Molecular and physiological analysis of chilling tolerance in maize

Research Collection

Doctoral Thesis

Molecular and physiological analysis of chilling tolerance inmaize (Zea mays L.) seedlings

Author(s): Biradar, Sunil Kumar

Publication Date: 2008

Permanent Link: https://doi.org/10.3929/ethz-a-005786359

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Diss. ETH No. 18028

Molecular and physiological analysis of chilling tolerance in maize (Zea mays L.) seedlings

A dissertation submitted to the

ETH ZURICH for the degree of

DOCTOR OF SCIENCES

presented by

SUNIL KUMAR BIRADAR M.Sc. in Agricultural Sciences, University of Agricultural Science, Dharwad, India

born 1. June 1976

Citizen of India

accepted on the recommendation of

Prof. Dr. P. Stamp, examiner

Dr. C. Sautter, co-examiner

Dr. J. Leipner, co-examiner

Zurich, 2008

I

Table of contents

Summary .................................................................................................................................. IV

Zusammenfassung...................................................................................................................VII

List of abbreviations.................................................................................................................. X

1 General Introduction........................................................................................................... 1

1.1 Aims of the study ....................................................................................................... 5

2 Re-analysis of a major QTL for chilling tolerance of photosynthesis based on an improved map of the ETH-DL3 × ETH-DH7 population.................................................. 7

2.1 Introduction ................................................................................................................ 7

2.2 Materials and Methods ............................................................................................... 7

2.2.1 Resource population and phenotypic data.............................................................. 7

2.2.2 Selection and development of molecular markers ................................................. 8

2.2.3 DNA markers assay................................................................................................ 8

2.2.4 QTL analysis .......................................................................................................... 9

2.3 Results and Discussion............................................................................................... 9

3 Marker assisted introgression of a major QTL for chilling tolerance of photosynthesis into the recurrent parental line.......................................................................................... 13

3.1 Introduction .............................................................................................................. 13

3.2 Materials and methods ............................................................................................. 14

3.2.1 Plant material........................................................................................................ 14

3.2.2 Growth conditions ................................................................................................ 15

3.2.3 Measurement of physiological traits .................................................................... 15

3.2.4 DNA isolation and SSR marker assay.................................................................. 16

3.2.5 Marker assisted and phenotype based backcrossing ............................................ 17

3.2.6 Statistical analysis and QTL mapping.................................................................. 18

3.3 Results ...................................................................................................................... 18

3.3.1 Phenotypic analysis of chilling tolerance............................................................. 18

3.3.2 QTL consistency over generations....................................................................... 20

3.3.3 Confirmation of the additional QTL in the distal region of chromosome 6 in the BC3F2 population ................................................................................................. 21

3.3.4 Time course analysis of QTLs for photosynthetic traits ...................................... 22

3.4 Discussion ................................................................................................................ 24

4 Characterization of the photosynthetic apparatus of near isogenic lines (NILs) for a QTL related to chilling tolerance of photosynthesis ................................................................. 28

4.1 Introduction .............................................................................................................. 28

II

4.2 Materials and Methods ............................................................................................. 29

4.2.1 Plant material........................................................................................................ 29


4.2.3 Light response curves of gas-exchange and chlorophyll fluorescence parameters under controlled conditions.................................................................................. 30

4.2.4 Measurement of physiological traits under field conditions ................................ 31

4.2.5 Data analysis and statistic .................................................................................... 31

4.3 Results ...................................................................................................................... 31

4.3.1 Genotypic differences in the light response of CO2 assimilation, PS II functioning and energy dissipation.......................................................................................... 31

4.3.2 Field experiment................................................................................................... 34

4.4 Discussion ................................................................................................................ 35

5 Validation of the major QTL for chilling tolerance of photosynthesis in a dent × flint maize population .............................................................................................................. 38

5.1 Introduction .............................................................................................................. 38

5.2 Materials and Methods ............................................................................................. 39

5.2.1 Plant material........................................................................................................ 39


5.2.3 Measurement of chlorophyll fluorescence parameters......................................... 40

5.2.4 DNA isolation and SSR marker assay.................................................................. 40

5.2.5 Bulk segregant analysis........................................................................................ 40

5.2.6 QTL mapping ....................................................................................................... 40

5.3 Results ...................................................................................................................... 41

5.3.1 Phenotypic analysis .............................................................................................. 41

5.3.2 Bulk segregant analysis........................................................................................ 42

5.3.3 Construction of molecular marker map and analysis of QTL.............................. 43

5.4 Discussion ................................................................................................................ 44

6 Comparative mapping and identification of candidate genes for the major QTL for chilling tolerance of photosynthesis ................................................................................. 47

6.1 Introduction .............................................................................................................. 47

6.2 Material and Methods............................................................................................... 48

6.2.1 Comparative mapping .......................................................................................... 48

6.2.2 Bioinformatics...................................................................................................... 48

6.3 Results and Discussion............................................................................................. 48

7 General Conclusions......................................................................................................... 53

8 References ........................................................................................................................ 55

III

9 Appendix .......................................................................................................................... 64

Acknowledgements .................................................................................................................. 66

Curriculum Vitae...................................................................................................................... 68

IV

Summary

Maize (Zea mays L.) is a crop of tropical/subtropical origin. It requires a temperature of about

25-35 °C for growth and development. In temperate regions, however, maize is sown during

spring, where maize seedlings are often exposed to suboptimal temperature coupled with short

spells of chilling events. In particular, the growth and early development of the seedling may

be strongly affected by low temperature. A better understanding of the genetic and

physiological basis of the effect of low temperature on maize may help to alleviate the

problems associated with the chilling sensitivity of maize seedlings. The genetic basis of

chilling tolerance has been studied by analysis of the quantitative trait locus (QTL); several

QTLs for chilling tolerance of photosynthesis were identified in a Swiss dent maize mapping

population derived from the cross of the chilling sensitive line ETH-DL3 and the chilling

tolerant line ETH-DH7. In order to conduct an in-depth study of the major QTL for chilling

tolerance of photosynthesis, which was found in the above mentioned study in the telomeric

region of the long arm of chromosome 6, physiological and genetic analyses were conducted

in backcross populations and near isogenic lines (NILs) derived from the ETH-DL3 × ETH-

DH7 population as well as in an alternative F2 population. Based on the results, the aim was

to identify further potential candidate genes which may underlay this QTL for chilling

tolerance of photosynthesis.

In order to narrow down the confidence interval of the major QTL for chilling tolerance of

photosynthesis on chromosome 6, in a first step, 17 simple sequence repeat (SSR), 18 indel

polymorphism (IDP) and 21 newly designed SSR markers from BAC sequences, located in

the QTL region, were analyzed for parental polymorphism. Two polymorphic SSR markers

were identified. The re-analysis of the data of the previous QTL study with the two additional

polymorphic SSR markers revealed an additional QTL in the telomeric region of the long arm

of chromosome 6, which was also associated with chilling tolerance of photosynthesis. The

re-analysis showed that the first QTL (QTL-1) is flanked by the markers umc1859 and

bnlg1740, while the other, new, QTL (QTL-2) is flanked by markers bnlg1136 and umc1653.

In order to elucidate the physiological and genetic basis of the above mentioned two QTLs, a

marker assisted backcross breeding was carried out. The chromosomal region, which harbors

the QTLs for chilling tolerance of photosynthesis, was introgressed by marker assisted fore-

and back-ground selection from the chilling tolerant line ETH-DH7 into the chilling sensitive

V

ETH-DL3 background. The QTL analysis by single factor ANOVA demonstrated the stable

expression of the introgressed segment over backcrossed generations. The QTL analysis by

composite interval mapping in the BC3F2 population confirmed again the presence of two

separate QTLs in the introgressed segment of chromosome 6. QTL-1 was found right at the

SSR marker bnlg1740 and was associated with the maximal quantum efficiency of PS II

primary photochemistry (Fv/Fm), the minimal fluorescence (Fo), the operating quantum

efficiency of PS II (ΦPSII), the quantum efficiency of open PS II reaction centers (Fv'/Fm'), the

carbon exchange rate (CER) and leaf greenness. QTL-2, which is flanked by the markers

bnlg1136 and umc1653, was associated with Fo and Fv/Fm. Furthermore, a time course

analysis of the QTLs indicated a differential expression of both QTLs in different leaves and

in different temperature regimes. Significant QTL effects but only in leaves that developed at

suboptimal temperature indicate that both QTLs are involved in the development of a

functional photosynthetic apparatus at suboptimal temperature.

Near isogenic lines (NILs) to ETH-DL3, which carried the ETH-DH7 allele in the QTL

region, were developed from selected BC3F2 lines. Two NILs in the BC3F4 generation were

obtained and contained the target region including QTL-1 and QTL-2. The light response

curves of the parental lines and the NILs showed that the efficiency and capacity of

photosynthesis were affected by growth at suboptimal temperature; however, a more

substantial reduction was observed in ETH-DL3 compared to chilling tolerant parent ETH-

DH7 as well as the NILs. The higher photosynthetic efficiency of the NILs compared to ETH-

DL3 was due to high a ΦPSII, which in turn due to both a higher photochemical quenching

factor (qP) and, in particular, a higher Fv'/Fm'. This indicates that the NILs maintain a higher

fraction of open PS II reaction centers, which were characterized by higher quantum

efficiency. Furthermore, the parameter ΦNO, which is a measure of chilling-induced structural

alterations of the photosynthetic apparatus, was lower in the NILs compared to the chilling

sensitive line ETH-DL3. This finding proved further that the QTL on chromosome 6 plays a

role in the assembly of a functional photosynthetic machinery.

The importance of the telomeric region of chromosome 6 for chilling tolerance was studied in

an alternate F2 population, which was derived from the cross of the chilling sensitive dent

maize line ETH-DL7 and the chilling tolerant flint maize line ETH-FH6. Bulk segregant

analysis (BSA) was employed to quickly analyze the importance of chromosome 6 for

chilling tolerance of photosynthesis in this population. The QTL analysis revealed one QTL in

this region, between the SSR markers bnlg1740 and umc1897. Based on its position and its

behavior, this QTL seemed to correspond to QTL-1 of the ETH-DL3 × ETH-DH7 mapping

VI

population. The conservation of this QTL in different mapping populations indicated the

importance of this genomic region for the development of a functional photosynthetic

apparatus at suboptimal temperature.

Potential candidate genes were identified by in silico analysis of the QTL region, which

contains 232 expressed sequence tags (ESTs). A further search for homology against EST

sequences from publicly available maize cold stressed library, revealed 33 putative candidate

genes, which are induced under chilling stress conditions. Among these, three genes were

directly involved in photosynthesis. In particular, cab-m7, coding for a light harvesting

complex II protein and the gene of the protease DegP1, which is known to be involved in the

repair of PS II, explained the observed phenotype of the studied QTL. However, further fine

mapping and functional genomic experimentation will be required to clarify whether one of

these genes underlies of the QTL for chilling tolerance of photosynthesis on the long arm of

chromosome 6.

VII

Zusammenfassung

Mais (Zea mays L.) ist eine Kulturpflanze (sub)tropischen Ursprungs. Sie benötigt eine

Temperatur von ungefähr 25 bis 35 °C für ein optimales Wachstum und Entwicklung. Mais

wird jedoch in den gemässigten Zonen im Frühling gesät, wenn die Pflanzen suboptimalen

Temperaturen und kurzen Kälteeinbrüchen ausgesetzt sein können. Insbesondere das

Wachstum und die frühe Entwicklung der Sämlinge kann durch niedrige Temperaturen stark

gestört werden. Ein besseres Verständnis der genetischen und physiologischen Ursachen der

Effekte niedriger Temperatur auf Mais ist nötigt, um züchterisch eine verbesserte

Kühletoleranz der Maissämlinge zu erreichen. In einer vorherigen Studie wurde die

genetische Ursache der Kühletoleranz in einer Quantitative Trait Locus (QTL) Analyse

untersucht. Dabei wurden in einer Schweizer Zahnmaispopulation, welche aus der Kreuzung

der kühlesensitiven Inzuchtlinie ETH-DL3 und der kühletoleranten Linie ETH-DH7

hervorging, mehrer QTLs für die Kühletoleranz der Photosynthese identifiziert. Um diesen

QTL für die Kühletoleranz der Photosynthese, welcher in der oben genannten Population in

der telomerischen Region des langen Arms von Chromosom 6 gefunden wurde, besser zu

untersuchen, wurden physiologische und genetische Analysen in Rückkreuzungspopulationen

und nahe-isogene Linien (NILs) der ETH-DL3 × ETH-DH7 Population sowie in einer

alternativen F2-Population durchgeführt. Basierend auf den erhaltenen Ergebnissen sollten

potentielle Kandidatengene identifiziert werden, die möglicherweise diesem QTL für die

Kühletoleranz der Photosynthese unterliegen.

Mit dem Ziel das QTL-Konfidenzintervall einzugrenzen, wurden in einem ersten Schritt 17

Mikrosatelliten (SSR Marker), 18 indel Polymorphismus (IDP) Marker sowie 21 aus BAC

Sequenzen konstruierte SSR Marker, welche in der QTL-Region lokalisiert sind, auf elterliche

Polymorphismen untersucht. Dabei wurden zwei polymorphe SSR Marker identifiziert. Die

Reanalyse der Daten aus der vorherigen QTL-Studie mit den beiden neuen polymorphen SSR

Markern zeigte ein zusätzliches QTL in der telomerischen Region des langen Arms von

Chromosom 6 auf, welches mit der Kühletoleranz der Photosynthese assoziiert war. Die

Reanalyse ergab, dass das erste QTL (QTL-1) durch die Marker umc1859 und bnlg1740

flankiert ist, während das zweite QTL (QTL-2) zwischen den Markern bnlg1136 und

umc1653 liegt.

Markergestützte Rückkreuzungen wurden durchgeführt, um die physiologischen und gene-

tischen Ursachen der beiden oben genannten QTLs in grösserer Genauigkeit zu untersuchen.

VIII

Die chromosomale Region, welche den QTL für die Kühletoleranz der Photosynthese

beinhaltet, wurde durch markergestützte Selektion von der kühletoleranten Linie ETH-DH7 in

die kühlesensitive Linie ETH-DL3 eingefügt. Die QTL-Analyse mittels ANOVA zeigte eine

stabile Expression des eingefügten chromosomalen Segments in den verschiedenen Rück-

kreuzungsgenerationen. Die QTL-Analyse mittels Composite Interval Mapping in der BC3F2-

Generation bestätigte das Vorhandensein von zwei unabhängigen QTLs im eingefügten

Segment von Chromosom 6. Das QTL-1 wurde nahe des SSR Markers bnlg1740 lokalisiert

und war mit der maximalen Quanteneffizienz der primären Photochemie des Photosystem II

(Fv/Fm), der minimalen Fluoreszenz (Fo), der aktuellen Quanteneffizienz von Photosystem II

(ΦPSII), der Quanteneffizienz der offenen Photosystem II Reaktionszentren (Fv'/Fm'), dem

CO2-Austausch und der Blattgrüne assoziiert. QTL-2, welches durch die Marker bnlg1136

und umc1653 flankiert war, war mit Fo und Fv/Fm assoziiert. Das Vorhandensein von

signifikanten QTL-Effekten nur in Blättern, die sich unter suboptimaler Temperatur

entwickelten, deutet an, dass beide QTLs in der Entwicklung eines funktionsfähigen

photosynthetischen Apparates involviert sind.

Von ausgewählten BC3F2 Linien wurden NILs zu ETH-DL3 entwickelt, welche das ETH-

DH7-Allel in der QTL-Region besassen. Es wurden zwei NILs in der BC3F4-Generation

erhalten, die die Zielregion mit QTL-1 und QTL-2 enthielten. Die Lichtsättigungskurven der

elterlichen Linien und der NILs zeigten, dass sowohl die photosynthetische Effizienz als auch

deren Aktivität durch suboptimale Wachstumstemperatur beeinträchtigt war. Jedoch wurde

eine deutlichere Reduktion in ETH-DL3 im Vergleich zu ETH-DH7 und den NILs

beobachtet. Die höhere photosynthetische Effizienz der NILs im Vergleich zu ETH-DL3

beruhte auf eine höhere ΦPSII, die wiederum durch einen höheren Faktor der photochemischen

Löschung (qP) und insbesondere einer höheren Fv'/Fm' verursacht wurde. Diese Beobachtung

deutete daraufhin, dass die NILs einen grösseren Anteil an offenen PS II Reaktionszentren

aufweisen, die zu dem durch eine höhere Quanteneffizienz charakterisiert waren. Ausserdem

war der Parameter ΦNO, welcher ein Mass für kühleinduzierte Veränderungen in der Struktur

des photosynthetischen Apparates ist, in den NILs niedriger im Vergleich zur kühlesensitiven

Linie ETH-DL3. Diese Erkenntnis gab einen weiteren Beleg für die Rolle des QTLs auf

Chromosom 6 für den Aufbau einer funktionstüchtigen photosynthetischen Maschinerie.

Die Wichtigkeit der telomerischen Region von Chromosom 6 für die Kühletoleranz der

Photosynthese wurde auch in einer alternativen F2-Population, welche aus der Kreuzung

zwischen der kühlesensitiven Zahnmaislinie ETH-DL7 und der kühletoleranten Flintmaislinie

ETH-FH6 abgeleitet wurde, untersucht. Um möglichst schnell die Wichtigkeit von Chromo-

IX

som 6 für die Kühletoleranz der Photosynthese in dieser Population zu untersuchen, wurde

zunächst eine Bulk Segregant Analysis (BSA) durchgeführt. Die QTL-Analyse bestätigte

einen QTL in dieser chromosomalen Region, welcher zwischen den SSR Markern bnlg1740

und umc1897 lag. Basierend auf seine Position und Charakteristika, kann vermutet werden,

dass dieser QTL mit dem QTL-1 der ETH-DL3 × ETH-DH7 Population korrespondiert. Das

Vorhandensein dieses QTLs in verschiedenen Kartierungspopulationen unterstreicht die

Wichtigkeit dieser genomischen Region für die Entwicklung eines funktionsfähigen

photosynthetischen Apparates unter suboptimaler Temperatur.

Potentielle Kandidatengene wurden in der QTL-Region, welche 232 Expressed Sequence

Tags (ESTs) enthält, in silco identifiziert. Eine Homologiesuche für EST-Sequenzen in einer

öffentlichen cDNA-Bibliothek von kühlegestressten Mais ergab 33 putative Kandidatengene.

Unter diesen befanden sich drei Gene, welche direkt in der Photosynthese involviert sind.

Insbesondere cab-m7, welches für ein Lichtsammler-Chlorophyll a/b-Protein II kodiert, und

das Gen der DegP1 Protease, welche in der Wiederherstellung des Photosystems II involviert

ist, könnten gut den Phänotypen des untersuchten QTLs erklären. Dennoch muss berück-

sichtigt werden, dass eine bessere Kartierung und funktionelle genetische Beschreibung nötig

sind, um abzuklären, ob diese Gene dem QTL für Kühletoleranz der Photosynthese auf dem

langen Arm von Chromosom 6 zugrunde liegen.

X

List of abbreviations

Add Additive effect

ANOVA Analysis of variance

bp Base pairs

CER CO2 exchange rate

CIM Composite Interval Mapping

cM Centimorgan

DNA Deoxyribonucleic acid

dNTPs Deoxynucledotide 5’-triphosphate

Dom Dominant effect

EST Expressed sequence tags

Fo Minimal fluorescence in dark-adapted leaves

Fo' Minimal fluorescence in light-adapted leaves

Fm Maximal fluorescence in dark-adapted leaves

Fm' Maximal fluorescence in light-adapted leaves

Fv Variable fluorescence in dark-adapted leaves

F ' Actual fluorescence intensity at any given time

Fv/Fm Maximum quantum efficiency of PS II primary photochemistry

Fv'/Fm' Quantum efficiency of open PS II reaction centers

LOD Base 10 logarithm of the likelihood ration (LR)

MAS Marker assisted selection

NIL Near isogenic line

PCR Polymerase chain reaction

PS II Photosystem II

qP Photochemical quenching factor

QTL Quantitative Trait Locus

R2 Phenotypic variance explained by quantitative trait locus

SPAD Soil plant analyses development

SSR Simple sequence repeat

ΦNO Quantum efficiency for dissipation not by down regulation

ΦNPQ Quantum efficiency for dissipation by down regulation

ΦPSII Operating quantum efficiency of PS II

Chapter 1

1

1 General Introduction

Plant abiotic stress factors, such as cold, drought, light or salt, constitute major constraints to

agricultural production. Crop production is never free of such abiotic stress factors;

consequently, they can affect crop production in every season and in every crop worldwide.

Plants have evolved multiple, interconnected strategies that enable them to survive under

abiotic stress. However, these strategies are not well developed in most agricultural crops.

