Simple Sequence Repeat (SSR)

1. MICROSATELLITE
OR SIMPLE SEQUENCE REPEAT (SSR)
Prepared by:
Santosh Bhandari and Dhiraj k. Singh
Department Of Genetics and Plant Breeding

2. INTRODUCTION
MOLECULAR MARKERS are all loci markers related to DNA (sometimes biochemical or
morphological markers included).

3. DEFINITION
Simple Sequence Repeat (SSR):
• Simple sequence repeats (SSRs) or microsatellites are tandemly repeated mono-,
di-, tri-, tetra-, penta-, and hexa-nucleotide motifs (chromosomes). 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”.
Example:

4. Examples of SSR in some plants
Crops Repeats
Rice CA, GT, GA, AT, GGT
Maize CT, CA, AC, AG, GA
Wheat GA, GT, CT, CA, AT, GT
Barley AT, CT,TG,TCT, CTT, ATTT
Soybean AT, CT,TA, ATT, AAT,TAT,TAA,
CTT

5. Feature of SSR Marker
• 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.
• Typically, SSRs are developed for di-, tri-, and tetra-nucleotide repeat motifs
(chromosomes). CA and GA have been widely used in plants.
• SSR markers have been developed for a variety of tri- and tetra-nucleotide
repeats in plants.
• Tetra-nucleotide repeats have the potential to be very highly polymorphic.

6. Where are microsatellites found?

7. Where does the molecular marker come from?
• Mutation= heritable (at the cell level) changes in DNA sequence, regardless of
whether the change produces any detectable effect on a gene product. Mutations
are the source of new variation (polymorphism) upon which natural selection
works. Inherited mutations that are dispersed through population can become
polymorphisms.
• Polymorphism= presence in the same population of two or more alternative forms
of a DNA sequence, with the most common allele having a frequency of 99% or
less. Any two individuals have a polymorphic difference every 1,000-10,000 base
pairs.

8. How do microsatellites mutate?
1. Replication Slippage:
When the DNA replicates, the polymerase
loses track of its place, and either leaves out
repeat units or adds too many repeat units.
“Polymerase slippage” or “slipped-strand
mispairing.”
• A commonly observed replication error is the
replication slippage, which occurs at the
repetitive sequences when the new strand
impairs with the template strand. The
microsatellite polymorphism is mainly caused
by the replication slippage. If the mutation
occurs in a coding region, it could produce
abnormal proteins, leading to diseases.

9. 2. Unequal crossing-over during meiosis

10. SSR Marker Development
• Construction of a DNA library in which small pieces of DNA are inserted into a
cloning vector.
• Each individual plasmid, containing a different pieces of DNA, is then
"transformed" or inserted into E. coli cells.
• The plasmid vector with the inserted DNA multiplies many times within the E. coli
cell.
• The resulting collection of E. coli cells, each containing a plasmid with a different
pieces of DNA, is referred to as a plasmid library.
• Once the library is constructed, it is screened for plasmids that contain DNA with a
desired SSR motif such as (ATT)n, (AT)n, (CT)n, (CTT)n, etc.
• Plasmid clones that are determined to contain the desired motif are then isolated
so that the DNA sequence of the entire crop insert can be determined.

11. Continue…,
• DNA sequence determination can be performed on a Perkin-Elmer ABI 377
Automated DNA Sequencer.
• The raw sequence data from each plasmid inserted is analyzed using Perkin-Elmer
ABI Auto Assembler software.
• The determination of DNA sequence is important for two reasons-
i. it verifies the presence of the SSR in the DNA insert and
ii. it provides the exact DNA sequence on either side of the SSR, which is necessary to
construct primers.
• Clones that are unique (that have not previously identified and that possess an
SSR of sufficient length) are advanced to the next step of the SSR marker
development process.
• This is the selection of PCR primers to the regions flanking the SSR.

12. Continue..,
• Once the primers are synthesized, a number of additional tests are required
before they can be utilized to produce a useful SSR marker.
• To test their effectiveness, the primers are used in PCR amplifications of both their
original plasmid, upon which the DNA sequence was determined, and on crop of
interest.
• Primers that perform well, by producing a single clean product with both the
plasmid and related crop DNA are advanced for further testing.

14. ADVANTAGES

15. DISADVANTAGES
• The development of SSRs is labor intensive number in sequence-based SSR
development) .
• SSR marker development costs are very high.
• SSR markers are taxa specific.
• Start-up costs are high for automated SSR assay methods.
• Developing PCR multiplexes is difficult and expensive. Some
• markers may not multiplex.

16. APPLICATION

17. ANY QUERY