“Deepwater Rice” the Green Revolution Gene to Allow Rice Adaptation to Submergence

1/25/2019

International Plant & Animal Genome XXVII W900 / January 14, 2019 San Diego, CA, USA

An Ethylene-Gibberellin Relay Co-opts “Deepwater rice” the Green Revolution Gene to Allow Rice Adaptation to Submergence

Takeshi Kuroha (Tohoku Univ.) Susan McCouch (Cornell Univ.) Moto Ashikari (Nagoya Univ.)

Deepwater rice field in Thailand Deepwater rice field in Thailand

Deepwater response (Tank was filled to the brim at the first day) Water is essential for all living organisms, and plant lives around swamps and rivers has an advantage in terms of water availability

NASA IRRI

Excess water due to flooding has an adverse effect on the growth of most plants Non-Deepwater rice Deepwater rice Hattori et al 2009 Nature

Dry season Hollow structure in the stem is used for air flow

Snorkel

Node

Rainy season Internode

Node

There are plants that can adapt to periodic flooding Internode

http://angkorvat.jp Shallow Deepwater

1 1/25/2019

Previous model of internode elongation in deepwater rice Genome wide association study (GWAS) on deepwater response

Collaboration with Prof. Mochizuki (Kyushu Univ.) (Non-deepwater rice; Deepwater rice) Korea Deepwater rice and Dr. Ebana (NARO) Non-deepwater rice (1; 0)

Submergence Submergence Bhutan (1; 0) China Total 68 varieties (5; 0) (28 non-deepwater and 40 deepwater rice) Nepal Ethylene Ethylene Taiwan (1; 0) India (1; 0) accumulation accumulation (9; 3) Philippines (4; 0)

Vietnam OsEIL1a OsEIL1a (0; 8) Bangladesh (1; 20) Cambodia (1; 2) Myanmar Thailand SNORKEL1/ 2 SNORKEL1/2 Madagascar (2; 4) (0; 4) × (1; 0) TILS ? (Total internode length after one week submergence) Gibberellin accumulation

Water level Drowning Internode elongation Air Submergence for 1 week

Procedure of submergence treatment

Togo field in Nagoya Univ.

Outline

1. Identification of the gene for internode elongation under submergence in deepwater rice

2. Special function of Green revolution gene in deepwater rice to adapt periodical flooding

Deepwater rice field in Thailand

Explorations of regulatory factors for deepwater response Result of GWAS on deepwater response

Hattori et al. Breeding Sci. 2008 Collaboration with Prof. Tamiya (Tohoku Univ./RIKEN) Nagai et al. Breeding Sci. 2012 and Prof. Yamasaki (Kobe Univ.) Biparental linkage analysis Deepwater response of 68 varieties for GWAS chromosomes Sub: Submergence for 1 week 1 2 3 4 5 6 7 8 9 10 11 12 Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Air Sub Genotyping with genetic makers 3

and mapping 1

VS m 4 12 2 1 T65 NIAS37 NIAS44 NIAS45 NIAS01 DW470 DW311 DW414 DW426 Bhadua C9285 T65 C9285 SNORKEL1/2 (non-deepwater rice) (deepwater rice) Ethylene response factor TILS values of each variety Manhattan plot for trait of TILS Hattori et al Nature 2009 Genome wide association study (GWAS) (Deepwater rice) based on haplotype association analysis C9285 chromosomes 7 120 6 1 2 3 4 5 6 7 8 9 10 11 12 100

) ) 5 P P 80 ( 4

Genotyping with 10 44k DNA microarray 3 60 log and mapping – ? 2 TILS(cm) 40 (Non-deepwater rice) ・・・ 1 T65 20 0

0 1 2 3 4 5 6 7 8 9 10 11 12

T65

C9285

DW314 DW478 DW483 DW487 DW316 DW313 DW417 DW466 DW489 DW318 DW397 DW471 DW311 DW456 DW457 DW375 DW369

NIAS44 NIAS45 NIAS21 NIAS42 NIAS15 NIAS53 NIAS06 NIAS39 NIAS46 NIAS08 NIAS31 NIAS05 NIAS09 Bhadua Various non-deepwater and deepwater rice in Asia NIAS38 Varieties (68 lines) Chromosome Kuroha et al Science 2018

2 1/25/2019

Strong peak on chromosome 1 Polymorphisms in SD1 are associate with deepwater response

Collaboration with Prof. Tamiya (Tohoku Univ./RIKEN) 34.5 42.5 (Mb) Chr.1 and Prof. Yamasaki (Kobe Univ.) Nucleotide variation in SD1 gene of varieties in the GWAS Insertion Deletion Substitution GWAS 8 qTIL1C9285

Hattori et al 2008 The peak of GWAS on chr1 was Haplotype n

) ) 6 C9285 P P T65 4 ( located inside of the QTL (qTIL1 ), T65

10 aus 23 4 which was previously detected by C9285 log indica 25 – other biparental linkage analysis 14 2 C9285

0 T65 C9285 Interaction between TILS, SD1 haplotypes, 36 38 40 42 (Mb) and existence of SNORKEL1/2 1 2 3 4 5 6 7 8 9 101112 (chr.) 1 2 3 4 5 6 7 8 9 101112

3.7 Mb 4.0 Mb SNORKEL1/2 DWH (Deepwater rice specific haplotype) × Linkage mapping 0.35 Mb ・17 polymorphisms are associated with internode

NIL-12 NIL-1+12 elongation under submergence

HindIII Indel

Marker ・The DWH works in the presence of SNORKEL1/2

3379 3379

3561N

32k 3227SSR High resolution linkage mapping to enhance internode elongation 32 kb was performed 169 kb 148 kb C9285 Kuroha et al Science 2018 Kuroha et al Science 2018

High-resolution linkage analysis of the QTL on chromosome 1 New model of internode elongation in deepwater rice

Collaboration with Dr. Wu (NARO) Deepwater rice Chromosome 1 Chr. 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 Submergence

>1,000 recombination events Ethylene accumulation

Indel SD1

5.5 kb SD106

SD103 32k 3561N ? T65 C9285 NIL-1 OsEIL1a T65 C9285 NIL-1

Os01g0883800 ? 15 6 * Os01g0883900 SNORKEL1/2 DWH SD1 * *

10 * 4 transcript level transcript

SD1 (SEMI DWARF1) 5 2 SD1 ・Gene involved in modern breeding including the “Green Revolution”

