Genetic markers

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WELCOME
PRESENTED BY
R. ASHA
BAM-10-11
GENETIC MARKERS AND
PLANT GENETIC
RESOURCE
MANAGEMENT

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CONTENTS
1. Introduction
2. Kinds of plant genetic resources
3. Plant genetic resource management
4. Need of plant genetic resource management
5. How genetic markers are useful
6. Different marker techniques
7. Role of markers in plant genetic resource management
8. Conclusions

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• Germplasm of a crop may be defined as the sum total of
hereditary material i.e., all the alleles of various genes, present
in a crop species and its wild and weedy relatives. It is also
termed as Genetic resources.
• Represents entire genetic variability or diversity available in a
crop species.
• Plant genetic resources constitute an invaluable reservoir of
gene pools that are needed by plant breeders for development
of superior varieties.
• Basic material for launching a crop improvement programmes.

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Plant genetic resources are the building blocks andPlant genetic resources are the building blocks and
fundamental not only in crop improvementfundamental not only in crop improvement
programme, but also for the very survival of theprogramme, but also for the very survival of the
species in time and spacespecies in time and space..

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Plant genetic
Resources are
components of
biodiversity
which sustains
the humankind.

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Kinds of Plant Genetic Resources
a) Basic Genetic Resources
1. Wild taxa related to a crop species
2. Weedy forms
3. Land races or primitive cultivars
b) Derived Genetic Resources
4. Obsolete varieties
5. Breeding lines with particular genes and performances
6. Prebreeding materials
7. Advanced cultivars
8. Parents of hybrid varieties
9. Cytogenetic stocks/tester
10. Mutants

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Plant Genetic Resource Management
Plant genetic resource management or simply
germplasm management comprises 2 phases
1. Germplasm Conservation
2. Encouraging Utilization

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GERMPLASM
CONSERVATION

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1.Germplasm conservation
 Acquisition, or securing germplasm in situ or Ex situ
 Maintenance
- Monitoring and protecting germplasm in reserves or
storing it Ex situ under controlled conditions.
2.Encouraging utilization
Evaluation
Genetic enhancement

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NEED OF PLANT GENETIC
RESOURCE MANAGEMENT

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• Continued alteration of the environment by man has resulted
unprecedented loss of biodiversity.
• Many species have become extinct or are near extinction due
to destruction and fragmentation of their habitats.
• Consequent erosion of genetic diversity leads to reduced
resilience to environmental changes and altered ecosystem
processes.
• Therefore developing effective conservation strategies is of
fundamental importance (Newton et al., 1999)

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• The realization that the world was rapidly losing much of its
agrobiodiversity led to a global effort to collect and conserve
germplasm.
• An increasing awareness of the narrow genetic base of crops in
advanced agriculture and potential susceptibility to crop failures
further stimulated the efforts to collect & presents new challenges
to genebank managers to determine needs for new collections,
maintain existing collections, determine optimum regeneration
methods, characterize collections for useful agronomic traits,
classify the collections, and reduce the size of the working
collection to a manageable size (The Core collection concept:
Frankel 1984; Hamon et al., 1995).

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HOW GENETIC MARKERS ARE
USEFUL IN PLANT GENETIC
RESOURCE MANAGEMENT

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• The role of genetic markers in genetic enhancement is
considered in the context of germplasm management as a
whole (Duvick, 1990).
• Genetic markers may assist plant germplasm management,
which when properly conducted provides scientists with high
quality raw genetic materials for analysis and breeding.

