1. ESCOLA TÈCNICA SUPERIOR DENGINYERIA AGRÀRIA (ETSEA) www.icrea.es Dr. Gustavo A. Slafer Research Professor of ICREA Department of Crop & Forest Sciences University of Lleida - Centre UdL-IRTA Do yield QTLs mean anything? A crop-physiology perspective Gustavo A. Slafer & Ignacio Romagosa

2. Yield of cereals has been very strongly increased during the last half century (e.g. Calderini & Slafer, 1998. Field Crops Res., 57:335-347; Slafer & Peltonen-Sainio, 2001. Agric. Food Sci. Finland 10:121-131; Cassman et al., 2003. Annu. Rev. Environ. Resour. 28:315–58)Yield of cereals has been very strongly increased during the last half century (e.g. Calderini & Slafer, 1998. Field Crops Res., 57:335-347; Slafer & Peltonen-Sainio, 2001. Agric. Food Sci. Finland 10:121-131; Cassman et al., 2003. Annu. Rev. Environ. Resour. 28:315–58) This was due to genetic improvements in both yield potential and in resistance to diseases as well as to improvements in management (e.g. Slafer & Andrade, 1991. Euphytica, 58, 37-49; Austin, 1999. Crop Science 39:1604- 1610)This was due to genetic improvements in both yield potential and in resistance to diseases as well as to improvements in management (e.g. Slafer & Andrade, 1991. Euphytica, 58, 37-49; Austin, 1999. Crop Science 39:1604- 1610) Improved yield potential has concomitantly improved yield responsiveness to environment (Calderini & Slafer, 1999. Euphytica 107: 51–59)Improved yield potential has concomitantly improved yield responsiveness to environment (Calderini & Slafer, 1999. Euphytica 107: 51–59) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

3. Further improving yield potential seems not simple: it implies that future genetic improvement should be as efficient as it was in the past, but this time with cereal cropsFurther improving yield potential seems not simple: it implies that future genetic improvement should be as efficient as it was in the past, but this time with cereal crops -that have already received an intense breeding effort -that possess a relatively high yield level (which, in turn, is likely the reason why genetic increases in wheat yields are becoming increasingly harder to achieve; Reynolds et al., 1996; In Increasing Yield Potential in Wheat: Breaking the Barriers. Mexico DF: CIMMYT) -that have to be grown with agronomic practices more sustainable than those used in the past (i.e. part of the past yield gains related to the increased use of inputs, largely fertilizers and pesticides, must be replaced by breeding) In this context, it may be useful to complement traditional breeding with other approachesIn this context, it may be useful to complement traditional breeding with other approaches Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

4. As molecular biology has the potential to identify and map particular genes or QTLs related to any trait - even quite complex ones such as WUE, NUE or yield… … and its usefulness in the case of traits controlled by major (or few) genes is beyond any questioning It is tempting to believe that the issue will be resolved with molecular biology tools, by simply identifying Yield-QTLs and complement traditional breeding with marker assisted selection Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

5. However, when dealing with complex traits (such as yield) the effort of finding appropriate QTLs has experienced problems in accuracy, trustworthiness and applicability Most of these problems seem related to the dependence on the genetic background of the mapping population dependence on the genetic background of the mapping population strong G x E interaction when the QTL has been identified (e.g. ) strong G x E interaction when the QTL has been identified (e.g. Romagosa et al. 1996. Theoretical & Applied Genetics 93:30-37) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

6. Although I cannot discuss the issue further here… Ann. Appl. Biol. [2003] 142:117-128 Trends in Plant Science 9:70-75 Ann. Appl. Biol. 146:61–70 Current Opinion in Plant Biology 8:337-341 …I would like to strongly suggest that these two problems may be behind the scientific curiosity that while the literature Although I cannot discuss the issue further here… (but, if you are interested, see details behind this reasoning inGenetic basis of yield as viewed from a crop physiologists perspective in Ann. Appl. Biol. [2003] 142:117-128; and see also Sinclair et al., 2004. Trends in Plant Science 9:70-75; Slafer et al., 2005. Ann. Appl. Biol. 146:61–70; and Wollenweber et al., 2005. Current Opinion in Plant Biology 8:337-341) …I would like to strongly suggest that these two problems may be behind the scientific curiosity that while the literature.- is plenty of papers with mapped QTLs for yield.- is plenty of papers with mapped QTLs for yield (that might be easily introgressed in the breeding program by marker assisted selection).- is virtually empty, as far as I am aware, of clear successful case-stories in which a QTL for yield may work in different populations and environments to those in which it was mapped Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