Across a range of cropping systems around the world, abiotic stress is estimated to reduce

yields to less than half of that possible under ideal growing conditions (Tang and Boyer,

2002). Of all the abiotic stresses that curtail crop productivity, temperature extremes are the

most important players besides water shortage, governing natural distribution of species and

the yield potential of crops. Plants can be divided into species, which are chilling sensitive,

chilling tolerant but freezing sensitive, and freezing tolerant. Chilling tolerance at the

vegetative level is defined as the ability to withstand the development of chilling injury and to

resume normal growth upon return to a non-chilling temperature (Lyons, 1973). Chilling-

sensitive species exhibit a marked physiological dysfunction when exposed to chilling

temperatures (between 0 and 15 °C) beyond a certain period of time and, if maintained at

these temperatures, develop a variety of external symptoms (chilling injury) and eventually

die (Lyons, 1973). Like other cereal crops of tropical and subtropical origin (e.g. rice,

sorghum), maize is a typical chilling-sensitive species that upon exposure to chilling

temperatures, inhibits seedling establishment and photosynthetic activity (Leipner et al., 1999;

Stirling et al., 1991) with the result that productivity and yield stability are reduced (Carr and

Hough, 1978; Stamp, 1986).

Years of selective breeding have led to the emergence of maize genotypes exhibiting varying

degrees of adaptation to temperate climate due to late planting and breeding for early-

maturing hybrids. Agronomic progress was achieved mainly an avoidance mechanism i.e., the

shortening the plant’s life cycle. These strategies minimize the risk of losses in the field

(Stamp, 1986). However, until now, little true chilling tolerance has been obtained, even

though the genetic diversity in the level of adaptation to suboptimal temperature is high

among the various genotypes (Greaves, 1996). An improvement in chilling tolerance would

be favorable for earlier spring planting and would consequently, lead to higher yielding maize

hybrids (Lee et al., 2002). Furthermore, earlier sowing may be an important avoidance

General Introduction

2

strategy for drought-affected flowering when the usual flowering time in midsummer

coincides with dry soil conditions.

In Switzerland, maize is sown during spring, where the requirement of high temperature

maize is not always met during the early growth stages of the crop. The optimum temperature

range for germination, seedling growth and dry matter accumulation of maize is between 25

and 35 °C (Miedema, 1982). Suboptimal temperature coupled with short spells of chilling

events during early growth stages results in decreased productivity of the crop (Carr and

Hough, 1978), poor yield stability (Stamp, 1986) and various effects on a number of

physiological processes. Under field conditions, both germination and development of the

first few leaves often take place at suboptimal temperature. Despite an increase in mean

temperature, later growth also seems to be affected by chilling, probably due to the inability

of the plant to respond quickly to the favorable changes in the environment. The slow

recovery of leaves after low temperature stress might be due to the inability of chloroplasts to

attain high photosynthetic efficiency after cessation of the unfavorable conditions (Nie et al.,

1995).

Amongst the various effects of low temperature on the physiology of maize, the considerable

susceptibility of the photosynthetic apparatus to low temperature is considered to be of

particularly important (Baker et al., 1994). Photosynthetic activity of maize leaves decreases

substantially when plants are subjected to cold stress (Ying et al., 2000). The mechanism

responsible for the reduction of leaf photosynthesis at low temperatures is unclear, as stomatal

closure (Massacci et al., 1995), reduced chlorophyll content (Leipner et al., 1999), lower

ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity (Stamp, 1981) and

reduced quantum efficiency of photosystem II (ΦPSII) (Fracheboud et al., 1999; Leipner et al.,

1999) have all been proposed as possible causes. With regard to photosynthetic apparatus,

several impact options have been discussed. First, the photosynthetic apparatus is known to be

highly sensitive to low temperature-induced photoinhibition (Long et al., 1983); second,

leaves that develop at a temperature of 15 °C or below are characterized by a very low

efficiency and capacity of photosynthesis, altered composition of leaf pigment (Haldimann et

al., 1995) and impaired chloroplast development (Robertson et al., 1993). Above all,

photosynthesis is one of most dominant and important biochemical process in higher plants,

and a loss of its activity damages the physiological activity of the whole plant. Improved

chilling tolerance of the photosynthetic apparatus of maize may, therefore, contribute

substantially to the improvement of the performance of the crop in temperate regions by

increasing early vigor and extending the culture period.

Chapter 1

3

Although impressive progress in the understanding of chilling stress has been achieved at the

biochemical and physiological levels, the potential for genetic improvement of maize

production under chilling conditions is still not been fulfilled. Genetic improvement under

chilling stress should be accelerated by implementing new molecular tools and by increasing

amount of genetic information generated through molecular and biochemical approaches. The

genetic basis of differences in physiological processes i.e., in quantitative traits is usually

complex, because several genes are involved and the expression of these traits depends

strongly on environmental factors. Analysis of Quantitative trait loci (QTL) enables to unravel

the genetics of such quantitative traits. The analysis of the genes controlling a variation in trait

may enable the molecular identification of these genes and, subsequently, the processes that

the control.

Molecular markers and QTL analysis based on carefully managed replicated tests have the

potential to alleviate the problems associated with chilling stress. The identified molecular

markers linked to QTLs for chilling tolerance could be used to increase the efficiency of

breeding and the selection of maize germplasm with enhanced chilling tolerance. Such an

approach provides a more systematic way of identifying specific traits that contribute to

chilling tolerance. Moreover, further analyses of these traits could lead to a better

understanding of the biological basis of chilling tolerance. The development of molecular

genetic markers and their use in QTL analyses is an increasingly a common approach to the

evaluation of the inheritance and the feasibility of accelerating selection for complex

quantitative traits in crop plants. Molecular markers have been used successfully to identify

and characterize QTLs associated with several traits in maize including plant height and

maturity (Beavis et al., 1991), characters concerned with plant domestication (Doebley et al.,

1990; Doebley and Stec, 1991; Doebley and Stec, 1993), resistance to disease and insect

(Dingersdissen et al., 1996; Freymark et al., 1993; Pe et al., 1993; Perez-Brito et al., 2001),

drought tolerance (Ribaut et al., 1996) and grain yield and component characters of grain

yield (Mohammadi et al., 2003; Tuberosa et al., 2002b).

Based on quantitative trait loci approach, several QTLs for chilling tolerance of

photosynthesis have already been mapped (Fracheboud et al., 2002; Fracheboud et al., 2004;

Jompuk et al., 2005; Pimentel et al., 2005). It was shown that the major QTLs involved in the

chilling tolerance of seedlings are found in the field as well as under controlled chilling

conditions (Jompuk et al., 2005). This cleared the path towards pinpointing chromosomal

regions involved in chilling tolerance. Although these authors identified regions of the maize

genome that condition the expression of chilling tolerance, however, little information has


4

been provided about the expression of individual QTLs. Furthermore, quantitative traits were

usually observed at a fixed time point or stage of trait development (e.g. at fully developed

third leaf). Such QTL mapping strategy can only provide an estimate of the effects of

individual QTLs accumulated from the beginning of ontogenesis to the time of the

observations. According to developmental genetics, the development of a trait results from the

differential activity of many related QTLs. This suggests that different QTLs show different

expression dynamics during trait development, even though the final effects may be same.

Lastly, a lack precision mapping of QTLs was intrinsic for these studies.

Near-isogenic lines (NILs), differing with regard to QTLs for chilling tolerance, could

provide a valuable material for a more detailed study of the genetic basis of quantitative

tolerance. Such NILs not only provide a better estimate of the effect of single QTL alleles, but

also provide better insight into QTL × environment interactions. Furthermore, QTL-NILs may

provide a starting point for unraveling the genes underlying these loci and may be useful for

positional cloning. Conventionally such QTL-NILs were developed by backcross breeding.

Recurrent backcrossing is a traditional breeding method commonly employed to transfer

alleles at one or more loci from a donor to an elite variety (Bravo et al., 2003; Reyes-Valdés,

2000). Traditional backcrossing programs are based on the assumption that the proportion of

the recurrent parent genome is recovered at a rate of 1 - (½)t+1 for each t generation of

backcrossing (Reddy et al., 2001). Thus, the expected recovery of the recurrent parent genome

after six generations of backcrossing would be 99.2%, i.e., near-isogenic. However, any

specific backcross progeny will deviate from this due to chance (stochastic or non-random

positions of chiasmata) and/or linkage between a target gene from the donor parent and

nearby genes (Ribaut and Hoisington, 1998). For example, Young and Tanksley (1997) found

an introgressed segment as large as 4 centimorgan (cM) in tomato cultivars developed after 20

backcrosses, and one cultivar developed after 11 backcrosses still contained the entire

chromosome arm carrying the gene from the donor parent. Therefore, the two main

limitations of the backcrossing approach are: i). the number of generations, and, thus, the

time, necessary to achieve the introgression objective and, ii). the simultaneous transfer of

other genes flanking the target gene from the donor parent (linkage drag).

Among the genes, which were carried through linkage drag, some coded for agronomically

undesirable traits, such as low yield or low temperature sensitivity. Depending on the linkage

distances, the size of the flanking regions can be decreased by additional backcrossing (Färber

et al., 1997), although breeders do not have direct control over the size of the region or the

recombination breakpoints. During the past two decades, the ability to transfer target genomic

Chapter 1

5

regions increased by means of molecular markers, which were obtained by extensive genetic

mapping experiments. Molecular markers are a tool that can be used as chromosome

landmarks to facilitate the introgression of chromosome segments (genes) associated with

economically important traits by marker assisted backcrossing (MAB), also referred to as

marker assisted selection (MAS), marker assisted introgression or molecular breeding. There

is now a large amount of research that aims to identify genomic regions of interest, from

which MAB experiments are the next attractive step. Molecular markers do not require

genetic engineering and cultivars developed by MAB are not transgenic and, therefore, are

more easily accepted by the public than transgenic crops.

1.1 Aims of the study Among the several QTLs detected for chilling tolerance of photosynthesis in the ETH-DL3 ×

ETH-DH7 population, the QTL with the largest genetic influence on chilling tolerance of

photosynthesis was identified on the long arm of chromosome 6 at bin 6.07 (Fracheboud et

al., 2004; Jompuk et al., 2005). This QTL alone explained 37.4 percent of the phenotypic

variance of photoinhibition (Fv/Fm) at suboptimal temperature and was significantly involved

in the expression of other traits, such as minimal fluorescence of dark-adapted leaves (Fo),

quantum yield of electron transport at PS II (ΦPSII), trapping efficiency of PS II (Fv'/Fm'),

including the rate of carbon fixation (CER) and shoot dry matter accumulation (Fracheboud et

al., 2004). This QTL contributes substantially to the maintenance of photosynthesis across

cold environments, a process long considered to be essential for chilling tolerance, making it

of particular interest for improving the chilling tolerance of elite chilling sensitive germplasm.

However, before such a QTL can be used in applied breeding its precise position on the

genome must be determined and it is necessary to discover its expression in different

environments and plant tissues. For these reasons, it would be helpful to understand the

underlying molecular mechanisms. Therefore, in the present study, an attempt was made to

elucidate the molecular and physiological basis of the QTL for chilling tolerance of

photosynthesis located in the telomeric region of the long arm of chromosome 6. For such a

study, near isogenic lines have to be developed that differ only in the presence of the chilling

tolerance allele at the target QTL. At the same time, QTL effects must be verified in different

genetic backgrounds before the deployment of such a QTL in an applied breeding program.


6

Keeping the above mentioned issues in mind, the present study was carried out in order

• to refine the position of the QTL for chilling tolerance of photosynthesis on chromosome

6 (bin 6.07) on the genetic linkage map of the ETH-DL3 × ETH-DH7 population,

• to investigate stability of this QTL during introgression of the chilling tolerant allele into a

chilling sensitive background,

• to study its effects during leaf development at different temperatures,

• to investigate the physiological mechanism of this chilling tolerance allele in near isogenic

lines,

• to verify the QTL effects in an alternate genetic background

• and to identify putative candidate genes underlying this QTL.

Chapter 2

7

2 Re-analysis of a major QTL for chilling tolerance of photosynthesis based on an improved map of the ETH-DL3 × ETH-DH7 population

2.1 Introduction In a previous analysis, a QTL with large effects on the chilling tolerance of photosynthesis

was detected in a Swiss dent mapping population originating from the cross ETH-DL3 ×

ETH-DH7 (Fracheboud et al., 2004). It explains a large amount of phenotypic variation for

photosynthesis and chlorophyll fluorescence parameters and was detected on chromosome 6

at bin 6.07. However, due to the small number of markers used in the linkage analysis, the

position of the QTL is still vague within a large marker interval of approximately 54 cM. This

region, which is flanked by the SSR markers umc1859 and umc1653, is too large to be studied

effectively and must be refined to facilitate positional cloning of the underlying genes.

Fine mapping of QTLs can be achieved by increasing the marker density within the

chromosomal region of interest, by increasing the number of individuals for which phenotypic

information can be obtained, or by increasing the accuracy of assigning QTL genotypes

(Nezer et al., 2003). For this QTL, the most straightforward approach is to increase the

number of informative markers within the QTL interval, as this eliminates the need to produce

new F2:3 individuals or to score new phenotypes on the previously generated F2:3 population.

Recent advances in maize genome resources, including the development of physical maps and

the genome sequencing project, provide reagents for the targeted isolation of new markers.

This chapter describes the use of these resources to increase the marker density around bin

6.07, and, thereby, the map position of the major QTL for chilling tolerance of photosynthesis

was refined.

2.2 Materials and Methods 2.2.1 Resource population and phenotypic data The ETH-DL3 × ETH-DH7 population derived from the chilling sensitive inbred line ETH

DL3 and the chilling tolerant inbred line ETH DH7 was used in the present study (for details,

see Fracheboud et al., 2004). The phenotypic data for photosynthesis and the chlorophyll

fluorescence parameters of the ETH-DL3 × ETH-DH7 population in the F2:3 generation were

obtained from the Maize Genetics and Genomics Database (http://www.maizegdb.org/qtl-

data.php). The methods of measurements have been described by Fracheboud et al. (2004). In

Re-analysis of major chilling tolerance QTL

8

brief, the parameters were measured on the fully developed third leaves which developed

either at optimal (25/22 °C) or at suboptimal temperature (15/13 °C). The operating quantum

efficiency of photosystem II (ΦPSII), the efficiency of open PS II reaction centers (Fv'/Fm') and

the carbon exchange rate (CER) were measured using LI-6400 instrument equipped with an

LI-6400-40 pulse-amplitude modulation fluorometer (LI-COR, Lincoln, NE, USA). The

maximum quantum efficiency of PS II (Fv/Fm) was determined with a PAM-2000 fluorometer

(Walz, Effeltrich, Germany) after 30-60 min dark adaptation. Leaf greenness was measured

using a Minolta SPAD-502 chlorophyll meter (Minolta Corporation, Ramsey, Japan).

2.2.2 Selection and development of molecular markers Seventeen simple sequence repeat (SSR) and 18 indel polymorphism (IDP) markers, which

are located in the proximity of bin 6.07 close to the SSR marker bnlg1740, were selected

based on information provided by the IBM 2 neighbors map at the Maize Genetics and

Genomics Database (http://www.maizegdb.org). Additional 21 SSR markers were developed

from BAC sequences, which were located in the QTL confidence interval. The simple

sequence repeat identification tool (SSRIT) available at http://www.gramene.org was

employed to identify SSRs in the BAC sequences. The SSRIT program was run online and

the parameters were set for the detection of di-, tri-, tetra- and penta-nucleotide motifs with a

minimum of 10, 7, 5 and 4 repeats, respectively. The repeat region and surrounding flanking

sequence (ca. 200 bp on either side) were extracted and used for PCR primer design by the

program Primer3 available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

(Rozen and Skaletsky, 2000). The parameters were set as follows: primer product size range,

200-300 bp; primer optimum length, 20 bp; primer length, 18 bp; primer maximum length, 25

bp; primer optimum temperature, 55 °C; primer minimum temperature, 50 °C; primer

maximum temperature, 60 °C; primer maximum differential temperature, 3 °C; primer

minimum GC content, 50 percent; primer maximum GC content, 55 percent.

2.2.3 DNA markers assay The DNA of 254 F2 plants of the ETH-DL3 × ETH-DH7 population (Fracheboud et al., 2004)

was used for marker analysis. The PCR was carried out using 50 ng DNA as template, 5

pmoles of each primer, 0.2 mM dNTPs, 1 × PCR buffer and 1 U Taq DNA polymerase (New

England Biolab) in a total volume of 15 µl. Template DNA was initially denatured at 94 °C

for 5 min followed by 35 cycles of PCR amplification under the following cycling conditions:

30 sec denaturation at 94 °C, 30 sec primer annealing at 55 °C, 1 min extension at 72 °C. A

Chapter 2

9

final extension of 72 °C for 7 min was conducted. The PCR amplification was carried out in

an Eppendorf DNA Thermal cycler (Eppendorf, Hamburg, Germany) and the amplification

products were size fractioned in a 4 % high resolution agarose (Eurobio) as described by

Sambrook and Russell (2001). After electrophoresis, the gels were stained in 0.5 μg ml-1

ethidium bromide and visualized under ultraviolet light.

2.2.4 QTL analysis The genetic map was constructed using mapmaker 3.0 (Lander et al., 1987) with the Haldane

mapping function. QTL analysis based on this map was carried out using the QTL

Cartographer v.1.17b (Basten et al., 1994; Basten et al., 2003). The method of composite

interval mapping (CIM), model 6 of Zmapqtl program module, was used for mapping QTLs

and estimating their effects (Jansen and Stam, 1994; Zeng, 1994). Cofactors were chosen

according to the forward-backward method of stepwise regression at p(Fin) = p(Fout) < 0.01.

The chromosomal region was scanned at 2 cM; the window size was set at 30 cM.

2.3 Results and Discussion By taking advantage of recent developments in maize genome resources, including the high

resolution IBM2 2008 neighbors map and BAC fingerprint maps, an attempt was made to

saturate the confidence interval of the QTL for chilling tolerance of photosynthesis on the

long arm of chromosome 6 with additional SSR markers. Over 56 molecular markers located

between bin 6.06 and bin 6.08 were screened for parental polymorphism between ETH-DL3

and ETH-DH7. Of these markers, only two markers showed parental polymorphism. These

two polymorphic SSR markers, namely bnlg1136 and umc2234, were used to survey the

genomic DNA of the 254 F2 plants and a new genetic linkage map was constructed (Figure

2.1). The relatively low rate of polymorphism is attributed to the similar origin of the parental

lines; both parents were derived from the same Swiss Dent breeding population (Fracheboud

et al., 1999). Similarly, Fracheboud et al. (2004) found only 118 polymorphic markers from a

total of 1200 markers screened for parental polymorphism between ETH-DL3 and ETH-DH7

at the level of the whole genome.

Furthermore, a primer pair was designed (agpslzm), which amplifies the gene of the ADP-

glucose pyrophosphorylase that was previously proposed as a positional candidate gene of the

major QTL for chilling tolerance of photosynthesis on chromosome 6 (Fracheboud et al.,

2004; Jompuk et al., 2005). However, the linkage analysis in the F2 population revealed that


10

agpslzm, which is specific for the gene of ADP-glucose pyrophosphorylase in the leaf, is not

located on chromosome 6 but on chromosome 1 at 205.6 cM between mmc0041 (189.3 cM)

and bnlg1502 (249.2 cM). A re-analysis of the phenotypic data from Fracheboud et al. (2004)

revealed that agpslzm is closely located to QTLs for SPAD (205.3 cM), Fv/Fm (213.6 cM),

CER (221.6 cM) and ΦPSII (227.6 cM) in leaves that developed at suboptimal temperature.

Due to the closeness of agpslzm to the mentioned QTLs for photosynthesis related traits,

agpslzm can be considered as a potential positional candidate gene. However, it remains to be

open how ADP-glucose pyrophosphorylase, which plays a central role in the starch turn-over

within the chloroplast (Baroja-Fernández et al., 2001), affects the chlorophyll content under

chilling stress conditions.

In another analysis of the data of Fracheboud et al. (2004), two QTLs associated with

photosynthesis and chlorophyll fluorescence parameters were identified on the long arm of

chromosome 6 (Figure2.1, Table 2.1). The first QTL, which is located at 206 to 228 cM and is

flanked by the markers umc1859 and bnlg1740, was also detected in the original study

grown at 15 °C

LOD score

0 5 10 15

Fo

Fv/Fm

SPADΦPSII

Fv'/Fm'q

P

CER

grown at 25 °C

LOD score

0 5 10 15 20

Fo

Fv/Fm

SPADΦPSII

Fv'/Fm'q

P

CER

Chr 6

bnlg1043 0.0bnlg161 7.4

bnlg1867 46.3bnlg249 56.4

bnlg1188 59.2

umc1887 108.4bnlg1617 122.9bnlg1922 126.9

bnlg1732 175.8umc1859 186.2

bnlg1740 231.5bnlg1136 239.8

umc1653 255.4

umc2324 272.5

Figure 2.1: Linkage map and putative locations of QTLs on chromosome 6 for chlorophyll fluorescence parameters (Fo, Fv/Fm, ΦPSII, Fv'/Fm' and qP), leaf greenness (SPAD) and carbon exchange rate (CER) in seedlings of the ETH-DL3 × ETH-DH7 population in the F2:3generation grown at suboptimal (15 °C) and optimal temperature (25 °C). Phenotypic data were obtained from Fracheboud et al. (2004). The new markers are indicated by bold names and the threshold for the LOD score of 3.5 is shown.