・ 0 0 Loss-of-function of the gene leads semi-dwarf phenotype Relative 0 3 0 1 ・encodes a gibberellin biosynthesis enzyme, OsGA20ox2 After submergence (h) After ethylene treatment (h) Both submergence and ethylene induce SD1 expression WT sd1 Sasaki et al Nature 2002 in deepwater rice Internode elongation Kuroha et al Science 2018 Kuroha et al Science 2018

New model of internode elongation in deepwater rice Collaboration with Dr. Mitsuda, Dr. Ohme-Takagi, and Dr. Yanagisawa Deepwater rice submergence Submergence Outline Ethylene accumulation OsEIL1a fused with the repression domain inhibited SD1 induction under submergence 1. Identification of the gene for internode elongation ? OsEIL1a under submergence in deepwater rice

Suppressor ? × SNORKEL1/2 DWH SD1 2. Special function of Green revolution gene Relative LUC activity in deepwater rice to adapt periodical flooding Reporter 0 50 100

LUC NBS MYC (bp) Effector OsEIL1a -3,551 +30 pSD1-3551LUC pSD1T65 * T65 -3,153 +30 OsEIL1a directly activates the pSD1-3153LUC SD1 promoter in rice protoplast pSD1C9285 *

Deepwater rice field in Thailand Internode elongation Kuroha et al Science 2018

3 1/25/2019

New model of internode elongation in deepwater rice Origin of the DWH Collaboration with Dr. Sakakibara 0h 12h 24h Geographical distribution of DWH in wild and cultivated rice Deepwater rice GA1 GA4 O. rufipogon Submergence Bangladesh (n = 258) O. rufipogon

Ethylene accumulation non-DWH DWH Standing variation OsEIL1a DWH In South/Southeast Asia non-DWH Active gibberellins, GA1 and GA4, increased under submergence Suppressor ? Country having × DWH-positive accessions

) 60 SNORKEL1/2 DWH SD1 Artificial selection m × c 10.4

( in Bangladesh

g O. sativa n 40 Bangladesh

l O. sativa e (n = 235) Gibberellin accumulation d o non-DWH

e 20

GA1 GA n 4 i

1 ×1.0 DWH

m T 0 Deepwater rice

5 5 5 5

5 Promotion of 5

8 6 6 8 6 6 in Bangladesh

5 5 5

5 5

2 2

T T

8 8

6 6

8 internode elongation 6

2 2

T 2 2

T T

9 9

C C

C Control DWH Control GA GA GA1 GA4 1 4 non-DWH 2 weeks from germination under air condition, 10-5 M gibberellin treatment with 10-6 M uniconazole Internode elongation Deepwater rice cultivation GA4 strongly induces internode elongation in deepwater rice Kuroha et al Science 2018

New model of internode elongation in deepwater rice Contribution of SD1 gene to rice breeding for two different purposes

Non-deepwater rice Deepwater rice Amplification of an ethylene-gibberellin relay Submergence Submergence Internode elongation under submergence Green revolution

Ethylene accumulation Ethylene accumulation sd1 mutation DWH SD1 SD1 SD1 Loss of function Gain of function OsEIL1a OsEIL1a Ethylene- gibberellin relay Lodging sensitive SNORKEL1/2 × Suppressor ? Suppressor ? Survive Lodging resistant × under deep water SD1 SNORKEL1/2 DWH SD1 Drown under deep water ×

Gibberellin accumulation Gibberellin accumulation

GA1 GA4

Promotion of internode elongation Semi-dwarf rice Normal rice Deepwater rice Human selected two SD1 alleles with opposite function for agriculture Drowning Internode elongation

Haplotype network of the SD1 Acknowledgement Tohoku Univ. /RIKEN AIP Haplotype network of the SD1 with O. sativa and O. rufipogon Gen Tamiya Nagoya Univ. Tohoku Univ. Kyushu Univ. Keisuke Nagai Shinjiro Yamaguchi Toshihiro Mochizuki aus Subpopulation based on Rico Gamuyao (present: Kyoto Univ.) Univ. of Tokyo genome wide SNP data (present: Johns Hopkins Univ. Kiyoshi Mashiguchi Shuichi Yanagisawa indica School of Medicine) Yoshiya Seto aus RIKEN CSRS/Nagoya Univ. Japonica Tomoyuki Furuta (present: Meiji Univ.) DWH aro Hitoshi Sakakibara indica Masanari Nakamori Kazuhiko Nishitani tropical japonica RIKEN CSRS temperate japonica Takuya Kitaoka Ryusuke Yokoyama Mikiko Kojima admixed Keita Adachi O. rufipogon Kobe Univ. Anzu Minami Cornell Univ. Masanori Yamasaki Yoshinao Mori Diane R. Wang NIAS Deepwater rice (present: Univ. at Buffalo) in O. sativa Kaworu Ebana indica Jianzhong Wu aus JST CREST JICA-JST SATREPS AIST O. rufipogon Admixed (C9285) the Japan Advanced Plant Science Network Masaru Ohme-Takagi The Canon Foundation The DWH was found only in deepwatar rice within O. sativa and MEXT/JSPS KAKENHI (present: Saitama Univ.) a part of O. rufipogon accessions the NSF Graduate Research Fellowship Nobutaka Mitsuda USDA NIFA Kuroha et al Science 2018

4 1/25/2019

Admixture analysis in OsGA20ox2 region

Admixture analysis using a subset of diverse non-admixed O. sativa as

the reference panel (114 Japonica; 57 aus; 87 indica). Varieties harboring DWH harboring Varieties

The DWH is associated with indica or aus subpopulations of O. sativa The DWH was artificially selected for cultivation of japonica deepwater rice

The evolutionary history and domestication of SD1 Responsible gene for chr 1 QTL by linkage analysis

10cm Gain-of-function analysis Gain-of-function of SD1 Loss-of-function of SD1

Origin of japonica Chr 1 1cm 31

cultivated rice SNP2 (DWH) - Huang et al (2012) ・Domestication of japonica rice Seq ・DWH was used for domestication Introgression 5.5 kb SD1 Japonica 1cm of deepwater rice in Bangladesh (~1940) (weak enzymatic activity) (Induction under submergence) ・Green revolution (null allele) SD1 Lodging Survive Os01g0883900 aus/indica resistant Node under Sub-clone deep water Internode elongation after Elongated DWH 2-week submergence internode ancient standing variation in Southeast/South Asia. ＊Background: T65 with SNORKEL1/2 segment Node 40 DWH(+) O. rufipogon 30 Bangladesh 20

10 SD1 is responsible gene 0 Human selected different alleles with opposite function of the same gene for chr 1 QTL for agriculture history internode length Total (cm)

