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Hidden treasures: Precious stress-tolerance genes from low-yielding rice varieties

Sigrid Heuer
IRRI Thursday Seminar May 05 2011

Outline of presentation Introduction Principles of Marker Assisted Breeding Submergence tolerance: Sub1 and beyond Phosphorus-deficiency tolerance (Pup1) Pup1 candidate genes Pup1 breeding summery and outlook

Rice is life
Million tons milled rice
600

550

Additional rice needed: 116 million tons by 2035

500

450

400

2010 global rice production


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300

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Asia

Africa

Americas

Rest of World
A. Dobermann, IRRI 2010

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Produce more with less : HOW?


Two different major rice production systems: Irrigated
~60% of total rice area Asia (80 M ha*) ~ 70% of rice production constant water control high fertilizer inputs weed control (by flooding and herbicides) favorable conditions

Rainfed
~40% of total rice area Asia (60 M ha*) *Dawe et al 2010 ~ 25% of rice production no or little water control low to very low fertilizer inputs weed control difficult and labor intensive Often unfavorable conditions

Prevalent abiotic stresses:


drought submergence salinity

toxicities (Al, Fe)


acidity/alkalinity nutrient deficiencies (P).

Poor soils in poor countries


60% of rainfed-lowland rice in Asia is grown on problem soils
Nepal

NE India Bangladesh

India

Myanmar Thailand Vietnam Cambodia

climate change
Temperature

Sri Lanka

Indonesia

Water level
Adapted from Haefele and Hijmans, 2007

No progress in stress-prone environments without stresstolerant rice varieties

heat stress floods Snow cover other calamities droughts

The good news: Tolerant rice varieties do exist that can be used for breeding

The problem: Tolerant varieties have many adverse agronomic traits:


low yield long duration lodging grain shattering

tolerance
use tolerance only

Marker-assisted molecular breeding: Take the best and leave the rest
1. QTL mapping

tolerant

intolerant

chromosomal region where trait is located

= Quantitative Trait Locus (QTL)


intolerant tolerant

2. QTL fine mapping intolerant

tolerant

X
Markers for the region:
background

tolerant

tolerant
flanking QTL specific (foreground) flanking

At this point: Breeders are happy

and molecular biologists excited !

3. Gene identification

QTL projects at IRRI


Submergence (Sub1) Phosphorus deficiency tolerance (Pup1) Heat Salinity (SalTol) Anaerobic germination Drought (e.g., QTL12.1) Blast resistance (Pi40t) . FR13A: aus-type; Orissa Kasalath and Dular: aus-type, India N22; aus-type, India Pokkali: Sri Lanka or Bangladesh: aus introgression (K. McNally; pers com.) Kayan: aus-type, Bangladesh Vandana/Way Rarem: India O. australiensis: Wild rice

japonica

Nepal

NE India Bangladesh

aus

indica

India

Myanmar Thailand Vietnam Cambodia

Sri Lanka

Indonesia
Adapted from Londo et al 2006

Markers and phenotyping: Submergence tolerance


Chr. 9
RM316

Sub1

RM219 RM524

RM6051

RM434

RM242

Sub1
RM215

non-Sub1

Swarna TDK1 BR11 IR64 Samba Mahsuri

Sub1 mega varieties: Submergence screening at IRRI


Recovery of Sub1 varieties after 17 days complete submergence

IR64-Sub1 Samba M-Sub1 IR49830 (Sub1)

Samba-Mahsuri
IR42 IR64 IR64-Sub1

IR64 IR42

Samba Mahsuri
IR49830 (Sub1) IR49830 (Sub1) Samba-Mahsuri IR64 IR64-Sub1 IR42 IR64-Sub1 IR49830 (Sub1) Samba Mahsuri

Samba M-Sub1

IR42
IR49830 (Sub1)

Samba M -Sub1

IR64

Sub1-variety testing in farmers fields in India and Bangladesh


Swarna-Sub1 was first variety released (in India and the Philippines)

Farmers field in Rangpur (Bangladesh) 2008

Sub1 varieties

Seed increase, dissemination, and development of additional Sub1 varieties is ongoing


susceptible check farmers tolerant variety BRRI, Rangpur (Bangladesh) BR11-Sub1 Swarna Farmers field, Orissa (India)

SwarnaSub1

BR11
Septiningsih et al 2009, Annals of Botany Singh S et al 2009, Field Crops Res

Gayatri

SwarnaSub1

3. Tolerance gene identification

QTL
gene

Phosphorus deficiency tolerance: Pup1

Submergence tolerance: Sub1

Sub1 region derived from FR13A (tolerant)

Sub1 region in Nipponbare (intolerant)

Sub1B

Sub1C

Gene expression analysis

Sub1B

Sub1C

Sub1A

A single gene (SUB1A) confers tolerance of submergence

+Sub1 QTL
SUB1A, B or C?

