Marker Assisted Selection

(MAS)

1
  Deoxyribonucleic acid (DNA) is a molecule made
up of pairs of building blocks called nucleotides.

 DNA is packaged into chromosomes that are

located within the nucleus of all cells.

 Chromosomes contain stretches of DNA called

genes that code for amino acids that make proteins.

 The interaction and structure of proteins determine

the visible characteristics or phenotype of an
organism, while the genetic makeup of an organism
is called its genotype.
2
 The sequence of nucleotides that make up a gene
can differ among individuals.

 The different forms of a gene are called alleles.

 The DNA sequence of a gene inherited from each

parent may be identical, in which case the individual
is said to be homozygous for that trait. Or the
sequence of the gene from one of the parents may
be different, in which case the individual is said to
be heterozygous.

 Allele variations may differ in their DNA sequence

by as little as a single nucleotide.
3
  Genotyping means using laboratory methods to
determine the sequence of nucleotides in the DNA
from an individual, usually a specific gene.

 Marker assisted selection is the process of using the

results of DNA testing in the selection of individuals
to become parents for the next generations.

 The information from the DNA testing, combined

with the observed performance records for
individuals, is intended to improve the accuracy of
selection and increase the possibility of identifying
organisms carrying desirable and undesirable traits
at an earlier stage of development.
4
  Complex traits, including many of economic
importance, are controlled by many genes and are
influenced by the environment.

 It is important to combine DNA results with

performance and phenotype information to
maximize the effectiveness of selection for traits of
interest.

 Combining information from performance records

and genetic tests into the selection process will be
better than using performance, phenotype, and
markers separately.
5
 DNA sources
1. Genomic DNA from chromosomes: (Fragments
because usually too large to clone directly)
2. cDNA (complementary DNA): derived by action of
reverse transcriptase from (usually) mRNA template
3. chemically synthesized DNA molecules
(oligonucleotides)

6
 Types of markers

1. Morphological markers
• Seed color e.g. Kernel color in maize
• Function based e.g. Plant height
associated with salt tolerance in rice

7
Limitations
1. Most phenotypic markers are undesirable in
the final product (Yellow color in maize).

2. Dominance of the markers: homozygotes/

heterozygotes not distinguishable

3. Sometimes dependent on the environment for

expression e.g. Height of plants

8
2. Molecular markers
• Non-DNA such as isozyme markers: Restricted
due limited number of enzyme systems
available.

• DNA based markers: Markers based on the

differences in the DNA profiles of individuals.

9
 Some molecular markers are pieces of DNA
that have no know function or impact on
plant performance (Linked Markers):
• Detected via mapping.
• Linked markers are near the gene of interest
and are not part of the DNA of the gene.

 Other markers may involve the gene of

interest itself (Direct Markers):
• Based on part of the gene of interest.
• Hard to get but great once you have it.
10
 What is MAS?
 Concept of using molecular markers particularly
DNA based to detect and track presence of gene
transfer in breeding programs

 MAS works on the principle of linkage dis-equilibrium

where markers that are tightly linked to target genes
segregate together in a non random manner (due
linkage)

11
Advantages of MAS

12
 1. Improvement of response to selection (Rs)
2. Assays require small amount of tissue, therefore
no destructive sampling.
3. Use of codominant markers allows accurate
identification of individuals for scoring without
ambiguity
4. Multiple sampling for various QTLs is possible from
same DNA prep
5. Can assay for traits before they are expressed, e.g.
before flowering
6. Time saving.
13
14
 CONVENTIONAL PLANT BREEDING
P1 x P2
Recipient Donor

F1
large populations consisting of
F2 thousands of plants

PHENOTYPIC SELECTION

Phosphorus deficiency plot

Salinity screening in phytotron Bacterial blight screening
15
Glasshouse trials Field trials
 MARKER-ASSISTED BREEDING

P1 x P2
Susceptible Resistant

F1

F2 large populations consisting of

thousands of plants

MARKER-ASSISTED SELECTION (MAS)

Method whereby phenotypic selection is based on DNA markers 16

Overview of
‘marker (1) LEAF TISSUE
genotyping’ SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

17
 Requirements for a useful
molecular marker
1. Molecular markers must be tightly linked to a target
gene. The linkage must be really tight such that the
presence of the marker will reliably predict the
presence of the target gene.