Kuroha et al Science 2018

Distance tree analysis of diverse O. sativa subpopulation Chr1 QTL detected by biparental linckage analysis and GWAS

Distance tree of 480 varieties of Orizya sativa Collaboration with Prof. McCouch (Cornell Univ.) Marker or SNP including deepwater rice QTL for TIL RM9 qTIL1 Chromosome (2007) 1 2 3 4 5 6 7 8 9 10 11 12 DWH DWH japonica aus RM246

qTIL12 RM1183 (2007) SNOKEL1/2 (2009) qTIL1 RM7180 qTIL1 (2007) (2008) Subpopulations DWR1-B

indica Hattori et al (2007) Breeding Science aus GWAS id1024246 Hattori and Nagai et al (2008) Breeding Science aro id1024432 312kb DWR1-S indica tropical japonica DWH temperate japonica The peak of GWAS on chr1 was located inside of the RM6840 candidate region of the QTL named as qTIL1, which The DWH were present in only 14 Bangladesh deepwater rice varieties previously detected by biparental linkage analysis classified into indica, aus, or japonica.

5 1/25/2019

Scheme for production of QTL mapping population on chromosome 1 Internode elongation by near-constitutive expression of the SD1 gene

T65 C9285 NIL-12 NIL-1+12

Chr.1 2 3 4 5 6 7 8 910 1112 1 2 3 4 5 6 7 8 910 1112

P × SK1/2 #1 #2 #3 #6 #2 #3 #6 #14

SD1

Chr.12 Homozygote Chr.1 1 2 3 4 5 6 7 8 9 10 1112 Heterozygote OsACT1 F1 RM6386 RM7180 RM235 RM6840

Selfing 12 Chr.1 *

RM7180 F2 ・・・・ 8

RM6840

Selfing Selfing 4

Expected

segregation ratio 1 : 2 : 1 1 : 2 : 1 Total internode (cm) length internode Total RM7180 0 F3

RM6840

Transcriptional regulation of SD1 under submergence treatment

T65 C9285 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 5 6 7 8 9 101112 SD1 expression under submergence

× SNORKEL1/2

NIL-12 NIL-1+12 Basal part

1 cm

T65 C9285 0, 1, 3, 6, 12h after submergence

Deepwater rice showed strong induction of SD1 expression under submergence treatment Kuroha et al Science 2018

Expression analysis of submergence-induced SD1 transcription Principal component analysis (PCA) of the GWAS panel. in different tissue regions (I to III) above the top node (arrow)

15 Dot plots of TILS in the three groups l

e 0h 3h

t * p 1 cm III i

r 10 c

s * n

1 cm a

II r

e 5 v

0.5 cm i

I t a

l *

e R 0 I II III

6 1/25/2019

Gibberellin level after submergence in deepwater rice Geographical distribution of deepwater rice and cultivated rice

Collaboration with Ms. Kojima (RIKEN) and Gibberellin biosynthesis pathway Prof. Sakakibara (RIKEN/Nagoya Univ.) Cultivated rice（Oryza sativa） temperate GA - japonica 200 GA53 12 aldehyde aromatic 100

pmol/gFW GA13ox 0 GA GA aus

0 20 40 60 53 12 GA20ox (SD1) GA20ox (SD1) 45 After submergence (h) GA9 tropical 30 20 japonica GA20 15

pmol/gFW Wild rice 10 (O. rufipogon )

pmol/gFW 0 0 20 40 60 0 After submergence (h) indica 0 20 40 60 GA 20 GA9 15 After submergence (h) GA4 15 Deepwater rice GA3ox GA3ox 10 GA1 cultivation 10 Zhao et al Nature Comm. 2011, modified Active 5 GA GA pmol/gFW 1 gibberellins 4 Indica Japonica pmol/gFW 5 Although deepwater rice varieties are mainly , some have been classified as 0 with isozyme and microsatellite markers 0 Submergence increases levels of 0 20 40 60 0 20 40 60 After submergence (h)  We explored for origin of DWH to understand the evolutionary history of GA and GA in deepwater rice After submergence (h) 1 4 the deepwater response Ayano et al Plant Cell Environ. 2014

Enzymatic activity of SD1 in deepwater rice Search for origin of the DWH (Deepwater rice specific haplotype)

Collaboration with Gibberellin biosynthesis pathway Dr. Mashiguchi, Dr. Seto, and Prof. Yamaguchi Collaboration with Dr. Wang and (Tohoku Univ.) Prof. McCouch (Cornell Univ.)

GA12- aldehyde GA53  GA20 GA12  GA9 O. sativa (n = 149) GA13ox x19 68 varieties in the GWAS

Japonica and Indica group

;nM) 9 9 ;nM) GA GA 30 O. rufipogon (n = 34)

30 53 12 27.5

GA20ox (SD1)

20 20 GA20ox (SD1)

20 20 Resequecing data set

10 10 Comparison of polymorphisms in SD1 GA20 GA x1 9 among accessions 1.5

0.0 0.0 GA3ox GA3ox (GA product of Concentration 0.0 0.1 0

Concentration of product (GA product of Concentration 0 Active GA1 gibberellins GA4 O. sativa O. rufipogon Search for DWH-positive accessions Reaction (200µl): by haplotype network analysis 10 µg protein SD1 protein efficiently produces GA in deepwater rice Ishii et al Nature Genet. 2010 10 ng substrate 4 at 25oC for 10 min Kuroha et al Science 2018

Effect of GA1 and GA4 on internode elongation Collection of SNP data by DNA microarray analysis 60 Collaboration with Prof. McCouch (Cornell Univ.)

x10 Chromosome 480 varieties of Orizya sativa 1 2 3 4 5 6 7 8 9 10 11 12 including DNA microarray 40 deepwater rice

Genotyping of 20 ~34 k SNPs DNA Total internode length (cm)length internode Total Extraction

x1 10cm

T65 T65 T65 T65

T65

C9285 C9285

C9285 C9285 C9285 C9285

C9285 C9285 GA Control GA1 GA4 3 Control GA1 GA4 ・2 weeks from germination under air condition GA4 strongly induces internode elongation ・10-5 M gibberellin treatment in deepwater rice with 10-6 M uniconazole Kuroha et al Science 2018

7 1/25/2019

Interaction of internode elongation and SD1 induction in mapping lines Evolutionary history of deepwater rice proposed in this study

Promoter and cDNA GR 2nd intron Ancestor SK1/2 T65 C9285 Non-deepwater rice Deepwater rice Appearance of DWH

as ancient standing variation SD1 SNP1

SNP2 SD1 expression -

- Internode elongation FokI

Submergence Submergence W1/W4 W2/W3 7-day submergence under 3 hour submergence

13k N 13k

SD1

6k 6k

3561N SD1 W5/W6 3k Chr 1 O. rufipogon DWH GR GR Ethylene accumulation Ethylene accumulation promoter Total internode length (cm) Related expression… SK1/2 SK1/2 -15.9k -9.9k -2.7k 0 5 10 W4 -12.8k SD1 0 40 80 Loss of SK1/2 OsEIL1a OsEIL1a Ethylene- Selection of 0h gibberellin relay DWH Selection of Loss of SK1/2 SD1EQ 3h

Suppressor ? Suppressor ?

