Differences in the genetic control of pre-heading phases in barley and wheat, and relationships
with agronomic traits
Gisela Borràs Gelonch
Dipòsit Legal: L.1314-2013 http://hdl.handle.net/10803/120451
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llegit la tesi
DIFFERENCES IN THE GENETIC CONTROL OF PRE-HEADING PHASES IN BARLEY AND WHEAT AND RELATIONSHIPS WITH
AGRONOMIC TRAITS
Tesis doctoral
GISELA BORRÀS GELONCH
DIFFERENCES IN THE GENETIC CONTROL OF PRE-HEADING PHASES IN BARLEY AND WHEAT,
AND RELATIONSHIPS WITH AGRONOMIC TRAITS
Tesis doctoral
GISELA BORRÀS GELONCH
Directors: Ignacio Romagosa Clariana Gustavo A. Slafer Fred van Eeuwijk
Lleida, Abril 2013
A la meva mare
i AGRAÏMENTS
Agraeixo l’ajuda i col·laboració de totes aquelles persones que han fet possible la realització d’aquesta tesis. D’entrada agrair en especial a l’Ignacio Romagosa la co-direcció de la tesis, el seu temps i la seva ajuda malgrat totes les seves responsabilitats, i pels seus consells, l’haver facilitat en varis aspectes la realització dels assajos, l’oportunitat d’assistir en els cursos a Zaragoza i a un congrés, i la seva comprensió en alguns dels moments més difícils. Agraeixo també al co-director Fred van Eeuwijk i a en Marcos Malosetti de la Universitat de Wageningen la seva ajuda en els anàlisis estadístics, i per l’oportunitat d’assistir al curs d’estadística a Wageningen. També al director Gustavo A. Slafer per alguns dels seus suggeriments, i perquè de tot s’aprèn.
Vull agrair també en Richard A. Richards i Greg J. Rebetzke del CSIRO a Canberra per l’enriquidora estada a Austràlia, per la seva ajuda i la possibilitat de treballar en els seus assajos. També en especial l’Ana M. Casas de l’Estació Experimental Aula Dei del CSIC (Zaragoza), per la seva valuosa contribució, i junt amb l’Ernesto Igartua, perquè de cada viatge a Zaragoza i trobada amb ells s’aprenia moltíssim. Al Bill Thomas per la seva aportació i per facilitar-nos el mapa de SNPs d’Steptoe x Morex. Al Marco Denti per la gran dedicació en el seu treball i per la seva ajuda. A la Maria Bagà, el Josep A. Betbesé i la resta de tècnics de l’àrea de conreus extensius de l’IRTA (Lleida) per la seva ajuda, professionalitat inestimable i disponibilitat. L’ajuda de què vaig disposar per la presa de dades va ser més que insuficient pel que
“proposava” el director (G. Slafer) en moltes ocasions, i no em va estalviar les dures jornades al camp i al laboratori, el treballar la majoria de caps de setmana i els maratonians horaris dia rere dia, en detriment de la salut en alguns casos, i de la realització de la tesis dins un temps raonable (tot i que les dades pels capítols 5, 6, 7 i 8 després ja no interessaven). Malgrat això vull agrair als tècnics que han contribuït amb el seu treball, paciència i dedicació en algun moment o altre en aquesta tesis, ja que sense ells moltes dades tampoc s’haurien pogut agafar: la Mireia, la Mònica, la Noemí, el Said, la Susana, la Belén i l’Andreu, i d’altres que han treballat puntualment. Però molt especialment agraeixo a l’Alex Pswarayi, per les llargues hores del seu temps que va dedicar a ajudar-me a comptar fulles i fillols al camp al 2004, i per prendre les dades de collita al 2005. A ell, a la Júlia, la Virginia, la Isabel, la Pili i el Marco, i els esmentats abans, pels moments compartits, pel seu companyerisme i amistat, i per fer l’ambient de treball molt més agradable.
Agraeixo també als pares, en especial a la meva mare, a qui li hagués agradat veure que acabava, a la meva àvia, al Luis i a altres amics i familiars, els consells, la força per seguir endavant, els petits i grans detalls, i l’ajudar-me a riure’m de tot plegat.
I a d’altres que em deixo, que han ajudat o han facilitat la feina en algun moment d’una manera o altra, gràcies. També un record especial per en Lluis Torres, tot i que no vam coincidir gaire, per haver sigut un grandíssim professor d’estadística. Sense la bona base que ens va donar, tota l’estadística després s’hauria fet més àrida.
ii Part d’aquesta tesis a sigut possible gràcies a la beca FPU que em va concedir el Ministeri de Ciència e Innovació al 2004. També agrair al programa europeu ICA3-CT2002-10026 Mapping Adaptation of Barley to Drought Environments (MABDE) bona part del finançament dels assajos, i a d’altres projectes finançats pel Ministeri.
iii SUMMARY
Crop phenology is widely recognized as the most important factor determining adaptability of wheat and barley cultivars to particular environments. The genetic control of flowering time has been extensively studied, although much less is known about differences in the genetic control of pre-heading phases. The aim of the present thesis was studying the genetic control of the duration of different pre-heading phases (leaf and spikelet initiation, LS, and stem elongation, SE) and identifying particular loci responsible for independent genotypic variability in LS and SE, as well as studying the possible impact that changing the ratio SE/LS could have on traits related to leaf appearance, tillering and other agronomic traits such as biomass and N accumulation and yield components among others. Two barley and two wheat double- haploid populations were grown in several Mediterranean environments. The Steptoe x Morex population (S x M), which is known to segregate for some major genes regulating flowering time, was grown under two different sowing dates and two photoperiod treatments. Independent genotypic variability in the duration of LS and SE was found in each population, and several quantitative trait loci (QTLs) responsible for these differences were also identified in each of them. None of the QTLs with different effects between LS and SE in the barley population Henni x Meltan (H x M) seemed to be major genes. Some major flowering genes were responsible for part of the differences in the ratio SE/LS in the other populations. In the S x M experiment, in spite of significant genotype x environment and QTL x E effects for both LS and SE, differences between genotypes in the ratio SE/LS were quite well maintained across environments (h2 of 0.82). Shortening LS, so as to lengthen SE, would not have a negative impact on early vigour, although some tillering traits could result affected. For the QTL with the highest effects on the ratio SE/LS (significant mainly for SE on 2HS) in H x M, a higher ratio was associated to lower rate of tillering, maximum number of tillers, spikes/m2, grains/m2, grain yield, and biomass partitioning both at heading and at harvest. Some correlations between duration of pre-heading phases and several agronomic traits varied depending on the environment, which could suggest that increasing the ratio SE/LS might be more beneficial in some environments than others. Two of the most important QTLs for grain yield in H x M were not related with duration of pre-heading phases, but were associated to early vigour, in line with is importance under Mediterranean conditions, or with higher spikes/m2 and other tillering traits, in line with the importance of tillering in 2-rowed barley. In the S x M population most tillering traits co-localised with QTLs for durations of phases (mainly LS and HD), and three of the most relevant regions for these traits contained three of the most important QTLs for the ratio SE/LS. Other QTLs significant for the ratio SE/LS in both HxM and SxM were not associated to negative effects for the studied traits. Effects on important aspects as tillering, biomass partitioning and spikelet generation, among others, from particular loci responsible of genetic differences between LS and SE, as well as interactions with the environment, should be considered since they may offset the advantages that a higher ratio SE/LS could have on yield generation.
