Molecular Markers & Its application

• Genetic engineering and biotechnology

through the use of molecular markers like
isoenzymes, restriction fragment length
polymorphisms (RFLPs), random amplified
polymorphic DNAs (RAPDs), amplified
fragment length polymorphisms (AFLPs),
simple sequence repeats (SSRs), and
sequence–tagged sites (STSs) are providing
new tools for breeding of plant crops.
• What are molecular markers?
• -Heritable DNA sequence differences
(polymorphisms)
• – Phenotypically neutral, developmentally and
environmentally stable
• – Identified by techniques such as Southern
hybridizations or PCR
•
Uses of molecular markers

• – Application as a genetic tool for plant

genotyping and gene mapping
• -Application in agriculture
• •Marker-assisted breeding
• •Plant variety protection
• •Assessing genetic diversity
• – Applications in Human Health
• •Association with genetic-based diseases
• •Forensic studies
Types of Markers
• –Those detected by Southern Hybridizations
• RFLPs --Restriction Fragment Length
Polymorphisms
• VNTRs -- variable number of tandem repeats
(minisatellites)
• –Those detected by PCR-based methods
• •RAPD -- randomly amplified polymorphic DNA
• •AFLP -- amplification fragment length
polymorphism
• •CAPS -- cleaved amplified polymorphic site
• •SSR -- simple sequence repeats (microsatellites)
• •SNP -- single nucleotide polymorphisms
 Why we use molecular markers
• The best molecular markers are those that
distinguish multiple alleles per locus (i.e. are
highly polymorphic) and are co-dominant
(each allele can be observed)
• Advantages such as rapidity, non-
interference by the environment and
accuracy during selection have made the
marker-assisted selection as the most reliable
tool for identifying agronomically important
traits.
• Modern crop varieties have been developed
from plant populations that exhibiting genetic
variability.
• Hybrid cultivars are developed from a
population improved previously through
inbreeding and crossing.
• Selection, as a systematic process, consists of
identifying superior individuals co-existing
within the population under improvement,
and the best individuals are used as parents
for producing the next generation, which is
theoretically better than the original
population.
• In other words, the selection changes the genetic
structure of the populations as a result of
preserving superior alleles and discarding the
undesirable ones.
• Crop improvement by selection primarily
depends on
1. discovery and generation of genetic variability in
agronomic traits, and
2. precise selection of genotypes with favorable
characteristics, as a product of a recombination
among superior alleles at different loci .
• Additionally, in all breeding programs the
breeders are interested in maximizing the
efficiency of the selection that permits
differentiation of the best genotypes.
• In cases where the selection is based only upon
the phenotypic values, the breeders commonly
confront the problem of genotype environment
interaction (GEI), which may mask favorable
genotypes making selection more difficult.
• New exigencies and challenges for increasing
the yield of crops have led to the generation
of new genetic tools that have enhanced the
effectiveness of selection.
• Novel genetic tools make use of several
molecular marker techniques, which permits
establishment of linkage maps in crop plants.
• In addition, these linkage maps allow
breeders to conduct applied research to
identify, characterize, and use genetic
variability in economically important plants
• The main reasons supporting the utilization of
molecular markers in national breeding
programs are the heritability of the markers
(100 %), and their cost, which is potentially
lower than the conventional selection .
• Additionally, the proposed uses for the
molecular markers into plant improvement
are their application onto selection for traits
with low heritability, identification of
resistance or tolerance for biotic or abiotic
stresses in plants, and the gene introgression
coming from native or exotic germplasm .
• Other uses include estimation of genetic
relatedness among accessions, cultivar
description and the identification of
quantitative trait loci (QTL) linked to
molecular markers, which control important
agronomic traits .
 What is QTL
• Quantitative traits refer to phenotypes
(characteristics) that vary in degree and can be
attributed to polygenic effects, i.e., product of two or
more genes, and their environment.
• Quantitative trait loci (QTLs) are stretches of DNA
containing or linked to the genes that underlie a
quantitative trait.
• Mapping regions of the genome that contain genes
involved in specifying a quantitative trait is done using
molecular tags such as AFLP or, more commonly SNPs.
This is an early step in identifying and sequencing the
actual genes underlying trait variation.
Molecular Markers in Crop Improvement
• Conventional crop improvement has
developed a large number of varieties, which
have been generated following strategies
based on the phenotypic values and in some
cases by using the progeny tests.
• Genotypic and environmental effects produce
phenotypic values over genotypes in plants
and other living organisms.
• Strategies using best phenotypes to infer the
best genotypes are commonly less efficient
than the schemes performing progeny tests.
These strategies spend more time on
selection, and consequently it is necessary to
invest major economic resources.
(disadvantage)
• On the other hand, it is convenient to
recognize the effects of quantitative or
qualitative genes on phenotypes.
• In general, it can be assumed that if the
substitution effect of an allele on the
phenotypic expression is small, the trait is
classified as quantitative, in turn, if the effect
is large the characteristic is considered as
qualitative.
• The selection using molecular markers can be
enhanced, and the efficiency of the molecular
marker will be higher if the markers are
closely linked to the genes controlling
quantitative and qualitative characters.
• Progress in crop improvement has been
reached by manipulating the genetic variation
within populations.
 Molecular markers & Crop
Improvements

