New approach for rice improvement using a pleiotropic QTL gene for lodging resistance and yield

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1 Received 28 My 21 Accepted 4 Nov 21 Pulished 3 Nov 21 DOI: 1.138/ncomms1132 New pproch for rice improvement using pleiotropic QTL gene for lodging resistnce nd yield Tiichiro Ookw 1, Tokunori Hoo 2, Mshiro Yno 3, Kzums Murt 4, Tsuyu Ando, Hiroko Miur 1, Kenji Asno 2, Yusuke Ochii 1, Myuko Iked 2, Ryoichi Nishitni 1, Tkeshi Eitni 4, Hidenou Ozki 4, Enrique R. Angeles 2, Tdshi Hirsw 1 & Mkoto Mtsuok 2 The use of fertilizer results in tll rice plnts tht re susceptile to lodging nd results in reduced plnt yields. In this study, using chromosome segment sustitution lines, we identified n effective quntittive trit loci (QTL) for culm strength, which ws designted STRONG CULM2 (SCM2). Positionl cloning of the gene reveled tht SCM2 ws identicl to ABERRANT PANICLE ORGANIZATION1 (APO1), gene previously reported to control pnicle structure. A nerisogenic line crrying SCM2 showed enhnced culm strength nd incresed spikelet numer ecuse of the pleiotropic effects of the gene. Although SCM2 is gin-of-function mutnt of APO1, it does not hve the negtive effects reported for APO1 overexpression mutnts, such s decresed pnicle numer nd norml spikelet morphology. The identifiction of lodgingresistnce genes y QTL nlysis comined with positionl cloning is useful pproch for improving lodging resistnce nd overll productivity in rice. 1 Grdute School of Agriculture, Tokyo University of Agriculture nd Technology, Fuchu, Tokyo , Jpn. 2 Bioscience nd Biotechnology Center, Ngoy University, Ngoy, Aichi , Jpn. 3 QTL Genomics Reserch Center, Ntionl Institute of Agroiologicl Sciences, Tsuku, Irki 3-862, Jpn. 4 Agriculturl Reserch Institute, Toym Agriculturl, Forestry & Fisheries Reserch Center, Toym, Toym , Jpn. Rice Genome Reserch Deprtment, Institute of the Society for Techno-Innovtion of Agriculture, Forestry, nd Fisheries, Tsuku, Irki 3-84, Jpn. Correspondence nd requests for mterils should e ddressed to T.O. (emil: ookw@cc.tut.c.jp). nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

2 nture communictions DOI: 1.138/ncomms1132 Food scrcity is ecoming serious glol prolem ecuse of the constntly incresing world popultion nd the effects of glol climte chnge on food production 1,2. In the 196s, remrkle success ws ttined in incresing grin production through the development nd widespred doption of high-yielding, semidwrf vrieties of whet nd rice. Extensive cultivtion of these vrieties led to mjor increse in grin production, consequently verting predicted lrge-scle fmine. This phenomenl chievement ws referred to s the Green Revolution 3. A mjor fctor in the Green Revolution ws the widespred use of fertilizers in the cultivtion of high-yielding, semidwrf vrieties. Supplementl fertiliztion is not only essentil for incresing grin yield, ut lso promotes stem nd lef elongtion, resulting in n overll increse in plnt height. Under hevy fertiliztion, trditionl vrieties of whet nd rice grow excessively tll nd re susceptile to lodging, which reks their sl culms nd results in significnt yield loss. In contrst, dwrf vrieties respond to high fertilizer inputs nd produce more grin ecuse their short stture mkes them resistnt to lodging. Furthermore, semidwrf vrieties hve high tillering ility nd hrvest index. These re the mjor resons why the Green Revolution douled the yield in oth whet nd rice 6. The success of the Green Revolution convinced mny reeders of the suitility of semidwrf vrieties for incresed crop productivity, nd mny improved semidwrf vrieties hve een developed nd cultivted worldwide 7 9. However, despite the short stture conferred y the semidwrf1 (sd1) gene, lodging remins prolem in mny of the improved rice vrieties. In some whet cultivrs crrying the mjor dwrfing genes Rht1 nd Rht2, lodging ws lso oserved 1. Furthermore, hevy yield losses due to lodging hve een oserved in other crops In n effort to vert nother looming food crisis, rice reeders re developing new vrieties with incresed plnt iomss nd hrvest index to further increse the grin yield. Recently, severl genes ssocited with grin yield hve een identified, including GN1A 16, GS3 17, DEP1 18 nd WFP 19. However, to sustin this reeding ojective, it is lso necessry to develop new strtegies to improve lodging resistnce in rice. Insted of relying solely on the sd1 gene, mjor genes nd quntittive trit loci (QTL) controlling culm strength cn e used in developing improved rice vrieties. However, genes ssocited with culm strength re yet to e identified nd isolted. In this study, QTL nlysis comined with positionl cloning led to the identifiction nd isoltion of mjor QTL conferring lodging resistnce in rice. A ner-isogenic line (NIL) crrying this gene showed enhnced culm strength nd incresed spikelet numer due to pleiotropic effects. These results suggest tht reeding rice vrieties with high grin yield nd enhnced lodging resistnce cn now e pproched using this pleiotropic QTL gene or other genes hving similr functions. Results Culm morphology nd lodging resistnce in chromosome segment sustitution lines. To isolte genes conferring lodging resistnce, the improved indic rice (Oryz stiv) vriety,, ws selected s the donor prent, s it produces very strong culms compred with the typicl jponic vrieties. Furthermore, chromosome segment