Inheritnce of rice cold tolernce t the seedling stge 669 Inheritnce of rice cold tolernce t the seedling stge Rent Pereir d Cruz 1 *; Ismel Tigo de Lim Durte 2 ; Croline Cbreir 3 1 IRGA Estção Experimentl do Arroz, Seção de Melhormento Genético, Av. Bonifácio Crvlho Bernrdes, 1494, C.P. 29 94930-030 Cchoeirinh, RS Brsil. 2 UFRGS Progrm de Pós-Grdução em Fitotecni, Av. Bento Gonçlves, 7712 91540-000 Porto Alegre, RS Brsil. 3 Unisinos Grdução em Ciêncis Biológics, Av. Unisinos, 950 93022-000 São Leopoldo, RS Brsil. *Corresponding uthor <rpdcruz@hotmil.com> ABSTRACT: Rice (Oryz stiv L.) cold tolernce t the initil stges of development is highly desirble trit to be incorported into the stte of Rio Grnde do Sul, Brzil, cultivrs, but selection for this trit must be performed under controlled temperture conditions, which limits the number of lines tht cn be evluted. Knowledge of the inheritnce of this trit is importnt to define breeding strtegies. So the im of this pper ws to study the genetic bsis of rice cold tolernce t the vegettive stge. Six genotypes with constrsting cold tolernce rections were crossed in dillel scheme without the reciprocls. The prents nd the F 1 nd F 2 genertions were cultivted in greenhouse until the V 4 stge, when they were submitted to 10 C for ten dys nd evluted for plnt survivl fter seven dys of recovery under norml temperture. The results obtined by the dillel nlysis of the F 1 genertion indicted significnce of both dditive nd non-dditive effects, but the generl combining bility ws more importnt. The evlution of the F 2 genertion reveled oligogenic inheritnce with one or two dominnt lleles responsible for cold tolernce in the cold tolernt prents nd two complementry genes with recessive lleles segregting in the crosses involving sensitive nd intermedite genotypes. Key words: Oryz stiv L., genetics, low temperture, vegettive stge Hernç d tolerânci do rroz o frio no estádio de plântul RESUMO: A tolerânci o frio nos estádios iniciis de desenvolvimento do rroz (Oryz stiv L.) é um crcterístic ltmente desejável pr incorporr em cultivres do Rio Grnde do Sul (RS), porém seleção pr est crcterístic deve ser feit sob condições de tempertur controld, o que limit o número de linhs que pode ser vlid. O conhecimento d hernç dest crcterístic é importnte pr definir estrtégis de melhormento genético, ssim o objetivo do presente trblho é estudr bse genétic d tolerânci o frio no estádio vegettivo do rroz. Seis genótipos com reções contrstntes de tolerânci o frio form cruzdos num esquem dilélico sem os recíprocos. Os genitores e s gerções F 1 e F 2 form cultivdos em cs-de-vegetção té o estádio V 4, qundo form submetidos 10 C por dez dis e vlidos qunto à sobrevivênci de plnts pós sete dis de recuperção sob tempertur norml. Os resultdos obtidos pel nálise dilélic d gerção F 1 indicrm significânci tnto dos efeitos ditivos qunto não-ditivos, ms cpcidde gerl de combinção foi mis importnte. A vlição d gerção F 2 revelou um hernç oligogênic com um ou dois lelos dominntes responsáveis pel tolerânci o frio nos genitores tolerntes e dois genes complementres com lelos recessivos segregndo nos cruzmentos envolvendo genótipos sensíveis e intermediários. Plvrs-chve: Oryz stiv L., genétic, tempertur bix, estádio vegettivo Introduction In the stte of Rio Grnde do Sul (RS), the Southern most stte of Brzil, irrigted rice (Oryz stiv L.) crop is grown nnully in n re of bout one million h. Due to the subtropicl climte in this region, mny res re prone to cold wether dmge in some stge of development. It is estimted tht cold tempertures (below 20 C) occur once every three yers in RS, cusing yield losses of round 20% (Terres nd Glli, 1985). The rice breeding progrm conducted by the Rio Grnde do Sul Rice Institute (IRGA) is working towrds developing rice cultivrs with cold tolernce t the initil stges of development. Selection for cold tolernce is performed under controlled temperture conditions nd number of methodologies