Abstract. of the QTL of parameters of response curves provides a promising alternative for dealing with the genetic variability of adaptive traits.

Transcription

1 Journl of Experimentl Botny, Vol. 55, No. 47, Wter-Sving Agriculture Specil Issue, pp , November 24 doi:1.193/jxb/erh2 Advnce Access publiction 3 July, 24 Deling with the genotype3environment interction vi modelling pproch: comprison of QTLs of mize lef length or width with QTLs of model prmeters Mtthieu Reymond*, Bertrnd Muller nd Frncxois Trdieu INRA ENSAM1, Lbortoire d Ecophysiologie des Plntes sous Stress Environnementux, 2, Plce Pierre Vil, F-346 Montpellier cedex 1, Frnce Received 8 December 23; Accepted 14 My 24 Abstrct Quntittive genetics of dptive trits is mde difficult by the genotype3environment interction. A clssicl ssumption is tht QTLs identified in both stressed nd control conditions correspond to constitutive trits wheres those identified only in stressed tretments re stress-specific nd correspond to dptive trits. This hypothesis ws tested by compring, in the sme set of experiments, two wys of nlysing the genetic vribility of the responses of mize lef growth to wter deficit. One QTL detection ws bsed on rw phenotypic trits (length nd width of lef 6) of 1 recombinnt inbred lines (RILs) in four experiments with either well-wtered or stressing conditions in the field or in the greenhouse. Another detection followed method proposed recently which consists of nlysing intrinsic responses of the sme RILs to environmentl conditions, determined jointly over severl experiments. QTLs of three responses were considered: (i) lef elongtion rte per unit therml time in the bsence of stress, (ii) its response to evportive demnd in well-wtered plnts, nd (iii) its response to soil wter sttus in the bsence of evportive demnd. The QTL of lef length differed between experiments, but coloclized in seven cses out of 13 with QTLs of the intrinsic lef elongtion rte, even in experiments with stressing conditions. No colocliztion ws found between QTLs of lef length under wter deficit nd QTLs of responses to ir or soil wter sttus. By contrst, QTLs of lef width coloclized in ll experiments, regrdless of environmentl conditions. The clssicl method of identifying the QTL of constitutive versus dptive trits therefore did not pply to the experiments presented here. It is suggested tht identifiction of the QTL of prmeters of response curves provides promising lterntive for deling with the genetic vribility of dptive trits. Key words: Genotype3environment interction, lef growth, QTL, wter deficit. Introduction The concepts of dptive versus constitutive chrcters differ ccording to the scientific communities which use them. When pplied to gene expression or protein mounts, constitutive chrcter is supposed to be independent of environmentl conditions, while the opposite pplies to n dptive chrcter (Seki et l., 22). When pplied to phenotypic trits, the definition usully shifts towrds the existence or non-existence of genotype3environment (G3E) interction on the considered trit with positive effect on yield (Blum, 1996). An dptive trit is then defined s n ltertion in plnt structure or function which improves the behviour under stress of the considered genotype (e.g. reduction in trnspirtion rte, llowing the plnts to conserve wter through to the end of the crop cycle). Conversely, constitutive trit is either unffected by environmentl conditions, or is ffected by similr mounts in ll studied genotypes (no G3E interction). Although it does not respond to wter stress, constitutive trit cn bring comprtive dvntge under wter deficit (e.g. trnspirtion efficiency under well-wtered conditions, deep root system, or erly vigour; Richrds et l., 22). Breeding for constitutive trits hs yielded severl success stories. QTLs of deep rooting coloclize with QTLs of yield under wter deficit (Tuberos et l., 22); improving * Present ddress: CEA LBDP. Lbortoire de Biologie et du Développement des Plntes. Cdrche, Frnce. y To whom correspondence should be ddressed. Fx: E-mil: frncois.trdieu@ensm.inr.fr Journl of Experimentl Botny, Vol. 55, No. 47, ª Society for Experimentl Biology 24; ll rights reserved

2 2462 Reymond et l. wter-use efficiency of well-wtered plnts increses whet yield under severe wter deficit (Condon et l., 22). By contrst, breeders re often reluctnt to consider dptive trits ssocited with lrge built-in G3E interction which lowers heritbility. A clssicl wy to identify QTLs of dptive trits is to set up network of experiments with contrsting environmentl conditions nd identify QTLs in ll experiments seprtely. It is then ssumed tht QTLs identified in both stressed nd control conditions will correspond to constitutive trits, wheres those identified in stressed tretments only will be stress-specific nd will correspond to dptive trits (Prioul et l., 1997; Ribut et l., 1997; Frcheboud et l., 22). This is bsed on the hypothesis tht no QTLs re detected in stressed tretments, but re missed in control tretments for sttisticl resons. This hypothesis cnnot be considered s likely, thereby leding to n overestimtion of the frequency of dptive QTLs. An lterntive method hs recently been proposed (Reymond et l., 23), bsed on the fct tht lthough n dptive trit chnges with environmentl conditions, it frequently follows reproducible behviour. For instnce, lef elongtion rte chnges with meristem temperture, but follows tight reltionship with it provided tht plnts experience no wter or nutrient deficits, nd no evportive demnd. Under these circumstnces, this reltionship pplies to different experimentl conditions (Ben Hj Slh nd Trdieu, 1995, for mize; Grnier et l., 22, for Arbidopsis thlin). In the sme wy, the responses of mize lef elongtion rte to evportive demnd nd to soil wter sttus re stble chrcteristics of genotype, which pply to field s well s controlled conditions (Trdieu et l., 2). An dptive trit, with G3E interction, cn therefore be linked to stble underlying chrcteristics of genotypes, independent of experimentl conditions. The uthors of this study hve proposed tht genetic nlysis could be crried out on these stble chrcteristics tht describes the responses of genotypes to environmentl conditions (Reymond et l., 23). The purpose of the present pper ws to compre the two bove-mentioned methods on the sme dt set, in the cse of lef growth. First, QTLs of finl lef