Chapter 2

11

(Fracheboud et al., 2004). The second new QTL was detected at 244 to 254 cM in the region

where additional markers were mapped. This QTL is flanked by markers bnlg1136 and

umc1653. For clarity, the QTL at 206 to 228 cM will be designated QTL-1 and the QTL at

244 to 254 cM will be named as QTL-2. The possibility still remains that the newly identified

QTL is linked to the first QTL, given the shape of the test statistic curves (Figure 2.1).

However, the large distance between the two QTLs suggests that they are separate.

Furthermore, the two QTLs have distinct characteristics in respect to the analyzed traits. In

general, QTL-2 showed lower effects than QTL-1. However, while the additivity and the

phenotypic variance (R2) of Fv/Fm, Fo and Fv'/Fm' were only little lower for QTL-2 compared

to QTL-1, they were considerably lower for ΦPSII and CER. This was observed for seedlings

grown at 15 °C as well as for plants that grown at 25 °C.

Table 2.1: Main characteristics of significant QTLs on the long arm of chromosome 6 for photosynthetic traits in seedlings of the F2:3 population grown at suboptimal (15 °C) and optimal temperature (25 °C) recalculated from Fracheboud et al. (2004).

Trait cM Marker interval LOD score Add Dom R2 (%)

grown at 15 °C

Fv/Fm 224 umc1859-bnlg1740 18.51 0.032 0.017 39.0 246 bnlg1136-umc1653 16.07 0.025 0.020 29.4

Fo 222 umc1859-bnlg1740 17.37 -0.054 -0.026 35.5 248 bnlg1136-umc1653 15.82 -0.039 -0.039 27.7

ΦPSII 212 umc1859-bnlg1740 6.18 0.024 -0.013 24.8 254 bnlg1136-umc1653 6.54 0.012 0.014 10.6

Fv'/Fm' 228 umc1859-bnlg1740 4.94 0.019 0.005 8.4 254 bnlg1136-umc1653 5.97 0.015 0.019 9.6

CER 206 umc1859-bnlg1740 5.20 0.56 -0.21 15.1 255 bnlg1136-umc1653 2.81 0.11 0.42 4.4

grown at 25 °C

Fo 224 umc1859-bnlg1740 12.64 -0.004 -0.003 21.3 244 bnlg1136-umc1653 11.59 -0.003 -0.002 16.3

ΦPSII 214 umc1859-bnlg1740 6.05 0.013 0.008 21.8 250 bnlg1136-umc1653 4.99 0.009 0.003 9.6

Fv'/Fm' 216 umc1859-bnlg1740 10.01 0.013 0.012 37.1 250 bnlg1136-umc1653 8.67 0.010 0.005 18.2

cM = Position of the peak of the QTL in centimorgan; Add = additive effect of the ETH-DH7 allele; Dom = dominant effect of the ETH-DH7 allele; R2 = phenotypic variance as explained by QTL.


12

In order to characterize these two QTLs in more detail and to unravel their function, further

experiments are necessary and should include newly developed plant material, which makes it

possible to focus better on this genomic region of chromosome 6.

Chapter 3

13

3 Marker assisted introgression of a major QTL for chilling tolerance of photosynthesis into the recurrent parental line

3.1 Introduction In maize, suboptimal temperature coupled with short spells of chilling events during early

growth stages result in decreased productivity (Carr and Hough, 1978) and poor yield stability

(Stamp, 1986) and affects several physiological processes. Among the various effects of low

temperature on the physiology of maize, high susceptibility of the photosynthetic apparatus to

low temperature is considered to be of particular importance (Baker et al., 1994). This is due

to the photosynthetic apparatus of maize is known to be highly sensitive to low temperature-

induced photoinhibition (Long et al., 1983) and, leaves that develop at a temperature of 15 °C

or below are characterized by a very low photosynthetic capacity, altered leaf pigment

composition (Haldimann et al., 1995) and impaired chloroplast development (Robertson et al.,

1993). Improved chilling tolerance of the photosynthetic apparatus of maize may, therefore,

contribute substantially to an overall improvement in early vigor in temperate regions.

In previous studies, an F2:3 population, derived from the cross between ETH-DH7 and ETH-

DL3 was genetically analyzed (Fracheboud et al., 2004; Jompuk et al., 2005). It was

demonstrated that chromosome 6 harbored a major QTL, which was involved not only in the

development of a functional photosynthetic apparatus but also in its protection against low

temperature stress. A QTL re-analysis employing an improved linkage map of chromosome 6

indicated that probably two separate QTLs are located in this chromosomal region (Chapter

2). In order to study the genetic and physiological mechanisms of this/these QTL(s) in more

detail, it is desirable to conduct a backcross breeding program to introgress this chromosomal

region in an isogenic background. Different approaches have been described. For QTLs with

large effects, the most common approach is to introgress individual QTL alleles into

homozygous genetic backgrounds through multiple generations of backcrossing (e.g. Kole et

al., 2001; Li et al., 2001; Lin et al., 2000; Monforte and Tanksley, 2000; Van Berloo et al.,

2001; Vladutu et al., 1999). This can lead to qualitative segregation of phenotypic effects,

which can be used for fine mapping and, consequently, to clone the QTL of interest (Yano,

2001). However, introgression of QTLs through conventional backcrossing is time consuming

and laborious. The use of molecular markers to construct molecular genetic linkage maps

(Phillips and Vasil, 1994) and to detect marker-trait associations (Kearsey and Farquhar,

Marker assisted introgression of major chilling tolerance QTL

14

1998) has been exploited extensively in recent years. The information provided by marker-

trait associations can be used as a tool for crop improvement thorough the use of marker

assisted selection (MAS) of favorable alleles. Valuable information can be provided by the

screening of backcross populations with molecular markers of known position on a genetic

map. Not only can the origin of the QTL allele be determined, but also the rest of the genome,

both linked and unlinked to the QTL, can be monitored. Hence, molecular markers assist

backcross breeding by reducing the number of generations required to introgress QTLs into a

near isogenic background, in particular, by deploying DNA markers for selection of target

QTLs and of the recurrent parent genome (RPG) selection during backcross breeding

(Fracheboud et al., 2002; Frisch and Melchinger, 2001). Thus, marker assisted selection of

whole genome in favor of the recurrent parent alleles will help to recover the recurrent

genotype at a much faster pace than can be achieved by conventional backcrossing (Bouchez

et al., 2002; Hospital et al., 1992; Tanksley and Nelson, 1996).

The aim of this study was to elucidate the physiological and genetic basis of the major QTL

region for chilling tolerance of photosynthesis on the long arm of chromosome 6. The study

was carried out on a series of backcross populations derived from the cross between the

chilling sensitive parent, ETH-DL3, and the chilling tolerant parent, ETH-DH7. Important

goals were: (1) to assess the stability of the QTL expression during different generations of

marker assisted backcross breeding, (2) to assess the QTL dynamics in different leaves and

under different temperature regimes and (3) to find further evidence of the presence of two

separate QTLs in this genomic region.

3.2 Materials and methods 3.2.1 Plant material The maize genotypes ETH-DH7 and ETH-DL3 were used as the donor and the recurrent

parent, respectively. ETH-DH7 is a chilling tolerant inbred line, which maintains higher

photosynthetic activity at suboptimal growth temperature, whereas the inbred line ETH-DL3

shows poor photosynthetic activity when grown under suboptimal temperature (Fracheboud et

al., 2004). The two parental lines were originally obtained by divergent selection from a Swiss

dent maize breeding population using the chlorophyll fluorescence as a selection tool

(Fracheboud et al., 1999). The F1 plants were crossed with ETH-DL3 to produce BC1F1

plants. To select chilling-tolerant BC1F1 plants, seedlings were grown at suboptimal

temperature and the operating quantum efficiency of photosystem II (ΦPSII) was measured on

Chapter 3

15

the fully developed third leaf at 6 °C under a PPFD of 50 µmol m-2 s-1. The BC1F1 plants,

which had a high average of ΦPSII were selected for further crossing. Starting from the BC2F1

generation, marker assisted selection was carried out to develop the BC3F2 population.

3.2.2 Growth conditions The experiments were carried out in growth chambers (PGW36, Conviron, Winnipeg,

Canada). Seeds were allowed to germinate in 0.75 l pots containing a commercial mixture of

soil, peat and compost (Topf und Pikiererde 140, Ricoter, Aarberg, Switzerland) and grew

until 1-leaf stage at 25/22 °C (day/night) temperature, 12 h photoperiod with a light intensity

of 400 µmol m-2 s-1 and a relative humidity of 60/70 % (day/night). About six days after

seeding, when the 1-leaf was fully emerged, the seedlings were transferred to suboptimal

growth temperature (15/13 °C, day/night) for 15 days until the third leaf was fully developed.

The other conditions were the same. The plants were watered and fertilized with half-strength

Wuxal nutrient solution (0.2 %) as required.

Furthermore, a time-course experiment was conducted using seedlings of the BC3F2

generation. After growth at suboptimal temperature (15/13 °C), the temperature was

decreased to 10/10 °C (day/night) during the dark period for two days followed by recovery

for two days at 25/23 °C (day/night); recovery started at the beginning of the light period. The

light intensity was 400 µmol m-2 s-1 with a photoperiod of 12 hours and a relative humidity of

60/70 % (day/night).

3.2.3 Measurement of physiological traits Measurements of photosynthesis and chlorophyll fluorescence parameters were carried out

with an infrared gas analyzer equipped with a pulse-amplitude modulation fluorometer (LI-

6400, LI-COR, Lincoln, NE, USA). The conditions in the measuring chamber of the LI-6400

were the same as the growth conditions. The operating quantum yield of photosystem II

(ΦPSII), the efficiency of open PS II reaction centers (Fv'/Fm'), the photochemical quenching

factor (qP) and the carbon exchange rate (CER) were measured simultaneously on the middle

part of the third leaf. After determining the carbon exchange rate and the steady state

fluorescence (F '), a one second saturation flash (> 8000 µmol m-2 s1) was applied to

determine the maximum fluorescence in the light (Fm'). The actinic light was then turned off,

and the leaf was subjected to far red light for 3 seconds to determine the ground fluorescence

of light adapted leaves (Fo').


16

The maximum quantum efficiency of PS II primary photochemistry (Fv/Fm) was determined

with a PAM-2000 fluorometer (Walz, Effeltrich, Germany) after 30-60 minutes of dark

adaptation by applying a one-second saturation flash (> 8000 µmol m-2 s-1).

To measure leaf greenness, three measurements in the middle section of the third leaf were

made and averaged for each plant using a SPAD-502 chlorophyll meter (Minolta Corporation,

Ramsey, Japan).

To analyze the time-course of QTLs in the BC3F2 population, Fv/Fm and ΦPSII were

determined with the PAM-2000 fluorometer. The ΦPSII measurements were conducted under

growth conditions and Fv/Fm was determined after at least 30 minutes of dark adaptation.

3.2.4 DNA isolation and SSR marker assay The DNA was isolated from 3 cm leaf segments according to Zheng et al. (1995). The leaves

of the maize seedlings were harvested and homogenized immediately in a chilled mortar in

400 µl extraction buffer (50 mM TRIS-HCl, pH 8.0; 25 mM EDTA; 300 mM NaCl; 1 %

[w/v] SDS). 400 µl homogenate were extracted with 400 µl phenol:chloroform (24:1). After

centrifugation (10000 rpm for 10 minutes), the aqueous supernatant was transferred to another

1.5 ml tube, and DNA was precipitated with 97 % ethanol. After drying in air, the DNA was

re-suspended in 200 µl water.

Aliquots of 2 µl DNA solution (50 ng µl-1) were taken for PCR along with 5 pmoles of each

primer, 0.2 mM dNTPs, 1 × PCR buffer and 1 U Taq DNA polymerase (New England

Biolab) in a total volume of 15 µl. Template DNA was initially denatured at 94 °C for 5 min

followed by 35 cycles of PCR amplification under the following conditions: 30 second

denaturation at 94 °C, 30 second primer annealing at 55 °C, 1 minute extension at 72 °C. A

final extension of 72 °C for 7 minutes was conducted. The PCR amplification was carried out

in an Eppendorf DNA Thermal cycler (Eppendorf, Hamburg, Germany) and the amplification

products were size fractioned in a 4 % high resolution agarose (Eurobio) as described in

Sambrook and Russell (2001). After electrophoresis, the gels were stained in 0.5 μg ml-1

ethidium bromide and visualized under ultraviolet light.

To genotype the BC3F2 plants, genomic DNA was isolated from 25 day old plants according

to the method of Dellaporta et al. (1983). The PCR was carried out using 50 ng DNA as

template for amplification; the other conditions were the same as above.

Chapter 3

17

3.2.5 Marker assisted and phenotype based backcrossing The BC3F2 generation was generated by several steps of backcrossing with the recurrent

parent (ETH-DL3) and selection of plants based on analysis of the phenotype and by marker

assisted selection. For phenotypic selection, seedlings that had developed at suboptimal

temperature were screened for ΦPSII and Fv/Fm as described above. The schematic illustration

of the marker assisted backcross breeding is shown in Figure 3.1.

3.2.5.1 Foreground selection

Three SSR markers namely bnlg1740 linked to the studied QTL (as per LOD score) and

umc1859 and umc1653 located on right- and left-hand side of the confidence interval, were

used to select the favorable ETH-DH7 allele at the target QTL in the BC2F1 generation. In the

BC3F1 generation, bnlg1740 and umc1653 were used to select for the favorable alleles of

ETH-DH7 at the target QTL and to reduce linkage drag around the introgressed region;

recombination was allowed on the right hand side of the confidence interval by selecting

plants having the ETH-DL3 allele for the SSR marker umc1859. In order to reduce the

amount of plant material, a sequential genotyping approach was followed in both generations.

Initially, the plants were genotyped with SSR marker bnlg1740. Recombinant plants were

selected and genotyped further with umc1859 and umc1653.

ETH-DL3 × ETH-DH7

ETH-DL3 × F1

ETH-DL3 × BC1F1

BC3F1

BC3F2

The two inbred lines were crossed to generate the F1 generation.

One F1 plant was backcrossed to the recurrent parent (ETH-DL3) to generate the BC1F1generation.

The BC1F1 plants were screened for ΦPSII and Fv/Fm. Four BC1F1 plants having high ΦPSIIand Fv/Fmwere selected and backcrossed to the recurrent parent.

Foreground selection was conducted with markers linked to the target QTL as well as by phenotyping for ΦPSII and Fv/Fm.Background selection was conducted with markers linked to non-target QTLs.A single plant was selected based on the marker profile and the highest level of ΦPSII and Fv/Fm and backcrossed to the recurrent parent.

Foreground selection was conducted with markers linked to the target QTL as well as by phenotyping for ΦPSII and Fv/Fm.Background selection was conducted with markers linked to non-target QTLs and with markers located in the proximal and telomeric regions of the chromosomes.A single plant was selected based on the marker profile and the highest level of ΦPSII and Fv/Fm and selfed.

ETH-DL3 × BC2F1

Figure 3.1: Schematic illustration of the marker assisted backcross breeding.


18

3.2.5.2 Background selection

Based on the results obtained by Fracheboud et al. (2004), to cull out non-target QTLs, 9 SSR

markers linked to the respective non-target QTLs were chosen: bnlg1564 (QTL for leaf

greenness at chromosome 1), bnlg1502 (QTL for ΦPSII on chromosome 1), dupssr21,

bnlg2248 and bnlg1148 (QTLs for CER, leaf greenness and Fv'/Fm' on chromosome 2),

bnlg1447 and bnlg1019 (QTLs for Fv'/Fm' and CER on chromosome 3), phi072 (QTL for

Fv'/Fm' on chromosome 4) and bnlg1031 (QTL for Fo and Fm on chromosome 8). For

background selection in the BC3F1 generation, 35 SSR markers were chosen which were

located in the proximal and in the distal regions of the maize chromosomes. To produce the

BC3F2 population, a single BC3F1 plant was selected, which recovered 96 percent of the

parental genome and which had the highest level of chilling-tolerance of photosynthesis as

assessed by chlorophyll fluorescence analysis.

3.2.6 Statistical analysis and QTL mapping Pearson’s correlation coefficients between traits were computed according to the CORR

procedure in SAS (v. 9.0, SAS Institute Inc., USA).

Single factor ANOVA was employed to determine the association between marker genotypic

class means and the phenotype to assess the stability of the QTL during backcrossing.

In the BC3F2 population, the genetic map was constructed using mapmaker 3.0 (Lander et al.,

1987) with the Haldane mapping function based on the genotypic data of 176 BC3F2 plants.

The QTL analyses were carried out using QTL Cartographer v.1.17b (Basten et al., 1994;

Basten et al., 2003). The method of composite interval mapping (CIM), model 6 of the

Zmapqtl program module, was chosen for mapping QTLs and estimating their effects (Jansen

and Stam, 1994; Zeng, 1994). Cofactors were chosen according to the forward-backward

method of stepwise regression at p(Fin) = p(Fout) < 0.01. The chromosomal region was

scanned at 2 cM; the window size was set at 30 cM (see also Chapter 2).

3.3 Results 3.3.1 Phenotypic analysis of chilling tolerance The superior chilling tolerance of ETH-DH7 over ETH-DL3 in seedlings grown at suboptimal

temperature was obvious (Table 3.1). Compared to ETH-DL3, ETH-DH7 was characterized

by a higher maximum quantum efficiency of PS II primary photochemistry (Fv/Fm), a lower

minimal fluorescence in dark adapted leaves (Fo), a higher operating quantum efficiency of

Chapter 3

19

PS II (ΦPSII), a higher efficiency of open PS II reaction centers (Fv'/Fm'), a higher carbon

exchange rate (CER) and a higher leaf greenness (SPAD) in seedlings grown at suboptimal

temperature. These differences between the parents were significant. The average values of

the studied traits during the different generations of backcrossing were in between those of the

parents. Table 3.1 gives a representative example of the average values of the phenotypic data

in the BC3F2. The distribution of all the traits showed transgressive segregation in both

directions (data not shown).

Highly significant phenotypic correlations were detected among the eight traits in all three

Table 3.1: Statistics of traits of the parental lines, ETH-DL3 and ETH-DH7, and seedlings of the BC3F2 backcross populations derived from the cross ETH-DL3 × ETH-DH7 grown at suboptimal temperature. Values are means ± SD.

Trait ETH-DL3 ETH-DH7 BC3F2

Fv/Fm 0.641 ± 0.025 0.732 ± 0.034 *** 0.709 ± 0.042

Fo 0.195 ± 0.034 0.143 ± 0.015 *** 0.158 ± 0.027

Fm 0.552 ± 0.101 0.547 ± 0.052 ns 0.546 ± 0.048

ΦPSII 0.246 ± 0.095 0.371 ± 0.041 ** 0.300 ± 0.080

Fv'/Fm' 0.320 ± 0.112 0.461 ± 0.055 ** 0.374 ± 0.086

qP 0.778 ± 0.037 0.802 ± 0.036 ns 0.793 ± 0.050

CER 4.8 ± 2.1 8.3 ± 1.8 ** 6.3 ± 2.5

SPAD 18.7 ± 2.9 36.1 ± 5.1 *** 30.3 ± 6.2

Significant differences at *** = P < 0.001, ** = P < 0.01, * = P < 0.05 and ns = non significant based on t-test.

Table 3.2: Phenotypic correlation coefficients (r) between the investigated traits in seedlings of the BC3F2 population grown at suboptimal temperature.

Trait Fv/Fm Fo Fm ΦPSII Fv'/Fm' qP

Fo -0.77 ***

Fm 0.07 ns 0.44 ***

ΦPSII 0.43 *** -0.40 *** -0.17 *

Fv'/Fm' 0.43 *** -0.40 *** -0.18 * 0.99 ***

qP 0.21 ** -0.09 ns 0.02 ns 0.32 *** 0.30 ***

CER 0.28 ** -0.20 ns -0.02 ns 0.71 *** 0.68 *** 0.16 *

SPAD 0.58 *** -0.59 *** -0.19 * 0.53 ** 0.52 *** 0.22 **

Significant differences at *** = P < 0.001, ** = P < 0.01, * = P < 0.05 and ns = non significant based on t-test.