sustitution lines (CSSLs) from cross etween, nd the jponic vriety (s the recurrent prent) is ville 2. produces out 3 grins per pnicle, yet its plnt rchitecture is cple of supporting hevy pnicles (Fig. 1). In fct, culm dimeter nd culm wll thickness of re much wider nd thicker t the centre of internodes thn tht of jponic vrieties such s nd (Fig. 1). The ending moment t reking (M), prmeter for the physicl strength of the culm, ws mesured using the fourth internode of the plnt. The M vlue of ws much higher thn tht of nd (Fig. 2). The M vlue cn e further divided into two prmeters: the section modulus (SM), which is directly influenced y the culm morphology (for exmple, dimeter nd wll thickness); nd ending stress (BS), which is function of culm cell wll components, such s cellulose nd lignin. To determine the mechnism ehind the high M vlue of, SM nd BS were mesured (Figs 1c nd 2). The SM of ws over two times greter thn tht of the two jponic vrieties. This very high SM vlue of cn e ttriuted to its lrge nd thick culm (Fig. 1). On the other hnd, the BS of ws lower thn tht of the jponic vrieties (Figs 1c nd 2), which indictes tht the composition of the culm in is inferior to tht in the jponic vrieties with c Bending stress (g mm 2 ) 2, 1, 1, V IV III II I Bending moment t reking (g cm) 2, 1, 1, Figure 1 Plnt morphology nd culm strength. () Plnt morphology of nd jponic vrieties nd. () Cross-sections of the culm from the first internode (I: pnicle-neck internode) to the fifth internode (V: sl internode). Scle r: 2 mm. (c) Reltionship etween section modulus nd ending stress. Curved lines indicte the ending moment of the fourth internode t reking. Ornge circle:, red tringle:, lue tringle:, green circle:. nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

3 nture communictions DOI: 1.138/ncomms1132 ARTICLE Bending moment t reking (g cm) c Bending stress (g mm 2 ) 2, 1,8 1,6 1,4 1,2 1, ,8 1,6 1,4 1,2 1, Figure 2 Bending moment t reking, section modulus nd ending stress. () Comprison of the ending moment t reking of the fourth internodes t 2 dys fter heding etween,, nd. () The section modulus of the fourth internodes t 2 dys fter heding. (c) The ending stress of the fourth internodes t 2 dys fter heding. Ech column represents men ± s.d. P <.1, P <.1 nd P <. versus (Dunnett s multiple comprison test, three replictes). respect to lodging resistnce. Actully,, nd most jponic vrieties hve high BS vlues ecuse of elevted levels of lignin nd cellulose in the culms 21. These oservtions clerly indicte tht lodging resistnce in jponic vrieties cn e improved y incorporting the gene from tht increses the culm dimeter. To loclize the loci responsile for high SM in, the M nd SM vlues of the CSSLs were mesured in 2 nd 26 (Fig. 3). Both prmeters were clerly ffected y the environment (growth conditions such s wether nd soil), s most of the lines showed significntly higher vlues thn the originl vriety () in 1 yer ut not the other yer. However, two CSSLs (SL43 nd SL42) showed significntly higher M nd SM vlues thn in oth yers. Thus, the structure of the fourth internode of these lines ws exmined (Supplementry Fig. S1). Compred with, SL43 hd thicker culm wlls wheres SL42 hd wider culm dimeters. SL43 nd SL42 crry genome segment of chromosome (chr.) 1 nd 6 from, respectively. Thus, the QTL locted on chr. 1 is effective for incresing culm wll thickness, wheres the QTL on chr. 6 increses culm dimeter. The QTL on chr. 1 nd 6 were designted s STRONG CULM1 (SCM1) nd STRONG CULM2 (SCM2), respectively. Isoltion nd chrcteriztion of SCM2. For enhnced lodging resistnce in jponic vrieties, incresing the culm dimeter is etter thn incresing the culm wll thickness. The SM of hollow culm is clculted y formule tht considers culm dimeter (d) nd culm wll thickness (t) 22 s follows: 4 4 SM = Π( d ( d 2t) )/ 32 d (1) Apprently, culm dimeter is more effective in incresing M nd SM thn the culm wll thickness, nd requires less investment of iomss (Supplementry Fig. S2). Thus, wider culm dimeter with thinner culm wll mximizes lodging resistnce without reducing yield potentil. Hence, the isoltion nd further chrcteriztion of SCM2 ws pursued insted of SCM1. As the ultimte gol of this study is to utilize lodging-resistnce genes in rice reeding progrmmes, NIL with the introgressed SCM2 locus of ws developed. hs een the most populr vriety in Jpn for mny decdes, nd lodging is the most serious prolem in the cultivtion of this vriety. Using BC F 2 popultion generted from the cross etween nd its sustitution line (SL) crrying the QTL SCM2 region in ( cm of chr. 6), segregtion nlysis for SM ws conducted (Fig. 4). The SM vlues of the segregting F 2 popultion rnged from.9 to 1.3 mm 3. The SM distriution in homozygous, heterozygous nd homozygous lleles ws mm 3 (men of 7.6 mm 3 ), mm 3 (men of 11.2) nd mm 3 (men of 12.1), respectively. These results indicte tht the strong culm conferred y SCM2 is inherited in semidominnt mnner. It lso shows the oscure difference in the phenotype of plnts representing the different genotypes, thus rendering the phenotyping of SCM2 difficult nd sensitive nlysis. A stepwise positionl cloning of SCM2 (Fig. ) ws performed for mpping experiments. For the precise phenotyping of SM, 2 SLs crrying homozygous lleles of the trget region from 6 BC F 3 plnts were selected through mrker-ssisted selection (MAS). Ech of these lines ws phenotyped for the trit nd compred with for two growing sesons. First, using the 2 SLs (BC F 4 ), SCM2 ws mpped to the long-rm region of chr. 6. After using 2, selfed progenies of heterozygous for fine mpping, the cndidte region ws loclized within 1 k. In this region, there is only one gene, ABERRANT PANICLE ORGANIZATION1 (APO1), which encodes n F-ox-contining protein involved in controlling the rchis rnch numer of the pnicle 23. The sequences of APO1 in nd were compred nd were found to contin nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