length nd width were determined individully in four experiments, three in the greenhouse with contrsting soil wter sttus nd one in the field with well-wtered plnts subjected to high evportive demnd. Second, QTLs of the response of lef elongtion rte to environmentl conditions were determined in the sme experiments, tking dvntge of shortterm vritions of elongtion rte within nd between experiments. The first method therefore considered dptive trits independently in severl experiments, while the second considered tht the vrition of these trits with environmentl conditions were intrinsic chrcteristics of ech genotype. In prticulr, the im ws (i) to test whether QTLs of prmeters of response curves coloclized with QTLs of rw phenotypic trits, nd (ii) to test the vlidity of the hypothesis tht QTLs of dptive trits re specific to experiments in stressed conditions, while QTLs detected in both control nd stress conditions would correspond to constitutive trits. Mterils nd methods Genetic mteril The mpping popultion used in this study consisted of recombinnt inbred lines (RILs) with six genertions of self pollintion, derived from cross between two prentl lines, F-2 (n erly French flint) nd Io ( lte North Americn semi-dent considered s droughttolernt). A totl of 145 RILs ws produced nd 152 RFLP probes were used for mpping these RILs (Cusse et l., 1996). QTLs were identified in subset of 1 rndomly chosen RILs. Experiments Field experiment: One experiment ws crried out ner Montpellier, southern Frnce (FC2, Tble 1). Seeds were sown in pirs on 5 July 1999 under mobile shelter tht llowed ir temperture nd ir vpour pressure deficit to be modified. Plnts were thinned when lef 3 emerged, leving five plnts per RIL. RILs were sptilly orgnized in sequences of 1 plnts of different RILs, ech sequence being distributed t rndom in the field. The soil ws wtered twice week, with mounts greter thn the Penmn evpotrnspirtion during the sme periods so plnts experienced no wter deficit. Air temperture nd reltive humidity were mesured every 2 s (HMP35A Visl Oy, Helsinki, Finlnd). The temperture of the meristemtic zone ws mesured with fine copper-constntn thermocouple (.4 mm dimeter) locted inside the stem in the meristemtic zone. Light ws mesured continuously using PPFD sensor (LI-19SB, Li-Cor, Lincoln, NB, USA). All tempertures referred to herefter re meristem tempertures. All dt of temperture, PPFD, nd reltive humidity were verged nd stored every 6 s in dt logger (Cmpbell Scientific, LTD- CR1 Wiring Tble 1. Environmentl conditions during experiments Experiment Min chrcteristic Growing period Night temperture (8C) VPD b (kp) Lef W w c (MP) FC2 (field) Well-wtered, high VPD July >ÿ.8 GC2 (greenhouse) Well-wtered, low VPD June ÿ.3 GS1 (greenhouse) Mild wter deficit Mrch ÿ.11 GS2 (greenhouse) Wter deficit October ÿ.23 Rnge of men tempertures corresponding to one night during the growing period of lef 6. b Dytime meristem-to-ir VPD, clculted s in eqution 1, verged over the growing period of lef 6. c Lef W w, predwn lef wter potentil verged over the growing period of lef 6.

3 Deling with the genotype3environment interction vi modelling pproch 2463 Pnel, Shepshed, Leicestershire, Englnd). Vpour pressure difference between meristem nd ir (VPD, kp) ws clculted s the difference between sturtion vpour pressures t meristem temperture nd t ir dew point temperture. An equivlent VPD ws clculted for 1 d periods by cumulting mesured VPDs fter correction for chnges in stomtl conductnce due to diurnl vritions of PPFD (Reymond et l., 23 for detil). Briefly, the men VPDs t ech time step of 6 s were multiplied by coefficient (k i ) which ws nd 1, respectively, t PPFDs of nd 5 mmol m ÿ2 s ÿ1, proportionl to PPFD between these two vlues, nd equl to 1 for PPFDs bove 5 mmol m ÿ2 s ÿ1. VPD eq = + n ðvpd i 3k i Þ=n i=1 where VPD eq is the VPD corresponding to the considered dy period nd n the number of steps. Therml time of period (t th ; 8C d) ws clculted by cumulting nd integrting, t ech time step, the differences between the men meristem temperture (T i ) nd the x- intercept of the reltionship between meristem temperture nd lef elongtion rte. t th = + n ðt i ÿ T Þ=ð6=36324Þ i=1 Meristem tempertures verged during night periods rnged from C (during nights where plnts were in open ir) to 23 8C (during nights when the mobile shelter ws plced bove plnts). Dytime tempertures rnged from 17 to 34 8C. VPD eq, estimted s in eqution 2, rnged between 1 nd 2.8 kp. Low VPDs were obtined during three nights by sprying wter on the soil with the shelter closed. The verticl position of the tip of the sixth leves ws mesured twice dy on four plnts per RIL, in the morning (5. 7. h solr time) nd in the evening ( h) during the period elpsed between ppernces of leves 6 nd 8, during which the elongtion rte of lef 6 is constnt with time under constnt temperture. The position of the lef tip ws mesured using ruler ttched to 2.5 m horizontl br, itself fixed on verticl metl sticks permnently left in the soil. Lef elongtion rte ws clculted individully for ech of the four replictes of ech RIL, during dys nd nights. Ech elongtion rte, corresponding to one plnt for one dy or one night, ws considered s n individul dt point, nd ws nlysed s function of mesured environmentl conditions mesured on the sme plnt during the sme period. The finl length nd width of the sixth lef were mesured on four plnts per RIL, using ruler fter the ligule hd ppered. Greenhouse experiment, well-wtered plnts: One experiment ws crried out in the greenhouse in well-wtered conditions (Experiment GC2, Tble 1). On 1 June 2, seeds were plced t.25 m depth in columns (.15 m dimeter,.4 m height) contining 4:6 mixture (v/v) of lomy soil nd n orgnic compost. Six seeds per RIL were sown in pirs nd thinned to three plnts when lef 3 emerged. Ech column contined three different RILs nd ws locted t rndom in three blocks. The soil ws mintined t retention cpcity, by dily wtering with modified one-tenth strength Hoglnd solution corrected with minor nutrients. Columns were individully weighed every third dy in order to check tht the soil wter content ws 35 4% of dry soil, corresponding to predwn lef wter potentil higher thn ÿ.1 MP. Meristem temperture ws mesured nd VPD eq ws estimted s in the field experiment. Rnges of ech environmentl vrible were obtined by mnipultion of climte inside the greenhouse. On two