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So, which of you plants

are resistant to fungal

diseases?

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I am!

I am!I am!

I am!I am!

I am!

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Marker Assisted Selection:

Variation of traditional plant breeding wherein DNA

sequence differences are used to identify plant varieties that

carry the desired traits.

Used when the trait of interest is present within the gene

pool of the crop of interest.

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Most promising application of DNA markers for cultivar

development.

On the detection of polymorphisms in the DNA sequence.

Not affected by environmental conditions

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PCR

Polymerase Chain Reaction.

Used to amplify DNA via enzymatic replication.

Allows a small amount of DNA to be used for

analysis.

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Gel Electrophoresis

Allows separation of PCR product.

DNA inserted into agarose gel.

DNA fragments travel with current.

Smaller fragments will travel faster than largefragments.

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The other markers used are.

AFLP Markers

SCAR Markers

RFLP Markers

SSR Markers

RAPD Markers

SNP Markers

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Feature RFLPs RAPDs AFLPs SSRs SNPs

DNA required 10 0.02 0.5-1.0 0.05 0.05

DNA quality High High Moderate Moderate High

PCR-based No Yes Yes Yes Yes

Number of

polymorph locianalyzed

1.0-3.0 1.5-50 20-100 1.0-3.0 1.0

Ease of use Not easy Easy Easy Easy Easy

Amenable to

automation

Low Moderate Moderate High High

Reproducibility High Unreliable High High High

Development cost Low Low Moderate High High

Cost per analysis High Low Moderate Low Low

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A. Conventional selection is based on direct measurement of importanttraits, such as yield, maturity, or disease resistance.

B. In marker-assisted selection, plants are selected based on molecular

marker patterns known to be associated with the traits of interest.

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Traditional Selection

Infect Plants

Eliminate

SusceptiblePlants

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Marker Assisted Selection

S R R R S R S

Hundreds of seedlingswith the desired trait

can be selected by a

single person utilizing

MAS.

R= Resistant

S= Susceptible

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(1) LEAF TISSUE

SAMPLING

(2) DNA EXTRACTION(F2)

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Development of markers

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PCR-based DNA markers Generated by using Polymerase Chain Reaction

Preferred markers due to technical simplicity and cost

GEL ELECTROPHORESIS

Agarose or Acrylamide gels

PCR

PCR Buffer +

MgCl2 +

dNTPS +

Taq +

Primers +

DNA template

THERMAL CYCLING

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Agarose gel electrophoresis

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

UV light

UV transilluminator

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Backcrossing using Marker Assisted Selection

Generations of breeding

XA

AA A A AA

marker

A Gene ofinterest

Eliminateindividualswithout marker

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Markers mustbe polymorphic

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

RM84 RM296

P1 P2

P1 P2

Not polymorphic Polymorphic!

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Marker-assisted backcrossing (MAB)

MAB has several advantages over conventionalbackcrossing:

Effective selection of target loci

Minimize linkage drag

Accelerated recovery of recurrent parent

1 2 3 4

Target

locus

1 2 3 4

RECOMBINANT

SELECTION

1 2 3 4

BACKGROUND

SELECTION

TARGET LOCUS

SELECTION

FOREGROUND

SELECTION

BACKGROUND SELECTION

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F2

F1Gene A + B

P1Gene A

x P1Gene B

MAS

Select F2 plants that have Gene

A and Gene B

Genotypes

P1: AAbb P2: aaBB

F1: AaBb

F2AB Ab aB ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Process of combining several genes, usually from 2 different parents,

together into a single genotype

x

Breeding plan

Hittalmani et al. (2000). Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in riceTheor. Appl.

Genet. 100: 1121-1128

Liu et al. (2000). Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant Breeding 119:21-24.

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MAB: 1st level of selectionforeground

selection

Selection for target gene or QTL

Useful for traits that are difficult to

evaluate

Also useful for recessive genes

1 2 3 4

Target locus

TARGET LOCUS SELECTION

FOREGROUND SELECTION

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MAB: 2ND LEVEL OF SELECTION -

RECOMBINANT SELECTION

Use flanking markers to selectrecombinants between the targetlocus and flanking marker

Linkage drag is minimized

Require large population sizes

depends on distance of flankingmarkers from target locus

RECOMBINANT SELECTION

1 2 3 4

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Donor/F1BC1

c

BC3 BC10

TARGET

LOCUS

RECURRENT PARENT

CHROMOSOME

DONOR CHROMOSOME

TARGET

LOCUS

LIN

KED

DONOR

GENES

Concept of linkage drag

Large amounts of donor chromosome remain even after many

backcrosses

Undesirable due to other donor genes that negatively affect

agronomic performance

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MAB: 3RD LEVEL OF SELECTION -

BACKGROUND SELECTION

Use unlinked markers to select

against donor.

