Download Introduction to Molecular Markers and their

Introduction to Molecular Markers and
their Application in Biotechnology
Molecular Markers
„
1. What are molecular markers?
– Introduction to technology
– Description of types of markers
„
2. Uses of molecular markers
– Application as a genetic tool for plant genotyping and gene
mapping
– Application in agriculture
•Marker-assisted breeding
•Plant variety protection
•Assessing genetic diversity
– Applications in Human Health
•Association with genetic-based diseases
•Forensic studies
Molecular Markers
„1.
What are molecular markers?
– Heritable DNA sequence differences (polymorphisms)
– Phenotypically neutral, developmentally and environmentally stable
– Identified by techniques such as Southern hybridizations or PCR
„2.
Types of Markers
–Those detected by Southern Hybridizations
•RFLPs --Restriction Fragment Length Polymorphisms
–VNTRs -- variable number of tandem repeats (minisatellites)
–Those detected by PCR-based methods
•RAPD -- randomly amplified polymorphic DNA
•AFLP -- amplification fragment length polymorphism
•CAPS -- cleaved amplified polymorphic site
•SSR -- simple sequence repeats (microsatellites)
•SNP -- single nucleotide polymorphisms
The best molecular markers are those that distinguish
multiple alleles per locus (i.e. are highly polymorphic) and
are co-dominant (each allele can be observed)
•RFLP- a site in a genome where the distance between two restriction sites
varies among different individuals. These sites are identified by restriction enzyme
digests of chromosomal DNA, and the use of Southern blotting to identify the specific
fragments. Requires a radioactive probe!
•CAPS-a site in the genome polymorphic for the presence or absence of a
restriction enzyme site. Detected by PCR and restriction enzyme digests on gels.
•SSR-a site in the genome that contains many short tandem repeat sequences
(microsatellites). These sites are usually in the size range of 100-500 base pairs
composed of dinucleotide and trinucleotide repeats. They are very polymorphic,
scattered through out genomes. Genomes typically contain 1,000s of SSRs! They are
detected by PCR using primers flanking the repeats and resolved on gels.
•SNP-a single nucleotide difference in the sequence of a gene or segment of the
genome. There are typically tens of 1,000s of SNPs and a variety of methods for
analyzing them, including highly automated/high throughput procedures with
simultaneous scoring of many markers. Detection of SNPs can be done without gels.
RFLPs can arise through point mutations, deletions, insertions, etc.
RE
Allele A
RE
RE
Allele B
RE
probe
probe
polymorphism
•A deletion between two
restriction enzyme sites
RE
AA
BB
RE
RE
RE
AB
RE
AA
BB
AB
•An insertion between two restriction enzyme sites
RE
transposon
RE
RE
RE
RFLPs
Restriction Fragment Length Polymorphism (RFLP)
probe binding site
CHR 1:
5Õ EcoRI
↓
EcoRI 3Õ
NNNG|AATTCNNN-------NNNG|AATTCNNN
NNNCTTAA|GNNN-------NNNCTTAA|GNNN
3Õ
5Õ
probe binding site
CHR 2:
5Õ EcoRI
↓
EcoRI
3Õ
NNNG|AATTCNNN-------------------NNNG|AATTCNNN
NNNCTTAA|GNNN-------------------NNNCTTAA|GNNN
3Õ
5Õ
allele A
allele B
allele C
Genomic DNA cut by EcoRI, DNA run
on gel, blotted, and hybridized to probe
Polymorphism: fragments of different
lengths migrate differently in the gel
RFLPs
Allele B
Allele A
RE
RE
RE
RE
RE
probe
probe
polymorphism
•Southern blot
AA
BB
AB
•Allele “B” (RFLP “B”) appears
associated with the phenotype!
•But not completely! #12 must be a
recombinant between the phenotypic locus
and the RFLP “B”
AB
BB
*
P1
P2
*
1
2
*
3
4
5
6
*
*
7
8
9
10
*
*
11
12
RFLPs
Allele B
Allele A
RE
RE
RE
RE
RE
probe
probe
•Southern blot
AA
BB
RE
AB
Allele B
RE
*
RE
RE
RE
Allele A
*
P1
P2
*
1
2
*
3
4
5
6
*
*
7
8
9
10
*
*
11
12
Review of Genetic Linkage and
Recombination
Genes that are close together on a
chromosome tend to be inherited together.