nights, plnts were covered by m plstic shelter nd ir temperture ws lowered by two ir conditioners, llowing meristem temperture to be decresed to C. The sme shelter ws used on the other two nights, but the ir ws heted so tht meristem ð1þ ð2þ temperture reched 26.1 nd C, respectively. Finlly, ll plnts were moved on two non-consecutive nights into growth chmber in order to mesure lef elongtion rte t low meristem temperture (15.1 nd C on the two nights). Dytime VPD ws vried, either by turning off the wter of the ir-cooling system (2 3.5 kp) or by leving the wter circulting ( kp). Low VPDs were obtined by sprying wter on the soil (.2 1 kp). An exmple of the timecourse of environmentl conditions is presented in Fig. 1. The position of the tip of lef 6 ws mesured t lest twice dy (t h nd h solr time) on three plnts per RIL, during the period tht elpsed between the ppernces of leves 6 nd 8, with ruler fixed on the top of the columns. Lef elongtion rte ws clculted individully for ech plnt during dys nd nights nd processed s in the field experiment. The finl length nd width of the sixth lef were mesured on three plnts per RIL, s in the field experiment. Greenhouse experiments, wter-deficient plnts: Two experiments were crried out with soil wter deficit (Experiments GS1 nd GS2, Tble 1). Plnts were sown with the sme procedure s in Exp GC2, on 1 Mrch nd 6 October 2. While filling columns, soil smple ws tken from every second column to determine the initil soil wter content. It ws confirmed tht soil wter content ws similr in ll columns nd homogeneous within ech column (not shown). Soil wter content ws determined fterwrds by weighing columns every dy. Differences in weight were ttributed to chnges in soil wter content, s plnt weight ws negligible. A wter-relese curve ws obtined by coupling men soil wter content of the column to predwn lef wter potentil in the rnge of wter Meristem Temperture ( C) PPFD (µmol.m 2.S 1 ) VPD m (kp) Lef Elongtion Rte (mm.h 1 ) :3 b d Night 1 Dy 1 Night 2 c 7:45 11: 16:15 2: Time (hh:mm) Fig. 1. Time-course of lef elongtion rte of five mize RILs during 36 h climtic sequence in the greenhouse in the bsence of soil wter deficit (Experiment GC2). () Meristem temperture. (b) Incident light (PPFD). (c) Meristem-to-ir VPD (VPD m ). (d) Lef elongtion rte. Verticl lines indicte time of mesurements. Ech symbol corresponds to one RIL. During night 1, meristem temperture ws incresed by plcing the plnts under shelter nd heting the ir. During night 2, meristem temperture ws decresed by plcing the plnts under shelter nd cooling the ir. 7:45

4 2464 Reymond et l. potentils from ÿ.5 to ÿ1.5 MP. This procedure llowed the predwn lef wter potentil of every plnt in the experiments to be estimted dily. Irrigtion ws stopped when lef 5 ppered. A soil wter potentil of ÿ.3 MP ws reched in 3 6 d, depending on the lef re of the RIL under considertion (Fig. 2). Soil wter sttus ws then controlled in such wy tht ech plnt experienced vriety of predwn lef wter potentils rnging from ÿ.3 to ÿ.25 MP in Experiment GS1. This rnge ws lrger in Experiment GS2, from ÿ.1 to ÿ.6 MP. Columns were prtilly rewtered t the end of the growing period of lef 6 nd second sequence of dehydrtion ws followed. Light, meristem or ir tempertures, nd meristem-toir VPD were mesured, s in Experiment GC2. Lef elongtion rte finl length nd width of the sixth lef were mesured in three plnts per RIL, s in Experiment GC2. Meristem Temperture ( C) Predwn Lef Wter Potentil (MP) Lef Elongtion Rte (mm.h 1 ) Night # b c : : : : : Time (hh:mm) Fig. 2. Time-course of lef elongtion rte of five mize RILs during five consecutive nights in the greenhouse with incresing soil wter deficit (Experiment GS2). () Meristem temperture. (b) Predwn lef wter potentil. (c) Lef elongtion rte mesured over 12 h periods. Ech symbol corresponds to one RIL. Genetic nlysis QTL detections were performed with two methods, either s presented in Reymond et l. (23) by using the Sttisticl Anlysis System softwre (SAS Institute Inc., Cry, NC, USA), or by using the PLABQTL softwre (Utz nd Melchinger, 2). In the first method, QTLs were detected by composite intervl mpping with investigtion of episttic interctions. Cofctors were first chosen using stepwise regression between the studied trit nd the llele vlue t ech mrker. A bckwrd regression ws then crried out in ech chromosome to limit the number of cofctors. The presence of mineffect QTLs ws tested every 5 cm between the 152 mrkers (445 positions on the genome) using multiple regression with the cofctors. For tht, the llele vlue ws determined every 5 cm s the probbility of occurrence of llele F-2 t this position ccording to the llelic vlue t flnking mrkers. When the tested position on the genome ws close to cofctor (61 cm), the effect of this cofctor ws removed. Becuse the theoreticl distribution of the test sttistics (the F of the liner regression) ws unknown for multiple regression with cofctors, the threshold vlue ws determined by 1 permuttions (Churchill nd Doerge, 1994). In the lst step, regression ws crried out between the studied trit nd ll combintions of two positions on the genome, tking into ccount the min-effect QTLs previously determined. This llowed determintion of episttic QTLs. In order to strengthen the genetic nlysis, nd to test low-effect, episttic QTLs, second detection ws crried out with n independent method. This method (PLABQTL) ws bsed essentilly on the sme principles, with composite intervl mpping with cofctors either chosen by the softwre, or imposed externlly. In order to llow better comprison between methods, the sme cofctors s in the first method were used. The min difference with the first method is tht episttic interction between QTLs ws determined by PLABQTL only with QTLs tht hd first been detected s min-effect QTLs. The two methods of QTL detection were first pplied to lef length or width in individul experiments, by tking into ccount the men vlue of the three or four plnts of one RIL. They were lso pplied to the prmeters of the reltionships between individul elongtion rtes corresponding to one plnt for one dy or night, nd environmentl conditions mesured on the sme plnt during the sme period (see Results). In both methods, the prtil r 2 of ech ws estimted fter ccounting for the effects of ll other QTLs found for the sme trit. Brod-sense heritbility ws estimted ccording to Gllis (199): h 2 = r 2 G =ðr2 G + r2 E Þ where r 2 G is the genetic vrince nd r2 E the environmentl vrince. It ws estimted on the three plnts of one RIL. Anlysis of vrince nd sttisticl nlyses were crried out by using the SAS nd R softwres (R Development Core Tem, 23). Results Vribility between experiment of finl lef length nd width, GE interction The length of lef 6 hd lrge vribility in the whole set of dt, rnging from 17 to 8 cm (Figs 3, 4). An nlysis of vrince showed significnt effects of experiments, genotypes, nd the interction between them. Mximum lengths were observed in Experiment GC2, in which plnts were well-wtered in the greenhouse (Figs 3, 4b). The two levels of wter deficit decresed lef length, by 1 nd 29 cm on verge in Experiments GS1 nd GS2 (Figs 3, 4c). The field experiment GC2 with well-wtered plnts but high VPD showed lef length intermedite between Experiments GS1 nd GS2, on verge 25 cm shorter thn in Experiment GC2 (Figs 3, 4b). Differences were therefore lrger between the two experiments with well-wtered plnts in the field nd in the growth chmber thn between growth-chmber experiments with two levels of wter deficit. Consistent with significnt experiment3genotype interction, the rnking of RILs ccording to lef length differed between experiments, with incresing differences when the difference in environmentl conditions incresed (Fig. 5). It ws well conserved between greenhouse experiments with no or low wter deficit (GC2 nd GS1), or between experiments with well-wtered plnts in field or greenhouse (GC2 nd FC2, r 2 =.53, not shown). It ws looser between the greenhouse experiments with or without severe wter deficit (GC2 nd GS2, GS1 nd GS2) nd independent between the field ð3þ

5 Length cm Deling with the genotype3environment interction vi modelling pproch 2465 experiment with high VPD nd the greenhouse experiments with soil wter deficit (FC2 nd GC2). The three individul vlues of lef length corresponding to one RIL in one experiment were closely relted to their men vlue (Fig. 4). This resulted in high heritbilities in individul experiments with no or moderte wter deficits (h 2 =.75,.78, nd.73 in experiments FC2, GC2, GS1, respectively), suggesting tht reproducibility ws high in these experiments. In the sme wy, the correltions between men vlues of lengths mesured on leves 6 nd 7 were high in experiments GS2 nd FC2 (r 2 =.84 nd.93). Heritbility ws lower in experiment GS2, with more severe wter deficit (h 2 =.39, Fig. 4c), nd the correltion between lengths of leves 6 nd 7 ws lso lower thn in other experiments (r 2 =.69). This difference ws probbly due to the fct tht, during sequence of wter deficit, ech plnt replicte depleted soil wter t different rte, depending on its lef re nd tht of its neighbours in the sme column. Plnts were therefore subjected to different soil wter sttus. This cn be visulized in Fig. 2, in which differences in predwn lef wter potentil lrger thn.1 MP were observed between individul plnts of RILs t given time. Lef width ws less ffected by the tested environmentl conditions thn lef length. It only differed significntly in Experiment GS2 compred with the other three tretments (Figs 3, 4e, f). The effects of genotype, experiments, nd genotype3experiment interction were significnt, lthough the ltter hd reltively low contribution to the totl vrince compred to the cse of lef length. The correltions between lef widths mesured in Experiments GS2, GS1, nd FC2 were consistently higher thn those corresponding to lef length, especilly in the comprison between Experiments GS2 nd FC2 (Fig. 5). Heritbility ws high in ll individul experiments (Fig. 4e, f), confirmed by the correltion between the widths of leves 6 nd 7(r 2 =.87,.72, nd.58 in Experiments GC2, GS2, nd FC2, respectively). Width cm 4 2 FC2 GC2 GS1 GS2 Experiment Fig. 3. Box plots of the length nd width of lef 6 in the four experiments performed in the field (FC2) or the greenhouse with well-wtered plnts (GC2), or with two levels of wter deficit (GS1, GS2). Ech box represents the qurtile bove nd below the medin vlue. Verticl brs represent minimum nd mximum vlues except when the ltter re wy from the medin by more thn 1.5 times the first qurtile. Vlues out of this rnge re presented s circles. Differences in the men vlues of lef length nd rnkings of RILs between experiments cn be predicted from differences in environmentl conditions In the exmples presented in Fig. 6, the differences in finl length observed between greenhouse nd field experiments with well-wtered plnts rnged from 12 to 31 cm, depending on the RIL. Consistent with erlier studies (Ben Hj Slh nd Trdieu, 1997; Reymond et l., 23), differences in length my be cused by differences in evportive demnd during lef elongtion. VPDs, verged over the whole period of lef growth, were.6 nd 1.6 kp in the greenhouse nd field experiments, respectively. This corresponded to VPDs higher thn 3 kp in most fternoons of the field experiment. According to the model of Reymond et l. (23), pplied to the three presented RILs with their respective mesured sensitivities to VPD, this difference in evportive demnd would result in differences in lef length of cm. Thus, the effect of evportive demnd ccounted for lrge prt of the difference between greenhouse nd field experiments. Differences between RILs in the responses of finl lef length to evportive demnd were therefore of the sme order of mgnitude s differences in finl length between RILs in given experiment. This chnged the rnking of RILs lef length between experiments nd explined the significnt genotype3experiment interction. The sme chnge in rnking of genotypes ws observed on shorter timescles. When plnts were subjected to chnging evportive demnd (Fig. 1), ll RILs hd lef elongtion rte which decresed during the dy nd incresed during the fternoon, following chnges in VPD. The rnking of RILs ws similr during the two consecutive nights, but ws not conserved during the dy period with highest evportive demnd ( h), due to different sensitivities to evportive demnd. A similr nlysis ws crried out for the effect of soil wter sttus on finl lef length (Fig. 6b). Lef lengths were compred in Experiments GC2, GS1, nd GS2, in which men predwn lef wter potentils verged during the period of lef growth were ÿ.3, ÿ.11, nd ÿ.23 MP, respectively, with some differences between RILs, nd men VDP ws low throughout the experiments (Tble 1).