20

backcross populations. In Table 3.2, the correlation coefficient values for each pair-wise

combination of traits in the BC3F2 population are shown. Correlation coefficients ranged

from -0.02 between Fm and CER to 0.99 between ΦPSII and Fv'/Fm'. Positive correlation

coefficients were observed between all the traits except for Fo and Fm which were negatively

correlated with all other traits.

3.3.2 QTL consistency over generations Of the eight analyzed traits, six traits with the exception of CER in the BC2F1 population

showed significant differences among the genotypic groups for the SSR marker bnlg1740 in

all three backcross populations (Table 3.3). The other significant traits were Fv/Fm, Fo, ΦPSII,

Fv'/Fm' and SPAD. The proportion of phenotypic variance contributed by the genotypic

differences (R2) ranged from 5.6 % (SPAD) to 51.9 % (Fv/Fm) in the BC2F1 population, 6.2 %

(CER) to 41.8 % (Fv/Fm) in BC3F1 population and 6.6 % (CER) to 46.1 % (Fv/Fm) in the

BC3F2 population indicating the stability of QTL expression for these traits. The chilling

tolerant parent ETH-DH7 at this marker contributed to an increase of Fv/Fm, ΦPSII, Fv'/Fm',

CER, SPAD and a decrease in Fo in all three backcross populations.

Table 3.3: Stability of QTL expression of photosynthetic traits in seedlings of the three backcross population grown at suboptimal temperature based on single marker analysis of the SSR marker bnlg1740.

BC2F1 BC3F1 BC3F2 Trait F value a R2 (%) b F value R2 (%) F value R2 (%)

Fv/Fm 167.02 ** 51.9 87.03 ** 41.8 71.52 *** 46.1

Fo 143.19 ** 48.0 50.89 ** 29.6 26.59 *** 24.3

Fm 3.60 ns - 5.29 ns - 4.27 ns -

ΦPSII 26.87 ** 14.8 21.89 ** 15.0 9.91 *** 10.6

Fv'/Fm' 39.45 ** 20.3 17.84 ** 12.9 10.46 *** 11.1

qP 1.41 ns - 1.35 ns - 3.14 ns -

CER 1.19 ns - 8.01 ** 6.2 5.92 ** 6.6

SPAD 9.17 ** 5.6 53.78 ** 30.8 25.61 *** 23.5

a = tests of the phenotypic differences between the two genotypic groups in BC2F1, BC2F1 and three genotype groups in BC3F2; *, P < 0.05; **, P < 0.01; ***, P < 0.001; b = phenotypic variance as explained by the QTL.

Chapter 3

21

3.3.3 Confirmation of the additional QTL in the distal region of chromosome 6 in the BC3F2 population

Using the SSR markers, bnlg1740 and umc1653, as well as the two new markers, bnlg1136

and umc2324 (Chapter 2), the 176 plants of the BC3F2 population were genotyped and a new

genetic linkage map was constructed (Figure 3.2). The order of the SSR markers was the same

as in the F2 population but the genetic distances between them was higher in the BC3F2 than

in the F2 population. This genetic linkage map was used to analyze QTLs by composite

interval mapping of the traits of leaves that developed at suboptimal temperature. The QTL

analysis confirmed the presence of the QTL-1, which was involved in the variation of Fv/Fm,

Fo, ΦPSII, Fv'/Fm', CER and SPAD. The QTL-1 was localized at the SSR marker bnlg1740

(Table 3.4). An increase in Fv/Fm, ΦPSII, Fv'/Fm', CER and SPAD and a decrease in Fo were

due to the allelic contribution of the chilling tolerant parent ETH-DH7. The magnitude of the

phenotypic variation (R2) explained by this QTL ranged from 6.5% for CER to 47.3% for

F2 BC3F2bnlg1043bnlg161

bnlg1867bnlg249bnlg1188

umc1887bnlg1617bnlg1922

bnlg1732umc1859

bnlg1740bnlg1136

umc1653

umc2324

7.4

39.0

10.12.8

47.7

12.63.6

49.6

10.4

38.0

8.416.8

16.0

bnlg1740bnlg1136

umc1653

umc2324

10.5

19.4

26.8

F2 BC3F2bnlg1043bnlg161

bnlg1867bnlg249bnlg1188

umc1887bnlg1617bnlg1922

bnlg1732umc1859

bnlg1740bnlg1136

umc1653

umc2324

7.4

39.0

10.12.8

47.7

12.63.6

49.6

10.4

38.0

8.416.8

16.0

bnlg1740bnlg1136

umc1653

umc2324

10.5

19.4

26.8

Figure 3.2: Genetic linkage map of the introgressed segment of chromosome 6 in the BC3F2population in relation to the genetic linkage map of the whole chromosome 6 in the F2population. The additional SSR markers are indicated by bold letters.


22

Fv/Fm, while the LOD score ranged from 2.53 for CER to 22.66 for Fv/Fm. Furthermore, the

QTL analysis identified the second QTL (QTL-2) in the introgressed region flanked by

umc1653 and umc2324 and 45 cM distant from the first QTL at bnlg1740. This QTL-2

controlled the traits Fo and Fv/Fm and explained 45.5 and 24.8% of the trait variation,

respectively.

3.3.4 Time course analysis of QTLs for photosynthetic traits The time course experiment was designed to assess the effects of both QTLs in different

leaves and under different temperature regimes. The time course of Fv/Fm and ΦPSII in the

parental lines and in the BC3F2 population are shown in Figure 3.3. In the first leaf, which

developed at 25 °C, Fv/Fm and ΦPSII of the parental lines measured at 25 °C were nearly

identical. A decrease in growth and measuring temperature to 15 °C resulted in a substantial

reduction in Fv/Fm and ΦPSII. However, starting from day 3 after transfer to 15 °C, Fv/Fm and

ΦPSII of second and third leaves of ETH-DH7 were significantly (P < 0.01) higher compared

to ETH-DL3. These differences in Fv/Fm and ΦPSII between the parents were smaller in the

second leaf, which had only partially developed at 15 °C, than in the third leaf that fully

developed at 15 °C. Furthermore, the third leaf was characterized by a lower ΦPSII than the

second leaf, especially in the chilling sensitive genotype ETH-DL3.

The significant differences between the parental lines were also observed when the

temperature was lowered further to 10 °C for two days. This decrease in temperature resulted

in a marked decrease in ΦPSII and Fv/Fm. Both parameters recovered when the seedlings were

Table 3.4: Main characteristics of QTLs for photosynthetic traits in the BC3F2 population with an LOD score above a threshold of 2.5.

Trait cM Interval LOD score Add Dom R2 (%)

Fv/Fm 0.0 bnlg1740 - bnlg1136 22.66 0.042 0.030 47.3 44.8 umc1653 - umc2324 5.32 0.031 0.020 24.8

Fo 0.0 bnlg1740 - bnlg1136 8.50 -0.020 -0.023 39.7 44.8 umc1653 - umc2324 5.81 -0.025 -0.024 45.5

ΦPSII 0.0 bnlg1740 - bnlg1136 4.47 0.036 0.035 11.4

Fv'/Fm' 0.0 bnlg1740 - bnlg1136 4.76 0.040 0.041 12.1

CER 0.0 bnlg1740 - bnlg1136 2.53 0.9 0.7 6.5

SPAD 0.0 bnlg1740 - bnlg1136 10.27 4.1 4.1 23.8

cM = Position of the peak of the QTL in centimorgan; Add = additive effect of the ETH-DH7 allele; Dom = dominant effect of the ETH-DH7 allele; R2 = phenotypic variance as explained by QTL.

Chapter 3

23

transferred to 25 °C. Most notable was that ETH-DH7 recovered the initial values of Fv/Fm

and ΦPSII while this was not the case in ETH-DL3.

The performance of the both traits of the BC3F2 population was skewed towards the chilling

tolerant parent ETH-DH7 with regard to the second leaf and was between the corresponding

values of the two parents with regard to the third leaf during growth at 15 °C. In this state,

transgressive segregation occurred in both directions of the parents for Fv/Fm and ΦPSII in the

second leaf as well as in the third leaf at 15 °C (data not shown). During cold stress at 10 °C

F v/Fm

0.0

0.2

0.4

0.6

0.8

ΦPS

II

0.0

0.2

0.4

0.6

10°C 25°C15°C25°C10°C 25°C15°C25°C

ETH-DH7BC3F2ETH-DL3

leaf 1 2 3

LOD

(Fv/F

m)

0

5

10

15

20QTL-1QTL-2

leaf 1 2 3

LOD

(ΦP

SII)

0

5

10

15

20

time (days)

0 5 10 15 20

R2 (F

v/Fm)

0

10

20

30

40

50

time (days)

0 5 10 15 20R

2 (ΦPS

II)0

10

20

30

40

50QTL-1QTL-2

leaf 1 2 3

Figure 3.3: Time course of Fv/Fm and ΦPSII in seedlings of ETH-DH7 (white symbols), ETH-DL3 (black symbols) and of the BC3F2 population (grey symbols) and time course of LOD scores and R2 values for QTL-1 (black symbols) and QTL-2 (white symbols).


24

and during recovery at 25 °C, the mean values of the BC3F2 population were towards the

chilling tolerant parent ETH-DH7 for Fv/Fm and towards ETH-DL3 for ΦPSII.

The QTL analysis revealed that QTL-1 had not been not expressed in the first leaf (day 0;

25 °C) and in the second leaf (day 3 to 9 after transfer to 15 °C) (Figure 3.3). In the third leaf,

QTL-1 was expressed at all the time points at 15 °C for Fv/Fm and ΦPSII. Furthermore, QTL-1

consistently explained the variation for Fv/Fm even during cold stress at 10 °C and during

recovery phase at 25 °C. Its expression was below the threshold level for ΦPSII (Figure 2.3).

The LOD score and the R2 value for Fv/Fm showed a strong increase during growth of the

third leaf at suboptimal temperature. Similar to QTL-1, QTL-2 was not expressed in the first

and second leaves. However, QTL-2 was found for Fv/Fm in the third leaf at 15 and at 10 °C.

During the recovery phase at 25 °C, QTL-2 was below the threshold. The QTL-2 expression

(R2) for Fv/Fm changed as the third leaf developed. For ΦPSII, the QTL-2 was below the

threshold level irrespective of the temperature regime or leaf.

3.4 Discussion The main focus of this study was to understand the physiological and genetic basis of chilling

tolerance of photosynthesis. Previous studies identified several QTLs for chilling tolerance of

photosynthesis in maize seedlings (Fracheboud et al., 2004; Jompuk et al., 2005). The major

QTL for chilling tolerance of photosynthesis was located on chromosome 6 (bin 6.07); it

explained 37.4 % of the phenotypic variance in the chronic photoinhibition at low temperature

and was significantly involved in the expression of other traits, such as minimal fluorescence

of dark-adapted leaves (Fo), operating quantum efficiency of PS II (ΦPSII), carbon exchange

rate (CER) and accumulation of shoot dry matter (Fracheboud et al., 2004). However, this

QTL did not have a significant effect on flowering or yield parameters (Leipner et al., 2008).

For an in-depth analysis of the genetic and physiological basis of this QTL, it is required

necessary to develop a population segregating only for this QTL. In the present study, the

introgression of the chilling tolerant allele from ETH-DH7 at this QTL into the chilling

sensitive line ETH-DL3 was accomplished by marker assisted selection (MAS). Theoretical

studies have shown that the potential gain from MAS depends on the genetic distance

between the QTL and the linked molecular marker(s), the number of markers used per QTL

for MAS and the relative size of the phenotypic effect of the QTL (Bouchez et al., 2002;

Kraja and Dudley, 2000; Lander et al., 1987). In the present study, three SSR markers, namely

bnlg1740, linked to QTL as per LOD score, and umc1859 and umc1653, which are located on

Chapter 3

25

the right- and left-hand sides of the confidence interval (Fracheboud et al., 2004; Jompuk et

al., 2005), were useful for selecting the target QTL and for verifying the integrity of the QTL

during backcrossing. Numerous studies also demonstrated a successful MAS program by

taking the QTL confidence interval into account (Bouchez et al., 2002; Singh et al., 2001;

Thabuis et al., 2004). Marker assisted background selection to cull out non-target QTLs from

ETH-DH7 and to select a maximum of the recurrent parental genome were successfully

achieved in the BC2F1 and BC3F1 progenies. Previous theoretical (Bouchez et al., 2002; Hillel

et al., 1990; Visscher et al., 1996) and experimental (Bouchez et al., 2002; Ragot et al., 1995;

Tajima et al., 2002; Thabuis et al., 2004) studies have shown that background selection is an

efficient method.

QTL detection by means of single factor ANOVA and composite interval mapping

demonstrated that the introgressed segment of chromosome 6 of the chilling tolerant line

ETH-DH7 into the chilling sensitive line ETH-DL3 was associated with a significant increase

in Fv/Fm, ΦPSII, Fv'/Fm', CER and SPAD and a decrease in Fo in three backcross populations.

This shows a stable QTL expression of all the traits (with the exception of CER in BC2F1)

over backcross generations as was also shown in other studies (cf. Chaïb et al., 2006; Mutlu et

al., 2005). Discrepancy in QTL expression for CER in the BC2F1 population was attributed to

an experimental error or QTL × genetic background interaction. Thus, the present results and

those of previous studies (Fracheboud et al., 2004; Jompuk et al., 2005) support the location

of the QTL on chromosome 6 (bin 6.07) and its pleiotropic nature; it simultaneously controls

Fv/Fm, Fo, ΦPSII, Fv'/Fm', CER and SPAD. As described in Chapter 2, an additional QTL was

identified for Fv/Fm and Fo in the introgressed region of chromosome 6. This QTL was not

identified in previous studies (Fracheboud et al., 2004; Jompuk et al., 2005), probably due to

the insufficient number of markers in this region.

The significant reduction in Fv/Fm in the maize seedlings is a well documented in plants under

severe stress. The decrease in Fv/Fm is a measure of the decline of the potential maximum

quantum yield of PS II (Björkman and Demmig, 1987; Genty et al., 1989). This decline in

Fv/Fm in genotypes that do not carry the chilling tolerance allele from ETH-DH7 at bin 6.07

suggests that the integrity of PS II was altered and indicates photoinhibition of photosynthesis

(Long et al., 1994). The decrease in the Fv/Fm of genotypes without ETH-DH7 allele at both

QTLs was due to an increase in Fo which may have been caused by one of the following: a) a

functional dissociation of the antenna complex from the PS II reaction center (Armond et al.,

1978; Kitao and Lei, 2007), b) damage to the D1 protein of the PS II reaction center resulting

in an impairment of electron transport from QA to QB (Gilmore et al., 1996), or c)


26

chlororespiration, a non-photochemical reduction on QA by reducing equivalents supplied in

the dark from a stromal pool of NAD(P)H (Horváth et al., 2000). The increase in Fv/Fm due to

the ETH-DH7 allele was associated with an increase in the operating quantum efficiency of

PS II (ΦPSII) at QTL-1. The increase in ΦPSII was due to an increase in Fv'/Fm' whilst qP was

not affected. This indicates a higher efficiency of capture of excitation energy by open PS II

reaction centers without a change in the redox state of QA in the genotypes holding the ETH-

DH7 allele. Moreover, the low SPAD and CER values of chilling sensitive parent and chilling

sensitive genotypes of backcross populations also reflected a disturbance in the assembly of

the photosynthetic apparatus. The favorable allele in the chilling tolerant parent ETH-DH7

and in the chilling tolerant genotypes of the BC3F2 population at QTL-1 seems to be involved

in the development of a functional photosynthetic apparatus at suboptimal temperature or in

its protection. The QTL effects were not detected for leaf greenness at this locus in previous

studies (Fracheboud et al., 2004; Jompuk et al., 2005). On the one hand, this may be due to

high phenotypic variance caused by multiple genomic regions, which differ between

genotypes of the F2:3 population since several segregating QTLs contribute to phenotypic

differences between them. In contrast, in a NIL population, the phenotypic variation observed

between genotypes can be assigned directly to the distinct genomic regions introgressed in an

otherwise similar genetic background (Nadeau et al., 2000). On the other hand, the number of

QTLs detected in common mapping populations for the same trait can vary considerably

among experiments (Kearsey and Farquhar, 1998; Tanksley and Nelson, 1996) and is often

underestimated. The analysis of NILs carrying small introgressions of a donor genome, for

example, revealed 23 QTLs for single trait in an introgression line population of Lycopersicon

pennellii (Eshed and Zamir, 1995), whereas only four to 13 QTLs were detected when

segregating populations were used (Bernacchi et al., 1998b; Saranga et al., 2004; Tanksley

and Nelson, 1996). Similarly, it was found that NILs had significant affects on traits that were

undetected in the previous BC3 population from which the NILs were developed (Bernacchi et

al., 1998a; Bernacchi et al., 1998b). The results of the current study support the latter

observations.

Many QTL mapping studies have been limited with regard to the performance of a trait

observed at a fixed time point or stage of ontogenesis. Such a QTL mapping estimates the

effects of QTL accumulated from the beginning of ontogenesis to the time of observation, but

the development of trait involves differential activity of many related QTLs. This means that

the dynamics of expression of different QTLs may differ during trait development, even

though they may have the same end effect. To this end, an experiment was carried out to

Chapter 3

27

determine the genetic and physiological functions of the QTL region on chromosome 6 in

different leaves and under different temperature regimes. The results showed that the

expression of QTL-1 and QTL-2 was not detected in the first and second leaves, but it was

expressed in the third leaf. A similar response was found for the major QTL for ΦPSII, which

was located on chromosome 3, in the Ac7643 × Ac7729 mapping population (Fracheboud et

al., 2002). The differential expression of these QTLs in different leaves may be due to the

different developmental history of the leaves. While the first leaf was fully developed at

optimal temperature, the second leaf grew in both temperature conditions; it achieved

approximately 50 % of its final size at 25 °C. The development of the third leaf and its

complete expansion occurred under suboptimal temperature. The differential expression of

QTL may be due to differences in the growth rate and the expansion of leaf as a result of the

temperature decrease in the root zone where the shoot apex is located (Engels, 1994; Stone et

al., 1999). The chilling induced growth retardation of leaves seems to be caused by an

increase in the duration of the cell cycle and by a reduction in cell production (Rymen et al.,

2007). Furthermore, differences in the photosynthetic performance of the genotypes are

smaller when the plants were grown at 25 °C and transferred directly to the cold than when

plants grew at 15 °C (Fracheboud et al., 1999; Fracheboud et al., 2002). Thus, significant

effects of QTL in the third leaf only indicate that both QTLs play a major role in the

development of the photosynthetic apparatus under suboptimal temperature.

Finally, it is necessary to point out that the concept of QTL expression studied here was only

at the level of the phenotype. Revealing its relationship to the molecular or biochemical

process is a future task for developmental quantitative genetics. Based on results of such a

study the dynamics of QTL expression at the molecular or biochemical level could be

deduced.

Characterization of NILs

28

4 Characterization of the photosynthetic apparatus of near isogenic lines (NILs) for a QTL related to chilling tolerance of photosynthesis

4.1 Introduction Suboptimal temperature is particularly deleterious in the early growth stages of maize. It

induces many physiological, biochemical and molecular responses in which photosynthesis is

one of the primary physiological targets (Baker and Nie, 1994; Fryer et al., 1998; Leipner et

al., 1999; Marocco et al., 2005). Experimental results indicated that suboptimal temperature

has a negative effect on the development of the photosynthetic apparatus (Nie and Baker,

1991). Years of selective breeding have led to the emergence of genotypes exhibiting varying

degrees of adaptation to temperate climate. However, this was achieved mainly by shortening

the plant's life cycle while the reasons for the varied chilling-sensitivities of the individual

selected maize lines are poorly understood.

Several QTLs for chilling tolerance in maize, mainly with respect to photosynthesis have been

identified (Fracheboud et al., 2002; Fracheboud et al., 2004; Jompuk et al., 2005). To study

the genetic and physiological basis of these QTLs in more detail, it is advantageous to

develop lines that differ for a single QTL and are identical for the remaining genome. Such

near isogenic lines (NILs) not only provide a better estimate of the effect of the single QTL

alleles, but also serve as a starting point for unraveling the underlying functional genes and

may be useful for positional cloning (Frary et al., 2000; Fridman et al., 2000; Salvi and

Tuberosa, 2005). They have the advantage is that the genetic background noise is eliminated,

giving the QTL the characteristic of a qualitative gene. Approaches utilizing NILs to

accelerate QTL discovery, validation and germplasm improvement have been proposed

(Kaeppler et al., 1993; Saranga et al., 2004; Tuinstra et al., 1997). In addition, the availability

of NILs for specific QTL effects on a number of traits facilitates the elaboration of models

and hypotheses on their cause-effect relationships (Tuberosa et al., 2002a).

