Bacterial Blight of Rice - IRRI Rice Knowledge Bank

Bacterial Blight of Rice - IRRI Rice Knowledge Bank

Planning Breeding Programs for Impact Breeding for Resistance to Diseases Bacterial blight Blast Learning Objectives To predict durability of R genes To explain pathogen population structure To discuss breeding strategies for diseases where major genes are effective To associate known sequences of candidate genes to phenotypes of germplasm and breeding pedigrees To discuss the possibility to relate QTLs to candidate genes and metabolic pathways IRRI: Planning Breeding Programs for Impact Aim for durable and broad-spectrum disease resistance Blast Bacterial blight IRRI: Planning Breeding Programs for Impact Overall Strategy & Target Understand genetic variability and population structure of the pathogen Study pathogen adaptation & epidemiological consequences Identify effective R-gene combinations against local populations Determine quality of resistance genes Incorporation of resistance genes into high-yielding local cultivars Gene deployment quality geographic area time Stabilize pathogen evolution in agronomic time frame (5-10 yrs) sustain productivity reduce pesticide use

IRRI: Planning Breeding Programs for Impact Bacterial blight of rice Bacterial blight of rice Reduction in photosynthetic area Reduction in 1000 grain weight Empty grains 20 50% yield loss reported IRRI: Planning Breeding Programs for Impact Bacterial blight syndrome Kresek or wilting Leaf blight phase Pale yellow leaf IRRI: Planning Breeding Programs for Impact The Infection Process Bacteria multiply rapidly, 108-109 cfu/ml 24 hrs after inoculation. IRRI: Planning Breeding Programs for Impact Distribution and frequency of Xoo races Virulence frequency (%) 100 Race 1 Race 2 Race 3 Race 4 Race 5 Race 6 80 60 40 20 0 1972-74 1975-79 1980 1981 1982 1983 1984

1985 1986 1988 Year Virulence frequency of Xoo races from 1972-1988 IRRI: Planning Breeding Programs for Impact Distribution and frequency of Xoo races IRRI: Planning Breeding Programs for Impact Proposed evolutionary pathways among races 1, 3, and 9 of X. oryzae pv. oryzae Race 9a S on Xa7 Race 3 R on Xa7 S on Xa4 XorI PCR type C-01 lacks 4.2 kb BamHI fragment Races 9b & c have 4.2 kb BamHI fragment Race 9b MS on Xa7 Race 9c S on Xa7 Race 1 MS on Xa7 R on Xa4 XorI + PCR type C-05 Race 9d

MS on Xa7 S on Xa4 lacks 4.2 kb BamHI fragment R = resistant MS = moderately susceptible S = susceptible Interaction between rice and Xoo R-Gene Race 1 2 3 4 5 6 7 8 9 10 IR24 S S S S S S S S

S S Xa4 R S S I R S R R S R Xa10 S R S S R S R S S S xa5 R R

R S R S R R R R Xa14 S S S S R S S R S S Xa7 I R R S R S

R R S I Xa 21 R R R R R R R R I S R = resistant (<5 cm); S = susceptible (>10 cm); I = intermediate (5-10 cm) IRRI: Planning Breeding Programs for Impact Gene-for-gene interaction between host and pathogen Pathogen genotypes One pair of loci AA Aa aa rr Host genotypes Rr RR = R (incompatible) = S (compatible)

Classification of cultivar-pathogen interactions Class 1 2 3 4 C1 C2 Result C1 C2 P1 Uniform P2 P1 P-differential P2 P1 C-differential P2 P1 P2 Strongly interactive IRRI: Planning Breeding Programs for Impact Classification of cultivar-pathogen interactions Class 5 5 C1 C2 C1 C2 C1 C2 C1 C2

P1 P1 P2 P2 P1 P1 P2 P2 = R (incompatible) Weakly interactive = S (compatible) Zadoks & Schein (1979) IRRI: Planning Breeding Programs for Impact HR versus VR Resistant Maritta Higher HR Susceptible Kennebec Resistant Susceptible Blight (P. infestans) races (Van der Plank, 1963) Host-Pathogen Interactions Plant cell wall Pathogen Ge El n e pr ici t or od uc

t avr gen e Lignin RO ROH Receptor signal cascade Activate Defense Genes Peroxidase HO HO 2 (peroxidase, chitinase glucanases, phytoalexins, lignin enzymes, etc.) oxidase (adapted from Leach & White, 1996 Annu Rev Phytopathol) O2 membrane Bacterial blight R genes, their donor cultivars, and chromosome location R-gene Donor Chrom R-gene Donor Chrom Xa1 Kogyoku

4 Xa16 Te-tep - Xa2 Tetep 4 Xa17 Asominori - Xa3 Wase Aikoku 11 Xa18 IR24, Toynishiki - Xa4 TKM6 11 xa19 XM5 - Xa5 DZ192 5 xa20 XM6

- Xa7 DV85 6 Xa21 O. longistaminata 11 xa8 PI231129 7 Xa22(t) Zhachanglong - Xa10 CAS 209 11 Xa23 Oryza rufipogon - Xa11 IR8 - xa24(t) DV86 - Xa12

Kogyoku 4 xa25 Nep Bha Bong To - xa13 BJ1 8 Xa26 Arai Raj - Xa14 TN1 - xa27 Lota Sail - xa15 XM41 - Xa? Oryza minuta - IRRI: Planning Breeding Programs for Impact INGER Nurseries Breeding scheme to develop

varieties resistant to BB Improved Germplasm from IRRI & National Programs IRRI Germplasm (GRC) Initial Screening for BB Resistance BB-GSN (re-testing of selected entries) Hybridization (Plant Breeding) Single or Multiple crosses for different ecosystems (Plant Breeding) F2 Populations in field screening for BB Resistance (Plt Breeding & Plt Path) Pedigree Nurseries Screening for R to BB & other diseases (Plt Breeding & Plt Path) Screening RYT & OYT for R to BB & other diseases (Plt Breeding & Plt Path) INGER Nurseries for Disease Resistance in Field or GH (Plant Pathology) Types/Forms of resistance (Plant Pathology) Resistance to specific races Resistance at different growth stages Genetic studies for BB resistance (Plt. Breeding & Plt. Path) Improved Sources

of BB Resistance Evaluation of improved materials from Natl Program IRRI Scheme for screening resistance to bacterial blight and blast Improved Germplasm/ NILs/IRRI Germplasm/ Wild rice accessions Resistance to BB & Blast (EPPD) Hybridization (Indica & NPTs) (PBGB) Genetic studies (PBGB & EPPD) Single or multiple crosses for different ecosystems (PBGB) Transgenics/ Parents for Hybrids/ Alien Introgression Lines (PBGB) F2 populations field screening (PBGB & EPPD) Pedigree nurseries screening (PBGB & EPPD) RYT & OYT screening (PBGB & EPPD) Improved classical plant types/NPTs Resistance to specific diseases (EPPD)

Improved sources of disease resistance or elite lines for release by NARES Clippers & clipping inoculation Clippers Field inoculation with clippers IRRI: Planning Breeding Programs for Impact Greenhouse/ screenhouse incn Scoring system for BB Greenhouse test Lesion length Description (cm) Field test (Breeding lines) Scale % DLA Desciption 0-5 R 1 1-5 R >5-10 MR 3 6-12 MR >10-15 MS

