand loci per assay. DArT
assays generate whole genome fingerprints by scoring the presence versus
absence of DNA fragments in genomic representations generated from genomic
DNA samples through the process of complexity reduction. DArT has been
developed as a hybridisation-based alternative to the majority of current gel-
based marker technologies. It can provide from hundreds to tens of thousands of
highly reliable markers for any species (Jaccoud et al., 2001). An important step
of this technology is a step called “genome complexity reduction” which increases
genomic representation by reducing repetitive sequence that are abundant in the
eukaryotes. Another advantage of DArT markers is that their sequence is easily
accessible compared to AFLP, making DArT a better choice (James et al., 2008).
Restriction Site-Associated DNA (RAD)

Another high throughput method is Restriction site-Associated DNA (RAD)
procedure which involves digesting DNA with a particular restriction enzyme,
ligating biotinylated adapters to the overhangs, randomly shearing the DNA into
fragments much smaller than the average distance between restriction sites, and
isolating the biotinylated fragments using streptavidin beads. These fragments will
contain the genomic sequences that flank the restriction sites. These fragments are
then released from the streptavidin beads by restriction digestion at the original
restriction sites. The polymorphic loci, if present in any individual sample, will
not contain the tags for that loci in the purified RAD tag samples, thus resulting in
differential hybridization patterns of RAD tags on a microarray when compared
with other samples. However, the RAD tags that are present in all the individuals
of a population will not serve as an informative marker. Therefore, subtractive
hybridization of RAD tags with cDNA is used to enrich for sample-specific RAD
tags to produce a large number of informative markers. RAD-tag clone libraries
are generated from these enriched samples and are used as templates for PCR. The
products of the PCR amplification are used to produce RAD marker microarrays
(Miller et al., 2007a). RAD specifically isolates DNA tags directly flanking the
restriction sites of a particular restriction enzyme throughout the genome. More
recently, the RAD tag isolation procedure has been modified for use with high-
throughput sequencing on Illumina® platform, which has the benefit of greatly
reduced raw error rates and high throughput (Baird et al., 2008). The new procedure
involves digesting DNA with a particular restriction enzyme (SbfI, NsiI etc),
ligating the first adapter, called P1, to the overhangs, randomly shearing the DNA
into fragments much smaller than the average distance between restriction sites,
preparing the sheared ends into blunt ends and ligating the second adapter (P2),
and then using PCR to specifically amplify fragments that contain both adapters.
Importantly, the first adapter contains a short DNA sequence barcode, called MID
(Molecular IDentifier), which allows to pool different DNA samples with different
barcodes and to track each sample when they are sequenced in the same reaction
(Baird et al., 2008).
Single Feature Polymorphism (SFP)

Another high throughput method is single feature polymorphism (SFP) which is
done by labelling target genomic DNA and hybridizing to arrayed oligonucleotide
probes that are complementary to indel loci. The SFPs can be discovered through
sequence alignments or by hybridization of genomic DNA with whole genome
microarrays. Each SFP is scored by the presence or absence of a hybridization
signal with its corresponding oligonucleotide probe on the array. Both spotted
oligonucleotides and Affymetrix-type arrays have been used in SFP (Cui et al.,
2005). Borevitz et al. (2003) coined the term “single feature polymorphism or SFP”
and demonstrated that this approach can be applied to organisms with somewhat
larger genomes.
Internal Transcribed Spacer (ITS)

Nucleolar ribosomal DNA contains two internally transcribed spacers: ITS-1,
located between the small subunit and 5.8S rRNA cistronic regions, and ITS-2
which is located between the large subunit and 5.8S rRNA cistronic regions. The
two spacers and the 5.8S subunit are collectively called the Internal Transcribed
Spacer (ITS) region. The ITS regions are believed to be fast evolving and therefore
may vary in length and sequences. The flanking regions of the ITS are usually
used to design universal PCR primers to enable easy amplification of ITS regions.
The number of copies of rDNA repeats is up to 30000 per cell (Dubouzet and
Shinoda, 1999). The ITS region evolves much more rapidly than other conserved
regions of rDNA, this makes the ITS region an interesting subject for evolutionary,
phylogenetic and bio-geographic investigations (Baldwin et al., 1995). The ITS
region is highly conserved intra-specifically, but variable between different
species, this makes ITS a suitable systematic molecular tool (Bruns et al., 1991;
Hillis and Dixon, 1991). The genetic diversity studies using ITS can be used by
either direct sequencing of the region from different individuals followed by tree
construction based on sequence comparison. The other method is by measuring
sequence variation by restriction digestion of the ITS region, which can be used
towards taxonomic goals. The ITS region was successfully used for the diagnostics
and quick identification of cyst nematodes (Subbotin et al., 2000). Comparisons of
PCR-RFLP profiles and sequences of the ITS-rDNA of unknown nematodes with
those published or deposited in GenBank facilitate quick identification of most
species of cyst nematodes (Subbotin et al., 2000; Subbotin et al., 2001).
Conserved DNA-Derived Polymorphism (CDDP)