SD1 SD1 SK1/2 DWH DWHGR GR DWHGR GR EQ (GR or EQ) (GR) O. sativa SK1/2 SK1/2 SK1/2 SK1/2 SK1/2

Gibberellin accumulation Gibberellin accumulation

GA1 GA4

Promotion of internode elongation ・There is correlation between internode exon1 exon2 exon3 elongation and SD1 induction

indica, aus indica indica indica Subgroup Japonica Drowning Internode elongation admix aus aus aus ・The promoter region up to -13 kb is essential for induction by submergence Deepwater rice Non-deepwater rice

Relationship between internode elongation and OsGA20ox2 induction in various lines Evaluation of DWH in O. rufipogon

Standing variation Introgression from O. sativa Non-deepwater rice Deepwater rice

150 non-DWH non-DWH non-DWH O. rufipogon O. rufipogon

Internode elongation 100 DWH DWH Total submergence for 7 days 50 Artificial selection Introgression

internode (cm)length internode 0 O. sativa DWH O. sativa DWH 100 0h 3h non-DWH non-DWH

Induction of Color: nucleotide variations 50

OsGA20ox2 expression DWH DWH Relative submergence for 3 hours O. rufipogon DWH High O. rufipogon DWH Low

expression expression level DWH 0 DWH diversity DWH DWH diversity DWH DWH

non-DWH non-DWH O. rufipogon non-DWH High O. rufipogon non-DWH High non-DWH non-DWH non-DWH diversity non-DWH diversity Internode elongation and OsGA20ox2 induction are non-DWH non-DWH correlated to each other in various lines

Evaluation of DWH in O. rufipogon OsEIL1a is predominantly expressed in stem of deepwater rice

Standing variation Introgression from O. sativa

Expression level of OsEIL genes non-DWH non-DWH Phylogenetic tree of EIN-3 LIKE proteins DWH Rice: OsEILs Stem under submergence for 0, 1, or 3 hours Arabidopsis: EIN3/EILs DWH

Ethylene signaling T65 C9285 genomic

rufipogon rufipogon T65 C9285 DWH

0h 1h 3h 0h 1h 3h O. DWH O. ≈ 1 < 1

OsEIL1a non-DWH non-DWH Nucleotide diversity of diversity Nucleotide Nucleotide diversity of diversity Nucleotide 2 Mb 2 Mb OsEIL2 OsGA20ox2 OsEIL3 OsGA20ox2 Color: nucleotide variations OsEIL3 OsEIL4 DWH DWH O. rufipogon DWH High O. rufipogon DWH Low OsEIL5 DWH DWH diversity DWH DWH diversity OsACT1 DWH DWH

non-DWH non-DWH *OsEIL1b does not exist in T65 and C9285 genome O. rufipogon non-DWH High O. rufipogon non-DWH High non-DWH non-DWH non-DWH diversity non-DWH diversity OsEIL1a was used for transactivation analysis non-DWH non-DWH

8 1/25/2019

DWH is ancient standing variation What is the evolutionary history of genes for deepwater response?

Nucleotide diversity of 2 Mb region on 108 accessions of O. rufipogon were compared. Evolutionary history of SNORKEL genes

2 Mb Wild rice species possess the SNORKEL genes, genes may have been acquired before or during wild rice species divergence. OsGA20ox2

Standing variation non-deepwater rice DWH - deepwater rice non-DWH πDWH O. rufipogon πnon DWH DWH ≈ 1 Wild rice species non-DWH non-DWH O. sativa Hattori et al 2009 DWH What is the evolutionary history of the DWH in OsGA20ox2 ?

Analysis on genome-wide relationships of DWH and Ratio of nucleotide diversity ) ( diversity of nucleotide Ratio globally diverse O. sativa and wild rice (O. rufipogon)

DWH is ancient standing variation Elongation of plant body for adaptation to flooding

Nucleotide diversity of 2 Mb region on 108 accessions of O. rufipogon were analyzed.

2 Mb Standing variation Elongation of coleoptile in rice seedlings under ethylene treatment in darkness Submergence-induced OsGA20ox2 non-DWH petiole elongation in Rumex palustris O. rufipogon DWH Inhibition Promotion Inhibition A B Ethylene Air Submergence

. Air 1 non-DWH Ethylene

3 Air

. W O. sativa

1 1

7 Ethylene

, DWH

r Air

. ≈ 1

(

)

W DWH

(

8 DWH . High

0 DWH diversity DWH Arabidopsis Rice Brachypodium Rumex palustris

Quantiles non-DWH 0%: 0.77 25%: 0.92 non-DWH High Yang et al 2015 Mol Plant van Veen et al 2013 Plant Cell 50%: 1.00 75%: 1.09 non-DWH diversity DWH 100%: 1.38 DWH1/2 non-DWH nonDWH3-DWH recombinant/heterorecomb./het

６ leaf stage：３Traits １０leaf stage：６Traits

Trait Description Leaf stage

PHSY Plant height 13 days after submergence 6

TILSY Total Internode length 13 days after submergence 6

NEISY Number of elongated internode 13 days after submergence 6

PHA Plant height before submergence 10 The DWH is derived from an ancient PHS Plant height 7 days after submergence 10 TILA Total Internode length before submergence 10

standing variation in TILS Total Internode length 7 days after submergence 10 Southeast/South Asia NEIA Number of elongated internode before submergence 10 NEIS Number of elongated internode 7 days after submergence 10

9 1/25/2019

Transient assay for transactivation of SD1 promoter in rice mesophyll protoplast

B MYC A HBT NOS ter MYC NBS (89 bp) NOS ter NBS LUC 1 641 (A.A.) HBT NOS ter OsEIL1a OsEIL1a -1,500 +30 (bp) pSD1C9285 NOS ter pSD1C9285 LUC 1 318 HBT NOS ter OsERF1 OsERF1 ATG