Transformation as a tool to validate candidate genes


intolerant check
(Liaogeng)

Function of SUB1A: suppression of growth tolerant check (IR40931-26) under submergence Ubi:SUB1A-1
(T1)

Sub1 QTL
+ + +

Deep water rice: Escape response

Kende et al (1998)

Xu et al 2006, Nature Fukao et al 2006, Plant Cell Singh N et al 2010, Theor Appl Genetic Bailey-Serres et al 2010, Rice

Sub1 QTL: Three types tolerant indica (e.g., FR13A) intolerant indica (e.g., IR64) intolerant japonica
(e.g., Nipponbare)
Sub1A-1 Sub1A-2 Sub1B-1 Sub1B-n Sub1B-n Sub1C-1 Sub1C-n Sub1C-n

specific markers for SUB1A 1: SUB1A present ?

2: Tolerant or intolerant version of SUB1A (A1 or A2) ?

intolerant allele SUB1A-2

tolerant-specific SUB1A-1

Moving beyond Sub1: Screening for non-Sub1 tolerance


Sub1 facilitates survival for ~14 d - - - Can we enhance this furher? Screening and genotyping of >100 rice accessions
8080

IR40931 (tolerant check)

plant survival (%)

plant survival plant survival (%)

6060 4040 2020

SUB1A-1 SUB1A-2 SUB1A absent (A0)

IR42 (intolerant check)


0
0

00

50 50

shoot elongation (%)

shoot elongation (%)

100 100

150 150

200 200

Genotype and phenotype do not always associate


intolerant tolerant tolerant-specific SUB1A-1

Gene expression analysis:


Submergence tolerance is associated with high SUB1A gene expression

SUB1A-1 not expressed

SUB1A-2 (intolerant) high expression

Sub1 donor

Thats what followed


Intolerant (Teqing, SUB1A-2)
GARC SORLIP2AT/SiteII
DOF Myc Myc Myb DOF

GCC box
W-box
TA GARE TA Myb DOF

W-Box

Myb

W-box GAMYB

bidirectional promoter light regulation?

TATA TTS
(Inr: CATCA)

CGCG-box SR1-calmodulin binding

Start

ethylene response

GA response

5UTR: sugar, GA, and Ca2+ response?

Tolerant (FR13A, SUB1A-1)

Start

Tolerant (Goda Heenati, Kurkaruppan)


Start

More on this another time

SUB1A spatial expression (promoter::GUS studies)


T1 seedlings leaf collar leaves

non-submerged non-subm.

submerged

shoot meristem

submerged leaf base

roots

Singh N et al 2010, Theoret Appl Genet

meristem elongation differentiation

Singh N et al unpublished

Sub1 and drought ???

Fukao et al 2011, Plant Cell Sub1

Function of Sub1: Keep meristem alive under stress?

The major QTL Phosphorus uptake 1 (Pup1): confusion and progress


Reasons for Phosphorus (P) deficiency:

1. Low endogenous P in soil 2. Farmers dont apply fertilizer 3. Phosphorus is present in the soil but unavailable
Fixation of P is wide-spread in problem soils
Nepal

NE India
Bangladesh

India

Myanmar Thailand Vietnam Cambodia

Sri Lanka

+P
Indonesia

Pup1 mapping (Matthias Wissuwa; JIRCAS)


Pup1 near isogenic lines
14 12 P uptake (mg plant-1) 10 8 6 4 2

intermediate QTL

Screening Nipponbare of 30 rice accessions NIL C443 Kasalath

Nipponbare

M. Wissuwa, JIRCAS

P-uptake: dry weight: tiller number:

LOD 10.7 (28%) LOD 10.5 (27%) LOD 7.9 (21%) +P 13.7 13.9 10.9 -P 1.8 3.2 3.2

Pup1

P uptake
(mg root weight-1)

Nipponbare NIL-C443 Kasalath

Wissuwa et al 1998; Theor Appl Genet Wissuwa and Ae 2001; Plant Breeding

Studies on root-response to Phosphorus-deficiency


Induction of root growth Root hair growth (Arabidopsis) +P P Cluster roots

adapted from Zhang et al 2003, J Exp Bot

Pi

Mycorrhiza

Lambers et al 2006, Annals Bot

Organic acid exudation

Photo: M. Wissuwa

Molecular studies on plant response to Phosphorus deficiency


Up-regulation of high-affinity P-transporters

Many other studies.