5 13-25 MS >15-20 S 7 26-50 S >20 HS 9 >50 HS IRRI: Planning Breeding Programs for Impact Types of resistance Seedling resistance Partial resistance Moderate susceptibility High susceptibility Adult plant resistance IRRI: Planning Breeding Programs for Impact Resistance of BB NILs and pyramids to contemporary Xoo from IRBB7 and IRBB21 NIL/ Pyramid Race 1 3 9

10 IR24 S S S S Xa4 R S S R xa5 R R R R xa13 S S S S Xa 21 R R R R

Xa4/xa5 R R R R Xa4/Xa21 R R R R xa5/Xa21 R R R R xa13/Xa21 R M R S Xa4/xa5/xa13/Xa21 R R R R Ogawa et al., 1990; Huang et al., 1995 IRRI: Planning Breeding Programs for Impact

Markers available for BB Xa-genes Distance (cM) References RM144 - Carrillo et al 11 Npb181 1.7 Ma Bo-Jun et al, 1999 xa5 5 RG556 0-1 McCouch et al, 1991 Xa7 6 P5 0 xa13 8 RG136 3.8 Zhang et al, 1996 Xa21 11

pTA248, Kinase domain 0-1, 0 Ronald et al, 1992 Gene Chrom Xa3 11 Xa4 Linked marker IRRI: Planning Breeding Programs for Impact Porter et al. Map of avrXa7 BamHI MS S MS 9d S 9b 9c kb Reaction to IRBB7 9a Predicting durability of R genes 10 BamHI 5 1 kb 4

3 4.2 kb Mutations in avrXa7-fragment of PXO2684 (Race 9c) BamHI BamHI NLS AD 1 kb WT PXO1865(r3) & PXO0314(r9b) nt aa GAA TTC GAA GCC CGC TAC GGA E F E A R Y E MT PXO2684(r9c) nt GAA C TC GAA GCC CGC GGT GGA aa E L E A GR E IRRI: Planning Breeding Programs for Impact How does the pathogen adapt to Xa7? Strain Aggressiveness 4.2 kb Occurrence 9a

Low No Once (94) 9c Low Yes Once (94) 9b Moderate Yes Throughout (93-99) IRRI: Planning Breeding Programs for Impact Mutations in avrXa7 allele ranged from a single base pair change to multiple mutations spread throughout the alleles TTSS secretion signal AvrXa7 APAEWDEVQ PXO0314 . . . . . . . . PXO348 . . . . C . . . PXO441 . . . . C . . . PXO448 . . . . C . . . PXO356 . . . . . . Xoo . . StrainPXO357 . . . . . . . . PXO0314PXO557 . . . . . . . . Homolog . . . . . . PXO348 .A PXO441 PXO448 Repeat region

. . . . . . . . TVAVKYQHIITALP LTEARELRG E . .... ... ........ .. .... .. . .... ... . . . .T. . D . . . R.... . .... ... . . . .T. . D . . . R.... . .... ... . . . .T. . D . . . R.... . ... . . . .T. . D . . G.... R.... . ... Central repeatsGstructure . . . .T. . D . . .... R.... . ... . . . .T. . D . . G.... R.... . . D. . . . .T. . . . . . . . G. . . . .. LZ NLS AAD MTQFEMSRH STVMWEQD . . ... .. ... .. L. ... . . ... .. ... .. . . ...

. . ... .. ... .. . . ... . . ... .. ... .. . . ... . . . . G . . . .. . . . . . . . . . . . G . . . .. . . . . . . . . . . . G . . . .. . . . . . . . . . ..G...N PXO356 PXO357 PXO557 Adaptation of Xoo to Xa7 rice fields may be more complex than just alteration at the avrXa7 allele. avrXa7 mutant allele M1 (25.5) M2 (22.5) M3 (26.5) M4 (25.5) Ponciano et al., 2004 Adaptation of pathogen to host resistance Xa7 is a good gene for breeding programs - due to fitness penalty associated with avrXa7 mutation Prediction of durability - should not be based only on detection of virulent strains - but should include an understanding of the consequences of adaptation Pathogen may overcome the fitness penalty - by accumulating aggressiveness through recombination or mutation IRRI: Planning Breeding Programs for Impact Fitness penalty associated with loss of function of avrXa7 = sufficient to prevent BB epidemics on rice lines with Xa7 6 years later

Virulence of Xoo Population: Calauan Lesion length (cm) 15 10 5 BB7 LL 0 1993 1994 1995 Year 1998 1999 IRRI: Planning Breeding Programs for Impact Linholm et al. R gene pyramids developed through MAS Race NIL/ Pyramid IR24 1 3 9 10 Multiple R genes combined into S S S one line S Xa4 R S S R Pyramids with different combinations

ofRXa4, xa5, xa5 R R Xa7,R xa13, and Xa21 also available Xa7 I R I R Donors for disease R breeding Xa 21 R R R programR Xa4/xa5/Xa7 R R R R Tool to evaluate the predictability Xa4/Xa7/ of R gene durability for R R deployment R R development and of Xa21 cultivars carrying single and multiple genes xa5/Xa7/Xa21 R R R R Xa4/xa5/Xa7/ xa13/Xa21 R R R R Multiple R genes combined into 1 line Pyramids with different combinations of Xa4, xa5,

Xa7, xa13, and Xa21 also available Donors for disease R breeding program Tool to evaluate predictability R gene durability for development & deployment of cultivars carrying single & multiple genes IRRI: Planning Breeding Programs for Impact Do rice lines containing combinations of R genes confer more resistance and are more durable than rice lines with single R gene? 60 50 Xa4/5/7 IRBB4 IR24 IRBB7 Xa4/7/21 IRBB21 Xa4/7/5/21 Sta. Cruz 40 % Diseaed Leaf Area 30 20 10 0 10-03-02 6.0 5.0 10-09-02 10-17-02 Calauan 4.0

3.0 East 2.0 1.0 0.0 10-29-02 11-05-02 11-13-02 Scoring Date 11-21-02 Linholm et al. Application in breeding programs Via integration of pathogen population analysis & microbial genetics + efficient plant breeding towards a sustainable manipulation of host resistance in disease control IRRI: Planning Breeding Programs for Impact Resistance of Classical Elite Lines to Xoo 30 25 20 IR24 Lesion length (cm) 15 Elite lines 10 0 4 lines 5 0 PXO61

PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 30 25 20 IRBB4 15 10 5 0 Elite lines PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 Xa4 289 lines PXO546 PXO341

30 25 20 IRBB4 15 Elite lines 10 5 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 Bacterial blight races IRRI: Planning Breeding Programs for Impact Xa + ? 67 lines Resistance of Classical Elite Lines to Xoo 30 Lesion length (cm) 25 20 BB52 15 Elite Lines 10 X4/Xa21 6 lines 5 0

PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 30 xa5/Xa21 25 20 BB54 15 Elite Lines 10 5 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 Bacterial blight races IRRI: Planning Breeding Programs for Impact 1 line

Pedigree analysis (ICIS) Xa4 IR747 (TKM6) xa5 IR1545-339 Xa7 IRBB7 Xa21 O. barthii (O. barthii is the progenitor of O. longistaminata and the O. barthii allele is synonymous to Xa21) IRRI: Planning Breeding Programs for Impact Reaction of selected NPTs to Xoo Lines with Xa4/xa5/Xa7/xa13 Lesion length (cm) IR 72225-20-3-2-3 30 25 20 15 10 5 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280