This technique, developed by Collard and Mackill (2009a), uses short primers to
generate useful genetic markers across functional domains of well-characterized
plant genes. It targets short conserved gene sequences present in the plant genome
in multiple copies. Primers are specifically designed to anneal to these genes to
generate polymorphic banding patterns detected on agarose gels. Collard and
Mackill (2009a) described a set of primers that target well characterized plant genes
involved in responses to abiotic and biotic stress or plant development. CDDP can
easily generate functional markers (FM) related to a given plant phenotype, which
is advantageous in many genetic studies. The conserved nature of priming sites in
these gene regions makes the technique transferable to a wide variety of species.
Since highly conserved DNA regions share the same priming site, but differ in their
genomic distribution, variation can be detected as length polymorphism within
these regions. The technique is based on single long primer amplifications with
a high annealing temperature, which improves reproducibility. CDDP employs a
single primer in the PCR reaction to amplify polymorphic regions representing
DNA stretches between two identical or very similar conserved primer binding
sites (Poczai et al., 2011).
Cytochrome P450 Based Analogues (PBA)

Cytochrome P450 (Cyp450) mono-oxygenases are widely found in animals,
plants and microorganisms (Shalk et al., 1999). Sequence diversity of P450 gene-
analogs in different plant species has been studied and it has been reported that
such analogs can be used as genetic markers for diversity studies in plants, at both
functional and genome-wide scales (Somerville and Somerville, 1999). Based on
these findings the technique uses Cytochrome P450 Based Analog (PBA) markers
to create polymorphic fingerprints to characterize genetic diversity within and
among populations of a wide variety of organisms (Yamanaka et al., 2003). In
the method developed by Yamanaka et al. (2003), universal primer pairs were
designed to anneal to specific conserved exon regions of Cyp450 genes. Forward
and reverse primers flanking the intron regions were then used to initiate PCR
amplification. Based on the random distribution of Cyp450 genes in the genome,
the resulting banding patterns will reflect polymorphism based on the variation
found across the targeted (pseudo)genes.
Start Codon Targeted (SCoT) Polymorphism

A novel marker system called Start Codon Targeted Polymorphism (SCoT) was
described by Collard and Mackill (2009b), based on the observation that the short
conserved regions of plant genes are surrounded by the ATG translation start codon
(Sawant et al., 1999). The technique uses single primers designed to anneal to the
flanking regions of the ATG initiation codon on both DNA strands. The generated
amplicons are possibly distributed within gene regions that contain genes on both
plus and minus DNA strands. The utility of primer pairs in SCoTs was advocated
by Gorji et al. (2011). The SCoT markers are generally reproducible, and it is
suggested that the primer length and annealing temperature are not the sole factors
determining reproducibility (Gorji et al., 2011). They are dominant markers,
however, while a number of co-dominant markers are also generated during
amplification, and thus they could be used for genetic diversity analyses (Collard
and Mackill, 2009b).
Sequence-Related Amplified Polymorphism (SRAP) and

Targeted Region Amplified Polymorphism (TRAP)
The Sequence-Related Amplified Polymorphism technique (SRAP), developed
by Li and Quiros (2001), also uses arbitrary primers (17-21 bases) to generate
a specific banding pattern. The forward primers are designed to contain GC-rich
sequences near the 3’-end, while reverse primers contain AT-rich sequences at the
3’-end. This is based on the rationale that protein coding regions tend to contain
GC-rich codons, while 3’UTRs frequently consist of AT-stretches (Lin et al.,
1999). The technique has gained popularity due to several advantages: (i) a large
number of polymorphic fragments could amplified in each reaction, (ii) no need

for prior information about sequences, (iii) primers can be applied to any species,
(iv) cost effective and easy to perform, (v) reproducibility is high, and vi) PCR
products can be directly sequenced using the original primers without cloning.
These advantages have led to its widespread application for genetic diversity
analysis in diverse organismslike Salvia miltiorrhiza, Ganoderma and Celosia
argentea (Sun et al., 2006; Feng et al., 2009; Song et al., 2010). The Targeted
Region Amplified Polymorphism technique (TRAP) developed by Hu and Vick
(2003) is similar to SRAP, but it incorporates the advantage of the availability of
sequence information i.e., it employs known sequences.
Conserved Region Amplification Polymorphism (CoRAP)

The CoRAP technique (Wang et al., 2009) is based on the use of two primers, a
fixed and an arbitrary one, to detect polymorphism. The technique is similar to
TRAP but differs in the incorporation of sequence motifs in the arbitrary primer.
Fixed primers derived from ESTs will have a specific binding site in the exon of the
target sequence, while the arbitrary primers will bind to most of the introns during
the PCR amplifications. If the distribution of these gene elements allows successful
PCR, banding patterns resulting from a specific fingerprint will be amplified. In-
dels in these regions will certainly generate different distribution of amplified
production. The closer the genetic relationship between the two individuals, the
more similar the corresponding band patterns of the amplified PCR products will
be (Wang et al., 2009).
Mobile element based molecular markers