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WHAT ARE THE DIFFERENT
MARKER TECHNIQUES
GENERALLY USED

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Markers
Any identifiable mark on an object
Represent genetic difference between individual organisms
or species
Act as signs or flags
Located in close proximity to genes (tightly linked)
Genetic markers are tags for genes
Markers should be discrete and heritable

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TYPES
PHENOTYPE
PROTEIN
RNA
DNA
Biochemical markers
DNA Markers
Morphological Markers

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Morphological
marker
Morphological
level
Protein
marker
Protein level
DNA
level
DNA
marker

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Morphological Markers
 Visually characterized phenotypic characters
 Flower colour ,seed shape , growth habit, pigmentation
 Germplasm characterization
 Indirect selection –
Purple Coleoptiles – BPH resistance in Rice

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Morphological markers

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Advantages:
 Inexpensive to score,
Amenable to experiments in natural populations
Disadvantages:
Visible polymorphisms relatively rare.
Most genetic variation not so easily observed (Variants are
ambiguous)
Genetic basis of variation can be complex, and is not necessarily
easy to determine

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Limitations
 Environment influence and influenced by many genes
 Do not represent the genome adequately
 No stable inheritance( Need repeated measures)

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Biochemical markers
 Isozymes – Allelic variants of enzymes
(Enzymes that differ in amino acid sequences but
catalyze the same chemical changes)
 Detected by electrophoresis and specific staining
 ELISA (Enzyme Linked Immunosorbant Assay)
 Viral disease identification
Example:
Wheat Bread making Quality- Gluten by SDS – PAGE
Rice Cooking Quality – Amylose

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Protein Allozymes:
Electrophoretic variants of proteins produced
by different alleles at protein-coding genes.
Protein Electrophoresis Gel
Total protein

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Selected
samples
Proteins
Western
Blot
Biochemical Markers

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Advantages:
• Inexpensive;
• Markers are co-dominant.
Disadvantages:
• Only reveals small proportion of DNA
variation.
• Many DNA variants do not result in
changes in amino acid sequence (e.g.,
synonymous substitutions).
• Some changes in amino acid sequence
do not result in changes in mobility on
the gel.
Using Protein Polymorphism

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Limitations
Low level of polymorphism
Expressed at protein/ amino acid
level
Environmental influence

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DNA Markers
 Identifiable DNA sequences found at specific
locations
 Located in the non coding region of DNA
 Do not have any biological effect
 Follows standard laws of inheritance
Markers
Polymorphic Monomorphic
1. Co dominant
2. Dominant
MK1
MK2
Gene
Chromosome

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B
A)
P1 F1 P2
Polymorphic Monomorphic
Codominant Dominant
Markers at DNA level
31

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Co dominant Dominant
Discriminate b/w homozygous & No discrimination
heterozygous
No progeny testing Progeny testing

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MOLECULAR MARKERS
 Molecular markers are based on the identification of
polymorphisms in DNA.
 They have been termed as Molecular Markers
(Tanksley 1983)
 Molecular marker is a DNA sequence readily detected and
whose inheritance can be easily found.

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Molecular Basis of DNA Markers
Base substitution
Insertion
Deletion
Inversion
Duplication
Translocation
Methylation
34

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MOLECULAR BASIS OF DNA MARKERSMOLECULAR BASIS OF DNA MARKERS
GAATTCGAATTC
CTTAAGCTTAAG
GAATTCGAATTC
CTTAAGCTTAAG
11)) Gain or loss of a restriction site, or PCR priming siteGain or loss of a restriction site, or PCR priming site
RFLP, AFLP, CAPSRFLP, AFLP, CAPS
GAATTCGAATTC
CTTAAGCTTAAG
GACTTCGACTTC
CTGAAGCTGAAG
GAATTCGAATTC
CTTAAGCTTAAG
RAPD, AP-PCR, DAFRAPD, AP-PCR, DAF
22) Insertion or deletion between restriction or priming sites) Insertion or deletion between restriction or priming sites
GAATTCGAATTC
CTTAAGCTTAAG
GAATTCGAATTC
CTTAAGCTTAAG
RFLP, AFLP, CAPSRFLP, AFLP, CAPS
RAPD, AP-PCR, DAFRAPD, AP-PCR, DAF
GAATTCGAATTC
CTTAAGCTTAAG
GAATTCGAATTC
CTTAAGCTTAAG
35