PXO339 PXO546 IR 72976-AC 1 30 25 20 15 10 5 0 PXO61 PXO79 PXO71 PXO99 PXO280 Bacterial blight races PXO546 PX0341 Reaction of selected NPTs to Xoo IR 73449-24-3-2-2 30 Lesion length (cm) 25 20 15 10 5 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PX0341 Xa4/Xa7/xa13 IR 71693-197-4-4 30 25 20 15 10 5 0 PXO61

PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PX0341 Xa4/xa5/Xa7 Reaction of selected NPTs to 10 Xoo races and their genotypes Line Genotype Reaction to Xoo Genetic Background IR44962, Shen Nung 89366,Ketan Lumbu, Sengkeu IR 72164 - 348-6-2-2-2 Xa4 RSSSRRSRRSSR IR 70559-AC 5 Xa4 SSRRSSSSSRSS IR 71698-193-3-2-1 Xa4 + ? RSSSRRSRRRSR Shen Nung 89 - 366, Bali Ontjer, IR64 IR76905-8-1 Xa4/Xa7 RRRRRSSRRRRR Shen Nung 89-366, Ketan Lumbu, IRBB59 IR 69125-35-3-1-1 Xa4/Xa7 SSRSSSSSSRSR Shen Nung 89-366, Ketan Lumbu, Gundil Kuning IR76907-12-20

xa5/Xa7 SSRSSSSSSRSR Shen Nung 89- 366, Genjah Wangkal, IRBB59 IR76909-15-1 Xa7 Shen Nung 89 366, Genjah Wangkal, IRBB59 SSRSSSSSSRSS Shen Nung 89 - 366, Jimbrug, Ketan Lumbu Reaction of tropical japonica cultivars to Xoo Lesion length (cm) NOGO-BELE Xa4/Xa7 50 40 30 20 10 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 RIBON xa5/Xa7 50 40 30

20 10 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 KETAN LUMBU Xa4/xa5/Xa7 50 40 30 20 10 0 PXO61 PXO86 PXO79 PXO340 PXO71 PXO112 PXO99 PXO145 PXO280 PXO339 PXO546 PXO341 Bacterial blight races Possible mechanisms for variations 1. Gene expression 2. Allelic diversity among LRR-domains of R genes Xa21 gene family consist of 6 genes Xa21D has same spectrum of resistance but confer partial resistance only (Wang et al., 1998) IRRI: Planning Breeding Programs for Impact Possible mechanisms for variations 3. Modifier genes

Modifier gene - a gene that modifies the phenotype of another gene (Weaver et al., 1992) Arabidopsis RPS2 function in Col-1 variety but not in Po-1 Po-1 has RPS2 that function in other genetic background (Banerjee et al., 2001) 4. Quantitative traits Traits usually affected by many genes and many environmental factors IRRI: Planning Breeding Programs for Impact IR 67017-13-3-3 Basmatiderived lines Season 1 IR 71730-51-2 Basmati 370 x IRBB60 (Xa4, xa5,xa13, Xa21) Season 2 F1 Season 3 F2 MAS and phenotype Season 4 F3 Phenotype Season 5

F4 Phenotype Season 6 F5 MAS and phenotype Season 7 F6 Phenotype Season 8 F7 MAS and phenotype Season 9 F8 Aroma evaluation, 2AP tests and QTL analysis F5 Basmati-derived line (IR71730-51-2 x IRBB60) carrying Xa4, xa5, xa13 and Xa21 using MAS IRRI: Planning Breeding Programs for Impact Begum, Virk, et al. Basmati-derived lines carrying two to four combinations of BB R-genes with and without fgr gene for aroma using MAS Line A55225 A55241 A55231 A55232 A55236 A55244 A55245 A55246 A55247 Pedigree IR77542-40-3-3-2-1

IR77542-237-2-2-1-1 IR77542-198-1-2-3-1 IR77542-198-1-2-3-2 IR77542-220-2-2-3-3 IR77542-270-3-2-1-1 IR77542-270-3-2-1-2 IR77542-270-3-2-1-3 IR77542-284-1-2-2-1 Xa4 RR RR RR RR RR RR RR RR RR xa5 xa13 Xa21 rr RR RR rr RR RR RR rr RR RR rr RR RR RR RR RR RR RR RR RR RR RR RR RR rr rr RR fgr + + + + + - Begum, Virk, et al. IRRI: Planning Breeding Programs for Impact Asian Rice Biotechnology Network Cultivar development incorporating BB R

genes using MarkerAided Selection G e n e P y r a m id s Xa4, xa5, xa13, Xa21 + Xa7 IR 6 4 (x a 5 , X a 7 , X a 2 1 ) RIFCB Indonesia Released in 2002 Angke (Bio-1) = IR64 (Xa4+xa5) Conde (Bio-2) = IR64 (Xa4+Xa7) IR 6 4 , H y b rid r ic e lin e s (X a 4 ,x a 5 ,X a 7 ,X a 2 1 ) PR 106 S w a r n a , IR 6 4 (X a 4 ,x a 5 ,x a 1 3 ,X a 2 1 ) (X a 4 ,x a 5 ,x a 1 3 ,X a 2 1 ) PhilRice Philippines PAU India Released stop gap var AR32-19-3-3 AR32-19-3-4 = IR64 (Xa4+xa5+Xa21) IRRI: Planning Breeding Programs for Impact CRRI India IR64(Xa4+xa5) Bio-1 1 kb IRBB24 IR64 IRBB7 IRBB5 IRBB5 CBB7 S1033

IRBB7 IR24 IR64 Angke 1 kb IR64 Development of IR64 MAS elite lines with BB R-genes in Indonesia, CRIFC, 1999 R/ R/ S R RR R S R R S S S IR64 IR64 (Xa4+Xa7) Bio-2 IRRI: Planning Breeding Programs for Impact Disease Incide(% ) IR64 MAS elite lines with bacterial blight R-genes released in Indonesia in 2002, CRIFC & RIFCB Angk e 80 70 60 50 40 30 20 10 Cianjur, West Java, 2001 - IR64+xa5 (Bio-1) Bustamam et al. Conde IRRI: Planning Breeding Programs for Impact

IR64+Xa7 (Bio-2) Asian Rice Biotechnology Network MAS-improved pyramided IR64 with xa5, Xa7 and Xa21 IRRI: Planning Breeding Programs for Impact Asian Rice Biotechnology Network Marker-aided selection (MAS)-improved varieties developed by NARES teams from Philippines, Indonesia, India and China, 2002-2003 Country Background commercial/ Yield standard Released (R) / Near-release (NR) + Introgressed gene(s) Philippines IR64 IR64 IR64 BPI Ri10 BPI Ri10 PSB Rc28 IR64 IR64 IR64 PR106 PR106 PR106 Zhong 9A/Zhonghui 218 II-3A/Zhonghui 218 Shanyou 46 Indonesia India China Yield (t/ha) Gain over yield std (%) AR32-19-3-2 (xa5/Xa21)(NR) AR32-19-3-3 (xa5, Xa21) (NR)

