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AN ASSIGNMENT ON

APPLICATION OF MOLECULAR MARKERS IN FISHERIES

INTRODUCTION:

All organisms are subject to mutations because of normal cellular operations or

interactions with the environment, leading to genetic variation (polymorphism).

Genetic variation in a species enhances the capability of organism to adapt to

changing environment and is necessary for survival of the species. In conjunction

with other evolutionary forces like selection and genetic drift, genetic variation arises

between individuals leading to differentiation at the level of population, species and

higher order taxonomic groups. Molecular genetic markers are powerful tools to

detect genetic uniqueness of individuals, populations or species. These markers

have revolutionized the analytical power, necessary to explore the genetic diversity.

The conclusion from genetic diversity data has varied application in research on

evolution, conservation and management of natural resources and genetic

improvement programs etc.

MOLECULAR MARKER:

A molecular marker is a DNA sequence in the genome which can be located and

identified. As a result of genetic alterations (mutations, insertions, deletions), the

base composition at a particular location of the genome may be different in different

plants.

These differences, collectively called as polymorphisms can be mapped and

identified. Plant breeders always prefer to detect the gene as the molecular marker,

although this is not always possible. The alternative is to have markers which are

closely associated with genes and inherited together.

MOLECULAR MARKERS ARE OF TWO TYPES:

1. Based on nucleic acid (DNA) hybridization (non-PCR based approaches).

2. Based on PCR amplification (PCR-based approaches).

MARKERS BASED ON DNA HYBRIDIZATION:

The DNA piece can be cloned, and allowed to hybridize with the genomic DNA which

can be detected. Marker-based DNA hybridization is widely used. The major

limitation of this approach is that it requires large quantities of DNA and the use of

radioactivity (labeled probes).

Example: Restriction fragment length polymorphism (RFLP)

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MARKERS BASED ON PCR AMPLIFICATION:

Polymerase chain reaction (PCR) is a novel technique for the amplification of

selected regions of DNA. The advantage with PCR is that even a minute quantity of

DNA can be amplified. Thus, PCR-based molecular markers require only a small

quantity of DNA to start with.

PCR-BASED MARKERS MAY BE DIVIDED INTO TWO TYPES:

1. Locus non-specific markers e.g. random amplified polymorphic DNA (RAPD);

amplified fragment length polymorphism (AFLP).

2. Locus specific markers e.g. simple sequence repeats (SSR); single nucleotide

polymorphism (SNP).

APPLICATION OF MOLECULAR MARKERS FOR IDENTIFICATION OF FISH

SPECIES:

The inter-specific genetic divergence established through species specific

diagnostic molecular markers provides precise knowledge on phylogenetic

relationships and also resolve taxonomic ambiguities.

These markers can be used to detect hybrid and introgressed or backcrossed

individuals, distinguish early life history stage of morphologically close species

both in hatchery and in natural populations. Species-specific allozyme

markers have been identified in many fishes.

Specific diagnostic allozyme loci were used for different species: apache trout

(Oncorhynchus apache), cutthroat (Oncorhynchus clarki) and rainbow trout

(Oncorhynchus mykiss) and Gambusia affinis and G. holbrooki. Allozyme

markers have also been used for individual classification in cyprinid species

Zacco pachycephalus and Z. platypus in cyprinodontid species V. letourneuxi

and V. hispanica in mullets Mullus barbatus and M. surmuletus and hake

species Merluccius australis and M.hubbsi.

Species-specific diagnostic RAPD fingerprints were generated in several fish

species and their taxonomic relationship has been analyzed.

The RAPD-PCR technique was employed to identify three endemic

morphologically similar Spanish species of Barbus: Barbus bocagei, B.

graellsii and B. sclateri that have similar morphologies.

RAPD markers were characterized to identify five species of family

Cyprinidae: Chondrostoma lemmingii, Leuciscus pyrenaicus, Barbus bocagei,

Barbus comizo, all endemic in the Iberian Peninsula, and introduced Alburnus

alburnus, for studying genetic relationship and diversities in four species of

Indian Major carps (family Cyprinidae): rohu (Labeo rohita), kalbasu (L.

calbasu), catla (Catla catla) and mrigal (Cirrhinus mrigala) [89], for

identification of three eel species, A. japonica, A. australis and A. bicolor and

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to estimate the population structure and phylogenetic relationships among the

eight species of the genus Barbus .