The development of near isogenic lines (NILs) requires a controlled program of backcrossing,

ensuring that only the target QTL is present while the rest of the genome is inherited from the

recurrent parent. Phenotypic selection for good agronomic performance has always been

practiced along with backcross selection (Bravo et al., 2003). Genotypic selection by

monitoring the parental origin of alleles using molecular markers throughout the genome

during backcrossing was originally proposed by Young and Tanksley (1997) and was later

Chapter 4

29

referred as background selection (Bouchez et al., 2002). Screening plants with molecular

markers allows a controlled backcrossing program, and it enables to efficiently select against

the donor genome (Bouchez et al., 2002; Tajima et al., 2002; Thabuis et al., 2004).

In the previous chapter (Chapter 3), the effect of the major QTL region for chilling tolerance

of photosynthesis on chromosome 6 (bin 6.07) was confirmed by using a BC3F2 population.

This study, aimed at developing NILs for this chromosomal region in order to assess possible

effects of the introgressed segment on chilling tolerance. As a consequence, it was focused to

investigate the physiological mechanism underlying the QTL and finally to investigate the

physiological impact of the favorable QTL allele on chilling tolerance of seedlings at

suboptimal temperature under controlled as well as under field conditions.

4.2 Materials and Methods 4.2.1 Plant material Near isogenic lines (NILs) to ETH-DL3 were developed from selected BC3F2 lines of the

QTL mapping population (see Chapter 3). The SSR markers bnlg1740, bngl1139, umc1653

and umc2324 were used to select for the donor parental (ETH-DH7) allele in a homozygous

condition in the target region of chromosome 6 (bin 6.07) as well as to select against ETH-

DH7 in non target region using additional 35 SSR markers. The selected lines were selfed

twice; two NILs, namely NIL152 and NIL146 were recovered; they contained the target

region including QTL-1 and QTL-2 and the recurrent parental (ETH-DL3) alleles at 59 co-

dominant SSR markers used for background selection. The maize genotypes ETH-DH7 and

ETH-DL3 and their two NILs (NIL152 and NIL146) were used for this study.

4.2.2 Growth conditions Plants were grown in growth chambers (PGW36, Conviron, Winnipeg, Canada) in 0.75 l pots

containing a commercial mixture of soil, peat and compost (Topf und Pikiererde 140, Ricoter,

Aarberg, Switzerland). The seeds germinated and grew until the first leaf stage at 25/22 °C

(day/night), 12 h photoperiod with a light intensity of 400 µmol m-2 s-1 and a relative humidity

of 60/70 % (day/night). About six days after sowing, when the first leaf was fully emerged,

half of the seedlings were transferred to suboptimal growth temperature (15/13 °C, day/night)

for 15 days until the third leaf was fully developed; the other half was kept at optimal

temperature (25/22 °C) for seven days until they reached the same physiological age. The


30

plants were watered and fertilized with half-strength Wuxal nutrient solution (0.2 %) as

required.

Furthermore, the plant material was grown under field conditions at the experimental station

of the Institute of Plant Sciences of the ETH in Eschikon near Zurich (47°26' N, 8°40' E and

550 m above sea level) in 2007. Seedlings were grew until the first leaf stage in growth

chambers (PGW36, Conviron, Winnipeg, Canada) at 25/22 °C (day/night) temperature, 12 h

photoperiod with a light intensity of 400 µmol m-2 s-1 and a relative humidity of 60/70 %

(day/night). Six day old seedlings were transferred to the field on 3 May 2007. Twenty

seedlings per genotype were planted in two rows. The distance between the rows was 75 cm

and the distance between the plants in the rows was 15 cm. The soil was an Eutric Cambisol

(FAO classification) with a clay loam (CL) texture and a low content of organic matter (3%)

(Richner et al., 1996).

4.2.3 Light response curves of gas-exchange and chlorophyll fluorescence parameters under controlled conditions

To obtain light response curves of parental lines and NILs, the carbon exchange rate and the

chlorophyll fluorescence were monitored with a portable photosynthesis system (LI-6400)

equipped with a LI-6400-40 pulse-amplitude modulation fluorometer (LI-COR, Lincoln, NE,

USA). The maximum quantum efficiency of PS II primary photochemistry (Fv/Fm) and dark

respiration were determined after 15 min of dark adaptation. After determining of Fv/Fm and

dark respiration, plants adapted to the light for five minutes at the lowest light level intensity.

At each light intensity, the leaf adapted for 5 to 8 minutes to the new light intensity until the

variation in ΔCO2 + ΔH2O + Δflow was less than 0.2 %.

The maximum fluorescence in the dark-adapted (Fm) and in the light-adapted (Fm') state was

determined by applying a 0.8 s saturating flash (> 8000 µmol m-2 s-1). The Fo' was calculated

according to the equation of Oxborough and Baker (1997). The operating quantum yield of

photosystem II photochemistry (ΦPSII) was calculated according to Genty et al. (1989). The

quantum efficiency of dissipation by down-regulation was calculated as ΦNPQ = 1 - ΦPSII -

1/(Fm/Fm' + (((Fm'-F ')Fo') / ((Fm'-Fo')F ')) × (Fm/Fo-1)), and the quantum efficiency of other

non-photochemical losses (non-light induced, basal or dark, quenching process) as ΦNO =1 -

ΦPSII - ΦNPQ (Kramer et al., 2004). For nomenclature of the fluorescence states and parameters

see Rosenqvist and van Kooten (2003). The light response curves were fitted according to

Norman et al. (1992) using the Photosynthesis program V.1.0 (LI-COR).

Chapter 4

31

4.2.4 Measurement of physiological traits under field conditions Photosynthesis and chlorophyll fluorescence parameters were measured on fully developed

third leaves of randomly selected seedlings using infrared gas analyzer equipped with a pulse-

amplitude modulation fluorometer (LI-6400, LI-COR, Lincoln, USA). The light intensity was

400 µmol m-2 s-1, the temperature of the sample chamber was 15 °C and humidity and CO2

concentration were the same as the ambient conditions. The quantum yield of electron

transport at PS II (ΦPSII), the efficiency of open PS II reaction centers (Fv'/Fm'), photochemical

quenching factor (qP) and carbon exchange rate (CER) were measured when the variation of

ΔCO2 + ΔH2O + Δflow was below 0.2 %.

The maximum quantum efficiency of PS II (Fv/Fm) was determined with a PAM-2000 (Walz,

Effeltrich, Germany) fluorometer after 30 min dark adaptation by applying a one-second

saturation flash (> 8000 µmol m-2 s-1).

To measure greenness of the leaves, three measurements were made n the middle section of

the third leaf and averaged for each plant using a Minolta SPAD-502 chlorophyll meter

(Minolta Corporation, Ramsey, Japan).

4.2.5 Data analysis and statistic Mean and standard deviation were estimated for each genotype. Statistical comparison of the

means was done according to the Tukey-Kramer test where the P value was less than 0.05 and

was considered to be significant.

4.3 Results 4.3.1 Genotypic differences in the light response of CO2 assimilation, PS II

functioning and energy dissipation The maximum quantum efficiency of PS II primary photochemistry (Fv/Fm) showed no

significant differences between the genotypes at optimal temperature (Table 4.1). However,

significant differences among the genotypes were observed in seedlings that developed at

suboptimal temperature (15 °C). In the chilling sensitive genotype ETH-DL3, the Fv/Fm

declined most by 52%, whereas the Fv/Fm of NIL152, NIL146 and ETH-DH7 declined by 22,

29 and 16% respectively, when grown at 15 °C in comparison to seedlings that grown at

25 °C.


32

Figure 4.1 shows the light response curves for photosynthesis constructed from data of

genotypes grown at optimal (25 °C) and at suboptimal temperature (15 °C). The parameters,

which define the fitted curves, are summarized in Table 4.2. The rate of CO2 assimilation

(CER) of the genotypes grown at optimal temperature (25 °C) was nearly identical.

Significant differences were observed when genotypes were grown at 15 °C and at light

intensities higher than 1200 µmol m-2 s-1 (for statistical analysis see Annex Table A.1). The

chilling tolerant parent ETH-DH7 and NIL152 exhibited higher rates of CO2 assimilation than

the chilling sensitive parent ETH-DL3. Substantial decrease in the initial slope (Φ), the

convexity (Ø) of the light response curve and the light-saturated rate of photosynthesis (Pmax)

were observed at 15 °C for all genotypes. The high Φ and the low Ø for NIL152 compared to

the other genotypes grown at 15 °C may have been resulted from a bias in the fitting of three

parameters the light response curve to data that resided mainly in the lower light intensity

range. The chilling tolerant parent ETH-DH7 and NIL146 had higher absolute values for all

the three parameters at 15 °C. The operating efficiency of the light harvesting of PS II (ΦPSII)

was affected by genotype and light intensity. Under optimal temperature (25 °C),

Table 4.2: Parameters of the photosynthetic light response curve fitted to data from 25 °C and 15 °C grown plants. The fitting was conducted based on the average values for each genotype.

Genotype Trait Growth temperature ETH-DH7 NIL146 NIL152 ETH-DL3

Φ 25 °C 0.065 0.073 0.067 0.065 15 °C 0.033 0.023 0.063 0.019

Ø 25 °C 1.491 1.249 1.818 1.252 15 °C 1.093 1.052 0.515 1.027

Pmax 25 °C 31.0 39.3 28.8 44.1 15 °C 10.7 7.1 15.0 2.5

Table 4.1: Fv/Fm of genotype grown at 25 °C and 15 °C. Values are means ± SD of four replications. Significant differences (P ≤ 0.05) for pair-wise comparisons within a growth temperature are indicated by different letters.

Genotype Growth temperature ETH-DH7 NIL146 NIL152 ETH-DL3

25 °C 0.758 ± 0.023 a 0.761 ± 0.024 a 0.748 ± 0.012 a 0.782 ± 0.001 a

15 °C 0.658 ± 0.034 a 0.541 ± 0.069 b 0.589 ± 0.063 a 0.382 ± 0.036 c

Chapter 4

33

PPFD (µmol m-2 s-1)

0 400 800 1200 1600

ΦN

PQ

0.0

0.2

0.4

0.6

PPFD (µmol m-2 s-1)

0 400 800 1200 1600

ΦN

O

0.0

0.2

0.4

0.6

CER

(µm

ol m

-2 s

-1)

0

10

20

30

40

ΦPS

II

0.0

0.2

0.4

0.6

0.8F v'/

F m'

0.0

0.2

0.4

0.6

0.8

q P

0.0

0.2

0.4

0.6

0.8

1.0

ETH-DH7NIL146NIL152ETH-DL3

25 / 15 °C

Figure 4.1: Light response curves of carbon exchange rate (CER), operating quantum efficiency of PS II (ΦPSII), efficiency of open PS II reaction centers (Fv'/Fm'), photochemical quenching factor (qP), quantum efficiency for dissipation by down regulation (ΦNPQ), and quantum efficiency of other non-photochemical losses (ΦNO) in the chilling tolerant line ETH-DH7, chilling sensitive line ETH-DL3 and the near isogenic lines NIL152 and NIL146. Values are means ± SD of four replications.


34

all genotypes were having nearly equal ΦPSII values at low light intensity (Figure 4.1).

However, at higher light intensity (≥ 800 µmol m-2 s-1), the chilling sensitive parent ETH-DL3

had significantly higher values of ΦPSII compared to ETH-DH7 and NIL152. The higher

operating efficiency of light harvesting of PS II (ΦPSII) at higher light intensity was due to

higher quantum efficiency of open PS II centers (Fv'/Fm') and photochemical factor (qP). The

energy dissipation via regulated mechanism (ΦNPQ) and non-regulated processes (ΦNO) were

nearly the same in all the genotypes grown at optimal temperature. However, at suboptimal

temperature (15 °C), the chilling tolerant parent ETH-DH7 and the near isogenic lines

NIL152 and NIL146 had significantly higher ΦPSII values than ETH-DL3 at all light

intensities (Figure 4.1). The decrease in the ΦPSII of ETH-DL3 was associated with a decrease

in the quantum efficiency of open PS II centers (Fv'/Fm') as well as in the photochemical

factor, qP. The excess of absorbed light energy in the chilling sensitive parent ETH-DL3 was

dissipated to a significantly greater extent by non-regulatory process (ΦNO) than in the chilling

tolerant parental line ETH-DH7 and in the near isogenic lines NIL152 and NIL146. The ΦNPQ

was higher at 15 °C than at 25 °C at all the light intensities. However, there were no

significant differences among the genotypes (see also Annex Table A.1).

4.3.2 Field experiment Under field conditions, the seedlings experienced a mean temperature of 13.5 °C for 15 days

between the planting of seedlings and time of measurements. During the time of measurement

(18 May 2007), the temperature was about 13 °C. At, the leaves of the s this temperature,

seedlings had a low Fv/Fm, ΦPSII, Fv'/Fm', qP and CER (Table 4.3), which were comparable to

Table 4.3: Chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, Fv'/Fm', qP), carbon exchange rate (CER) and leaf greenness (SPAD) of two near isogenic lines and the parental lines ETH DH7 and ETH-DL3 under field conditions. The data are the mean values ± SD of five replications. Significant differences (P ≤ 0.05) for pair-wise comparisons are indicated by different letters.

Trait ETH-DH7 NIL146 NIL152 ETH-DL3

Fv/Fm 0.696 ± 0.020 a 0.525 ± 0.014 a 0.689 ± 0.015 a 0.382 ± 0.060 b

ΦPSII 0.159 ± 0.042 a 0.141 ± 0.009 ab 0.169 ± 0.029 a 0.113 ± 0.024 b

Fv'/Fm' 0.234 ± 0.044 a 0.214 ± 0.013 ab 0.251 ± 0.033 a 0.171 ± 0.032 b

qP 0.668 ± 0.058 a 0.658 ± 0.018 a 0.673 ± 0.043 a 0.659 ± 0.025 a

CER 03.9 ± 1.2 a 03.2 ± 0.4 ab 04.2 ± 0.7 a 02.1 ± 0.8 b

SPAD 25.4 ± 0.9 a 18.0 ± 0.6 ab 23.3 ± 2.2 a 12.6 ± 1.2 b

Chapter 4

35

the seedlings grown at 15 °C under controlled conditions (Table 4.1, Figure 4.1). With the

exception of qP, all the other traits were significantly different with higher values for ETH-

DH7 and NIL152 and low values for ETH-DL3. NIL146 was in between these two groups

(Table 4.3).

4.4 Discussion In the present study, the development of near isogenic lines (NILs) was accelerated by marker

assisted selection. Furthermore, the sequential genotyping approach used here considerably

lowered the costs of experiment and proved to be successful for QTL transfer as was also

shown for major genes (Frisch and Melchinger, 2001). The two NILs developed here

recovered the recipient genotype at all selected loci but contained the chromosomal segment

carrying QTL(s) for chilling tolerance of photosynthesis from the tolerant donor parent, ETH-

DH7. Since more and more recombination events accumulate over generations, the number of

donor segments spread over the genome increases as their length decreases. Therefore, an

increasing number of markers was needed to control all the potential recombinations during

the latter generations of backcross breeding. A higher marker density in the BC3F1 and BC3F2

generation was useful in eliminating these donor segments more rapidly as was also

demonstrated by Hospital et al. (2002). Homozygous alleles of the recurrent parent at all the

SSR loci indicated complete recovery of the recurrent parental genome.

In the previous QTL analyses of the ETH-DH7 × ETH-DL3 population in the F2:3

(Fracheboud et al., 2004; Jompuk et al., 2005) and BC3F2 generation (Chapter 3) it was found

that the major QTL on chromosome 6 was associated with changes in Fv/Fm suggesting a

lower photosynthetic efficiency of lines carrying the allele from ETH-DL3. The change in

Fv/Fm, which was accompanied with an opposite change in Fo, indicates structural alterations

in the photosystem II. In the above studies, the photosynthesis and chlorophyll fluorescence

were analyzed only at the light intensity of 400 µmol m-2 s-1, at which plants were grown,

thus, it was not known whether the QTL on chromosome 6 affects the dark-reaction of

photosynthesis as well. In order to throw light on this, it is necessary to analyze the plant

material by conducting light response curves. However this is not feasible in mapping

populations, NILs were developed, which carry the allele of the chilling tolerant parent ETH-

DH7 at the target locus while the remaining genome was recovered completely from the

recurrent parent as mentioned above. The light response curves of the parental lines and the

NILs revealed that the efficiency as well as the capacity of photosynthesis were affected by


36

the growth temperature. Suboptimal temperature resulted in inhibition of the photosynthetic

carbon metabolism of all the genotypes, but a more substantial reduction was found in ETH-

DL3 than for chilling tolerant parent ETH-DH7 and in the NILs. Furthermore, the results

demonstrated that suboptimal temperature was associated with a decrease in the values of all

the parameters that describe the light response curve. This indicates that the efficiency as well

as the capacity of photosynthesis were affected by suboptimal growth temperature. Such

changes are symptomatic of chilling induced damages, either due to perturbation in the D1

protein turn-over or due to developmental effects on the photosynthetic apparatus (Baker and

Ort, 1992; Stirling et al., 1993). Chilling-induced decrease in the of Φ and Pmax of maize have

been well documented (Baker et al., 1988; Long et al., 1983; Stirling et al., 1991). The higher

photosynthetic efficiency of the NILs compared to ETH-DL3 was due to a high efficiency of

the operating quantum efficiency of PS II (ΦPSII), which in turn was due to both higher qP and

higher Fv'/Fm'. This indicates that the NILs maintained a higher fraction of open PS II reaction

centers, which had a higher quantum efficiency. Consequently, a greater percentage of

absorbed quanta was converted into chemical energy. The remaining quanta were dissipated

as heat (and fluorescence). Since the sum of the quantum yields of photochemistry, heat

dissipation and chlorophyll fluorescence are unity, it was expected that genotypes with a low

ΦPSII would be characterized by a higher amount of heat dissipation. In this study, the lake

antenna model of Kramer et al. (2004) was employed to unravel the different compounds of

the dissipative mechanisms. Although suboptimal growth temperature induced energy

dissipation via the non-photochemical pathway (ΦNPQ), there were no significant differences

in ΦNPQ between genotypes despite differences in ΦPSII. However, seedlings of the chilling

sensitive genotype (ETH-DL3) grown at suboptimal temperature, were characterized by a

greater loss of energy (ΦNO), which was not attributed to down-regulation, indicating chilling-

induced constitutive structural alterations of the photosynthetic apparatus. Since the ΦNO

values of NILs and in ETH-DH7 was similar, it is concluded that the gene(s) behind the

QTL(s) on the long arm of chromosome 6 are directly or indirectly responsible for the

assembly or disassembly of the photosynthetic apparatus. This seems at first to in contrast

with the finding that this QTL also had an effect on the dark reaction of photosynthesis as

shown by the higher photosynthetic capacity of the NILs compared to ETH-DL3. Low growth

temperature decreases the amount of photosynthetic apparatus in a given leaf area (Baker and

Nie, 1994), which results in a lower ratio of photosystems to chlorophyll (Nie and Baker,

1991). The decrease in photochemical reaction centers per unit chlorophyll will inevitably

result in reductions in the quantum yield of ATP and reductants production by thylakoids and

Chapter 4

37

in a contribution to the decreased efficiency of light utilization for carbon assimilation (Baker

and Nie, 1994).

Another important finding of the present study is the confirmation of the effect of the QTL

under field conditions. The NILs exhibited a higher level of chilling tolerance in the field than

the chilling sensitive parent, ETH-DL3. The leaves of the NILs maintained a higher Fv/Fm,

ΦPSII, Fv'/Fm', CER and leaf greenness. This demonstrated that the introgressed allele from

ETH-DH7 at this QTL contributed to the development of functional chloroplasts and their

protection also under field conditions was expected from the previous QTL analysis of the F2:3

population (Jompuk et al., 2005).

Based on the above results it is clear that the introgression of the favorable allele from ETH-

DH7 into the near isogenic lines at chromosome 6 (bin 6.07) contributed towards the

development of a functional photosynthetic apparatus with a high quantum efficiency of PS II

for utilization and dissipation of light under suboptimal growth temperature. This may protect

the photosynthetic apparatus from further adverse effects of low temperature and might be

responsible for the higher photosynthetic activity of genotypes carrying the allele of ETH-

DH7 on chromosome 6. As was found in virescent mutants of maize, disturbances in the

development of the photosynthetic apparatus also have an effect on the photosynthetic activity

(on a chlorophyll basis) under non-limiting light conditions (Chollet and Paolillo, 1972).

Thus, the genes underlying the QTL must be involved in the development of the

photosynthetic apparatus and its protection at low temperature.