AR32-19-3-4 (xa5/Xa21)(NR) AR32-4-3-1 (xa5/Xa21) (NR) AR32-4-58-2 (xa5/Xa21) (NR) Yield standard Angke (Bio-1) (Xa4/xa5) (R) Conde (Bio-2) (Xa4/Xa7) (R) Yield standard (Xa4) IET17948 (xa5/xa13/Xa21) (NR) IET17949 (xa5/xa13/Xa21) (NR) Yield standard Hybrid Guofeng No. 2 (Xa21) (HR, NR) 5.1 6.7 6.1 6.0 6.5 5.1 5.4 5.4 4.5 8.2 7.9 6.7 7.8 0 31.4 19.6 17.6 27.5 20.0 20.0 22.4 17.9 11.4 Hybrid II You 218 (Xa21) (HR, R) Yield standard 8.3 7.0 18.6 - IRRI: Planning Breeding Programs for Impact Can anyone share how bacterial blight is being treated in their breeding program? IRRI: Planning Breeding Programs for Impact

Rice Blast Pyricularia oryzae Pyricularia grisea (anamorph) Magnaporthe grisea (teleomorph) Yield losses up to 50-85% reported Leaf blast Collar blast IRRI: Planning Breeding Programs for Impact Neck blast Node blast Blast infection structures Sporulating lesion Conidia IRRI: Planning Breeding Programs for Impact Structure of blast pathogen populations in three different ecologies in India ALMORA (hill region) HAZARIBAG (rainfed upland) HYDERABAD (irrigated) IRRI: Planning Breeding Programs for Impact Requirements in breeding for resistance to rice blast Diverse resistant sources Systematic evaluation schemes Suitable test environments IRRI: Planning Breeding Programs for Impact

Blast Nursery layout spreader rows test materials spreader rows IRRI: Planning Breeding Programs for Impact Scoring system for blast Scale Description 1 Small brown specks of pin-point size 2 Small roundish to slightly elongated, necrotic gray spots, about 1-2 mm in diameter, with a distinct brown margin. Lesions are mostly found on lower leaves 3 Lesion type is the same as in 2,but significant number of lesion are on upper leaves 4 Typical susceptible blast lesions, 3 mm or longer, infecting less than 4% of leaf area 5 Typical susceptible blast lesions, 3 mm or longer, infecting less than 4-10% of leaf area 6 Typical susceptible blast lesions, 3 mm or longer, infecting less than 11-25% of leaf area 7 Typical susceptible blast lesions, 3 mm or longer, infecting less than 26-50% of leaf area 8 Typical susceptible blast lesions, 3 mm or longer, infecting less than 51-75% of leaf area, many leaves dead 9 Typical susceptible blast lesions, 3 mm or longer, infecting more than 75% of the leaf area IRRI: Planning Breeding Programs for Impact

Blast R genes and their chromosomal locations in rice Locus Pi-a Pi-b (pi-s_ Pi-f Pi-I Pi-k (Pi-k, Pi-km, Pi-kk, Pikp) Pi-ta (=sl) M-Pi-z Pi-z Pi-is-I (Rb-4) Pi-se-1 (Rb-1) Pi(t) Pi-?(t) Pi-1(t) Pi-2(t) Pi-3(t) Pi-4(t) Pi-5(t) Pi-6(t) Pi-7(t) Pi-zh(t) Phenotype / product Chromosome P.o. resistance-a P.o. resistance-b P.o. resistance-f P.o. resistance-i P.o. resistance-k 11 2 11 6 11 P.o. resistance-ta P.o. resistance-z P.o resistance-z P.o. resistance-is P.o. resistance-se P.o. resistance P.o. resistance P.o. resistance-1 P.o. resistance-2 P.o. resistance-3 P.o. resistance-4 P.o. resistance-5

P.o. resistance-6 P.o. resistance-7 P.o. resistance-zh 9 or 12? 11 6 11 11 4 4 11 6 6 12 4 12 11 8 (McCouch et al., 1994) Monogenic lines developed for blast resistance (Y. Fukuta) Entry No. Target Designation Gene Gen. Donors IRBL 1 IRBLa-A Pia BC1F14 AICHI ASAHI 2 IRBLa-C Pia BC1F14

CO 39 3 IRBLi-F5 Pii BC1F14 FUJISAKA 5 4 IRBLks-F5 Pik-s BC1F14 FUJISAKA 5 5 IRBLks-S Pik-s BC1F14 SHIN 2 6 IRBLk-ka Pik BC1F13 KANTO 51 7 IRBLkp-K60 Pik-p BC1F12 K 60

8 IRBLkh-K3 Pik-h BC1F9 K3 9 IRBLz-Fu Piz BC1F14 FUKUNISHIKI 10 IRBLz5-CA Piz5 BC3F12 C101A51 11 IRBLzt-T Piz-t BC1F14 TORIDE 1 12 IRBLta-K1 Pita BC2F12 K1 13 IRBLta-CT2

Pita BC3F12 C105TTP2L9 14 IRBLb-B Pib BC1F9 BL 1 15 IRBLt-K59 Pit BC2F9 K 59 Similar gene name = Pi 2(t) = Pi 4(t) Contd Entry No. Designation Target Gene Gen. Donors 18 IRBL1-CL Pi1

BC3F12 C101LAC 19 IRBL3-CP4 Pi3 BC2F12 C104PKT 20 IRBL5-M Pi5(t) BC3F12 RIL 249 (Moro.) 21 IRBL7-M Pi7(t) BC3F12 RIL 29 (Moro.) 22 IRBL9-W Pi9 BC3F12 WHD-1S-75-1-127 23 IRBL12-M Pi12(t) BC2F12 RIL 10

24 IRBL19-A Pi19 BC1F11 AICHI ASAHI 25 IRBLkm-Ts Pik-m BC1F10 TSUYUAKE 26 IRBL20-IR24 Pi20 BC1F10 ARL 24 27 IRBLta2-Pi Pita2 BC1F8 Pi No. 4 28 IRBLta2-Re Pita2 BC1F10 REIHO 29

IRBLta-CP1 Pita BC5F10 C101PKT 30 IRBL11-Zh Pi11(t) BC2F12 ZHAIYEQING 31 IRBLz5-CA(R) Piz5 BC5F10 C101A51 LTH Similar gene name Markers available for blast Pi- genes R-gene Chromosome tagged Linked marker Distance (cM) 11 r10 - Pi2 6

RG64 P to kinase 2.8 - Pi9 6 RG16 - Pi1 IRRI: Planning Breeding Programs for Impact Development of blast resistant rice cultivars by Asian Rice Biotechnology Network via MAS RR ABC D E F G H I J K L MNO P Q 400 300 400 300 200 100 15 0 ss I II III Pto-Kinase motif Pi 1 Pi 2 Kalinga III CR203 KDML 105 WayRarem CRRI AGI DOA CRIFC

India VietnamThailandIndonesia HR versus VR Resistant Maritta Higher HR Susceptible Kennebec Resistant Susceptible Blight (P. infestans) races (Van der Plank, 1963) Rice-M. grisea rice interactions H2O2 accumulation 24 h resistant 48 h susceptible IRRI: Planning Breeding Programs for Impact M. Yang Will major R genes work for blast? High degree of pathogen variability Even though some good genes (e.g., Pi-2/Pi-1 combination), major R genes alone too risky Preferred strategies: strong layer of quantitative resistance Add quality major genes on top Diversify the use of resistance IRRI: Planning Breeding Programs for Impact Candidate genes Definition: DNA sequences that likely correspond to a specific trait based on a known biochemical pathway or DNA similarity to other functional genes Approaches: Relate sequences to known mapped phenotypes Relate sequences to mutations Associate sequences to phenotypes of germplasm and breeding pedigrees

