This document summarizes three case studies on using marker-assisted breeding techniques: 1) Introgressing rice QTLs controlling root traits from donor Azucena into recipient Kalinga III. Five target QTLs were introgressed over three backcrosses using foreground, background, and recombinant selection with RFLPs and SSRs. 2) Introgressing the submergence tolerance Sub1 QTL from donor IR49830 into popular rice variety Swarna. The QTL was introgressed over three backcrosses and a BC3F2 line identified with minimal donor DNA. 3) Introgressing drought tolerance QTLs from donor CML247 into

1. Application of Molecular Markers
in Marker Assisted Breeding
(MAB)
Case studies
Marker assisted backcrossing methods to transfer of
Nutritional traits in Maize
Presented by,
Sandesh,G.M
2016610811
TNAU,

2.  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.

3. Plant Breeding Approach
Classic Breeding
Main Street
Molecular
breeding
Abiotic and biotic resistance breeding
(disease/pest resistance, drought and salt tolerance)
Parent selection and progeny testing
Marker-assisted selection (MAS)
Genome-wide selection (GWS)
Marker-assisted backcross breeding (MABB)
QTL-based and genome-wide predictive breeding
P1 x P2 F1 F8-10F6-7F4-5F3F2
Cultivar
variety
Release
Parent selection
Predictive breeding
True/false,
self testing
MAS
for simple traits
Preliminary
Final Yield Test
P1
x
BC1F1
Backcross breeding
MAS
for quantitative traits
Genotyping by sequencing (GBS)
RAD-seq and RNA-seq
SNP discovery and validation
QTL mapping and association analysis
Candidate gene identified and clone

4. BACKCROSSIN
G
 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-cross.

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.

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

8. Problems of
conventional
backcrossing
 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.

9.  Selfing after
alternative backcross
generation requires more
number of generations to
select genotype with target
gene having maximum
recurrent parent
background.

10. 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

12. 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

13. MOLECULAR
MARKERS
 Not affected by environment.
 Not stage specific.
 Not tissue specific.
 More precise.
 Acts as proxies and helps in indirect selection.

14. MARKER
ASSISTED
SELECTION
 Markers 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.

15. Principle of
MAB
 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 parts in
the next generation of a genetic improvement programme.

16. Success
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 markers used, and available
technical facilities.(Weeden et al., 1992;Francia et al.,
2005).

17. Markers can
be used to
 Control the target gene (foreground
selection).
 Control the genetic background
(background selection).
 Control the linkage drag (recombinant
selection).

18. MARKER
ASSISTED
BACKCROSS
BREEDING
MABB
3 STAGES
1
2
3

19. MABC
1st stages
 Stage I involves selection of a subset of plants that carry desirable
allele from the donor parent. This will be done by genotyping
BC1F1 plants for target gene or QTL using linked marker, the
process referred to as foreground selection.

20. MABC
2nd stages
 Stage II involves further selection among those of BC1F1 plants,
which show heterozygosity in the target gene or QTL region.
 The BC1F1 that have fixed alleles may not have recombination in
that region so that the size of the introgression can not be reduced.
Hence, the BC1F1 progeny that is heterozygous for the target locus
will be selected, the process referred to recombinant selection .

21. MABC
3rd stages
 Stage III involves final selection of BC1F1 that carry maximum
proportion of recurrent parent genome.
 This will be done by genotyping the selected backcross progenies
using genome wide markers, the process referred to background
selection

22. FOREGROUND
SELECTION
 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

23. FOREGROUND
SELECTION

24. 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 diseases races and/ or insect biotypes and
pyramid them into a single line through MAS.

25. 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.

26. BACKGROUND
SELECTION
• In other words, markers that can be used to select against the donor genome.
• The use of background selection during MAB to accelerate the recovery of recurrent
parent genome with additional genes has been referred to as ‘complete line conversion’
(Ribaut et al. 2002).

27. 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’.

28. 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

29. ADVANTAGES
 When phenotypic screening is expensive, difficult or impossible.
 When the trait is of low heredity (incorporating genes that are
highly affected by environment).
 When the selected trait is expressed late in plant development,
like fruit and flower or adult characters in species with a juvenile
period.
 For incorporating genes for resistance to diseases or pests that
cannot be easily screening for due to special requirement for the
gene to be expressed.
 When the target 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.
APPLICATIONS
OF MABB

30. Reasonsfor
unexpected
resultsinMAB
 The putative QTL may be 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).

31. CASE STUDY

32. Marker-assistedselectiontointrogressriceQTLscontrollingroottraitinto
anIndianuplandricevariety

33. Selection of
parents and
target QTLs
 Recipient parent : Kalinga III (Indica
rice)
 Donor parent : Azucena (Japoca rice)
 Total QTLs : 5
 Root QTLs : QTL2, QTL7, QTL9, QTL11
 Aroma QTls :QTL8

34. Backcrossing
and selection
Positive selection was for Azucena
alleles and negative selection
was for Kalinga III.

35. PYRAMID
CROSSINGAND
SELECTION

36. Foreground
selection for
target QTLs
 RFLPs were used for first 3 back crosses.
 RFLPs that had mapped in Kalinga III x
Azucena population and flanked, or were
within the regions containing the target
QTLs.
 In addition, the RFLP marker C750 was
used, it was polymorphic between
Azucena and Kalinga III but not between
Bala and Azucena and mapped near to
QTL9.

37. Selection after 3rd Backcross as done using flexible and cheaper
PCR-based SSRs.

38. RECOMBINANT
SELECTION
 Negative selection was performed at these QTLs for Azucena alleles
because these alleles had negative effect which are linked to target
QTLs.

39. background
 SSR and RFLP (bold) markers are shown in their relative order along chromosomes according to comparative mapping
using http://www.gramene.org. The five target chromosome segments are boxed

40. Greenhouse
and field
experiments
 Extensive greenhouse and field experiments were
carried out at UAS(B)
1. To identify chromosomal regions from Azucena that
delayed anthesis so that they could be selected
against.
2. 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.

41. Conclusion
 Five target regions is probably the limit of efficient
MABC breeding (Hospital and Chacosset 1887; Servin et
al. 2004), but they successfully selected an ideotype with
all five regions from Azucena introgressed into the
predominantly Kalinga III genetic backgroung, with
almost complete line conversion.
 The work would have less tedious if they,
1. Started with SSRs as number of assays would be less.
2. Started with pre-existing RILs.
3. Tested more line at each backcross generation.

42. Case 2

43. 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.

44. Screening for submergence tolerance

45. Amajor 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

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

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

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

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

50. Seed increase of tolerant BC2F2
plant

51. 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

52. Time frame for “enhancing” mega-varieties
May need to continue until
BC3F2
• Name of process:
“variety enhancement” (by D.
Mackill)
• Process also called “line
conversion” (Ribaut et al.
2002)

53. Swarna with Sub1

54. Graphical
genotype of
Swarna-Sub1
BC3F2 line
Approximately 2.9 MB of donor DNA

55. Swarna 246-237
Percent chalky grains
Chalk(0-10%)=84.9
Chalk(10-25%)=9.1
Chalk(25-50%)=3.5
Chalk(>75%)=2.1
Chalk(0-10%)=93.3
Chalk(10-25%)=2.3
Chalk(25-50%)=3.7
Chalk(>75%)=0.8
Average length=0.2mm Average length=0.2mm
Average width=2.3mm Average width=2.2mm
Amylose content (%)=25
Gel temperature=HI/I
Gel consistency=98
Amylose content (%)=25
Gel temperature=I
Gel consistency=92

56. CONCLUSION
 A BC3F2 double recombinant plant was identified
that was homozygous for all Swarna type alleles
except for an approximately 2.3–3.4 Mb region
surrounding the Sub1 locus.
 The results showed that the mega variety Swarna
could be efficiently converted to a submergence
tolerant variety in three backcross generations,
involving a time of two to three years.

57. Case 3

58. Selection of
parents and
target QTLs
 Recipient parent : Ac7643
 Donor parent : CML247
 CML247 is an elite tropical inbred line developed by CIMMYT
and often used as a tester because of its outstanding combining
ability and good yield per se under well-watered conditions.
 Under water stress, CML247 displays a very large male–
female flowering asynchrony (also known as ASI or anthesis–
silking interval) and, as a consequence, is very susceptible to
drought.
The drought adaptation MABC experiment in tropical maize
was initiated in 1994

59.  F2:3 segregating population with 234 individuals
was developed from the F1 between Ac7643 and
CML247. F1 plants were also crossed to CML247 to
generate a BC1F1 population.
 All phenotypic evaluations were conducted during
the dry winter season (November–April) in
Tlaltizapan, Mexico.

60.  A genetic map was constructed from polymorphic loci
using MAPMAKER (Lander et al., 1987).
 Five QTLs for ASI, located on chromosomes 1, 2, 3, 8,
and 10, were selected.
 A cluster of QTLs for flowering traits and yield
components was identified on chromosome 3.

61.  Then plants were genotyped at four pairs of restriction
fragment length polymorphism (RFLP) markers flanking
ASI QTLs located on chromosomes 1, 2, 8, and 10, plus
one RFLP marker within the ASI QTL on chromosome 3.
 Plants displaying heterozygous genotypes at these
markers were selected. Finally, for the second BC and the
two self-pollinations, 60 polymorphic RFLP markers
distributed throughout the genome were used to recover
recurrent parent genotype at non-target regions.
 Plants with the highest proportion of CML247 alleles at
these markers were selected.

62.  After the four MABC cycles, the 70 BC2F3 individuals
presenting the closest allelic composition at target and
non-target loci compared with the target genotype were
crossed to two CIMMYT testers, CML254 and CML274.
 Ten individual CML247 plants were also crossed to the
same two testers to produce 10 control hybrids.
 From those 70 genotypes, 30 were selected based on
their agronomic traits and yield performance. Doing so
allowed capturing favourable alleles from Ac7643
outside the target loci and removing the genotypes
presenting negative epistatic effects.

63. MAB in
Maize
Marker assisted back crossing to transfer nutritional traits in
Maize

64.  The temperate maize inbred line Hp321-1 carrying the
favorable alleles crtRB1-50TE-2 and crtRB1-30TE-1 of the
crtRB1 gene (ProVA concentration= 9.74 lg g-1) was used as
the donor parent(male).
 Two tropical QPM maize inbred lines,CML161 and CML171,
were used as the recurrent female parents.
 At the lcyE locus, there is no polymorphism among Hp321-1,
CML161, and CML171.

65. Foreground
selection
 Foreground selection (FS) refers to selection of
favorable alleles (crtRB1-50TE-2 and crtRB1-30TE-1)
for higher ProVA concentration.
 Polymerase chain reaction (PCR) amplification of the
functional marker for
 crtRB1-50TE-2 was done using the forward primer
50-TTAGAGCCTCGACCCTCTGTG-30 and the
reverse primer 50-AATCCCTTTCCATGTTACGC-30.
 For crtRB1-30TE-1, the forward primer was
50 -ACACCACATGGACAAGTTCG-30
 The reverse primers were
50-ACACTCTGGCCCATGAACAC-30 and
50-ACAGCAATACAGGGGACCAG-30 (Yan et al. 2010).

66. Background
selection
 Two DNA bulks were constructed by mixing equal
amounts of DNA from five plants of the donor parent
(Hp321-1) and five plants of each recurrent parent
CML161 and CML171 for screening parental
polymorphic SSR markers.
 A total of 98 polymorphic SSR markers between
Hp321-1 and CML161
 89 polymorphic SSR markers between Hp321-1
andCML171, evenly distributed on 10 maize
chromosomes
 These were used for genotyping the FS-selected
individuals of the BC1F1, BC2F1, and BC2F2
populations developed with CML161 and CML171.

67.  Increase in ProVA concentrations in the QPM maize through molecular marker-assisted foreground and
background selections.
 A. The increase in ProVA concentration in the CML161 population.
 B. The increase in ProVA concentration in the CML171 population

68.  The two functional markers of crtRB1 gene were
polymorphic between the donor parent (Hp321-1) and
the recurrent parents CML161 and CML171.
 The donor parent Hp321-1 had the favorable allele
crtRB1-50TE-2 with a 600-bp band, while unfavorable
allele crtRB1-50TE-1 had an 800-bp band corresponding
to the recurrent parents CML161 and CML171.
 Furthermore, the donor parent Hp321-1 carried the
favorable allele crtRB1-30TE-2 with a 543-bp band.
crtRB1 3’TE gene Specific marker

69.  The polymorphic markers were codominant, which rendered them usable for the MAB
of the corresponding target genes.
 The results demonstrated that favorable alleles crtRB1-50TE-2 and crtRB1-30TE-1 for
high ProVA concentration were successfully transferred into the two QPM maize lines.

70. REFERENCE
S
 Molecular Markers and Marker-Assisted Breeding in Plants,
by Guo-Liang Jiang.
 Marker-assisted selection to improve drought adaptation in
maize: the backcross approach, perspectives, limitations, and
alternatives, Jean-Marcel Ribaut1 and Michel Ragot, Journal
of Experimental Botany, Vol. 58, No. 2, pp. 351–360, 2007
 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, Theor Appl Genet (2007) 115:767–
776
 Introgression of the crtRB1 gene into quality protein maize
inbred lines using molecular markers , Li Liu . Daniel Jeffers .
Yudong Zhang . Meiling Ding . Wei Chen . Manjit S. Kang .
Xingming Fan, Mol Breeding (2015) 35:154