iv RESUM
La fenologia del cultiu està àmpliament reconeguda com el factor més important que determina l'adaptació de cultivars de blat i ordi a ambients determinats. El control genètic de la floració ha sigut extensament estudiat, encara que se sap molt menys sobre les diferències en el control genètic de diferents fases en pre- espigat. L'objectiu d'aquesta tesi va ser estudiar el control genètic de la durada de diferents fases de pre- espigat (iniciació de fulles i espiguetes, LS, i encanyat, SE) e identificar loci particulars responsables de la variabilitat genotípica independent en LS i SE, així com estudiar el possible impacte que el canvi en la ràtio SE/LS podria tenir en caràcters relacionats amb l'aparició de fulles i fillols, i altres característiques agronòmiques com l’acumulació de biomassa i N, i components del rendiment, entre d'altres. Dues poblacions de doble-haploides d'ordi i dos de blat es van conrear en diversos ambients mediterranis. La població d'ordi Steptoe x Morex (S x M), que se sap que segrega per alguns gens importants de floració, es va conrear en dues dates de sembra diferents i dos tractaments de fotoperíode. Es va identificar variabilitat genotípica independent entre LS i SE a cada població, i diversos loci de caràcters quantitatius (QTLs) responsables d'aquestes diferències també van ser identificats en cadascuna d'elles. Cap dels QTLs amb efectes diferents entre LS i SE a la població d'ordi Henni x Meltan (H x M) semblen ser gens majors de floració coneguts. Alguns gens majors de floració foren responsables de part de les diferències en la ràtio SE/LS en les altres poblacions En l'experiment amb S x M, tot i les interaccions significatives genotip x ambient i QTL x E, tant per LS com per ES, les diferències entre genotips pel ràtio SE/LS es mantingueren bastant entre ambients (h2 de 0,82). Escurçar LS, per tal d'allargar SE, no tindria un impacte negatiu en el vigor inicial, encara que alguns caràcters de l’afillolament podrien resultar afectats. Per al QTL amb els majors efectes sobre la ràtio SE/LS (significatiu sobretot per SE en 2HS) en H x M, una major ràtio SE/LS estava associada a una reducció en la tasa d’afillolament, el nombre màxim de fillols, espigues/m2 , grans/m2, el rendiment de gra, i la partició de biomassa tant a espigat com a collita. Algunes correlacions entre la durada de fases en pre-espigat i diversos caràcters agronòmics variaren en funció de l'ambient, el que podria suggerir que l'augment en la ràtio SE/LS podria ser més beneficiós en alguns ambients que en d’altres. Dos dels QTLs més importants per rendiment de gra en H x M no estigueren relacionats amb la durada de fases en pre-espigat, sinó amb el vigor inicial, en línia amb la seva importància en condicions mediterrànies, o amb més espigues/m2 i altres caràcters d’afillolament, d'acord amb la seva importància en l'ordi de dues carreres. En la població S x M la majoria de caràcters relacionats amb l’afillolament co- localitzaren amb QTLs per la duració de fases (principalment LS i HD), i tres de les regions més importants per aquests caràcters contenien tres dels QTLs més importants per la ràtio SE/LS. Altres QTLs per la ràtio SE/LS en SxM i HxM no estigueren relacionats amb efectes negatius sobre els caràcters estudiats. Els possibles efectes de loci particulars responsables de les diferències genètiques entre LS i SE, sobre aspectes importants com la partició de la biomassa, l’afillolament, i la generació d’espiguetes, entre d'altres, així com les interaccions amb l’ambient, s'haurien de tenir també en compte, ja que poden contrarestar els avantatges que una major ràtio SE/LS podria tenir en la generació del rendiment.
v RESUMEN
La fenología del cultivo está ampliamente reconocida como el factor más importante que determina la adaptación de cultivares de trigo y cebada en ambientes determinados. El control genético de la floración ha sido extensamente estudiado, aunque se sabe mucho menos acerca de las diferencias en el control genético de distintas fases en pre-espigado. El objetivo de la presente tesis fue estudiar el control genético de la duración de diferentes fases de pre-espigado (iniciación de hojas y espiguillas, LS, y encañado, SE) e identificar loci particulares responsables de la variabilidad genotípica independiente en LS y SE, así como estudiar el posible impacto que el cambio en el ratio SE/LS podría tener en caracteres relacionados con la aparición de hojas, hijuelos y otras características agronómicas como la acumulación de biomasa y N, y los componentes del rendimiento, entre otros. Dos poblaciones de doble-haploides de cebada y dos de trigo se cultivaron en varios ambientes mediterráneos. La población de cebada Steptoe x Morex (S x M), que se sabe que segrega para algunos genes importantes de floración, se cultivó en dos fechas de siembra diferentes y dos tratamientos de fotoperiodo. Se identificó variabilidad genotípica independiente entre LS y SE en cada población, y varios loci de caracteres cuantitativos (QTLs) responsables de estas diferencias también fueron identificadas en cada una de ellas. Ninguno de los QTL con efectos diferentes entre LS y SE en la población de cebada Henni x Meltan (H x M) parecen ser genes mayores de floración conocidos.
Algunos genes mayores de floración fueron responsables de parte de las diferencias en el ratio SE/LS en las otras poblaciones. En el experimento con S x M, a pesar de significativas interacciones genotipo x ambiente y QTL x E, tanto para LS como para SE, las diferencias entre genotipos para el ratio SE/LS se mantuvieron bastante entre ambientes (h2 de 0,82). Acortar LS, con el fin de alargar SE, no tendría un impacto negativo en el vigor inicial, aunque algunos caracteres del ahijamiento podrían resultar afectados.