1 256 HBT NOS ter SK1 SK1

1 258 HBT NOS ter SK2 SK2

C Relative LUC activity 0 30 60

MYC NBS OsEIL1a NBS:LUCLUC Position from OsERF1 ATG (bp) SK1 SK2 -1,500 +30

pOsGA20oxLUC * pSD1C9285 2:LUC

cis-element for activation of OsGA20ox2 promoter by rice EIN3 Transactivation of SD1 promoter by chemical induction of OsEIL1a protein Relative LUC activity NBS (35S minimal promoter) ATG 0 5 10 15 LUC -74 +30 NBS MYC OsEIL1a-MYC LUC pSD1-74 -111 A -129 LUC pSD1-111 -147 LUC pSD1-129 1 485 (A.A.) -1,500 (bp) LUC pSD1-147

35S NOS ter LUC pSD1-1500 OsEIL1a-VP16-GR OsEIL1a VP16 GR pOsGA20ox2 (C9285) promoter

0 50 100 150 200 -129 -111 -94 NBS pSD1-129 CTCCCCTGTTACAAATACCCCACCCTCCTGCCCAGApSD1-129 M1 CTCGAGTGTTACAAATACCCCACCCTCCTGCCCAGApSD1-129M1 M2 CTCCTCGAGTACAAATACCCCACCCTCCTGCCCAGApSD1-129M2 Relative LUC activity M3 CTCCCCCTCGAGAAATACCCCACCCTCCTGCCCAGApSD1-129M3 B pSD1-129M4 0 5 10 M4 CTCCCCTGTCTCGAGTACCCCACCCTCCTGCCCAGA M5 CTCCCCTGTTACCTCGAGCCCACCCTCCTGCCCAGApSD1-129M5 MYC M6 CTCCCCTGTTACAAACTCGAGACCCTCCTGCCCAGApSD1-129M6 OsEIL1a-MYC M7 CTCCCCTGTTACAAATACCTCGAGCTCCTGCCCAGApSD1-129M7 NBS NBS LUC - DEX Position from + DEX -120 bp ~ -112 bp 0 5 10 15 ATG (bp) -129 -108 -1,500 +30 NBS MYC OsEIL1a-MYC LUCpOsGA20ox2 (129-108)x4 CTCCCCTGTTACAAATACCCCA x4+minimal promoter pSD1C9285 * (129-108)x16 CTCCCCTGTTACAAATACCCCA x16+minimal promoter

cis-element is located in -120 bp to -112 bp region upstream of OsGA20ox2 gene

EIN3 1 M------MFNEMGMCGNMDF------FSSGS---LGEVDFC-PVP OsEIL1a 1 MMGGGLVMDQGMMFPGVHNFVDLLQQNGGDKNLGFGALVPQTSSGEQCVMGEGDLVDPPP OsEIL1b 1 MMGGGLVMDQGMMFPGVHNFVDLLQQNGGDKNLGFGALVPQTSSGEQCVMGEGDLVDPPP

EIN3 30 QAEPDSIVEDDYTDDEIDVDELERRMWRDKMRLKRLKE--QDKGKEGV-----DAAKQRQ OsEIL1a 61 ESFPDA-GEDDSDDDVEDIEELERRMWRDRMKLKRLKELQLSRGKDPAGGVVGDPSKPRQ OsEIL1b 61 ESFPDA-GEDDSDDDVEDIEELERRMWRDRMKLKRLKELQLSRGKDPAGGVVGDPSKPRQ

EIN3 83 SQEQARRKKMSRAQDGILKYMLKMMEVCKAQGFVYGIIPENGKPVTGASDNLREWWKDKV Fig. S27. cis-regulatory element for binding of OsEIL1a to the SD1 promoter. OsEIL1a 120 SQEQARRKKMSRAQDGILKYMLKMMEVCRAQGFVYGIIPEKGKPVSGASDNLRGWWKEKV OsEIL1b 120DNA SQEQ AbindingRRKKMSRA QdomainDGILKYML ofKMM OsEIL1aEVCRAQGFV YproteinGIIPEKGK forPVS GtransactivationASDNLRGWWKEKV of SD1 promoter Position from ATG (bp) pSD1 Relative LUC activity A -123 -111 -108 -94 EIN3 143 RFDRNGPAAITKYAQAENNIPGIHEGNNPIGPTPHTLQELQDTTLGSLLSALMQHCDPPQR -129 OsEIL1a 180 RFDRNGPAAIAKYQADNAVPGFESELASGTGSPHSLQELQDTTLGSLLSALMQHCDPPQR 0 100 200 OsEIL1b 180 RFDRNGPAAIAKYQADNAVPGFESELASGTGSPHSLQELQDTTLGSLLSALMQHCDPPQR NBS (A.A.) MYC OsEIL1a CTCCCCTGTTACAAATACCCCACCCTCCTGCCCAGA * EIN3 203 RFPLEKGVPPPWWPNGKEIN3EDWWP227QLGLPKDQGPAPYKKPHDLKKAWKVGVLTAVIKHMFPD 263 WT OsEIL1a 240 RYPLEKGVPPPWWPTOsEIL1aGDEET65WWP264ELGIPKDQGPPPYKKPHDLKKAWKVSVLTAVIKHMSPD 300 CTCGAGTGTTACAAATACCCCACCCTCCTGCCCAGA OsEIL1b 240 RYPLEKGVPPPWWPTOsEIL1aGDEEC9285WWP264ELGIPKDQGPPPYKKPHDLKKAWKVSVLTAVIKHMSPD 300 * CTCCTCGAGTACAAATACCCCACCCTCCTGCCCAGA m1 * CTCCCCCTCGAGAAATACCCCACCCTCCTGCCCAGA m2 EIN3 263 IAKIRKLVRQSKCLQDKMTAKESATWLAIINQEESLARELYPESCPPLSLSGGSCSLLMN OsEIL1a 300 IEKIRRLVRQSKCLQDKMTAKEISTWLAVVKQEEELYLKLNPGARPPAPTGGITSAISFN CTCCCCTGTCTCGAGTACCCCACCCTCCTGCCCAGA OsEIL1b 300 IEKIRRLVRQSKCLQDKMTAKEISTWLAVVKQEEELYLKLNPGARPPAPTGGITSAISFN m3 CTCCCCTGTTACCTCGAGCCCACCCTCCTGCCCAGA * B Relative LUC activity m4 Effector CTCCCCTGTTACAAACTCGAGACCCTCCTGCCCAGA 0 50 100 EIN3 323 -DCSQYDVEGFEKESH1Y E V E E L K P 485 E K V 641MN (A.A.)SSNFGMVAKMHD----FPVKEEVPAGNSEFM * OsEIL1a 360 ASSSEYDVDVVDDCKGDEAGNQKAVVVADPTAFNLGAAMLNDKFLMPASMKEEATDVEFI CTCCCCTGTTACAAATACCTCGAGCTCCTGCCCAGA m5 OsEIL1b 360 ASSSEYDVDVVDDCKGDEAGNQKAVVVADPTAFNLGMYCAAMYCMLNDKFLMPASMKEEATDVNBSEFI MYC * m6 pOsGA20ox2pSD1C9285 OsEIL1aOsEIL1a * EIN3 378 RKRKPN-RDLNTIMDRTVFTOsEIL1aCENLGCAHMYCSEISRGFLDRNSRDNHQLACPH*RDSRLPYGAA m7 OsEIL1a 420 QKRSASGAEPELMLNNRVYTCHNVQCPHSDYGYGFLDRNARNSHQYTCKYNDPLQ----- OsEIL1b 420 QKRSASGAEPELMLNNRVYTCHNVQCPHMYCSDYGYGF(1L-485)D(1-485)RNARNSHQYTCKYNDPLQ----- K282N B Relative LUC activity MYC (K282N)(K282N) EIN3 437 PSRFHVNEVKPVVGFPQPRPVNSVAQPI-DLTGIVPEDGQKMISELMSMYDRNVQSNQTS Position from 0 5 10 15 OsEIL1a 475 ----QSTENKPSPPAIFPATYNTPNQALNNLDFGLPMDGQRSITELMNMYDNNFVANKN- ATG (bp) NBS OsEIL1b 475 ----QSTENKPSPPAIFPATYNTPNQALVP16NNLDFOsEIL1a-VP16OsEIL1aGLPMD-VP16GQRSITELMNMYDNNFVANKN- LUC NBS * -129 -108 MYC OsEIL1a (1-485)-VP16 EIN3 496 MVMENQSVSLLQ-PTVHNH----QEHLQVP16FPGNM(1V-E485)GS--VP16--FFEDLNIPNRANNNNSSNNQ (129-108)LUC x4 * ×4 * OsEIL1a 530 --LSNDNATIMERPNAVNPRK282NIQIEEGFFGQGSGIGGSNGGVFEDVNGMMQQPQQTTPAQQ -129 -108 OsEIL1b 530 --LSNDNATIMERPNAVNPRIQIEEGFFGQGSG(K282N)-VP16(K282N)IGGS-NVP16GGVFEDVNGMMQQPQQTTPAQQ VP16 (129-108)LUC x16 * ×16 * EIN3 548 TFFQGNNNNNNVFKFDTADHNNFEAAHNNNNNSSGNRFQLVFDSTPF------DMASF pSD1 OsEIL1a 588 QFF------IRDDTPFGNQMGDINGASEF OsEIL1b 588 QFF------IRDDTPFGNQMGDINGASEF