Paszkowski et al 2002; PNAS

Morcuende et al (2007), Plant Cell Environment

Hammond et al (2004) Annals of Botany Amtmann and Blatt (2009) New Phytologist

P-responsive genes in rice: No candidate gene in Pup1 region


Chr. 1 Chr. 2 Chr. 3 Chr. 4 Chr. 5 Chr. 6

intermediate QTL

OsPTF1
Yi et al (2005) Plant Physiology

Pup1
Chr. 7 Chr. 8 Chr. 9 Chr. 10 Chr. 11 Chr. 12

Pup1
1 M P 100 M P
major QTL

Heuer et al 2009, J Plant Biotech

Sequencing of Pup1 in the tolerant parent Kasalath: A complex hot spot of transposon integration

Pup1 region

Nipponbare (154 kb) 54 % TEs

LEGEND Percent identity Connecting lines

Kasalath (278 kb) 54.6 % TEs

Heuer et al (2009), Plant Biotechnology Journal

93-11 (742 bp) 45.2 % TEs


Heuer et al 2009, J Plant Biotech

68 predicted genes with RiceGAAS (incl. TEs) many unclear gene models due to transposable elements no P-transporter or P-uptake related gene

Finding the hidden treasure: Candidate gene identification and validation

Nipponbare

1 29

49

53
67

Kasalath

4, 5

20

DUF568

dirigent gene (PupK-21n)

hypothetical protein

Zn knuckle and Zn finger protein

aspartic proteinase

fatty acid DOX/ unknown protein

protein kinase

Transgenic approach to validate gene function


1. Knock out gene function in tolerant donor (Kasalath) P-uptake (mg plant-1) P uptake (mg)
1.2 1.0
P uptake (mg)

gene #1

1.2 1.0 0.8 0.6 0.4 0.2 0.0

gene #2

0.8 0.6 0.4 0.2 0.0 Kasalath RNAi-36a RNAi-35 RNAi-36 transgenic event null

Kasalath

21-1null

21-9

21-11

21-4null

transgenic event

Data: M. Wissuwa, unpublished

2. Express gene in intolerant variety (IR64) Phenotyping in P-deficient soil

Effect of Pup1 gene #1 on root and shoot growth


19A
1 T 2 3 T 4 5 T 6 1 T 2 -

20A
3 T 4 5 T 6 1 T 2 -

21A
3 T 4 5 T 6 1 T 2 3 T

5A
4 5 T 6 1 T 2 -

14A
3 T 4 5 T 6 water -

Transgene expression high expression


1 copy 1 copy 1 copy

low expression
2 copies

no expression
2 copies

NT

NT

NT

NT T

NT T

NT

NT

NT

NT

NT

Effect of Pup1 gene #1 on grain yield

Grain plant grain weightweight -1

16 16.000 14 14.000
12.000 12 10.000 10 8.000 8 6.000 6 4.000 4 2.000 2 0.000 0 -P -P 21A -P -P -P 19A -P -P -P 20A

P soil +P fertilizer

P soil

T NT

-P +P +P +P +P +P +P +P +P +P +P +P +P +P +P +P +P +P +P

21A 21A

19A 19A

20A 20A

10A

Protein kinases are regulatory proteins that activate or suppress function of other proteins

Pi Pi Pi Pi Pi Pi Pi
Pup1 kinase

Pi
Pi Pi Pi

Pi
Pup1 kinase

Pi
Pi

Pi Pi Pi

Microarray study to identify Pup1-related genes:

NO phosphorus-response gene
sugar metabolism ?

Latest preliminary data !

phenotype of gene #1 in culture solution +P

Non transgenic control (#10)

#19

#20

#22

#10

transgenic

Is Pup1 in the end an early-vigorous-root-growth QTL and P-uptake is simply a matter of having more roots early on ???