hve lredy been developed nd used for this purpose (Cruz nd Milch, 2000). Determintion of the most effective genertion to select for cold tolernce is crucil for the efficiency of these indirect selection schemes. In this context, knowledge of the inheritnce of cold tolernce t the vegettive stge is importnt to define when selection is likely to be more efficient, whether in erly or dvnced genertions of the breeding progrm. Conventionl genetic studies of rice cold tolernce t the vegettive stge indicted simple nd oligogenic inheritnce when evluted by mesuring chlorosis nd yellowing of the leves (Kwk et l., 1984; Ngmine nd Nkghr, 1991). Recently moleculr studies hve lso found mjor quntittive tri loci (QTLs) ssocited Sci. Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010
670 Cruz et l. with cold tolernce t seedling stge (Andy nd Mckill, 2003; Jing et l., 2008; Lou et l., 2007; Zhng et l., 2005). However, continuous distribution of cold tolernce in the indic jponic popultions together with findings of minor QTLs explining prt of the vrition points to more complex inheritnce for cold tolernce t seedling stge (Hn et l., 2005; Lou et l., 2007; Qin et l., 2000; Zhng et l., 2005). Such studies hve not been performed with Brzilin genotypes yet, nd s the number of genes nd gene ction depend on the genetic mteril studied, it is of fundmentl importnce to know the genetic bsis of cold tolernce of breeding lines nd cultivrs currently used in our breeding progrms in order to estblish more efficient breeding strtegy under controlled temperture conditions. Mteril nd Methods The experiments were conducted under greenhouse nd controlled temperture room in Cchoeirinh, RS, Brzil (29 57 S; 51º06 W). Six rice genotypes were used s prents in complete dillel crossing scheme, without the reciprocls, mounting 15 combintions. Three of them belong to the jponic subspecies nd re cold tolernt (Rizbel, Aln nd ) nd the other three belong to the indic subspecies nd hve intermedite or sensitive rection to cold temperture t seedling stge (INIA Olimr, -20-4-3-3V nd IRGA 424). For ese of use, from now on the line - 20-4-3-3V will be referred to s only. The crosses were performed in the 2007/08 growing seson nd on the following winter seson some of the F 1 seeds were cultivted in 5 L plstic pots filled with field soil nd kept in greenhouse (28 C) in order to obtin the F 2 seeds. Experiment I ws conducted from June to July 2008. Seeds of the six prents nd of the 15 F 1 hybrids obtined in the prtil dillel were sowed in Petri dishes with two pieces of moist germintion pper nd set to germinte t 28 C in BOD chmber. After five to seven dys they were trnsplnted to trys filled with orgnic soil nd kept in greenhouse with controlled temperture (28 C ± 2 C). The experimentl design ws completely rndomized blocks with four replictions nd ten plnts of ech prent nd F 1 hybrid per repliction. Plnts were kept t the greenhouse until V 3 -V 4 stge (Counce et l., 2000) when they were tken to controlled temperture nd rtificilly lighted room t 10 C for ten dys. After this period the plnts were tken bck to the greenhouse nd evluted for percentge of plnt survivl fter seven dys of recovery under norml temperture (28 C). Dt for the prents nd F 1 s were submitted to nlysis of vrince nd comprison of mens using SNK test (α = 0.05), ll performed in the SAS progrm (SAS Institute, 2000). Dillel nlysis ws performed using the GENES softwre (Cruz, 2006) ccording to the model proposed by Griffing (1956), method 2, considering fixed model without the reciprocls. Qudrtic components for the fixed model were estimted ccording to Cruz nd Regzzi (1994): φ g = GMS - EMS / p + 2 φ s = SMS - EMS where φ g = qudrtic component ssocited to the generl combining bility (GCA); φ s = qudrtic component ssocited to the specific combining bility (SCA); GMS = GCA Men Squre; EMS = Error Men Squre; SMS = SCA Men Squre, nd p = number of prents in the dillel. Experiment II ws performed