6 2466 Reymond et l. Individul vlue (cm) Width Length Experiments with Experiments with All experiments well-wtered plnts Soil wter deficit 1 b c 8 GC2 GS1 6 FC2 4 GS d e f Men vlue of RIL in the considered experiment (cm) Men vlue of RIL in ll experiments (cm) As mentioned bove, predwn lef wter potentil hd n pprecible vribility between plnts of given RIL becuse plnt trnspirtion scenrios differed between plnts (Fig. 2). In spite of this vribility, it cn be seen in Fig. 6b tht the studied RILs hd pprecible differences in sensitivity of lef length to soil wter deficit, with effects rnging from cm, i.e. similr to the differences between RILs in given experiment. Tken together, these results suggest (i) tht individul experiments were reproducible in terms of finl lef length, lthough with lower heritbility in the experiment with high wter deficit due to differences in wter sttus between plnts of n RIL t given time; (ii) tht the rnking of RILs Men vlue of RIL in ll experiments (cm) Fig. 4. ( c) Individul vlues of finl lef length, nd (d f) individul vlues of lef width, corresponding to ech plnt replicte of RILs in ech experiment, plotted ginst men vlues of the sme RIL. In pnels (, d) individul vlues in one experiment re plotted ginst the men vlue of the RIL in the sme experiment. In pnels (b, c, e, f), individul vlues re plotted ginst the men vlue of the RIL verged over the four experiments. Open circle, Experiment GC2; open dimond, Experiment FC2; open tringle, Experiment GS1; open squre, Experiment GS2. Lef length (cm) GS2 Lef width (cm) GS r 2 = GC2 r 2 =.35 r 2 =.46 r 2 =.44 r 2 =.32 r 2 =.58 GS1 r 2 =.11 FC2 Length or width of lef 6 (cm) GS1 r 2 = Fig. 5. Reltionship between men vlues of lef length or width between experiments. Low vlues of r 2 indicte high G3E interction. GS1 GC2 on lef length differed between experiments, both in wellwtered nd wter-deficit conditions; (iii) tht differences between experiments in rnkings of RILs were t lest in prt due to contrsting sensitivities to evportive demnd nd to soil wter sttus. QTLs of lef length differed in the four experiments while QTLs of width were common Fourteen QTLs of length were detected with the first method of QTL detection (6, 1, 3, nd 4 in Experiments GC2, FC2, GS1, nd GS2, respectively, Tble 2). Two were detected s min effects, nd 12 s interctions

7 Reduction in finl lef length (GC2 fixed t ) (cm) Deling with the genotype3environment interction vi modelling pproch 2467 Men VPD during lef elongtion period (kp) Men Predwn Lef Wter Potentil during lef elongtion period (MP) Field vs Green house Well wtered vs Wter deficit b Fig. 6. Reduction of finl lef length compred to those in Experiment GC2 (well-wtered plnts with low evportive demnd). Three different RILs re presented (closed squre, tringle, circle). () Experiments without soil wter deficit but contrsting evportive demnd (GC2 nd FC2). The men equivlent VPD ws clculted in both experiments over the whole period of lef growth ccording to eqution 1. (b) Experiments with contrsting soil wter sttus nd low evportive demnd (GC2, GS1, nd GS2). The men soil wter potentil ws clculted in the three experiments over the whole period of lef growth. between two loci ( epistsis ). The second method of QTL detection confirmed the two min-effect QTLs (LOD scores >3). Among the other 12 QTLs, it detected 4 QTLs s min-effect (LOD scores >3), 4 s tendencies (LOD scores of 2 3), nd 4 were not confirmed. In the following, only QTLs detected with both methods were kept. Three QTLs re lso displyed becuse they hd LOD scores higher thn 3.5 with the second method. This double nlysis provided 13 QTL positions (Fig. 7), with one cluster on chromosome 2 corresponding to three experiments with no or low soil wter deficit (Experiments GS1, GC2, nd FC2) nd two clusters corresponding to experiments with low or severe soil wter deficit (Experiments GS1 nd GS2), one on chromosome 6 nd one on chromosome 3. Clusters of QTLs therefore occurred for groups of experiments in which the rnking of QTLs ws reltively well conserved (Fig. 5). The other six QTL positions corresponded to individul experiments. QTLs were lso detected on the differences in length observed between two experiments (GC2 GS2 nd GC2 FC2). Anlyses of both rw nd normlized difference (divided by the length in Experiment GC2) provided no new informtion, with three QTLs lredy detected on chromosomes 2, 3, nd 8. Lef width hd common QTL in the four experiments, locted on chromosome 5 between 125 nd 135 cm (Tble 2; Fig. 7). Other QTLs were detected by the first method s interctions between loci, but were not confirmed by the second method. This common position of one QTL for the four experiments is consistent with the reltively low G3E interction observed in Fig. 5. An lterntive method: QTLs of prmeters of responses to soil wter deficit nd evportive demnd The rtionle for this method (Reymond et l., 23) is the desire to tke dvntge of ll sources of vrition of both soil wter deficit nd VPD, between experiments, between consecutive dys of n experiment, nd between plnts of RIL. The method consists in building response curves of lef elongtion rte to environmentl conditions t timescle of severl hours. Becuse mize lef elongtion rte is stble for 5 7 d fter lef ppernce, it is possible to consider chnges in lef elongtion rte with environmentl conditions s reproducible events, on which response curves re built (Trdieu, 23). Fluctutions of lef elongtion rte were synthesized by responses to three environmentl conditions, nmely meristem temperture, lef-to-ir VPD, nd predwn lef wter potentil. The response of lef elongtion rte to meristem temperture ws considered during night periods in experiments without soil wter deficit, when evportive demnd ws null nd lef elongtion rte only depended on meristem