QTL analysis in a dent × flint mapping population

38

5 Validation of the major QTL for chilling tolerance of photosynthesis in a dent × flint maize population

5.1 Introduction A major limitation of the applications of identified QTLs in breeding programs is the lack of

consistency of the effects of QTL in different genetic background and/or across environments

(Kandemir et al., 2000; Lecomte et al., 2004). It is particularly relevant when evaluating QTL

effects for abiotic stress which are largely affected by many loci as well as by several

environmental factors. Thus, it is important to confirm QTL effects before implementing

MAS in breeding programs (Melchinger et al., 1998) or attempting to clone a QTL (Mackay,

2001). An accurate evaluation of the effects of a QTL is also beneficial before its cloning, a

resource-demanding undertaking, which can be facilitated when the effects of the QTL show

limited interactions with the environment (Salvi and Tuberosa, 2005). Furthermore, a QTL

with consistent effects across genetic backgrounds has a greater breeding value, particularly

when the favorable allele is not present in the elite germplasm. Results from QTL

confirmation experiments will generate new knowledge about the limitations and strengths of

using QTL in the MAS program. The plethora of QTL data will aid programs to improve

plants and future genomic studies but only when identified the QTL are proven to be real.

The conservation of QTL in different mapping populations exposed to similar stress

conditions indicates the importance of specific genomic regions in the stress response. Several

QTLs for chilling tolerance of photosynthesis in maize were identified in recent studies

(Fracheboud et al., 2002; Fracheboud et al., 2004; Jompuk et al., 2005; Pimentel et al., 2005).

In the ETH-DL3 × ETH-DH7 population, which is the object of this study, the major QTL for

chilling tolerance of photosynthesis was identified on the long arm of chromosome 6

(Fracheboud et al., 2004; Jompuk et al., 2005). Furthermore, a QTL for the minimal

chlorophyll fluorescence (Fo) was found near to this position in seedlings of the IBM302

mapping population (O. Guerra-Peraza, ETH Zurich, personal communication). However,

these QTLs on chromosome 6 were not co-localized with any other QTL for similar traits,

neither in the Ac7643 × Ac7729 (Fracheboud et al., 2002) nor in the W6786 × IL731A

mapping population (Pimentel et al., 2005). To obtain additional information about the

extension of this QTL within the maize germplasm, it is necessary to study additional

mapping populations. However, it will take considerable amount of time to develop,

genotype, and adequately phenotype additional mapping populations. An alternative to the

Chapter 5

39

conventional mapping of an entire population is the bulked segregant analysis, which is a

rapid method for confirming the presence of QTL quickly in other genetic background by

using two bulks of phenotypic extremes for the trait (Michelmore et al., 1991).

In view of these considerations, the present study was undertaken to evaluate the effects of the

QTL on chromosome 6 (bin 6.07) in an alternate F2 population derived from the cross ETH-

DL7 × ETH-FH6 by employing DNA pooling and selective genotyping of selected

individuals.

5.2 Materials and Methods 5.2.1 Plant material One hundred and seventy nine F2 lines, derived from a cross between ETH-DL7 and ETH-

FH6 were used in this study. The two parental lines were originally obtained by divergent

selection from a Swiss dent and flint maize breeding population using chlorophyll

fluorescence as the selection tool (Fracheboud et al., 1999). ETH-FH6 is a chilling tolerant

line of the flint maize type, which maintains higher photosynthetic activity at suboptimal

growth temperature, whereas ETH-DL7 is of the dent type and the photosynthetic activity of

which is poor at suboptimal temperature (Hund et al., 2005). The initial screening of 19

inbred lines, which were derived by divergent selection from Swiss dent and flint maize

breeding populations (Fracheboud et al., 1999) by means of SSR markers linked to the major

chilling tolerance QTLs located on chromosomes 2 and 6 (Fracheboud et al., 2004; Jompuk et

al., 2005), revealed high polymorphism between ETH-DL7 and ETH-FH6 at these loci.

Hence, the population based on these two lines was selected to confirm QTL for chilling

tolerance of photosynthesis in the present study.

5.2.2 Growth conditions Seedlings were germinated in 0.75 l pots containing a commercial mixture of soil, peat and

compost (Topf und Pikiererde 140, Ricoter, Aarberg, Switzerland) and grew at 25/22 °C

(day/night) temperature until the first leaf stage. Six days after sowing, the seedlings were

transferred to suboptimal growth temperature (15/13 °C, day/night) for 15 days. Starting from

the dark period between day 15 and 16, the temperature was decreased to 5/5 °C (day/night)

for two days followed by a recovery treatment for two days at 25/23°C (day/night); the

recovery was started at the beginning of the light period. Throughout the experiment, the light

intensity was 400 µmol m-2 s-1, the photoperiod of 12 hours and the relative humidity 60/70 %


40

(day/night). The plants were watered and fertilized with half-strength Wuxal nutrient solution

(0.2 %) as required. For further details see Chapter 3.

5.2.3 Measurement of chlorophyll fluorescence parameters The chlorophyll fluorescence parameters: minimum fluorescence (Fo), maximum fluorescence

(Fm), variable fluorescence (Fv) and maximum quantum efficiency of PS II (Fv/Fm) were

monitored with a PAM-2000 fluorometer (Walz, Effeltrich, Germany) after 30-60 min dark

adaptation. For details see Chapter 3.

5.2.4 DNA isolation and SSR marker assay DNA was isolated from 25 day old plants according to the method of Dellaporta et al. (1983)

(see also Chapter 3). The PCR using primers of publicly available SSR markers was

conducted as described in Chapter 3.

5.2.5 Bulk segregant analysis Phenotypic extreme plants were selected based on average ranking based on all the

measurements of the trait. This was done by ranking each Fv/Fm measurement. The plants

with highest and lowest average ranking based on all the measurements were considered

phenotypic extremes. Equal amounts of DNA from the phenotypic extremes were used to

constitute the bulks. The size of the bulks differed with 5, 10 and 15 individuals per bulk

(Govindaraj et al., 2005).

5.2.6 QTL mapping The genetic map was constructed using Mapmaker 3.0 (Lander et al., 1987) with the Haldane

mapping function. Based on this map, QTL analysis was carried out using QTL Cartographer

v.1.17b (Basten et al., 1994; Basten et al., 2003). The method of composite interval mapping

(CIM), model 6 of the Zmapqtl program module, was used to map QTLs and estimate their

effects (Jansen and Stam, 1994; Zeng, 1994), as described in Chapter 3.

Chapter 5

41

5.3 Results 5.3.1 Phenotypic analysis The time course for chlorophyll fluorescence parameters of the parental lines and the F2 plants

of the ETH-DL7 × ETH-FH6 population during growth at 15 °C, exposure to 5 °C and

recovery at 25 °C are presented in Figure 5.1. Throughout the experiment, all the leaves of

Leaf 4Leaf 2

F v/F

m

0.0

0.2

0.4

0.6

0.8

ETH-FH6 F2 populationETH-DL7

Leaf 315 5 25 15 5 25 15 5 25

F o

0.0

0.1

0.2

0.3

0.4

0.5

15

Time after transfer to 15°C (days)

5 10 15

F m

0.0

0.2

0.4

0.6

0.8

10 15

Figure 5.1: Time course of Fv/Fm, Fo and Fm in seedlings of ETH-FH6 (white symbols), ETH-DL7 (black symbols) and of the F2 population (grey symbols) in leaf 2 (circles), 3 (squares) and 4 (triangles) during growth at 15 °C, exposure to 5 °C and recovery at 25 °C. Values are means ± SD of 5 (parental lines) and 179 (F2 plants) replications, respectively.


42

ETH-FH6 had higher Fv/Fm values compared to ETH-DL7. The lower Fv/Fm of ETH-DL7

than ETH-FH6 was due to a higher Fo, while Fm was similar in both genotypes. The decrease

in growth temperature from 15 to 5 °C resulted in a substantial reduction in Fv/Fm in both

parental lines. However, the decrease in the Fv/Fm in ETH-FH6 was less pronounced than in

ETH-DL7, especially in leaves that developed at suboptimal temperature. The decrease in

Fv/Fm during exposure to 5 °C was accompanied by a considerable decrease in Fm and a slight

increase in Fo. The variance analysis revealed significant differences between the two parental

lines, ETH-FH6 and ETH-DL7, in Fv/Fm and Fo, but no significant difference in Fm (data not

shown). The response of Fv/Fm of seedlings of the F2 population to the different temperatures

was similar to that of the chilling tolerant genotype ETH-FH6, indicating that the major QTL

for chilling tolerance of photosynthesis is dominant in this population. Transgressive

segregation occurred in both directions (data not shown) implying that different positive

alleles for Fv/Fm exist in both parental lines and that a recombination of these positive alleles

would produce genotypes with enhanced tolerance to photoinhibition at 5 °C.

5.3.2 Bulk segregant analysis In order to identify polymorphic markers, a total of 300 SSR markers were used to screen the

parental lines, ETH-FH6 and ETH-DL7. Ninety SSR markers were found to be polymorphic

between the two parental lines and were used for the bulk segregant analysis. Among these 90

markers, only eight were putatively discriminating between the bulked extremes. These

markers were located on chromosomes 1, 2, 3 and 6. For an example, Figure 5.2 shows the

result of the bulk segregant analysis with the SSR markers bnlg1740 and umc1859. The

difference between the bulked extremes was clearer when the bulk included five individuals

bnlg1740

ETH

-DL7

ETH

-FH

6

5 10 15

L H L H L H

Bulk of

umc1859

ETH

-DL7

ETH

-FH

6

5 10 15

L H L H L H

Bulk of

bnlg1740

ETH

-DL7

ETH

-FH

6

5 10 15

L H L H L H

Bulk of

umc1859

ETH

-DL7

ETH

-FH

6

5 10 15

L H L H L H

Bulk of

Figure 5.2: Profile of the markers umc1859 (bin 6.06) and bnlg1740 (bin 6.07) for DNA bulks of the 5, 10 and 15 phenotypic extremes for Fv/Fm. L, F2 bulks with low Fv/Fm; H, F2bulks with high Fv/Fm.

Chapter 5

43

rather than 15. Based on the analysis of bulked extremes, the eight identified SSR markers

were used to establish the association between marker and Fv/Fm. The screening of the F2

individuals revealed a significant association between Fv/Fm and the SSR marker bnlg1740

indicating that a major QTL is located close to this position (Table 5.1).

5.3.3 Construction of molecular marker map and analysis of QTL In order to precisely map the QTL around bnlg1740 and to asses its genetic effect, additional

polymorphic markers including, 4 SSR and 1 IDP marker, were used to construct a linkage

Table 5.1: Markers associated with Fv/Fm of the 3rd leaf on day 15 based on one way ANOVA. The critical F value was 3.142.

Marker Allele Fv/Fm ANOVA result

Name Bin Name Count Mean Variance F calc. a P

mmc0011 1.06 HH 10 0.664 0.0019 0.312 0.7329 HL 38 0.666 0.0016 LL 18 0.658 0.0010

umc1306 1.09 HH 12 0.663 0.0027 2.046 0.1376 HL 43 0.659 0.0012 LL 11 0.684 0.0009

umc1797 1.12 HH 16 0.673 0.0007 0.731 0.4850 HL 40 0.663 0.0016 LL 10 0.655 0.0020

dupssr21 2.06 HH 14 0.663 0.0011 0.344 0.7098 HL 27 0.668 0.0018 LL 25 0.659 0.0015

phi127 2.07 HH 12 0.674 0.0014 0.563 0.5719 HL 38 0.661 0.0013 LL 25 0.659 0.0015

bnlg1447 3.03 HH 12 0.639 0.0020 1.973 0.146 HL 42 0.663 0.0019 LL 24 0.664 0.0007

umc1859 6.06 HH 13 0.671 0.0022 2.472 0.0925 HL 34 0.670 0.0011 LL 19 0.647 0.0015

bnlg1740 6.07 HH 20 0.676 0.0007 8.824 0.0004 HL 31 0.671 0.0012 LL 15 0.631 0.0019

a Significant at 1 % confidence level. LL, homozygous F2 lines inheriting allele from ETH-DL7; HH, homo-zygous F2 lines inherited allele from ETH-FH6; HL, heterozygous F2 lines inherited allele from both the parents.


44

map covering a length of 48.4 cM (Figure 5.3). Analysis of the marker and phenotypic data of

179 F2 lines were analyzed by composite interval mapping revealed a QTL for Fv/Fm and Fo.

It is probably located between bnlg1740 and umc1897, 2.8 cM from bnlg1740. The QTL

analysis revealed differential expression of the QTL across the three leaves (Figure 5.4). For

Fv/Fm and Fo in leaves at 15 °C, the QTL was insignificant in the second leaf but was strong

in the third and fourth leaves. The favorable allele (high Fv/Fm and low Fo) was inherited from

ETH-FH6. The QTL was also present for Fv/Fm and Fo in the third and fourth leaf at 5 °C and

25 °C but with a lower LOD score than at 15 °C, especially for Fv/Fm. For Fm, the LOD score

was always below the threshold of 3.5.

5.4 Discussion Prior using QTLs to improve elite germplasm through marker assisted breeding program, it is

necessary to confirm the significance of a QTL in other mapping populations and, thereby, to

identify the genotype carrying the most favorable allele for the trait of interest. With respect

to the latter, it was found that maize of the flint type is characterized by better chilling

tolerance than dent maize (e.g. Dolstra et al., 1988) and, therefore, might contain interesting

alleles to improve the chilling tolerance of maize seedlings. Hence, an attempt was made to

verify and characterize the effects of the major QTL for chilling tolerance of photosynthesis,

which was found to be located on chromosome 6 (bin 6.07) in the ETH-DL3 × ETH-DH7

population, in an independent F2 population derived from the cross of a chilling tolerant flint

line (ETH-FH6) and a chilling sensitive dent line (ETH-DL7). In order to avoid the

genotyping of the entire F2 population with polymorphic markers, a bulk segregant analysis

umc1063

umc1897

bnlg1740

AY109996

umc1653

14.9

13.3

5.2

14.9

Figure 5.3: Segmental map of the QTL region on chromosome 6 of the ETH-DL7 × ETH-FH6 population.

Chapter 5

45

(BSA) was conducted to identify genetic loci associated with photoinhibition during chilling

stress. In this method, the DNA of the phenotypic extremes for Fv/Fm was bulked as reported

by Zhang et al. (1994). A preliminary analysis involved a subset of F2 individuals with

phenotypic extremes; out of eight SSR markers, which differentiated the DNA bulks, only

one SSR maker, namely bnlg1740, significantly co-segregated with Fv/Fm. Nevertheless, the

identification of only one QTL based on BSA approach does not preclude the existence of

other loci of minor effect that BSA was not detected due to its low statistical power

(Missiaggia et al., 2005; Wang and Paterson, 1994). The QTL analysis of the 179 F2

individuals with five molecular markers localized in the putative QTL region confirmed the

15 5 25 15 5 25 15 5 25Leaf 4Leaf 2

F v/Fm (L

OD

, R2 )

0

10

20

30LOD scoreR2 (%)

Leaf 3

F o (LO

D, R

2 )

0

10

20

30

15

Time after transfer to 15°C (days)

5 10 15

F m (L

OD

, R2 )

0

10

20

30

10 15

Figure 5.4: Time course of the LOD score (closed symbols) and the phenotypic variance (R2

in %; open symbols) of the QTL at chromosome 6 for Fv/Fm, Fo and Fm in leaf 2 (circles), 3 (squares) and 4 (triangles) of seedlings of the ETH-DL7 × ETH-FH6 population during growth at 15 °C, exposure to 5 °C and recovery at 25 °C.


46

presence of this QTL near to SSR marker bnlg1740, similar to QTL-1 of the ETH-DL3 ×

ETH-DH7 population (see Chapters 2 and 3). Furthermore, the time course of this QTL

revealed very similar effects as found for QTL-1 in the ETH-DL3 × ETH-DH7 BC3F2

population (Chapter 3). Both populations expressed the QTL only in leaves that developed at

suboptimal temperature; furthermore the QTL was less important in both populations when

seedlings were exposed to more severe chilling/cold stress. Given the similar position and the

behavior of the QTL, it is assumed that the same gene (or cluster of genes) may underlie it.

Since ETH-DL3 and ETH-DL7 were derived from the same breeding population (Fracheboud

et al., 1999), the QTL was expressed in both populations due to a similar defect in the ETH-

DL7 and ETH-DL3 allele. a QTL for a higher chilling sensitivity. On the other hand, common

gene chilling tolerance may present across dent and flint lines, regulating the key step in the

development of functional photosynthetic apparatus under chilling stress conditions.

Whatever the cause, the conservation of this QTL in different mapping populations indicates

the importance of this genomic region for the development of a functional photosynthetic

apparatus at suboptimal temperature. However, this QTL was not found in earlier studies with

Ac7643 × Ac7729 (Fracheboud et al., 2002) and the SL × TH populations (Presterl et al.,

2007). Inconsistent QTL expression among mapping populations may have several reasons.

For example, the QTL for chilling tolerance of photosynthesis may exist in the Ac7643 ×

Ac7729 population but was not detected due to the very low marker resolution of the QTL

region in this population. In the SL × TH population, the traits measured for chilling tolerance

were virtually different from the traits measured in the ETH-DL3 × ETH-DH7 population in

which QTL was detected.

The results of the current study indicates that the SSR marker bnlg1740, which is closely

linked to the QTL on chromosome 6, should enable marker assisted selection for chilling

tolerance of photosynthesis if the correct genetic material is used. Since it was shown that

three markers are sufficient to control the QTL confidence interval by minimizing the risk of

"losing" the target allele (Bouchez et al., 2002), the use of the additional flanking markers,

umc1897 and AY10996, is advisable for marker assisted selection of this QTL. Nevertheless,

other monomorphic markers flanking the QTL in this mapping population may be useful for

screening the major QTL when they are polymorphic in the targeted populations.

Chapter 6

47

6 Comparative mapping and identification of candidate genes for the major QTL for chilling tolerance of photosynthesis

6.1 Introduction Chilling tolerance is a complex trait and seems to be controlled by multiple genes in maize

(Fracheboud et al., 2002; Fracheboud et al., 2004; Hund et al., 2005; Jompuk et al., 2005;

Pimentel et al., 2005; Presterl et al., 2007). Thus far, four mapping populations have been

used to identify the QTLs for chilling tolerance in maize. Co-localization of QTLs related to

chilling tolerance of photosynthesis have been proposed for the centromeric region of

chromosome 2 between the ETH-DL3 × ETH-DH7 and the Ac7643 × Ac7729 mapping

population (Fracheboud et al., 2004). This region on chromosome 2 was also found to be

involved in the chilling tolerance of photosynthesis in a part-exotic maize population (J.

Leipner, ETH Zurich, personal communication). With respect to the major QTL for chilling

tolerance of photosynthesis in the ETH-DL3 × ETH-DH7 population, which is localized on

chromosome 6, co-localization has been identified with the ETH-DL7 × ETH-FH6 population

(Chapter 5). Identification of co-localization not only provide information about the

importance of a QTL across different genetic backgrounds, but is also a good starting point

for identifying positional candidate genes and for narrowing the genetic region in which the

underlying gene(s) may be located, especially when high resolution mapping populations are

used.

Positional cloning of a QTL is a time consuming procedure and success is uncertain. This

procedure usually requires fine scale mapping and near isogenic lines of the individual QTL.

As an alternative, the candidate gene approach is a promising method for QTL cloning

especially when genes are known to control the same trait in other species (Collins et al.,

2008). To reduce the number of potential positional and functional candidate genes, results of

expression studies can be employed. The rationale behind this is that chilling induced genes

may ameliorate chilling stress, which implies that chilling induced genes may be genes for

tolerance to chilling. Various techniques have been used to identify chilling induced genes

and a number of these genes have been isolated from maize (e.g. Anderson et al., 1994;

Nguyen et al., 2009; Prasad et al., 1994).

This chapter described an attempt to exploit new data sources to identify putative candidate

genes underlying the QTL on chromosome 6. This was achieved by anchoring both ETH

Comparative mapping and identification of candidate genes

48

mapping populations to maize physical and sequence maps, and putative candidate genes

were identified based on a publicly available cold stress cDNA library.

6.2 Material and Methods 6.2.1 Comparative mapping The QTL mapping data based on the ETH-DL3 × ETH-DH7 (Chapter 2), the ETH-DL7 ×

ETH-FH6 (Chapter 5) and the IBM302 (B73 × Mo17) populations (O. Guerra-Peraza, ETH

Zurich, personal communication) were used for comparative mapping. To identify the map

region containing overlapping QTLs across populations, the maps were merged with common

anchor markers.

