MARKER-ASSISTED BACKCROSSING

Definition:

Marker assisted selection (MAS) refers to the use of DNA markers that are tightly-linked to target loci as a substitute for or to assist phenotypic screening

Assumption: DNA markers can reliably predict phenotype

Backcross breeding (MAB) is a well-known procedure for the introgression

of a target gene from a donor line into the genomic background of a

recipient line.

The objective is to reduce the donor genome content (DGC) of the

progenies by repeated back-crosses to the recipient line.

Marker-assisted backcross is of great practical interest in applied breeding

schemes either to manipulate ‘classical’ genes between elite lines or from

genetic resources, or to manipulate transgenic constructions.

Principles and Requirements for MAB:

MAB is the process of using the results of DNA tests to assist in the selection of

individuals to become the parents in the next generation of a genetic improvement

program.

It is an approach that has been developed to avoid problems connected with

conventional plant breeding by changing the selection criteria from selection of

phenotypes towards selection of genes that control traits of interest, either directly or

indirectly.

Molecular markers are clearly not influenced by environment (unaffected by the

conditions in which the plants are grown) and are detectable at all stages of plant growth.

With the availability of an array of molecular markers ( Semagn et al., 2006a for review)

and genetic maps,

MAB has become possible both for traits governed by single gene and quantitative

trait loci (QTLs) (Francia et al.,2005).

F2

P2

F1

P1 x

large populations consisting of thousands

of plants

PHENOTYPIC SELECTION

Field trialsGlasshouse trials

DonorRecipient

CONVENTIONAL PLANT BREEDING

Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

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

The success of MAB depends upon:

•The distance between the closest markers and the target gene,

• Number of target genes to be transferred,

• Genetic base of the trait,

• Number of individuals that can be analyzed and the genetic background in which the

target gene has to be transferred,

•The type of molecular marker(s) used, and available technical facilities (Weeden et al.,

1992; Francia et al., 2005).

• Identification of molecular markers that should co-segregate or be closely linked with

the desired trait is a critical step for the success of MAB.

• The most favorable case for MAB is when the molecular marker is located directly

within the gene of interest (direct markers).

•MAB conducted using direct markers is called gene assisted selection (Dekkers03).

The lower the genetic distance between the marker and the gene, the more reliable is the application of the marker in MAB because only in few cases will the selected marker allele be separated from the desired trait by a recombination event.

The presence of a tight linkage between desirable trait(s) and a molecular marker(s) may be useful in MAB to increase gain from selection.

In backcross breeding, markers can be used to:

i) Control the target gene (Foreground selection)

ii) Control the genetic background (Background selection).

iii) Control the linkage drag (Recombinant selection)

1) Foreground selection:

•select for marker allele of donor genotype/Target gene

•close linkage between marker loci and target loci is essential

This may be particularly useful for traits that have laborious or time-consuming phenotypic screening procedures .

Background selection:

The second level of MAB involves selecting BC progeny with the greatest proportion of recurrent parent (RP) genome, using markers that are unlinked to

the target locus—refer to this as ‘background selection’.

Background markers are markers that are unlinked to the target gene/QTL on

all other chromosomes,

In other words, markers that can be used to select against the donor genome.

The use of background selection during MAB to accelerate the development of an RP with an additional (or a few) genes has been referred to as ‘complete line conversion’ (Ribaut et al. 2002).

The third level involves selecting BC progeny with the target gene and recombination events between the target locus and linked flanking markers—refer to this as ‘recombinant selection’.

The purpose of recombinant selection is to reduce the size of the donor chromosome segment containing the target locus (i.e. size of the introgression).

This is important because the rate of decrease of this donor fragment is slower than it leads to as ‘linkage drag’ (Hospital 2005).

RECOMBINANT SELECTION:

Marker assisted back cross breeding schematic representation:

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Overview of ‘marker

genotyping’

Considerations for using DNA

markers in plant breeding

• Technical methodology

– simple or complicated?

• Reliability

• Degree of polymorphism

• DNA quality and quantity required

• Cost**

• Available resources

– Equipment, technical expertise

Markers must be

tightly-linked to target loci!