IRRI: Planning Breeding Programs for Impact Candidate gene analysis of quantitative disease resistance in wheat Disease Candidate gene Chromosome Phenotypic effect (%) Tan spot Oxalate oxidase, ion channel regulator 1A 58 Leaf rust Peroxidase 2B DR gene clusters: catalase, thaumatin, chitinase 2BS 31 Powdery mildew, karnal bunt, stem rust Oxalate oxidase, thaumatin, chalcone synthase, chitinase Minor QTL Faris et al., 1999 TAG 98:219-225 IRRI: Planning Breeding Programs for Impact Candidate genes, clone designation, source & predicted function or pathways used in study (partial list) (http://www.ksu.edu/ksudgc) Gene categories Accession

number NBS-LRR AF 032688- Clone designation R1-R15 Predicted function or pathway Plant source Resistance gene analog Rice RP1 a-d Resistance gene analog Maize AF 032702 rNBS 1-69 Rp1 Peroxidase AF014467 POX22.3 Multiple Rice Aldose reductase X 57526 pg2269 Phenylpropanoid pathway Barley Dihydrofolate reductase AF 013488 ZmDRTs

Amino acid metabolism Maize Oxalate oxidase Y 14203 PHvOXOa Generation of active oxygen species Barley Oxalate oxidase-like X 93171 pBH6-903 Generation of active oxygen species Barley hsp-70-like mRRI11 mRRI11 PR Protein Maize Hv14-3-3a X 62388 pHv1433a PR Protein Barley Chitinase Type ll X 78671 HvCht2a PR Protein

Barley IRRI: Planning Breeding Programs for Impact 2 3 143.9 cM 204.5 cM XLRin12A1 RG437 XLRfrI7 PK1K2I1 RG544 b9 RG171, NLRin12I3 RG157 ChitinaseII Hv1433c PalI RZ318 XLRin12I2 XLRin12A5 RZ58 CDO686 Amy1AC RG95 RG654 RG256 XLRfrI5 RZ123 RZ213 Pi-b RG520 8 7 118.5 cM 163.1 cM RG104 RG348 RZ329 RZ892 RG100 RG191 RZ678

S1AS1A3 A18A1120 RG511 A5J560 TGMS12 A10K250 Pi-11(t) AG8-Aro RZ617 RG978 XLRfrI1 rNBS53 rNBS52 rNBS28 RG1 S1AS1I2 Amy3DE RG773 RZ488 XLRin12I1 RG477 RZ284 RZ394 PK1K2A3 r9 rNBS37 rNBS17 RG179 RZ403 pRD10A Oxalate oxidase CDO337 RZ519 RZ448 S2AS3I2 Pgi1 CDO87 RG910 PLD5 rNBS61 RG418A 153.9 cM RG769

Thaumatin1 RZ574 11 Oxalate Oxidase-Like NLRin12I2 NLRin12I5 PGMS07 PK1K2A1 XLRin12A6 S2AS3A3 rNBS23 r7 S2AS3I4 RZ66 AC5 RG418B Amp2 rNBS35 rNBS36 CDO59 RG711 CDO99 Est9 Pi-17(t) RZ337B QTL for disease resistance rNBS54 PK1K2I5 Putative for BB resistance CDO497 CDO418 Blast DLA Peroxidase POX22.3 RZ978 CDO38 RG351 Blast lesion number Blast lesion size 12 M-Pi z Pi-se-1 Pi-is-1 CDO127 RZ638

114.9 cM RZ816 RZ400 RG118 Adh1 S2AS3A1 rNBS8 S2AS3I1 RG1094 r6b Pi-a RG247 Npb44 XLRin12A4 RG167 NLRfrA2 ZmDRTSc Pi-7(t) r11 r4 r12 r6a Xa21 Rp1d Rp1e RG103 rNBS10 ZmDRTSd Xa10 ZmDRTSe RG1109 rNBS55 r2, r3, r5, r10 Npb186 Xa4 rNBS38 Xa3 OS-JAMyb Pi-1 RZ536 XLRfrA6 Pi-k, Pi-f BBphen DH map (IR64 Sheath blight resistance x Azucena) Neck blast resistance RG574 NLRfrA6 S2AS3A2 Pi-6(t)

RG341 rNBS63b Pi-ta PK1K2A2 AF6 ZmDRTSb S2AS3A5 PK1K2I4 rNBS14 RG457 Sdh1 Bph9 bph2 Bph1 mRGH CDO344 RG901 RG463 RG958 XLRfrI2 RG181 PCR markers: R gene analogs RFLP markers: R gene analogs RFLP markers: Defense response genes Ramalingam et al., MPMI SHZ2 LTH Four Steps to move useful alleles from a durably-blast resistant variety SHZ-2 to a popular variety 0 . 0 11 Chr 2 b2(NBSr4(NBSLRR) RG634 LRR) .1 1. Identify donor with non-racespecific resistance

35 4 XLRfr-6 RM3 .09. . RM 1 XLRfr-5 4 341 1. 24 XLRfr-11 4. 0 1.1 14-3-3 7.37 protein-A Chitinase 13. RG 2a-A 5 3. RM 13 5 26 3 9 XLRfr 1.7 0. 3 XLRfr-15 41.2 16 RM2 08 Chr 7 PK1K2 -6 Chr RZ143 8 21.4 Chr 10 RM333 XLRfr-13 XLRfr-10

6.7 2.3 15.5 9.7 XLRfrRG21 9.0 7.2 184-A 3.7 7.9 43. 4.3 PK1K2 r14-A (NBS17.1 RG5 12.4 LRR) -12 6 22.3 11 9.5 XLRfr3.5 14.5 0.3 XLRfr 0.0 12 PK1K2-53.3 5.2 Oxalate XLRfr-211.3 NLRin 5.0 RG3 6.3 3.8 RG678 -8 Pi-GD3.8 2.8 oxidase 2.6 3.5 6.5 0 v-6 12.6 1(t) PK1K2-8 0.3 PK1K 1.9 XLRfr-14 Chitinase 3.3 4.5 4.1

RG97 RG1034 18.6 2-7 2a-B 8 19.9 RG1 4.2 18.2 Dehydrin 17.9 RG6 37.8 1.2 50NLRinv-8 1.4 11.9 PK1K2 1.5 0.7 -16 0.0 2.8 RG5 Peroxidase 6.2 98 19.1 3.9 7.7 RZ811 CDO98 RZ625 RZ400 Chr 12 RGH CDO459 RM19-A 12.2 26.5 4.2 0.0 7.9 15.2 12.0 RZ PR-1 PK1K2-2 r6-C (NBS-LRR) 892 4.5

RM216 PK1K2-17 0.2.4 r8 (NBSXLRfr-17 8.6 5 LRR) XLRfr-19 4.8 23.8 (RM222) XLRfr-21 0. 5 XLRfr-20 18.9 r7 (NBS-LRR) NLRinv-1 3.3 r6-A (NBS-LRR) 2.7 b4-A (NBS-LRR) 5.9 NLRinv-3 7.1 Chitinase r16 (NBS-LRR) Pi-GD-2(t) 2b r14-B (NBS-LRR) 6.1 RG235 RM19-C RG574A RM247 RG RZ397 81 Rp1CRG869 RM179 XLRf Pi-GD-3(t) r-9 NLRinv-5 RM277 PK1K2-13 RM260 RZ RG413 76 RM313 3. Candidate defense genes associated with QTL 20 18 16