Para el QTL con los mayores efectos en el ratio SE/LS (significativo sobre todo para SE en 2HS) en H x M, un mayor ratio SE/LS estuvo asociado a una reducción en la tasa de ahijamiento, el número máximo de hijuelos, espigas/m2, granos/m2, el rendimiento de grano, y la partición de biomasa tanto en espigado como en cosecha. Algunas correlaciones entre la duración de fases en pre-espigado y varios caracteres agronómicos variaron en función del ambiente, lo que podría sugerir que el aumento en el ratio SE/LS podría ser más beneficioso en algunos ambientes que otros. Dos de los QTLs más importantes para rendimiento de grano en H x M no estuvieron relacionados con la duración de fases en pre-espigado, sino con el vigor inicial, en línea con su importancia en condiciones mediterráneas, o con más espigas/m2 y otros caracteres del ahijamiento, en consonancia con la importancia de éste en la cebada de dos carreras. En la población SxM la mayoría de caracteres relacionados con el ahijamiento co-localizaron con QTLs para la duración de fases (principalmente LS y HD), y 3 de las regiones más significativas para estos caracteres incluían 3 de los QTLs más importantes para el ratio SE/LS. Otros QTLs para el ratio SE/LS en HxM y SxM no estuvieron asociados a efectos negativos en los caracteres estudiados. Los posibles efectos de loci particulares responsables de las diferencias genéticas entre LS y SE, sobre aspectos importantes como la partición de la biomasa, el ahijamiento, y la generación de espiguillas, entre otros, así como las
vi interacciones con el medio ambiente, se deberían tener también en cuenta, ya que pueden contrarrestar las ventajas que un mayor ratio SE/LS podría tener en la generación del rendimiento.
vii INDEX
Agraïments ...i
Summary ...ii
Resum ...iii
Resumen ...iv
Index...vii
General Introduction ...2
Objectives ...15
Chapter 1: Genetic variability in duration of pre-heading phases and relationships with leaf appearance and tillering dynamics in a barley population ...26
Chapter 2: Genetic control of pre-heading phases and other traits related to development in a double-haploid barley (Hordeum vulgare L.) population ...38
Chapter 3: Genetic control of pre-heading phases in the Steptoe x Morex barley population under different conditions of photoperiod and temperature ...52
Chapter 4: Genetic control of duration of pre-anthesis phases in wheat (Triticum Aestivum L.) and relationships to leaf appearance, tillering, and dry matter accumulation ...72
Chapter 5: Yield generation and nitrogen dynamics in a modern 2-rowed barley population, and relationships with duration of pre-heading phases: I. Biomass and nitrogen accumulation ...100
Chapter 6: Yield generation and nitrogen dynamics in a modern 2-rowed barley population, and relationships with duration of pre-heading phases: II. Biomass and nitrogen partitioning, nitrogen remobilization and plant height ...136
Chapter 7: Yield generation and nitrogen dynamics in a modern 2-rowed barley population, and relationships with duration of pre-heading phases: III. Yield components ...156
Chapter 8: Relationships between duration of pre-heading phases and leaf appearance, tillering, fertile florets, grains per spike and other agronomic traits in the Steptoe x Morex population...184
General discussion...226
Conclusions ...246
Annex ...250
“Si no te comes la sopa te llevará el coco..”
“...Los macarras de la moral..”
“Quien se sale del rebaño, destierro y excomunión.”
Joan Manuel Serrat, 1998
Los macarras de la moral, Sombras de la China
General Introduction and Objectives
2
GENERAL INTRODUCTION
Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), both long-day species from the Triticeae tribe, are two of the most ancient crops (von Bothmer et al. 2003) and two of the most important temperate cereals in the world, with a cultivated area of c. 220 and 50 million ha respectively (FAO 2013, data for 2011). Nowadays both crops are cultivated in regions far away from their centre (or centres) of origin, in a wide range of environmental conditions, which is possible in part due to the wide differences between cultivars in developmental patterns and genes controlling flowering time in response to environmental cues (Worland 1996, von Bothmer et al. 2003, Igartua et al. 2008).
Development has been widely recognised as the most important factor determining adaptation and crop performance, particularly in determinate species as small-grain cereals. It allows matching crop phenology with availability of resources to maximise crop growth, and avoiding abiotic stresses due to climatic conditions (Richards 1991, Worland 1996). Under Mediterranean conditions the occurrence of frosts in late spring and terminal drought at the end of the season, together with the amount and distribution of rainfall, are the main factors that define critical periods for sowing and for phenological events, such as the transition of apices from vegetative to reproductive and flowering time (Richards 1991, van Oosterom and Acevedo 1992, Igartua et al. 2008).
In Spain barley is still the first ranking crop in devoted arable land and in total production (2.7 million ha and 8.3 million t), whereas less area is dedicated to wheat, which is usually grown under more favourable conditions of rainfall than barley (2 million ha and 6.9 million t; FAO 2013, data for 2011). The climate of the main regions of Spain where barley and wheat are grown can be defined in general as Mediterranean, although there are important differences from one region to the other, as the distribution and amount of rainfall, and temperature regimes (Yahiaoui 2006, Igartua et al. 2008).
The importance of flowering time as a key adaptative trait has been shown through numerous studies: For example, with the different and rapid shifts in heading time, or in vernalization and photoperiod responses, due to only natural selection, when the same bulk population is grown under contrasting environments (Danquah and Barrett 2002, Goldringer et al. 2006); when comparing several genotypes under different sowing dates (e.g. Young and Elliott 1994, Gómez-Macpherson and Richards 1997, Kirby et al. 1999);
when studying the contrasting developmental patterns of genotypes adapted to particular regions (van Oosterom and Acevedo 1992, Lasa et al. 2001); in retrospective studies showing changes in heading date over time due to breeding, particularly in areas where the crop was introduced more recently (e.g. bread wheat in Australia, Richards 1991; durum wheat in Spain, Álvaro et al. 2008); or in studies showing that differences amongst genotypes in heading are an important source of genotype by environment interactions for grain yield (Romagosa et al. 1996, Voltas et al. 2002, Igartua et al. 2008, Francia et al. 2011).
3 At the gene or marker level, the importance of flowering time in adaptability is shown for example through the geographical distribution of alleles of major genes such as PPD and VRN in wheat and barley (Worland 1996, Yahiaoui 2006, Cockram et al. 2007, Jones et al. 2008, Casas et al. 2011); and through the collocation of QTLs for heading with QTLs for yield or other important agronomic characters in many studies (e.g. Hayes et al 1993, Tinker et al. 1996, Li et al. 2006, Cuesta-Marcos et al. 2009), which may help in some cases to define an optimal window for heading and combination of alleles for major genes, in the tested environments (Cuesta-Marcos et al. 2009). In some of these studies, QTLs with strong effects on heading also co-located with some of the QTLs for yield that exhibited the strongest QTL by environment interactions (Hayes et al. 1993 and Romagosa et al. 1996, Tinker et al. 1996, Francia et al. 2011).
Many of these studies are focused on heading time. Depending on the purpose of the study, it may be interesting considering different pre-heading phases, and the different developmental processes and responses to the environment that occur in each one (e.g. Gómez-Macpherson et al. 1997). Studying the genetic control of different pre-heading phases could bring a better understanding of crop development patterns and more tools for fine-tunning it. For example some adaptative characters, such as the avoidance of late frosts in spring, are better assessed by knowing the duration of the phase from sowing to the onset of spikelet differentiation rather than total time to anthesis (e.g. Limin et al. 2007). On the other hand recent studies have shown that a great part of the effect on yield from QTLs for heading (underlying QTLxE for yield) can be explained by the different sensitivity of the alleles to environmental conditions such as temperature during different phases of the crop cycle (Malosetti et al. 2004, Francia et al. 2011). Moreover it has been suggested that optimising the partitioning of time to heading, changing duration of different pre- heading phases. could be used for increasing grain yield potential of small-grain cereals (Appleyard et al.
1982, Kitchen and Rasmusson 1983, Slafer et al. 2005).
1.- Changing duration of pre-heading phases to increase yield potential
Wheat and barley breeding for yield was greatly successful during the last century, mainly thanks to the improvement of harvest index through reductions in plant height, although with some exceptions (Abeledo et al. 2002). This, in turn, allowed unconsciously improving assimilates partitioning to the spikes (Fischer 2007). It seems unlikely that further reductions in plant height maintain increases in yield potential, since most modern cultivars are within the optimal height to maximise grain yield and near the theoretical limit for biomass partitioning. Therefore improvements in yield potential, in general, should be achieved by increasing crop biomass (Fischer 2007 and references therein).
4
Floret initiation (wheat)
Development processes Sw00 10Em 12 13/21FI DR14/22 15/24 30TS/Aw/MNP31 37 39 45 Hd55 An BGF65 71 PM92 Zadoks stages
Leaf initation Spikelet initiation Spikelet differentiation
Grain set
Floret survival
Leafinit.
Leaf appearance
Tiller appearance Tillers survival
Plants/m2
Grains/spike Spikes/plant
Grain weight
Grains/m2
Spike Max. rate Grain
Stem Max. rate
Grain fill.
Yield components
Growth
(Barley)
5 Figure 1. Schematic diagram of wheat and barley development showing different stages, periods in which some developmental processes occur and periods in which yield components are determined.
Sw, sowing; E, seedling emergence; FI, beginning of floral initiation or ‘collar’ stage; DR, double ridge; TS, terminal spikelet (for wheat); Aw, awn primordia stage (in most advanced spikelets), for barley; MNP, maximum number of primordia, for barley; Hd, heading; An, anthesis; BGF, beginning of grain filling; PM, physiological maturity. Underlined, apex stages. Apex development does not necessarily match with the same Zadoks stages included in the figure, apart of An, BGF, PM, and TS or Aw respect to Z30 when the latter is determined as the onset of stem elongation (first node elongated c. 1 cm, short before TS or Aw, Kirby et al. 1994). Adapted from Slafer and Rawson (1994) and García del Moral et al. (2002).
In most countries yield improvements in both crops were achieved mainly through increases in grain number/m2, also with exceptions, particularly in barley, for which grain weight was also improved in some cases (Abeledo et al. 2002). In general, both wheat and barley are more sink than source limited, although a co-limitation by both has been reported in barley under Mediterranean conditions, particularly under severe drought stress limiting biomass accumulation before heading (Voltas et al. 1997). Therefore, in general, it seems more effective improving yield potential through increasing grain number/m2 than grain weight (Fischer 2008). However grain number /m2 is a trait as complex as yield itself and more difficult to assess (Slafer 2003).
From the perspective of development, yield is formed throughout the crop cycle from sowing to harvest through the formation of different structures and yield components (Figure 1), although some developmental phases are more critical than others in determining grain number/m2 and final yield (Fischer 1985, Kirby 1988, Slafer and Rawson 1994). Some authors identified independent variability between the leaf initiation, the spikelet initiation and the spikelet differentiation phases amongst genotypes and suggested that optimising the partitioning of total time to heading between different pre- heading phases could be used for improving yield (Halloran and Pennell 1982 in wheat, Appleyard et al.
1982 and Kitchen and Rasmusson 1983 in barley and references therein). The spikelet differentiation phase or stem elongation phase (SE, from now on) and first days of grain filling when grain setting occurs are the most critical in the determination of the final number of grains/m2. During the last part of SE floret death coincides with the rapid growth of both stem and spike in competition for assimilates (Fischer 1985, Kirby 1988, Fischer 2007). Considering this, extending the SE has been proposed as a way to increase assimilates availability and floret survival, and thereby, grains/m2 and yield potential of small-grain cereals. One of the pre-requisites is that flowering time should not be modified, since this is a key adaptative trait often already optimised (or to be optimised) in any breeding program (Slafer et al. 2005).
Thus, longer SE should be achieved at the expense of the leaf and spikelet initiation phase (LS, from now on). Another question is how the increase in assimilate availability due to extending duration of SE is partitioned into spikes and stems biomass, that is, if there could be any negative impact on harvest index (Araus et al. 2008).
6
Some works have shown that duration of the stem elongation phase, spike weight at anthesis, number of fertile florets at anthesis and grains per spike is reduced when the same wheat genotype sensitive to photoperiod during SE is grown under a extended photoperiod treatment starting at terminal spikelet (Miralles et al. 2000, González et al. 2003a, González et al. 2003b). The authors suggested that the reduction in the number of grains per spike was the result of a reduction in duration of SE and assimilates availability for spikes growth. However González et al. 2003a and 2003b concluded that effects on spikelet fertility and grains/spike could be due in part to direct photoperiod effects (i.e. others than due to increased assimilate availability). In another study, González et al. (2005a and 2005b) compared the extended photoperiod treatment with a shading treatment to see if effects on spikelet fertility were similar and due to differences in assimilate availability. They concluded that effects were likely associated to increased assimilate availability, although some relevant differences between the photoperiod and the shading treatments were found (different effects on stems dry weight, dynamics of floret abortion and position of the florets aborted in the spike, etc.), which suggests that effects on spikelet fertility could be due also in part to direct photoperiod effects. In any case, further studies are needed to assess the potential of lengthening SE on yield generation for breeding.