from Jnury to July 2009. Pre-germinted seeds of the prents nd of 10 F 2 popultions were trnsplnted to trys filled with orgnic soil nd kept in greenhouse with controlled temperture (28 C ± 2 C). A vrible number of F 2 plnts ws evluted per popultion, depending on the vilbility of seed. In generl, for ech F 2 popultion three trys of 150 cells were used, with 130 F 2 plnts nd 10 plnts for ech prentl genotype per try. Only ten F 2 popultions were evluted in this experiment due to lck of enough F 2 seeds for the remining five popultions of the dillel. The conduction of the experiment nd cold tolernce evlution were performed s described for experiment I. The prents were evluted by n nlysis of vrince of the percentge of plnt survivl in completely rndomized design with vrible number of replictions, depending on the number of crosses they were included. Comprison of mens ws mde by the lest squre test. All these nlysis were performed in the SAS progrm (SAS Institute, 2000). The F 2 popultions were evluted by counting the number of surviving nd non-surviving plnts in ech nd the dt ws submitted to the Chi-Squre test to verify djustment to the theoreticl proportions expected for one nd two independent genes nd two complementry genes segregting. Chi-Squre vlues were obtined through the following formul: χ 2 = Σ (F o F e ) 2 / F e, where F o = observed frequency for ech clss; nd F e = expected frequency for ech clss, bsed on the Mendelin proportion. Results Anlysis of vrince showed highly significnt vrition mong replictions, genotypes, Generl Combining Ability (GCA) nd Specific Combining Ability (SCA) for percentge of plnt survivl (Tble 1). The significnce of GCA nd SCA indictes tht the prents differed in reltion to the generl combining bility (GCA) nd the hybrids presented distinct specific combining bilities (SCA) (Tble 1). It lso indictes tht both dditive nd non-dditive genetic effects re involved in the inheritnce of the trit. However, the higher mgnitude of the GCA component mens tht dditive gene ction is predominnt over non-dditivity (Tble 1). On the other hnd, qudrtic component ssocited to SCA presented higher mgnitude thn the Sci. Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010
Inheritnce of rice cold tolernce t the seedling stge 671 Tble 1 Anlysis of vrince for percentge of plnt survivl from cold tretment evluted in dillel involving six prents nd 15 F 1 hybrids without the reciprocls. Source of vrition Degrees of freedom Men Squre Replictions 3 968.17* * Tretments 20 2191.48* * GCA 5 5152.82* * SCA 15 1204.36* Error 58 242.98 R 2 = 0.76 φg 1 = 613.73 CV(%) = 19.1 φs 2 = 961.38 Key: GCA = generl combining bility; SCA = specific combining bility. ** Significnt (α = 0.01). 1 Qudrtic component ssocited with the GCA. 2 Qudrtic component ssocited with the SCA. one ssocited to GCA (Tble 1). In the fixed model, the qudrtic components express the genetic vribility of the genotypes studied (Cruz nd Regzzi, 1994). The superiority of the qudrtic component ssocited to SCA is observed in previous selected genetic mteril, in which the differentil for dditive effects my be reduced. This is the cse here, where prentl genotypes re very contrsting nd belong to two subspecies tht hve developed dpttion to specific environments, like cold temperture tolernce in the cse of jponic. So, these ones hve, in fct, been previously selected for cold tolernce s they originted from colder regions. There ws lrge vrition mong prentl genotypes (Tble 2), with the three jponic genotypes presenting the higher percentge of survivl, ll clssified s tolernt to cold t seedling stge. Among indic genotypes, INIA Olimr presented n intermedite behviour nd nd sensitive rection (Tble 2). Regrding the F 1 hybrids, the mjority presented tolernt rection, with more thn 80% of plnt survivl. This clerly demonstrtes dominnt gene ction for cold tolernce s evluted by the percentge of plnt survivl in these genotypes. The only exception were the two