Similarity No. of RI lines 14 12 A 10 8 6 B C 4 2 0 1 3 8 13 18 23 28 33 38 43 48 % Diseased leaf area 2. Assay quantitative resistance without major R genes Liu et al. 2004 Mol Plant-Microbe Interact 4. Candidate gene-aided backcrossing and validation in the field

Five defense related (DR) genes associated with blast resistance QTL in SHZ-2 X LTH recombinant inbred lines Phenotypic effects in disease nurseries in 3 locations DR Gene Chr. Guangdong IRRI Cavinti, Phil. Chitinase 2a 2 6.7* 5.0* 5.0* 14-3-3 protein 2 7.1* 5.0* 4.0* Dehydrin (Esi 18.5) 7 14.9**** 25.6* 16.7* Oxalete oxidase 8 41.2**** 12.1*

27.0* PR-1 10 13.8**** 10.7* 16.9* IRRI: Planning Breeding Programs for Impact Liu Bin et al. 2004 MPMI Candidate defense response (DR) genes contribute to quantitative resistance against rice blast 100 More DR genes Less disease 60 20 0 0 1 2 3 4 Number 5 of Defens e Respon Genes in se 101 RI Li nes GD-GH IRRI-BN GD-BN IRRI-BN Ca-BN IRRI: Planning Breeding Programs for Impact

ca t io n 40 Lo % DLA 80 Liu et al., 2004, MPMI Advanced backcross lines selected by candidate genes: broad-spectrum quantitative resistance to blast disease SHZ Susceptible recurrent parent Texianzhan-13, 90% neck blast donor Dendrogram from SSR fingerprints BC3 lines Elite backcross line (#116) Favorable alleles at five candidate defense gene loci (chitinase, PR-1,oxalate oxidase, dehydrin, 14-3-3 protein) Quantitative resistance to leaf and neck blast disease High quality rice Resistant BC3 -line (#116) Bin Liu et al. Vandana x Moroberekan: Putative QTL for blast partial resistance to PO6-6 in BC3F3 lines Trait DLA (%)

LN LS Marker Candidate Gene Source R2 P RGA8-4 NBS-LRR Flax 11.79 9.36 0.0030 RM215 SSR Rice 9.19 7.09 0.0096 CG10d Oxalate oxidase Barley 28.65 27.7 0.0001 CG17

Hv1433 Barley 14.07 11.3 0.0013 RGA1-10 LRR Rice 9.39 7.25 0.0089 RM21 SSR Rice 9.09 6.90 0.0100 RM168 SSR Rice 10.73 8.42 0.0050 RM250 SSR Rice

9.55 7.39 0.0082 TV (%) (%) DLA = % Diseased Leaf Area, LN = Lesion Number, LS = Lesion Size b Total variation explained by the traits c The model included 7 markers at P = 0.05 a F 59.49 34.54 J. Wu et al., 2004 Distribution of % DLA(A), lesion number(B) and lesion size(C) in the VxM BC3F3 lines inoculated with PO 6-6 A 2 4 6 8 10 12 14 16 % Diseased Leaf Area

20 B No. of lines 15 10 5 No. of lines No. of lines 30 25 20 15 10 5 0 20 15 C 10 5 0 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 0 1 2 3 4 5 6 7 Number of lesion size

8 9 10 Susceptible lesion size IRRI: Planning Breeding Programs for Impact 5 % DLA 100 90 80 70 60 50 40 30 20 10 0 VM34 CO39 VM37 VM32 Vandana VM46 VM76 Progress of percent DLA in selected lines of BC3F3 in blast nursery VM15 VM14 14 16 19 22 25

Moro 28 Relationship between % DLA of BC3F3 VxM lines in GH (PO6-6) & Blast nursery % DLA in GH (PO6-6) Days after sowing 16 14 12 10 8 6 4 2 0 0 20 40 60 80 % DLA in blast nursery 100 Phenotypic selection of BC3F5 Vandana x Moroberekan, IRRI & Cavinti Line SRS S S S P S PRRS PR SR BC3F4 lines, IRRI Blast Nursery *In India, have partial R across screening sites; V4M-5-3-B has good phenotypic acceptability M. Variar Neckblast** Seedling blast* (% Incidence) (%DLA) Blast

GH Cavinti Nursery (PO6-6) V4M-5-3-B* V4M-6-1-B* V4M-10-1-B V4M-14-1-B V4M-15-3-B V4M-19-1-B V4M-42-2-B V4M-52-2-B V4M-53-1-B V4M-60-2-B V4M-63-1-B V4M-70-1-B V4M-74-1-B V4M-75-1-B V4M-82-2-B V4M-83-2-B 3.7 3.1 6.1 0.7 1.0 1.4 0.7 9.2 1.2 1.8 9.6 5.6 8.4 8.1 8.5 9.2 * BC3F4 gen 21.9 13.9 53.9 4.2 9.2 17.2 6.4 30.9 15.5 25.3 47.8 31.1

31.5 36.6 17.0 35.9 33.0 26.0 67.0 4.8 36.8 52.4 28.6 20.0 42.9 42.9 30.0 71.4 76.2 47.6 23.8 19.0 ** BC3F5 gen Phenotypic selection of BC3F5 Vandana x Moroberekan, IRRI & Cavinti Selected F4 lines from intermated BC3F5 V x M lines, WS 2003 F2 F4 SRS S S S P S P RRS P S R BC3F5 intermated V x M partially R lines IRRI: Planning Breeding Programs for Impact Moroberekan Seedling blast severity of selected 60 F4 lines of BC3F5 Vandana x Moroberekan lines in Almora, India and Cavinti, Phil. Cavinti (SES, 0-9) 9 6 3

0 0 3 6 Almora (SES, 0-9) IRRI: Planning Breeding Programs for Impact 9 Identify consensus candidate genes Upland rice Vandana x Moroberekan: blast Irrigated rice SHZ-2: blast (Wu et al., 2004,TAG) (Liu et al., 2004, MPMI) NBS-LRR (rice, barley) NBS-LRR (rice) Aldose reductase NBS-LRR (maize, barley) Oxalate Oxidase Dihydrofolate reductasethymidylate synthase DH Rice IR64 x Azucena: BB, blast, ShB (Ramalingam et al., 2003, MPMI) Peroxidase Chitinase Ion channel Regulator PR1 Thaumatin Catalase SOD PAL

Wheat: tan spot and/or leaf rust (Faris et al. 1999, TAG) 1st generation stress response array containing rice and maize cDNA clones 350 X. oryzae-induced cDNAs (J. Leach, KSU) 100 rice NBS-LRR sequences (JEL, KSU) 215 blast- or JA-induced cDNAs (Yinong Yang, U. Arkansas) 460 cDNAs from maize suppression subtractive hybridization libraries (Scot Hulbert, KSU) Control sequences from rice and human Microarray hybridization, scanning, & analysis IRRI: Planning Breeding Programs for Impact Gene Candidate genes potentially involved in disease resistance Bioprocess Functional evidence Oxalate oxidase/germin like protein Aspartyl protease (Esi-18) 14-3-3 PR-1 Oxidative burst, signaling, structure Early salt-induced Signal cascade Stress response PBZ (PR10a) Could have ribonuclease activity Gene cluster as a disease resistance QTL