Validation of Pup1 in different environments and genetic backgrounds


Nipponbare-Pup1 in P-deficient soil: IRRI and JIRCAS 115 DAS (harvest)
66 55

grain weight (g plant-1) 4.0


4.0

+P

grain weight (g plant-1)


-P-P

3.5 3.5
grain weight (g plant-1)

4 3

-P
+P

+P Series1
ns
ns ns ns
plant P content (mg plant -1)

P content mg plant-1
21 18 15 12 9 6 3

ns

ns

ns ns

ns

ns

grain weight (g plant-1)

44
33 22 11 00
14-4 14-6

-P
ns

+P

3.0

3.0 2.5 2.0 1.5

ns

a a b a

2 2.5

2.0 1.0 1
0.5

b
NB NB

a b

1.5 0.0

+ +P
14-4

14-6

14-4 14-6

+ -P
14-4

14-6 1.0
0.5 0.0

24-6 24-6

28-10 28-10

24-4 24-4

6-4 6-4

14-4 14-4

0
NB NB 24-6 24-6 28-10 28-10 24-4 24-4 6-4 6-4 14-4 14-4

-Pup1

+Pup1

-Pup1
24-4 6-4 14-4

+Pup1

IRRI

JIRCAS (Japan)
NB 24-6 28-10

Chin et al 2010, Theor Appl Genet

Problem: Nipponbare doesnt grow well under short-day conditions

development of indica-Pup1
molecular markers

Pup1 gene-based markers

markers distributed over entire Pup1 region dominant and co-dominant

co-dominant

dominant

Pup1 markers survey of IRRI breeding lines and diverse varieties


96.2 K1 116.6 K5 170.0 K20-1Mse 170.1 K20-2Bsp 202.8 K29-1 204.5 K29-2 205.2 K29-3 262.2 K41 267.6 K42 269.0 K43 274.2 K45 276.0 K46-1 276.5 K46-2 283.2 K48 301.1 K52 325.0 K59

Ecosystem Subspecies Breeding history

Haplotype I

+Pup1

mainly rainfed-adapted

Haplotype II

0.1

Pup1
Haplotype III

mainly modern, irrigated

Chin et al 2011, under revision Plant Phys (special issue)

Field performance of diverse germplasm with and without Pup1


IRRI upland farm experiments
drought
2007

well watered +P

-P

grain weight plant-1 (g)

41 DAS

2008

-P

-P -P

45 DAS

plant height (cm)

IR64-Pup1 and IR74-Pup1

Marker-assisted selection

Pup1 lines >90% identical to recipient parent

- Seed increase - Field evaluation: Starting next season

Pup1 introgression into Indonesian upland varieties


(collaboration with ICABIOGRAD)

P+
Grain weight per plant (g)
30 25 20 15

Situ Bagendit-Pup1
no Pup1 alleles

Batur-Pup1
some Pup1 alleles

Dodokan-Pup1
most Pup1 alleles

Dodokan 10 5 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Situ Bagendit Batur

Grain weight per plant (g)

low P

Summery
QTLs are regions on chromosomes where tolerance genes are located Molecular markers facilitate breeding targeted at these regions (take the best and leave the rest) Tolerance genes are specific to the tolerant parent and sometimes absent in modern varieties (e.g., Sub1, Pup1) The Pup1 protein-kinase gene has a large effect on root growth and yield Indica-Pup1 lines for field evaluations

Whats next? QTL P-use efficiency for intensive systems Move beyond Pup1: Enhance gallery (reduce P-fertilizer inputs) Sequencing of tolerant varieties (QTL mapping at the computer ?) cold salt flood drought heat BPH Blast P Pup1/drought
flood/salt

Streamline QTL identification and candidate gene validation

Abdel Ismail

Sub1

Julia Bailey-Serres

Dave Mackill Pam Ronalds

Special thanks to

Inez Slamet Loedin, Rudi Trijatmiko, + team Kenong Xu Trang TM Dang Endang Septiningsih (Septi) Gina Vergara
for their support during the transformation experiments

Darlene Sanchez Namrata Singh

Pup1
Rico Gamuyao
Masako Akutso
Jonghwa Park

Cheryl Dalid

JIRCAS:

JH Chin

Juan Pariasca Tanaka

Rani Lorie

Anthony

Matthias Wissuwa

NIAS: Masahiro Yano


ICABIOGAD: M. Bustamam J. Prasetiyono IRRI: Stephan Haefele et al Arvind Kumar MPI-Golm: Bernd Mueller-Roeber Lutz Neumetzler University Milano: Paolo Pesaresi

Thanks to our donors Pup1: BMZ + Sub1: BMZ, BMGF, IRRI

Thank you for your attention !