Large variation in mtDNA sequences among species can be utilized to

produce species-specific markers. Since the structures of mitochondrial RNA

genes (tRNA and rRNA) and the functional molecule of the 16S rRNA are

highly conserved among the animal taxa that are related even distantly,

change of even few nucleotides in such a gene between closely related taxa

might indicate a substantial degree of genetic divergence.

Mt-DNA sequences have been used as useful marker for species-specific

identification in many fishes.

GENETIC VARIATION AND POPULATION STRUCTURE STUDY IN NATURAL

POPULATIONS:

Molecular markers provide direct assessment of pattern and distribution of

genetic variation thus helping in answering, “if the population is single unit or

composed of subunits”.

Several evolutionary forces affect the amount and distribution of genetic

variation among populations and thereby population differentiation.

Geographic distance and physical barriers enhance reproductive isolation by

limiting the migration and increase genetic differentiation between

populations.

Impact of migration and gene flow on genetic differentiation also depends

upon effective size of receiving population and number of migrants.

Increased computational power and mathematical models have enhanced the

scope of conclusions that can be drawn out of genotype data generated

through molecular markers.

Some of the possibilities are assignment of migrants, determination of genetic

bottleneck, effective breeding population estimates besides genetic variation

and differentiation estimations. Fifteen random primers were used to analyze

the genome DNA of Jian carp (Cyprinus carpio varjian) by the RAPD

technique.

Study on cold tolerant traits for common carp Cyprinus carpio was conducted

by Chang and nine RAPD-PCR markers associated with cold tolerance of

common carp were identified.

The genetic diversity has been studied using RAPD markers in Carassius

auratus, Epinephelus merra population and Solea solea. Genetic variation

have been assessed with Allozyme and RAPD markers on Mullus surmuletus

L., and three species of Pimelodidae catfish. Population structure has been

examined using microsatellite markers of sockeye salmon, Chinook salmon

and Arctic charr populations.

Genetic variation have been assessed using microsatellite genetic markers to

identify the population structure of brook charr, Salvelinus fontinalis and 14

populations of northern pike (Esox lucius) in the North Central United States

and in six populations from Quebec, Alaska, Siberia, and Finland.

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(Reg No:12-05-2859, From Introduction To Genetic Variation And

Population Structures Study In Natural Population)

COMPARISON OF GENETIC VARIATION BETWEEN WILD AND HATCHERY

POPULATIONS:

Molecular markers also find application in aquaculture to assess loss of

genetic variation in hatcheries through, comparison of variation estimates

between hatchery stocks and wild counterparts.

The information is useful obtained in monitoring farmed stocks against

inbreeding loss and to plan genetic up gradation programs.

A major aspect such studies address is concerned with the assessment of

farm escapes into the natural population and introgression of wild genome.

Brook trout Salvelinus fontinalis from unstocked waters, naturalized lakes, and

hatcheries were analyzed electrophoretically for allozyme expression.

All wild-unstocked samples were highly differentiated populations and

significantly different from each other and from hatchery samples.

Genetic diversity was investigated using microsatellites between farmed and

wild populations of Atlantic salmon. Farmed salmon showed less genetic

variability than natural source population in terms of allelic diversity.

Variation in allozymes and three microsatellite loci was assessed in

populations of wild and cultured stocks of Sparus aurata and Sparius auratus.

The microsatellite heterozygosity values were high in wild, but lower in the

cultured samples.

DNA MARKER DEVELOPMENT IN AQUACULTURE

SPECIES, STRAIN, AND HYBRID IDENTIFICATION:

Genetic identification of species or strains is sometimes required in an

aquaculture setting. Because of the major genetic differences among most

species, their identification using DNA markers is relatively straightforward.

RFLP, RAPD, AFLP, and microsatellite markers are all applicable, but RAPD

analysis probably provides the least expensive, yet reliable identification of

species if no prior molecular information is available.

Each species will generally exhibit a RAPD profile with unique binding

patterns, and a simple comparison of profiles generated using one or two

primers should be sufficient for species identification.

