Principles of SSRs

Plant Breeding Unit

FAO/IAEA Agriculture and Biotechnology Laboratory
International Atomic Energy Agency
Vienna and Seibersdorf, Austria
Simple Sequence Repeat Markers
•First referred to as microsatellites
•Also called
• SSLP (Simple Sequence Length Polymorphisms)
• SSR (Simple Sequence Repeats)
• STMS (Sequence Tagged Microsatellites)
•Short tandem repeat motifs usually 1-6
bps of nucleotides in length
• ……..GAGAGAGAGAGAGA.…..
• ……..CCACCACCACCACCA.....
• ……..GATAGATAGATAGATA…..
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 SSR loci

(CA)n

(CT)n

(CA)n

(GA)n

CIAT -
BRU
Food and Agriculture Organization of the United Nations International Atomic Energy Agency
 SSR loci
• Are evolutionarily very unstable
owing to the repetitive nature of the
sequences

• They tend to mutate relatively often

• Ubiquitous to all eukaryotic genomes

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
 SSR loci
• This means that such DNA regions are
very good genetic markers because
they would show polymorphism between
closely related genotypes

• Flanking regions highly conserved,

making them suitable for PCR primer
pair design (to amplify the
intervening repeat loci)

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
 SSRs as markers
• Ifthe flanking DNA sequences on each
side of a SSR is known then it is
relatively easy to synthesize a primer
for each side of the locus

• Suchprimer pairs specifically amplify

the SSR in a simple PCR reaction

• Thelength of the amplified fragment can

then be compared for different genotypes
after electrophoresis

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 SSRs in PCR
• With known sequences for
the flanking regions,
specific primers (18-25
bp) can be designed

• These anneal on each

side of the
microsatellite on
opposite DNA strands

• With such a pair of

primers it is easy to
amplify the
microsatellite in a PCR
reaction

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 SSRs in PCR
•SSR locus amplified
from line L1 has one
extra repeat (3
nucleotides) compared
to its allele from
line L2.

•Since both alleles

amplify they are both
visible in the hybrid
(codominance)

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 SSR Markers
• Highly variable SSR loci on account
of the number of repeat units found

• Highly heterozygous, codominant and

PCR-based

• SSRs though expensive to generate are

cheap to use

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 SSR Markers
……. making them the molecular
markers of choice for:

•Genetic mapping

•Population genetic studies

•Germplasm characterization

•Marker-aided selection
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 Development of SSR
• Enriched Libraries
•Whole genomic libraries
•Expression (cDNA) libraries
•BAC, YAC libraries
• Database (In silico)
•Genbank
•Sequencing projects
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 SSR Discovery
5’ GTGTGTGTGTGTG 3’ Construction of small insert
CACACACACACACA libraries enriched for
repeat units

screening with a synthetic

labeled oligonucleotide
repeat

Sequencing of
positive clones

SSR analyses

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 SSR Evaluation
GENOTYPE 1 GENOTYPE 2
(CT)25 (CT)18

PCR

Control 1 2
800
50
0
PAGE 200
10
0

50

CIAT -
BRU

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Genetic Mapping of SSR Markers

TMS 30572

CM2177-2
F1 Progenies F1 Progenies

Silver stained polyacrylamide gel showing a unique

allele (arrowed) in the male parent (TMS 30572) of the
mapping progeny

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 Genetic Mapping of SSR Markers
TMS 30572
CM 2177-2

Mapping Progeny

Ethidium bromide stained metaphor agarose gel showing

the inheritance of a unique allele (arrowed) amongst
mapping population

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Automated Fluorescent SSR Analysis

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SSR Marker-aided Genetic Analysis
Tagging gene involved in resistance to
 cassava mosaic disease (CMD)

CMD Resistant F1 progeny

RP/SP/RB/SB

200bp
CMD Susceptible F1 progeny
175bp
150bp

SSR Analysis of 80 Genotype Bulks of

TMS30555 X TME3 Mapping Population CIAT
BRU
-

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
 Analytical procedures
• Extraction of DNA
• Amplification of DNA fragments by PCR
• Separation ofthe polymorphisms by
polyacrylamide or agarose gel-
electrophoresis
• Visualizationof the polymorphisms by
autoradiography, silver staining or
fluorescence (polyacrylamide gels, capillary
system), or ethidium-bromide staining and
ultraviolet light (agarose gels)

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 Main Requirements
• Thermocycler
• Setup for gel-electrophoresis

• Facilities for radioactive labeling

• Ultraviolet light source

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 Advantages
• Lowquantities of DNA template required
(10-100 ng per reaction)

• High genomic abundance

• Random distribution throughout the
genome

• High levels of polymorphism (alleles)

• Bandprofiles can be interpreted in

terms of loci and alleles

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 Advantages
• Codominance of alleles
• Allele sizes can be determinedwith an
accuracy of 1 bp, allowing accurate
comparison across different gels
• High reproducibility
• Different microsatellitesmay be
multiplexed in PCR or on gel
• Wide range of applications
• Amenable to automation
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 Disadvantages
• High development costs where primers are not yet
available
• Heterozygotes may be misclassified as
homozygotes when null-alleles occur due to
mutation in the primer annealing sites
• Stutter bands on gels may complicate accurate
scoring of polymorphisms
• Underlying mutation model (infinite alleles
model or stepwise mutation model) largely
unknown
• Homoplasy due to different forward and backward
mutations may underestimate genetic divergence

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