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GENETIC MARKERS

IN PLANT BREEDING

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Marker

  • Gene of known function and location
  • Gene that allows studying the inheritance of that gene
  • Genetic information resides in the genome

Genetic Marker

  • Any phenotypic difference controlled by genes, that can be used for studying recombination processes or selection of a more or less closely associated target gene
  • Anything in the genome that is variable and can be used to compare individuals
  • Detectable allelic variation on a chromosome can be a phenotype, can also be a unique detectable sequence of DNA

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Genetic Marker

  • Morphological marker
  • Molecular marker

1. Protein marker

2. DNA marker

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Morphological Marker

hulled naked

Black white

  • Phenotypic markers
  • Naked eye marker

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Readily detectable sequence of protein or DNA that are closely linked to a gene locus and/or a morphological or other characters of a plant

Readily detectable sequence of protein or DNA whose inheritance can be monitored and associated with the trait inheritance independently from the environment

Molecular Markers

Types:

a) protein polymorphisms

b) DNA polymorphisms

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Molecular markers

Resolution power

  • allozymes (protein-electrophoresis)
  • RAPD�(random amplified polymorphic DNA)
  • AFLP�(Amplified Fragment Length Polymorphism)
  • Multi-locus fingerprints (RFLP)
  • Microsatellites (SSRs)
  • Sequencing (SNPs)

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DNA

Gene A

Gene B

AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAA

AAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG

MFG

1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag gcgggcgtcg ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc cattcgcctg 181 ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc cagagtgctt gatgccatgc tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361 agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta 541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt ggaggacagc tttgtggag

MFG

DNA Marker

M1

M2

Readily detectable sequence of DNA whose inheritance can be monitored and associated with the trait inheritance

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Hybridization based markers

Examine differences in size of specific DNA restriction fragments

Usually performed on total cellular genome

Require pure, high molecular weight DNA and probe

DNA Marker

  1. Hybridization molecular based markers
  2. PCR molecular based markers

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DNA/DNA Hybridization

Denaturation

Elevated temperature

Known DNA sequence

Restriction Fragment Length Polymorphism

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RFLP techniques

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3

6

2

6

1

2

4

3

5

4

5

1

MFG

RFLP Polymorphisms interpretation

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Advantages and disadvantages

  • Advantages
    • Reproducible
    • Co-dominant
    • Simple
  • Disadvantages
    • Time consuming
    • Expensive
    • Use of radioactive probes

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Polymerase Chain Reaction

Powerful technique for amplifying DNA

Amplified DNA are then separated by gel electrophoresis

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PCR Based markers

  • Sequencing (SNPs)
  • Microsatellites (SSR)
  • AFLP (Amplified Fragment Length Polymorphism)
  • RAPD (random amplified polymorphic DNA)

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RAPD Markers

DNA markers which developed by amplifying random sequence of specific markers through the used of random primers

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RAPD

Disadvantages:

  • Dominant markers
  • Reproducibility problems

Advantages:

  • Amplifies anonymous stretches of DNA using arbitrary primers
  • Fast and easy method for detecting polymorphisms

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RAPD Markers

  • RAPD markers need to be converted to stable PCR markers.
  • The polymorphic RAPD marker band is isolated from the gel
  • It is used a template and re-PCRed
  • The new PCR product is cloned and sequenced
  • Once the sequence is determined, new longer and specific primers can be designed

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RAPD Polymorphisms among landraces of sorghum

M

Sequences of 10-mer

RAPD primers

Name Sequence

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OP A08 5’ –GTGACGTAGG- 3’

OP A15 5’ –TTCCGAACCC- 3’

OP A 17 5’ –GACCGCTTGT- 3’

OP A19 5’ –CAAACGTCGG- 3’

OP D02 5’ –GGACCCAACC- 3’

RAPD gel configuration

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AFLP Markers

  • Most complex of marker technologies
  • Involves cleavage of DNA with two different enzymes
  • Involves ligation of specific linker pairs to the digested DNA
  • Subsets of the DNA are then amplified by PCR
  • The PCR products are then separated on acrylamide gel
  • 128 linker combinations are readily available
  • Therefore 128 subsets can be amplified
  • Patented technology

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AFLP Markers

  • Technically demanding
  • Reliable and stable
  • Moderate cost
  • Need to use different kits adapted to the size of the genome being analyzed.
  • Like RAPD markers need to be converted to quick and easy PCR based marker

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SSR (Simple sequence repeat)

DNA markers which developed by amplifying microsatellite in the genome

Sequence Primer

ACTGTCGACACACACACACACGCTAGCT (AC)7

TGACAGCTGTGTGTGTGTGTGCGATCGA

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ACTGTCGACACACACACACACACGCTAGCT (AC)8

TGACAGCTGTGTGTGTGTGTGTGCGATCGA

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ACTGTCGACACACACACACACACACACGCTAGCT (AC)10

TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGA

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ACTGTCGACACACACACACACACACACACACGCTAGCT (AC)12

TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA

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AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG

P1

AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG

P2

P1

P2

SSR polymorphisms

Gel configuration

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SNPs

(Single Nucleotide Polymorphisms)

  • Any two unrelated individuals differ by one base pair every 1,000 or so, referred to as SNPs.
  • Many SNPs have no effect on cell function and therefore can be used as molecular markers.

Hybridization using fluorescent dyes

SNPs on a DNA strand

DNA markers which their polymorphism can be determined by single nucleotide difference

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DNA sequencing

Sequencing gel

Sequencer

Sequencing graph

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Genetic marker characteristics

Characteristics

Morphological markers

Protein markers

RFLP markers

RAPD markers

SSR markers

Number of loci

Limited

Limited

Almost unlimited

Unlimited

High

Inheritance

Dominant

Codominant

Codominant

Dominant

Codominant

Positive features

Visible

Easy to detect

Utilized before the latest technologies were available

Quick assays with many markers

Well distributed within the genome, many polymorphism

Negative features

Possibly negative linkage to other characters

Possibly tissue specific

Radioactivity requirements, rather expensive

High basic investment

Long development of the markers, expensive

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Polymorphism

-Parent 1 : one band

-Parent 2 : a smaller band

-Offspring 1 : heterozygote = both bands

-Offspring 2 : homozygote parent 1

Polymorphism

Parent 1 : one band

-Parent 2 : no band

-Offspring 1 : homozygote parent 1

-Offspring 2 : ????

P 2

P 1

O 2

O 1

Gel configuration

Co-dominant marker

P 2

Gel configuration

P 1

O 1

O 2

Dominant marker

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  • Polymorphic
  • Co-dominant inheritance
  • Occurs throughout the genome
  • Reproducible
  • Easy, fast and cheap to detect
  • Selectivity neutral
  • High resolution with large number of samples

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Desirable properties