7. It seems clear that we can map virtually any trait but when it comes to yield (or any other complex trait, e.g. WUE, NUE, tolerance to complex abiotic stresses) the direct identification of QTLs in a mapping population doesnt seem quite useful in practice … … unless the identification of the Yield-QTL is just the first step in a top-down approach to identify relatively simple traits related to yield (though in this case, chances are that the GxE interaction for these traits may be similar than that for yield) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

8. The problem with the top-down approach is that any trait that might be putatively associated with yield but whose magnitude of effect may be relatively small would hardly be uncovered. This may be irrelevant for a crop with little selection pressure imposed in the past, but for most important crops we need fine-tuning for further rising yield For uncovering these possible traits a bottom-up approach might be rewarding, but avoiding the failures of the past physiological attempts based on traits at a far lower level of organisation to that of crop yield, assuming that the interactions among traits at different levels of organisation might be negligible… For uncovering these possible traits a bottom-up approach might be rewarding, but avoiding the failures of the past physiological attempts based on traits at a far lower level of organisation to that of crop yield, assuming –perhaps unconsciously- that the interactions among traits at different levels of organisation might be negligible… Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

9. Figures from Structuralism by Jean Piaget Passioura (1979) Accountability, Philosophy and Plant Physiology Search 10:347-350 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

10. Would it be possible to identify traits that being simple (controlled by major -or at least few- genes) would be almost unequivocally related to yield (naturally under in field conditions) ? As yield is strongly related to number of grains per m 2 (grain weight seem to be strongy sink-limited during the effective grain filling period, at least in healthy crops) I will (i) describe very briefly a major physiological attribute (almost) universally related to grain number and and (ii) Discuss some approaches to identify genetic bases for this physiological determinant of grain number (and yield) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

11. Sw Em At DR FI BGF TS Timing when yield is mostly affected Number of grains per uinit land area (% unstressed) Anthesis Stem Spike Stem or Spike dry matter Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

12. Spike weight at anthesis (g m -2 ) Fertile florets or grains (m -2 ) Due to radiation levels Fischer, 1985 (Mexico-Australia); Thorne & Wood, 1987 (United Kingdom); Savin & Slafer, 1991 (Argentina); Abbate et al., 1995 (Argentina, Southern wheat belt); Demontes-Meinard et al., 1999 (France) Slafer et al 2005, Ann Appl Biol 146,61-70 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

13. Anthesis Stem Spike Stem or Spike dry matter Spike growth rate duration Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

14. or Stem or Spike dry matter The growth of the spikes in this very short window of time is so relevant that most of the breeding success on improving wheat yields were based on improving this trait (e.g. Slafer et al, 1994; Calderini et al., 1999). G Yield G Yield H Index H Index G Number G Number SDW anth SDW anth (due to smaller stems) Height! Height! Traditional cultivar Modern cultivar Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

15. Spike weight at anthesis (g m -2 ) Fertile florets or grains (m -2 ) Due to radiation levels Fischer, 1985; Thorne & Wood, 1987; Savin & Slafer, 1991; Abbate et al., 1995; Demontes- Meinard et al., 1999 Due to genetics (semidwarf vs tall cvs.) Brooking & Kirby, 1981; Stockman et al., 1983; Miralles et al., 1998 Slafer et al 2005, Ann Appl Biol 146,61-70 Due to genetics (old vs modern cvs.) Siddique et al., 1989; Slafer & Andrade, 1993 Slafer et al., 1994 (modern-old x shading treatments) Due to genetics (Introgression of Lr19 from A. elongatum) Reynolds et al., 2001 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

16. or Stem or Spike dry matter Traditional cultivar Modern cultivar G Yield G Yield H Index H Index G Number G Number SDW anth SDW anth (due to smaller stems) Height! Height! Modern cvs already have optimum stature (e.g. Richards 1992; Miralles & Slafer, 1995) Modern cvs already have HI close to maximum (e.g. review by Calderini, Reynolds & Slafer, 1999) If further genetic gains in number of grains per m 2 are attempted, which would be sound as even modern cultivars are mostly sink- limited during grain filling, future breeding must find alternatives to biomass partitioning between the growing stems and spikes Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

17. Yield Spike Dry Weight (Anthesis) Spike DW Grain # Grain number m -2 Grain # Grain Yield Breeding effects Most management effects Length of the growth period Crop growth rate Partitioning to growing spikes Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