4 nture communictions DOI: 1.138/ncomms1132 Bending moment t reking (g cm) SCM1 SCM2 SL41 SL42 SL43 SL44 SL4 SL46 SL48 SL49 SL41 SL411 SL412 SL413 SL414 SL41 SL416 SL417 SL418 SL419 SL42 SL421 SL422 SL423 SL424 SL42 SL426 SL427 SL428 SL429 SL43 SL431 SL432 SL433 SL434 SL43 SL436 SL437 SL438 SL439 Bending moment t reking (g cm) SCM 1 SCM 2 SL41 SL42 SL43 SL44 SL4 SL46 SL49 SL413 SL414 SL419 SL42 SL421 SL424 SL427 SL429 SL43 SL432 SL433 c 2 d SCM1 SCM SCM1 SCM2 SL41 SL42 SL43 SL44 SL4 SL46 SL48 SL49 SL41 SL411 SL412 SL413 SL414 SL41 SL416 SL417 SL418 SL419 SL42 SL421 SL422 SL423 SL424 SL42 SL426 SL427 SL428 SL429 SL43 SL431 SL432 SL433 SL434 SL43 SL436 SL437 SL438 SL439 SL41 SL42 SL43 SL44 SL4 SL46 SL49 SL413 SL414 SL419 SL42 SL421 SL424 SL427 SL429 SL43 SL432 SL433 Figure 3 Culm strength nd culm thickness in CSSLs. () The ending moment t reking of the fourth internodes t 2 dys fter heding in 2. () The ending moment t reking of the fourth internodes t 2 dys fter heding in 26. (c) The section modulus of the fourth internodes t 2 dys fter heding in 2. (d) The section modulus of the fourth internodes t 2 dys fter heding in 26. Ech column represents men ± s.d. Blck rs: significnt t P <.1; grey rs: significnt t P <.1 versus (Dunnett s multiple comprison test, three replictes). Blue rs: (recurrent prent), ornge rs: (donor prent). insertions, deletions, single-nucleotide polymorphisms nd insertion of lrge retrotrnsposon (Fig. ). Any of these vritions my hve cused the differences in APO1 function in the two vrieties. To confirm tht APO1 corresponds to SCM2, genomic DNA frgment contining the APO1 locus of ws introduced into the jponic vriety Nipponre through Agrocterium-medited trnsformtion. The APO1 trnsgenic plnts showed wider culm dimeter t internodes s compred with the prentl vriety (Fig.,c), indicting tht APO1 regultes the dimeter of culm internodes. Interestingly, the trnsgenic plnts lso showed thicker culm wll thn the prentl vriety Nipponre (Fig. ), suggesting tht SCM2 lso regultes culm wll thickness, lthough no difference in culm wll thickness etween SL42 nd the originl vriety ws oserved (Supplementry Fig. S1). On the sis of the oservtions of APO1-overexpressing mutnts 24, different levels of APO1 expression hd vrying effects on the phenotype of plnt orgns, especilly the rice pnicle. As plnts trnsformed with genomic DNA contining the APO1 locus my not e identicl, the difference in phenotype etween the introgressed nd trnsgenic lines my e explined y differences in APO1 expression levels. Evlution of SCM2 for lodging resistnce. To evlute the effect of SCM2 on lodging resistnce, n NIL crrying 484-k region from the distl prt of chr. 6 of (; Fig. d) ws developed. There ws no difference in the flowering time etween nd. The APO1 expression levels in, nd were compred (Fig. e). The APO1 expression level in ws similr to in most of the plnt orgns. However, in the inflorescence pex t 3 nd 2 dys efore heding, the APO1 expression level ws out 2 3 times higher in nd s compred with. The culm dimeter of nd ws lso compred. In, the dimeters of the minor nd mjor xes in ll the internodes were significntly wider thn in (Figs 1 nd 6). Consequently, the SM of ws lso higher thn tht of (Fig. 2). The BS of nd ws higher thn tht of (Figs 1c nd 2), ut the M vlue of the culm ws sximilr to (Fig. 2). The size of the inflorescence meristem of ws pprently lrger thn tht of (Fig. 7,), nd the numer of prenchym cells in the sl internode of ws out 38% higher thn tht in (Fig. 7c f). These oservtions suggest tht the high APO1 expression level in the inflorescence pex of cuses the incresed culm dimeter y promoting ctive cell division in the meristem (see Discussion). To determine the usefulness of SCM2 in prcticl reeding, the performnce of the gene ws further evluted under field nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

5 nture communictions DOI: 1.138/ncomms1132 ARTICLE c d 8 6 (7.6 mm 3 ) Numer of plnts 4 2 (24.1 mm 3 ) ~6 6~7 7~8 8~9 9~1 1~11 11~12 12~13 13~14 14~1 1~16 16~17 17~18 18~19 19~2 2~21 21~22 22~23 23~24 24~2 Figure 4 Frequency distriution of section modulus in BC F 2 popultion. ( c) Chromosome mps of (), Koshi-QTL SCM2 heterozygous () nd Koshi-QTL SCM2 homozygous (c). Blck rs: cm of the sixth chromosome. (d) Frequency distriution of the section modulus in BC F 2 popultion produced y crossing etween nd its sustitution line crrying the QTL SCM2 region ( cm of the sixth chromosome). Ornge, lue nd red rs indicte the numer of plnts crrying the homozygous, heterozygous nd homozygous llele t the QTL SCM2 region (18.6 cm), respectively. conditions. Figure 8 shows the overll plnt stture of, nd crrying the null llele of sd1 (ref. 16), which ws widely used in the Green Revolution of rice 8. ws similr in plnt height to. On the other hnd, NILsd1 ws shorter ecuse of deficiency