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• Co-dominant inheritance
• Highly polymorphic.
• Multi-functional.
• High reproducibility
• Frequent occurrence
• No environmental influence
• Ability to be automated.
• Easy access and exchange
PROPERTIES OF A GOOD MARKER
Co dominant Dominant
Within population sex-linked visible
polymorphism(STAG BEETLE)

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RFLP (1975)
Minisatellites
Microsatellites
RAPD (1990)
STS/SCAR (1991)
ISSR (1994)
AFLP (1995)
SNP (1999)
INDEL (1999)
Pre Genome
Sequencing
Post Genome
Sequencing
Before PCR boom
After PCR boom
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RFLP : The variation(s) in the length of DNA fragments produced by a
specific restriction endonuclease from genomic DNAs of two or
more individuals of a species

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(Devos, K. M. and M. D.Gale,2000.)
Restriction Fragment Length Polymorphism
(RFLP)

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Uses of RFLP
Direct identification of genotype in environment
independent manner .
They are co dominant markers & simple as no sequence
specific information is required.
Indirect selection using qualitative traits.
Tagging of monogenic traits with RFLP markers
Indirect selection using quantitative trait loci.

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AFLP :
Any difference
between corresponding
DNA fragments from two
organisms A and B, that is
detected by the amplified
restriction length
polymorphism.

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RAPD
Any DNA segment that is amplified using short
oligodeoxynucleotide primers of arbitrary nucleotide
sequence (amplifiers) and polymerase chain reaction
procedures. (Kahl,2001).
Laboratory steps are:
Isolating DNA
PCR reaction with a primer
Separating DNA fragments by gel electrophoresis
Visualizing DNA fragments, using ethidium bromide

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RAPD technology
A B C
Genomic DNA
+
Taq polymerase
+
Arbitrary primers
A
+
Nucleotides
+
Buffer
PCR
(under relaxed conditions)

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Advantages of RAPD
 small amount of DNA (15-25ng)
 Non radioactive assay
 Thermocycler- Agarose gel
 No probe is required,
 Efficient screening for DNA sequence –based polymorphism
at many loci
 It does not involve blotting or hybridization steps

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Limitations of RAPD
They are not co- dominant markers
The primers -short,
Sensitive to changes in PCR condition, resulting in changes to
some of amplified fragments

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APPLICATIONS:
 Construction of genetic maps
 Mapping of traits
 Indirect selection of segregating population
 Analysis of genetic structure of population
 Finger printing.
 Identification of somatic hybrids

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OTHER TYPES OF MARKERSSCAR ( Sequenced Charecterised Amplified Region)
Desired RAPD marker can be increased by sequencing its
termini and designing a pair of longer primer (24 bp long)
This is for specific amplification of RAPD marker
More reproducible
Used to develop +/- arrays

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CAPS (cleaved amplified polymorphic sequence)
Here specific primers are used to amplify a sequence that
can be genotyped by RFLP assay.
These are codominatant (design of primers needs
sequence information)
Has the advantage of RFLP assay avoiding southern blot
analysis
Also called as PCR – RFLP

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SNP : Any polymorphism between two genomes that is based on
a single nucleotide exchange, small deletion or insertion.
STS : It is a general term given to a marker that defined by its
primer sequences
SSR: Any one of the series of very short (2-10bp) middle,
repetitive, tandemly arranged, highly variable DNA sequences
dispersed through out plant, human and animal genome.

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ROLE IN PLANT GENETIC RESOURCE MANAGEMENT
A. Genetic Markers And Systematic Relationships
B. Acquisition/Distribution Of Collected Material
C. Maintenance Of The Genetic Integrity Of Accessions
D. Utilization Of Genetic Resources

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Genetic Markers and
Systematic Relationships

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Genetic Markers and Systematic Relationships
Systematics is defined as the scientific study of types of organisms
and of any and all relationships among the organisms (Simpson, 1961 )
one of the most important roles of genetic markers in plant
germplasm management is elucidating the systematic and
characteristic genetic profiles of germplasm.
Youssef et al. (2011) studied the phylogenetic relationships among
eight sorghum genotypes using RAPD markers and reported that
different levels of genetic similarity between them.

