Introduction Backcross breeding & its types Marker assisted breeding Marker assisted backcross breeding (MABC) Main strategies Advantages over conventional breeding Case studies Future outlook Conclusion

1. Marker Assisted Backcross Breeding (MABC)
in Crop Improvement

2. ●Introduction
●Backcross breeding & its types
●Marker assisted breeding
●Marker assisted backcross breeding (MABC)
●Main strategies
●Advantages over conventional breeding
●Case studies
●Future outlook
●Conclusion
Flow of seminar

3. ● Backcross breeding is a well-known procedure for the introgression of a target gene f
● The objective is to reduce the donor genome content (DGC) of the progenies by repe
Backcrossing
6

4. Factors necessary for backcross method:
(i) Recurrent parent Selection
(ii) Screening for target trait
(iii) Number of backcrosses
●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.
Backcrossing is used

5. Stepwise transfer Simultaneous transfer Stepwise but parallel transfer
Types of backcrossing when more than one gene is to be
transferred from different sources

6. Conventional Backcross breeding
P1
P1
P1
BC1F
1
P1
F 1
P2
BC2F
1
X
X
X
P1
P1
BC3F
1
P1
X
X
BC4F
1
BC5F
1
BC6F
1
X
X
BC5- 98.4: 1.6
BC6- 99.2: 0.8

7. General equation for average of the recurrent parent:
1-(1/2)n+1
Where, n is the number of backcrosses to the recurrent parent.
For f2, n=0
for BC1, n=1
for BC2, n=2
for BC3, n=3 etc.,

8. Problems of conventional backcrossing
• Requires large number of plants for selection
• Introgression of quantitative traits is nearly not possible.
• Recovery of recipient genome is less efficient.
• Poses difficulties in negative selection of undesirable genes (linkage drag
problems)
• Cannot select the plants until the traits are expressed.
• Phenocopy effect hinders the selection.
• Recessive traits take more time to transfer

9. Marker assisted breeding:
application of molecular biotechnological tools, in combination with
linkage maps and genomics, to improve plant or animal traits on the
basis of genotypic assays.

10. Marker assisted backcrossing
• 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 other than the trait of interest.
• Markers enable the pyramiding of resistance genes; they enable the incorporation of
alleles at multiple loci each of which confers resistance to the same race of pathogen,
which is difficult to do traditionally because one locus masks the presence of the others.

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
Principle of MAB:

12. 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
Factors responsible for the efficiency of MABC are-
• Size of population for each backcross generation.
• Markers distance from locus.
• Number of markers is used in each selection process.

13. 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)

14. ✓Also known as positive selection- Proposed by Tanksley
Foreground selection refers to the use of markers that are tightly linked to the gene of
interest in order to select for the target allele or gene (associate a molecular marker with
the target trait by some genetic mapping method).
Foreground selection:
1 2 3 4
Target locus
TARGET LOCUS
SELECTION
FOREGROUND SELECTION

15. The objective is to maintain the target locus in a heterozygous state (one
donor allele and one RP allele) until the final backcross is completed.
Then, the selected plants are self-pollinated and progeny plants identified that
are homozygous for the donor allele.
• 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.
• Selection for target gene or QTL
• Useful for traits that are difficult to evaluate
• Also useful for recessive genes

16. Selecting backcross progeny with the target gene and tightly-
linked flanking markers is termed 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).
•Require large population sizes
•By using markers that flank a target gene , linkage drag can be
minimized.
RECOMBINANT
SELECTION
1 2 3 4
Recombinant Selection

17. 1 2 3 4
BACKGROUND
SELECTION
➢ Negative selection- Proposed by Takeuchi
➢ 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.
➢ Background selection refers to the use of tightly linked flanking
markers for recombinant selection and unlinked markers to
select RP.
➢ This is extremely useful because the RP recovery can be greatly
accelerated.
Background Selection

18. • With conventional backcrossing, it takes a minimum of six BC generations to
recover the RP and there may still be several donor chromosome fragments unlinked
to the target gene.
• The use of background selection during MABC to accelerate the development of an
RP with an additional one or more genes has been referred to as ‘variety
development or enhancement’ and ‘complete line conversion’
• Accelerates the recovery of the recurrent parent genome
• Savings of 2, 3 or even 4 backcross generations

19. Schematic representation of development of resistant rice variety
through marker-assisted backcrossing (MABC).

20. Schematic representation of selection of heterozygous carrying
resistance gene based on genotyping analysis resembling RP
genome at BC1F1.

21. Schematic representation of selection of heterozygous carrying resistance
gene based on genotyping analysis resembling RP genome at BC2F1

22. Schematic representation of selection of homozygous
plants for the donor allele

23. Schematic representation of transferring undesirable genes
with target gene

24. Schematic representation of difference between
conventional backcrossing and marker-assisted
backcrossing

25. Comparison of conventional and Marker-Assisted backcrossing

26. Comparing the expected recovery of recurrent parent in
conventional and Marker-Assisted backcrossing in subsequent
generation.
% of recurrent parent (RP)
Backcross generation Number of Generation
Marker-Assisted
backcross
Conventional
backcross
BC1 70 79.0 75.0
BC2 100 92.2 87.5
BC3 150 98.0 93.7
BC4 300 99.0 96.9
Hospital, 2003

27. Case studies

28. Super Annigeri 1 and improved JG 74: two Fusarium wilt-resistant
introgression lines developed using marker-assisted backcrossing approach in
chickpea (Cicer arietinum)
Need to develop high yielding chickpea cultivars with FW resistance.
Development of Super Annigeri 1 and improved JG 74 with enhanced yield and resistance to
FW using MABC approach.
Materials :
Recipient parents – Annigeri 1 and JG74 ( susceptible to FW race 4)
Donor parent – WR 315 (resistant to FW race 4)
Mannur et al ., 2019