in gierellin synthesis 8,2. Apprently, the resistnce of ginst the ending type of lodging ws improved. The mesured vlue for the lodgingresistnce fctor (clr) of the line showed tht it ws similr or slightly etter thn (Fig. 8). In the utumn of 29, typhoon hit the experimentl field where nd were plnted. Although there ws severe lodging of plnts in the plot, the plnts were not ffected y the strong wind nd remined stnding (Fig. 8c). This unexpected oservtion strongly demonstrtes the effectiveness of SCM2 in enhncing lodging resistnce of jponic vriety. It lso shows the potentil of utilizing lodging-resistnce gene(s) s new pproch in reeding rice vrieties for improved lodging resistnce. Effects of SCM2 on crop productivity., nd were lso evluted for the grin yield, nd two importnt components of yield, the totl iomss nd spikelet numer. The totl iomss of nd ws similr, wheres produced 87% less iomss thn owing to its shorter height (Fig. 8d). More importntly, the grin yield per hectre of ws significntly higher thn tht of nd (Fig. 8e). These differences in grin yield depend minly on spikelet numers; for exmple, plnts produced more spikelets per pnicle thn (for exmple, 188 per pnicle for s compred with 16 for ; Fig. 8f,g). Consequently, SCM2 incresed the spikelet numer of y out 2%. This increse in spikelet numer y SCM2 ws cused minly y the increse in the secondry rnch numer of the pnicle (Fig. 8h,i). The secondry rnch numer per pnicle of ws out 38, wheres hd out 3 (Fig. 8i), resulting in n increse of 2%. The increse in the secondry rnching of the pnicle nd spikelet numer in is positive, pleiotropic effect of APO1. Indeed, the trnsgenic plnts crrying the APO1 gene showed 2% increse in the spikelet numer reltive to the prentl vriety, Nipponre (Supplementry Fig. S3), lthough trnsgenic plnts grown in the greenhouse produced fewer spikelets thn plnts grown in the field. In contrst to, productivity of ws considerly reduced in oth totl iomss nd spikelet numer (Fig. 8d,g). Previously, two ppers reported n incresed numer of spikelets in APO1-overexpressing mutnts, po1-d nd Ur1, ut with drmtic decrese in the pnicle numer per plnt 24,26. As this negtive effect of APO1 on pnicle numer per plnt decreses the crop productivity 26, we compred the pnicle numer per plnt of nd NILsd1 with (Fig. 8j). The pnicle numer per plnt in nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

6 nture communictions DOI: 1.138/ncomms1132 V IV III II I 6S RM6274 Centromere 1 SCM RM264 6L Prentl Nipponre Nipponre trnsformed with APO1 gene SCM InDel9 SCM InDel RAP2 locus 28,34 k 28,3 k 28,36 k 28,37 k 28,38 k Os6g6648 Os6g66 Os6g661 Os6g66 RAP2 trnscript (representtive cdna) AK11988 AK69711 AK1133 AK73262 Predicted locus Os6g6649 Os6g661 Os6g664 Os6g664 Os6g662 c Dimeter of mjor nd minor xes (mm) d Os6g664 (APO1) 2.6 k +1 k 3 k 2 k 1 k. k 9. k insertion Retrotrnsposon (Ty3-gypsy suclss) 1 p deletion 2 p deletion 12 p V I G R insertion 9 p deletion e Reltive expression level of APO Lef Root VSA IFA3 IFA2 Lemm Mture Anther Pistil stem Figure Loction nd gene structure of SCM2. () Loction of SCM2 nd comprison of the gene sequence of APO1 etween nd. The SCM2 locus ws predicted etween the mrkers RM639 nd RM9 on chromosome 6. Course mpping nrrowed the SCM2 locus etween RM246 nd RM262. The cndidte region of SCM2 ws nrrowed down to the 1 k etween InDel2 nd InDel8 y fine mpping. The numer of recominnts etween moleculr mrkers is indicted elow the mps. Nucleotide replcements re shown in ginst. () Culm cross-sections from the first (I: pnicle-neck internode) to the fifth (V: sl internode) internodes in Nipponre trnsformed with the APO1 locus nd n empty vector. Scle r: 2 mm. (c) The dimeter of mjor nd minor xes from the first to the fifth internode of the min culms etween the APO1 trnsgenic nd control plnts. Ech column represents men ± s.d. (n = ); P <.1 nd P <. (Student s t-test). (d) Grphicl genotype of on rice chromosomes. The red r indictes the chromosome segment (484 k) of including APO1. (e) Expression nlysis y rel-time PCR of APO1 in vrious orgns. VSA, vegettive shoot pex, IFA3, inflorescence pex t 3 dys efore heding, IFA2, inflorescence pex t 2 dys efore heding. Ech column represents men ± s.d. (three replictes); P <.1; P <. versus (Dunnett s multiple comprison test). nd ws similr, wheres hd slightly more pnicles thn, proly ecuse of its short stture. Thus, in contrst to previously reported APO1-overexpressing mutnts, shows positive effect of APO1, tht is, n increse in the spikelet numer per pnicle without decrese in the pnicle numer (see Discussion). Therefore, SCM2 confers lodging resistnce nd lso increses grin yield. Discussion To dte, short plnt stture hs een the mjor trget for improvement of lodging resistnce in rice ecuse it confers lower susceptiility to lodging nd higher hrvest index. In fct, the use of semidwrf genes such s sd1 nd Rht1 successfully eliminted the lodging prolem in rice nd whet, respectively, nd lso incresed the crop productivity. However, in jponic vrieties tht normlly produce wek nd fine culms, sd1 is ineffective in enhncing the culm strength. Hence, the lodging resistnce of these vrieties hs een improved y decresing the length nd weight of the oveground prts of the plnt 27. The use of lodging-resistnce genes in ddition to the dwrfing gene is promising new pproch in improving lodging resistnce nd further incresing