6.2.2 Bioinformatics To identify candidate genes, expressed sequence tags (ESTs), which are located in the QTL

region, were searched on the Gramene Curated AGI FPC Oct 2004 physical map

(http://www.gramene.org). A homology search was made with the obtained ESTs from the

above map against EST sequences from a cold stress cDNA library, which is available at the

National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). This

cDNA library was constructed from seedlings of the chilling tolerant inbred line CO328 (Lee

et al., 2002) exposed to low temperature by the Eastern Cereal and Oilseed Research Centre,

Ottawa, Canada (Singh et al., 2001). In brief, maize seedlings at the 4-leaf stage were exposed

to low temperature in combination with high light intensity (10 °C / 700-800 µmol m-2 s-1) for

4 days and a normalized cDNA library was constructed from mRNA obtained from the cold

stressed plants; furthermore, this cDNA library contained expressed sequence tags (ESTs)

which were obtained from seedlings exposed to gradual cooling down to 5 °C at low light

intensity (125 µmol m-2 s-1).

For ESTs of unknown function, a homology search was made using the basic local alignment

search tool at the NCBI was conducted by employing the blastx algorithm.

6.3 Results and Discussion QTL analyses of the ETH-DL3 × ETH-DH7 mapping population in its F2:3 generation have

revealed that the long arm of chromosome 6 harbors at least one major QTL for chilling

tolerance of photosynthesis (Chapter 2, Fracheboud et al., 2004; Jompuk et al., 2005). The

QTL analysis of this population in the BC3F2 generation gave further evidence that this

Chapter 6

49

chromosomal region contains two separate QTLs, namely QTL-1 near SSR marker bnlg1740

and QTL-2 between bnlg1136 and umc1653 (Chapter 3). Mapping of these QTLs and

comparison with the QTL analysis of the ETH-DL7 × ETH-FH6 population (Chapter 5)

indicate that the QTL found in the ETH-DL7 × ETH-FH6 population corresponds to QTL-1

in the ETH-DL3 × ETH-DH7 population, as shown for the trait minimal chlorophyll

fluorescence (Fo) in Figure 6.1. A comparison with the results of a QTL analysis using the

IBM302 population (O. Guerra-Peraza, ETH Zurich, personal communication) showed a co-

localization of a QTL for Fo in seedlings grown at 24/22 °C (day/night) with QTL-1 of the

ETH-DL3 × ETH-DH7 population as well as the QTL found in the ETH-DL7 × ETH-FH6

population (Figure 6.1). However, the QTL in the IBM302 population was further located

away from the SSR marker bnlg1740 than was the case in the other two mapping populations.

The SSR markers bnlg1136 and umc1653, which flank QTL-2 in the ETH-DL7 × ETH-FH6

population, are mapped in a region in which a QTL for Fo in seedlings of the IBM302

population grown at 17/13 °C and at 17/6 °C was identified. Although these QTLs overlap in

the three populations, it still remains to be determined whether they are controlled by the

same gene.

In order to identify potential candidate genes for this QTL region, the flanking SSR markers

Fo (17/6°C)Fo (17/13°C)

Fo (25°C)Fo (15°C)

bnlg

1732

umc1

859

bnlg

1740

bnlg

1136

umc1

653

umc2

324

Fo (15°C)

umc1

063

umc1

897

bnlg

1740

AY1

0999

6

umc1

653

Fo (24/22°C)

bnl

g173

2

um

c185

9

um

c106

3

bnl

g174

0

bnl

g113

6 u

mc1

653

ETH-DL7 × ETH-FH6

ETH-DL3 × ETH-DH7

IBM302

QTL-1 QTL-2

Figure 6.1: Comparative mapping of QTLs for Fo on the distal part of the long arm of chromosome 6 in the ETH-DL7 × ETH-FH6 (Chapter 5), the IBM302 (O. Guerra-Perza, personal communication) and the ETH-DL3 × ETH-DH7 mapping population (Chapter 2). The colored bars show the range of LOD score values from 0 in blue to 6 (ETH-DL7 × ETH-FH6), 10 (IBM302) and 17.5 (ETH-DL3 × ETH-DH7) in red.


50

umc1859 and umc2324 were anchored to the Gramene Curated AGI FPC Oct 2004 physical

map. This region contained three EST contigs (ctg279, ctg280 and ctg281) and several BACs,

yielding a contiguous sequence region of around 8.5 Mbp within which 232 ESTs were

identified. In order to identify the putative candidate genes, a homology search was made with

the obtained 232 EST sequences with the EST sequences from the cold stress library (Singh et

al., 2001). This analysis revealed 33 putative cold induced ESTs, which are located in the

QTL region (Table 6.1).

Among these ESTs, several are involved in the catabolic metabolism. Pyruvate kinase is an

enzyme involved in glycolysis, while NAD-dependent isocitrate dehydrogenase, ATP citrate

lyase and aconitate hydratase are found in the tricarboxylic acid cycle. It was shown for

aconitate hydratase that it is highly sensitive to ROS and is down-regulated in Arabidopsis

during oxidative stress (Sweetlove et al., 2002). Although these enzymes play major roles in

the plant’s metabolism, it is unlikely that they are involved in the expression of the studied

QTL and may not be associated with chilling tolerance for photosynthesis.

Potential candidate genes that may explain the QTL for chilling tolerance of photosynthesis

are the proteins and enzymes which are directly involved in photosynthesis. One of these is

the gene for phosphoribulokinase, an enzyme which is part of the Calvin cycle and catalyzes

the generation of ribulose 1,5-bisphosphate. This gene was also found to be cold-induced in

one of the parental lines of the ETH dent population, namely ETH-DH7 (Nguyen et al., 2009).

However, its position on the map makes it less likely to be the underlying gene of one of the

QTLs on chromosome 6.

The gene psaH, which is located in the close vicinity of marker umc1897 and codes for

PS I-H subunit of PS I. It was shown for Arabidopsis that, in psaH-less mutants, energy can

not be transferred from PS II to PS I and that state transition is impaired (Lunde et al., 2000).

However, due to the fact that PS I is more stable than PS II when photoinhibition occurs

(Powles, 1984), thus, psaH may not be the candidate gene for the QTL.

Following page:

Table 6.1: List of ESTs in the QTL region between SSR marker umc1859 and umc2324 which show homology to ESTs from cold stress (CS) library of Singh et al. (2001) as well as their potential gene product identified by similarity search using the blastx algorithm. The positions of the ESTs and SSR markers correspond to the Gramene Curated AGI FPC Oct 2004 physical map.

Marker/EST Search result to CS EST lib. Search result by blastx Name Position Name E-value Annotation (Species) Accession E-value

umc1859 147509600 PCO083425 147509600 BG320414 9·e-12 Senescence-associated protein (O. sativa) ABF94900 1·e-123 PCO134372 151258100 BG320123 0 Hypothetical protein (O. sativa) EAZ13676 5·e-105 PCO125467 151782400 BG320691 3·e-53 Probable zinc finger protein (O. sativa) AAX92933 5·e-43 CL2757_5 151963700 BG320586 0 S-adenosylmethionine synthetase (O. sativa) CAJ45486 0 PCO079415 152012700 BG319765 0 Phosphoribulokinase (O. sativa) NP849912 6·e-146 PCO142167 152027400 BG321117 2·e-05 Zea root preferential 4 (Z. mays) NP_001066698 2·e-61 CL1479_1 153860000 BG321211 1·e-62 Zmhox1a homeobox protein (Z. mays) NP_001105447 0 CL34961_1 154041300 BG319996 0 Unknown (Z. mays) 2·e-31 CL187_5 154423500 BG320953 2·e-46 Hypothetical protein (O. sativa) EAZ35078 6·e-48 CL1166_1 154555800 BG321034 1·e-26 Cellulose synthase-1 (Z. mays) NP_001104954 0 PCO068622 154874300 BG320162 0 Putative 60S ribosomal protein L28 (O. sativa) BAD22882 5·e-66

umc1063 155080100 PCO089133 155246700 BG319632 1·e-154 Phosphatidylinositol transfer protein (O. sativa) BAD07999 0 PCO088717 156383500 BG321165 0 Hypothetical protein (O. sativa) EAY95622 2·e-11 PCO129762 156711800 BG320677 4·e-72 NAD-dependent isocitrate dehydrogenase (Z. mays) AAV39277 4·e-113 PCO099414 157045000 BG319800 0 Photosystem I complex PsaH subunit precursor (Z. mays) NP_001104905 0

umc1897 157089100 PCO073638 157299800 BG320981 1·e-163 Pyruvate kinase (A. thaliana) NP_566976 0 PCO150712 157397800 BG319952 0 Zein-alpha precursor (Z. mays) NP_001105941 0

bnlg1740 157608500 PCO080711 157657500 BG320382 0 ATP citrate lyase A-3 (A. thaliana) NP_172414 0 PCO099268 157760400 BG321261 0 Unknown (Z. mays) ACF78700 1·e-131 CL187_1 157843700 BG321115 0 Light harvesting chlorophyll a/b binding protein m7 (Z. mays) X53398 0 CL1288_1 157985800 BG320858 0 Thymidylate synthase (Z. mays) NP_001104916 0 PCO060210 157985800 BG319847 9·e-25 No similarity - - PCO068697 158167100 BG319945 0 Putative aconitate hydratase 1 (S. bicolor) ABE77202 0 CL2997_-2 158897200 BG320282 7·e-39 Hypothetical protein (O. sativa) EAZ28425 1·e-01 CL4121_1 158902100 BG320164 2·e-16 Mitogen-activated protein kinase (O. sativa) AAT39148 7·e-132

bnlg1136 158911900 umc1653 159156900

PCO086864 159524400 BG320128 0 Putative lipid transfer protein (O. sativa) BAD73499 8·e-38 PCO111918 159759600 BG319860 0 Putative polyprenyl diphosphate synthase (O. sativa) AAT44203 3·e-135 PCO060490 159769400 BG320274 0 Hypothetical protein (O. sativa) EAZ37586 2·e-44 PCO129004 159945800 BG320318 2·e-19 Putative adhesion regulating molecule family (O. sativa) NP_001056509 2·e-139

umc2324 162116500


52

The position of the cab-m7 gene is very close to the SSR marker bnlg1740; codes for a light

harvesting chlorophyll a/b binding protein. The cab-m7 codes for the LHC II proteins

Lhcbm7, which is strongly preferentially expressed in maize mesophyll cells upon

illumination (Becker et al., 1992). In maize seedlings, growth at suboptimal temperature

induces changes in the chlorophyll a/b ratio, indicating a change in the proportion of LHC II

to the reaction center compared to the situation at optimal growth temperature (Haldimann et

al., 1995). It is interesting that, a QTL for the chlorophyll a/b ratio was identified near to

marker bnlg1740 in the ETH-DL3 × ETH-DH7 mapping population when seedlings grew at

suboptimal temperature (J. Leipner, ETH Zurich, personal communication). This QTL was

characterized by negative additivity, meaning that the allele of ETH-DH7 responded to a

lower chlorophyll a/b ratio and, consequently, to a greater proportion of the light harvesting

complex compared to the core complex; which could be explained by a differential expression

of cab-m7. Moreover, co-localization of Lhcb genes with QTLs for cold-induced photo-

inhibition in maize were also reported (Pimentel et al., 2005). However, without knowing the

exact function of Lhcbm7, in particular with respect to the regulation of photosynthesis, it is

only a speculation as to how Lhcbm7 affects the efficiency and capacity of the photosynthetic

apparatus of maize seedlings grown at suboptimal temperature.

Among the ESTs within the QTL region, another candidate gene was identified, which is not

present in the cold stress cDNA library. This EST (PCO153568) is located between bnlg1740

and bnlg1136 and shows homology (E-value of 7·e-39) with the serine protease DegP1 from

Arabidopsis (NP_189431). It was well documented that DegP1 plays an important role in the

repair of degraded D1 protein of the PS II reaction center (Kapri-Pardes et al., 2007) and,

therefore, could explain the changes in Fv/Fm since this parameter is strongly affected by

integrity of the D1 protein (Salonen et al., 1998).

It is already difficult to verify or identify putative candidate genes for chilling tolerance with

known functions; these problems increase, of course, with putative candidate genes of still

unknown functions in chilling tolerance. Clearly, for such a gene, fine mapping and functional

genomic experimentation will be required to clarify their candidacy.

Chapter 7

53

7 General Conclusions

The goal of the present study was to obtain a better understand of the significance of the

telomeric region of chromosome 6 for chilling tolerance of photosynthesis both on the

molecular and a physiological level. In a previous QTL analysis, Fracheboud et al. (2004)

identified a major QTL for chilling tolerance of photosynthesis on chromosome 6 at bin 6.07

in seedlings of the Swiss dent mapping population ETH-DL3 × ETH-DH7 grown at

suboptimal temperature under controlled conditions. Using the same population, Jompuk et al.

(2005) found a stable expression of this major QTL in varying cool temperature conditions in

the field. However, this QTL has been roughly placed on the linkage map with a large

confidence interval. Mapping with additional SSR markers in the QTL region revealed the

presence of another QTL in this region (Chapter 2). The considerable distance between the

QTLs as well as their different effects on the traits indicated that these two QTLs are separate.

The introgression of the QTL region of chromosome 6 (bin 6.07) of the chilling tolerant line

ETH-DH7 into the chilling sensitive line ETH-DL3 by marker assisted backcross breeding

using SSR markers for fore- and background selection was found to be efficient in

introgressing the desired allele into the recurrent parent. In the BC3F2 generation itself, plants

contained the ETH-DH7 allele at the target region while at the remaining genome was

completely recovered from ETH-DL3 (Chapter 3). In traditional backcross breeding, such a

complete recovery of the recurrent parent genome is possible only in the BC6 generation,

showing that the marker assisted selection significantly accelerated the development of near

isogenic lines.

The QTL analyses of the BC2F1, BC3F1 and BC3F2 generation confirmed that the introgressed

segment from ETH-DH7 at chromosome 6 had a stable effect on the chilling tolerance over

backcrossed generations.

The QTL analysis of the BC2F3 generation by composite interval mapping confirmed again

the presence of two separate QTLs in the telomeric region of chromosome 6 (Chapter 3).

Furthermore, the QTL mapping showed significant effects of QTL-1 on Fv/Fm, Fo, ΦPSII,

Fv'/Fm', CER and SPAD while QTL-2 was associated only with the traits Fv/Fm and Fo. The

QTL analysis in the BC3F2 generation revealed that QTL-1 had also an effect on the leaf

greenness (SPAD), which was not the case in the F2:3 population (Fracheboud et al., 2004;

Jompuk et al., 2005). This might be due to the fact that QTL analyses of a particular QTL in

General Conclusions

54

advanced backcrossed generations, such as BC3F2, have the advantage that they are un

affected by other QTLs.

The time course analysis showed that the QTLs were not expressed in leaves, which

developed at optimal growth temperature but were strongly expressed in leaves that

developed at suboptimal temperature (Chapter 3). The effects of the QTLs were weaker when

seedlings were exposed to more severe cold stress as well as during recovery at optimal

temperature. The differential behavior of the QTLs in different leaves indicated that these

QTLs were involved in the development of a functional photosynthetic apparatus at

suboptimal temperature. This conclusion is supported by the finding that near isogenic lines

(NILs), exhibited a much lower value of the quantum efficiency of light regulated by non-

photochemical losses (ΦNO), a measure of structural changes to the photosynthetic machinery

(Chapter 4). It seems that these changes resulted in a higher quantum efficiency of the open

PS II reaction centers (Fv'/Fm') and, partially higher fraction of open reaction PSII centers,

indicated by qP. Both resulted in higher operating quantum efficiency of PS II (ΦPSII) and

together with a greater amount of chlorophyll in a higher photosynthetic activity.

The time course analysis of the QTL, which was found in the dent × flint (ETH-DL7 × ETH-

FH6) mapping population at the same map position as QTL-1 in the ETH-DL3 × ETH-DH7

population, further supports the conclusion that these QTLs in the telomeric region of

chromosome 6 were involved in the development of a functional photosynthetic machinery at

low temperature (Chapter 5).

The co localization of the QTL region by comparative mapping of the ETH-DL3 × ETH-DH7

with the ETH-DL7 × ETH-FH6 and the IBM302 population allowed to perform an in silico

analysis, to identify putative candidate genes underlying these QTLs (Chapter 6). In

particular, two candidate genes in the close vicinity of the QTL peak explained the observed

phenotype. One of these candidate genes, namely cab-m7, codes for a light harvesting

complex II protein and may directly influence the composition of the photosynthetic

apparatus. The other candidate gene showed homology with the gene for the DegP1 protease

in Arabidopsis. This protein is involved in the turnover of D1 protein during photoinhibition,

an essential process for maintaining a functional PS II. The identification of these candidate

genes may be helpful in the future for cloning this QTL; a QTL that seems to be present

across the maize germplasm.

55

8 References

Anderson, M.D., Prasad, T.K., Martin, B.A., Stewart, C.R. 1994. Differential gene expression in chilling-acclimated maize seedlings and evidence for the involvement of abscisic acid in chilling tolerance. Plant Physiology 105: 331-339.

Armond, P.A., Schreiber, U., Björkman, O. 1978. Photosynthetic acclimation to temperature in desert shrub, Larrea divaricata II. Light harvesting efficiency and electron transport. Plant Physiology 61: 411-415.

Baker, N.R., Long, S.P., Ort, D.R. 1988. Photosynthesis and temperature with particular reference to effects on quantum yield. In: Long, S.P., Woodward, F.I. (eds) Plants and Temperature, pp. 347-375. Company of Biologists, Cambridge.

Baker, N.R., Ort, D.R. 1992. Light and crop photosynthetic performance. In: Baker, N.R., Thomas, H. (eds) Crop Photosynthesis: Spatial and Temporal Determinants, pp. 289-312. Elsevier Science Publishers, Amsterdam.

Baker, N.R., Farage, P.K., Stirling, C.M., Long, S.P. 1994. Photoinhibition of crop photosynthesis in the field at low temperature. In: Baker, N.R., Bowyer, J.R. (eds) Photoinhibition of photosynthesis: From molecular mechanisms to the field, pp. 349-363. Bios Scientific Publishers, Oxford.

Baker, N.R., Nie, G.Y. 1994. Chilling sensitivity of photosynthesis in miaze. In: Bajaj, Y.B.S. (ed) Biotechnology in Agriculture and Forestry: Maize, pp. 465-481. Springer, Berlin.

Baroja-Fernández, E., Muñoz, F.J., Akazawa, T., Pozueta-Romero, J. 2001. Reappraisal of the currently revailing model of starch biosynthesis in photosynthetic tissues: a proposal involving the cytosolic production of ADP-glucose by sucrose synthase and occurrence of cyclic turnover of starch in the chloroplast. Plant and Cell Physiology 42: 1311-1320.

Basten, C.J., Weir, B.S., Zeng, Z.-B. 1994. Zmap-a QTL cartographer. In: Smith, C., Gavora, J.S., Chesnais, B.B.J., Fairfull, W., Gibson, J.P., Kennedy, B.W., Burnside, E.B. (eds) Proceedings of the 5th World Congress on Genetics Applied to Livestock Production: Computing Strategies and Software, pp. 65-66, Guelph, Ontario, Canada.

Basten, C.J., Weir, B.S., Zeng, Z.-B. 2003. QTL cartographer; a reference manual and tutorial for QTL mapping. Department of Statistics, North Carolina State University, Raleigh, North Carolina, USA.

Beavis, W.D., Grant, D., Albertsen, M., Fincher, R. 1991. Quantitative trait loci for plant height in four maize populations and their associations with qualitative genetic loci. Theoretical and Applied Genetics 83: 141-145.

Becker, T.W., Templeman, T.S., Viret, J.F., Bogorad, L. 1992. The cab-m7 gene: a light-inducible, mesophyll-specific gene of maize. Plant Molecular Biology 20: 49-60.

Bernacchi, D., Beck-Bunn, T., Emmatty, D., Eshed, Y., Inai, S., Lopez, J., Petiard, V., Sayama, H., Uhlig, J., Zamir, D., Tanksley, S.D. 1998a. Advanced backcross QTL analysis of tomato. II. Evaluation of near-isogenic lines carrying single-donor introgressions for desirable wild QTL-alleles derived from Lycopersicon hirsutum and L. pimpinellifolium. Theoretical and Applied Genetics 97: 170-180.

56

Bernacchi, D., Beck-Bunn, T., Eshed, Y., Lopez, J., Petiard, V., Uhlig, J., Zamir, D., Tanksley, S.D. 1998b. Advanced backcross QTL analysis in tomato. I. Identification of QTLs for traits of agronomic importance from Lycopersicon hirsutum. Theoretical and Applied Genetics 97: 381-397.

Björkman, O., Demmig, B. 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170.

Bouchez, A., Hospital, F., Causse, M., Gallais, A., Charcosset, A. 2002. Marker-assisted introgression of favorable alleles at quantitative trait loci between maize elite lines. Genetics 162: 1945-1959.

Bravo, L.A., Gallardo, J., Navarrete, A., Olave, N., Martinez, J., Alberdi, M., Close, T.J., Corcuera, L.J. 2003. Cryoprotective activity of a cold-induced dehydrin purified from barley. Physiologia Plantarum 118: 262-269.