29. Annigeri 1
BC1F1
WR 315
BC2F1
X
X
BC2F2
BC2F3
X
F1
Annigeri 1
Annigeri 1
BC2F4 in wilt sick plot
X
X
X
Hybridity confirmation
42 plants heterozygous for FG
markers
38 SSRs for BGS, 80-87% RPGR
67 plants heterozygous for FG
markers
35 SSRs for BGS, 90-95% RPGR
119 Plants showed resistance to
FW
resistant lines with higher yield
Multi-locational phenotyping
2 superior lines
Mannur et al ., 2019
Phenotyped for FW, resistant plants
were obtained

30. Phenotypic evaluation for Fusarium Wilt disease
Mannur et al ., 2019

31. JG 74
BC1F1
WR 315
BC2F1
X
X
BC3F1
BC3F2
X
F1
Phenotyped for FW, 44 resistant plants
were obtained
BC3F4 in wilt sick plot
X
X
Hybridity confirmation
3 plants heterozygous for FG markers
15 plants heterozygous for FG markers
42 SSRs for BGS, 52-97% RPGR
JG 74 type plants were selected
resistant lines with recurrent parent
type
2 superior lines
JG 74315-14 (MLT)
JG 74
JG 74
X
JG 74
BC3F3
X
4 plants heterozygous for FG markers
JG 74315-2 & 14 evaluated
Mannur et al ., 2019

32. Marker-Assisted Backcrossing to Introgress Resistance to Fusarium Wilt Race 1
and Ascochyta Blight in C 214, an Elite Cultivar of Chickpea
Varshney et al ., 2014
Materials:
2 parallel crossing;
C214 – recurrent parent ( susceptible to FW and AB disease)
WR 315 – donor parent ( resistant to FW disease)
ILC3279 – donor parent ( resistant to AB disease)

33. C 214
BC1F1
WR 315
BC2F1
X
X
BC3F1
BC3F2
X
F1
BC3F4
X
X
Hybridity confirmation
41 plants heterozygous for FG
markers
30 plants heterozygous for FG
markers40 SSRs for BGS, 90-98%RPGR
98% RPGR
homozygous plants with >98%rpgr
C 214
C 214
X
C 214
BC3F3
X
Fusarium Wilt
Varshney et al ., 2014
30 plants heterozygous for FGmarkers
32 SSR markers BGS, 89-95% RPGR

34. Screening of marker-assisted backcrossing (MABC) lines for
resistance to Fusarium wilt

35. Disease reaction of parental and BC3F4 lines resistance to Fusarium oxysporum.
Lines FW incidence (%) Disease reaction†
Parental lines
C 214 (recurrent parent) 54.50 susceptible
WR 315 (donor parent) 6 resistant
MABC lines‡
ICCX-100175-349-2-2 0 resistant
ICCX-100175-389-3-2 5 resistant
ICCX-100175-382-4-6 20 resistant

36. C 214
BC1F1
IL 3279
BC2F1
X
X
BC3F1
BC3F2
X
F1
BC3F4
X
X
Hybridity confirmation
2 plants heterozygous for FG markers
38 plants heterozygous for FG
markers
43 SSRs for BGS, 80-90% RPGR
homozygous plants with >90%rpgr
C 214
C 214
X
C 214
BC3F3
X
46 plants heterozygous for FG
markers
29 SSR markers BGS, 80-87% RPGR
Ascochyta
Blight
Varshney et al ., 2014

37. Screening of marker-assisted backcrossing (MABC) lines for resistance to
Ascochyta blight.

38. Disease reaction of parental and BC3F4 lines resistance to Ascochyta blight
(AB).
Lines AB score Disease reaction
Parental lines
C 214 (recurrent parent) 7 susceptible
ILC 3279 (donor parent) 4 moderately resistant
MABC lines
ICCX-100176-421-1-11 3 resistant
ICCX-100176-421-1-12 2 resistant
ICCX-100176-470-2-5 2 resistant
ICCX-100176-470-2-7 2 resistant
ICCX-100176-470-2-16 3 resistant
ICCX-100176-470-3-1 2 resistant
ICCX-100176-470-3-3 3 resistant

39. Marker-Assisted Backcrossing to develop an Elite Cytoplasmic Male Sterility
line in Rice
To transfer CMS from IR68897 A line to Yosen B as a putative maintainer line,
with the ultimate goal of application of the resultant final CMS line in hybrid rice
seed production.
Materials:
Yosen B line – potential maintainer line for rice WA-CMS ( paternal recipient
parent)
CMS IR68897 A –maternal CMS donor parent
Conducted Pollen fertility test from F1 generation to BC4F1 progenies.
Foreground selection by using mitochondrial WA-CMS-specific marker for
validating
the transfer of CMS from donor to F1 hybrid and subsequent backcross progenies.
Ahmadikhan et al ., 2015

40. IR68897-A
BC1F1
F 1
Yosen-B
2 BC2F1
X
X
X
1 BC3F1
BC4F1
X
X
Yosen-B
Yosen-B
Yosen-B
Yosen-B
Tracing
transfer of
CMS
higher RPG
higher RPG
4 CMS plants with
>98%RPG

41. Future prospects
An efficient cost effective MABC technology must be developed that will allow breeders
to assess the genotype across the full genome and to recombine genes of agronomic
importance from diverse sources. The most significant cost prior MABC is the
development of genetic linkage map for the species of interest.

42. MABC approach plays a vital role for basic research applications to develop new
and advance varieties with much greater precision than conventional backcrossing. It has
generated good deal of expectation, which in some cases let to over/optimize and in other
to disappointment because many of the expectation have not been realized.
Conclusion