18. Anthesis Sw Em At DR FI BGF TS Spike dry matter Stem dry matter Number of grains per uinit land area (% unstressed) Growth Growth Partitioning Partitioning SDW anth SDW anth GNumber GNumber Photoperiod sensitivity? Intrinsic earliness? Length of critical phase Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

19. Variation in stem elongation phase (independent of cycle length) Slafer, 2003. Ann. Appl. Biol., 142:117-128 0200400600800100012001400 BUCK CHAMBERGO KLEIN DON ENRIQUE Wheat Short cycle 1600 BONAER. ALAZAN KLEIN ESTRELLA Wheat Long cycle Thermal time from seedling emergence (°C d) 05001000150020002500 Kernich et al. (1997) Aust. J. Agric Res PROCTOR WEEAH SCHOONER TRIUMPH Barley Short cycle Barley Long cycle Whitechurch et al. under revision Differences may be due to photoperiod or earliness per se Sowing-jointing Stem Elongation Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

20. We conducted several studies manipulating photoperiod throughout the season for a single sowing date in whichWe conducted several studies manipulating photoperiod throughout the season for a single sowing date in which (i) sensitivity of the length of the stem elongation was clear, and(i) sensitivity of the length of the stem elongation was clear, and (ii) changes in number of fertile florets or grains were associated with changes in duration of stem elongation(ii) changes in number of fertile florets or grains were associated with changes in duration of stem elongation Slafer & Rawson, 1995 J Expt Bot, 46:1877-1886, Slafer & Rawson, 1996. Field Crops Res, 46:1-13, Slafer & Rawson, 1997. Aust J Plant Physiol, 24:151-158, Whitechurch & Slafer, 2001. Euphytica, 118:47-51, Miralles, Ferro & Slafer, 2001. Field Crops Res, 71:211-223, Slafer et al., 2001. Euphytica, 119:191-197 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

21. Then we determined (in a phytotron) that direct photoperiod effects on this phase were likely and that these effects were correlated to changes in number of fertile florets at anthesis (Then we determined (in a phytotron) that direct photoperiod effects on this phase were likely and that these effects were correlated to changes in number of fertile florets at anthesis (Miralles & Richards, 2000. Ann Bot, 85:655-663 Miralles, Richards & Slafer, 2000. Aust J Plant Physiol, 27:931-940) Gabriela Abeledo and more recently Fernanda González studied these effects in field plotsGabriela Abeledo (Slafer and Abeledo, unpublished) and more recently Fernanda González (e.g. González, Slafer & Miralles, 2003. Field Crops Res.) studied these effects in field plots Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

22. Extended photoperiod only during TSI-Anthesis (+6 h) or natural (+0 h) throughout, under field conditions (González, Slafer & Miralles, FCR, 2003). Study included 15 or 50 d of vernalizaton but BM is insensitive 0 0.1 0.2 0.3 0.4 40060080010001200 +6 +0 Thermal time (ºCd) Dry matter (g/spike) Buck Manantial 020040060080010001200 50 d +0 +6 Thermal time (ºCd) 15 d +0 +6 TSI Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

23. 0 5 10 15 20 25 0246 Fertile florets Spikelet position within the spike González, Slafer & Miralles, 2003, Field Crops Res 81:29-38 0 200 400 600 +0 +6 Stem elongation (ºCd) Photoperiod extension (h) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

24. 020406080100120140 TR-TSTS-Ant C TR-TS TS-Ant C TS-An Sw Days after transplant Vazquez et al. 2005, 2006 (M Sci. Thesis candidate) ppd-D1 ppd-B1 ppd-D1 Ppd-B1 Ppd-D1 ppd-B1

25. Spike weight at anthesis (g m -2 ) Fertile florets or grains (m -2 ) Due to photoperiod effects on the length of stem elongation Miralles et al., 2000 ; Slafer et al., 2001; González et al., 2003; González et al., 2005 Due to radiation levels Fischer, 1985; Thorne & Wood, 1987; Savin & Slafer, 1991; Abbate et al., 1995; Demontes-Meinard et al., 1999 Due to genetics Brooking & Kirby, 1981; Stockman et al., 1983; Siddique et al., 1989; Slafer & Andrade, 1993; Miralles et al., 1998; Reynolds et al., 2001 Slafer et al 2005, Ann Appl Biol 146,61-70 Slafer et al., 1994 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

