genetic marker (1)

Genetic Markers

Variation

The differences that distinguish one individualfrom another are encoded in the individual’sgenetic material, the deoxyribonucleic acid(DNA). DNA is packaged in chromosome pairs,one coming from each parent. The genes, whichcontrol a plant’s characteristics, are located onspecific segments of each chromosome.

Marker

Genetic markers

Genetic markers are the biological features thatare determined by allelic forms of genes orgenetic loci and can be transmitted from onegeneration to another, and thus they can beused as experimental probes or tags to keeptrack of an individual, a tissue, a cell, a nucleus,a chromosome or a gene.

Genetic Markers

• represent genetic differences between individual organisms or species

• do not represent the target genes themselves but act as ‘signs’ or ‘flags

• located in close proximity to genes (i.e. tightly linked) may be referred to as gene ‘tags

• do not affect the phenotype of the trait of interest because they are located only near or ‘linked’ to genes controlling the trait

• occupy specific genomic positions within chromosomes (like genes) called ‘loci’ (singular ‘locus’)

Classification of Genetic Markers

• Classical markers• Morphological markers,

• Cytological markers

• Biochemical markers

• DNA markers• RFLP, AFLP, RAPD, SSR, SNP

Morphological markers:

• visible traits, such as leaf shape, flower color,pod color, seed color, seed shape, awn typeand length, fruit shape, stem length

• Some of these markers are linked with otheragronomic traits and thus can be used asindirect selection criteria in practical breeding

Example of morphological markers

• Mendelian characters

• In wheat breeding, the dwarfism governed bygene Rht10 was introgressed into Taigunuclear male-sterile wheat by backcrossingand a tight linkage was generated betweenRht10 and the male-sterility gene Ta1

• Then the dwarfism was used as the marker foridentification and selection of the male-sterileplants in breeding populations

Drawbacks of morphological markers

• Limited in numbers

• many of these markers are not associated withimportant economic traits (e.g. yield andquality)

• influenced by environmental factors or thedevelopmental stage of the plant

• However, despite these limitations,morphological markers have been extremelyuseful to plant breeders

Cytological markers:

• In cytology, the structural features ofchromosomes can be shown by chromosomekaryotype and bands. The banding patterns,displayed in color, width, order and position,reveal the difference in distributions ofeuchromatin and heterochromatin

Biochemical/protein markers:

• Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution

Drawbacks of biochemical markers

• Limited in numbers

• influenced by environmental factors or the developmental stage of the plant

• However, despite these limitations, biochemical markers have been extremely useful to plant breeders

Molecular/DNA markers

DNA markers are defined as a fragment of DNA revealingmutations/variations, which can be used to detectpolymorphism between different genotypes or alleles ofa gene for a particular sequence of DNA in a populationor gene pool. Such fragments are associated with acertain location within the genome and may be detectedby means of certain molecular technology.

DNA marker is a small region of DNA sequence showingpolymorphism (base deletion, insertion and substitution)between different individuals

Defined

• RFLPs are differences in restriction fragment

lengths caused by SNPs or INDELs that create or

abolish restriction endonuclease recognition sites.

RFLP assays are performed by hybridizing a

chemically labelled DNA probe to a Southern blot of

DNA digested with a restriction endonuclease.

Restriction Fragment Length Polymorphisms

(RFLPs)

RFLPs

• Restriction fragment length polymorphism

• Co-dominant

• Requires:single copy DNA probeRestriction enzymeSouthern blottingDNA polymorphism

RAPDs

• Randomly

amplified

polymorphic DNA

• Based on a 10 bp

single arbitrary

primer

• Cheap, easy

• Insufficient

reproducible maize lines; only primer 2 and 5 demonstrate polymorphism

10/8/2017

RAPD – dominant marker

AB

10/8/2017

AFLP: Major Steps• Restriction endonuclease digestion of genomic

DNA and ligation of specific adapters

• Amplification of the restriction fragments by PCR

using primer pairs containing common sequences

of the adapter and two or three arbitrary

Nucleotides

• Analysis of the amplified fragments using gel

electrophoresis

10/8/2017

AFLPs: amplified fragment length

Polymorphisms

•A combination of PCR and RFLP

•Informative fingerprints of amplified

fragments

10/8/2017 NIBGE Ph.D lecture

AFLP-Major Steps

Amplified Fragment Length Polymorphism

• Genomic DNA double digests with a 4-cutter

(MseI) and a 6-cutter (EcoR1)

• Ligate adapters to the EcoR1 and MseI RE sites

• Primers complementary to Adapters with selective

nucleotides at 3’ ends and perform PCR

amplification

• Separate DNA fragments on high-resolution gels

• After detection, screen for band polymorphisms

10/8/2017

AFLP: Restriction and Ligation to Adapters

10/8/2017

AFLP: Pre-Selective Amplification

Primer (+ 1) for pre-selective amplification

10/8/2017

AFLP: Selective Amplification

Primer (+ 3) for selective amplification

10/8/2017

10/8/2017

AFLP – band polymorphisms

Simple Sequence Repeats (SSRs)

Defined

• Simple sequence repeats (SSRs) or microsatellites

are tandemly repeated mono-,di-, tri-, tetra-, penta-,

and hexa-nucleotide motifs.

SSR length polymorphisms are caused by differences

in the number of repeats.

• SSR loci are “individually amplified by PCR using

pairs of oligonucleotide primers specific to unique DNA

sequences flanking the SSR sequence”.

Why Have SSRs Had Such a Large Impact

on Genomics?

• SSRs tend to be highly polymorphic.

• SSRs are highly abundant and randomly dispersed

throughout most genomes.

• Most SSR markers are co-dominant and locus

specific.

• Genotyping throughput is high and can be

automated.

SSR / microsatellite

1. Isolation of DNA fragments (vectors) containing a simple sequence repeat(microsatellite), e.g.

[AT]n [GC]n, [CGA]n, [GATA]n

1. Sequencing regions flanking the SSR

2. Designing primers for border sequences

3. Testing in population for duplications andSSR polymorphism

SSR - methodolgyGenotype A

Genotype A Genotype B

PCR amplification with

radiolabelled nucleoltide

Polyacrylamide Gel Electrophoresis

of PCR products and autoradiography

[AT]18

[AT]22

[AT]18

[AT]22

Genotype B

SSRs display multiple alleles

Distribution of SNP

SINGLE NUCLEOTIDE POLYMORPHISM

Distribution of SNP

SNP

HUMAN SNP DISTRIBUTION

Most common changes

Transitions:

Purines to Purines

Pyrimines to Pyrimidines

Transversion:

Purines to Pyrimidines

Pyrimidines to Purinces

Single-base insertions & deletion

(indel)

Distribution of SNP

HOW SNP ARE INDICATED

Distribution of SNP

Criteria for ideal DNA markers

• selectively neutral because they are usually located in non-coding regions of DNA• High level of polymorphism• unlimited in number and are not affected by environmental factors and/or the

developmental stage of the plant• Even distribution across the whole genome (not clustered in certain regions)• Co-dominance in expression (so that heterozygotes can be distinguished from

homozygotes)• Clear distinct allelic features (so that the different alleles can be easily identified)• Single copy and no pleiotropic effect• Low cost to use (or cost-efficient marker development and genotyping)• Easy assay/detection and automation• High availability (un-restricted use) and suitability to be duplicated/multiplexed (so

that the data can be accumulated and shared between laboratories)• No detrimental effect on phenotype• However, no molecular markers fulfill all these characteristics. Researchers choose

the molecular marker according to their need and availability

genetic marker (1)

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