As disease resistance QTL As disease resistance QTL As disease resistance QTL Pathogen induced defense gene Rice peroxidase 22.3 Oxidation of organic and inorganic substrates at the expense of H2O2 Pathogen induced defense gene Heat shock protein 90 Stress response Pathogen induced defense gene Putative 2-dehydro-3deoxyphosphoheptonate aldolase Aromatic amino acid synthesis Pathogen induced defense gene Thaumatin-like pathogenesis related protein Glyoxalase 1(Oryza sativa) S-adenosyl L-homocystein hydrolase Binding to D-glucans of the type commonly found in fungal cell walls Detoxification of the cytotoxic metabolite methylglyoxal that can be produced by increased levels of glycolysis under conditions of stress Cytokinin-binding protein CBP57 (cytokinin-mediated signal transduction) Stress response Stress response

Pathogen induced defense gene Genome scan of Vandana/Moroberekan intercross progenies using SSRs Oxalate oxidaselike proteins Eukaryotic aspartyl protease Putative oxalate oxidases Chromosome 3 Chromosome 7 Chromosome 8 Legend: 1= Moroberekan; 2= Vandana; 3= IR78221-19-6-56; 4= IR78222-20-7148; 5= IR78222-20-1A-7; 6= IR78224-22-2-59; 7= IR78224-22-2-114; 8= IR78222-20-1A-18NB; 9= IR78222-20-2-7NB Heterozygous loci are colored green 3: IR78221-19-6-56 4: IR78222-20-7-1485: IR78222-20-1A-7 6: IR78224-22-2-59 7:IR78224-22-2-114 (R) (R) (S) (HS) (HS) Leaf blast in Almora, India, Sept 2004 Putative oxalate oxidase Chromosome 3 Co39 (S ck) Local check 3: IR78221-19-6-56 4: IR78222-20-7-148 2: Vandana Genome scan of Vandana-Moroberekan wilting intercross progenies using SSRs for oxalate Crop stand in Hazaribag, India under drought condition under 10 d without rain

(10 d no rain). Blast pressure lower than in Almora, oxidases (OsGLPs) Sept 2004 Yield during DS drought condition at IRRI and seedling blast infection at 2 sites of selected 60 intercrossed Vandana x Moroberekan lines, 2004 Cavinti Almora Seedling blast (SES, 0-9) 9 Vandana 6 3 IR78221 19-6-99 IR78221 19-6-56 IR78221 19-6-7 IR78221 19-6-33 IR78221 19-6-90 IR78221 19-6-3 IR78222 20-7-128 0 0 30 60 90 Yield under drought IRRI: Planning Breeding Programs for Impact 120 150 R. Lafitte, E. Javier et al. Chrom 2 Chrom 3 Oxalate oxidase Chitinase MR S S S R S S S S S S S S S S S R S S S S S S S S S S S S S R R R R R R R R R R R S R S R R R R

MR S S S R S S S S S S S S S S S R S S S S S S S S S S S S S R R R R R R R R R R R S R S R R R R Oxalate oxidaselike protein Thaumatin Peroxidase MR S S S R S S S S S S S S S S S R S S S S S S S S S S S S S R R R R R R R R R R R S R S R R R R MR S S S R S S S S S S S S S S S R S S S S S S S S S S S S S R R R R R R R R R R R S R S R R R R Chrom 7 Chrom 8 Candidate gene alleles contrib by Moroberekan (+) in blast resistant F5 V x M intercrosses Candidate Gene IR7822119-6-3-B IR7822119-6-7-B IR78221-196-33-B IR7822119-6-56B IR7822 1-19-690-B IR7822 1-19-699-B IR7822220-7128-B IR7822220-7148-1-B IR7822220-7148-2-B IR7822220-7-1483-B IR7822 2-20-747-B Vandana Chitinase - -

- + - - + + + + - - Oxalate Oxidase + + + + -/+ + - + + + - - Aldose reductase - - -

- - - - - - + - - Thaumatin (Chr6) - - - - - - - - - - - - HSP90 + + -

- - - - - + + - - Thaumatin (Chr7) - - - - - - - - - - - - Eukaryotic aspartyl protease - - -

- - - - - - - - - Deoxyphosphohept onate aldolase - - - - - - - -/+ -/+ - - - Peroxidase - + +

+ + + + - - - + - Oxalate Oxidaselike - + -/+ + + - + + + + - - PR1 - - - -

- - + - + - + - PBZ - - - - - - - - - - - - Seedling blast (Philippines) 1 1 1 1

1 1 1 2 2 2 5 7 Seedling blast ( Almora, India) 4 2 4 3 4 3 4 4 4 4 5 8.5 Evidence for the role of oxalate oxidase in resistance to plant pathogens 3 8 RG104 RG348 Increased oxalate oxidase activity in

barley infected with powdery mildew (Zhou et al., 1995) Induction of germin gene expression in wheat infected with powdery mildew (Hurkman and Tanaka, 1996) Association of oxalate oxidase to partial blast resistance in Vandana x Moroberekan population (Wu et al., 2004) RZ329 RZ892 RG100 RG191 RZ678 RZ574 RZ284 RZ394 PK1K2A3 R9 LP37 LP17 RG179 RZ403 pRD10A Oxalate oxidase CDO337 RZ519 RZ448 S1AS1A3 A18A1120 Oxalate Oxidase-Like A5J560 TGMS12 A10K250 Pi 11(t) AG8-Aro RZ617 RG978 XLRfrI1 LP53 LP52 LP28 RG1 S1AS1I2 Amy3DE S2AS3I4 RZ66

AC5 RG418B Amp2 LP35 CDO99 S2AS3I2 Pgi1 CDO87 RG910 PLD5 LP61 RG418A Ramalingam et al, 2003 Identifying oxalate oxidases in rice The TIGR Whole Rice Genome Annotation DB was searched for sequences similar to barley mRNA sequence for oxalate oxidase Y14203 All the sequences related to oxalate oxidase (e.g. germin-like proteins, cupins) were extracted from the database A phylogenetic tree of rice oxalate oxidase sequences was constructed using ClustalX IRRI: Planning Breeding Programs for Impact Spherulin1a|AAA29982.1| Spherulin1b|P09351| 1000 OsGLP36 OsGLP16 1000 OsGLP35 799 1000 454 660 546 996 996 526 759

1000 729 OsGLP03 OsGLP14 OsGLP15 OsGLP10 1000 OsGLP12 999 OsGLP11 865 OsGLP13 OsGLP01 OsGLP18 OsGLP02 OsGLP05 OsGLP04 OsGLP09 OsGLP17 OsGLP06 998 981 1000 840 Spherulin1a|AAA29982.1| 998 Germin-like proteins in plants Ryegras|CAD43309.1| Hv|Oxox|CAA74595.1 wheat|CAD89357.1| 588 976 751 1000 OsGLP10 1000 OsGLP13 OsGLP12

937 1000 At|BAB10832.1| 1000 At|AAM98218.1| 712 999 Hv|Oxox-like|CAA63659.1 1000 1000 0.1 0.1 991 705 OsGLP11 997 999 1000 OsGLP07 OsGLP08 OsGLP37 529 OsGLP40 1000 OsGLP38 808 OsGLP39 OsGLP33 OsGLP21 1000 1000 OsGLP25 947 OsGLP22 1000 OsGLP26 OsGLP19

