Credit ge netic marker .pptx

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WELCOME

PRESENTED BYR. ASHABAM-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 and Plant genetic resources are the building blocks and fundamental not only in crop improvement fundamental not only in crop improvement programme, but also for the very survival of the programme, 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 Resourcesa) Basic Genetic Resources 1. Wild taxa related to a crop species 2. Weedy forms 3. Land races or primitive cultivarsb) 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

Morphologicallevel

Proteinmarker

Protein level

DNAlevel

DNAmarker

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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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BA)

P1 F1 P2

Polymorphic Monomorphic

Codominant Dominant

Markers at DNA level

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Co dominant Dominant

Discriminate b/w homozygous & No discrimination heterozygousNo 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

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MOLECULAR BASIS OF DNA MARKERSMOLECULAR BASIS OF DNA MARKERS

GAATTCGAATTCCTTAAGCTTAAG


11) ) Gain or loss of a restriction site, or PCR priming site Gain or loss of a restriction site, or PCR priming site

RFLP, AFLP, CAPSRFLP, AFLP, CAPS


GACTTCGACTTCCTGAAGCTGAAG


RAPD, AP-PCR, DAFRAPD, AP-PCR, DAF

22) Insertion or deletion between restriction or priming sites) Insertion or deletion between restriction or priming sitesGAATTCGAATTCCTTAAGCTTAAG


RFLP, AFLP, CAPSRFLP, AFLP, CAPSRAPD, AP-PCR, DAFRAPD, AP-PCR, DAF



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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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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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RAPDAny 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 technologyA 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 codominant 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 Accessions1. 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.

http://www.agrsci.unibo.it/~frascaro/References/Refs_S-Z.html

http://www.agrsci.unibo.it/~frascaro/References/Refs_S-Z.html

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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

scenario.

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Name of centre Group leader

Markers used

Plant system

Area of research

1 Tata Energy Research Institute, NewDelhi

M. S.Lakshmikumaran

RFLP, RAPD, SSR, AFLP, SAMPL

neem, withania, Brassica, poplar

DNA fingerprinting, Germplasm characterization, Diversity study, Gene tagging

2 International Centre for GeneticEngineering & Biotechnology, NewDelhi

Madan Mohan S. Nair

RFLP, RAPD, AFLP

, Rice Gene tagging,Physical mapping,

3 Ch. Charan Singh University, Meerut 12 8 20

P. K. GuptaH. 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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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 fingerprinting, Diversity study

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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 fingerprinting, 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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Markers used

Plant system

Area of research

8 Centre for Cellular & Molecular Biology, Hyderabad

Ramesh Agarwal

AFLP Rice Diversity study

9 University of Delhi (South CampusNew Delhi & North Campus, Delhi)

Deepak Pental, S. N. Raina

RFLP,AFLP, RAPD,

Mustard, Vigna

DNA fingerprinting, Diversity study, Gene tagging,

10 Jawaharlal Nehru University, New Delhi

K. C. Upadhyaya

AFLP Chickpea Diversity

study

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Name of centre

Group leader

Markers 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 Amaranthus

Diversity study

15 International Crops Research Institutefor the Semi-Arid Tropics, Patancheru

S. Sivaramakrishnan, CT Hash, J.Kumar

RFLP, RAPD, isozymes, AFLP, SSR

Pulses, millets

Germplasm characterisation,diversity study,characterisation 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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Credit ge netic marker .pptx