Progress and expected impacts of Saltol and Pup1 QTLs on rice

improvement

IRRI

GCP Project #2

Revitalizing Marginal Lands: Discovery of Genes for Tolerance of Saline and P-Deficient Soils to Enhance and Sustain Productivity

Project Breeding ObjectivesPrecision mapping of Saltol & Pup1 loci Develop/validate markers for both QTLs & a MAB system for introgressing them into popular varieties

and elite lines

Identify and validate the candidate genes involved at the two loci Functional confirmation and assessment of positive and any negative impacts using NILsStrengthen the capacity of NARES partners in MAB and other relevant technologies

Salinity: Crop type and target RegionsSalinity affect > 21 m ha in S and SE rice areas and considerably reduce productivityTarget areas:

Salt affected coastal areas (coastal Bangladesh) Inland saline and alkaline areas

Salt stress in rice

Germinationtolerant

emergence of radicle

& primary leaf

Seedlingsensitive

Flowering/pollinationsensitive

stem elongationpanicle exertionsensitive

grain ripeningtolerant

Active tilleringtolerant

panicle initiation Sensitive

Tolerance at seedling stage does not

ensure tolerance during reproduction

Phosphorus deficiency: Crop type and target Regions

Phosphorus deficiency affects about 50% of rice areasTarget areas:

Uplands of South and South East Asia (Indonesia)Other P-deficient soils (acid, alkaline, peat soils)Drought-prone areas

Outputs and productsA Marker Aided Breeding system to incorporate major QTLs associated with tolerance Better understanding of tolerance mechanismsEfficient large-scale phenotyping systemTrained NARES capable of applying MAB package in their breedingPopular varieties tolerant to salinity and phosphorus deficiency developed and disseminated in target areas

QTL Ch R2 LOD Interval

Saltol 1 ~ 65% >8 ~ 1.2 Mb

Pup1 12 ~ 80% 16.5 ~ 0.3 Mb

Major QTLs identified for both stresses

+P -P

P-defSalinity

A MAB system for Pup1 and Saltol

1 2 3 4

Target gene

Foreground markers

MAB strategy

Target gene + flanking markers

1 2 3 4

Recombinant markers

1 2 3 4

Background markers

> 20-fold difference in P uptake with traditional varieties being superior

Mapping population developed from Nipponbare x Kasalath and used in QTL mapping

Nipponbare

Kasalath

0

2

4

6

8

10

12

14

P up

take

(mg

plan

t-1)