EIN3 600 DYRDDMSMPGVVGTMDGMQ-QKQQDVSIWF---- OsEIL1a 611 RFGSGFNMSGAVEYPGAMQGQQKNDGSNWYY--- OsEIL1b 611 RFGSGFNMSGAVEYPGAMQGQQKNDGASEFEELE

10 1/25/2019

Binding assay with OsEIL1b protein and SD1 promoter in vitro Relationship between TILS value and Haplogroup 6 of SD1 or presence/absence of SK1/2 in the GWAS panel

4 4 5 1 Relative LUC activity = = GST OsEIL1b B n n A 160 120 b 0 5 10 15

. 140 3

6 1 - Hap-6 100

120 9 + Hap-6

pSD1 .

) MYC 6

)

80 a

8 4

NBS m

. NBS 100 4

From ATG (bp) 2 5

-129 . -147 -108 OsEIL1b (

(

* .

9 0

5 A 5 . 60 80 S

Biotin I

60 2

7 8

T .

. . 1

6 40

. .

9 6

4 . 2

. 2

4 8

2 2

. .

C9285 .

8 .

. 3 .

3 2

pSD1C9285 6 8

2 . 8

-123 -111

pSD1 9 2

0 2

40 1 2 2 7

. .

8 4

1 0

2 9 1

. . 1 .

1 .

3 9 .

2 1 .

. .

7 7

1 .

7 7 3

* 0

0 0

1 8

1 1

0 20

1 3 .

1 1 1

1 1

1 9

20 4

0 0 0 0 0 0 0 0 0

...... OsEIL1a 1 MMGGGLVMDQGMMFPGVHNFVDLLQQNGGDKNLGFGALVPQTSSGEQCVMGEGDLVDPPP 60 1 0 0 0 0 0 0 0 0 0 0

OsEIL1b 1 MMGGGLVMDQGMMFPGVHNFVDLLQQNGGDKNLGFGALVPQTSSGEQCVMGEGDLVDPPP 60 0

3 5 5 a 6 9 9 5 4 7 9 6 4 4 1 0 2 7 8 8 9 6 2 6 7 3 6 7 5 6 6 5 4 8 8 9 3 4 7

9 4 5 8 4 3 3 6 9 1 6 2 3 5 7 2 0 2 1 5 9 5 1 4 7 8

1 4

C 2 8 2 u 2 6 6 7 3 5 1 5 1 6 7 7 1 9 1 2 8 5 2 6 1 1 3 2 6 1 2 8 1 3 7 7 8 8 8

0 0 0 3 0 3 1 4 4 3 0 0 5 5 1 4 1 4 4 2 3 1 4 4 4 3 3

4 2 4 d 4 3 4 3 4 4 3 4 4 4 4 4 3 3 3 4 4 3 3 4 4 3 3 3 3 3 4 3 4 4 4 4 4 4

S S S S S S S S S S S S S S S S S S S S S S S S S S S

OsEIL1a 61 ESFPDAGEDDSDDDVEDIEELERRMWRDRMKLKRLKELQLSRGKDPAGGVVGDPSKPRQS 120 9 a Hap-6 -

A A A A A A A A A A A A A A A A A A A A A A A A A A

W +

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

h I I I I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D

D OsEIL1b 61 ESFPDAGEDDSDDDVEDIEELERRMWRDRMKLKRLKELQLSRGKDPAGGVVGDPSKPRQS B

120 N N N N N N N N N N N N N N N N N N N N N N N N N N OsEIL1a 121 QEQARRKKMSRAQDGILKYMLKMMEVCRAQGFVYGIIPEKGKPVSGASDNLRGWWKEKVR 180 OsEIL1b 121 QEQARRKKMSRAQDGILKYMLKMMEVCRAQGFVYGIIPEKGKPVSGASDNLRGWWKEKVR 180 Varieties 6 2 OsEIL1a 181 FDRNGPAAIAKYQADNAVPGFESELASGTGSPHSLQELQDTTLGSLLSALMQHCDPPQRR 240 4 2 = OsEIL1b 181 FDRNGPAAIAKYQADNAVPGFESELASGTGSPHSLQELQDTTLGSLLSALMQHCDPPQRR 240 = n n 160 OsEIL1a 241 YPLEKGVPPPWWPTGDEEWWPELGIPKDQGPPPYKKPHDLKKAWKVSVLTAVIKHMSPDI 300 120 b

