Description of molecular

marker types
silvio.salvi@unibo.it

WUEMED training course

June 5-10, 2006
 Classes of molecular markers

Class Level of analysis

• Morphological • Phenotype

• Biochemical • Gene product

• Molecular • DNA sequence

Main molecular marker types AFLP

RFLP RAPD

SSR
Southern blotting

Da: Brown TA, Genomes, BIOS

 RFLP
(Restriction Fragment Length Polymorphism)

Da: Cooper GM, The Cell, Sinauer

PCR (Polymerase Chain Reaction)

Da: Alberts et al., Molecular biology of the cell, 4a

ed.
SSR (simple sequence repeat) aka SSLP, STR,...
Sequenza
Primer F microsatellite
Plant A ACACACACACAC
TGTGTGTGTGTG
Primer R

Plant B ACACACAC
TGTGTGTG

Ax B
Scala

B
Direzione di
elettroforesi
Example of application of SSR marker

F1 (P1 x P2)
Sample of F2 plants derived from the cross P1 x P2

Ladder
P1
P2
High concentration (3%) gel electrophoresis.
Marker umc1001. Maize.
RAPD (Random Amplified Polymorphic DNA)
Williams et al., 1990 NAR 18, 6531

Pianta A Pianta B

Genomic
PCR PCR
DNA
YES YES

Primer RAPD
PCR
X Sequence
PCR NO polymorphism at
YES priming site
ladder

Ax B
A

B
Example of RAPD analysis

Williams et al. 1990 NAR 18, 6531

 AFLP
(Amplified Fragment Length Polymorphism)
Separation with PAGE
Genomic DNA

(Double) restriction digestion

Ligation of oligo adapters

First PCR (pre-amplification)

Second PCR (selective PCR),

with labelled primers
 SNP (Single Nucleotide Polymorphisms)
The relatively large availability of DNA sequence information
enable to identify a large number of SNP which are the most
abundant type of variation among individuals of the same
species.

Rafalski, 2002, Curr Opin Plant Biol. 5:94-100

Syvanen AC, Nat Rev Genet 2001
 !

" # " $
High-density Affymetrix yeast tiling microarrays (YTMs) with overlapping 25-
nucleotide oligomers spaced an average of 5 base pairs (bp) apart to provide
complete and 5-fold redundant coverage of the entire S. cerevisiae genome.
Do we need so many markers?
• A) Mapping populations for gene/QTL
mapping

• B) Genome-wide association mapping

 Current trends
• Small volumes, high multiplexing, no
electrophoresis
• Reduction of costs (also because PCR
patents are expiring)
• The gap between genotyping and
sequencing is narrowing
 Genotyping or sequencing?

"$1,000 Genome" NHGRI Grants

NHGRI's "Revolutionary Genome Sequencing Technologies"

grants have as their goal the development of breakthrough
technologies that will enable a human-sized genome to be
sequenced for $1,000 or less.
www.genome.gov/15015208
Genotyping or sequencing?

Moore’s law and DNA sequencing

Shendure et al., 2004 Nat Rev Genet 5, 335

 Selected references
• Collard B.C.Y., Jahufer M.Z.Z., Brouwer J.B., Pang E.C.K. (2005) An introduction to
markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop
improvement: The basic concepts Euphytica 142: 169–196
• JONES N, OUGHAM H and THOMAS H (1997) Markers and mapping: we are all
geneticists now. New Phytol. (1997), 137, 165-177
• Kumar LS (1999) DNA markers in plant improvement: An overview. Biotechnology
Advances 17, 143–182
• Rafalski A. (2002) Applications of single nucleotide polymorphisms in crop genetics.
Curr Opin Plant Biol. 5:94-100.