PLANT MOLECULAR MARKERS

SIR SOHAIL AHMED

Course: Agriculture Science / Agriculture
Biotechnology
SZABIST
 GENETIC DIVERSITY

 Assessment Genetic diversity in a crop species is fundamental to its improvements.

 Genetic variability is considered the reservoir that plant breeders fall upon in their continuous
strive to develop improved varieties and hybrids.

 Knowledge of germplasm diversity and of relationship among elite breeding materials has a
significant impact improvement of crop plants.

 This information is useful in planning crosses for hybrid and line development, in assigning lines to
heterotic groups.
 WHAT IS MARKER?

Marker- that mark, tag or identify.

Marker can be Morphological marker (size, shape, color etc.), Biochemical

markers (isozymes, proteins) and Genetic markers (DNA level).

Genetic marker- an allelic difference / variation at a given locus in the genome

that can be observed at the level of morphology.

 MARKER – WHAT IT’S FOR?

•To select plant traits and develop new varieties

•Markers linked to gene of interest
•Enabling use of valuable gene (nutritional and non-nutritional factors)
•Evergreen Revolution (Borlaug, 2001; Swaminathan, 2007).
•Assist in conventional breeding
•Gene pyramid
 THE 'PERFECT MARKER'

Polymorphic
Reproducible
Easy to use and economical
High-throughput
- automation
- combination of different markers in one reaction
 TYPE OF MARKERS

There are four type of markers.

• Morphological Markers

• Cytological Markers

• Protein Markers

• Molecular Markers
 MORPHOLOGICAL MARKERS

These are related to shape, size, colour and surface of various plants parts.

• Such characters used for the varietal identification.

Advantages:
• Readily available.
• Usually require only simple equipment.
• Form the most direct measure of phenotype.
Disadvantages:
• Require expertise on crop or species.
• Subject to environmental influences.
• Limited in number.
 HERBARIUM COLLECTION
Recording their identity and establishing permanent preserved for study
 CYTOLOGICAL MARKER

Markers that are related to variation in chromosome number, shape, size and banding
pattern are referred to as cytological markers. In Other words, it refers to the
chromosomal banding produced by different stains; for example, G banding.
1. Readily available
2. Requires small equipment’s
1. Limited in number

2. They exhibit less polymorphism, need experts.

 PROTEIN (BIOCHEMICAL) MARKERS
Available since 1950's.
Such markers are related to the variations in protein and amino acid banding pattern.
Gel electrophoretic studies used for identification of biochemical markers.
e.g.- Peroxidase, Acid Phosphate, Esterase etc.

Advantages:
Require simple equipment.
A vigorous complement to the morphological assessment of variation.

Disadvantages:
Subject to environmental influences.
Limited in number.
 MOLECULAR MARKERS

Reflect heritable differences in homologous DNA sequences among individuals.

They may be due to:

• Base pair changes.
• Rearrangements (translocation or inversion).
• Insertions or deletions.
• Variation in the number of tandem repeats.
 ADVANTAGES OF MOLECULAR MARKERS

 Detectable in all tissues, at all ages.

 Long shelf life of the DNA samples.

 Ubiquitous.

 Stably inherited.

 Multiple alleles for each marker.

 Devoid of pleiotropic effects.

 TYPES OF MOLECULAR AMARKER

Molecular marker are further divided in to two categories

1. Hybridization based marker (Non PCR based)

2. PCR based marker

 HYBRIDIZATION BASED MARKER

The variation in the length of the DNA fragments generated by specific restriction
endonucleases in two or more individuals is due to the difference in the pattern of
occurrence to specific restriction sites (of the corresponding restriction endonucleases)
on the genomic DNA detected in the hybridization experiment and thus the markers
are called hybridization based markers

Example : Restriction Fragment Length Polymorphism (RFLP)

 POLYMERASE CHAIN REACTION BASE MARKERS

PCR based marker Markers which Shows polymorphism based on the PCR amplification

is Called PCR based marker

Example :
Simple Sequence Repeat or microsatellites (SSR)
Cleaved Amplified Polymorphic Sequences (CAPS)
Amplified Length Polymorphism (AFLP)
Sequence Characterized Amplified Regions (SCARS)
Expressed Sequence Tags (ESTS)
Single Nucleotide Polymorphism (SNPS)
 HYBRIDIZATION BASED (NON-PCR) TECHNIQUE
(Restriction Fragment Length Polymorphism Analysis)

Genetic markers resulting from the variation or change in the length of

defined DNA fragments produced by digestion of the DNA sample with
restriction endonucleases
 STEPS FOR RFLP

Electrophoretic comparison of the size of defined restriction fragments derived from

genomic DNA
1. Isolate high quality DNA
2. Digest with a combination of restriction enzymes (e.g. TaqI,MspI,BmHI,XbaI,PstI)
3. Fractionate digested samples by electrophoresis
4.Transfer fragments to membrane

5.Hybridize with radioactively labeled DNA probe(s); detect by autoradiography. Can

also use non-radioactive labeling systems
 APPLICATIONS OF RFLP

Intraspecific level or among closely related taxa.

Presence and absence of fragments resulting from changes in recognition sites are

used for identifying species or populations.

Estimating genetic distance and fingerprinting.

Forensic - biological parentage, paternity cases.

Disease status.

Genetic mapping.
 ADVANTAGE OF RFLP

High reproducibility

Show codominant alleles

Detect coupling phase of DNA

Reliable marker in linkage and breeding analysis

Easily determine a linked trait present in both

homozygous and heterozygous .

 LIMITATION OF RFLP

Require large quantities of high molecular weight DNA (5- 10 Np).

Expensive process

Time consuming

Labor intensive

Radioactive probe
QUESTIONS, ANSWER & FEEDBACK