BY R.

Dhivya bharathi

So, which of you plants are resistant to fungal diseases?

I am!
I am! I am!

I am!

I am!

I am!

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.

Most promising application of DNA markers for cultivar development. On the detection of polymorphisms in the DNA sequence. Not affected by environmental conditions

PCR
Polymerase Chain Reaction. Used to amplify DNA via enzymatic replication. Allows a small amount of DNA to be used for analysis.

Gel Electrophoresis
Allows separation of PCR product. DNA inserted into agarose gel. DNA fragments travel with current. Smaller fragments will travel faster than large fragments.

The other markers used are.

AFLP Markers

SCAR Markers
RFLP Markers

SSR Markers
RAPD Markers

SNP Markers

Feature
DNA required DNA quality PCR-based Number of

RFLPs
10 High No

RAPDs
0.02 High Yes

AFLPs
0.5-1.0 Moderate Yes

SSRs
0.05 Moderate Yes

SNPs
0.05 High Yes

polymorph loci
analyzed Ease of use Amenable to automation Reproducibility Development cost Cost per analysis

1.0-3.0

1.5-50

20-100

1.0-3.0

1.0

Not easy

Easy

Easy

Easy

Easy

Low
High Low High

Moderate
Unreliable Low Low

Moderate
High Moderate Moderate

High
High High Low

High
High High Low

A. Conventional selection is based on direct measurement of important traits, 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.

Traditional Selection
Infect Plants

Eliminate Susceptible Plants

Marker Assisted Selection

Hundreds of seedlings with the desired trait can be selected by a single person utilizing MAS.

S
R= Resistant S= Susceptible

Development of markers

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION(F2)

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

PCR-based DNA markers

Generated by using Polymerase Chain Reaction Preferred markers due to technical simplicity and cost
PCR Buffer +
MgCl2 + dNTPS + Taq +

PCR

Primers +
DNA template

THERMAL CYCLING

GEL ELECTROPHORESIS
Agarose or Acrylamide gels

Agarose gel electrophoresis

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

UV transilluminator UV light

Backcrossing using Marker Assisted Selection

X
A

Generations of breeding
marker A Gene of interest Eliminate individuals without marker

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!

Marker-assisted backcrossing (MAB)

MAB has several advantages over conventional backcrossing:
Effective selection of target loci Minimize linkage drag Accelerated recovery of recurrent parent
1 2 3 4 1 2 3 4
1 2 3 4

Target locus

TARGET LOCUS SELECTION

FOREGROUND SELECTION

RECOMBINANT SELECTION

BACKGROUND SELECTION

BACKGROUND SELECTION

 Process of combining several genes, usually from 2 different parents, together into a single genotype
Breeding plan Genotypes

P1 x Gene A

P1 Gene B

P1: AAbb

P2: aaBB

F1 Gene A + B

F1: AaBb

F2 MAS

F2
AB Ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Select F2 plants that have Gene A and Gene B

aB ab

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.

MAB: 1st level of selection foreground 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

MAB: 2ND LEVEL OF SELECTION RECOMBINANT SELECTION

Use flanking markers to select recombinants between the target locus and flanking marker Linkage drag is minimized Require large population sizes
depends on distance of flanking markers from target locus
RECOMBINANT SELECTION
1 2 3 4

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

TARGET LOCUS

c
TARGET LOCUS Donor/F1 BC1 BC3
RECURRENT PARENT CHROMOSOME DONOR CHROMOSOME

BC10

LINKED DONOR GENES

MAB: 3RD LEVEL OF SELECTION BACKGROUND SELECTION

Use unlinked markers to select against donor. Accelerates the recovery of the recurrent parent genome.
BACKGROUND SELECTION
1 2 3 4

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

CONVENTIONAL BACKCROSSING

MARKER-ASSISTED BACKCROSSING

P1 x P2 P1 x F1 BC1
VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT

P1 x P2 P1 x F1 BC1
USE 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

BC2

Materials and Methods:- Common peas

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.

Breeding scheme to develop Multiple Disease resistance parents to Angular leaf spot, Anthracnose, Pythium root rot and BCMV/BCMNV

Trait
ALS

Markers
OPE4709 PF9250

Source
MEX 54 G1o474 RWR719

Pythium root rot

PYAA19

PYB08 Anthracnose SAS-13

SBB-14 SH-18

RWR719 G2333
G2333,AB136 G2333

Scab-resistant apple -- markers tightly linked to the Vf resistance 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.

DNA marker-assisted selection was used to pyramid four bacterial blight resistance genes, Xa-4, xa-5, xa-13 and 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-5 and 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.

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

Marker-directed phenotyping
(Also
Recurrent Parent

called tandem selection)

Donor Parent

P1 (S) x P2 (R)

F1 (R) x P1 (S)

BC1F1 phenotypes: R and S

Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping

MARKER-ASSISTED SELECTION (MAS)

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

SAVE TIME & REDUCE COSTS PHENOTYPIC SELECTION

*Especially for quality traits*

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

Important attributes of markers include: Ease of use Small amount of DNA required Repeatability of results High rate of polymorphism Occurrence throughout the genome

Contd.
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.

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 which is seldom reported.

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

Low reliability of markers to determine phenotype.