A marker is a DNA sequence which serves as a signpost/flag post linked to the trait/gene of interest and is co-inherited along with the trait Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation

1. Marker-assisted introgression of biotic
stress resistance genes in rice
Rice
R.M. Sundaram
Directorate of Rice Research
Hyderabad 500030
Bacterial blight rms_28@rediffmail.com Blast

2. Outline of the presentation……
 Need for application of biotechnological tools in
rice, particularly for biotic stress resistance
 Molecular marker assisted selection: An
introduction
 Application of MAS for developing bacterial blight
resistant rice: Success stories
 Scope of MAS: some thoughts

3. Rice - Indian perspective
 Staple food for two thirds of the population
 contributing 20-25% of GDP
 Coverage ~44 m.ha
 22% of cropped area
 Accounts for 43% of food production
 Current production ~ 95 Mt
 Demand ~ 130 Mt by 2025 AD.
 How can we meet this demand?

4. Biotic stresses of rice: A major threat to production
 Biotic stresses limit rice yields upto 30%
 Major biotic stresses
Insects - BPH and Gall midge
Diseases - Bacterial leaf blight and blast
 Even though resistance breeding is successful, breakdown of single
gene conferred resistance is a major concern
 Changing pest and disease scenario: Need to deploy „suitable‟ genes or
gene combinations

5. Changing pest and disease scenario
necessitates an active breeding
programme for introgression of biotic
stress resistance genes into elite rice
varieties and hybrids…

6. Need for Biotechnological applications
 Conventional breeding – led to green revolution era
 But, can conventional breeding alone meet future rice
production targets? NO
 Need for judicious and pragmatic application of
biotechnological tools for rice improvement
 Biotechnology can be helpful to
(i) increase productivity levels beyond yield barriers
(ii) to sustain productivity (by limiting damage due to
biotic and abiotic stresses)

7. Biotechnology can enhance breeding efficiency
through
i. Precise manipulation of target trait without any disruption (or
with minimal disruption) to non-target sites of the genome (i.e.,
increasing the efficiency of selection through MAS)
ii. Transfer of economically important traits/genes across species
and genera into the rice gene pool (i.e., broadening the genetic
base through genetic engineering)

8. What is a molecular marker?
A marker is a DNA sequence which serves as a signpost/flagpost
linked to the trait/gene of interest and is co-inherited along with
the trait.
- Gene of interest
1 cM
- Marker
Presence of specific allele of marker = Presence of specific allele of target gene

9. Marker on one side of the gene (Ideal: 1-2 cM from the gene)
- Gene of interest
1 cM
- Marker

10. Flanking markers (Ideal: 5-10 cM on either side of the gene)
- Gene of interest
5 cM 5 cM
- Marker

11. Application of DNA markers
 Seed purity testing
 Varietal fingerprinting and patenting
 Tagging, mapping and pyramiding major
genes for pest and disease resistance
 Pyramiding QTL‟s of agronomic importance like Yield,
abiotic stress tolerance etc.
 Map based cloning
 Testing of GMO‟s and GMO products through PCR based
markers

12. Marker assisted breeding can reduce the time taken
for breeding significantly
Traditional breeding marker assisted breeding
10,000 breeding 10,000 breeding
lines lines
Molecular
500 -----Year 1----- 500 Marker analysis
Field testing 100 -----Year2---- 20 Field testing
20 Seed increase
Seed increase ----Year3-----
Commercial launch
----Year4---- Of 4 or 5 varieties
Commercial launch
Of 4 or 5 varieties ----Year5----

13. Pyramiding multiple resistance genes in a single cultivar
Bacterial blight
resistance gene
Blast resistance gene
BPH resistance gene
Gall midge Elite cultivar
resistance gene
Elite cultivar
Desired allele of
with multiple
„target‟ gene biotic stress
linked to resistance
molecular marker

14. Usefulness of MAS – Pyramiding multiple genes
Indian Success stories
 Bacterial leaf blight (BLB) resistance genes
- Xa21, xa13, xa5 (DRR, PAU & IARI)
 Blast resistance genes
- Pi1, Pi2 (CRRI & UAS)
 Multiple resistance genes – Gall midge and BLB (DRR)
 Seed purity analysis – Hybrids (DRR) and Inbreds (TNAU)

15. Success story
Marker assisted introgression of Bacterial blight resistance into
Samba Mahsuri and Triguna (A DRR-CCMB Collaboration)

16. Bacterial blight (BB)
 BB caused by Xanthomonas oryzae pv. oryzae
(Xoo) is wide spread in irrigated and lowland
rice ecosystem. Growing resistant varieties is
the only possible way to tackle the disease.
 More than 30 genes (Xa1, Xa2……..Xa30)
characterized. Genotypes carrying single
resistant gene have shown susceptibility at
different locations
 Introgression of multiple R genes into the
genetic background of elite rice varieties with
the help of closely linked markers can ensure
durable resistance

17. Disease symptoms
An Xoo colony
Lesion
Control Infected

18.  Effective bactericides are not available for
controlling the disease
 Bacterium can overcome rice cultivars
containing single genes for resistance against
the disease
 Rice cultivars should be developed that contain
multiple genes for Bacterial Leaf Blight
Resistance

19.  What are the rice resistance genes that are
effective against Indian strains of Xoo?
 What is the genetic diversity within the
population of Xoo strains in India?

