Backcrossing in breeding

1. Marker-Assisted Backcrossing in breeding
Doctoral seminar
Presented by:-
Bineeta Devi
I.D. No.- 10PHGPB207
DEPARTMENT OF GENETICS AND PLANT BREEDING
ALLAHABAD SCHOOL OF AGRICULTURE
SAM HIGGINBOTTOM INSTITUTE OF AGRICULTURE,
TECHNOLOGY AND SCIENCES

2. Marker assisted backcrossing
There are three levels of selection in which markers
may be applied in backcross breeding.
 In the first level, markers may be used to screen for
the target trait, which may be useful for traits that have
laborious phenotypic screening procedures or recessive
alleles.
The second level of selection involves selecting
backcross progeny with the target gene and tightly-linked
flanking markers in order to minimize linkage drag. We
refer to this as ‘recombinant selection’.
 The third level of MAB involves selecting backcross
progeny (that have already been selected for the target
trait) with ‘background’ markers.
 In other words, markers can be used to select against
the donor genome, which may accelerate the recovery of
the recurrent parent genome.

3. Three levels of selection during marker-assisted backcrossing.
With conventional backcrossing, it takes a
minimum of five to six generations to recover the
recurrent parent. Data from simulation studies
suggests that at least two but possibly three or
even four backcross generations can be saved by
using markers.

4. Marker-Assisted Backcrossing
May improve efficiency in three ways:
1) If phenotyping is difficult
2) Markers can be used to select against the donor parent in the
region outside the target
3) Markers can be used to select rare progeny that result from
recombinations near the target gene
Marker assisted backcrossing uses DNA markers, which can be scored
as a dominant or codominant trait prior to flowering, to facilitate the
backcrossing program, saving time if progeny testing would need to be
conducted and saving resources if phenotyping is difficult.
 Markers can be used to select for the gene being introgressed into the
recurrent parent and to select against undesirable donor DNA.
Markers enable the pyramiding of resistance genes; that is, they enable
the incorporation of alleles at multiple loci each of which confers
resistance to the same race of pathogen.
This is difficult to do traditionally because one locus masks the
presence of the others.
The literature on marker-assisted backcrossing is increasingly large, as
new tweaks to methodology are developed and new scenarios are
encountered in different crop species.

5. Transfer of genes from DG to RG
- Foreground selection (for donor’s trait)
- Background selection (for host genome)
(i) 2 step selection (chromosome, genome)
(ii) 3-4 step selection (gene region arm
chromosome genome

6. Backcrossing strategy
Adopt backcrossing strategy for incorporating genes/QTLs
into ‘mega varieties’
Utilize DNA markers for backcrossing for greater efficiency
– marker assisted backcrossing (MAB)

7. 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

8. MAB: 1ST LEVEL OF SELECTION –
TARGET LOCUS 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
SELECT
IONFOREGR
OUND
SELECTI
ON

9. 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

10. 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.

11. 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

12. 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

13. 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

14. 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

15. 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

16.  Marker assisted pyramiding
Pyramiding is the process of simultaneously combining
multiple genes/QTLs together into a single genotype.
This is possible through conventional breeding but
extremely difficult or impossible at early generations. Using
conventional phenotypic selection, individual plants must be
phenotypically screened for all traits tested.
Therefore, it may be very difficult to assess plants from
certain population types (e.g. F2) or for traits with destructive
bioassays.
DNA markers may facilitate selection because DNA
marker assays are non-destructive and markers for multiple
specific genes/QTLs can be tested using a single DNA
sample without phenotyping.
The most widespread application for pyramiding has been
for combining multiple disease resistance genes in order to
develop durable disease resistance.

17. Marker assisted pyramiding of two disease resistance genes.
Note that homozygotes can be selected from the F2
population.

18. Early generation marker assisted selection
One of the most intuitive stages to use markers to select
plants is at an early generation (especially F2 or F3).
 The main advantage is that many plants with unwanted
gene combinations, especially those that lack essential
disease resistance traits and plant height, can be simply
discarded.
This has important consequences in the later stages of
the breeding program because the evaluation for other
traits can be more efficiently and cheaply designed for
fewer breeding lines (especially in terms of field space).

19. Early generation selection scheme (proposed by Ribaut & Betran
(1999). Note that many lines can be discarded in an early generation
which permits the evaluation of fewer lines in later generations.

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

21. Some considerations for MAB
• IRRI’s goal: several “enhanced Mega varieties”
• 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

22. A common application of marker-assisted backcrossing has been the
introgression of transgenes into an adapted variety or line (e.g.,
introgression of the Bt insect resistance transgene into different maize
genetic backgrounds). Often the target gene can be detected
phenotypically, and markers are used to select for the recurrent parent
genome. The technique has reportedly accelerated the recovery of the
recipient genome by about two backcross generations (Hospital, 2003).
In Australia, a marker linked (0.7 cM) to the Yd2 gene for resistance to
barley yellow dwarf virus was successfully used to select for resistance
in a barley backcross breeding scheme (Jefferies et al., 2003). Field test
data showed that BC2 F2-derived lines containing the linked marker had
fewer leaf symptoms and higher grain yield when infected by the virus
compared to lines lacking the marker.
Soybean yields were increased by using marker-assisted
backcrossing to introgress a yieldQTL from a wild accession into
commercial genetic backgrounds (Concibido et al., 2003). Although the
yield enhancement was observed in only two of six genetic
backgrounds, the study demonstrates the potential of incorporating wild
alleles with the assistance of markers.
Marker-assisted backcrossing for a single gene

23. Future of MAS in rice breeding
We believe that despite the relatively small adoption of
markers in rice breeding to date, there will be a greater level
of adoption within the next decade and beyond.
Factors that should lead to a greater adoption of MAS in rice
include:
•establishment of facilities for marker genotyping and staff
training within many rice breeding institutes in different
countries
•currently available (and constantly increasing) data on
genes/QTLs controlling traits and the identification of tightly-
linked markers
•development of effective strategies for using markers in
breeding
•establishment and curation of public databases for
QTL/marker data
•available resource for generating new markers from DNA
sequence data arising from rice genome sequencing and
research in functional genomics.

24. Achievements of MAS (details not being discussed)
• MAS Programs World-Wide: USA, Canada, Australia,
CIMMYT, IRRI - Many Cultivars Released
• MAS in India: Cultivars Released –
1. Rice (Improved PB-1; Improved Sambha Mahsuri;
2. Pearl-millet (HHB-67-2); 3. Maize (Vivek-QPM9)
• Submergence tolerance: Sub1A