crosses between sensitive prents, in which IRGA 424 ws one of them, tht presented intermedite rection (between 30 nd 70% of plnt survivl) (Tble 2). In bsolute terms, lmost ll F1 s presented higher plnt survivl thn both of the prents, indicting overdominnce for cold tolernce. The exceptions were the crosses Rizbel nd Rizbel. However, sttisticlly ll the F 1 s did not differ from one of the prents, indicting dominnce for cold tolernce (Tble 2). By these results it is evident the importnce of non-dditivity for cold tolernce t the vegettive stge. The prentl performnce per se seemed to be good index of their GCA effects for cold tolernce, s the jponic prents showed positive vlues for GCA effects nd the indic genotypes negtive vlues (Tble 3). The three tolernt genotypes hd significnt nd positive GCA estimtes, indicting tht ll of them present good combing bility to be used s donors to trnsfer cold tolernce to sensitive genotypes (Tble 3). Among the cold sensitive prents, ws the worst genotype s donor for this trit (Tble 3), s it cn be seen by its low plnt survivl rte (Tble 2). Tble 2 Percentge of plnt survivl from cold tretment for the six prents nd 15 F 1 hybrids. Rizbel Aln INIA Olimr Rizbel Aln INIA Olimr 1 92.6 97.5 100.0 100.0 92.2 92.2 1 Mens followed by the sme letter do not differ (SNK test, α = 0.05). * Significnt (α = 0.05). ** Significnt (α = 0.01). 80.7 b 97.5 97.5 97.5 97.5 83.0 b 91.4 92.5 97.5 52.9 bc 80.1 b 55.3 bc 30.6 c 43.1 c 10.3 d Tble 3 Generl combining bility (GCA) nd specific combining bility (SCA) effects of six rice genotypes nd their hybrids for percentge of plnt survivl fter cold tretment. Rizbel Aln INIA Olimr R izbel - 14.59* * -6.40* - 3.51 6.20* 6.09* 18.64* * 12.79* * A ln -19.90** - 2.71 6.99* 14.68* * 27.23* * 9.50* L 2825CA -16.82** 1.29 10.08* 27.63* * 9.11* I NIA Olimr - 8.50* 7.38* -4.87-0.59 I RGA 2852-14.43* - 9.38* -8.28* I RGA 424-29.63* * -20.83** GCA Sci. Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010
672 Cruz et l. The hybrids between jponic cold tolernt genotypes presented negtive vlues for SCA, indicting tht no increment in cold tolernce might be gined by crossing these genotypes. On the other hnd, the crosses involving jponic prent nd n indic one showed only positive effects for SCA, reveling existence of heterosis for plnt survivl (Tble 3). Most of them were significnt, with the exception of the cross INIA Olimr, indicting lmost no heterosis in this combintion. Among the jponic indic crosses the highest heterosis ws found in the combintions involving s the sensitive prent. Among the indic genotypes, the only combintion tht produced positive nd significnt SCA effects ws INIA Olimr, indicting tht this cross exhibits heterosis for cold tolernce. The SCA vlues for the prents hve genetic mening both in their signl s well s their mgnitude. The negtive vlue mens existence of unidirectionl dominnce devitions, so positive heterosis in hybrids between divergent prents. The high mgnitude mens high genetic divergence of genotype in reltion to the men of the others nd so, high heterosis in the hybrids. In the present cse ll prents presented negtive nd significnt SCA effects, with the highest mgnitude found in Aln nd. In fct these were the prents tht provided the highest positive heterosis considering ll hybrid combintions (Tble 3). Prentl mens in experiment II hd wide vrition from 2.5% of plnt survivl in to 97.7% in Rizbel (Tble 4). This cold rection is quite similr to the results obtined in experiment I (Tble 2), clssifying Rizbel, Aln nd s cold tolernt (more thn 70% of plnt survivl), INIA Olimr s intermedite (between 40 nd 70% of plnt survivl) nd nd s sensitive (below 40% of plnt survivl). Results reltive to the F 2 genertion reveled tht in popultions in which there ws t lest one cold tolernt prent (jponic jponic nd jponic indic), there ws predominnce of surviving plnts, indicting dominnce gene ction for tolernce is these crosses (Tble 5, Figures 1 nd 