Mobile element based markers have great potential as tools for investigating
aspects of molecular ecology, including population structure, conservation
genetics, the genetics of speciation, phylogeography and phylogeny (Ray, 2007).
One group of mobile elements, retro-transposons, provides an excellent basis for
the development of markers systems. Retro-transposons replicate like retroviruses
by successive transcription, reverse transcription and insertion of the new cDNA
copies back into the genome (Scheifele et al., 2009). The structure and replication
strategy of retro-transposons give them several advantages as markers (Kalendar
et al., 1999). Firstly, they are ubiquitous, present in high copy numbers and
widely dispersed on chromosomes and show insertional polymorphism (Kumar
et al., 1997). Secondly, active mobile elements produce new insertions in the
genome, leading to polymorphism (Kumar and Bennetzen, 1999). Thirdly, they
are widely distributed in the euchromatin domains of chromosomes, making it
possible to generate markers linked to a given phenotype (Kenward et al., 1999).
Fourthly, their ancestral state is known and stable and once the element is present
it will almost invariably remain there indefinitely (Shedlock and Okada, 2000).
Finally, mobile element based markers are co-dominant. There are several retro-
transposon-based markers available now-a-days like, Inter-Retro-transposon
Amplified Polymorphism (IRAP) and Retro-transposon-Microsatellite Amplified
Polymorphism (REMAP) which have been used in barley, wheat, oak, rice, banana,
Pisum and other important crops (Kalendar et al. 1999), Retro-transposon-Based
Insertion Polymorphisms (RBIP), in Pisum sativum (Flavell et al., 1998) and
Retro-transposon-based Sequence-Specific Amplification Polymorphism (SSAP)
in barley, pea, wheat and cashew (Grzebelus, 2006).
Analytical Tools to estimate Genetic Diversity

Polymorphism data can be scored in presence/absence matrices manually or with the
aid of specific software. The banding patterns produced, largely depending on the
nature of the methods and whether they generate dominant or co-dominant markers.
There are two features fingerprinting markers that considerably constrain how they
can be statistically analyzed. Firstly, polymorphic marker loci are generally scored
as two alleles, the “band-presence” allele and the “band absence” allele. However,
the large number of fingerprinting markers available across the genome, or the
specific exploration of multiple regions, can counteract this drawback. Secondly,
the banding patterns produced by each technique are treated as dominant markers
during the evaluation process, and the methods are described accordingly. Thus,
it is difficult to distinguish heterozygous individuals from homozygous ones with
respect to the band-presence allele.
Band-based approaches
The easiest way to analyze banding patterns and to measure the diversity represented
by them is to focus on band presence or absence and to compare it between samples.
This method is routinely used at the level of the individual, and produces distances
generated from the bands rather than taking into account the diversity of the
population. The disadvantage is that it treats the data as polymorphism produced
by the markers rather than describing the genetic diversity of the organism.
Measuring Polymorphism
A locus is considered polymorphic when the band is present at a frequency of
between 5% and 95%. The polymorphism represented in the banding patterns can
be extracted simply by observing the total number of polymorphic bands (PB) and
then calculating the percentage of polymorphic bands (PP) present in any individual
based on this number. This “band informativeness” (Ib) can be represented on a
scale ranging from 0 to 1 according to the formula: Ib = 1 – (2 × |0.5 – p|), where p
is the portion of genotypes containing the band.
Hardy-Weinberg equilibrium test (HWT)

Hardy- Weinberg equilibrium explains that the both gene and genotype frequencies
will be constant from generation to subsequent next generations (Lebate, 2000).
Chi-square test is useful for determining whether the allelic frequencies are in HW
equilibrium. The statistical test follows this formula (Guo and Thompson, 1992):
2
 O − Ei 
HWT= ∑  i 
 Ei 
when: HWT = Statistical test, Oi = Observed frequencies, Ei = Expected frequencies,
df = Degree of freedom. If X2 cal ≤ X2 tab then H0 hypothesis is accepted, it means
that allele frequencies for loci in a given population are HWT equilibrium, if X2 cal
≥ X2tab then H0 hypothesis is rejected (Hartl and Clark, 1997).
Average number of Alleles per locus

These measures provide complementary information to that polymorphism
(Kimura and Crow, 1964):
1 k
N = ∑ ni
k i =1
Where, k = Number of loci, ni = Number of alleles detected by locus. This parameter
has the best application in co-dominant markers.
Effective Number of Alleles (Ne)

The measure explains about the number of alleles that would be expected in a
locus in each population (Kimura and Crow, 1964):
 1 
Ae =  
 ∑ pa
k

2
 a=1 
Where, pa2 is the frequency of the ath of k alleles. By taking allele frequencies into
account, this descriptor of allelic richness is less sensitive to rare alleles.
Average Expected Heterozygosity (He)