Carr, M.K.V., Hough, M.N. 1978. The influence of climate on maize production in north-western Europe. In: Bunting, E.S., Pain, B.F., Phipps, R.H., Wikinson, J.M., Gunn, R.E. (eds) Forage maize: production and utilisation, pp. 15-56. Agricultural Research Council, London.

Chaïb, J., Lecomte, L., Buret, M., Causse, M. 2006. Stability over genetic backgrounds, generations and years of quantitative trait locus (QTLs) for organoleptic quality in tomato. Theoretical and Applied Genetics 112: 934-944.

Chollet, R., Paolillo, D.J.J. 1972. Greening in a virescent mutant of maize. Pigment, ultrastructure and gas exchange studies. Zeitschrift für Pflanzenphysiologie 68: 30-44.

Collins, N.C., Tardieu, F., Tuberosa, R. 2008. Quantitative trait loci and crop performance under abiotic stress: Where do we stand? Plant Physiology 147: 469-486.

Dellaporta, S.L., Wood, J., Hicks, J.B. 1983. A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter 1: 19-21.

Dingersdissen, A.L., Geiger, H.H., Lee, M., Schechert, A., Welz, H.G. 1996. Interval mapping of genes for quantitative resistance of maize to Setosphaeria turcica, cause of northern leaf blight, in tropical environment. Molecular Breeding 2: 143-156.

Doebley, J., Stec, A., Wendel, V., Edwards, W. 1990. Genetic and morphological analysis of a maize-teosinte F2 population: implications for the origin of maize. Proceedings of the National Academy of Sciences of the USA 87: 9888-9892.

Doebley, J., Stec, A. 1991. Genetic analysis of the morphological differences between maize and teosinte. Genetics 129: 285-295.

Doebley, J., Stec, A. 1993. Inheritance of morphological differences between maize and teosinte: comparison of results for two F2 populations. Genetics 134: 559-570.

Dolstra, O., Jongmans, M.A., de Jong, K. 1988. Improvement and significance of resistance to low-temperature damage in maize (Zea mays L.). I. Chlorosis resistance. Euphytica 39: 117-123.

Engels, C. 1994. Effect of root and shoot meristem temperature on shoot to root dry matter partitioning and the internal concentrations of nitrogen and carbohydrates in maize and wheat. Annals of Botany 73: 211-219.

Eshed, Y., Zamir, D. 1995. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141: 1147-1162.

57

Färber, A., Young, A.J., Ruban, A.V., Horton, P., Jahns, P. 1997. Dynamics of xanthophyll-cycle activity in different antenna subcomplexes in the photosynthetic membranes of higher plants. Plant Physiology 115: 1609-1618.

Fracheboud, Y., Haldimann, P., Leipner, J., Stamp, P. 1999. Chlorophyll fluorescence as a selection tool for cold tolerance of photosynthesis in maize (Zea mays L.). Journal of Experimental Botany 50: 1533-1540.

Fracheboud, Y., Ribaut, J.-M., Vargas, M., Messmer, R., Stamp, P. 2002. Identification of quantitative trait loci for cold-tolerance of photosynthesis in maize (Zea mays L.). Journal of Experimental Botany 53: 1967-1977.

Fracheboud, Y., Jompuk, C., Ribaut, J.-M., Stamp, P., Leipner, J. 2004. Genetic analysis of cold tolarnace of photosyntheis in miaze. Plant Molecular Biology 56: 241-253.

Frary, A., Nesbitt, T.C., Frary, A., Grandillo, S., Van der Knaap, E., Cong, B., Liu, J., Meller, J., Elber, R., Alpert, K.B., Tanksley, S.D. 2000. fw2.2: A quantitative trait locus key to the evolution of tomato fruit size. Science 289: 85-88.

Freymark, P.J., Lee, M., Woodmann, W.L., Martinson, C.A. 1993. Quantitative and qualitative trait loci affecting host response to Exserohilum turcicum in maize (Zea mays L.). Theoretical and Applied Genetics 87: 537-544.

Fridman, E., Pleban, T., Zamir, D. 2000. A recombination hotspot delimits a wild-species quantitative trait locus for tomato sugar content to 484 bp within an invertase gene Proceedings of the National Academy of Sciences of the USA 97: 4718-4723.

Frisch, M., Melchinger, A.E. 2001. Marker-assisted backcrossing for introgression of a recessive gene. Crop Science 41: 1485-1494.

Fryer, M.J., Andrews, J.R., Oxborough, K., Blowers, D.A., Baker, N.R. 1998. Relationship between CO2 assimilation, photosynthetic electron transport, and active O2 metabolism in leaves of maize in the field during periods of low temperature. Plant Physiology 116: 571-580.

Genty, B., Briantais, J.-M., Baker, N.R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 87-92.

Gilmore, A.M., Hazlett, T.L., Debrunner, P.G., Govindjee 1996. Comparative time-resolved photosystem II chlorophyll a fluorescence analyses reveal distinctive differences between photoinhibitory reaction center damage and xanthophyll cycle-dependent energy dissipation. Photochemistry and Photobiology 64: 552-563.

Govindaraj, P., Arumugachamy, S., Maheswaran, M. 2005. Bulked segregant analysis to detect main effect QTL associated with grain quality parameters in Basmati 370/ASD 16 cross cross in rice (Oryza sativa L) using SSR markers. Euphytica 144: 61-68.

Greaves, J.A. 1996. Improving suboptimal temperature tolerance in maize - The search for variation. Journal of Experimental Botany 47: 307-323.

Haldimann, P., Fracheboud, Y., Stamp, P. 1995. Carotenoid composition in Zea mays developed at sub-optimal temperature and different light intensities. Physiologia Plantarum 95: 409-414.

Hillel, J., Schaap, T., Haberfeld, A., Jeffreys, A.J., Plotzky, Y., Cahaner, A., Lavi, U. 1990. DNA fingerprint applied to gene introgression breeding programs. Genetics 124: 783-789.

58

Horváth, E.M., Peter, S.O., Joët, T., Rumeau, D., Cournac, L., Horváth, G.V., Kavanagh, T.A., Schäfer, C., Peltier, G., Medgyesy, P. 2000. Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure. Plant Physiology 123: 1337-1349.

Hospital, F., Chevalet, C., Mulsant, P. 1992. Using markers in gene introgression breeding programs. Genetics 132: 1199-1210.

Hund, A., Frascaroli, E., Leipner, J., Jompuk, C., Stamp, P., Fracheboud, Y. 2005. Cold tolerance of the photosynthetic apparatus: pleitropic relationship between photosynthetic performance and specific leaf area of maize seedlings. Molecular Breeding 16: 321-331.

Jansen, R.C., Stam, P. 1994. High resolution of quantitative traits into multiple loci via interval mapping. Genetics 136: 1447-1455.

Jompuk, C., Fracheboud, Y., Stamp, P., Leipner, J. 2005. Mapping of quantitative trait loci associated with chilling tolerance in maize (Zea mays L.) seedlings grown under field conditions. Journal of Experimental Botany 56: 1153-1163.

Kaeppler, S.M., Phillips, R.L., Kim, T.S. 1993. Use of near-isogenic lines derived by backcrossing or selfing to map qualitative traits Theoretical and Applied Genetics 87: 233-237.

Kandemir, N., Jones, B.L., Wesenberg, D.M., Ullrich, S.E., Kleinhofs, A. 2000. Marker-assisted analysis of three grain yield QTL in barley (Hordeum vulgare L.) using near isogenic lines. Molecular Breeding 6: 157-167.

Kapri-Pardes, E., Naveh, L., Adam, Z. 2007. The thylakoid lumen protease Deg1 is involved in the repair of photosystem II from photoinhibition in Arabidopsis. Plant Cell 19: 1039-1047.

Kearsey, M.J., Farquhar, A.G.L. 1998. QTL analysis in plants:where are we now? Heredity 80: 137-142.

Kitao, M., Lei, T.T. 2007. Circumvention of over-excitation of PSII by maintaining electron transport rate in leaves of four cotton genotypes developed under long-term drought. Plant Biology 9: 69-76.

Kole, C., Quijada, P., Michaels, S.D., Amasino, R.M., Osborn, T.C. 2001. Evidence for homology of flowering-time genes VFR2 from Brassica rapa and FLC from Arabidopsis thaliana. Theoretical and Applied Genetics 102: 425-430.

Kraja, A.T., Dudley, J.W. 2000. QTL analysis of two maize inbred line crosses. Maydica 45: 1-12.

Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G. 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynthesis Research 79: 209-218.

Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E., Newburg, L. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181.

Lecomte, L., Duffé, P., Buret, M., Servin, B., Hospital, F., Causse, M. 2004. Marker-assisted introgression of five QTLs controlling fruit quality traits into three tomato lines revealed interactions between QTLs and genetic backgrounds. Theoretical and Applied Genetics 109: 658-668.

59

Lee, E.A., Staebler, M.A., Tollenaar, M. 2002. Genetic variation in physiological discriminators for cold tolerance - early autotrophic phase of maize development. Crop Science 42: 1919-1929.

Leipner, J., Fracheboud, Y., Stamp, P. 1999. Effect of growing season on the photosynthetic apparatus and leaf antioxidative defenses in two maize genotypes of different chilling tolerance. Environmental and Experimental Botany 42: 129-139.

Leipner, J., Jompuk, C., Camp, K.-H., Stamp, P., Fracheboud, Y. 2008. QTL studies reveal little relevance of chilling-related seedling traits for yield in maize. Theoretical and Applied Genetics 116: 555-562.

Li, Z., Jakkula, L., Hussey, R.S., Tamulonis, J.P. 2001. SSR mapping and confirmation of the QTL from PI96354 conditioning soybean resistance to southern root-knot nematode. Theoretical and Applied Genetics 103: 1167-1173.

Lin, H.X., Yamamoto, T., Sasaki, T., Yano, M. 2000. Characterization and detection of epistatic interactions of 3 QTLs, Hd1, Hd2, and Hd3, controlling heading date in rice using nearly isogenic lines. Theoretical and Applied Genetics 101: 1021-1028.

Long, S.P., East, T.M., Baker, N.R. 1983. Chilling damage to photosynthesis in young Zea mays. I. Effects of light and temperature variation on photosynthetic CO2 assimilation. Journal of Experimental Botany 34: 177-188.

Long, S.P., Humphries, S., Falkowski, P.G. 1994. Photoinhibition of photosynthesis in nature. Annual Review of Plant Physiology and Plant Molecular Biology 45: 633-662.

Lunde, C., Jensen, P.E., Haldrup, A., Knoetzel, J., Scheller, H.V. 2000. The PSI-H subunit of photosystem I is essential for state transitions in plant photosynthesis. Nature 408: 613-615.

Lyons, J.M. 1973. Chilling injury in plants. Annual Review of Plant Physiology 124: 445-466.

Mackay, T.F.C. 2001. Quantitative trait loci in Drosophila. Nature Reviews Genetics 2: 11-20.

Marocco, A., Lorenzoni, C., Fracheboud, Y. 2005. Chilling stress in maize. Maydica 50: 571-580.

Massacci, A., Iannelli, M.A., Pietrini, F., Loreto, F. 1995. The effect of growth at low temperature on photosynthetic characteristics and mechanisms of photoprotection of maize leaves. Journal of Experimental Botany 46: 119-127.

Melchinger, A.E., Utz, H.F., Schön, C.C. 1998. Quantitative trait locus (QTL) mapping using different testers and independent population samples in maize reveals low power of QTL detection and large bias in estimates of QTL effects. Genetics 149: 383-403.

Michelmore, R.W., Paran, I., Kesseli, R.V. 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences of the USA 88: 9828-9832.

Miedema, P. 1982. The effects of low temperature on Zea mays L. Advances in Agronomy 35: 93-129.

Missiaggia, A.A., Piacezziand, A.L., Grattapaglia, D. 2005. Genetic mapping of Eef1, a major effect QTL for early flowering in Eucalyptus grandis. Tree Genetics & Genomes 1: 79-84.

60

Mohammadi, S.A., Prasanna, B.M., Singh, N.N. 2003. Sequential path model for determining interrelationships among grain yield and related characters in maize. Crop Science 43: 1690-1697.

Monforte, A.J., Tanksley, S.D. 2000. Fine mapping of a quantitative trait locus (QTL) from Lycopersicon hirsutum chromosome 1 affecting fruit characteristics and agronomic traits:breaking linkage among QTLs affecting different traits and dissection of heterosis for yield. Theoretical and Applied Genetics 100: 471-479.

Mutlu, N., Miklas, J., Reiser, J., Coyne, D.P. 2005. Backcross breeding for improved resistance to common bacterial blight in pinto bean (Phaseolus vulgaris L.). Plant Breeding 124: 282-287.

Nadeau, J.H., Singer, J.B., Matin, A., Lander, E.S. 2000. Analysing complex genetic traits with chromosome substitution strains. Nature Genetics 24: 221-225.

Nezer, C., Collette, C., Moreau, L., Brouwers, B., Kim, J., Giuffra, E., Buys, N., Andersson, L., Georges, M. 2003. Haplotype sharing refines the location of an imprinted quantitative trait locus with major effect on muscle mass to a 250-kb chromosome segment containing the porcine IGF2 gene. Genetics 165: 277-285.

Nguyen, H.T., Leipner, J., Stamp, P., Guerra-Peraza, O. 2009. Low temperature stress in maize (Zea mays L.) induces genes involved in photosynthesis and signal transduction as studied by suppression subtractive hybridization. Plant Physiology and Biochemistry 47: 116-122.

Nie, G.-Y., Baker, N.R. 1991. Modifications to thylakoid composition during development of maize leaves at low growth temperatures. Plant Physiology 95: 184-191.

Nie, G.-Y., Robertson, E.J., Freyer, M.J., Leech, R.M., Baker, N.R. 1995. Response of the photosynthetic apparatus in maize leaves grown at low temperature on transfer to normal growth temperature. Plant, Cell and Environment 18: 1-12.

Norman, J.M., Welles, J.M., McDermitt, D.K. 1992. Estimating canopy light-use and transpiration efficiency from leaf measurements. Licor Application Note #105.

Oxborough, K., Baker, N.R. 1997. Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components - calculation of qP and Fv'/Fm' without measuring Fo'. Photosynthesis Research 54: 135-142.

Pe, M.E., Gianfranceschi, L., Taramino, G., Tarchini, R., Angilini, P., Dani, M., Binelli, G. 1993. Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize. Molecular & General Genetics 241: 11-16.

Perez-Brito, S., Jeffers, D., Gonzalez-de-Leon, D., Khairallah, M., Cortes-Cruz, G., Velasquez-Cardelas, G., Azpiroz-Rivero, S., Srinivasan, G. 2001. QTL mapping of Fusarium moniliforme ear rot resistance in highland maize, Mexico. Agrociencia 35: 181-196.

Phillips, R.L., Vasil, I.K. 1994. DNA-based markers in plants. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Pimentel, C., Davey, P.A., Juvik, J.A., Long, S.P. 2005. Gene loci in maize influencing susceptibility to chilling dependent photoinhibition of photosynthesis. Photosynthesis Research 85: 319-326.

61

Powles, S.B. 1984. Photoinhibition of photosynthesis induced by visible light. Annual Review of Plant Physiology and Plant Molecular Biology 35: 15-44.

Prasad, T.K., Anderson, M.D., Martin, B.A., Stewart, C.R. 1994. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6: 65-74.

Presterl, T., Ouzunova, M., Schmidt, W., Möller, E.M., Röber, F.K., Knaak, C., Ernst, K., Westhoff, P., Geiger, H.H. 2007. Quantitative trait loci for early plant vigour of maize grown in chilly environments. Theoretical and Applied Genetics 114: 1059-1070.

Ragot, M., Sisco, P.H., Hoisington, D.A., Stuber, C.W. 1995. Molecular-marker-mediated characterization of favorable exotic alleles at quantitative trait loci in maize. Crop Science 35: 1306-1315.

Reddy, A.R., Ramakrishna, W., Sekhar, A.C., Ithal, N., Babu, P.R., Bonaldo, M.F., Soares, M.B., Bennetzen, J.L. 2001. Novel genes are enriched in normalized cDNA libraries from drought-stressed seedlings of rice (Oryza sativa L. subsp. indica cv. Nagina 22). Genome 45: 204-211.

Reyes-Valdés, M.H. 2000. A model for marker-based selection in gene introgression breeding programs. Crop Science 40: 91-98.

Ribaut, J.-M., Hoisington, D.A., Deutsch, J.A., Jiang, C., Gonzalez-de-Leon, D. 1996. Identification of quantitative trait loci under drought conditions in tropical maize. 1. Flowering parameters and the anthesis-silking interval. Theoretical and Applied Genetics 92: 905-914.

Ribaut, J.M., Hoisington, D. 1998. Marker assisted selection: new tools and strategies. Trends in Plant Science 3: 236-239.

Richner, W., Soldati, A., Stamp, P. 1996. Shoot to root relations in field-grown maize seedlings. Agronomy Journal 88: 56-61.

Robertson, E.J., Baker, N.R., Leech, R.M. 1993. Chlorophyll thylakoid protein changes induced by low growth temperature in maize revealed by immunocytology. Plant, Cell and Environment 16: 809-818.

Rosenqvist, E., Van Kooten, O. 2003. Chlorophyll fluorescence: a general description and nomenclature. In: DeEll, J.R., Toivonen, P.M.A. (eds) Practical Applications of Chlorophyll Fluorescence in Plant Biology, pp. 31-77. Kluwer Academic Publishers, Dordrecht.

Rozen, S., Skaletsky, H.J. 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz, S., Misener, S. (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp. 365-386. Humana Press, Totowa, NJ.

Rymen, B., Fiorani, F., Kartal, F., Vandepoele, K., Inzé, D., Beemster, G.T.S. 2007. Cold nights impair leaf growth and cell cycle progression in maize through transcriptional changes of cell cycle genes. Plant Physiology 143: 1429-1438.

Salonen, M., Aro, E.-M., Rintamäki, E. 1998. Reversible phosphorylation and turnover of the D1 protein under various redox states of Photosystem II induced by low temperature photoinhibition. Photosynthesis Research 58: 143-151.

Salvi, S., Tuberosa, R. 2005. To clone or not to clone plant QTLs: present and future challenges. Trends in Plant Science 10: 297-304.

62

Sambrook, J., Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

Saranga, Y., Jiang, C.-X., Wright, R.J., Yakir, D., Paterson, A.H. 2004. Genetic dissection of cotton physiological responses to arid conditions and their inter-relationships with productivity. Plant, Cell and Environment 27: 263-277.

Singh, J.A., Wakui, K., Couroux, P., De Moors, A., Harris, L.J., Hattori, J.I., Ouellet, T., Robert, L.S., Sprott, D., Tinker, N.A.: Expressed sequence tags from cold-stressed maize seedlings. GenBank, National Center for Biotechnology Information (2001).

Stamp, P. 1981. Activities of PEP and RuBP carboxylase and pigment content in leaves of maize seedlings in relation to genotype and growing temperature. Journal of Plant Breeding 86: 20-32.

Stamp, P. 1986. Chilling stress in maize. In: Dolstra, O., Miedema, P. (eds) Breeding of silage maize, pp. 43-50. Eucarpia Proceeding, Wageningen.

Stirling, C.M., Nie, G.-Y., Aguilera, C., Nugawela, A., Long, S.P., Baker, N.R. 1991. Photosynthetic productivity of an immature maize crop: changes in quantum yield of CO2 assimilation, conversion efficiency and thylakoid proteins. Plant, Cell and Environment 14: 947-954.

Stirling, C.M., Rodrigo, V.H., Emberru, J. 1993. Chilling and photosynthetic productivity of field grown maize (Zea mays); changes in the parameters of the light-response curve, canopy leaf CO2 assimilation rate and crop radiation-use efficiency. Photosynthesis Research 38: 125-133.

Stone, P.J., Sorensen, I.B., Jamieson, P.D. 1999. Effect of soil temperature on phenology, canopy development, biomass and yield of maize in a cool-temperate climate. Field Crops Research 63: 169-178.

Sweetlove, L.J., Heazlewood, J.L., Herald, V., Holtzapffel, R., Day, D.A., Leaver, C.J., Millar, A.H. 2002. The impact of oxidative stress on Arabidopsis mitochondria. The Plant Journal 32: 891-904.

Tajima, T., Noguchi, S., Tanoue, W., Negishi, H., Nakakawaji, T., Ohno, T. 2002. Background selection using DNA markers in backcross breeding program for potato virus Y resistance of tobacco. Breeding Science 52: 253-257.

Tang, A.-C., Boyer, J.S. 2002. Growth-induced water potentials and the growth of maize leaves. Journal of Experimental Botany 53: 489-503.

Tanksley, S.D., Nelson, J.C. 1996. