A new gene for broad-spectrum resistance to rice blast

1
A new gene for broad-spectrum resistance to rice
blast
K. K. Jena International Rice Research
Institute www.irri.org
2
Outline

• Background
• New source of resistance gene
• High-resolution mapping
• MAS validity and spectrum of resistance
• Future opportunities

3
Blast epidemic in Korea (1984-1985)
4
Breakdown of cultivars with major blast
resistance genes
Pib, Pia
Pia, -
pathogen change
Pib, -
Pib, Pi5
Pib, Pita, Pi9
Need broad-spectrum durable resistance gene
5
Status of blast resistant varieties released in
Japan
No. of years from release to breakdown
Varieties
Genotype/ R gene
Pik Pia, Pik Pia, Pik Pik Pik Pia, Pik-m Pia,
Pita Piz Pii, Piz Pia, Pii, Pik, Pib Pik, Piz
1 - 6 2 2 2 2 3 2 - 7 5 1 2 3
Kusabue Yuukara Teine Ugonishiki Tachihonami Mineh
ikari Shimokita Fukunishiki Yamatenishiki Hamaasah
i Akiyutaka
6
Objectives

• To identify and fine map a new gene for blast
  disease resistance using genomic tools and blast
  isolates of South Korea
• To study validation of DNA markers linked to the
  new resistance gene and spectrum of blast
  resistance in rice improvement

7
Gene mapping and Breeding Strategies
Recurrent parent (S) X
Donor parent (R)
Fine mapping of the R gene
1. Association analysis (ANOVA) F2 Linkage
map skeleton F23 Phenotyping (QT
data) 2. e-landing on Nipponbare ORFs
More F2s High density map (target locus)
R BCs MAS validity test 3.
Comparative analysis and gene cloning (gene
function)
F1 x RP (MAS)
?B1F1 x RP Dual selection ? B2F1 x
RP Dual selection ? B3F1 (Self)
(MAS) ? B3F2 - - - - - - - - - - - - - - - - - -
- - - - - - - - - Selection of
Background Agronomic traits selection
B3Fn (R)
Homozygous for R allele
8
The New R gene Source IR65482-4-136-2-2
Disease reactions of varieties elite lines
tested
Odaebyeo
Suwon365
Sangjubyeo
Jinbubyeo
IR31917- 45-3-2
IR65482- 4-136-2-2
Isolates
3
4
3
2
3
0
84-419 (KI315b)
2
0
2
4
4
1
86-228 (KI1117)
3
4
4
3
4
0
87-134 (KJ107)
4
0
2
2
5
0
89-001 (KJ401)
4
5
4
4
5
1
93-064 (KJ203)
4
5
4
5
5
0
00-272 (KJ301)
3
1
5
3
3
1
R88-02 (KI313)
NT
NT
NT
5
4
0
01-01 (KJ105a)
Strong resistance (0,1), Moderate resistance
(2,3), Susceptible (4,5), Not tested (NT)
9
Identification of Blast R gene Source

• Screening with 15 isolates

Lineages from MGR586 fingerprints on collections
of M. grisea isolates
No. of genotypes tested 1 indica, 12 INTs, 19
Korean japonica
Score (1 5)
Genotypes
Japonica (S)
Donor Line
1
IR65482-4-136-2-2
5
IR65482-7-216-1-2
5
Sangjubyeo
5
Jinbubyeo
5
Chucheongbyeo
10
Bl-R gene Homologue Polymorphism
11
Sequence Homology between the R line and Pita
gene
Resistance level of Pita
Pita is not the driving force of the
broad-spectrum resistance
12
Phenotyping of F23 (Isolate KJ105a)
Individual based scoring (QT data acquisition)
0 (R) 5 (S)
F23 progenies
Jinbubyeo
IR65482-4 -136-2-2
13
Preliminary Mapping ofthe R-gene using
association analysis
Chromosome 6S
ANOVA on S2539 locus No. of F23 lines ? N88,
Mean 1.03 Homo for A ? N14, Mean 3.25 Homo
for B ? N28, Mean 0.11 Hetero ? N46,
Mean 0.96 Degree of Dominance 0.46
33 anchors to tag Ch 6S as the nest of Bl-R gene
Prompt testing the putative chromosomal region
14
Major Bl-R genes on Chromosome 6 short arm
Chromosome 6
RM133
Gene
DNA Marker
Chromosome 6S
Marker
RZ398
cM
z4792, z60510
Piz
RG64
RM5963
Pizt
z4792, z60510
RM527
7.2
RG468
Piz5 ( Pi2 )
e-landing
RG64-SAP
Pi9
S2539
pBA14, pB8
RM454
2.7
RM527
Bl-R gene rich region
4.4
RM343
RM3330
Broad spectrum of resistance
1.1
RM5745
RG653
Tightly linked to each other
RG433
Demands comparative analysis ? DNA level
Pathotyping
15
e-Landing on Nipponbare Reference Genome
Nipponbare (31.20Mb, 125cM)
9.16Mb (54.1 cM)
Ch 6S
P0038C05
Marker
F-value ( R2 )
cM
P0450D12
RM5963
35.1 (0.46)
12 BAC or PACs
OSJNBa0063H02
OSJNBa0021H05
7.2
P0491D10
1.9 Mb
S2539
84.4 (0.67)
P052B12
2.7
4.6 cM
P0649C11
RM527
84.7 (0.67)
B1197G05 B1026E06
4.4
B1066D09
RM3330
60.8 (0.59)
1.1
RM5745
46.7 (0.53)
P0036B02
OSJNBa0055N24
11.07Mb (58.7 cM)
16
Tagging Novel Blast R gene via e-Landing SNP
Markers
Chromosome 6 (31.2Mb)
9.86Mb
OSJNBa0063H02
cM
F-value ( R2)
OSJNBa0021H052
RM5963
35.2 (0.46)
P0491D10
7.2
P052B12
S2539
84.4 (0.67)
2.7
P0649C11(6 NBS-LRRs)
84.7 (0.67)
RM527
B1197G05
1.1
9871.T7E
94.1 (0.69)
3.3
B1066D09
60.8 (0.59)
RM3330
1.1
P0036B02
46.7 (0.53)
RM5745
OSJNBa0055N24
11.1Mb
17
Inside PAC clone, P0649C11
P0649C11 (Acc. AP005659 , 138.8Kb)
18
Tagged sites of genes by Bl-R gene
markers(Mirror map)
e-Landing using primer sequences
ORF searching rice genome annotation (TIGR)
19
Features of 9871.t7 on P0649C11 (138,870bp)
20
Comparative Analysis by usingAnnotated ORF
derived Primer sets
STS primer sets for 6 NBS-LRRs
ID Lines cultivars Genes 1. IR31917-45-3-2 Pib 2
. IR65482-4-136-2-2 Pib, Pita, R 3. Jinbubyeo Pi
z 4. Junambyeo Unknown 5. Australiensis R 6. LTH
None 7. IRBLz5-CA Piz5 Pi2(t) 8. IRBLzt-T
Pizt 9. IRBLz-Fu Piz 10. IRBL9-W Pi9
Clues on DNA marker analysis Jinbubyeo Piz Piz
and Pi9 susceptible Piz5and Pizt resistant
to screening isolate 01-01 (KJ105a)
21
Strategies for Allele Differentiation

• High stringency PCR ? Annealing Temp 65oC
• Careful primer design to generate clear single
  band
• Apply frequent cutters to detect SNPs

RG64-SAP
9871.T14E23
22
Differentiation of Pi40 allele types
Example of S2539
Six restriction enzymes (AluI, HaeIII, HinfI,
RsaI, TaqI, Tsp509I) and high resolution PAGE
analysis
23
9871.T7 as the Putative ORF for Pi40
DP IR65482-4-136-2-2 RP Jinbubyeo Aus O.
australiensis
Pizt and Pi40 (IR65482-4-136-2-2) are
undifferentiated
24
Determined detailed haplotypes of nine genotypes
on the near-centromeric region of chromosome 6
Locus Line discrimination Line discrimination RPD DOR RP1 RP2 LTH Piz-5 Piz-t Piz Pi9
Locus Method Type RPD DOR RP1 RP2 LTH Piz-5 Piz-t Piz Pi9
S2539 CAPs 3 A B A B A C B A B
RG64-SAP CAPs 3 A B A C A B C A B
RM0527 SSR 4 A B C B A B D C D
9871.T5I1 CAPs 1 a a A a A A A A a
9871.T5E2 CAPs 1 A A A A A A A A A
9871.T7E CAPs 6 A B C D D B B E -
9871.T7E2b CAPs 6 a B C D D E B F f
9871.T8I12 CAPs 2 - A A - A a A a -
9871.T8E3 CAPs 4 A B C A C D B C C
9871.T9I1 CAPs 2 A A B A A A A B a
9871.T9E3 STS 2 A B A A B B B A A
9871.T10I2 STS 2 A A A A B A A A A
9871.T10E4 CAPs 2 A A B A A A A B A
zt6057-Nip STS 3 - A B A A A A B B
9871.T14I1E2 CAPs 4 - A B C B A A B B
9871.T14E23 CAPs 6 - A B C D A A B E
pBA14 CAPs 3 A A A - B - - - B
RM3330 SSR 5 A A B C B A D B E
RPD IR31917-45-3-2, DOR IR65482-4-136-2-2,
RP1 Jinbubyeo, RP2 Junambyeo
25
9871.T7E as the DNA Marker for Tagging the Novel
Bl-R gene, Pi40
9871.T7E / Tsp509I
Pi40
26
Multiple Sequence Alignments and deduced
Phylogenetic Tree on 9871.T7E locus
27
Broad-spectrum blast resistance
Pathotyping with different virulent isolates
Pizt
Pi40
RPJinbubyeo DPDonor Parent RResistant
SSusceptible
28
MAS for blast resistance with Pi40 gene
Elite cultivars
QTLs for Agronomic traits
IR65482-4-136-2-2
Japonica/Indica with Pi40
(Pi40, Pib, Pita)
29
Durable blast resistance testing of Pi40 using
sequential planting methods
Pi40 (Chr.6)
Primer 9871.T7E
P1 P2
BC progenies
Pii
Piz
Piz5
Pizt
Pit
Pi1
Pi5
Pi9
Pita2
Pib
Pi5
Pi9
Pita
LTH
A B C
P1 Junambyeo (Recurrent), P2
IR65482-4-136-2-2 (Donor)
30
Confirmation of BC progenies having Pi40(t) gene
RP DP BP
CK
Pib
Pita
Pi40(t) (9871.T7E2b)
DP IR65482-4-136-2-2 RP Junambyeo
BP Backcross progenies CK Check DNA
31
Durability of monogenic near-isogenic lines
(NILS)
120
Pi-9
Pi-5
100
Pi-ta(K1)
Pi-zt
80
Pi-i
D LA()
Pi-3
Pi-b
60
Pi-ks
Pi-19
40
Pi-t
Pi-ta2
20
Palgong
Ilpum
0
1st
2nd
3th
4th
5th
6th
7th
No. of planting
32
Reaction of IRBL9-W (Pi-9) monogenic rice line to
Korean blast isolates
LTH
IRBL9-W
33
Progress of blast reaction to different R genes
tested by sequential planting method
100
LTH
JB, IP LTH Pii, Piz Pit, Pita
IP
80
JN
60
JB
Diseased leaf area ()
JN, Pi1 Pizt
40
Durable resistance
PG, Pi5
PG, Pi5 BP, DP Piz5
20
DP, BP Piz5
0
1st
2nd
3rd
4th
5th
6th
7th
Sequential planting time
IP Ilpumbyeo, JB Jinbubyeo, JN Junambyeo, PG
Palgongbyeo DP IR65482-4-136-2-2, BP Backcross
progenies
34
MAS Validity Test on Bl-R BC Progenies
Segregating BC2F4 families with KJ105a (01-01)
Resistance gene donor IR65482-4-136-2-2
- 15 families (15 28 seedlings per family)
- Mean R level 1.4 2.5 DNA Marker
9871.T7E - Among resistant seedlings, 94
were randomly selected
Genotypes ? 57 BB type , 37 AB type, 0 AA
type
35
DP
BC3F4
RP
Plant type of BC progenies with Pi40 gene
(center), recurrent parent (RP), and donor parent
(DP).
36
Grain Pictures and field blast test of Derived
Progenies (12) with parents
Rough rice
Brown rice
Milled rice
1
2
RP
DP
Field blast resistance test
R (2)
DP
R (1)
S
RP
37
Blast Reaction of BC lines in Northeastern China
S
R
R
Limgang
Nampyong
38
Opportunities through Pi40 gene

• Integration of Pi40 gene into indica and japonica
  elite cultivars using MAS strategy
• Sharing the donor indica line and new breeding
  lines with NARS
• world-wide (temperate as well as tropical
  countries) through
• Temperate Rice Research Consortium (TRRC) and
  INGER for
• durable blast resistance
• Cloning and characterization of Pi40 gene for
  understanding its
• structure and function

Map-based cloning of Pi40 gene
Identify candidate gene(s) ? Gene function and
Expression analysis for blast resistance
Integration into a wide range of cultivars 4.
Study genealogy of the Pi40 gene in different
wild Oryza species and other grasses
Pi40
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Conclusions

• A major dominant gene from the exotic breeding
  line, IR65482-4-136-2-2 confers strong resistance
  to Korean and Philippines blast isolates.
• A preliminary linkage map constructed from the
  Jinbubyeo x IR65482-4-136-2-2 mapping population,
  located the new R gene (Pi40) on the short arm of
  chromosome 6.
• Fine mapping by e-Landing on Nipponbare genome
  sequence and haplotyping of LTH monogenic blast
  differential lines identified a putative physical
  region of 9.1Mb, and a PAC clone, P0649C11 that
  contains 6 NBS-LRR homologues.
• Of the 6 NBS-LRR specific primers, the primer
  9871.T7E2b differentiated earlier reported R
  genes, Piz, Piz5, Pi9 and confirmed the Pi40 gene
  as a homologue from O. australiensis (EE genome).
• Pathotyping with virulent Korean and Philippine
  isolates differentiated the Pi40 from Pizt and
  confirmed broad-spectrum of blast resistance. The
  gene has been MAS validated for application in
  rice breeding.

Pi40
40
Acknowledgments

• Rural Development Administration (RDA), Korea
• for financial support of this project
• IRRI RDA
• Dr. Darshan S. Brar Dr. Ho-Yeong Kim
• Dr. David J. Mackill Dr. Seong-Sook Han
• Dr. C. Vera Cruz Dr. Yeon-Gyu Kim
• Dr. Byung-Run Kim
• Dr. Ji-Ung Jeung
• Dr. Jung-Pil Suh
• Dr. Kyu-Seong Lee

41
Kshirod K. Jena

• Current position
• Senior scientist, plant breeding, Plant Breeding,
  Genetics and Biotechnology (PBGB) Division, IRRI,
  since 2001
• Country representative for Korea, IRRI, since
  2001
• Education
• PhD, Plant breeding and genetics, Utkal Univ.,
  Bhubaneswar, and Central Rice Research Institute
  (CRRI), Cuttack, India, 1983
• MSc, Plant genetics and cytogenetics, Utkal
  Univ., 1976
• BSc, Botany, Honors with distinction, Utkal
  Univ., 1974
• Previous professional activities
• McNamara post-doctoral fellow, PBGB, IRRI,
  1983-85
• Collaborative research scientist, plant breeding,
  PBGB, IRRI, 1985-89
• Visiting faculty, Dept. of Botany and Crop
  Sciences, Univ. of Georgia, USA, 1989-92
• Associate professor, Dept. of Genetics, Osmania
  Univ., Hyderabad, India, 1992-95
• Research coordinator and head, Barwale
  Foundation, Hyderabad, India, 1995-2001