Findings by other authors seem to support an advantage of extending SE. Van Oosterom and Acevedo (1992) showed that well adapted barley landraces in environments in Syria with mild winters had an earlier transition of the apex from vegetative to reproductive, compared to landraces from more severe winters, which could suggest that an earlier onset of stem elongation could be beneficial whenever frosts are not very limiting. Otherwise these landraces could have kept a later apex transition to ensure that no frost damage occurs in any year. Instead, they combine an earlier transition of the apex with cold resistance (van Oosterom and Acevedo 1992). Francia et al. (2011) observed negative and positive correlations with LS and SE, respectively, with grain yield in the Nure x Tremois barley population, when adjusting both phases by time to heading (HD), using partial correlations. Kitchen and Rasmusson (1983) reported positive correlations between spikelet primordia number and survival, and final grains/spike with both duration of spikelet initiation and spikelet differentiation phases, in a set of 10 cultivars (in the case of spikelet survival, only significant with the spikelet differentiation phase). However both phases were strongly and positively correlated in this set of cultivars (although not with the leaf initiation phase), and thus, it is not clear to what extent differences in final grains/spike were due to duration of one phase or the other. Final grains/spike was positively correlated with both number of spikelets initiated and spikelet survival with very similar coefficients (0.80-0.81), and the two latter were not correlated (estimated from data in Kitchen and Rasmusson 1983).
Kernich et al. (1997) suggested that there could be more potential for increasing number of grains/spike by improving floret survival than by increasing number of floret primordia initiated, since differences between genotypes for the latter were much smaller in their study. On the other hand other authors found large differences in the number of spikelet primordia initiated between barley genotypes. Apart of the
7 already mentioned work by Kitchen and Rasmusson (1983), Appleyard et al. (1982) found large differences in the number of spikelet primordia initiated in a set of 13 2-rowed barley lines. These differences were strongly related to the duration of the spikelet initiation phase, and to important differences in final number of grains/spike, although the proportion of aborted spikelets was also greater in lines with the highest number of primordia initiated. More recently, Lewis et al. (2008) found that increased number of grains/spike was associated to higher number of spikelets initiated, which in turn was associated to longer duration of the leaf and spikelet initiation phases, not to SE, comparing two alleles of the gene Eps-Am1 from wild and cultivated genotypes of T.monococcum. In order to not compromise spikelet initiation, a possibility would be reducing only the leaf initiation phase, if independent variability were identified as in Kitchen and Rasmusson (1983). On the other hand, the number of spikelets initiated may depend not only on genotypic differences in duration of the phase, but also in its rate of initiation (McMaster 1997 and references therein).
2.- Environmental and genetic control of developmental time
The three major factors determining differences among genotypes in flowering time are the different genotypic responses to photoperiod and vernalisation, and differences in intrinsic earliness or earliness per se (García del Moral et al. 2002). Further evidence from recent studies with the gene Eps-Am1 (Lewis et al. 2008) support findings by other authors showing that, within the term earliness per se, there are large genotypic differences in the response to non-vernalising temperatures or temperature per se (e.g.
Ellis et al. 1988, Slafer 1996). The wide genotypic differences in response to these factors are considered as responsible for the spread of wheat and barley worldwide, to a wide range of environmental conditions (Worland 1996, Igartua et al. 2008). Responses in the duration of a particular phase to these factors can be illustrated by models as in Figure 2.
2.1 - Vernalization
Wheat and barley, as many other long-day species, include genotypes which are sensitive to vernalization (Roberts et al. 1988). The range of responses between genotypes in both species is very high, from those that do not respond or little, to those that do not flower until a certain period of exposure to cold temperatures is satisfied, being the most effective, within the range c. 0-10 ºC (McMaster 1997, García del Moral et al. 2002, and several references therein). Depending on their vernalisation requirement varieties are considered as winter, facultative or spring types (Worland et al. 1996, Karsai et al. 2001).
Nowadays the classification of genotypes is also made with molecular markers to characterise alleles for the major vernalisation genes.
8
Figure 2. Schematic representation of changes in the duration and in the rate of development for a particular developmental phase, in response to photoperiod and vernalization (A and B) and to temperature per se (C and D) (adapted from García del Moral et al. 2002 and Miralles et al. 2003).
Vernalization affects the moment of transition of the apices from vegetative to reproductive, and thereby, affects mainly duration of the leaf initiation phase (although direct responses have been also observed in some wheat genotypes in the spikelet initiation phase; Slafer and Rawson 1994). However it can affect indirectly also duration of the pre-heading phases after floral initiation, e.g. changing sensitivity to photoperiod, through more leaves generated that will have to appear, and/or increasing phyllochron of later leaves (Ellis et al. 1988, Slafer and Rawson 1994, González et al. 2002). Responses to vernalisation not only differ between genotypes during the vegetative phase, but there may be also genotypic differences in the responses observed later, and these responses may be partially independent of responses observed in the vegetative phase (Slafer and Rawson 1994).
2.2 - Photoperiod
Photoperiod and vernalisation responses complement each other since both are mechanisms to prevent flowering and the transition of the apex from vegetative to reproductive under unfavourable conditions.
Responses to photoperiod vary also greatly among genotypes both in wheat and barley (Slafer and Rawson 1994 and references therein for wheat, Karsai et al. 2001, Boyd et al. 2003 for barley).
Genotypes can differ in the critical and optimum photoperiods and in the slope of the relationship D)
Durationof thephase(d)
Duration of photoperiod (h) or vernalization (d) treatments
Sensitivity
Optimal duration Intrinsic earliness for
a given temperature
A)
Rateof developmentfor thephase(1/d)
Duration of photoperiod (h) or vernalization (d) treatments
B)
Temperature ºC
Durationof thephase(d)
Optimum T
C)
Rateof developmentfor thephase(1/d)
Temperature ºC 1/(ºCd) =
1/(Duration of the phase in Thermal time)
BaseT
Supra-optimal temperatures
Optimum T
9 between rate of development and photoperiod (e.g. Roberts et al. 1988 for barley, Slafer and Rawson 1996 for wheat). Barley, as many other crops, may have two periods in which plants do not respond to photoperiod: after seedling, called juvenile phase or pre-inductive period, and the post-inductive period just prior to heading. Barley genotypes may differ also in both periods and in the length of the inductive period between them (Roberts et al. 1988). These periods of non-responsiveness have not been found in wheat (Slafer and Rawson 1994).
Considering different pre-heading phases, it is important to highlight, in the context of the present thesis, that i) barley and wheat genotypes can respond to photoperiod not only during the leaf initiation phase but also during the spikelet initiation and differentiation phases (Roberts et al. 1988, Slafer and Rawson 1994, Miralles and Richards 2000 and several references in both); ii) there are genotypic differences in the response to photoperiod in each sub-phase, and responses during the spikelet differentiation phase can be even greater than in previous phases (Slafer and Rawson 1994, Slafer and Rawson 1996, Kernich et al.
1997, González et al. 2002); and relevantly iii) these responses in each phase are largely independent, beyond any ‘memory’ effect (studies with reciprocal photoperiod transfers as in Kernich et al. 1997, Miralles and Richards 2000, or when photoperiod treatments are applied only after terminal spikelet, e.g.
González et al. 2002).