OsEIL1b 241 YPLEKGVPPPWWPTGDEEWWPELGIPKDQGPPPYKKPHDLKKAWKVSVLTAVIKHMSPDI 300 8

140 1

1 - SK1/2

0 360 1 100 OsEIL1a 301 EKIRRLVRQSKCLQDKMTAKEISTWLAVVKQEEELYLKLNPGARPPAPTGGITSAISFNA 1

B 9

OsEIL1b 301 EKIRRLVRQSKCLQDKMTAKEISTWLAVVKQEEELYLKLNPGARPPAPTGGITSAISFNA 360 120 . + SK1/2

)

4 80

OsEIL1a 361 SSSEYDVDVVDDCKGDEAGNQKAVVVADPTAFNLGAAMLNDKFLMPASMKEEATDVEFIQ 420 4

6 m 100

9 5 OsEIL1b 361 SSSEYDVDVVDDCKGDEAGNQKAVVVADPTAFNLGAAMLNDKFLMPASMKEEATDVEFIQ 420 8

7 . .

(

9 9

6 5 80 . 60

OsEIL1a 421 KRSASGAEPELMLNNRVYTCHNVQCPHSDYGYGFLDRNARNSHQYTCKYNDPLQQSTENK 480

2 OsEIL1b 421 KRSASGAEPELMLNNRVYTCHNVQCPHSDYGYGFLDRNARNSHQYTCKYNDPLQQSTENK

8 480

2 60

. a

4 . 2

2 .

. .

OsEIL1a 481 PSPPAIFPATYNTPNQALNNLDFGLPMDGQRSITELMNMYDNNFVANKNLSNDNATIMER 540 0

4 4

2 8 2 9

1 .

40 0 7 0

8 .

1 4

9 OsEIL1b 481 PSPPAIFPATYNTPNQALNNLDFGLPMDGQRSITELMNMYDNNFVANKNLSNDNATIMER 2 1

. .

. 540 1

1 .

1 7 3

7 3 0

8 20

1 1 1 5

. 7

. 3 20 4

. OsEIL1a 541 PNAVNPRIQIEEGFFGQGSGIGGSNGGVFEDVNGMMQQPQQTTPAQQQFFIRDDTPFGNQ 600 6

0 0 0 0 0 0 0 0 0

...... OsEIL1b 541 PNAVNPRIQIEEGFFGQGSGIGGSNGGVFEDVNGMMQQPQQTTPAQQQFFIRDDTPFGNQ 600 1 0 0 0 0 0 0 0 0 0 0

3 5 a 6 9 9 5 4 7 9 6 4 4 4 1 0 2 7 8 8 9 6 2 6 7 3 6 7 6 6 5 4 8 8 9 3 4 7

1 9 4 5 1 8 4 3 3 6 9 1 6 2 3 5 7 2 0 2 1 9 5 1 4 7 8

OsEIL1a 601 MGDINGASEFRFGSGFNMSGAVEYPGAMQGQQKNDGSNWYY--- 641 5

2 2 u 2 6 6 7 3 5 1 5 1 6 6 7 7 1 9 1 2 8 5 2 6 1 1 3 2 1 2 8 1 3 7 7 8 8 8

0 0 0 3 0 3 1 4 4 3 0 0 5 5 1 4 1 4 4 2 1 4 4 4 3 3

4 4 d 4 3 4 3 4 4 3 4 4 3 4 4 4 3 3 3 4 4 3 3 4 4 3 3 3 3 3 4 3 4 4 4 4 4 4

S S S S S S S S S S S S S S S S S S S S S S S S S S

OsEIL1b 601 MGDINGASEFRFGSGFNMSGAVEYPGAMQGQQKNDGASEFEELE S

644 9 a SK1/2 - +

A A A A A A A A A A A A A A A A A A A A A A A A A A

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

I I I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D

N N N N N N N N N N N N N N N N N N N N N N N N N N

N Varieties

D E 2 2 4 1 5 9 = = = = n n n Total F3 F5 C 160 n [kDa] 120 c Relative alpha signal

140 3

1 6 - Hap-6 - SK1/2

0 1 2 3 4 1 0 100

1 150

120 9

. - Hap-6 + SK1/2

)

) 80

Control 4 + Hap-6 - SK1/2 b

100 .

100 7

7 NBS c

6 NBS (

(

9 + Hap-6 + SK1/2 9 60

5 0

80 6

GST-OsEIL1b .

. 4

2 75 8

4 T

60 4 a

4 7 8 4 40

. . .

6 1

1 9 6

3 .

4 5

4 2

3 2 2

. 2 .

. 3 .

2 4

2 9

EBS 2 5 EBS 9 a

8 .

2 2

. .

40 1

9 8

. .

3 9 1 0

2 1

1 2

. . .

7 4

1 4

2 5

7 7 3

3 20

. .

1 0 0 0

1 1

1 1 * 0

1 0

0 . 7

1 1 1

1 5

1 9

. 3

4 6

20 .

0 0 0 0 0 0 0 0 0

1 50 ...... pOsGA20ox2pSD1 0 0 0 0 0 0 0 0 0 0

5 4

3 5 a 6 9 9 5 4 7 9 6 4 4 4 1 0 2 7 8 8 9 6 2 6 7 3 6 7 5 8 8 9 3 4 7

1 6

1 9 4 5 1 8 4 3 6 9 1 6 2 3 5 7 2 0 2 1 5 9 5 7 8

2 2 u 2 6 6 7 3 5 1 5 1 6 6 7 7 1 9 1 2 8 5 2 6 1 1 3 2 8 3 7 7 8 8 8

0 0 0 3 0 3 4 4 3 0 0 5 5 1 4 1 4 4 2 3 1 4 3 3

2 3

4 4 d 4 3 4 3 4 4 3 4 4 3 4 4 4 3 3 3 4 4 3 3 4 4 3 3 3 4 4 4 4 4 4 4

S S S S S S S S S S S S S S S S S S S S S S S S Hap-6 - - + +

(-147 to -108) S

9 * a

A A A A A A A A A A A A A A A A A A A A A A A A

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D

N N N N N N N N N N N N N N N N N N N N N N N N

N 37 N SK1/2 - + - + Varieties

Positive effect of both DWH and SNORKEL1/2 on TIL

DWH n = 53 n = 14 160

8 120

3 *

1 140 .