2. The marker should be able to predict the presence

of the target gene in most if not all genetic
backgrounds.

18
 MAS

Marker 1 (more tightly

linked than 2)
1

Target gene

19
 Markers must be
tightly-linked to target loci!
• Ideally markers should be <5 cM from a gene or QTL
RELIABILITY FOR
SELECTION
Marker A

QTL Using marker A only:

5 cM
1 – rA = ~95%

Marker A Marker B
Using markers A and B:
QTL
5 cM 5 cM 1 - 2 rArB = ~99.5%

• Using a pair of flanking markers can greatly improve

reliability but increases time and cost
20
 Marker Plant A Plant B Plant C -

Monomorphic bands

Polymorphic bands

Presens of a band, ”1” Absence of a band, ”0”

21
Markers must be polymorphic
RM84 RM296
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

P1 P2
P1 P2

Not polymorphic Polymorphic!

22
 DNA extractions

Mortar and pestles

Porcelain grinding plates

LEAF SAMPLING
Wheat seedling tissue sampling in
Southern Queensland, Australia.

23
DNA EXTRACTIONS
 PCR-based DNA markers
• Generated by using Polymerase Chain Reaction
• Preferred markers due to technical simplicity and cost

PCR Buffer +
MgCl2 +
dNTPS + PCR
Taq +
Primers +
DNA template

THERMAL CYCLING

GEL ELECTROPHORESIS
24
Agarose or Acrylamide gels
Agarose gel electrophoresis

UV transilluminator
UV light

25
A woman is uncertain which of two men is the father of her child. DNA
typing is carried out on blood from the child (C), the mother (M), and each of
the two males (A and B), using probes for a highly polymorphic DNA marker
on two different chromosomes (“locus 1” and “locus 2”). The result is shown
in the accompanying diagram.

Can either male be excluded as the possible father?

Explain your reasoning.
26
DNA replication in natural systems requires:
1. A source of the nucleotides adenine (A), cytosine
(C), thymine (T), and guanine (G).

2. The DNA polymerase (DNA synthesis enzyme).

3. A short RNA molecule (primer).

4. A DNA strand to be copied.

5. Proper reaction conditions (pH, temperature).

27
Is there any differences between PCR and
mechanisms of the natural replication system?

1. DNA primers are used instead of the RNA primer

found in the natural system.
2. Magnesium ions that play a role in DNA replication
are added to the reaction mixture.

3. A DNA polymerase enzyme that can withstand high

temperatures, such as Taq, is used.

4. A reaction buffer is used to establish the correct

conditions for the DNA polymerase to work.
28
 Conditions under which MAS is valuable
1. Low heritability traits

2. Traits too expensive to score:Soybean Cyst

Nematode (SCN) resistance. Young (1999)

3. Recessive genes:Pyramiding of dominant and

recessive genes conferring resistance to important crop
diseases which would otherwise be very difficult

4. Multiple genes (Quantitative traits): QTLs underlying

phenotypic and physiological traits can be traced using
markers. Although QTL mapping is tedious, markers
once identified can be used fast and accurately to detect
the QTLs of interest. 29
5. Quarantine: No need to grow plants to screen for viral
diseases that can not be visually detected, and small
tissues can be used for DNA typing.

30
 Limitations of MAS
1. Cost of equipment, reagents and personnel.

2. Data collected in the field is assumed to be normally

distributed, but usually is not.

3. Integration of the DNA information into existing systems

is difficult.