2.3- Temperature and intrinsic earliness
The effect of temperature (without considering vernalizing effects) is considered universal. It affects all genotypes and every developmental phase, from emergence to maturity (McMaster 1997, García del Moral et al. 2002). Linear relationships are observed with rate of development but not with duration of particular phases (Figure 1; Roberts et al. 1988). On the other hand, genotypic differences in intrinsic earliness or earliness per se are usually measured as differences when genotypes are grown under saturating photoperiod and vernalising conditions (e.g. Karsai et al. 2001). There are several other definitions in the literature about intrinsic earliness which may be confusing and misleading, depending on the assumptions made (Slafer 1996). i) Intrinsic earliness is, at least partially, dependent of environmental conditions, since genotypes may exhibit large differences in their responses to temperature, amongst which, differences in base and optimum temperatures (Ellis et al. 1988 for barley, Slafer 1996 for wheat, and several references therein in both). Relevantly for the hypothesis of the present thesis: ii) This sensitivity is not constant throughout development. Duration of phases after floral initiation are differently sensitive to temperature respect the previous phase, with both base and optimum temperatures tending to increase, as development advances (Slafer and Rawson 1995, Gómez- Macpherson and Richards 1997 and references in both). iii) Moreover there may be genotypic differences in the response to temperature in each sub-phase. Interestingly, responses to temperature after floral initiation may be independent of those in previous phases among genotypes (Slafer and Rawson 1995, Slafer 1996 and other references therein). These differences are not kept in calendar time, neither in
10
thermal time, across different temperature conditions, since genotypes also differ in their base and optimum temperatures in each phase (Slafer and Rawson 1995, Slafer 1996). Therefore an important component of what is called intrinsic earliness can be related to responses to temperature, although this do not exclude that there may be other genotypic factors independent of environment differing amongst genotypes (Slafer 1996).
2.4- Other factors
These main factors do not affect development independently, but they interact between them in a more or less extent, and the level of interaction of responses to these factors can also differ amongst genotypes (Roberts et al. 1988, Ellis et al. 1988, Slafer and Rawson 1994). Some of these interactions reported affecting durations of phases, found in both barley and wheat, are the following ones: i) Some barley and wheat genotypes can complement their vernalization requirements by exposure to short photoperiods (short-day vernalization), since floral initiation is more advanced under short than long photoperiods under a given vernalisation treatment (Roberts et al. 1988, Slafer and Rawson 1994); ii) Vernalization and photoperiod responses interact largely in those genotypes that exhibit response to vernalisation, that is, the response to photoperiod is much smaller in genotypes that have not satisfied their vernalisation requirements respect those that have (Ellis et al. 1988, Worland 1996). Interactions between photoperiod and vernalisation do not follow always the same pattern amongst genotypes (e.g. Figure 5 in Slafer and Rawson 1994). iii) Temperature also modifies photoperiod responses (Ellis et al. 1988, Slafer and Rawson 1996). Within a genotype these interactions between temperature and photoperiod are quantitative, although the level of interactions differs markedly amongst genotypes (Ellis et al. 1988, Slafer and Rawson 1996).
Apart of these interactions, other environmental factors may affect duration of flowering time, although their effects on particular pre-heading phases are usually much lower and are less studied. It is not clear either if there could be differences amongst genotypes in the response to these factors. Among them water stress is the most widely reported, in general hastening phenological time (few days), and it seems that effects are not constant throughout development, being higher at reproductive stages (McMaster 1997 and references therein). Other factors reported to have small effects on phenological time are nutrient availability (although some authors also reported no significant effects) and CO2 concentration (McMaster 1997 and references therein). Gibberellins have also a role promoting flowering time, although it is much less understood in wheat and barley compared to Arabidopsis (Winfield et al. 2009).
2.5- Genetic control of flowering time
In the last decade, candidate genes have been identified for major loci controlling flowering time in barley and wheat. The vernalization genes VRN-H1 and its homologues VRN-A1, VRN-B1 and VRN-D1 in wheat are MADS-box transcription factors similar to APETALA1 in Arabidopsis (Yan et al. 2003, Trevaskis et
11 al. 2003, Fu et al. 2005). HvZCCT and TaZCCT are the candidate genes for VRN-H2 and its wheat homologue VRN-Am2, respectively (Yan et al. 2004, Distelfeld et al. 2009). The alleles at these loci and their interactions determine sensitivity to vernalisation (e.g. Yan et al. 2004, Zitzewitz et al. 2005). VRN- H3 and its homologues VRN-A3, VRN-B3 and VRN-D3 are FT-like genes, and they also interact with PPD and VRN genes (Yan et al. 2006, Faure et al. 2007, Bonnin et al. 2008).
The photoperiod responsive gene PPD-H1 in barley and its wheat homologues PPD-D1, PPD-B1 and PPD-A1 are PRR-like genes (Turner et al. 2005, Beales et al. 2007, Wilhelm et al. 2009). In both species the photoperiod responsive allele accelerates flowering under long day conditions. However in barley the greatest differences between sensitive and insensitive alleles are found under long photoperiod conditions, with the dominant allele being sensitive, whereas in wheat, the greatest differences are observed under short photoperiod conditions and the dominant alleles confer insensitivity (Worland 1996, Laurie et al.
2004, Jones et al. 2008). HvFT3 is the candidate gene for Ppd-H2 in barley, also related to photoperiod, whose active allele is expressed and accelerates flowering under short photoperiod or low latitudes (Laurie et al. 1995, Faure et al. 2007, Kikuchi et al. 2009).
Other reported genes that determine differences in heading time in barley are the called ‘earliness per se’
loci (eps) identified by Laurie et al. (1995), the series of Eam loci (Franckowiak 1997, Lundqvist et al.
1997, Stracke and Börner 1998, Börner et al. 2002) and the gene HvAP2 (Chen et al. 2009). However, except the latter, no candidate genes have been found yet for them, and their role is much less clear. In wheat, other less characterised loci have also been identified, as the gene Eps-2B on 2BS (Scarth and Law 1983, Shindo et al. 2003); Eps-Am on 1AL (the only found determining differences in responses to temperature, Lewis et al. 2008); VRN-D4 close to the centromere in 5D (Yoshida et al. 2010), and other earliness per se genes on 5AL (Kato et al. 2002).
Additionally other loci have been found to have an effect on heading time in different regions than the loci mentioned above, although most of them with smaller effects: by the use of aneuploids in wheat (Worland 1996, Law and Worland 1997) or through QTL mapping, both in barley (e.g. Hayes et al. 1993, Bezant et al. 1996, Tinker et al. 1996, Baum et al. 2003, Li et al. 2006) and wheat (e.g. Sourdille et al.
