“SMART breeding: A non-invasive biotechnology alternative to genetic engineering of plant

varieties”

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Pavan. RPh. D Scholar

Department of Genetics and Plant BreedingUniversity of Agricultural Sciences

Bengaluru-65

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Key challenges

Feeding the world within the carrying capacity of

planet earth (2x more with 2x less)

Improve food security, safety and quality

Increase the production per ha

Reduce input at the same time

Use biomass for bio-fuels and green chemistry while

securing food production

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Accelerating The Breeding Cycle

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Breeder’s equation

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Selection Intensity

Large F2 populations

Big screening nurseries

Many crosses / populations

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Selection Accuracy

Many replicated trials

National/International trials

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Genetic Variance

Bring in new genes not present in current program

Nachum Kedar, Israeli Scientist

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Years per Cycle

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Running short of time……….!!

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Use of molecular markers in selection

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How molecular markers server our purpose?

Simpler than phenotypic screening, which can save

time, resources and effort

Selection can be carried out at the seedling stage

Single plants can be selected

Shortens the breeding cycle

Selection of drought tolerant plant without drought

No question of biosafety and bioethics

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Defined as DNA sequence with a known location on

a chromosome.

Molecular marker

Qualities of a Suitable molecular marker are:

1) Must be polymorphic

2) Co-dominant inheritance

3) Randomly and frequently distributed throughout the

genome

4) Easy and cheap to detect

5) Easily reproducible

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FIRST GENERATION DNA MARKERS

Year Acronym Nomenclature Reference

1974 RFLP Restrition Fragment Length Polymorphism Grodzicker et al. (1974)

1985 VNTR Variable Number Tandem Repeats Jeffreys et al. (1985)

1989 SSCP Single Stranded Conformational Polymorphism Orita et al. (1989)

1989 STS Sequence Tagged Site Olsen et al. (1989)

SECOND GENERATION DNA MARKERS

1990 RAPD Randomly Amplified Polymorphic DNA Williams et al. (1990)

1992 CAPS Cleaved Amplified Polymorphic Sequence Akopyanz et al. (1992)

1992 SSR Simple Sequence Repeats Akkaya et al. (1992)

1993 SCAR Sequence Characterized Amplified Region Paran and Michelmore (1993)

NEW GENERATION DNA MARKERS

1994 ISSR Inter Simple Sequence Repeats Zietkiewicz et al (1994)

1994 SNP Single Nucleotide Polymorphisms Jordan and Humphries (1994)

1995 AFLP Amplified Fragment Length Polymorphism Vos et al. (1995)

1996 ISTR Inverse Sequence-Tagged Repeats Rhode (1996)

1997 S-SAP Sequence-Specific Amplified Polymorphism Waugh et al. (1997)

1998 RBIP Retrotransposon Based Insertional Polymorphism Flavell et al. (1998)

1999 REMAP Retrotransposon-Microsatellite Amplified Polymorphism Kalendar et al. (1999).

2001 SRAP Sequence-related amplified polymorphism Li and Quiros (2001)1515

Different types of molecular marker profiles

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Marker assisted selection (MAS):

Defined as Phenotype is selected based on the

genotype of the marker.

Reliability: Markers should be tightly linked to target

loci, preferably less than 5 cM genetic distance. DNA

quantity and quality.

DNA quality and quantity

Level of polymorphism: it should discriminate

between different genotypes

Cost

Assumption

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F2

P2

F1

P1 x

large populations consisting of thousands

of plants

ResistantSusceptible

MARKER-ASSISTED SELECTION (MAS)

MARKER-ASSISTED BREEDING

Method whereby phenotypic selection is based on DNA markers

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Marker development ‘pipeline’

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APPLICATIONS OF MAS IN PLANT BREEDING

1. Marker assisted evaluation of breeding material

2. Marker-assisted backcrossing

3. Marker-assisted pyramiding

4. Early generation marker-assisted selection and

5. Combined marker-assisted selection

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1. Marker-assisted evaluation of breeding material

Cultivar identity/assessment of ‘purity

Assessment of genetic diversity and parental selection

Study of heterosis

Identification of genomic regions under selection

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2. Marker-assisted backcrossing (MAB)

Advantages over conventional backcrossing:

◦ Minimize linkage drag

◦ Accelerated recovery of recurrent parent- Young & Tanksley (1989)

◦ Effective selection of target loci- Tanksely (1983)

FOREGROUND

SELECTIONBACKGROUND SELECTION

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Foreground selection

Proposed by Tanksely (1983)

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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Donor/F1 BC1

c

BC3 BC10

TARGET

LOCUS

RECURRENT PARENT

CHROMOSOME

DONOR

CHROMOSOME

TARGET

LOCUS

LIN

KE

D D

ON

OR

GE

NE

S

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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• Markers can be used to greatly minimize the amount of donor chromosome….but how?

Marker-assisted backcrossing

F1

c

BC1 BC2

TARGET

GENE

Conventional backcrossing

F1 BC1

c

BC2

c

BC3 BC10 BC20

TARGET

GENE

Reduces the number of breeding cycles

(Ribaut and Hoisington, 1998 )

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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

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Background selection

Proposed by Young and Tanksley

(1989) and termed by Hospital and

Charcosset (1997)

Accelerates the recovery of the

recurrent parent genome

Savings of 3 to 4 backcross

generations

1 2 3 4

BACKGROUND

SELECTION

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Ideal Markers must be

Ideally markers should be <5 cM from a gene or QTL

tightly-linked to target loci!

Marker A

QTL5 cM

RELIABILITY FOR

SELECTION

Using marker A only:

~95%

rA

QTL

Marker B

4 cM

Using marker B only

~96%

rB

Marker A

QTL

Marker B

5 cM 4 cM

Using markers A and B:

~99.5%

rA rB

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Markers must be polymorphic

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

P1 P2

P1 P2

Not polymorphic Polymorphic!