productivity in rice. Before this study, lodging-resistnce genes hd not een identified. It is possile tht the genes involved in strong nd/or thick culm lso regulte the overll development of the culm, nd often cuse negtive pleiotropic effects on the development of other orgns, such s the lef, flower nd grin. Thus, we performed QTL nlysis for lodging resistnce using high-yielding rice vriety with strong, thick culms. From, n improved high-yielding indic vriety with strong nd thick culms, we isolted QTL tht effectively controls culm strength. This QTL, which ws locted on chr. 6 nd designted s SCM2, lso increses the spikelet numer of the pnicle s pleiotropic effect. NIL introgressed with SCM2 from showed significntly enhnced culm strength, incresed spikelet numer nd grin yield. This result shows tht the gene cn e effective in improving lodging resistnce, nd yield in jponic vrieties. It lso indictes tht the use of genes involved in culm strength cn nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

7 nture communictions DOI: 1.138/ncomms1132 ARTICLE 7 Dimeter of mjor nd minor xes (mm) V Minor V Mjor IV Minor IV Mjor III Minor III Mjor II Minor II Mjor I Minor I Mjor Neck minor Neck mjor IV III II I Figure 6 Culm dimeter nd section modulus in in comprison with nd. () Comprisons of dimeter in mjor nd minor xes from the neck node nd the first (I: pnicle-neck internode) to the fifth (V: sl internode) internodes mong, nd. Ornge rs:, lue rs:, red rs:. Ech column represents men ± s.d. (three replictes). () Comprisons of section modulus from the first to the fourth internodes mong, nd. Ornge rs:, lue rs:, red rs:. Ech column represents men ± s.d. (three replictes). P <.1; P <.1; P <. versus (Dunnett s multiple comprison test). e used in reeding oth jponic nd indic vrieties for incresed lodging resistnce. Although SCM2 is common mong indic vrieties, other QTL hving similr ction s SCM2 (for exmple, weker lleles of Ur1) cn e identified nd used in indic reeding. Positionl cloning nd sequencing of SCM2 reveled tht the gene is identicl to APO1, which encodes n F-ox protein orthologous to Aridopsis UFO 23. Through detiled nlysis of the APO1- overexpressing mutnt, po1-d, Iked-Kwmitsu et l. 24 reported tht APO1 regultes the cell prolifertion rte in the inflorescence meristem. They further oserved tht high expression level of APO1 increses the size of the inflorescence meristem, resulting in n incresed numer of spikelets. The isoltion of po1-deficient mutnts, which produce smller inflorescence meristem 23, supports positive role for APO1 in cell prolifertion. Although previous studies on APO1 24,26,28 focused on its effect on pnicle structure, the incresed cell division in the inflorescence meristem lso impcts the size of the sl prt of the inflorescence meristem (Fig. 7); s consequence, culm size is lso ffected ecuse culm cells re produced from this region 29. Thus, ny increse in the cell prolifertion rte in the inflorescence meristem inevitly leds to n increse in the spikelet numer nd culm dimeter, oth of which re fvourle trits in terms of crop productivity. nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

8 nture communictions DOI: 1.138/ncomms1132 f 1, Numer of prenchym cells 8, 6, 4, 2, c d e Figure 7 Morphologicl chrcteristics of the shoot pex nd the sl internode. (, ) The longitudinl sections stined with propidiumiodide of the shoot pex region of () nd () t 3 dys efore heding. The rrows indicte the dimeter size t the sl prt of pnicle for ech plnt. Scle r: 1 µm. (c e) The cross-sections stined with crystl violet of the fourth internodes in (c), (d) nd (e) t 2 dys fter heding. Scle r: 1 µm. (f) The numer of prenchym cells in the cross-section re of the fourth internode of in comprison with nd. Ech column represents men ± s.d. (three replictes). P <.1; P <. versus (Dunnett s multiple comprison test). Why re overexpressing mutnts of APO1 not used for prcticl reeding? Muri nd Iizw 3 oserved n incresed spikelet numer in the APO1-overexpressing mutnt, Ur1, nd exmined the possiility of mutting the gene to further increse the grin yield. According to their results, the numer of spikelets per pnicle of plnts crrying homozygous or heterozygous Ur1 ws out 2, which is 1% more thn the originl vriety. However, the numer of pnicles per plnt ws reduced to out 7% in oth genotypes; thus, the spikelet numer did not differ significntly etween the mutnts nd the wild vriety. Moreover, the Ur1 plnts exhiited decresed kernel weight nd spikelet fertility nd norml pnicle morphology 31, especilly t lower tempertures 32. The Hokkido Ntionl Agriculturl Experiment Sttion in Jpn lso ttempted to develop high-yielding cultivrs in its reeding progrm using the Ur1 mutnt, ut filed ecuse of the norml phenotypes mentioned ove. Similr undesirle ut more severe normlities were oserved in trnsgenic plnts overproducing APO1 under the control of culiflower mosic virus 3S or mize UBIQUITIN promoter. These trnsgenic plnts produced n excessively rnched inflorescence without florets, nd tiller outgrowth ws suppressed 24. Agin, the severity of phenotype normlities exhiited y the plnts ws dependent on APO1-expression level, confirming tht the APO1 ffects the morphology of the pnicle. When overexpressing mutnts nd were compred, APO1 expression ws higher in APO1-overexpressing mutnts in vrious plnt orgns, including the inflorescence