temperture. This resulted in liner reltionships which were common, for ech RIL, to severl experiments nd llowed the clcultion of lef elongtion rte per unit therml time (Fig. 8, d; see Trdieu et l., 2 for detil). Expressed in this wy, lef elongtion rte becomes independent of temperture in the studied rnge, nd is common to severl experiments. Elongtion rte per unit therml time, termed prmeter herefter, therefore defines the intrinsic cpcity of the considered RIL to elongte in the bsence of stress (expressed in cm 8Cd ÿ1 ). It ws clculted here with dt from Experiment GC2, nd from dt collected t the beginning of Experiments GS1 nd GS2 while predwn lef wter potentil ws still higher thn ÿ.8 MP. Dt originting from nother greenhouse experiment fitted in the sme regression. The response to evportive demnd ws considered during dy periods without soil wter deficit, while meristem temperture, light intensity, nd evportive demnd fluctuted with time (Fig. 1). It ws shown erlier tht light intensity hs no direct effect on lef elongtion rte t this

8 2468 Reymond et l. Tble 2. QTL detected by composite intervl mpping for ech trit L, length of the sixth lef, W, width of the sixth lef, in experiments GC2, GS1, GS2, nd FC2., b, c: prmeters of the response to environmentl conditions, see eqution 3 nd Fig. 8. QTLs were detected with first method (SAS procedure), nd confirmed by second method (PLABQTL). The results of the second method re either confirmtion of the first method (**, QTL confirmed with LOD>3; *, LOD from 2 3; ÿ, not confirmed), or new QTLs with n LOD score>3.5 presented in itlics. Trit Type b Chr c Mrker d Distnce e Effect f LOD Second method g L GC2 E1 3 gsy46 56 ÿ E1 8 bnl ** E2 2 gsy199_ptk * E2 3 gsy298c_pmg 1 E3 4 umc * E3 9 gsy33_tdr3 16 L FC2 M 5 incw1_cellw 11 ÿ ** N 7 csu ** N 2 gsy237_agp ** L GS1 M 6 gsy325c_bt ** E1 2 gsy18_gut ** E1 7 gsy345_o2 N 3 umc92 22 ÿ ** L GS2 E1 4 gsy431_pdsi ** E1 6 gsy236_agp1 234 * E2 3 umc1 36 ÿ ** E2 9 bnl * W GC2 M 5 pl1_phenyl 132 ÿ ** E1 4 umc ÿ E1 1 slt1 76 W FC2 M 5 pl1_phenyl 132 ÿ ** W GS1 E1 5 pl1_phenyl 127 ÿ ** E1 5 umc68 25 E2 8 sps E2 1 bnl3-4 5 W GS2 M 5 pl1_phenyl 127 ÿ ** M 2 gsy348c_lvr1 3 ÿ ** M 2 gsy134_glr ** M 4 gsy431_pdsi ** E1 8 sc179_cs 5 ÿ * E1 9 csu E2 6 gsy236_agp * E2 7 csu81 1 E3 4 umc ** E3 9 bnl b M 1 kn1 193 ÿ ** M 8 umc ** E1 4 gsy431_pdsi 59 ÿ * E1 5 umc43 84 E2 2 umc44b 14 ÿ E2 7 gsy345_o2 c M 2 umc6 5 ÿ * E1 1 dh1_lcohol 198 ÿ E1 6 umc85 58 Considered trit in given experiment (L or W) or clculted over ll experiments (, b, c). b Min effect QTL (M) or epistsis (E1, E2, etc.) in the first method of QTL detection, or QTL detected in the second method only (N). c Chromosome number. d Closest left mrker. e Genetic distnce of the QTL from the top of the chromosome. f Additive effect of the llele of prent F-2. g Confirmtion by PLABQTL. timescle, nd essentilly cts vi its effect on lef-to-ir VPD (Ben Hj Slh nd Trdieu, 1996). The sensitivity to evportive demnd ws estimted vi the slope of the response curve of lef elongtion rte, expressed per unit therml time, to meristem-to-ir VPD. Common liner reltionships pplied to the four experiments in the field nd the greenhouse. For ech RIL, the sensitivity to evportive demnd could therefore be considered s common to the four considered experiments, s shown for two RILs in Fig. 8b, e. It ws clculted by common regression over the whole dt set, nd termed prmeter b herefter (cm 8Cd ÿ1 kp ÿ1 ). The response to soil wter deficit ws nlysed during night periods. The slope of the response curve of

9 Position cm b b c c bb Fig. 7. QTLs of finl lef length (open circle, dimond, tringle, squre for experiments GC2, FC2, GS1, nd GS2, respectively) nd QTLs of prmeters of lef elongtion model (, intrinsic elongtion rte; b, slope of the response of lef elongtion rte to meristem-to-ir VPD, b x-intercept of the sme reltionship; c, slope of the response of lef elongtion rte to predwn lef wter potentil, c x-intercept of the sme reltionship). The QTL of lef width common to the four experiments is lso presented (inverted grey-filled tringle). Brs on chromosome indictes positions of mrkers. For lef length nd prmeter, symbols re locted on the right side of the chromosome if the llele F-2 increses the vlue of the trit. For prmeters b nd c, symbols re locted on the right side of the chromosome if the llele F-2 decreses the sensitivity of lef elongtion rte to the considered environmentl condition. lef elongtion rte to predwn lef wter potentil ws clculted jointly in Experiments GC2, GS1, nd GS2. A common liner reltionship pplied to the experiments crried out with or without soil wter deficit, s shown for two RILs in Fig. 8c, f. The slope of this reltionship defines the sensitivity of the considered RIL to soil wter deficit over the whole set of dt, nd is termed prmeter c herefter (cm 8C d ÿ1 MP ÿ1 ). It ws shown erlier (Trdieu et l., 2; Reymond et l., 23) tht the three responses cn be combined in model with three prmeters : dl=dt = ðt ÿ T Þð + bvpdÿcwþ where dl/dt is lef elongtion rte, T is meristem temperture, nd T is the x-intercept of the reltionship between meristem temperture nd lef elongtion rte, which ws common to ll RILs in the considered set of dt. The prmeters, b, nd c re defined s bove. Deling with the genotype3environment interction vi modelling pproch 2469 ð4þ QTLs of prmeter of the model coloclize with QTLs of finl lef length The bility of RIL to elongte in optimum conditions (prmeter ) ws ccounted for by nine significnt QTLs in the first method of QTL detection (Tble 2, Fig. 7). Three were detected s min-effect QTLs nd confirmed by the second method of detection (two on chromosome 2 nd one on chromosome 4). Six QTLs were detected by the first method s interctions between two loci, mong which one ws confirmed s min-effect QTL by the