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B. ACQUISITION/DISTRIBUTION OF COLLECTED
MATERIAL
1. Assessing Collection Gaps and Redundancies
2. Sampling Strategies
3. Assembly of Core Collections
4. Characterizing Newly Acquired Germplasm

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1. Assessing Collection Gaps and Redundancies
 Variety of genetic markers are useful in assessments of how
completely a germplasm collection.
 The fingerprints developed by these markers employed to
verify synonymy and thus reduce duplication in collections ,to
note misidentifications and to understand the breadth and
gaps in holdings

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S. No. Name of the Group with
Year
Crop Marker (s)
Used
1 Treuren et al., (2010) Lettuce AFLP
2 Treuren et al., (2008) Perennial Kale Microsatellites
3 Sretenovic et al., (2008) Wild Lactuca AFLP
4 Treuren et al., (2004) Potato AFLP
5 Treuren et al., (2001) Flax AFLP

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2. Sampling Strategies
 Effective approach for optimizing sampling strategies involves
graphing the amount of genetic polymorphism (as determined by
genetic markers) in a sample against the sample size.
Hintum et al., (1995) developed optimal sampling strategies
in Barley by comparing the alternative methods for composing a core
collection using Isozyme markers & stated that clustering on the
basis of location of collection site proved to be best followed by
qualitative descriptive data, where as based on quantitative data did
not improve sampling efficiency.

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3. Assembly of core collections
• Genetic marker data may be instrumental for assembling a
collection with maximum allelic diversity.
• To facilitate utilization, core collections have been developed by
genebanks, following the concept developed by Frankel (1984).
• Treuren et al. (2006) asssemble core collection in Barley using AFLP
• Hintum et al. (1994) in Barley using Isozymes

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4. Characterizing Newly Acquired Germplasm
Genetic markers provide key information for designing and
implementing new in situ or ex situ germplasm management
programs for newly acquired germplasm.
Genetic markers can characterize the genetic profiles and
population genetic structure of newly acquired germplasm as a
prelude to ex situ management per se.

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Marco Pessoa et al. (2007) used a set of multiplex panels of
microsatellite markers for rapid molecular characterisation of rice
accessions.
They studied a collection of 548 accessions ,Pairwise genetic
distances were estimated &classified into two main
clusters,corresponding to materials with a possible indica and japonica
genetic backgrounds .
Allelic frequencies were estimated &taken as a reference for
comparision. The results showed that all 63 samples of the minor
cluster would be more probably described as possessing an indica
background.out of total accessions 485 samples were japonica.

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C. Maintenance of The Genetic Integrity of Accessions
1. Maintaining Trueness –To-type
a) Morphological Traits
b) Isozymes, seed Proteins & DNA Markers
c) Secondary Metabolites
d) Comparative Studies
e) Pollination Control Methods
2. Monitoring Shifts in Population Genetic Structure in
Heterogenous Germplasm
3. Monitoring Genetic Shifts Caused by Differential Viability in
Storage
4. Monitoring Genetic Shifts Caused by In Vitro Culture
5. Monitoring Germplasm Viability and Health

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1.Maintaining Trueness –to-Type
Genetic markers have
_ frequently documented outcrossing rates under
defined conditions of cultivation and
_have measured how effectively various
managerial methods maintain true-to-type populations (i.e.,
Accession integrity)
 Various DNA markers are particularly valuable for
identifying specific clones and monitoring their trueness-to-
type during regeneration (Thomas et al., 1993).

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• Iqbal et al.,(2010) carried out SSR analysis in 16 genotypes
of Sunflower for hybrid identification and to determine purity
among them,
of 20 specific SSR primers 18 authenticated the
purity of these hybrids.

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Comparative studies
 In some cases more than one type of genetic markers has
documented outcrossing or other genetic changes resulting
from seed regeneration.
Pollination controlling methods
 Genetic markers evaluated the efficacy of caging and bagging
for controlling pollen flow in germplasm plantings.

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2.Monitoring shifts in population genetic structure in
heterogeneous germplasm
Genetic markers have demonstrated that
genotypic frequencies in a homozygous, heterogeneous
germplasm mixture may shift dramatically after just a few
regeneration cycles through the differential viability of certain
genotypes.

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3.Monitoring in genetic shifts caused by Differential
viability in Storage:
• The genetic profiles of germplasm accessions can change
during the course of medium or long term storage.
• Storage effects fall into
The occurrence of mutations
The occurrence of chromosomal aberrations
Shifts in gene frequencies resulting from differential
genotypic viability in heterogeneous populations

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4.Monitoring Genetic Shifts Caused by In Vitro Culture
The genetic stability of germplasm maintained in tissue
culture (in vitro) has generally been monitored with
karyotypic markers .
Other genetic markers ,such as isozymes , cp DNA ,and n DNA
have detected point mutations or chromosomal aberrations
in such cultures.

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5.Monitoring Germplasm Viability and Health
 ELISA is a disease detection procedure based on protein antibody
markers diagnostic for plant pathogen genotypes or phenotypes.
 The ELISA protocol and other recently developed technologies
involving DNA and RNA hybridisation can help monitor the health
of plant germplasm collections through disease indexing
 These techniques are often combined with in vitro culture to
produce disease -free propagules.
This was exploited in Papaya at Hawai for the identification and
conservation of germplasm.

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Utilization Of Plant Genetic Resources
1. Developing Optimal Utilization Strategies From Genetic
Marker Data
2. Exploiting Associations Among Traits Of Interest And Genetic
Markers
3. Genetic Enhancement

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1.Developing Optimal Utilisation Strategies from
Genetic Marker Data
 Genetic Markers help In optimizing germplasm utilisation
strategies by
 identifying novel alleles of agronomically valuable
traits with relatively low heritabilities .
Incorporating these valuable traits into breeding
populations .

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• Jaemin cho et al. (2011) tagged SNP markers for gland
morphogenesis in cotton.
• Mariza et al. (2002) used different molecular techniques
(AFLP, SSR, RAPD) for the identification of genetic
characteristics in Maize.

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2.Exploiting Associations among Traits of Interest and Genetic
Markers
 Genetic markers exploit valuable traits when the markers and traits
are in tight linkage (i.e., associated genetically)
 Some favorable genes may be masked or swamped by more
dominant deleterious genes.
 The most valuable contribution of genetic markers to germplasm
utilization may be the efficient detection of these valuable latent
genes.
Jaemin cho et al. (2011) tagged SNP markers for gland
morphogenesis in cotton.

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3.Genetic Enhancement
 Genetic Enhancement may involve adapting alien material to
local conditions without eliminating its essential genetic
contributions (i.e., genetic diversity), termed “Base-
broadening" by Simmonds (1993) because it widens the
locally-adapted genetic base for crops.
 Genetic markers currently facilitate introgressing specific
high-value traits into adapted, elite germplasm in many
breeding programs.

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• Genetic Markers may facilitate genetic enhancement,
sometimes termed pre-breeding
 by identifying novel (relative to the germplasm in
common use) alleles of valuable polygenic traits with
relatively low heritabilities
 Sometimes by helping to incorporate these latent traits
into breeding populations
CGIAR Institutes (CIMMYT in Wheat and Maize, IRRI in Rice, CIP
in Potato) have initiated the efforts towards pre-breeding for
important alleles to meet the biotic and abiotic stresses and
also to improve the yield levels in the climate change