2000, Shindo et al. 2003, Kuchel et al. 2006, Griffiths et al. 2009). Furthermore some semidwarfing genes are reported to have effects on heading time in barley (the allele that reduces plant height slightly delays heading time, e.g. some erectum, ert genes, or the denso gene, Thomas et al. 1995, Forster et al. 2007; see also descriptions for genes in the Barley Genetic Stocks AceDB Database http://ace.untamo.net/ or through Graingenes, http://wheat.pw.usda.gov/ ). All these studies would confirm that the genetic control of heading time is under a strong but complex genetic control (Worland 1996, Law and Worland 1997).
Despite particular VRN and PPD alleles are more frequent in some geographical areas, variation amongst genotypes has been found within regions, so it is possible finding different combinations of VRN and PPD alleles in successful genotypes well adapted to particular regions, which would reinforce the idea
12
that several other genes and their combined effects can be important in the control of flowering time (Cockram et al. 2007b, Eagles et al. 2009).
3.-- Genetic control of different pre-flowering phases
Up to section 2, the motivations for studying different pre-flowering phases and their genetic control have been presented, while section 2 presents some evidences of differences in this genetic control between sub-phases, that is, the different genotypic sensitivities to vernalisation, photoperiod and temperature in phases after floral initiation independent of the previous phase. There are other evidences from studies exploring phenotypic variability, although very few from studies identifying particular genetic factors responsible for these differences. Independent phenotypic variability in the duration of pre-anthesis phases has been observed among different genotypes, both in wheat and barley, within 2-row and 6-row types, and amongst cultivars or lines derived from different crosses (Halloran and Pennell 1982, Appleyard et al. 1982 Kitchen and Rasmusson 1983, Kernich et al. 1995, Kernich et al. 1997, Whitechurch et al. 2007). Durations of the leaf initiation, spikelet initiation and spikelet differentiation (or stem elongation) phases were not significantly correlated in several cases and in those that found significant correlations, the degree and the direction of the correlations varied between studies.
Despite the genetic control of flowering time has been extensively studied, the genetic control of different pre-anthesis sub-phases (others than total time to heading, and from sowing to the transition of the apex from vegetative to reproductive in some cases) has received little attention. There are several studies comparing wheat substitution lines, single chromosome recombinant lines or near isogenic lines (differing in PPD alleles) showing differences between genotypes in the duration of pre-heading phases and in the response to photoperiod of each sub-phase. However these differences could not be attributed to particular major PPD genes, as results differed depending on both genetic background and environmental conditions (see results and comparative review by González et al. 2005c). Recently Lewis et al. (2008) found that alleles of a cultivar and a wild line of Triticum monoccocum for Eps-Am had large effects on duration of LS (due to different sensitivity to temperature per se), but no effect on SE. Zhou et al. (2001), using a QTL approach in rice, found some independent QTL for the duration of the vegetative and reproductive phases, either by different magnitude of QTL effects or by opposite allele effects between phases. Nevertheless further studies should be required in wheat and barley for identifying particular genetic factors controlling durations of different pre-heading phases, and for exploring to what extent duration of pre-heading phases could be changed independently (without modifying total time to heading).
Another question that arises from section 2 is in what extent differences in the partitioning of time to heading in sub-phases could be maintained across different photoperiod and temperature conditions (the
13 degree of genotype x environment interaction), given that durations of phases and genetic control of flowering are highly dependent on them. This may sound a bit contradictory. Many of the studies cited in section 2 compared a wide range of photoperiod and temperature conditions, including sometimes extreme treatments, and several of them were carried out under controlled and constant conditions (which is necessary for the hypothesis tested e.g. different sensitivities in each phase, determination of optimum and base temperatures, ceiling and critical photoperiods, etc.). However in some cases they may give an unrealistic idea of GxE interactions in most growing conditions for the partitioning of time to heading in different sub-phases.
Correlations between environments for heading time in DH-populations are still very high when including a relatively wide range of latitudes and temperature conditions (Teulat et al. 2001, Granada vs.
Montpellier, Moralejo et al. 2004, Catalonia vs. Scotland). Other studies found also high correlations for heading time between sowing dates, although differences between genotypes tended to be reduced as conditions became more inductive (Gómez-Macpherson et al. 1997, Kirby et al. 1999). On the other hand, in the two latter studies the differences amongst genotypes in each pre-heading phase (genotypes ranking for duration of the leaf initiation, spikelet initiation (LS as the sum of both) and stem elongation (SE) phases, and also for the ratio SE/LS) were much less maintained than for heading (estimated from data presented in Gómez-Macpherson et al. 1997 and Kirby et al. 1999). Nevertheless they compared genotypes with very contrasting responses to vernalisation and photoperiod, and in the case of Kirby et al.
1999, very contrasting sowing dates. Further studies should be required for identifying genetic factors for a different partitioning of heading time in sub-phases, and for exploring if some of these differences (if found) could be maintained across different temperature and photoperiod conditions, within the range in which barley and wheat are most commonly grown. The latter would be also interesting in breeding since it would increase the suitability of the genotypes selected for a wider range of environments (e.g.
fluctuations from year to year in temperature at a given site, changes in sowing dates due to rainfalls that delay them in autumn, or differences in temperature and photoperiod across a range of sites).
4.- Leaf appearance, tillering and internode elongation.
Another important aspect to take into account when considering changing duration of pre-heading phases is the possible impact on several processes related to the phenology of the crop, as leaf appearance, tillering and internode elongation. It has been shown that these processes are highly coordinated between them and with duration of pre-heading phases both in barley and wheat (Kirby et al. 1985 and 1994, Kirby 1988, Hay and Kirby 1991, McMaster 1997). Leaf appearance and tillering are two important developmental processes in the formation of the crop canopy and in the determination of the potential number of spikes/plant and spikes/m2 at harvest. Both are closely related between them and to the duration of LS (Kirby et al. 1985, Hay and Kirby 1991, McMaster 1997, García del Moral et al. 2002, see
14
also Figure 1). Time to heading depends strongly on the rate of leaf appearance and on total number of leaves on the main shoot which in turn depends on the duration of the leaf initiation phase and the rate of leaf initiation (García del Moral et al. 2002, McMaster 1997). Number of tillers is determined mainly up to the onset of stem elongation, reaches a maximum around then and may become stable for some time.