2). On the other hnd, in the sensitive intermedite crosses (indic indic) there were more non-surviving plnts thn surviving, nd in the sensitive sensitive cross (indic indic) there were no surviving plnts t ll (Tble 5 nd Figure 1). Among the cold tolernt prents, the popultion Aln Rizbel presented one dominnt llele segregting for cold tolernce nd Rizbel presented two independent genes with dominnt lleles segregting for this trit. The popultion Aln did not fit into ny of the genetic hypothesis tested (Tble 5). The crosses involving tolernt sensitive genotypes showed two independent genes with dominnt lleles segregting for cold tolernce (Tble 5). The cultivr INIA Olimr presents two gene difference from the sensitive genotypes. In the popultion INIA Olimr there re two independent genes with recessive lleles segregting for cold tolernce nd in INIA Olimr there re two complementry genes with recessive lleles segregting for cold tolernce (Tble 5). The genetic nlysis evi- Tble 4 Percentge of plnt survivl of the prents used in the study of the inheritnce of cold tolernce t the seedling stge of rice. G enotype Men of plnt survivl (%) Rizbel Aln INIA Olimr 97.7 83.3 b 81.8 b 56.6 c 22.2 d 2.5 e Mens followed by the sme letter do not differ (lest squre test, α = 0.05) Tble 5 Number of surviving nd non-surviving plnts, totl number of plnts nd djustment of the Chi-Squre test for 10 F 2 popultions of rice evluted for cold tolernce t seedling stge. Popultion Surviving Number of plnts Non- Surviving Chi-Squre Totl 3: 1 15: 1 9: 7 A ln (T) Rizbel (T) 277 107 384 1. 7 306.1* * 39.4* * A ln (T) (T) 334 39 373 42.1* * 11.3* * 168.0* * R izbel (T) (T) 365 13 378 93.7* * 5. 1 249.6* * A ln (T) (S) 363 17 380 85.4* * 2. 0 238.2* * A ln (T) (S) 367 17 384 86.7* * 2. 2 241.3* * R izbel (T) (S) 231 6 237 63.8* * 5. 6 163.6* * L 2825CA (T) (S) 368 17 385 87.0* * 2. 2 242.1* * I RGA 424 (S) INIA Olimr (I) 12 321 333 905.3* * 4. 1 219.0* * I RGA 2852 (S) (S) 0 358 358 1074.0* * 23.4* * 279.6* * I RGA 2852 (S) INIA Olimr (I) 114 127 241 98.6* 696.8* * 1. 4 χ 2 = 6.64. ** Significnt (α = 0.01)..01(1) Sci. Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010
673 Inheritnce of rice cold tolernce t the seedling stge Discussion Figure 1 Sttus of the F2 genertion of the indic indic crosses (left) nd of the jponic jponic crosses (right) seven dys fter recovery in greenhouse (28 C) from the cold tretment of 10 C for ten dys t the V3 stge. Figure 2 Sttus of the F2 genertion of the indic jponic crosses seven dys fter recovery in greenhouse (28 C) from the cold tretment of 10 C for ten dys t the V3 stge. denced monogenic nd oligogenic inheritnce for cold tolernce t seedling stge in rice, with one or two genes segregting in the popultions studied (Tble 5). Cold tolernce t the seedling stge hs simple genetic bsis in the popultions studied, with dominnt lleles in the cold tolernt prents nd recessive lleles in the intermedite genotype INIA Olimr. The complementry genes segregting in the cross INIA Olimr indictes tht might possess recessive llele for cold tolernce s well. In this study rice cold tolernce t seedling stge ws evluted by the percentge of survivl fter exposing plnts t the V3 stge to cold temperture (10 C) for ten dys. In geneticl studies the mode of inheritnce determined is vlid only for the evluted trit, the geneticl mteril studied nd, in this cse, the intensity nd durtion of stress imposed (Cruz nd Milch, 2000). In the present cse, cold tolernce hs simple genetic bsis with both dditive nd dominnce gene ction involved (Tbles 1 nd 5). Similr results were lso obtined in genetic studies performed under controlled temperture conditions (Kwk et l., 1984; Kw nd Khush, 1986; Ngmine nd Nkghr, 1991; Shhi nd Khush, 1986), like we hve done here, indicting fesibility of selection for cold tolernce under these conditions. Moleculr pproches such s QTL mpping hve been employed to study the genetic rchitecture of importnt gronomic trits. In