Average expected heterozygosity (He) is the probability that at a single locus in a
diploid organism, any two alleles are chosen at random and are different from each
other. It is an indicator of genetic diversity in a population and expressed as:
1 m
∑ ∑
k
He = 1− pa 2
m i =1 a =1
Where, m number of analyzed loci. The range of this parameter from 0-1 and it is
maximized when there are many alleles at equal frequency.
Shannon’s Information Index (I)

Shannon’s information index is a diversity coefficient widely used to quantify the
degree, if bad polymorphism provided by banding patterns (Lewontin, 1972). It
can be calculated as I = –∑ pilog2pi , where I is diversity and pi is the frequency of
a particular RAPD band. It is assumed that (i) the population is large, (ii) random
mating takes place, (iii) the population is isolated from others, and (iv) the effects
of migration, mutation and selection are not relevant. Allele-frequency statistics
are commonly supplemented with another estimation that allows for the distortion
from HWE. These methods depend on the extraction of allelic frequencies from the
data, which can be accomplished using a number of different procedures (Lynch
and Milligan, 1994; Stewart and Excoffier, 1996). The value of the Shannon index
obtained from the data usually falls between 1.5 and 3.5, and sometimes surpasses
4 (Margalef, 1972). It is only when there are a huge number of species in the
samples that have high values are produced. The fact that the Shannon index is so
narrowly constrained in most cases can make interpretation difficult. Therefore,
some investigators sidestep the problem by using eI instead of I (Kaiser et al.,
2000).
Allelic Diversity (A)

One of the easiest ways to measure genetic diversity is to quantify the number
of alleles present. Allelic diversity (A) is the average number of alleles per locus
and is used to describe genetic diversity. When there is more than one locus, A is
calculated as the number of alleles averaged across all loci: A = ni / nl, where ni is
the total number of alleles over all loci and nl is the number of loci.
Effective Population size (Ne) and Minimum Viable population

(MVP)
The concept of effective population size (Ne) plays an important role to measure of
the rate of genetic drift, the rate of genetic diversity loss and increase of inbreeding
within a population. The Ne defines the number of individuals that would give rise
to the calculated inbreeding coefficient, loss of heterozygosity, or variance in allele
frequency if they behaved in the manner of an idealized population (Frankham et
al., 2002). In other words, it is the size of an idealized population that would lose
genetic diversity (or become inbred) at the same rate as the actual population. It
is a value which depicts the general core number individuals in a population who
contribute offspring to the next generation. The effective population size is usually
smaller than the absolute population size. The major factors effecting Ne are (i)
fluctuating population size, (ii) breeding sex ratio, (iii) overlap of generations and
(iv) spatial dispersion. In should be noted that the effective size of a population is
an idealized number, since many calculations depend on the genetic parameters
used and on the reference generation. Therefore, a single population may have
many different effective sizes which are biologically meaningful but distinct from
each other.
The demographic stochasticity and genetic drift can negatively affect small
populations. Demographic stochasticity leads to the random extinction of small
populations, while genetic drift can cause a reduction of genetic diversity within a
population. These factors can interact in an extinction vortex (Figure 2) that can
eventually lead to the extinction of a population (Primack, 2000). Shaffer (1981)
proposed the concept of the Minimum Viable Population (MVP) by defining it
as the smallest isolated population (of a given species in a given habitat) having
a 99% chance of remaining in existence for 1,000 years, despite the effects of
demographic stochasticity, genetic drift, environmental stochasticity and natural
catastrophes (Shaffer, 1981). Populations smaller than the MVP are considered to
be at significant risk of entering into the extinction vortex and becoming extinct, so
a conservation program can be considered successful only if it raises the effective
population size above the MVP.
Figure 2. Extinction vortex. Population size decreases in a positive feedback loop,

eventually resulting in the extinction of the population (modified from Primack,
2000).
Heterozygosity (H)
Heterozygosity can be regarded as the average portion of loci with two different
alleles at a single locus within an individual. It is commonly extended to the
whole or a sub-portion of an entire population and differentiated into observed and
expected heterozygosity. Expected heterozygosity (HE) or Nei’s gene diversity (D)
is the expected probability that an individual will be heterozygous at a given locus,
in multi-locus systems over the assayed loci. In other words, it is the estimated
fraction of all individuals that would be heterozygous for any randomly chosen
locus (Nei, 1973). It is often calculated based on the square root of the frequency
of the null (recessive) allele as follows:
H E =1 − ∑ i pi2
n
where pi is the frequency of the ith allele. Observed heterozygosity (HO) is the
portion of genes that are heterozygous in a population. It is calculated for each locus
as the total number of heterozygotes divided by sample size. Typically values for
HE and HO range from 0 (no heterozygosity) to nearly 1 (a large number of equally
frequent alleles). Expected heterozygosity is usually calculated when describing
genetic diversity, as it is less sensitive to sample size than observed heterozygosity.
If HO and HE are similar (they do not differ significantly) mating in the populations
is approximately random. If HO is less then HE, the population is inbreeding; if HO
exceeds HE, the population has a mating system avoiding inbreeding.
F-statistics
In population genetics, the most widely applied measurements besides
heterozygosity are F-statistics, or fixation indices. These were originally designed
to measure the amount of allelic fixation by genetic drift. F-statistics are used
to describe the structure of the population at different levels. It can describe the
inbreeding (coefficient) of an individual relative to the total population (FIT) or the
inbreeding (coefficient) of an individual relative to the subpopulation (FIS), and
can also express the “fixation” (index) resulting from comparing subpopulations to
the total population (FST) (Wright, 1977). In other words, different F-coefficients
explain the correlation of genes within individuals over all populations (FIT),
the correlation of genes of within individuals within populations (FIS), and the
correlation of genes of different individuals in the same population (FST). For a
given species this can be interpreted as the overall inbreeding (FIT), the inbreeding
within taxa (FIS) and the coefficient of co-ancestry (FST), which provides an
estimate of inter-specific genetic differentiation. The F-statistics are related to
heterozygosity and genetic drift. Since inbreeding increases the frequency of
homozygotes, as a consequence, it decreases the frequency of heterozygotes. In
other words, population substructure results in a reduction of heterozygosity. The

general result of genetic drift in small populations is a deficiency of heterozygotes
and an excess of homozygotes. Moreover, mating between relatives (inbreeding)
and genetic drift are related phenomena, which allow us to determine fixation
indices when expected and observed heterozygosity are known. Thus the three
indexes can be calculated as: FIT = 1 – (HI/HT); FIS = 1 – (HI/HS) and FST = 1 –
(HS/HT); where HI is the average observed heterozygosity within each population,
HS is the average expected heterozygosity of subpopulations, assuming random
mating within each population, and HT is the expected heterozygosity of the total
population, assuming random mating within subpopulations and no divergence
of allele frequencies among subpopulations. Fixation indices are interrelated as
follows: 1 – FIT = (1 – FST) (1 – FIS) (Wright, 1977). It varies from 0 (no isolation)
to 1 (complete isolation), but in the case of a bias towards polymorphic loci, lower
values (e.g. 0.5) may indicate complete isolation between populations.
Gene Flow (Nm)

The parameter of gene flow (Nm) is the product of the effective size of individual
populations (N) and the rate of migration among them (m). However, it is difficult
to measure the rate of gene flow directly by tracking individuals and also to infer
its value mathematically. However, commonly used approach is to make an
estimation based on the degree of genetic differentiation among populations by
calculating F-statistics. FST is related to population size and migration rate, based
on a standard island model, which is not applicable to every natural population,
but provides an easy way to calculate gene flow as the following: Nm = (1/FST –
1) (McDonald and McDermott,1993). Gene flow among fragmented populations
is related to dispersal ability, so it is expected to provide information about the
dispersal ability of the organism and the degree of isolation among populations.
Shannon’s Evenness measure

The degree of evenness in species abundance is calculated by the Shannon’s
evenness measure. The maximum diversity (Hmax) could occur when all species has
equal abundance, in other words if H’=Hmax =lnS. The ratio of observed diversity
to maximum diversity can, therefore, be used to measure evenness (J’) (Pielou,
1975) therefore, J’= H’/Hmax = H’/ln S.
Heip’s Index of Evenness

Heip (1974) opined that the evenness measure should be independent of species
richness and that they should have a low value in contexts where evenness in
very low. The proposed measured was EHeip= (eH’ - 1)/ (S-1). The minimum value
of Heip’s measure is 0 and that it registers 0.006 when an extremely uneven
community is used (Smith and Wilson, 1996).
SHE Analysis
Shannon’s index describes two aspects of diversity: species richness and evenness.
Sometimes this notion makes interpretation difficult because the increase in
diversity may arise either as a result of greater richness or greater evenness or both.
Buzas and Hayek (1996) and Hayek and Buzas (1997) pointed out that Shannon’s
index is simply the sum of the natural log of evenness value (lnE) (where E= eH/S)
and natural log of species richness (lnS). This decomposes the Shannon’s index
into its two components H’= lnE + lnS. This allows the investigators to interpret
the changes in diversity pattern in a spatial or temporal scale. The SHE analysis
describes a relationship between the S (species richness), H (diversity measured by
Shannon’s index) and E (evenness). When a large number of samples are counted
or estimated there are five scenarios possible (Hayek and Buzas, 1997):
i. S1=S2, H1=H2, E1=E2; identical richness, evenness and relative

abundance.
ii. S1=S2, H1≠H2, E1≠E2; richness remains constant but evenness changes.
iii. S1≠S2, H1=H2, E1≠E2; H remains constant because changes in S and E
offset each other.
iv. S1≠S2, H1≠H2, E1=E2; E remains constant but S and therefore H changes
v. S1≠S2, H≠H2, E1≠E2, H changes because differences in S and E do not
offset each other.
Phylogenetic Diversity
Besides estimation of genetic diversity, it is also obvious in molecular ecological
studies to represent the genetic structure of the population(s) examined. Such
information is extremely valuable because it can address many important issues
such as estimation of migration pattern, identification of conservation units, or
resolution of biogeographical patterns (Mantel et al., 2005). When geographical
locations are available for populations and sampling allows the detection of
spatial structure, then tree-based methods should be considered. These methods
allow graphical representation of the relatedness of individuals and exploration of
information on the spatial structure of populations. Therefore, the results can be
evaluated in the light recent dispersal or/and other biogeographical patterns, genetic
differentiation or even migration-drift equilibrium. The simultaneous evaluation
of the results of tree-based methods coupled with the distribution of individual
markers within and among identified groups provides some information on the
distribution of genetic diversity (Bonin et al., 2007). Knowledge of phylogenetic
structure of genetic variation in populations is essential for understanding what
the genetic diversity within a given species is likely to represent as part of the total
diversity (Pleijel and Rouse, 2000).
Faith (1992) showed how the sum of the branch lengths on a minimum-spanning
path between any set of terminals on a cladogram could be used to calculate a
metric he termed Phylogenetic Diversity (PD) for that set. PD is important not
only because species in isolated, monotypic lineages usually represent much
more unique genetic diversity than ones in younger, more speciose groups, but
also because the very nature of these species means that they may actually be
disproportionately threatened by extinction (Purvis et al., 2000). Isaac et al.
(2007) devised a measure they termed Evolutionary Distinctiveness (ED), which
represents the relative contribution of an individual species to a clade’s PD. They
further showed how extinction risk could be combined with ED to identify species
that are both Evolutionarily Distinct and Globally Endangered (EDGE) species.
Hierarchical Gene Diversity Analyses

One of the easiest approaches to define the Evolutionary Significant Unit (ESU) is
to ascertain the hierarchical gene diversity analysis and it is based on that species
is situated at a spatially genetic hierarchical structure. Therefore, genetic diversity
can be partitioned into within population and among population components and
determine where biologically significant breaks occur into the genetic diversity.
Hierarchical Populations
Structure
First Order A and B
First Order C and D
Second Order A, B and C
Second Order D, E and F
Third Order A, B, C and

D, E, F
Fourth Order A, B, C, D,
E, F and G
Figure 3: Genetic hierarchical model of seven different populations. The

populations are arranged in a hierarchical order, viz., first order, second order, third
order and fourth order populations. The different shaded areas represent different
orders of the hierarchy. The triangles indicate the collection spots and curved line
indicate the river streams (adapted from Meffe and Carroll, 1997).
At the lowest level of hierarchy inbreeding individuals are genetically more similar.
As move through the hierarchy the genetic differences among more geographically
separated populations are greater (Meffe and Carroll, 1997). Genetic hierarchy
occurs because the divergence component of diversity (Dpt) can be subdivided into
geographic hierarchy in the distribution of species. Therefore, genetic divergence
exists between different populations because of different level of gene flow among
them. Populations that are geographically proximate show continuous gene flow
and are genetically more similar than the more distant populations. Thus a species
can be visualized as having a spatial genetic architecture. Perhaps the clearer picture
of genetic and geographic hierarchy is observed in riverine species because river
forms a geographic structure with natural hierarchy (Meffe and Carroll, 1997).
The river forms the first order, second order and so on hierarchical structures. The
first order stream joins to form second order streams, and the second order streams
join to form the third order streams and so on. In figure 3, the first order hierarchy
constitutes the A and B populations, and C and D populations. The second order
hierarchy constitutes the A, B and C populations, and D, E and F populations. The
third order hierarchy constitutes the A, B, C and D, E, F populations. Whereas the
fourth order hierarchy constitute the A, B, C, D, E, F and G populations (Figure 3).
Therefore, the overall diversity of any river stream is determined by calculating
genetic diversity and genetic divergence of the individual hierarchical order of
the river stream and finally adding the values of genetic diversity and genetic
divergence of the individual first, second, third and fourth hierarchical orders.
Table 1: List of some freely available software used to analyze genetic diversity.
Software OS Analyses performed Source Reference
CERVUS Windows Estimates parentage inference and Freeware from Marshall et