Then it decreases when the period of maximum growth of stems and spikes start with competition for available assimilates (Kirby et al. 1985, Hay and Kirby 1991, García del Moral et al. 2002). Thus, shortening LS could reduce the maximum number of tillers, although the onset and the rate of tillering may be as important as the duration of LS (or more) in determining genotypic differences in the tillering capacity (Kirby et al. 1985, van Oosteom and Acevedo, 1992). The main factors that affect duration of phases are the most important for leaf initiation and leaf appearance as well, but they act in different ways on each of them. On the other hand tillering traits are also influenced by other important factors such as nutrient and water availability, particularly those related to tiller survival (McMaster 1997, García del Moral et al. 2002 and several references in both). It is not clear either if there could be any negative impact on early vigour, which has been shown a beneficial trait under some Mediterranean conditions (Richards et al. 2002), and it could be associated to some leaf appearance and tillering traits. Large genetic variability can be found in both wheat and barley for phyllochron and number of leaves (Kirby et al. 1985, Frank and Bauer 1995, Dofing 1999, McMaster 1997), in some tillering traits (Kirby et al. 1985, van Oosterom and Acevedo 1992, García del Moral et al. 2002, McMaster 1997), and in other traits related to early vigour (Richards et al. 2002). However there are few studies about the genetic control of traits related to leaf and tillering appearance and the potential impact of changing duration of pre-heading phases on these traits.
Internode elongation is also closely related with leaf appearance. The number of internodes depends on the number of leaves that appear after the onset of stem elongation, and the time course of each internode elongation is strongly related with the rate of leaf appearance (Kirby 1988, Kirby et al. 1994). Therefore extending duration of SE could have an effect on plant height, although the latter depends on the rate of length-growth among other factors (Kirby et al. 1994). Coleman et al. (2001) found important pleiotropic effects on plant height at stem elongation and anthesis, from two QTLs with very strong effects on heading time (likely Ppd-D1 and Ppd-B1), with the same direction of allele effects. On the other hand QTLs with strong effects on total time to heading have been associated with reduced peduncle elongation (from the allele the increases time to heading, e.g. Kandemir et al. 2000, on the Ppd-H1 region). Final plant height depends on numerous other factors, and many genes or QTLs for this trait have been found independent of time to heading (others than those mentioned in section 2.5). Possible pleiotropic effects on plant height from QTLs with strong effects on heading time must not be confounded with those from some semidwarf genes (see section 2.5), which have opposite allele effects between plant height and heading time, and are probably involved in different physiological processes.
15 All in all it seems that changing duration of pre-heading phases could probably have effects on other traits. However little is known about i) particular loci determining genotypic differences on several traits related to leaf appearance and tillering as phyllocrhon, the onset and the rate of tillering, and the maximum number of tillers; ii) the possible impact of particular loci determining differences in the duration of LS and SE on several traits related to leaf appearance, tillering and internode elongation, and if it is possible finding independent genetic variability between duration of phases and these traits; and iii) the possible overall impact of different QTLs for LS and SE on yield and its components.
5.- Objectives
Given the importance of phenology on many aspects of crop development and growth in general, the important role that different pre-heading phases have on yield generation, and the scarce number of works devoted to study the genetic control of different pre-heading phases, the first main objectives of the present thesis were:
i) Assessing to what extent variability amongst genotypes in duration of SE is independent from variability in LS, in different barley and wheat double-haploid populations: chapter 1, 3 and 4;
ii) If such independent variability is found, identifying particular loci determining differences in the duration of each pre-heading phase (chapters 2, 3 and 4);
iii) If these loci are identified, studying to what extent effects on different pre-heading phases are maintained under different photoperiod and temperature conditions, i.e. assessing genotype and QTL by environment interactions in the Steptoe x Morex population (chapter 3).
iv) Studying if some of the loci identified in chapters 2, 3 and 4 for duration of pre-heading phases could be major known genes (chapters 2, 3 and 4).
Considering the relevance of the stem elongation phase (SE, or spikelet differentiation phase) in yield formation highlighted by some authors (see section 1 of the present introduction), and the difficulty of assessing collar stage in a large number of genotypes, the leaf initiation and spikelet initiation phases are considered together (LS). The genetic control of pre-heading phases was studied using two different double-haploid barley populations (chapters 1, 2 and 3) and two wheat populations (chapter 4), grown in different environments each one. The approach consisted on studying the genotypic variability in each population by estimating genotypic means and heritabilities using mixed models; and the genetic control of each phase, by using whole genome QTL analyses to relate the phenotypic data obtained in this thesis with genotypic-marker data available for each population. In chapter 3 one of the barley populations (Steptoe x Morex) was grown under four field environments differing in photoperiod and temperature
16
conditions (two photoperiod treatments and two sowing dates), using the same approach but with more emphasis on the study of genotype by environment and QTL by environment interactions.
In order to asses if some QTLs for duration of phases could be major well-known genes, in chapter 2 specific polymorphisms for candidate genes and for some markers were screened. In chapter 3, the Steptoe x Morex population is known to segregate for several major genes, and in chapter 4 the two wheat populations are known to segregate for Ppd-D1 (for which a SNP marker was included in the two linkage maps).
Additionally, a selection of pairs of DH-lines from the Henni x Meltan population, with similar time to heading but different partitioning in LS and SE were grown in the same environments than in chapter 3.
The objective in this annex was studying in more detail differences in the duration of SE and LS for each pair of genotypes, by following the time course of apex development from just before the onset of stem elongation until heading (spikelet differentiation); and to assess if differences between LS and SE were maintained under different photoperiod and temperature conditions. Results are presented only as a complement for the general discussion.
Given the close relatedness between leaf appearance, tillering, internode elongation, and their relationships with duration of phases (see section 4), and the effects that duration of phases may have on some yield components (see section 1), the second general objective of the thesis was assessing the potential effects of changing duration of pre-heading phases on traits related to leaf appearance, tillering, plant height components, yield and yield components, and can be sub-divided in the following objectives:
i) Studying the genetic control of several traits related to leaf appearance and tillering (chapters 1,2, 4 and 8), among which phyllochron, the onset and the rate of tillering, and maximum number of tillers for which little is known about their genetic control.
ii) Assessing to what extent the genetic control of these traits is independent of the genetic control of duration of pre-heading phases (chapters 1, 2, 4 and 8); and
iii) Studying possible effects of changing duration of pre-heading phases on biomass and nitrogen accumulation, biomass and N partitioning (chapters 5 and 6), plant height, yield and yield components (chapters 6, 7 and 8).
The approach was the same as for duration of phases for studying the genotypic variability and the genetic control of each trait. For assessing relationships between duration of pre-heading phases and the rest of traits, the approach consisted on estimating correlations between them, and studying the co- localisation of QTLs for several traits. Chapters 5, 6 and 7 are interrelated and each one is a part of the same work, about the effects of duration of pre-heading phases on several agronomic characters in the Henni x Meltan population, and data was obtained from the same field experiments. In chapter 8 the
17 second objective (mainly i and ii, and part of iii) is addressed in the Steptoe x Morex population. Despite the limitations of the QTL analyses using biparental populations in distinguishing if effects come from linkage or pleiotropy (Thomas 2003), it will be shown that several of the QTLs co-localising on the same region in some cases are very likely pleiotropic.
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