Species identification is often required for determining whether fish stocks are

pure species or hybrids, a problem often seen in tilapia. Both RAPD and

AFLP analyses can provide rapid solutions; the dominant nature of these

markers means that hybrid fish should have a gel profile that combines the

unique dominant bands from each parent.

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Use of both RAPDs and AFLPs for the analysis of situations involving

hybridization and introgression beyond the F1 generation is more

complicated, however, due to the unresolved nature of the dominant bands so

that breeding studies to determine the Mendelian inheritance of species-

specific bands becomes mandatory.

Strain identification is more complicated, since fixed, strain-specific markers

are not usually available for strains within a species.

The amount of genetic variation among strains may be limited, and may

require DNA markers and techniques with higher resolution than traditional

markers such as allozymes, RFLPs, or RAPDs. Both microsatellites and

AFLPs have been shown to provide sufficient power for the determination of

strains in aquaculture fish species. The use of allele frequency analysis

across multiple microsatellite loci is a powerful approach for delineation of

individual strains.

Allele frequencies for each microsatellite locus are estimated for each strain

involved and those microsatellites that have highly differential allele

frequencies among strains are used for strain identification.

This approach has been used in strain identification of catfish. For more

technical details, readers are also referred to similar applications in

microorganisms. In contrast to microsatellites, AFLP markers typically have

only two alleles per locus, and are treated as dominant markers with either the

presence or absence of the band.

The large number of loci typically generated simultaneously in an AFLP

analysis makes up for the lack of large numbers of alleles per locus.

As in the case of microsatellites, allele frequencies of multiple loci are

combined to delineate strains. The AFLP approach has been used to identify

strains of common carp and channel catfish.

(Reg No:12-05-2835, From Comparison of Genetic Variation Between Wild and

Hatchery Populations to DNA Marker Development In Aquaculture)

GENETIC DIVERSITY AND RESOURCE ANALYSIS OF AQUACULTURE

STOCKS:

In spite of a long aquaculture history, aquaculture broodstocks are not well

characterized genetically. Many strains/lines of a specific aquaculture species

may be used, and the precise genetic relationship among strains is most often

unknown. Questions arise as to how diverse is the genetic background of

aquaculture broodstocks and how the domestic stocks differ from their wild

counterparts.

As with strain identification, analyses of genetic diversity and resources

require methodologies that exhibit high powers of resolution to reveal genetic

variations among the stocks.

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Traditionally, allozymes and mtDNA have been most frequently used in fishes,

but their differentiating power is limited compared to more recently developed

markers such as RAPDs, AFLPs, and microsatellites (as discussed earlier

under the sections dealing with marker types).

Among these newer marker systems, RAPDs have the least differentiation

power.

AFLPs and microsatellites should be highly powerful in revealing genetic

diversities. In the author’s experience, AFLP markers have proven very useful

for genetic diversity analyses because of the large number of loci that can be

screened simultaneously.

Bagley et al. (2001) compared the use of RAPDs and AFLPs in rainbow trout

and concluded that AFLP is the method of choice for the analysis of genetic

diversity.

PARENTAL ASSIGNMENTS AND REPRODUCTIVE CONTRIBUTION:

Fishes have some of the most complex mating systems known in the animal

kingdom.

Effective methods of traceability are required for basic research, different

types of aquaculture operations, and to control the trade in aquatic animals

and products. With the advent of powerful genetic markers and an emerging

mathematical framework to calculate parentage, it is now possible to analyze

genetic relatedness and inheritance in these systems.

For parental assignments, microsatellites provide the best results, since

genetic variation among individuals is extremely high with microsatellites,

while polymorphism in other types of markers is generally low among

individuals of the same strain.

The large numbers of alleles and high levels of detectable polymorphism

exhibited by microsatellites make obtaining unique genotypes for every

individual in a study feasible. Of course, the larger the population, the greater

the number of required microsatellite loci.

(Reg No:12-05-2864, From Genetic Diversity and Resource Analysis Of

Aquaculture Stocks To Parental Assignments And Reproductive Contribution)

DNA MARKERS, QUANTITATIVE TRAIT LOCI (QTL), AND MARKER-ASSISTED

SELECTION (MAS):

Benefits of the expanded molecular genetics technologies currently available

include their potential for greatly enhancing traditional breeding techniques.