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

• Using a pair of flanking markers can greatly improve reliability but increases time and cost

Marker A

QTL5 cM

RELIABILITY FOR SELECTION

Using marker A only:

1 – rA = ~95%

Marker A

QTL

Marker B

5 cM 5 cM

Using markers A and B:

1 - 2 rArB = ~99.5%

Markers must be 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!

Advantages of MAB:

• When phenotypic screening is expensive, difficult or impossible.

• When the trait is of low heritability (incorporating genes that are highly affected by environment).

• When the selected trait is expressed late in plant development, like fruit and flower features or adult characters in species with a juvenile period.

• For incorporating genes for resistance to diseases or pests that cannot be easily screened for due to special requirement for the gene to be expressed.

• When the expression of the target gene is recessive.

• To accumulate multiple genes for one or more traits within the same cultivar, a process called gene pyramiding

Reasons for unexpected results in MAB:

•The putative QTL may be a false positive.

•QTL and environmental interactions (Ribaut et al.,)

•Epistasis between QTLs and QTL and genetic background.

•QTL contain several genes and recombination between those genes would modify the effect of the introgressed segment (Eshed and zamir, 1995;Monna et al.,2002)

Donor/F1 BC1

c

BC3 BC10

TARGET LOCUS

RECURRENT PARENT CHROMOSOME

DONOR CHROMOSOME

TARGET LOCUS

LIN

KED

DO

NO

R

GEN

ES

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

Conventional backcrossing

Marker-assisted backcrossing

F1 BC1

c

BC2

c

BC3 BC10 BC20

F1

c

BC1 BC2

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

TARGET GENE

TARGET GENE

Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239.

Some considerations for MAB

• Main considerations:

– Cost

– Labour

– Resources

– Efficiency

– Timeframe

• Strategies for optimization of MAB process important

– Number of BC generations

– Reducing marker data points (MDP)

– Strategies for 2 or more genes/QTLs

IRRI MAB CASE STUDY

3. Marker-assisted backcrossing for

submergence tolerance in rice

David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar, Darlene

Sanchez, Endang Septiningsih & Abdel Ismail

Photo by Abdel Ismail

Abiotic stresses are major constraints to

rice production in SE Asia

• Rice is often grown in unfavourable environments in Asia

• Major abiotic constraints include:– Drought

– Submergence

– Salinity

– Phosphorus deficiency

• High priority at IRRI• Sources of tolerance for all traits in germplasm and

major QTLs and tightly-linked DNA markers have been identified for several traits

‘Mega varieties’

• Many popular and widely-grown rice varieties - “Mega varieties”– Extremely popular with farmers

• Traditional varieties with levels of abiotic stress tolerance exist however, farmers are reluctant to use other varieties– poor agronomic and quality

characteristics

BR11 Bangladesh

CR1009 India

IR64 All Asia

KDML105 Thailand

Mahsuri India

MTU1010 India

RD6 Thailand

Samba

Mahsuri

India

Swarna India,

Bangladesh

1-10 Million hectares

Conventional backcrossingx P2P1

DonorElite cultivar

Desirable trait

e.g. disease resistance

• High yielding

• Susceptible for 1 trait

• Called recurrent parent (RP)

P1 x F1

P1 x BC1

P1 x BC2

P1 x BC3

P1 x BC4

P1 x BC5

P1 x BC6

BC6F2

Visually select BC1 progeny that resemble RP

Discard ~50% BC1

Repeat process until BC6

Recurrent parent genome recovered

Additional backcrosses may be required due to linkage drag

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

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

‘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

References:

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

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)

• Important when donor is a traditional variety

RECOMBINANT SELECTION

1 2 3 4

OR

Step 1 – select target locus

Step 2 – select recombinant on either side of target locus

BC1

OR

BC2

Step 4 – select for other recombinant on either side of target locus

Step 3 – select target locus again

* *

* Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2

MAB: 3RD LEVEL OF SELECTION -

BACKGROUND SELECTION

• Use unlinked markers to

select against donor

• Accelerates the recovery of

the recurrent parent genome

• Savings of 2, 3 or even 4

backcross generations may

be possible

1 2 3 4

BACKGROUND SELECTION

Background selection

Percentage of RP genome after backcrossing

Theoretical proportion of the recurrent parent genome is given by the formula:

Where n = number of backcrosses, assuming large population sizes

2n+1 - 1

2n+1

Important concept: although the average percentage of the recurrent parent is 75% for BC1, some individual plants possess more or less RP than others

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

BC1

USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME

BC2

Breeding for submergence tolerance

• Large areas of rainfed lowland rice have short-term submergence (eastern India to SE Asia); > 10 m ha

• Even favorable areas have short-term flooding problems in some years

• Distinguished from other types of flooding tolerance

– elongation ability

– anaerobic germination tolerance

A major QTL on chrom. 9 for

submergence tolerance – Sub1 QTL

1 2 3 4 5 6 7 8 9

0

5

10

15

20

Submergence tolerance score

IR40931-26 PI543851

Segregation in an F3 population

0 10 20 30 40

LOD score

50cM

100cM

150cM

OPN4

OPAB16

C1232

RZ698

OPS14RG553

R1016RZ206

RZ422

C985

RG570

RG451

RZ404

Sub-1(t)

1200

850

900

OPH7950

OPQ1600

Xu and Mackill (1996) Mol Breed 2: 219

Make the backcrosses

SwarnaPopular variety

X

IR49830Sub1 donor

F1 X

Swarna

BC1F1

Seeding BC1F1s

Pre-germinate the F1 seeds and seedthem in the seedboxes

Collect the leaf samples - 10 days after

transplanting for marker analysis

Genotyping to select the BC1F1 plants with

a desired character for crosses

Seed increase of tolerant BC2F2

plant

Selection for Swarna+Sub1

Swarna/IR49830 F1

Swarna

BC1F1697 plants

Plant #242

Swarna

376 had Sub121 recombinantSelect plant with fewest donor alleles

158 had Sub15 recombinant

SwarnaPlant #227

BC3F118 plants

1 plant Sub1 with2 donor segments

BC2F1320 plants

Plants #246 and #81

Plant 237BC2F2

BC2F2937 plants

Swarna with Sub1

It is predominantly derived from the initial variety, or from a variety that is

itself predominantly derived from the initial variety, while retaining the

expression of the essential characteristics that result from the genotype or

combination of genotypes of the initial variety,

It is clearly distinguishable from the initial variety and

except for the differences which result from the act of derivation, it

conforms to the initial variety in the expression of the essential

characteristics that result from the genotype or combination of genotypes of

the initial variety.

Phenotypic similarity:

The phenotype of the EDV must be distinct to the phenotype

of the initial variety (according to DUS characteristics) The

initial variety must be protected by PBR.

Genotypic similarity:

The genotype of EDV must widely conform to the genotype

of the initial variety.(estimated through Molecular DNA

analysis Definition of Minimal Distances)

Through back cross method:

Backcrossing is an established breeding method to introduce specific traits (e. g. resistances) into

a certain variety or breeding line. Is it technical feasible to develop„Me too“ varieties through a

continued backcross program? (Self incompatibilty, inbreeding depression)

After 3 rounds of backcrossing the genome of the progeny is almost identical to the backcross

parent

• An introduction to markers, quantitative trait loci (QTL) mapping and

marker-assisted selection for crop improvement: The basic concepts

• Marker assisted backcross breeding to improve cooking quality traits in

Myanmar rice cultivar Manawthukha Myint Yi a,b, Khin Than Nwea,

Apichart Vanavichit b, Witith Chai-arree c, Theerayut Toojinda b,*

• A marker-assisted backcross approach for developing submergence-

tolerant rice cultivars C. N. Neeraja · R. Maghirang-Rodriguez · A.

Pamplona · S. Heuer · B. C. Y. Collard · E. M. Septiningsih · G. Vergara ·

D. Sanchez · K. Xu · A. M. Ismail · D. J. Mackill

• Progress and prospects of marker assisted backcrossing as a tool in crop

breeding programmes-k semegn et al.,

• Marker assisted selection for plant breeding-P.K Gupta

•Essentially derived varieties (edv)

position of ciopora

january 2008