second method (chromosome 4) nd two s tendencies. The other three QTLs in interction could not be confirmed by the second nlysis. As bove, only QTLs detected by both methods were kept in the nlysis. Among the six resulting QTLs of prmeter, five were locted t less thn 15 cm from QTL of lef length (Fig. 7). Reciproclly, mong the 13 QTLs of lef length, seven were locted t less thn 15 cm from QTL of prmeter, two other QTLs of length on chromosome 2 were locted t greter distnce, nd four hd no colocliztion with QTL of prmeter. In ll cses, the llele for longer lef ws lso the llele for fster elongtion rte. Sensitivity to evportive demnd (prmeter b) hd six significnt QTLs in the first method (Tble 2; Fig. 7), mong which two were min effect QTLs, lso detected by the second method. The four QTLs detected s interctions between two loci by the first method were not confirmed. The two confirmed QTLs locted on chromosomes 1 nd 8 coloclized with significnt QTLs of the x-intercept of the response of lef elongtion rte to VPD, termed b, which represents the VPD tht would stop elongtion. One QTL of prmeter c ws detected s min effect in the first method, nd ws confirmed by the second method. Another QTL ws confirmed, of the x-intercept of the response of lef elongtion rte to predwn lef wter potentil, termed c, which represents the wter potentil tht would stop lef elongtion. Although some QTLs detected by the first method for prmeters b nd c coloclized with QTL of lef length (especilly on chromosome 5), this ws not the cse for ny of the QTLs detected by both methods. Further, no colocliztion ws detected between differences in lef length between experiments nd QTLs of prmeters b nd c. Discussion Adptive versus constitutive trits The concepts of dptive versus constitutive trits cn be discussed from the dt presented here, nd somewht chnge the definition by Blum (1996) given in the Introduction. Lef length hd the chrcteristics of n dptive trit, with G3E interction nd n instbility of QTLs which both incresed with differences in environmentl conditions between experiments (Figs 5, 6, 7). However,

10 Lef elongtion rte (mm. Cd 1 ) 247 Reymond et l..6 RIL D c..6 b RIL D19 c.4.2 d Meristem Temperture ( C) this instbility ws determined by responses of genotypes which were stble cross environments (Fig. 8). The responses of lef elongtion rte to evportive demnd (in the bsence of soil wter deficit) nd to soil wter sttus (in the bsence of evportive demnd) were common to severl experiments for ech genotype. They were therefore stble chrcteristics of ech genotype, without G3E interction nor chnges in rnking of genotypes between experiments. Differences in rnkings of RILs, which resulted in n instbility of QTLs of lef length, were linked to intrinsic differences in the sensitivities of RILs to evportive demnd or soil wter deficit. The cse of prmeter is prticulr. It cn be viewed s response to n environmentl condition, nmely meristem temperture, or s constitutive trit, the bility of the leves of given genotype to elongte in the bsence of stress. This second view is bsed on the fct tht elongtion rte, expressed in therml time, is independent of temperture. It is lso bsed on the fct tht the chnge with temperture of the rtes of processes such s tissue elongtion or cell division re mtched by those of the durtion of the sme processes, thereby hving negligible effect on the finl size or number of cells (Ben Hj Slh nd Trdieu, 1995; Trdieu et l., 2; Lfrge nd Trdieu, 22). Consistently, QTLs of prmeter coloclized with QTLs of lef lengths in severl experiments. Lef width lso hd severl chrcteristics of constitutive trit lthough it ws ffected by wter deficit. In effect, it e Meristem to ir VPD (kp) f.2.4 Predwn Lef Wter Potentil (MP) Fig. 8. Responses of lef elongtion rte per unit therml time to (, d) meristem temperture, (b, e) evportive demnd, nd (c, f) soil wter deficit, in two different RILs. (, d) Lef elongtion rte, mesured in the bsence of evportive demnd, plotted ginst meristem temperture. Individul results re pooled for better legibility. (b, e) Lef elongtion rte during dy periods plotted ginst meristem-to-ir VPD in well-wtered plnts. Night periods re considered s hving VPD of (eqution 1), nd individul results re pooled for better legibility. (c) Lef elongtion rte of night periods plotted ginst predwn lef wter potentil. Individul vlues re presented. Open circle, Experiment GC2 dy vlues; closed circle, Experiment GC2 night vlues; open dimond, Experiment FC2; open tringle, Experiment GS1; open squre, Experiment GS2; closed squre, Experiment GS2, second cycle of dehydrtion fter rewtering. hd reltively low G3E interction nd stble QTLs cross four experiments with contrsting environmentl conditions. The fct tht trit with high G3E interction cn be predicted from intrinsic chrcteristics of genotypes my open the wy to modelling of the interction (Trdieu, 23). Recently, the uthors hve combined the QTL models of the three prmeters, b, nd c with n ecophysiologicl model which predicts the lef elongtion rte of plnts subjected to ny climtic scenrio. This llowed prediction of the lef elongtion rte of new RILs of the sme mpping popultion, which were only known by their lleles t QTLs, under new climtic scenrios in growth chmber (Reymond et l., 23). RILs differed by their intrinsic elongtion rte nd by their sensitivity to evportive demnd, thereby hving chnged rnkings under different environmentl conditions. Predicted nd observed lef elongtion rtes were in good greement, so the G3E interction could be predicted over short timescles. The test remins to be done over longer period, in prticulr, tking into ccount the durtion of elongtion. Comprison of QTLs of finl lef size nd of prmeters of the model of elongtion rte The detection of QTLs with two different methods, both bsed on multiple regression, provided converging results. The min difference between the two methods is tht the