Accelerates the recovery of therecurrent parent genome.

Savings of 2, 3 or even 4backcross generations may be

possible

1 2 3 4

BACKGROUND SELECTION

CONVENTIONAL BACKCROSSING MARKER ASSISTED BACKCROSSING

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P1 x F1

P1 x P2

CONVENTIONAL BACKCROSSING

BC1

VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY

RESEMBLE RECURRENT PARENT

BC2

MARKER-ASSISTED BACKCROSSING

P1 x F1

P1 x P2

BC1USE BACKGROUND MARKERS TO SELECT PLANTS THAT HAVE

MOST RP MARKERS AND SMALLEST % OF DONOR GENOME

BC2

Comparison of conventional and marker-assisted backcrossing for recurrent parent recovery

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Single crosses between of screening up to 1500 F2 plants per cross MCM5001and MCM and bc- 3 genes for BCMV/BCMNV

G2333Co-4, Co-5 and Co-7 for resistance to anthracnose,

RWR719 and MLB-49-89A-Pythium root rot,

MEX54- phg for resistance to ALS.

DNA extracted from leaves of 2 week old F2 plants.

2mm discs used as templates in PCR reactions using specific molecular

markers.

Plants positive for 2-3 gene combination selected and double crosses

conducted.

Materials and Methods:- Common peas

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Breeding scheme to develop Multiple Disease resistance parents to Angular

leaf spot, Anthracnose, Pythium root rot and BCMV/BCMNV

T it M k S

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Trait Markers Source

ALS OPE4709 MEX 54

PF9250 G1o474

Pythium root rot PYAA19 RWR719

PYB08 RWR719

Anthracnose SAS-13 G2333

SBB-14 G2333,AB136

SH-18 G2333

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Scab-resistant apple -- markers tightly linked to the Vfresistance

gene.

Markers -- SCAR markers & RAPD markers.

AM19-SCAR is a co-dominant marker.

The availability of two co-dominant, tightly linked markers flanking

both sides of the resistance gene (AL07-SCAR and M18-CAPS) also

makes it easy to identify the seedlings homozygous for the resistance

gene.

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DNA marker-assisted selection was used to pyramid four bacterial

blight resistance genes, Xa-4, xa-5, xa-13and Xa-21.

To speed up the gene pyramiding process and to facilitate future

marker-aided selection, PCR markers is developed for the two recessive

genes, xa-5and xa-13, and used these to survey a range of rice

germplasm.

Aimed at transferring these bacterial blight resistance genes from one

varietal background to another.

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Combined approaches

In some cases, a combination of phenotypic screening and MAS

approach may be useful.

To maximize genetic gain (when some QTLs have been unidentified

from QTL mapping).

Level of recombination between marker and QTL (in other words

marker is not 100% accurate).

To reduce population sizes for traits where marker genotyping is

cheaper or easier than phenotypic screening

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Marker-directed phenotyping

BC1F1 phenotypes: R and S

P1 (S) x P2 (R)

F1(R) x P1(S)

RecurrentParent

DonorParent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

SAVE TIME & REDUCECOSTS

*Especially for quality traits*

MARKER-ASSISTED SELECTION (MAS)

PHENOTYPIC SELECTION

(Also called tandem selection)

Use when markers are not100% accurate or whenphenotypic screening is moreexpensive compared tomarker genotyping

References:

Han et al (1997). Molecular marker-assisted selection for malting quality traits in barley. Mol Breeding 6: 427-437.

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Important attributes of markers include:

Ease of use

Small amount of DNA required

Repeatability of results

High rate of polymorphism

Occurrence throughout the genome

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Simpler compared to phenotypic screening

Selection may be carried out at seedling stage

Single plants may be selected with high reliability.

Co-dominance - ability to detect both parental forms of a marker

in heterozygotes.

Contd.

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Drawbacks:

The equipment and consumables required to establish and

maintain a marker lab is considerable.

There is a large initial cost in the development of markers whichis seldom reported.

Source of gene is restricted to the gene pool of the species.

Low reliability of markers to determine phenotype.

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