• Molecular markers are being used for crop

improvement to achieve different objectives
such as measuring genetic diversity among
corn lines.
Marker-Assisted Selection (MAS)
• In plant breeding, MAS is a relatively new
concept, nevertheless the original selection
concept per se (by itself) has not changed,
that is, the purpose of the selection is to
search and preserve the best genotypes, but
using molecular markers.
• MAS can be used for manipulating both
qualitative and quantitative traits.
• A highly saturated marker linkage map is
necessary for effective marker based
selection.
• Basically, MAS consists of identifying
association between molecular markers and
genes, controlling agronomic traits, and using
these to improve lines or populations.
Comparison of Molecular Markers for Their
Advantages and Disadvantages.
• Type of markers : Restriction Fragment Length Polymorphism
(RFLP):
• Advantages
• -High genomic abundance
• -Co-dominant markers
• -Highly reproducible
• -Can use filters many times
• -Good genome coverage
• -Can be used across species
• -No sequence information
• -Can be used in plants reliably (well-tested)
• -Needed for map based cloning
•
• Disadvantages
• -Need large amount of good quality DNA
• -Laborious (compared to RAPD)
• -Difficult to automate
• -Need radioactive labeling
• -Cloning and characterization of probe are
required
 Randomly Amplified Polymorphic DNA (RAPD)

• Advantages:
• -High genomic abundance
• -Good genome coverage
• -No sequence information
• -Ideal for automation
• -Less amount of DNA (poor DNA acceptable)
• -No radioactive labeling
• -Relatively faster
• Disadvantages
• -No probe or primer information
• -Dominant markers
• -Not reproducible
• -Can not be used across species
• -Not very well-teste
 Simple Sequence Repeat (SSR)

• Advantages:
• -High genomic abundance
• -Highly reproducible
• -Fairly good genome coverage
• -High polymorphism
• -No radioactive labeling
• -Easy to automate
• -Multiple alleles
Disadvantages
• -Can not be used across species
• -Need sequence information
• -Not well-tested
 Amplified Fragment Length Polymorphism
(AFLP)
• Advantage:
• -High genomic abundance
• -High polymorphism
• -No need for sequence information
• -Can be used across species
• -Work with smaller RFLP fragments
• -Useful in preparing contig maps
• Disadvantage
• -Very tricky due to changes in
• patterns with respect to materials used
• -Cannot get consistent map (not reproducible)
• -Need to have very good primers
Sequence-Tagged Site (STS)
• Advantages:
• -Useful in preparing contig maps
• -No radioactive labeling
• -Fairly good genome coverage
• -Highly reproducible
• -Can use filters many times
• Disadvantage
• -Laborious
• -Cannot detect mutations out of the target
sites
• -Need sequence information
• -Cloning and characterization of probe are
required
• Disadvantage
• -Laborious
• -Limited in polymorphism
• -Expensive (each system is unique)
• -Have to know the location of the tissue
• -Not easily automated
• With respect to the application of MAS for
selecting quantitative traits, some researchers
have used molecular markers to locate loci
associated with quantitative characters.
• Studies in cereal crops have been carried out
to achieve the aim.