20. Xoo strains were collected from rice plants growing in
many different locations in India
These were subjected to DNA fingerprinting and
pathotype analysis
The analysis revealed presence of two main groups –
Group I and Group II among Indian pathotypes of Xoo.
Group I predominant in India

21. Rice resistance genes xa13 and Xa21 are effective
against both groups of Indian Xoo strains
_____________________________________________________
Xoo Rice resistance genes
Strain
Xa3 Xa4 xa5 Xa7 xa13 Xa21 TN-1
_____________________________________________________
Group I S S R/S S R R S
Group II S S S S R R S
_____________________________________________________
R= Resistant; S= Susceptible

22.  A single lineage of Xoo (Group I) is widely
distributed in India.
 Xa21 and xa13 rice resistance genes are
effective against both groups of Indian Xoo
strains
 The xa5 resistance gene provides a moderate
level of resistance against strains within the
widely distributed Xoo lineage

23.  A rice line called SS1113 that contains Xa21, xa13
and xa5 genes in the genetic background of PR106
has been developed at Punjab Agricultural
University, Ludhiana
 SS1113 is resistant to Bacterial Blight disease but
scores low on other characteristics that are favored
by farmers and consumers
 SS1113 – Can serve as a donor for BB resistance into
elite high yielding but Bacterial blight susceptible
rice cultivars

24. Samba Mahsuri (BPT5204)
 Samba Mahsuri (also called BPT5204) is a leading rice variety
of Andhra Pradesh and has been developed by scientists at the
Acharya N.G. Ranga Agricultural University, Hyderabad
 This variety is being cultivated in many other parts of India
because of its exceptional yield and quality characteristics. But
Samba Mahsuri is susceptible to several diseases including
Bacterial Blight (BB)
 Need for introgression of effecive BB resistance genes into
Samba Mahsuri background without loss of its unique quality
and yield characteristics

25. Marker assisted introgression of BB resistance genes
into Samba Mahsuri and Triguna
 Donor
SS1113 (with Xa21, xa5 & xa13)
 Recipient(s)
Samba Mahsuri, Triguna
 Linked markers (used for foreground selection)
Xa21 - pTA 248 (ALP marker)- 0.1 cM – Chr. 11L
xa13 - RG136 (CAPS marker)- 2.0 cM – Chr. 8L
xa5 - RG556 (CAPS marker)- 0.1 cM – Chr. 5S
 For accelerated recovery of recurrent parent genome
parental polymorphic rice microsatellite markers used
(background selection)

26. Approach for marker-assisted introgression
 Marker-assisted breeding/backcross breeding involving both
foreground and background selection
 Foreground selection: Selection for backcross plants possessing
target genes. Done using gene linked/functional markers to select
backcross plants possessing target genes (viz., Xa21, xa13 & xa5)
 Background selection: Selection for „foreground selection positive‟
backcross plants which possess maximum recurrent parent genome
introgression. Done using ~90 parental polymorphic SSR markers
distributed uniformly throughout the rice genome
 Backcrossing continued till BC4 generation and further selections
were done based on agromorphological traits

27. Schematic illustration of bacterial blight resistance gene transfer
programme using molecular markers
SS 1113
Samba Mahsuri
(xa5 xa13 & Xa21) X (AABBcc)
(aabbCC)
2000
F1 (AaBbCc)
aa = xa5 Marker-assisted
bb = xa13 Backcrossing
BC1F1
CC = Xa21 (AaBbCc)
BC2F1 (AaBbCc)
BC3F1 (AaBbCc)
BC4F1 (AaBbCc)
BC4F2 (aabbCC)
BC4F3; Evaluation of
2003-04
Resistance, yield, etc.

28. Foreground selection of BC1F1 progeny for target trait
Selection for Xa21
44/145
Selection for xa13
Selection for xa5 23/44
11/23

29. Number of triple „R‟ gene heterozygotes
amongst backcross progeny
Cross Progeny # of plants # of triple
stage scored heterozygotes
SS1113 X BC1F1 145 9
BPT5204
BC2F1 156 11
BC3F1 160 12
BC4F1 148 10

30. Markers used for background selection
1 2 3 4 5 6 7 8 9 10 11 12
RM3340 RM60 RM551 RM7029 RM508 RM295 RM337 RM316
RM6340 RM481 RM407 RM7545
RM485 RM589
xa5 RG556 RM3309 RM244
RM428 RM110 RM231 RM524
RM335
RM279 RM413 RM204 RM332
RM13 RM5556
RM402 RM320 RM247
RM261 RM6051 RM491
RM7488
RM174 RM346
RM490 RM3859 RM25 Xa21 RM536 RM511
RM583 RM169 RM434 RM271 pTA248
RM72 RM5590
RM324 RM7 RM331 RM269 RM287
RM339 RM242 RM304 RM229 RM463
RM561 RM541 xa13
RM24 RM119 RM163 RG136 RM107 RM5961
RM3628 RM294A
RM18
RM478 RM215
RM5919 RM206 RM17
RM5473 RM6070 RM591
RM246 RM470 RM178 RM248
RM5545
RM458
RM411 RM340 RM264
RM212 RM497 RM280
RM250 RM7324
RM186
RM482
RM207
RM442
RM85
Foreground selection: 3 markers
Background selection: ~ 90 SSR markers

31. Background selection of foreground selected BC2F1
plants using parental polymorphic SSR markers
1 2 3 4 5 6 7 8 9 10 11
RM19
RM201
1 = SS1113; 2 = Samba Mahsuri; 3 to 11 = BC2F1 lines

32. Estimation of recurrent parent genome
contribution to selected backcross progeny
Cross Progeny Cumulative
stage contribution
of recurrent parent
genome
SS1113 BC1F1 72%
X BPT5203
BC2F1 80%
BC3F1 89%
BC4F1 96%

33. Number of lines with multiple „R‟ gene combinations
Ser. Gene combinations SS1113 X Samba Mahsuri
# BC4F2
1 Xa21/Xa21 xa13/xa13 xa5/xa5 5
2 Xa21/Xa21 xa13/xa13 Xa5/Xa5 5
3 Xa21/Xa21 Xa13/Xa13 xa5/xa5 4
4 xa21/xa21 xa13/xa13 xa5/xa5 2
5 Xa21/Xa21 Xa13/Xa13 Xa5/Xa5 4
6 xa21/xa21 xa13/xa13 Xa5/Xa5 4
7 xa21/xa21 Xa13/Xa13 xa5/xa5 2

34.  At BC4F1, a line possessing the BB resistance genes along with
maximum recurrent parent genome introgression was selfed to
generated homozygous BC4F2 lines
 The lines were evaluated for their morphology, yield, quality and BB
resistance from BC4F2 generation onwards
 Four homozygous lines possessing high BB resistance, yield and
grain quality similar to Samba Mahsuri were nominated for All India
Coordinated Rice Improvement Programme (AICRIP) trials
- IET 19026
- IET 19045
- IET 19046
- IET 19590

35. Samba “The gene
Mahsuri pyramid
lines are
resistant to
bacterial blight
(BB)”
2 gene 1 gene
3 gene

36. The gene pyramided line IET19046 has shown
high BB resistance under AICRIP trials
Year Proposed Susceptible check Resistance check National
entry (TN1) (SS1113) resistance check
(Ajaya)
(IET 19046)
Total Number of locations tested 2005 13 13 13 13
Number of locations where the 9 0 9 8
entry showed high BLB resistance
(score < 5)
Number of locations where the 4 0 13 5
entry showed moderate BLB
resistance (score – 5)
Severity Index value 3.8 8.0 2.5 3.9
Total Number of locations tested 2006 18 18 18 18
Number of locations where the 14 0 14 11
entry showed high BLB resistance
(score < 5)
Number of locations where the 4 0 4 3
entry showed moderate BLB
resistance (score – 5)
Severity Index value 3.4 7.7 3.4 4.2

37. The three gene pyramided line IET19046 is
highly resistant to BB

38. The gene pyramided line IET19046 is highly resistant to
BB under field conditions
Very small lesion length under No damage under BB infestation
BB infestation

39. Yield levels of IET19046 was identical to Samba Mahsuri
under AICRIP trials
Year of No. of Improved Check 1 Check 2 Check 3 Qual. Qual. Var. Qual.
testing trials Samba (Samba (Donor (Local Var. 1 2 Var. 3
Mahsuri Mahsuri) parent) check) (IET (IET (IET
(IET19046) 19026) 19045) 19590)
Mean Yield
(kg/ha) K-2005 10 4727 4739 4486 4887 4597 4667 4574
K-2006 10 4542 4529 4344 4936 4625 5025 4910
Mean - 4635 4634 4415 4912 4611 4846 4742
% increase
or decrease K-2005 10 - -0.25 +5.37 -3.27 +2.83 1.29 3.34
over the
checks &
qualifying
varieties K-2006 10 - +0.29 +4.56 -7.98 -1.79 -9.61 -7.49
Frequency
in the top
group Pooled 20 10/20 10/20 6/20 9/20 10/20 10/20 10/20
(pooled for
2 years)

40. Improved Samba Mahsuri
 IET19046 has since been named as
Improved Samba Mahsuri and
released by Central Varietal
Release Committee as a possible
replacement of Samba Mahsuri in
BB endemic areas
 A success story of MAS in India
 Improved Samba Mahsuri doing
very well in farmers field. More
than 20 tonnes of seed sold by
DRR in 2008 Kharif alone
 Excellent example of Inter-
Institutional collaboration
(DRR-CCMB)

43. Development of high yielding BB resistant gene pyramids of „Triguna‟
Two lines: Trg12-5-
4-9 and Trg136-22-
3-5 nominated for
AICRIP trials (IVT-
IME), possess high
yield and BB
resistance (Xa21
and xa13)

44. Thank you