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Regularly affects 15 million hectares or more of rainfed

lowland rice areas in South and Southeast Asia.

An economic loss of up to one billion US dollars annually has

been estimated

A major QTL, Sub1 was fine mapped on chromosome 9 in

FR13A cultivar, IR49830-7 (Donor parent)

Swarna (Recipient parent), widely grown cultivar in India and

Bangladesh

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Foreground selection & recombination selectionRM219 – 3.4 cM/Sub1/ RM464A -0.7 cM,RM316- 1.5 cM

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(Neeraja et al., 2007)32

Screening for submergance tolerance

Fourteen- day-old seedlings were submerged for 14 days(BC1F2,

BC2F2 and BC3F2). The survival of plants was scored 14 days

after de-submergence (calculated as a percentage) for

confirmation of the presence of the Sub1 locus.

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Introgressed fragment containing Sub1 in a selected BC3F2 plant

BC3F2 plant (No. 227-9-407) Selected BC2F2 plant No. 246-237

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Submergence tolerance of Swarna-Sub1 (BC2 progeny)

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Salinity is the most common abiotic stresses affects rice

growth in all stages of crop, leads to the reduction in yield

Major salinity tolerance QTL, named Saltol, which responsible

for seedling-stage salinity tolerance was identified on the

short arm of chromosome 1

Used 368 SSR markers, 89 were polymorphic (8 in Saltol locus)

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Foreground selection

RM3412,

RM140,

RM493

Recombination selection

RM1287, RM10843, RM10852,

RM562 and RM7075

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3. Marker-assisted pyramiding

Widely used for combining multiple disease resistance

genes for specific races of a pathogen

Pyramiding is extremely difficult to achieve using

conventional methods

Important to develop ‘durable’ disease resistance

against different races of pathogen

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Marker Assisted Gene Pyramiding

Assembling multiple desirable genes from multiple parents into single

genotype

(Hospital et al., 2004)

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F2

F1

Gene A + B

P1

Gene A

x P1

Gene 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

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A. Stepwise transfer;B. Simultaneous transfer; C. Simultaneous and stepwise transfer

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Blast, caused by the fungus Magnaporthae grisea is

one of the most serious diseases of rice (Oryza

sativaL.) worldwide

Three major genes for blast resistance, Pi1, Piz-5

and Pita on chromosomes 11, 6, and 12, in three

NILs, C101LAC, C101A51 and C101PKT respectively.

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RG64 (SAP) and RG456:Piz-5

RZ536 :Pi1

RZ397 and RG869 for Pita

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The Xa21gene (resistance to bacterial blight), the

Bt fusion gene (for insect resistance) and the

chitinase gene-RC7 (for tolerance of sheath blight)

were combined in a single rice line by reciprocal

crossing of two transgenic homozygous IR72 lines.

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4. Early generation marker-assisted selection

MAS conducted at F2 or F3 stage

Very useful in self pollinated crops to fix alleles in their

homozygous state as early as possible

Allows breeders to focus attention on a lesser number of

high-priority lines in subsequent generations

Linkage between the marker and QTL is not very tight,

Disadvantage is the cost of genotyping a larger number

of plants.

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F2

P2

F1

P1 x

large populations (e.g. 2000 plants)

ResistantSusceptible

MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes

MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes

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Benefits: breeding program can be efficiently scaled down to focus on fewer lines

Ribaut & Betran (1999)

SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)

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5. Combined MAS approaches

In some cases, a combination of phenotypic screening

and MAS approach may be useful

1. To maximize genetic gain (when some QTLs have

remained unidentified)

2. Especially when large population sizes are used and trait

heritability is low

3. Low level of recombination between marker and QTL

4. 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)

Recurrent

Parent

Donor

Parent

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

SAVE TIME & REDUCE

COSTS

*Especially for quality traits*

MARKER-ASSISTED SELECTION (MAS)

PHENOTYPIC SELECTION

(Also called ‘tandem selection’)

• Use when markers are

not 100% accurate or

when phenotypic

screening is more

expensive compared to

marker genotyping

Han et al (1997). 57

REASONS TO EXPLAIN THE LOW IMPACT

OF

MARKER-ASSISTED SELECTION

1.Still at the early stages of DNA marker technology

development

Even though SSR markers were developed in 1990’s

the availability is less

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A significant gap between QTL discovery and their utilization in MAS is

depicted.

2. Marker-assisted selection results may not be

published

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3. Reliability and accuracy of QTL mapping

studies

No. of replication used to generate phenotypic

data

Population size

Sampling bias

Factors may affect the accuracy of a QTL mapping

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4. Insufficient linkage between marker and gene/QTL

5. Limited markers and limited polymorphism of

markers in breeding material

6. ‘Application gap’ between research laboratories

and plant breeding institutes

7. ‘Knowledge gap’ among molecular biologists, plant

breeders and other disciplines

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8. Effects of genetic background

QTLs identified in a particular mapping population

may not be effective in different backgrounds

(Liao et al.2001)

Only one of four root length QTLs were effective

when transferred by backcrossing into a new rice

variety.

Steele et al. (2006)

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9. Quantitative trait loci × environment interaction

The magnitude of effect and even direction of QTLs

may vary depending on environmental conditions

Li et al.2003

10. High cost of marker-assisted selection

The inheritance of the trait,

The method of phenotypic evaluation,

The cost of field and glasshouse trials and labour costs

Large initial capital investments on purchase of

equipment, and regular expenses for maintenance.

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Commercially available MAS varieties developed by public institution

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Examples of traits, for which marker assisted breeding is

being applied in rice

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Research projects for the nutritional improvement

of varieties by marker assisted selection

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