v. 1.0 estimates confidence, allele frequencies, http://helios.bto. al. (1998)
determine critical values of likelihood, ed.ac.uk/evolgen/
analyse parentage in populations. index.html
Relatedness Mac Estimates genetic relatedness, calculates Freeware from Queller and
v. 5.0.4 symmetrical and asymmetrical relatedness http://www.bioc. Goodnight
and jackknife standard errors. rice.edu/~kfg/ (1989)
GSoft.html
Analyse Mac Likelihood analysis, estimating Fst, Fis, Freeware from Barton and
v. 2.0 and standardized linkage disequilibrium, http://helios.bto. Baird (1998)
estimating variation between clines. ed.ac.uk/evolgen/
index.html
Arlequin Windows Calculates gene and nucleotide diversity, Freeware from Schneider et
v. 1.1 mismatch distribution, http://anthropolo al.(1997)
haplotype frequencies, linkage gie.unige.ch/
disequilibrium, tests of Hardy-Weinberg arlequin
equilibrium, neutrality tests, pairwise
genetic distances,
AMOVA.
DnaSP Windows DNA sequence variation within and Freeware from Rozas and
v. 2.52 between populations, also linkage http://www.bio Rozas (1995,
disequilibrium, recombination, gene flow, .ub.es/~julio/Dn 1997)
gene conversion parameters, and neutrality aSP.html
test.
Fstat DOS/ Gene diversity, computes jackknifing Freeware by Goudet
v. 1.2 Windows and bootstraping using a permutation writing to J. (1995)
algorithm. Goudetatjerome.
goudet@izea.
unil.ch
GDA Windows Standard gene diversity measures, Freeware; Weir (1996)
F-statistics, genetic distance http://chee.unm.
matrices, UPGMA and neighbor- edu/gda
joining dendrograms, exact tests for
disequilibrium
GENEPOP DOS Exact tests for Hardy-Weinberg Freeware from 3 Raymond
v. 3.1b equilibrium, population differentiation, ftp sites: and
genotypic disequilibrium, allele ftp://ftp.cefe.cnr Rousset
frequencies, Fst, and other correlations, s-op.fr/genepop/ (1995a, b)
linkage disequilibrium. ftp://ftp2.cefe.cn
rs-mop.fr/pub/pc/
msdos/genepop/
ftp://isem.isem.u
nivmontp2.fr/pub/
pc/genepop/
GENETIX Windows/NT Genetic distances, variability parameters, Freeware from Belkhir et al.
v. 3.3 Wright’s fixation indices, linkage http://www. (1998)
disequilibrium. univmontp2.
fr/~genetix/
genetix.htm
Migrlib Unix Estimates the pattern of migration in Freeware from Tufto et al.
v. 1.0 a subdivided population from genetic http://www.math (1996)
differences likelihood ratio tests between .ntnu.no/~jarlet/
alternative models such as the island migration
model and the stepping stone model.
PMLE12 Windows or Gene flow maximum likelihood. Freeware from Rannala and
v. 1.2 Mac For discrete generationisland model, http://mw511.biol. Hartigan
theta=2Nm. For acontinuous-generation berkeley.edu/ (1996)
islandmodel, theta is the ratio ofthe bruce/exec.html
immigration rate phi tothe individual birth
rate lambda.
POPGENE Windows Genetic diversity measures, tests of Hardy- Freeware from Yeh and
v. 1.21, Weinberg Equilibrium, F-statistics, genetic http://www.ualb Boyle
distances, UPGMA dendrogram, neutrality erta.ca/~fyeh/ (1997); Yeh
tests, linkage disequilibrium. index.htm et al.(1997)
RSTCALC Windows Population structure analyses, genetic Freeware from Goodman