meristem. Wheres the expression level of the mutnts ws reportedly much higher thn tht of wild-type plnts 24, APO1 expression in ws only out two-fold higher thn tht in the prentl vriety (Fig. e). In the cse of the Ur1 mutnt, the incresed level of expression of APO1 ws oserved in ll prts of the seedlings 1 dys fter germintion, wheres such upregultion ws not oserved in the vegettive tissues of (Fig. e). The expression of APO1 in the vegettive tissues of the Ur1 mutnt, therefore, my hve cused the suppression of tiller outgrowth, resulting in decresed pnicle numer per plnt. Previously, we predicted possile situtions in which conflicts etween gene function nd suitility of the trit ffect the overll cceptility of n llele for reeding 33. In this context, we discussed the possiility tht lleles with greter gin-of-function my cuse detrimentl side effects to the trit tht it is regultes. In such situtions, n llele hving more moderte level of function is preferle to those with gretly incresed function. For exmple, the APO1 lleles in the overexpressing mutnts exhiit lrge gin-of-function ctivity, with side effects not suitle for reeding. While these stronger lleles cn significntly increse the spikelet numer, they lso reduce the pnicle numer per plnt resulting in little or negtive gin in grin yield. In contrst, the QTL SCM2, weker llele of APO1, is more suitle for reeding s it increses spikelet numer without reducing pnicle numer. Identifying superior lleles of griculturlly importnt genes in nturl vrints cn e ccomplished through llele mining, n emerging new moleculr pproch in crop improvement. In the pst, rice geneticists nd reeders hve collected nd generted mny rice mutnt lines. However, most of these mutnts hve not een utilized in reeding progrms ecuse they convey undesirle or non-productive trits. However, the identifiction nd proven effectiveness of SCM2, wek llele of APO1, opens possiilities for finding superior nd more suitle lleles in nturl vrints. Thus, comintion of mutnt studies nd QTL nlysis nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

9 nture communictions DOI: 1.138/ncomms1132 ARTICLE d Totl iomss (t h 1 ) 1 1 e Grin yield (t h 1 ) clr vlue (g cm 1 ) c g Spikelet numer per pnicle h Primry rnch numer per pnicle 1 1 f i Secondry rnch numer per pnicle j Pnicle numer per plnt Figure 8 Lodging resistnce nd yield performnce of compred with nd. () Morphology of, nd t the ripening stge. () clr vlues. (c) Comprison of lodging in nd fter strong typhoon. (d, e) Comprison of totl iomss production (d) nd unhulled grin yield (e). (f) Pnicle structure of, nd. Scle rs: cm. (g i) Comprison of spikelet (g), primry rnch (h) nd secondry rnch (i) numers per pnicle oserved from the min stem of, nd. ( j) Pnicle numer per plnt mong, nd. Ech column represents men ± s.d. (three replictes). P <.1; P <.1; P <. versus (Dunnett s multiple comprison test). using nturl vrition re importnt strtegies for identifying genes tht cn further increse crop productivity. Methods Plnt cultivtion for precise phenotyping. Field experiments for the precise phenotyping of lodging-resistnce trits were conducted t the Experimentl Frm of Field Science Center, Tokyo University of Agriculture nd Technology, in Becuse the sme cultivtion prctice ws performed for ll yers, only the cultivtion methods for 29 re descried here. Seeds were sown in nursery oxes on 7 My 29. Seedlings t the fifth-lef stge were trnsplnted to pddy field on Tm River lluvil soil using one seedling per hill on 28 My 29. The plnting density ws 22.2 hills per m 2, with spcing of 1 3 cm. Mnure ws pplied t the rte of out 2 tons per hectre efore puddling, wheres complete fertilizer contining 14% ech of N, P 2 O nd K 2 O ws pplied s sl dressing t the rte of kg N, P nd K per hectre. The field ws mintined under sumerged conditions throughout the course of the experiments. The experiments were designed with three rndomly rrnged replictes (4 m 2 for ech plot). Precise phenotyping for lodging-resistnce trits. The physicl prmeters of the min culm, which re closely ssocited with lodging resistnce, were used for precise phenotyping. Eight min culms were smpled from ech plot to determine the verge lef numer, nd the ending lod t reking ws mesured t distnce of 4 cm etween two supporting points y the method of Ookw nd Ishihr 27 using universl testing mchine (Tensilon RTM-2, Orientec). The Tensilon is useful tool for precise evlution of lodging-resistnce trits in rice. Physicl prmeters were clculted y the following formul 27,34 : M = sectionmodulus endingstress. M is the ending moment of the sl internode t reking (g cm). Sectionmodulus( SM) = Π/ 32 ( )/ 1, where, 1 is the outer dimeter of the minor xis in n ovl cross-section, 1 is the outer dimeter of the mjor xis in n ovl cross-section, 2 is the inner dimeter of the minor xis in n ovl cross-section nd 2 is the inner dimeter of the mjor xis in n ovl cross-section. BS is mechnicl prmeter for lodging resistnce, which is influenced y the chemicl composition of the culm such s cellulose nd lignin (see Results). The lodging-resistnce fctor (clr), prmeter for evluting resistnce to ending type of lodging ws lso mesured. The clr vlue of the plnt ws determined following the method of Grfius nd Brown 3. QTL detection using CSSLs. Detection of QTL for lodging resistnce ws conducted in 2 nd 26. A set of 39 CSSLs derived from the ckcrossed progenies involving the jponic vriety (recurrent prent) nd the high-yielding nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