4. Linkage drag. As the marker distance from the target

gene increases, more of the donor DNA is retained in the
desired background resulting in need for more
backcrosses.
31
Types of DNA based Markers

32
 1. Hybridization based markers
• Restriction Fragment Length Polymorphisms (RFLPs)
where differences in the number and size of
fragments is analyzed

2. Polymerase Chain Reaction (PCR) based

• Randomly Amplified Polymorphic DNAs (RAPDs),
Single Sequence Repeats (SSRs).
• Amplified Fragment Length Polymorphic DNA
(AFLPs)
• Other variants such as SCAR, CAPS, SSCP e.t.c.

3. Sequence based markers

• Expressed Sequence Tags (ESTs),
• Single Nucleotide Polymorphism (SNPs)
33
 Restriction Fragment Length Polymorphisms
(RFLPs)

 The first type of DNA markers that were used for

genetic mapping were RFLPs.

 For instance a given restriction site may be present

in one line and not in the other.

34
 Procedure
 For detecting RFLPs involves the fragmentation of
genomic DNA by a Restriction enzyme, which can
recognize and cut DNA wherever a specific short
sequence occurs.
 The resulting DNA fragments are then separated by
length in agarose gel electrophoresis, and
transferred to a membrane via the
Southern blot procedure.
 Hybridization of the membrane to a labeled DNA
probe then determines the size of the fragments
which are complementary to the probe.
35
  An RFLP occurs when the size of a detected
fragment varies between individuals.
 Each fragment size is considered an allele, and can
be used in genetic analysis.

36
RFLP markers have several advantages:
1. They are co-dominant and unaffected by the
environment.

2. Any source DNA can be used for the analysis.

3. Many markers can be mapped in a population

that is not stressed by the effects of phenotypic
mutations.

A main disadvantage is that:

 RFLP mapping necessitates relatively large
amounts of DNA. 37
 Cleaved Amplified Polymorphic Sequences
(CAPS)
 The principle of CAPS markers is very similar to that of
RFLP markers.

 The main difference is that PCR is used instead of DNA blot

hybridisation to detect a restriction site polymorphism.

 A genomic DNA region is amplified by PCR using specific

primers and those amplified fragments are then digested
with a diagnostic restriction enzyme to reveal the
polymorphism.

 RFLP probes can be anonymous clones, CAPS markers

require sequence information to design the specific PCR
primers.
38
39
Advantages

1. CAPS markers are co-dominant.

2. Most CAPS genotypes are easily scored and

interpreted.

3. CAPS markers require only small quantities of

genomic DNA.

40
Random Amplified Polymorphic DNA (RAPD)

 RAPD markers are another type of PCR-based markers that

have been used for genetic mapping.

 This approach is based on the amplification of random DNA

segments with single primers of arbitrary nucleotide
sequence.

 The oligonucleotide (around 10-bp long) is used for PCR at

low annealing temperatures.

 When the oligonucleotide hybridises to both DNA strands at

sites within an appropriate distance from each other, the

41
  DNA region delimited by these two sites will be amplified.

 Small nucleotide changes (polymorphism) at one of the two

sites may prevent hybridisation of the oligonucleotide and
hence also prevent DNA amplification.

42
 Typically a RAPD primer will amplify a given fragment from
line A and not from line B.

 It will thus be impossible to distinguish an homozygous

individual AA from an heterozygous individual AB.

 In other words, RAPDs are dominant markers and are thus

less efficient than co-dominant markers in extracting
information from a given F2 population.

 Another limitation of RAPD markers is that because of the

low annealing temperatures used, the amplification of a
given polymorphic band seems to be highly sensitive to PCR
conditions and hence less consistently reproducible in
different laboratories.
43
 Advantages:

1. Random distribution throughout the genome

2. The requirement for small amount of DNA (5-20 ng)

3. Easy and quick to assay

4. The efficiency to generate a large number of

markers

5. Cost-effectiveness!

44
 Limitations

1. Dominant nature (heterozygous individuals can not

be separated from dominant homozygous)
2. Sensitivity to changes in reaction conditions, which
affects the reproducibility of banding patterns
3. The results are not easily reproducible between
laboratories

45
 Amplified Fragment Length Polymorphism
(AFLP)
 AFLP TM is a patented technology developed by KeyGene,
Wageningen, The Netherlands.