Kasalath

Nippobare

MAB for P-deficiency tolerance

1 0 c M

1

R 1 1 7

C 7 4 2

R 2 4 14C 8 6

C 8 1 3

R 2 4 17

C 1 3 70

C 1 2 2

R 8 8 6

C 8 0 8

R 1 4 85

R 2 6 35

R 1 9 28

C 1 7 8

R 2 3 29

R 2 1 0

C 1 2 11

C 9 5 5

C 8 8 5

R 1 9 44

R 1 6 13

C 9 7 0

C 1 6 1

C 1 1 2

2C 1 4 70

C 1 4 45

C 1 2 21

G 2 75

C 5 6 0

C 1 4 08

C 7 4 7

R 4 8 0A

C 4 2 4

R 1 8 26

R 2 6

G 1 32

C 7 7 7

R 1 8 43

R 7 1 2

G 2 27

C 3 6 5

C 1 3 2

G 1 32 7

C 4 2 1

G 3 65

R 2 5 10

R 4 1 8R 3 3 93

C 4 9 9

G 1 31 4 B

5C 5 9 7

R 8 3 0

R 3 1 66

R 1 8 38

C 2 4 9

R 5 6 6

R 2 5 58

R 1 4 36

R 2 2 89

C 1 2 68

R 1 5 53

C 6 2 4

C 1 2 8

C 1 0 18

C 4 6 6

C 2 4 6

R 5 2 1

C 1 2 30

G 1 45 8

R 3 7 2

3R 1 9 25

R 1 9 27

R 3 2 26

R 1 6 18

C 5 9 5

C 9 4 4

C 7 4 6

C 1 3 6

R 2 5 0R 1 9

G 3 32

C 8 0

C 1 6 77

C 1 1 35

R 3 1 56

C 1 4 88

C 6 3

C 5 6 3

C 2 5

C 5 1 5

C 3 6 1

R 2 1 70

4

R 1 4 27

C 1 0 16

G 2 35

R 5 1 4

C 1 1 00

R 1 7 83

R 3 7 4

C 3 3 5

C 5 1 3

R 9 3

C 8 9 1

C 9 7 5

C 7 3 4

R 2 8 8

C 9 4 6

R 1 8 54

R 2 3 73

C 4 4 5C 1 0 7

6R 2 8 69

R 1 9 62

C 1 9 1B

C 4 9 8

R 1 9 54

L6 88

C 1 4 78

R 2 1 71

R 2 1 23

R 2 6 54R 4 3 7

C 2 1 4G 1 22R 6 7 4

R 2 5 49

C 3 5 8

C 5 5 6

R 2 0 71

R 1 1R 1 8 88

R 1 6 08

G 2 00

R 2 1 47

C 6 0 7

R 1 1 67

1 0C 7 0 1

R 1 9 33

R 2 1 74

R 2 1 94

R 1 6 29

R 2 4 47

C 1 2 86

C 1 3 69

R 1 8 77

C 4 8 8

R 7 1 6

C 8 0 9

G 1 27

C 2 2 3

9C 7 1 1

R 1 1 64

R 1 6 87

C 1 4 54

G 1 03

R 7 9

R 1 7 51

G 3 85

R 2 2 72

R 2 6 38

C 6 0 9

C 1 2 63

C 5 7 0

C 5 0 6

G 2 93

1 1R 1 5 06

C 9 5 0C 8 2

G 3 76

G 1 46 5

C 5 0C 1 8 9C 1 1 72

S 2 2 6 0

G 2 57

R 7 2 8G 2 02

C 5 3 5

S 2 1 3 7

G 3 20

R 2 3 16

C 4 7 7

C 1 3 50

C 7 9 4A

R 2 2 53

R 7 7

R 3 2 02

R 1 4 66

C 3

G 4 4

8R 1 9 63

R 2 6 62

C 5 0 2

C 1 0 12 2

R 2 0 2

R 2 6 76G 1 87

G 1 07 3

R 7 2 7

C 3 4 7

R 2 3 81G 1 04

C 1 1 07

R 1 8 13C 1 1 21

R 9 0 2

C 1 6 6

C 9 0 5

C 3 9 0

R 1 9 43

7C 2 6 1

C 1 0 57

R 5 6 5

S 1 0 0 12

R 2 4 01

R 1 4 88

C 3 9

C 1 2 26

R 1 4 40

R 3 0 89

C 4 5 1

R 1 3 57

R 1 2 45

C 8 4 7

C 1 4 12

R 1 7 89

C 5 9 6

C 2 1 3

C 7 2 8

1 2C 9 0 1

G 1 40 6

C 1 0 69

R 1 7 09

R 2 7 08

G 2 14 0

C 4 4 3

R 6 1 7

R 3 3 75

R 2 6 72

C 1 3 36

R 6 4 2

C 1 1 16 A

C 7 3 2

G 1 93

G 2 4B

P uptake P use efficiency

Four QTLs for P uptake detected on chromosomes 2,6,10,12

The one on chromosome 12 is a major QTL (~13 cM)

Pup1Pup1

Fine mapping of Pup1 locus on chr

12

C901

C449

W326

C2808

G2140

C443S10520

G124AS2572

C732

12

C443 (50.5)

G124A (30.0)

S10520 (40.3)

S14025 (51.8)S10704 (49.3)

P96 (47.9)

C449 (72.5)

G2140 (63.7)

V124 (70.7)

S13126 (55.1)

S1436 (57.4)S13752 (56.0)

C61722 (58.9)

(cM)

NIL14-4

RM27815 (7.47)

RM27090 (12.20)RM28002 (12.95)

RM28073 (14.95)

RM28102 (15.91)RM465 (16.75)

RM511 (17.40)RM1261 (17.53)RM28195 (18.08)

RM519 (19.90)

NIL14-6

T S

(Mb in Nip.)

Pup1T5-4 (15.32)

Ba76H14_7154(15.47)

Pup1 fine-mapped to within 272 kb and sequencedCandidate genes short listed and are being analyzed through RNAi

and overexpression

Kasalath (272007bp)

Nipponbare (154071bp)

4/5T5-4 14

T5-4(15.32Mb)

18

22/24

26

67

Ba76H14_715443

over 95% 90~95% 80~90%Sequence similarity

21 3839

40 42

45 50 59

Ba76H14_7154(15.47Mb)

SSR3SSR3

Kasalath allele-specific dominant markers

to be developed for candidate genes

Candidate gene-specific Co-dominant markers

SSR markers developed

Gene-

& allele-specific markers for Pup1

Markers (SSRs, gene-specific) developed & are being used to transfer Pup1 into 3 popular upland varieties, Dodokan, Situ Bagendit, and Batur Nine sets of crosses madeBC1

F1

populations genotyped (Foreground, recombinant & background) and BC2

F1

developed for further analysis

1 2 3 4 5 6 7 8 9 10 11 12

= seedling salt tolerance

= Na+ exclusion

= Δ13C

Saltol

Saltol is fine-mapped, annotated genes are being further analyzed

MAB for salinity tolerance

Different types of markers were developed (SSRs, SFPs, gene-specific)

11.1 11.2 11.3 11.4 11.5 11.6

P0475H04

AP002871(32,660-143,337)

OsJNOa173H09OsJNBa0008D05

AP007204(1-134,776)

B1146F03

AP003206(1,288-137,047)

P0552C05

AP002873(69,518-136,586)

OsJNBb-0022N24

AP003567(60,200-124,062)

AP006856(33,026-39,054)