10 nture communictions DOI: 1.138/ncomms1132 indic vriety, 2 (donor prent), ws used for the experiment. A QTL ws considered to e present when the verge vlue of three replictes ws significntly different etween CSSL nd the recurrent prent in 2 yers, ccording to Dunnett s multiple comprison test. Mpping nd NIL production with SCM2. To dissect the puttive QTL for culm strength on chr. 6 of, BC F 2 popultion ws generted from cross etween nd its SL crrying the QTL SCM2 region. To mp SCM2, 2 plnts with recomintion in the vicinity of the QTL were identified from 6 BC F 3 plnts through MAS. Recominnt homozygous plnts (BC F 4 ) were then further selected from the selfed progenies of these 2 BC F 3 plnts on the sis of MAS nd were used to further define the QTL region. The phenotype of the recominnt homozygous lines ws compred with in 28 nd 29. Similrly, selected from the BC F 3 genertion using MAS ws used to evlute the effects of the QTL on lodging resistnce for two consecutive sesons. Genomic DNA ws extrcted from leves using the cethyltrimethylmmonium romide (CTAB) method 36, nd pure DNA smples were used for genotyping using single-sequence repet mrkers 37,38. Fine mpping nd positionl cloning of SCM2. A totl of 2, selfed progenies (BC F 3 ) of heterozygote derived from the cross were used for the comined nlysis of fine mpping nd positionl cloning of SCM2. Phenotyping of SCM2 ws performed using the BC F genertion. For fine mpping, single-sequence repet mrkerss nd newly developed InDel mrkers identified y using rice genome sequences in the NIAS nd STAFF dtses ( were used. The primer sets of the InDel mrkers used re listed in Supplementry Tle S1. A BAC clone (J18E9) covering the cndidte region ws selected from the BAC lirry nd sequenced. DNA frgments of (HABA61H13) nd within 1 k of the cndidte region were then compred. Trnsformtion. Full-length genomic DNA of APO1, including the nd 3 regions (1. k), ws inserted to the inry vector pbi-hm12, which contins hygromycin-resistnce gene. The DNA frgment ws introduced into the jponic vriety Nipponre y Agrocterium tumefciens-medited trnsformtion. More thn ten independent T plnts were generted nd five plnts were used for ssy. As control, trnsformnts contining n empty vector were included, nd used five control plnts in ech ssy. RNA isoltion nd expression nlysis. Totl RNA from vrious orgns ws prepred s descried y Smrook et l. 39 nd ws treted with DNse I. Firststrnd complementry DNA (cdna) ws synthesized from 1 µg of totl RNA using n Omniscript reverse trnscription kit (Qigen). Quntittive rel-time PCR ws performed using the SYBR Green PCR Mster Mix (Applied Biosystems) on n utomted DNA sequence detector system (Model 7; Applied Biosystems). The results were confirmed using three independent iologicl replictes. The UBC (AK9694; uiquitin-conjugting enzyme E2) gene from rice ws used s n internl stndrd for normlizing vritions in cdna concentrtion. The primer sets used for PCR re listed in Supplementry Tle S2. Oservtion for the longitudinl section of shoot pex. Tissue smples were fixed overnight in 4% (v/v) prformldehyde in 2 mm NPO 4 uffer, ph 7., t 4 C. Smples were then dehydrted in grded series of ethnol concentrtions, infiltrted with the polymethcryl resin Technovit 71 (Hereus Kulzer), followed y emedding nd polymeriztion in Technovit 71, nd were oserved with light microscope (BX-; Olympus). Oservtion of the cross-section of sl internode. The fourth internodes were smpled t 2 dys fter heding nd fixed in FAA fix solution (formlin (3%): cetic cid: lcohol (7%) = 1:1:18). Tissue smples were stined y.% crystl violet solution. The cross-section of internodes ws oserved with digitl microscope (SZX12; Olympus) nd the prenchym cells were counted using digitl imge processing softwre (Win-ROOF version 6.; Mitni). Sttisticl nlysis. Sttisticl comprison of multiple sets of dt ws crried out using Dunnett s multiple comprison test nd Tukey s significnt difference test. References 1. Brown, M. E. & Funk, C. C. Food security under climte chnge. Science 319, 8 81 (28). 2. Beddington, J. Food security: contriutions from science to new nd greener revolution. Phil. Trns. R. Soc. B 36, (21). 3. Hrgrove, T. R. & Cnill, V. L. The impct of semi-dwrf vrieties on Asin rice-reeding progrms. BioScience 29, (1979). 4. Dlrymple, D. G. Development nd Spred of High-Yielding Rice Vrieties in Developing Countries (Bur. Sci. Tech., Agency for Int. Dev., 1986).. Khush, G. S. Green revolution: prepring for the 21st century. Genome 42, (1999). 6. Khush, G. S. Modern vrieties their rel contriution to food supply nd equity. GeoJournl 3, (199). 7. Asno, K. et l. Genetics nd moleculr nlysis of utility of sd1 lleles in rice reeding. Breed Sci. 7, 3 8 (27). 8. Ashikri, M. et l. Loss-of-function of rice gierellin iosynthetic gene, GA2 oxidse (GA2ox-2), led to the rice green revolution. Breed Sci. 2, (22). 9. Peng, S. et l. Grin yield of rice cultivrs nd lines developed in the Philippines since Crop. Sci. 4, (2). 1. Berry, P. M., Spink, J. H., Gy, A. P. & Crigon, J. A comprison of root nd stem lodging risks mong winter whet cultivrs. J. Agric. Sci. 141, (23). 11. Berry, P. M. et l. Understnding nd reducing lodging in cerels. Adv. Agron. 84, (24). 