the cse of rice cold tolernce t the seedling stge rnge of stress conditions hs been pplied, with some of them relting mjor QTLs involved in cold tolernce (Andy nd Mckill, 2003; Lou et l., 2007) while others hve found both mjor nd minor QTLs controlling this trit (Jing et l., 2008; Zhng et l., 2005). All these results were obtined in recombinnt inbred lines (RILs) or double hploid (DH) popultions between indic jponic prents, such s in our study. The jponic prents contributed with mjor dominnt lleles for cold tolernce nd in the indic crosses recessive lleles nd episttic interction were detected (Tble 5). By evlution of seedling mortlity of 71 RILs, Jing et l. (2008) found three QTLs relted to cold tolernce, one mjor QTL contributed by the jponic prent nd two minor QTLs from the indic one. Episttic interctions hve lso been relted in the control of seedling cold tolernce in rice (Lou et l., 2007; Zhng et l., 2005) nd in low temperture germintion bility (Chen et l., 2006). The existence of segregtion in the crosses T T (Tble 5) indictes tht the cold tolernt genotypes studied crry different genes for cold tolernce. This mens tht these genes my probbly be grouped into single genotype, but for this moleculr mrkers re necessry in order to differentite the genes nd llow for selection. Approches like genetic trnsformtion nd gene expression hve shown tht mny different pthwys my be involved in rice cold tolernce t seedling stge, with genes for scobrte peroxidse (Sto et l., 2001), fructn synthesis (Kwkmi et l., 2008) nd trnscription fctor (Wng et l., 2003) ssocited with incresed levels of tolernce. Differences in the ctivity nd ccumultion of oxidtive enzymes nd sugrs were found between cold sensitive genotype nd cold tolernt one (Morsy et l., 2007), corroborting the relted bove. These results suggest tht mny different genes my be involved with rice seedling cold tolernce, s hs been Sci. Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010
674 Cruz et l. found in some QTL studies (Hn et l., 2005; Qin et l., 2000). But they do not exclude the possibility of one or more mjor genes being involved, s my be the cse with trnscription fctors. Dillel nlysis showed the importnce of both dditive nd non dditive gene ction in rice seedling cold tolernce. This hs lso been found in low temperture germintion studies (Cruz et l., 2006; Shrifi, 2008) where both GCA nd SCA were significnt. Similrly to the results reported here, those studies found tht GCA ws of higher mgnitude, suggesting the greter importnce of dditive effects. All this indictes tht selection for cold tolernce my be effective in crosses between contrsting genotypes like the indic jponic. The im of studying the genetic bsis of cold tolernce t seedling stge ws to estblish when to pply selection pressure, if in erly genertions of the breeding progrm or in dvnced ones. Selection pressure under controlled temperture conditions my be useful in erly genertions, s only two genes with dominnt lleles were found segregting in T S crosses. However, s the dominnt lleles were responsible for tolernce, progeny tests re necessry. The three jponic prents evluted re good donors for cold tolernce genes, s showed by their high GCA effects (Tble 3) nd s relted by Kw nd Khush (1986) prentl performnce per se ws good index of the GCA effects for cold tolernce. According to the SCA effects, the crosses involving ny of the jponic prents versus re the most fvourble ones to select superior genotypes. Acknowledgements To the Brzilin PIBIC/CNPq for the scholrships provided on this project. References Andy, V.C.; Mckill, D.J. 2003. Mpping of QTLs ssocited with cold tolernce during the vegettive stge in rice. Journl of Experimentl Botny 54: 2579-2585. Chen, L.; Lou, Q.; Sun, Z.; Xing, Y.; Yu, X.; Luo, L. 2006. QTL mpping of low temperture on germintion rte of rice. 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Agric. (Pircicb, Brz.), v.67, n.6, p.669-674, November/December 2010