Since most, if not all, performance and production traits are controlled by

multiple genes and therefore inherited as quantitative traits, analysis of their

associated quantitative trait loci (QTL) is emerging as a very important part of

aquaculture genetics/genomics. QTL are largely unidentified genes that affect

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performance traits (such as growth rate and disease resistance) that are

important to breeders.

Relative chromosomal positions of QTL in a species genome can be identified

in a two-step process that begins by constructing a genetic linkage map.

Genetic linkage maps are constructed by assigning (mapping out)

polymorphic DNA markers to chromosome configurations based on their

segregation relationships. This requires two elements: polymorphic DNA

markers, and families in which these markers segregate.

Once a linkage map has been constructed for a given species, it can be used

in combination with studies of breeding and assessment of quantitative traits

to identify markers that are closely associated (linked) to QTL of interest, thus

allowing the QTL to be positioned on the linkage map.

This information can then be used to aid aquaculture personnel in efficiently

crossing different strains of cultured species to maximize growth, disease

resistance, or some other desirable trait through marker assisted selection

(MAS). Typically, evenly spaced markers covering the entire genome are

selected for screening of trait-linked markers, and this process is known as a

genome scan of QTL.

Once the QTL are mapped to a chromosomal region, fine mapping can be

conducted using polymorphic markers near the chromosomal regions

containing the QTL.

A number of QTL have been mapped and characterized in aquaculture

species. In rainbow trout, QTL for upper thermal tolerance, spawning time,

and embryonic development rate have been mapped.

In tilapia, a collaborative group involving Israeli and US scientists has

developed a hybrid tilapia stock for the analysis of linkage mapping and QTL

analysis.

In addition, loci associated with deleterious alleles and distorted sex ratios,

QTL controlling body color and sex determination and QTL controlling a

number of biochemical parameters related to innate immunity response to

stress have been recently identified in tilapia. Several markers have been

identified in catfish that are linked to feed conversion efficiency.

Several groups are now working on QTL for disease resistance, and two

putative QTL have been identified to be associated with

resistance/susceptibility to infectious pancreatic necrosis virus (IPNV) in

rainbow trout.

With the availability of resource families and DNA markers, it is expected that

greater successes will be achieved in the near future in QTL mapping in

aquaculture species, which will eventually lead to marker-assisted selection

(MAS).

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FUTURE APPLICATIONS OF DNA MARKERS IN AQUACULTURE GENETICS:

In addition to genome mapping and the other applications discussed in this

review, DNA markers are likely to prove useful in many other aspects of

aquaculture.

The development and application of DNA marker technologies already

underway in other areas such as molecular systematics, population genetics,

evolutionary biology, molecular ecology, conservation genetics, and seafood

safety monitoring will undoubtedly impact the aquaculture industry in

unforeseen ways. Already, lessons learned from studies in population and

conservation genetics are changing the very role that hatcheries and

aquaculture play for augmentation and restoration of wild fish stocks such as

salmon and trout.

Advances in aquaculture genomics are also likely to affect other areas

utilizing molecular markers as well.

Although it may take some time to implement marker-assisted selection in

aquaculture, the techniques of genome mapping and QTL analysis used to

support MAS will eventually also be used to identify and clone genes that

could prove to be economically important outside of the aquaculture arena,

and find applications in medicine and other bio-related industries.

CONCLUSION:

The development of DNA-based genetic markers has had a revolutionary impact on

animal genetics. With DNA markers, it is theoretically possible to and exploit genetic

variation in the entire genome. Popular genetic markers in the aquaculture

community include allozymes, mitochondrial DNA, RFLP, RAPD, AFLP,

microsatellite, SNP, and EST markers. The application of DNA markers has rapid in

aquaculture Investigations of genetic variability and in breeding parentage

assignments, species and strain identification, and the construction of genetic

linkage maps for aquaculture species. Studies using these genetic markers Will

undoubtedly accelerate identification of involved in quantitative trait (QTL) for

marker-assisted selection.

(Reg No:12-05-2853, From DNA Markers, Quantitative Trait Loci (QTL), And

Marker-Assisted Selection (MAS) To Conclusion)

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