In the example shown at left, genes A and
B would tend to be inherited together
much more often than with gene C.
Gene C would be inherited with B slightly
more often than with A.
Recombination & Crossing Over
• Exchange of alleles, but not gene order,
between the two homologous
chromosomes in diploids during meiosis.
• Frequency of crossing-over increases
with distance between loci.
RFLPs
Allele B
Allele A
RE
RE
RE
RE
RE
probe
probe
•Southern blot
RE
AA
BB
Allele B
*
RE
AB
RE
RE
RE
Allele B
Allele A
RE
RE
RE
Allele A
*
P1
P2
*
1
2
*
3
4
5
6
*
*
7
8
9
10
*
*
11
12
RE
*
RE
*
CAPS (cleaved amplified polymorphic sequence) as a molecular marker.
RE
Allele A
RE
RE
RE
Allele B
RE
Sequence Specific Primers
polymorphism
•Amplify by PCR
•This procedure is much quicker than
doing a Southern hybridization, yet
yields the same information!!
•Digest with restriction enzyme
•Visualize on Agarose Gel
AA
BB
AB
SSR: simple sequence repeat
(length polymorphism)
PCR Primer
(CA)n
(CA)n+4
variation between strains in number of repeats
at a given locus
PCR yields products of different size:
Simple Sequence Repeat (SSR) or Microsatellite Markers
Polymorphism is based on the
number of times a simple
sequence of DNA, usually 2-3
base pairs, is repeated. The
variant alleles are probably
generated by “stuttering” of DNA
polymerase or repair enzymes
during DNA replication of
repeated sequences.
• Male parent (red square) contains alleles 5 and 2
• Female parent (black circle) contains alleles 6 and 3
• Progeny segregate for alleles 2, 3, 5, and 6
• One daughter is mated to a male containing another
allele (4), resulting in offspring heterozygous for
alleles 5 and 4.
Bottom line: interpretation of
data for RFLP markers and
microsatellites is the same, even
though the data is generated by
means of different techniques
(Southern blotting for RFLP, PCR
for microsatellite)
Molecular Markers
„
1. What are molecular markers?
– Introduction to technology
– Description of types of markers
„
2. Uses of molecular markers
– Application as a genetic tool for plant genotyping and gene
mapping
– Application in agriculture
•Marker assisted breeding
•Plant variety protection
•Assessing genetic diversity
– Applications in Human Health
•Association with genetic-based diseases
•Forensic studies
Genotyping Progeny in a Standard Genetic Cross
ƒAn example of a genetic cross
wild type X
(+/+)
X
F1
teosinte branched mutant
(tb1/tb1)
all wild type (+/tb1)
Normal : tb1
F2
3
: 1
(+/+ or +/tb1) : (tb1/tb1)
“Normals” are either +/+ or +/tb1, but
one can not distinguish between the two
genotypes because “+” is dominant to the
recessive “tb1”!
•BUT, a molecular marker can easily
distinguish between the genotypes!
Prepare DNA from leaves of each type and use molecular
marker to determine genotype!
•Physical Maps
•Molecular markers can be mapped relative to one another
•This leads to a “physical map” that illustrates the linkage relationships
between physical markers on each of the chromosomes
•Now, one can map a new mutation (morphological marker) relative to the
molecular markers by following linkage between the mutant phenotype and
polymorphisms in molecular markers.
Each chromosome is divided into bins
These bins are about 20 cM
Bins are defined by molecular markers
•Lets use molecular markers to find the
map location of a newly isolated mutant!
Map your mutant by comparing its inheritance relative
to the inheritance of mapped molecular markers
First, make a mapping population:
New mutant: tb1-like. This mutant was found in an EMS
screen for new mutants in maize inbred B73
tb
tb
In a B73 background
B1 B2 tb B3 B4
B1 B2 tb B3 B4
F1
x
x
Mo17
M1 M2 TB M3 M4
M1 M2 TB M3 M4
B1 B2 tb B3 B4
M1 M2 TB M3 M4
Generate Mapping Population
F1
F2
B1 B2 tb B3 B4
B1 B2 TB B3 B4
B1 B2 TB B3 B4
B1 B2 tb B3 B4
B1 B2 tb B3 B4
B1 B2 TB B3 B4
B1 B2 TB B3 B4
B1 B2 tb B3 B4
WT
1
WT
tb mutant
2
1
This population will segregate 3:1 for wild type and tb-like
plants.
