Within the last twenty years, molecular biology has revolutionized conventional breeding techniques in all areas. Biochemical and Molecular techniques have shortened the duration of breeding programs from years to months, weeks, or eliminated the need for them all together. The use of molecular markers in conventional breeding techniques has also improved the accuracy of crosses and allowed breeders to produce strains with combined traits that were impossible before the advent of DNA technology

1. LOGO
Gene pyr amiding for biotic resistance
through marker assisted selection

2. ADVENT OF MARKERS
 Molecular biology has revolutionized conventional
breeding techniques
 Molecular markers in conventional breeding has improved
the accuracy of crosses and allowed breeders to produce
strains with combined traits that were impossible before
the advent of DNA technology

3. Contd..,
 MARKERS
 MAS

4. Marker Assisted Selection (MAS)
 Exploitation of molecular markers linked to genetic factors
are useful in assessing the genotype which is the genetic
material for the development of new improved varieties.
 This led to the development (MAS)
 MAS– improves the efficiency with which breeders can
select plants with desirable combinations of genes.
 A marker is a “genetic tag”

5. BREEDER’S PERSPECTIVE
 Efficient and effective crop trait selection
 Avoid genotype-environment interaction
 Shorter time to bring new varieties to market
 The discovery of molecular markers associated with many
important agronomic triats has made marker-assisted
selection (MAS) reality for several crops.

6. CONVENTIONAL PLANT BREEDING
P1 x P2
Recipient Donor
F1
large populations consisting
F2 of thousands of plants
PHENOTYPIC SELECTION
Bacterial blight screening Phosphorus deficiency plot
Salinity screening in phytotron
Glasshouse trials Field trials

7. Resistance
Gene Susceptible Marker - What for?
Example:
A breeder aims to improve the resistance
of a cultivated form. Therefore, he/she
performs a cross between the susceptible
culitivated form with a wild form that
possess the required resistance.
However, at least 6 backcrossing steps are
necessary and the resistance is difficult to
detect.
The major goal is to reduce costs
associated with screening for traits

8. MARKER-ASSISTED BREEDING
P1 x P2
Susceptible Resistant
F1
large populations consisting of
F2 thousands of plants
MARKER-ASSISTED SELECTION (MAS)

9. MARKER ASSISTED PYRAMIDING
Breeding plan Genotypes
P1 P1 P1: AAbb x P2: aaBB
Gene A Gene B
F1 F1: AaBb
Gene A + B
F2
MAS
Select F2 plants that have Gene
A and Gene B
Fig: A simple scheme for marker assisted pyramiding of two different traits
(Hittalmani et al., 2000; Liu et al., 2000)

10. GENE PYRAMIDING -INTRODUCTION
 Pyramiding is the accumulation of genes into a single line
or cultivar.
 A pyramid could be constructed with major genes, minor
genes, defeated genes, effective genes, ineffective genes,
race-specific genes, non race-specific genes, or any other
type of host gene that confers resistance

11. Contd..,
 It includes
Stacking of traits
Stacking of events
Stacking of genes
 A genetically modified organism (GMO) and all subsequent
identical clones resulting from a transformation process
are called collectively a transformation event.
 If more than one gene from another organism has been
transferred, the created GMO has stacked genes (or
stacked traits), and is called a gene stacked event.

12. Contd..,
 Widely used for combining multiple disease resistance
genes for specific races of a pathogen
 Pyramiding is extremely difficult to achieve using
conventional methods
 Consider - phenotyping a single plant for multiple forms of
seedling resistance – almost impossible
 Important to develop ‘durable’ disease resistance against
different races
 Main use- to improve existing elite cultivar
 Eliminates extensive phenotyping
 Control linkage drag
 Reduces breeding duration

13. TYPES OF GENE PYRAMIDING
 Conventional technique
Serial gene pyramiding : Genes are deployed in same
plant one after other
 Molecular technique
Simultaneous gene pyramiding : Genes are deployed at a
time in a single plant

14. Gene-pyramiding scheme cumulating six
target genes

15. FIXATION STEPS
 Production of doubled haploids from root genotype
 Crossing the root genotype with a blank parent and selfing
the offspring
 Crossing the root genotype with one of the founding
parent
 Selfing the root genotype

16. ENHANCING RESISTANCE
 In Rice BPH resistance genes Bph1 and Bph2 were
LEVEL
pyramided
 Pyramided line shows resistance greater than the Bph2
single introgression line
(Sharma et al., 2004)
 Gall midge resistant genes Gm2 and Gm6 was pyramided
which shows non overlapping resistance to allopatric
biotypes
(Katiyar et al., 2001)
 In wheat, Russian wheat aphid resistant genes Dn2 and
Dn4 were pyramided

17. Durable resistance
 In cotton, Bt genes (Cry1Ab)+high terpenoid plant trait
pyramided line shows durable resistance to H. virescens
(Sachs et al., 1996)
 In soybean, two QTLs + Cry 1Ac increased resistance to
soybean looper and corn ear worm
(Walker et al., 2004)
 In rice, three BLB resistance genes Xa 5, Xa13 and Xa21
have been pyramided. (Joseph et al., 2004)
 In rice, transgenic pyramided IR 72 lines showed increased
resistance to BB (Xa21),yellow stem borer(Bt fusion gene)
and sheath blight( chitinase gene)
(Datta et al., 2004)

18. Pyramiding of genes
RICE PLANT
Xa 21 gene Chitinase gene B.t. gene
Bacterial resistance Sheath blight Insect resistance
(Datta, et. al., 2002)

19. Iterative Strategies
 Two or more transgenes can be sequentially introduced
into a plant by conventional iterative procedures
 Pyramided cry1Ac and cry1C Bt genes in broccoli
controlled diamond back moth
(Cao et al.,
2002)

20. Co-transformation with Multiple
 Most promising approach for the introduction of multiple
Transgenes
genes into plants
 Quick & Simple
 Tend to co-integrate at the same chromosomal position in
a high proportion of transgenics
 In Arabidopsis, six genes (including two selectable marker
genes) on two T-DNAs were deployed to produce
copolymer,
 The availability of metabolic precursors had to be
increased by redirecting intermediates, manipulating
Protein expressing and gene suppressing transgenes
(Baucher et al., 2003)

21. Expression Multiple Proteins From Single
Promoter
(Baucher et al., 2003)

22. Contd..,
 Chimeric polycistronic constructs that incorporate
internal ribosome entry sites (IRESs) from different
viruses
 Different protein sequences are connected in a single open
reading frame via short linker sequences
 A polyprotein incorporating coat proteins of tobacco
mosaic tobamovirus and soybean mosaic potyvirus was
expressed in tobacco to yield plants resistant to multiple
viruses

23. Chimeric Transgenes for Multiple Gene
Suppression
 Single transgenes can also be used to simultaneously
suppress multiple genes
 Containing fused sequences of several target genes under
the control of a single promoter
 But in most cases resulted in Co-suppression or post-
transcriptional gene silencing
(Baucher et al., 2003)

24. UD Y1
ST
CA SE

25. Introduction
 Cereal Cyst Nematode (CCN)-Heterodera avenae
 Two CCN genes (CreX and CreY) from Ae. variabilis in a wheat
background
 Employing MAS – comparison of two pyramided CCN resistance
level with parental single-gene recombinant lines.
 The development of markers is the best way to achieve the
pyramiding of genes conferring partial resistance in a single
recombinant line.

26. Materials and Methods
32 32
6 7

27. RAPD to SCAR MARKER
 RAPD, OpY16 linked to Rkn-mn1 converted to SCAR
(1060bp) ie SCAR Y16

29. RESULTS….
 The level of resistance observed suggested an additive effect
of the two genes in the pyramided line
 The identification of codominant markers linked to these
two genes facilitate gene pyramiding and rapid screening of
different genotype
 SCAR Y16 and OpR4-1600, the latter after transformation
into a SCAR, could be used as introgression markers to
pyramid CreY and CreX genes in different backgrounds

30. UD Y2
ST
CA SE

31. Introduction
 Soybean mosaic virus (SMV)
 Single-dominant resistance genes Rsv genes against strains of
SMV.
 Pyramiding respective Rsv genes from different loci (Rsv1, Rsv3,
and Rsv4) MAS - ideal -creating durable and wide spectrum
resistance to all strains of SMV.

32. Contd..,
 Two-gene and three-gene isolines of Rsv1Rsv3, Rsv1Rsv4 and
Rsv1Rsv3Rsv4, acted in a complementary manner, conferring
resistance against all strains of SMV, whereas isolines of
Rsv3Rsv4 displayed a late susceptible reaction to selected SMV
strains.
 We demonstrate with MAS and three near-isogenic lines, each
containing a different SMV-resistance gene, that pyramided lines
can be generated in a straightforward manner into two- or three-
gene–containing lines with high levels of resistance to SMV.

33. UD Y3
ST
CA SE

34. Introduction
 Barley Yellow Mosaic Virus disease caused by different
strains of BaYMV and BaMMV
 For pyramiding of resistance genes rym4, rym5, rym9 and
rym11, located on chromosomes3Hand4Hof barley using
two approaches
 doubled haploid lines (DHs) and marker assisted selection
procedures

35. TRANSGENIC APPROACH
UD Y4
ST
CA SE

36. Introduction
 Indica rice cultivars - cotransformed with genes
Rice chitinase (chi11)
Thaumatin-like protein (tlp) (fungal pathogens)
Serine-threonine kinase (Xa21) (bacterial blight resistance)
 Particle bombardment (Callus and imature embryo)
 Varieties – PB1, ASD16, ADT 38 , IR72 AND WHITE PONNI
 Putative transgenic lines analysed PCR, Southern Blot
hybridization and Western Blotting showed stable integration
and expression of the transgenes in a few independent
transgenic lines.

37. GENE OF INTEREST
Chi 11 t lp Xa 21 3 genes
pyramided
Se
Fu
Sh
Sh teria
ng
r-
bac
ea
thr
eat
al
lth
pat
eo
h b bligh
blig
ho
kin
ligh t
gen
l
ht
ase
s
t&
PUTATIVE
GENE SELECTION – RICE
TRANSGENIC
GENE 1 GENE 2 GENE 3
PLANT

38. GENE CONSTRUCT
1 2 3
p MKU-RF2 p GL2-ubi-tlp p C822
3.2kb cassette 3.1kb cassette 9.6kb cassette
1.1kbp 1.1kbp
rice chil 1 gene tlp gene
Ubiquitin promoter Ubiquitin promoter
NOS terminator NOS terminator
SELECTABLE MARKER – Hygromycin B

39. POLYMERASE CHAIN
REACTION

40. SOUTHERN BLOTTING
 Chi11 - genomic DNA - HindIII -to release 3.2 kbp chitinase
expression cassette and blotted onto the membrane
 α-32P dCTPlabeled 1.08 kbp chi11 coding sequence by digesting
pMKU-RF2 with PstI.
 tlp gene- the genomic DNA-HindIII -to release the 3.1 kbp
TLP expression cassette
 α-32P dCTP-labeled 1.1 kbp TLP coding sequence by digesting 3.1
kbp TLP expression cassette with BamHI.
 Xa21 -genomic DNA-EcoRV -to release 3.8 kbp Xa21
 Hybridized with α-32P dCTP-labeled 3.8 kbp Xa21 coding
sequence by digesting pC822 with EcoRV.

41. SOUTHERN BLOT
HYBRIDIZATION

42. WESTERN BLOT ANALYSIS

43. PROGENY ANALYSIS
Pyramided line SM-PB1-1 did not segregate for Xa21 in both T1
and T2 progenies
The influence of chi11 and/or tlp on Xa21-mediated bacterial
blight resistance could not be assessed

44. Global Status of Approved GM Crops with Stacked Genes
www.themegallery.com
(Halpin,Company Logo
2007)

45. 21 st Century Product Developement

46. LOGO

47. LOGO