This document discusses marker-assisted backcrossing (MAB) for introgressing traits from a donor parent into a recipient line. MAB uses DNA markers linked to target genes/QTLs to aid in selection. Markers can be used for foreground selection of target genes, background selection to recover the recipient genome, and recombinant selection to minimize linkage drag. A case study is described where MAB was used over multiple generations to introgress 5 drought resistance QTLs from a donor rice variety into a recipient variety. Through foreground, background, and recombinant selection using DNA markers, lines were developed with the target QTLs and most of the recipient genetic background.

1. MARKER-ASSISTED
BACKCROSSING
Anilkumar, C
PALB5062

2. FLOW OF PRESENTATION
• Introduction
• Utilization of markers in backcrossing
I. Foreground selection
II. Background selection
III. Recombinant selection
•Case study
•conclusion
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3. Introduction
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• Plant breeding is an art of
managing the variability.
• Existence of variability is the
pre-requisite for any kind of
breeding programme.
• Creation of variability can be
done through mutation,
hybridization, polyploidy and
genetic engineering.

4. Backcrossing
 Backcross breeding 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.
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5. Backcrossing is used
• To transfer a major gene
• In disease/pest resistance breeding
• To transfer alien cytoplasm or to transfer
cytoplasmic male sterility
• To transfer a transgene from already
developed transgenic line.
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6. Types of backcrossing when more than one
gene is to be transferred from different
sources
• Stepwise transfer
• Simultaneous transfer
• Stepwise but parallel transfer
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7.  Need to grow large number of plants for selection in
each generation of backcrossing.
 Introgression of quantitative traits is nearly not
possible.
 Recovery of recipient genome is less efficient.
 Poses difficulties in negative selection of undesirable
genes or avoiding the linkage drag problems.
Problems of conventional backcrossing
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8. • Selfing after alternative
backcross generation
requires more number of
generations to select
genotype with target
gene having maximum
recurrent parent
background.
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9.  Landmarks in genome
 Not affected by
environment
 Not stage specific
 Not tissue specific
 More precise
 Acts as proxies and helps in
indirect selection
Molecular Markers
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10. MARKER ASSISTED SELECTION
 Marker assisted selection refers to the use of DNA markers that
are tightly-linked to target loci as a surrogate to phenotypes.
 Assumption: DNA markers can reliably predict phenotype.
 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.
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11. 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.
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 programme.
The success of MAB depends upon:
Principle of MAB:
•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). 119/14/2016

12. 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)
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13. Foreground selection:
selection using single marker
Select for marker allele of donor genotype/target gene
Close linkage between marker locus and target locus is essential
The probability that the Q/Q genotype can be obtained through
selection of marker genotype M/M, that is, the probability for
selecting the correct individuals, is
P=(1-r)2
Where, r- recombination frequency of marker and gene
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14. This may be particularly useful for traits that have
laborious or time-consuming phenotypic screening
procedures .
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15. Foreground selection: selection
using multiple marker for
multiple targets
This is helpful in resistance breeding for
disease and pest resistance.
Marker-trait association can be used to
simultaneously select multiple resistances
from different disease races and/or insect
biotypes and pyramid them into a single
line through MAS.
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16. Background selection
o 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’.
o Background markers are markers that are unlinked to the
target gene/QTL on all other chromosomes,
o In other words, markers that can be used to select against
the donor genome.
o The use of background selection during MAB to accelerate the
recovery of recurrent parent genome with additional (or a few)
genes has been referred to as ‘complete line conversion’ (Ribaut et
al. 2002). 9/14/2016
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17. 9/14/2016
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18. 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
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19. 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. 9/14/2016 19

20. RECOMBINANT SELECTION:
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’.
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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. 9/14/2016
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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)
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23. Some considerations that
encourages 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
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25. Rice cultivation in eastern
part of India is purely
depends on rain. This
necessitates the cultivars to
be drought resistant.

26. Selection of parents and target QTLs
• Recipient parent: Kalinga III (Indica rice)
• Donor parent: Azucena (Japonica rice)
• Total QTLs: 5
• Root QTLs: QTL2, QTL7, QTL9, QTL11
• Aroma QTLs: QTL8
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27. BACKCROSSING AND SELECTION
:DEVELOPMENT OF NILS
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28. Pyramid crossing and selection
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29. Foreground selection for target QTLs
RFLPs were used for first 3 backcrosses.
Done using RFLPs that had mapped in Bala ×
Azucena population and flanked, or were within,
the regions containing the target QTLs.
In addition, the RFLP marker C570 was used.
C570 was polymorphic between Azucena and
Kalinga III but not between Bala and Azucena
and mapped near to QTL9.
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30. • Selection after 3rd backcross was done using
flexible and cheaper PCR-based SSRs.
QTLs Trait of QTL RFLPs SSRs
QTL 2 Root thickness, root penetration G39, G57, C601
RM6, RM221,
RM318
QTL 7
Deep root per shoot ratio, root
length, deep root weight per tiller
RG351, RG650,
C507
RM234, RM351
QTL 8 Aroma G1073 RM223
QTL 9 Deep root thickness G385, C570 RM 242
QTL 11 Root length, root penetration C189, G1465 RM229, RM206
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31. Recombinant selection:
selection for recurrent parent allele near QTLs
QTLs Trait of QTL RFLPs SSRs
QTL 2 Root thickness RG171, G45 -
QTL 5
Root length, root thickness
and penetration
RG13, C43 -
QTL 7 Flowering time - RM248
QTL 8 Osmotic adjustment G56 -
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Negative selection was performed at these QTLs for Azucena
alleles because these alleles had negative effect which are linked
to targeted QTLs.

32. Background selection:
selection for recurrent parent allele at other regions
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A- BC3F121-01-03
B- BC3F142-01-05
C-PY2F33-26-05-18

33. Greenhouse and field experiments
Extensive greenhouse and field experiments were carried
out at UAS(B)
I. To identify chromosomal regions from Azucena that
delayed anthesis so that they could be selected against.
II. To test the presence of pleiotropic or linkage drag
effects in developed NILs.
A segment on chromosome 1 that was introgressed
unintentionally that had a significant effect on grain width.
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34. Conclusion
 Five target regions is probably the limit of efficient
MABC breeding (Hospital and Chacosset 1997; Servin et
al. 2004), but they successfully selected an ideotype
with all five regions from Azucena introgressed into the
predominantly Kalinga III genetic background, with
almost complete line conversion.
 The work would have less tedious if they
i. Started with SSRs as number of assays would be less
ii. Started with pre-existing RILs
iii. Tested more lines at each backcross generation
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