marker assisted selection

1. MARKER-ASSISTED BACKCROSSING

2. 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

3.  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.

4. 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).

5. 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

6. 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

7. 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).

8. 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.

10. 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

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

12. 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).

14. 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).

15. RECOMBINANT SELECTION:

16. Marker assisted back cross breeding schematic representation:

17. (1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
Overview of
‘marker
genotyping’

18. 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

19. 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
QTL
5 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%

20. 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!

21. 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

22. 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)

23. Donor/F1 BC1
c
BC3 BC10
TARGET
LOCUS
RECURRENT PARENT
CHROMOSOME
DONOR CHROMOSOME
TARGET
LOCUS
LINKEDDONOR
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

24. 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.

25. 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

26. IRRI MAB CASE STUDY

27. 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

28. 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

29. ‘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

30. Conventional backcrossing
x 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

31. 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

32. 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

33. ‘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.

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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
OPS14
RG553
R1016
RZ206
RZ422
C985
RG570
RG451
RZ404
Sub-1(t)
1200
850
900
OPH7
950
OPQ1
600
Xu and Mackill (1996) Mol Breed 2: 219

41. Make the backcrosses
Swarna
Popular variety
X
IR49830
Sub1 donor
F1 X
Swarna
BC1F1

42. Seeding BC1F1s
Pre-germinate the F1 seeds and seed
them in the seedboxes

43. Collect the leaf samples - 10 days after
transplanting for marker analysis

44. Genotyping to select the BC1F1 plants with
a desired character for crosses

45. Seed increase of tolerant BC2F2
plant

46. Selection for Swarna+Sub1
Swarna/
IR49830 F1
Swarna
BC1F1
697 plants
Plant #242
Swarna
376 had Sub1
21 recombinant
Select plant with
fewest donor
alleles
158 had Sub1
5 recombinant
SwarnaPlant #227
BC3F1
18 plants
1 plant Sub1 with
2 donor segments
BC2F1
320 plants
Plants #246
and #81
Plant 237
BC2F2
BC2F2
937 plants

47. Swarna with Sub1

55. 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.

56. 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)

58. 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

59. • 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