RM10711(AGG)9

RM8094(AT)31

RM10719(CCG)8

RM3412(AG)17

S21150 E4175 C11732

LOC_Os01g19694

homeobox protein OSH1 (KNOX)

LOC_Os01g19770

stress-inducible membrane pore protein

LOC_Os01g19800

PIT1 (zinc-finger)

LOC_Os01g19850

cation-chloride co-transporter(CCC1-like)

LOC_Os01g19820

universal stress protein (ER6)

retrotransposons

LOC_Os01g19970

myb-like transcription factor

RM10720(AT)34

retrotransposons

retrotransposons

LOC_Os01g20160

cation transporter OsHKT8(SKC1)

RM10725(AC)25

retrotransposons

LOC_Os01g20720

NBS-LRR

retrotransposons

RM8094

RM3412

RM493

RM8115

RM562

RM7075

Centromere

RM10649

RM10927

10.3

15.3

RM243

RM3252

RM24

RM9

RM5461

RM7643

Chromosome 1

RM140

RM1287

10.4 Mb

15.3

SaltolQTL

10.9

11.3

11.5

12.2

12.7

14.6

15.1

13.7

12.1

13.8

SKC1

SalT

RM10720

RM10748

RM10800RM10793

RM10825

RM10655

RM10772RM10773

RM10829

RM10843

RM10852RM10864RM10871RM10890

RM10927

30 SSRs and gene-based

markers across the Saltol region

Saltolfine-map

RM10711RM10701RM10696RM10694

RM10713AP3206

Background markers developed for a set of recipient varieties

MAB scheme for SaltolRecipient varieties:

IR64 (widely grown), BR11 and BRRI dhan 28 (Bangladesh), swarna

Donors:Pokkali, FL478 and FL378

MAB is used to transfer Saltol:

Foreground and recombinant markers used for selection after each crossSSRs for background selection

BR28/FL478 BR28//

BC1 F1

Selected BC1 F1

BC1 F1

BC2 F1

Selected BC2 F1

BR28/

BC2 F1 BR28/

BC3

F2

X

BC3 F1

Selected BC3 F1

BC2

F2

Saltol MAB for BRRI dhan 28• Goal: Saltol QTL line with

BR28 background• Current status:

– SSR-selected BC2 F1 individuals were backcrossed to BR28

– 1,500 BC3 F1 plants now growing at IRRI

– Markers are being used to select individuals to advance to BC3 F2

• Confirmation: – BC3 F2 seeds will be used

to confirm the Saltol effect in the BR28 background

Saltol MAB for BR11

Goal: Saltol QTL line with BR11 backgroundCurrent status:

90 BC1 F1 individuals currently being screened for salinity tolerance Tolerant plants will be rescued and backcrossed to BR11 to BC2 F1

BC2 F1 plants will be genotyped with markers for foreground and background selection

Progress in MAB in BangladeshBR11 X FL378

BC1F1 (251)

F1 (185) X BR11

Selected BC1F1 (135)

FOREGROUND SELECTION(RM3412 & RM493)

Selected BC1F1 (39)

RECOMBINANT SELECTION( RM490 & RM7075)

BC1F1 (2/3) X BR11

BACKGROUND SELECTION( 60 SSR DISTRIBUTED WHOLE GENOME)

Out of 248 F1 185 were confirmed by using a primer Methionine Synthatase

BC2F1

Again do the similar selection scheme with more primers.

If recovery of RPG is > 90% then selfing of selected plants, otherwise, another backcross (BC3)

Parents:Parents:

Recurrent:BR11 (T.Aman/WS)BRRI dhan28 (Boro/DS)

DonorFL378 (NIL from IR29 x Pokkali)

Backcrosses: BRRIDNA isolation, marker analysis: DU

Training of NARES in MAS and other relevant technologies

Training workshop in MAB (2005)On-job trainingDegree training

Participatory varietal selection (PVS) networksINGERSalinity network Farmers field days

Out-scaling and up-scalingLarge-scale seed production and disseminationSupport and involvement of policy makers and development organizations

Transfer plans

MAB is efficient but expensiveCapacity of National programs in MAB

Additional degree training

In-country workshops

Infrastructure (equipments, storage etc)

Supplies: availability and funds

Constraints to plans of product transfer

Product deliveryProduction of sufficient certified seeds for PVS trialsOut-scaling on a large scale require varieties to be released firstNational policies for release of MAB products still the same as that for varieties developed conventionally

Constraints ….

Research Team

Abdelbagi IsmailMike Thomson Sigrid Heuer Dave Mackill Glenn GregorioRK SinghX. LuHei Leung

JIRCASM. WissuwaUCDEd BlumwaldEllen Tumimbang

IRRIIndonesiaM. BustamamM. SugionoJoko PrasetiyonoBangladeshZeba SerajMA Salam

NARES ARIs

USDAClyde WilsonLinghe

ZengABRII, IranG. HosseiniNIASM. Yano

KoreaJukon

KimUCRTim CloseHarkamal Walia Xinping Cui

Collaborators