v. 2.2 differentiation, and gene flow. Calculates http://helios.bto. (1997)
estimates of Rst, tests for significance and ed.ac.uk/evolgen
calculates 95% CI.
TFPGA Windows Calculates descriptive statistics, genetic Freeware from Miller (1997)
distances, and F-statistics. Performs tests http://herb.bio.
for Hardy-Weinberg equilibrium, exact nau.edu/~miller
tests for genetic differentiation,
Mantel tests, and UPGMAcluster analyses.
Bottleneck Windows Effective population size, the observed Freeware from Cornuet
v. 1.1.03 heterozygosity, expected heterozygosity, http://www.ensam. and Luikart
expected at mutation-drift equilibrium and inra.fr/~piry (1997)
assuming a given mutation model
Fluctuate Windows, Estimates the effective population size Freeware from Kuhner et al.
v. 1.50B Mac, UNIX and an exponential growth rate of a single http://evolution. (1995, 1998)
population using maximum likelihood and genetics.washin
Metropolis-Hastings importance sampling gton.edu/lamarc.
of coalescent genealogies. html
Migrate-0.4 Windows, Menu driven, character-based program that Freeware from Kuhner et al.
v. 0.4.3 Mac, finds 4+1maximum-likelihoodestimates http://evolution. (1995, 1998)
LINUX of population parameters for a two genetics.
population model. washington.edu/
lamarc.html
Migrate-n Windows, Menu driven, character based program Freeware from Kuhner et al.
v. Alpha- Mac, that finds n*nmaximum-likelihood http://evolution. (1995, 1998)
LINUX estimates of population parameters for genetics.
n-populationmodel. washington.edu/
lamarc.html
Recombine Windows95, Fits a model which has a single Freeware from Kuhner et al.
v. 1.0, NT, population of constant size with a single http://evolution. (1995, 1998)
Mac, recombination rate across all sites. It genetics.
LINUX estimates 4Nu and r, where N is the washington.edu/
effective population size, u is the neutral lamarc.html
mutation rate per site, and r is the ratio of
the per-site recombination rate to the per-
site mutation rate.
SITES DOS, Generates tables of polymorphic sites, Freeware from Hey and
v. 1.1 Mac indels, codon usage. Computes numbers http://heylab. Wakeley
of synonymous and replacement base rutgers.edu/index. (1997)
positions, pairwise sequence differences html#software Wakeley and
and GC content. Performs group Hey(1997)
comparisons and polymorphism analyses
and estimates historical population
parameters. Primarily intended for datasets
with multiple closely related sequences.
Conclusion
The study of genetic diversity is very much essential from the standpoint of
conservation and management purpose. Presently earth faces a huge threat to
genetic diversity, especially to several endemic and threatened taxa. The genetic
diversity of several species has gradually degraded and might be reaching a
critical condition in near future. Several anthropogenic and environmental
pressures are the main causative agents behind this phenomenon. Therefore, the
proper identification and estimation of genetic diversity of such critical/threatened
species is important for proper conservation and management purposes. Previously
developed molecular tools and statistical procedures and recent advances in
molecular genetics methods open a new door for better understanding the
particular conservation and management unit and allow investigators to answer
the empirical questions of genetic diversity regarding the conservation concern.
The recent advances in computational biology, mathematical biology, biostatistics
and bioinformatics have opened a new window for the researchers to investigate
the questions concerning genetic diversity and conservation in unequivocal way.
The above review gives a clearer insight into the importance and ecological
consequences of genetic diversity degradation. Moreover, it also gives some idea
regarding the basic molecular and analytical statistical tools used in the genetic
diversity study. Although not exhaustive, Table 1 lists some of the freely available
computer softwares, with their utilities and sources that implement analyses of
genetic diversity. Diversity clearly makes the world a more interesting place
and the idea that we should conserve genetic diversity is therefore tempting. Yet
when we imagine imposing genetic diversity to secure this same good, its value
is revealed as elusive, especially if we concede recognize it must be achieved at
the cost of the well being of some individuals whose existence has been used to
produce a benefit enjoyed mainly by others.
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Genetic Diversity: Its Importance and Measurements.

Chapter · October 2016

Tanmay Mukhopadhyay Soumen Bhattacharjee

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CONSERVING BIOLOGICAL DIVERSITY: A MULTISCALED APPROACH

Tanmay Mukhopadhyay1 and Soumen Bhattacharjee1*

which support the ecological consequences on genetic diversity. Information

Keywords: Ecology and genetic diversity, Genetic diversity indices, Genetic

Information about genetic diversity is necessary for the development of appropriate

Importance of Genetic Diversity

Effect on the individuals

Effect on the populations

Phenotypes and functions are dependent on genetic variability

Genetic diversity helps to adapt to environmental variability

environment, then a relatively narrow range of phenotypes might be optimally

Plant populations often include individuals with a range of phenological calendar.

Ecology and Genetic diversity

Figure 1: Processes underlying potential direct and indirect impacts of genetic

Secondly, there is a growing interest on the ecological effects of the variance

communities (Tovar-Sanchez and Oyama, 2006). Effects of genetic diversity on

Molecular tools to assess Genetic Diversity

Restriction Fragment Length Polymorphism (RFLP)

and transpositions which result in the loss or gain of a restriction endonuclease

Randomly Amplified Polymorphic DNA (RAPD or AP-PCR)

Inter-Simple Sequence Repeat (ISSR)

Simple Sequence Repeats (SSR) or Microsatellites

Amplified Fragment Length Polymorphisms (AFLP)

Single Nucleotide Polymorphism (SNP’s)

Diversity Array Technology (DArT)

Restriction Site-Associated DNA (RAD)

Single Feature Polymorphism (SFP)

Internal Transcribed Spacer (ITS)

Conserved DNA-Derived Polymorphism (CDDP)

Cytochrome P450 Based Analogues (PBA)

Start Codon Targeted (SCoT) Polymorphism

Sequence-Related Amplified Polymorphism (SRAP) and

number of polymorphic fragments could amplified in each reaction, (ii) no need

Conserved Region Amplification Polymorphism (CoRAP)

Mobile element based molecular markers

Analytical Tools to estimate Genetic Diversity

Hardy-Weinberg equilibrium test (HWT)

Average number of Alleles per locus

Effective Number of Alleles (Ne)

Average Expected Heterozygosity (He)

Shannon’s Information Index (I)

Allelic Diversity (A)

Effective Population size (Ne) and Minimum Viable population

Figure 2. Extinction vortex. Population size decreases in a positive feedback loop,

other words, population substructure results in a reduction of heterozygosity. The

Gene Flow (Nm)

Shannon’s Evenness measure

Heip’s Index of Evenness

community is used (Smith and Wilson, 1996).

i. S1=S2, H1=H2, E1=E2; identical richness, evenness and relative

diversity (Pleijel and Rouse, 2000).

Hierarchical Gene Diversity Analyses