OsGLP20 OsGLP24 1000 OsGLP34 OsGLP23 1000 OsGLP27 938 OsGLP28 759 OsGLP31 643 OsGLP32 541 OsGLP29 359 OsGLP30 adenosine diphosphate glucose pyrophosphatase Putative Nectarin1 precursor Transposon insertion Chr 8 OsGLP OsGLP28 OsGLP29 OsGLP17Indica|5852087|emb|CAB55394.1| OsGLP21 Wheat|6996619|gb|AAF34811.1|AF Phylogenetic relationships of rice germin-like proteins (GLP). Alignment of protein sequences and phylogenetic analyses were done using ClustalX. The tree was rooted with spherulin1A (AAA29982) and spherulin1b (P09351). Pairwise alignment of OsGLP11 vs OsGLP13 OsGLP11 MEHSFKTITAGVVFVVLLLQQAPVLIRATDADPLQDFCVADLDSKVTVNGHACKPASAAG OsGLP13 MEHSFKTIAAGVVIVVLLLQQAPVLIRATDADPLQDFCVADLDSKVTVNGHACKPASAAG OsGLP11 DEFLFSSKIATGGDVNANPNGSNVTELDVAEWPGVNTLGVSMNRVDFAPGGTNPPHVHPR OsGLP13 DEFLFSSKIATGGDVNANPNGSNVTELDVAEWPGVNTLGVSMNRVDFAPGGTNPPHVHPR OsGLP11 ATEVGIVLRGELLVGIIGTLDMGNRYYSKVVRAGETFVIPRGLMHFQFNVGKTEATMVVS OsGLP13 ATEVGIVLRGELLVGIIGTLDTGNRYYSKVVRAGETFVIPRGLMHFQFNVGKTEATMVVS Cis-acting

elements in OsGLP11 and OsGLP13 FNSQNPGIVFVPLTLFGSNPPIPTPVLVKALRVDTGVVELLKSKFTGGY OsGLP11 OsGLP13 FNSQNPGIVFVPLTLFGSNPPIPTPVLVKALRVDAGVVELLKSKFTGGY Cis element OsGLP11 OsGLP 13 ASF1MOTIFCAMV 1 0 WBOXATNPR1 1 1 WBOXHVISO1 3 0 IRRI: Planning Breeding Programs for Impact Detailed marker analysis of chromosome 3 loci with four putative oxalate oxidases (OsGLPs) PCR primers designed from the coding region of each gene. OsGLP10UP, OsGLP11UP, and OsGLP13UP primers designed from the 1000b upstream region of each gene. 1: Moroberekan 2: Vandana 22-2-114 3: IR78221-19-6-56 18NB 4: IR78222-20-7-148 5: IR78222-20-1A-7 OsGLPs 1 Chromosome 3

2 6: IR78224-22-2-59 7: IR782248: IR78222-20-1A9: IR78222-20-2-7NB Heterozygous loci Monomorphic markers 3 4 5 6 7 8 9 Almora, India: R S R R S HS HS - Cavinti, Phil : R R R R R R R S S Glazebrook, 1999, Current Opinion in Plant Biology, 2:280-286 Lesion mimic genes Ethylene JA HR SA PR genes (e.g. PR1), SAR IRRI: Planning Breeding Programs for Impact Resistance to rice blast and sheath blight of TXZ x SHZ2, CIAT Rice Blast BC Line Sh Blight Lineage 4 Lineage 5

Lineage 6 Isol 1 LT % LT % LT % %PAA 3,4 50 3,4 48 3,2,4 39 30 TXZ/SHZ2 Bc10-46 4 30 4 38 4 59 21 TXZ/SHZ2 Bc10-10 4 19

4,3 12 4,3 37 8 TXZ/SHZ2 Bc116 4 50 4,3 16 4 26 14 Shan Huang Zhan Blast: Lesion Type (1-2 = R, 3 = I, 4 = S); % Lesion Area Affected Sheath Blight: % PAA (% Plant area affected) IRRI: Planning Breeding Programs for Impact Are candidate defense related genes identified in elite germplasm with quantitative R in common with candidate genes shown in advanced breeding lines? Moroberekan or Shan huangzhan (SHZ) with partial R to blast X Backcross to high quality rice Vandana or Way Rarem or Ilpumbyeo BC3 lines in elite background Validate field performance of different candidate gene combinations

VARIETAL RELEASE Extending to other breeding populations BC2F3 lines of Oryzica Llanos 5 crossed to Way Rarem showing blast resistance used to diversify the varieties planted in farmers fields in Indonesia Susceptible lines in the same field were highly diseased in comparison to the BC2F3 lines IRRI: Planning Breeding Programs for Impact Can anyone share how bacterial blight is being treated in their breeding program? Can anyone describe some breeding strategies for diseases where major genes are effective? IRRI: Planning Breeding Programs for Impact Summary . . . By using known genes to predict functional diversity in the pathogen and how the pathogen responds to host genotypes, we were able to predict durability of R genes We are currently field testing combinations of R genes predicted to be durable (Xa7, xa5) and others (Xa4, Xa21, xa13) NARS breeding programs have developed and are beginning to release pyramided for disease IRRI: Planning Breeding Programs forgenes Impact Summary . . . Breeding for disease resistance should be complemented by knowledge of pathogen population structure: (a) allows to identify tester strains for screening breeding lines; (b) prerequisite for any gene deployment strategy Breeding strategies for diseases where major genes are effective, e.g. BB: gene pyramiding, or gene rotation (spatial & temporal deployment)

IRRI: Planning Breeding Programs for Impact Summary . . . We have associated known sequences of candidate genes to phenotypes of germplasm and breeding pedigrees Breeding strategies being adopted to develop varieties with broad spectrum resistance to blast: (a) combining different mechanisms of quantitative R, (b) pyramiding effective major genes Using the available genetic and bioinformatic resources for rice coupled with efficient phenotyping tools, it is possible to relate QTLs to candidate genes and metabolic pathways IRRI: Planning Breeding Programs for Impact Future prospects Further analysis of gene expressions of functional candidate genes (USAID Linkage project) Northern analysis/RT-PCR expression analysis of candidate genes in rice by RNAi Recurrent selection to increase the resistance of the lines to biotic stresses (GenCP) Development of NILs for blast QTL (GenCP) IRRI: Planning Breeding Programs for Impact Future prospects SNP analysis of effective candidate genes in resistant donors (RDA-IRRI) Combining blast resistance and phosphorus tolerance (GenCP, A. Ismail & M. Wissuwa) Combining blast resistance and drought tolerance (R. Lafitte, E. Javier) Technology development for MAS application (G-CP) IRRI: Planning Breeding Programs for Impact Contributors IRRI: I. Oa, M. Reveche, G. Carrillo, J. Wu, B. Liu, S. Begum, N. Sugiyama, R. Mauleon, M. Bernardo, M. Laza, E. Javier, B. Courtois (CIRAD), H. Leung CRURRS & VPKAS, India: M. Variar, J.C. Bhatt, R. Babu RDA: S.S. Han, J. Rho, Y.C. Cho, CRIFC: Suwarno, E. Soenarjo, M. Bustamam Kansas State U: S. Hulbert, J. Bai Colorado State U: J.E. Leach University of Guelph: P.H. Goodwin ARBN Members: PhilRice, ICABGRRD, PAU, CRRI, AGI

NIPP, DOA, CNRRI IRRI: Planning Breeding Programs for Impact

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  • Chapter 3

    Chapter 3

    Arial Calibri ch01_temp Chapter 3: Using Variables and Constants Creating Variables and Named Constants Lesson A Objectives Creating Variables and Named Constants Lesson A Objectives (continued) Previewing the Completed Application Previewing the Completed Application (continued) Using Variables to Store Information...