0 - DWH

120 .

)

6 . + DWH

c .

. 100

( 3

80 .

. .

6 .

7 7

TIL (cm) TIL

. .

6 9

3 3

TIL (cm) TIL

. 5

3 2

. .

2 .

2 2

2 5

1 8 2

2 2 .

2 .

9 .

40 9 1

. .

8 . 0

. 9

2 2

2 1

. 5

1 .

4 . .

7 7

3 .

3 3

0 0

1 1

. .

1 5

1 .

1 1

9 9

3 3

4 0 20 . .

0 0 0 0 0 0 0 0 0

......

0 0 0 0 0 0 0 0 0

3 5 5 a 6 9 9 5 4 7 9 6 4 4 9 4 5 4 1 0 1 2 8 4 7 3 8 3 6 8 9 6 9 1 6 2 3 2 5 7 6 2 7 0 2 1 5 9 5 3 1 6 4 7 6 6 5 4 8 8 9 3 4 7 7 8

2 8 2 u 2 6 6 7 3 5 1 5 1 6 6 7 7 1 9 1 2 8 5 2 6 1 1 3 2 1 2 8 1 3 7 7 8 8 8

0 0 0 3 0 3 1 4 4 3 0 0 5 5 1 4 1 4 4 2 3 1 4 4 4 3 3

3 4 2 4 d 4 3 4 3 4 4 3 4 4 3 _ _ _ 4 4 4 _ 3 _ _ 3 _ 3 _ _ 4 4 3 _ _ _ _ _ 3 _ _ 4 _ 4 ______3 _ 3 _ 3 3 3 4 3 4 4 4 4 4 4 _ _ -A +B

9 a

S S S S S S S S S S S S S S S S S S S S S S S S S S S DWH

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

A A A A A A A A A A A A A A A A A A A A A A A A A A A

I I I I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D

N N N N N N N N N N N N N N N N N N N N N N N N N N N Varieties used for GWAS

SNORKEL1/2

160

n = 46 n = 21

140 2

1 6 - SK1/2 120

120 9

)

0 4 + SK1/2

3 100

(

6 80

T 8

_ 60 .

6 .

6 . 7

TIL (cm) TIL

3 1

5 TIL (cm) TIL

2 .

3 8

2 .

0 .

4 9

2 2 2

. 5

8 4

2 .

. . . 9

2 8

2 40

9 .

40 1

1 1 0

7 9

2 8 3

7 7 7

. .

1 4 .

1 1 1

1 3

0 0

1 0

1 1 5

3 3

20 .

0 0 0 0 0 0 0 0 0

2 2

......

0 0 0 0 0 0 0 0 0

0 0

3 5 5 a 6 9 9 5 4 7 9 6 4 4 9 4 5 4 1 0 1 2 8 4 7 3 8 3 6 8 9 6 9 1 6 2 3 2 5 7 6 2 7 0 2 1 5 9 5 3 1 6 4 7 6 6 5 4 8 8 9 3 4 7 7 8

2 8 2 u 2 6 6 7 3 5 1 5 1 6 0 0 0 6 7 7 3 1 0 3 9 1 1 4 4 2 8 5 3 0 0 5 5 2 1 4 6 1 1 4 4 2 3 1 4 1 4 3 4 2 1 2 8 1 3 7 7 8 8 8 3 3

4 2 4 d 4 3 4 3 4 4 3 4 4 3 _ _ _ 4 4 4 _ 3 _ _ 3 _ 3 _ _ 4 4 3 _ _ _ _ _ 3 _ _ 4 _ 4 ______3 _ 3 _ 3 3 3 4 3 4 4 4 4 4 4 _ _

9 a

S S S S S S S S S S S S S S S S S S S S S S S S S S S

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

A A A A A A A A A A A A A A A A A A A A A A A A A A A

I I I I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D B A B N N N N N N N N N N N N N N N N N N N N N N N N N N N SK1/2 - + Accessions used for GWAS

Interaction between DWH and SNORKEL1/2 on TIL DWH and SNORKEL1/2 160

. 140 1

1 6 - DWH - SK1/2

120 . - SK1/2

) + DWH

. - DWH c + SK1/2

100

(

9 5

6 + DWH 5 + SK1/2

_ 60

6 .

9 8

3 1

O 5

0 8

. 0

3 TIL (cm) TIL

3 .

2 9

0 2 2 2

. . . 9

2 9 . 0

. 1

7 9

1 4 .

2 .

7 7 7

4 .

1 1 1 0

1 0

1 . .

9 3 3

5 4

. .

20 0 0 0 0 0 0 0 0 0

2 2

......

0 0 0 0 0 0 0 0 0

3 5 5 a 6 9 9 5 4 7 9 6 4 4 9 4 5 4 1 0 1 2 8 4 7 3 8 3 6 8 9 6 9 1 6 2 3 2 5 7 6 2 7 0 2 1 5 9 5 3 1 6 4 6 6 5 4 8 8 9 3 4 7 7 8

2 8 2 u 2 6 6 7 3 5 1 5 1 6 0 0 0 6 7 7 3 1 0 3 9 1 1 4 4 2 8 5 3 0 0 5 5 2 1 4 6 1 1 4 4 2 3 1 4 1 4 3 4 1 2 8 1 3 7 7 8 8 8 3 3

4 2 4 d 4 3 4 3 4 4 3 4 4 3 _ _ _ 4 4 4 _ 3 _ _ 3 _ 3 _ _ 4 4 3 _ _ _ _ _ 3 _ _ 4 _ 4 ______3 _ 3 _ 3 3 4 3 4 4 4 4 4 4 _ _

9 a

S S S S S S S S S S S S S S S S S S S S S S S S S S S

W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W

A A A A A A A A A A A A A A A A A A A A A A A A A A A

I I I I I I I I I I I I I I I I I I I I I I I I I I I

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D

N N N N N N N N N N N N N N N N N N N N N N N N N N N Varieties used for GWAS

n = 41 n = 5 n = 12 n = 9 120 c

0 1 ・Deepwater rice with high TIL have

0 808 b the DWH.

0 a

4 TIL (cm) TIL 40 ・ a The DWH has a positive effect on

0 2 TIL only in presence of SNORKEL1/2.

0 0 DWH - + - + AA BA AB BB SK1/2 - - + +