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Name of centre Group
leader
Markers
used
Plant
system
Area of research
1 Tata Energy Research
Institute, NewDelhi
M. S.
Lakshmi
kumara
n
RFLP,
RAPD, SSR,
AFLP,
SAMPL
neem,
withania,
Brassica,
poplar
DNA fingerprinting,
Germplasm
characterization,
Diversity study,
Gene tagging
2 International Centre for
Genetic
Engineering &
Biotechnology, New
Delhi
Madan
Mohan
S. Nair
RFLP,
RAPD, AFLP
, Rice Gene tagging,
Physical mapping,
3 Ch. Charan Singh
University, Meerut 12 8
20
P. K.
Gupta
H. S.
Balyan
RFLP, SSR,
STS, AFLP,
SAMPL,
EST
Wheat,
barley
DNA fingerprinting,,
Diversity study,
Gene
tagging,Genome
and QTL mapping,
Association analysis,
A list of institutes in India, involved in molecular
marker work in higher plants

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Name of centre Group
leader
Markers
used
Plant
system
Area of
research
4 National Chemical
Laboratory, Pune
P. K.
Ranjekar,
Vidya
Gupta
RAPD, ISSR,
SCAR
Wheat,
chickpea
Diversity
study,
Gene
tagging
5 National Research
Centre on Plant
DNA
Fingerprinting, New
Delhi
J. L.,
Karihaloo,
K. V. Bhatt
RAPD, SSR,
AFLP
All major
crops
DNA
fingerprinti
ng,
Diversity
study

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Name of centre Group
leader
Markers
used
Plant
system
Area of
research
6 National Research
Centre for Plant,
Biotechnology,
IARI, New Delhi
ISSR
T.
Mohapatra
RAPD, SSR,
AFLP,
Brassica,
rice
DNA
fingerprinti
ng,
Diversity
study,
Gene
tagging,
Genome
and QTL
mapping,
7 M. S. Swaminathan
Research,
Foundation,
Chennai
Ajay Parida RFLP,
RAPD,AFLP
Millets,
Mangroves
,
Diversity
study

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Name of centre Group
leader
Marker
s used
Plant
syste
m
Area of
research
8 Centre for Cellular &
Molecular
Biology, Hyderabad
Ramesh
Agarwal
AFLP Rice Diversity
study
9 University of Delhi
(South Campus
New Delhi & North
Campus, Delhi)
Deepak
Pental, S. N.
Raina
RFLP,AFL
P, RAPD,
Mustard
, Vigna
DNA
fingerprintin
g, Diversity
study, Gene
tagging,
10 Jawaharlal Nehru
University, New Delhi
K. C.
Upadhyaya
AFLP Chickpe
a Diversity
study

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Name of
centre
Group
leader
Mark
ers
used
Plant
system
Area of
research
11 M. S. University
of Baroda,
Baroda
B. B.
Chattoo
RAPD Rice Gene tagging
12 Agriculture
Research
Institute, Naini .
C. Kole RFLP Brassica Genome and
QTL mapping
13 University of
Ag. Sciences,
Bangalore
H. C.
Shashidhar
, Shailaja
Hittalmani
RAPD Rice Genome and
QTL mapping

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Name of
centre
Group
leader
Markers
used
Plant
system
Area of
research
14 National
Botanical
Research
Institute,
Luknow
Ranade Amaranthu
s
Diversity study
15 International
Crops
Research
Institute
for the Semi-
Arid Tropics,
Patancheru
S.
Sivaramakris
hnan, CT
Hash,
J.Kumar
RFLP, RAPD,
isozymes,
AFLP, SSR
Pulses,
millets
Germplasm
characterisation,d
iversity
study,characterisa
tion of
cytoplasmic male
sterility systems.
Source :Plant Cell, Tissue and Organ Culture 70: 229–234, 2002.

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CONCLUSIONS:
A wide variety of new molecular marker technologies are
available to assess genetic variation, and many of them are
increasingly being applied to complement traditional
approaches in germplasm and genebank management.
Genetic marker data will complement, not replace,
managerial experience with germplasm, prudent judgement,
and keen knowledge of a plant’s natural history.

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Genetic marker data should be weighed judiciously before
basing germplasm management decisions on them.
When exploited carefully, genetic markers do have
enormous, generally unrealised potential for optimising
germplasm conservation, especially by providing the precise
details of plant germplasm’s genetic architecture which are so
vital for effective and efficient germplasm management.

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