12. Bord, J. Reduced lodging for soyen in low plnt popultion is relted to light qulity. Crop. Sci. 41, (21). 13. Sherry, A. F.- G., Jmptong, C., Drrh, L. L. & Mcmullen, M. D. Quntittive trit locus nlysis of stlk strength in four mize popultions. Crop. Sci. 43, (23). 14. Tripthi, S. C., Syre, K. D. & Kul, J. N. Plnting systems on lodging ehvior, yield components, nd yield of irrigted spring red whet. Crop. Sci. 4, (2). 1. Ymmoto, H. & Iwy, K. Influence of typhoon on the pddy rice production in Kyushu nd Ymguchi Districts in 24 nd slty wind dmge of rice cused y Typhoon 418 (SONGDA). Jpn. J. Crop. Sci. 7, 3 41 (26). 16. Ashikri, M. et l. Cytokinin oxidse regultes rice grin production. Science 39, (2). 17. Fn, C. et l. GS3, mjor QTL for grin length nd weight nd minor QTL for grin width nd thickness in rice, encodes puttive trnsmemrne protein. Theor. Appl. Genet. 112, (26). 18. Hung, X. et l. Nturl vrition t the DEP1 locus enhnces grin yield in rice. Nt. Genet. 41, (29). 19. Miur, K. et l. OsSPL14 promotes pnicle rnching nd higher grin productivity in rice. Nt. Genet. 42, 4 49 (21). 2. Ando, T. et l. Genetic dissection nd pyrmiding of quntittive trits for pnicle rchitecture y using chromosoml segment sustitution lines in rice. Theor. Appl. Genet. 116, (28). 21. Ookw, T. & Ishihr, K. Vrietl difference of the cell wll components ffecting the ending stress of the culm in reltion to the lodging resistnce in pddy rice. Jpn. J. Crop. Sci. 62, (1993). 22. Berry, P. M., Sylvester-Brdley, R. & Berry, S. Ideotype design for lodging-resistnt whet. Euphytic 14, (27). 23. Iked, K. et l. Rice ABERRANT PANICLE ORGANIZATION1, encoding n F-ox protein, regultes meristem fte. Plnt J. 1, (27). 24. Iked-Kwktsu, K. et l. Expression level of ABERRANT PANICLE ORGANIZATION1 determines rice inflorescence form through control of cell prolifertion in the meristem. Plnt Physiol. 1, (29). 2. Sski, A. et l. A mutnt gierellin-synthesis gene in rice. Nture 6913, (22). 26. Muri, M. & Iizw, M. Effects of mjor genes controlling morphology of pnicle in rice. Breed Sci. 44, (1994). 27. Ookw, T. & Ishihr, K. Vrietl difference of physicl chrcteristics of the culm relted to lodging resistnce in pddy rice. Jpn. J. Crop. Sci. 61, (1992). 28. Tero, T., Ngt, K., Morino, K. & Hirose, T. A gene controlling the numer of primry rchis rnches lso controls the vsculr undle formtion nd hence is responsile to increse the hrvest index nd grin yield in rice. Theor. Appl. Genet. 12, (21). 29. Kwhr, H., Chonn, N. & Wd, K. Interreltion of the growth mong leves, pnicles nd internodes, nd histologicl oservtion on the meristem of culm. Proc. Crop. Sci. Soc. Jpn. 37, (1968). 3. Muri, M. & Iizw, M. Effects of mjor genes controlling morphology of pnicle in rice. Breed Sci. 44, (1994). 31. Kinoshit, T. & Tkhshi, M. The hundredth report of geneticl studies on rice plnts. Linkge studies nd future prospects. J. Fc. Agr. Hokkido Univ. 6, 1 61 (1991). 32. Muri, M., Kinoshit, T. & Tkhshi, M. Influences of cold wter for the chrcter expression of the six kinds of mjor genes responsile for pnicle type. Geneticl studies on rice plnt, LXXIX. Res. Bull. Univ. Frm Hokkido Univ. 22, (1981). 33. Tked, S. & Mtsuok, M. Genetic pproches to crop improvement: responding to environmentl nd popultion chnges. Nture 9, (28). 34. Ookw, T. et l. Biomss production nd lodging resistnce in Lef Str, new long-culm rice forge cultivr. Plnt Prod. Sci. 13, 6 64 (21). 3. Grfius, J. E. & Brown, H. M. Lodging resistnce in ots. Agron. J. 46, (194). 36. Murry, M. G. & Thompson, W. F. Rpid isoltion of high moleculr weight plnt DNA. Nucl. Acids Res. 8, (198). 37. McCouch, S. R. et l. Development nd mpping of 224 new SSR mrkers for rice (Oryz stiv L.). DNA Res. 9, (22). 38. Interntionl Rice Genome Sequencing Project. The mp-sed sequence of the rice genome. Nture 436, (2). 39. Smrook, J., Fritsch, E. F. & Mnitis, T. Moleculr Cloning: A Lortory Mnul (Cold Spring Hror Lortory Press, 1989). 1 nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved.

11 nture communictions DOI: 1.138/ncomms1132 ARTICLE Acknowledgments A genomic DNA clone contining APO1 in the inry vector pbi-hm12 ws provided y Dr H. Hirno t the University of Tokyo. This work ws supported y grnt from the Ministry of Agriculture, Forestry nd Fisheries, Jpn (Green Technology Project; QT-29 nd Genomics for Agriculturl Innovtion; IPG-3). Author contriutions T.O., H.M., Y.O., R.N. nd T.H. performed the phenotyping nd genotyping for lodging resistnce of the sustitution lines for QTL nlysis. M.Y. nd T.A. contriuted to the genotyping for fine mpping; K.M., T.E. nd H.O. generted the sustitution lines nd NILs; T.H., K.A., M.I., E.R.A. nd M.M. performed positionl cloning experiments, sequencing nd gin of function nlysis; T.O., M.M., E.R.A. nd T.H. wrote the pper. Additionl informtion Supplementry Informtion ccompnies this pper on nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is ville online t reprintsndpermissions/ How to cite this rticle: Ookw, T. et l. New pproch for rice improvement using pleiotropic QTL gene for lodging resistnce nd yield. Nt. Commun. 1:132 doi: 1.138/ncomms1132 (21). License: This work is licensed under Cretive Commons Attriution-NonCommercil- Shre Alike 3. Unported License. To view copy of this license, visit cretivecommons.org/licenses/y-nc-s/3./ nture communictions 1:132 DOI: 1.138/ncomms Mcmilln Pulishers Limited. All rights reserved. 11