26. Spike weight at anthesis (g m -2 ) Fertile florets or grains (m -2 ) Slafer et al 2005, Ann Appl Biol 146,61-70 Due to photoperiod sensitivity genes??? Due to photoperiod effects on the length of stem elongation Miralles et al., 2000 ; Slafer et al., 2001; González et al., 2003; González et al., 2005 Due to radiation levels Fischer, 1985; Thorne & Wood, 1987; Savin & Slafer, 1991; Abbate et al., 1995; Demontes-Meinard et al., 1999 Due to genetics Brooking & Kirby, 1981; Stockman et al., 1983; Siddique et al., 1989; Slafer & Andrade, 1993; Miralles et al., 1998; Reynolds et al., 2001 Slafer et al., 1994 Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

27. 6 8 10 12 14 16 18 Mercia isogenic lines (recessive, Ppd-D1 and Ppd-B1) R 2 = 0.80 p < 0.001 20406080100120140 Main shoot spike dry weight (g m -2 ) Number of fertile florets (10 -3 m -2 ) González, Slafer & Miralles, 2006, Euphytica Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

28. Ppd-D1 Yes Scarth et al., 1985 - (Ann. Bot.) Gonzalez et al., 2006 (Euphytica) No Foulkes et al., 2004 - (Euphytica) Ppd-B1 Yes Whitechurch & Slafer, 2001 (Euphytica) Yes Whitechurch & Slafer, 2002 (Field C. Res) No Gonzalez et al., 2006 (Euphytica) No Scarth et al., 1985 - (Ann. Bot.) Ppd-A1 No studies that I am aware Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

29. Lack of consistency may be due to interactions with genetic background interactions with environmental background (in which treatments were imposed) Different types of lines (with more or less contributions from other genes to the different phenotype) Lack of knowledge on the effects of Ppd alleles that have not been discovered yet but must be located in chromosomes 1 and 6, from evidences available on photoperiod sensitive genes in barley (Snape et al., 2001; Euphytica 119) Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

30. Genetic control of duration of stem elongation Population Oregon Wolfe Barley, 94 di-haploid lines, extreme morphological variation Glasshouse study under natural photoperiod Martí, Romagosa & Slafer, unpublished Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

31. Sowing-onset stem elongation Stem elongation 0 2 3 5 6 8 9 LOD 0 2 3 5 6 8 9 LOD Martí, Romagosa & Slafer, unpublished 1(7H) 2(2H) 3(3H) 4(4H) 5(1H) 6(6H) 7(5H) Chromosome Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

32. Genetic control of duration of stem elongation Population from Wageningen University, 120 di-haploid lines, parents differing in yield potential and yield stability (Meltan x Hennie) Field study in two contrasting conditions (Gimenells = irrigated and Foradada = rainfed) It was found, in both experiments, that the duration of phases occurring before or after the onset of stem elongation were controlled, in part, by independent QTLs, For the stem elongation phase we found a QTL located in chromosome 2 (LOD=4.5) that did not affect development before jointing…. But this is in conflict with results from the OWB population (chromosome 1), indicating that the genetic control may be more complex than I expected beforehand Borras, Slafer, Romagosa & van Eeuwijk, unpublished Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

33. î Identifying QTLs for yield or any complex trait may be only useful as an initial step for uncovering bases (genetic and physiological) useful for further rise productivity î This top-down approach to uncover useful bases might not identify traits that being putatively related to tield may still have a relatively small impact compared with that of large differences in biomass or partitioning î A bottom-up approach should only be followed with traits putatively related to the complex trait we are interested in, and only under realistic (field) conditions Conclusions Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

34. î One alternative might be increasing sensitivity to photoperiod in the late reproductive phase so that stem elongation becomes longer (combined with a shorter vegetative+early reproductive phases) then increasing growth when the inflorescences grow and reducing rate of degeneration of floret primordia î The alternative seemed likely from experiments manipulating either photoperiod or photoperiod-sensitivity genes î Although with only incipient results so far, it seemed that genetic control of duration of different pre-anthesis phases may be at least partially independent, though the genetic bases seems not simple Conclusions Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida

35. ACKNOWLEDGEMENTS L. Gabriela ABELEDO Eileen M. WHITECHURCH Fernanda G. GONZALEZ Daniel J. MIRALLES IgnacioROMAGOSA Jordi MARTI Gisela BORRAS Gustavo A. Slafer Centre UdL-IRTA Universitat de Lleida