You will isolate DNA from some number of mutant (tb) plants. Every
chromosome transmitted is potentially recombinant. Every
F2 mutant can be scored for 0/2, 1/2, or 2/2 recombinant chromosomes.
For each molecular marker to be tested for linkage with “tb” mutant, identify
a polymorphism between the B73 and MO17. Then score inheritance of the
polymorphism.
•You look to see if the polymorphism is inherited with (linked to)
your mutant.
B73
Mo17
tb 1
tb 2
tb 3
tb 4
tb 5
tb 6
tb 7
tb 8
tb 9
tb 10
tb 11
tb 12
tb 13
tb 14
tb 15
tb 16
tb 17
tb 18
tb 19
tb 20
If a molecular marker is very closely linked to the mutation
(tb), then those progeny homozygous for the mutation will
also be homozygous for the B73 polymorphism
B73
Mo17
tb 1
tb 2
tb 3
tb 4
tb 5
tb 6
tb 7
tb 8
tb 9
tb 10
tb 11
tb 12
tb 13
tb 14
tb 15
tb 16
tb 17
tb 18
tb 19
tb 20
If a molecular marker is unlinked to the mutation (tb), then
those progeny homozygous for the mutation are just as likely
to inherit the MO17 polymorphism as the B73 polymorphism
•20 recombinant chromosomes out of a total of 40 scored
•This marker is unlinked to the mutation!
B73
Mo17
tb 1
tb 2
tb 3
tb 4
tb 5
tb 6
tb 7
tb 8
tb 9
tb 10
tb 11
tb 12
tb 13
tb 14
tb 15
tb 16
tb 17
tb 18
tb 19
tb 20
If a molecular marker is linked to the mutation (tb), then those
progeny homozygous for the mutation are more likely to
inherit the B73 polymorphism then the MO17 polymorphism
•8 recombinant chromosomes out of a total of 40 scored
8/40 = 0.2 or 20% recombination or 20 map units (20 cM between
this marker and the mutation).
•You have found a linked molecular marker!!
B1 B2 tb B3 B4
B73
Mo17
tb 1
tb 2
tb 3
tb 4
tb 5
tb 6
tb 7
tb 8
tb 9
tb 10
tb 11
tb 12
tb 13
tb 14
tb 15
tb 16
tb 17
tb 18
tb 19
tb 20
If a molecular marker is linked to the mutation (tb), then those
progeny homozygous for the mutation are more likely to
inherit the B73 polymorphism then the MO17 polymorphism
•8 recombinant chromosomes out of a total of 40 scored
8/40 = 0.2 or 20% recombination or 20 map units (20 cM between
this marker and the mutation).
•You have found a linked molecular marker!!
B1 B2 tb B3 B4
mutants
Mo17
B73
B73
Mo17
Bulked Segregant Analysis
If a molecular marker is
unlinked to the mutation
Pooled
(bulked)
B73
Mo17 mutants
mutants
If a molecular marker is linked
to the mutation
F1
Pooled
(bulked)
normals
Pooled
(bulked)
mutants
B73
Mo17
Pooled
(bulked)
mutants
SNP: single nucleotide
polymorphisms
¾
Outgrowth of sequencing projects
™
Detection of SNPs can be done without gels:
highly automated/high throughput and/or
highly parallel (simultaneous scoring of
MANY markers)
Allele-Specific Extension & Identification in CE:
“Minisequencing” (ABI SNaPShotTM)
Degree of Multiplexing Depends on Resolution
dR6G
dR110
ABI SNaPshot® on 3130xl
Genotyping by SBE and Mass Spectrometry
PCR Amplification
SAP Treatment
Single Base Extension
Spot on 384-place Chips
MALDI-TOF Mass Spec
Other uses for Molecular Markers
•Plant Variety Protection
•Verify varietal identity, purity and stability
•Marker-Assisted Breeding
•For accelerated trait/transgene introgressions
•For introgression of quantitative traits
•Estimation of Genetic Variation
•For phylogenetic analysis
•For preservation of rare plant and animal species
•For management of wild species
•Plant Pathogen Identification
Molecular Markers in Human Health
•Identification of human genetic
diseases
•Sickel cell anemia
•Huntingtons disease
•Tay Sachs disease
•Cystic Fibrosis (and many more)
•Forensic Science
•Paternity Determinations
Introduction to Molecular Markers and
their Application in Biotechnology