 In this procedure, the genomic DNA is digested by two

different restriction enzymes, a rare cutter and a frequent
cutter.

 Double-stranded adapters are then ligated to the ends of the

restriction fragments.

 The fragments are then amplified by PCR using primers that

correspond to the adapter and restriction site sequences.

46
  These primers have additional nucleotides at the 3' ends
extending into the restriction fragments, in order to limit the
number of fragments that will be amplified.

 The AFLP products are detected by labelling one of the two

primers, and the labelled DNA fragments are separated by
electrophoresis in denaturing polyacrylamide gels (similar to
sequencing gels).

 Typically, 50 to 100 amplification products are detected in a

single lane. Polymorphic bands can be identified by
comparing the amplification products derived from two lines.

 Like RAPDs, AFLPs are typically dominant markers.

47
 The procedure of AFLP technique is
divided into three steps:
1. Digestion of total cellular DNA with one or more
restriction enzymes and ligation of restriction half-
site specific adaptors to all restriction fragments.
2. Selective amplification of some of these fragments
with two PCR primers that have corresponding
adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel
matrix, followed by visualisation of the band
pattern.
48
 Simple Sequence Repeats (SSR):
Microsatellites
 Regions of genome where a short (1-4 base) motif is
repeated many times (can be repeated 10 to 100 times)

 These microsatellite repeat sequences are usually

polymorphic in different lines because of variations in the
number of repeat units.

 These polymorphisms are called SSR, and can be

conveniently used as co-dominant genetic markers.

 As compared to CAPS markers, SSR offer the additional

advantage that they do not involve the use of restriction
endonucleases and thus avoid the problems associated with
partial digestions.
49
 One common example of a microsatellite is a (CA)
repeat.

 CA nucleotide repeats are very frequent in human

and other genomes, and present every few
thousand base pairs.

 Microsatellites developed for particular species can

often be applied to closely related species, but the
percentage of loci that successfully amplify may
decrease with increasing genetic distance.

50
Single Nucleotide Polymorphisms (SNPs)
Definition of SNP = Single Nucleotide Polymorphism: a single
base difference in DNA sequence among individuals.

 (SNP, pronounced snip), is a DNA sequence variation

occurring when a single nucleotide - A, T, C, or G - in the
genome (or other shared sequence) differs between
members of a species (or between paired chromosomes in
an individual).

51
DNA strand 1 differs from
DNA strand 2 at a single
base-pair location (a C/T
polymorphism).

In this case we say that

there are two alleles : C
and T.

52
Factors to consider when choosing markers

1. Abundance
– Dependent on frequency at which the marker
sites occur through out the genome.

2. Level of polymorphism
– Determined by rate of mutations in a loci e.g.
charges in a protein, number of repeats in a
core sequence in microsatellite, substitutions,
deletions, insertions etc.
53
3. Locus specificity
– Homology Vs Non homology of bands need to
be considered.

4. Codominance/dominance
– Allows differentiation of homozygotes and
heterozygotes and therefore determination of
genotypes and allele frequencies at a loci

54
5. Reproducibility
– Repeatability of findings over time and space.

6. Labor intensity and safety

– RFLPs Vs SSRs.

7. Technical demand
– Skills required, equipment needed
Hybridization >PCR-based

55
8. Operational costs
– Chemicals, supplies, visualization techniques
e.g. labeling, staining methods etc.

9. Development costs
– Construction of genomic libraries and
development of site specific PCR-primers for
SSRs and Probes for RFLPs, Sequencing is
expensive

56
10. Quantity of DNA required for analysis
– RFLPs = 5-10µg, PCR =5-100ng per reaction
– Important when only small tissues are
available.

11. Amenability to automation

– Increase in sample throughput
– Manually, 100 PCR reactions > 2hrs to set up;
Robot <20 min

57