11 first llowed clcultion of QTLs of interction between loci ( epistsy ) even if these QTLs did not pper s min effect. This method ws lso slightly more stringent thn the second one (PLABQTL), which only clculted mineffect QTLs. Overll, the decision to keep only the QTLs common to both methods in the nlysis ws dpted to the objective of this pper, comprison of QTLs. It results in fewer number of QTLs thn those published erlier (Reymond et l., 23), in which the min objective ws to get the best genetic model, which ws then checked ginst independent dt. The colocliztion of QTLs of finl lef length with those chrcterizing the model of lef growth is ressuring in terms of the bility of both methods to nlyse the genetic vribility of lef growth. However, the study presented here csts doubt on the method tht clssifies QTLs of rw phenotypic trits on the bsis of environmentl conditions during experiments. More thn hlf of the QTLs of finl lef length, determined in different experiments, coloclized with prmeter, the intrinsic lef elongtion rte, even in experiments with tmospheric or soil wter deficits. By contrst, prmeters of sensitivity to evportive demnd or to soil wter deficit hd no cler colocliztion with finl lef length in experiments with either high evportive demnd (FC2) or wter deficit (GS2). A first interprettion of this result could be tht the study filed to detect QTLs of prmeters of response curves in loci where QTLs of lef length of stressed plnts were identified. This is possible, due to the reltively low number of RILs tken into ccount in this nlysis, nd to the stringent nlysis with two different methods which reduced the number of QTLs. However, it is still the cse tht the clusters of QTLs of responses which were determined on chromosomes 1 nd 8 did not correspond to QTLs of lef length in stressed experiments, suggesting tht detection of QTLs of rw phenotypic trits (or even of their differences between experiments) my not be n pproprite method for identifiction of the zones of the genome involved in responses to environmentl conditions. The presence of strong QTL of lef width, stble between experiments nd unrelted to QTLs of lef length or of prmeters of the model, is n unexpected result of this study. It suggests tht lef width nd length hve different genetic determinnts. Responses to environmentl conditions lso differed between these two vribles, with low effect of moderte soil wter deficit nd low difference between greenhouse nd field experiments. This confirms n erlier study which suggested difference in response to incident light (Muller et l., 21): lef elongtion rte nd finl lef length were unffected by chnge in incident light, while lef width hd cler response to it. Although identifiction of QTLs of responses to environmentl conditions is more time-consuming thn nlysis of rw phenotypic trits such s lef length, lef re, or Deling with the genotype3environment interction vi modelling pproch 2471 yield, it hs severl dvntges. (i) The min dvntge is probbly tht this method brings results which chrcterize genotype per se, nd not the behviour of genotype in given environment. It is therefore possible to compre results of experiments crried out t different times nd in different experimentl conditions (field, controlled conditions). (ii) The nlysis presented here suggests tht this method might be more relible thn others for identifying QTLs of mechnisms of dpttion. (iii) The uthors hve shown erlier tht it llows the behviour of virtul RILs in series of environmentl conditions (Reymond et l., 23) to be predicted, including conditions which do not exist yet but cn be forecst in climte chnges scenrios. This could help selecting in silico the combintions of lleles of interest for such scenrios. Acknowledgements This work ws supported by the French progrmme of genomics Génoplnte. The uthors thnk Christelle Bencivenni for help in dt processing nd Clude Welcker for criticlly reding the mnuscript. Philippe Nudin nd Philippe Hmrd contributed to mesurements. References Ben Hj Slh H, Trdieu F Temperture ffects expnsion rte of mize leves without sptil distribution of cell length. Plnt Physiology 19, Ben Hj Slh H, Trdieu F Quntittive nlysis of the combined effects of temperture, evportive demnd nd light on lef elongtion rte in well-wtered field nd lbortory-grown mize plnts. Journl of Experimentl Botny 47, Ben Hj Slh H, Trdieu F Control of lef expnsion rte of droughted mize plnts under fluctuting evportive demnd. Plnt Physiology 114, Blum A Crop responses to drought nd the interprettion of dpttion. Plnt Growth Regultion 2, Cusse M, Sntoni S, Dmervl C, Murice A, Chrcosset A, Detrick J, de Vienne D A composite mp of expressed sequences in mize. Genome 39, Churchill CA, Doerge RW Empiricl threshold vlues for quntittive trit mpping. Technicl report of Biometricl Unit. Ithc, NY: Cornell University. Condon AG, Richrds RA, Rebetzke GJ, Frquhr GD. 22. Improving intrinsic wter-use efficiency nd crop yield. Crop Science 42, Frcheboud Y, Ribut JM, Vrgs M, Messmer R, Stmp P. 22. Identifiction of quntittive trit loci for cold tolernce of photosynthesis in mize (Ze mys L). Journl of Experimentl Botny 53, Gllis A Théorie de l sélection en méliortion des plntes. Pris: Msson. Grnier C, Mssonnet C, Turc O, Muller B, Chenu K, Trdieu F. 22. Individul lef development in Arbidopsis thlin: stble therml-time-bse progrm. Annls of Botny 89, Lfrge T, Trdieu F. 22. A model coordinting the elongtion of ll leves of sorghum cultivr, pplies to Mediterrnen nd Shelin conditions. Journl of Experimentl Botny 53, Muller B, Reymond M, Trdieu F. 21. The elongtion rte t the bse of mize lef shows n invrint pttern during both the

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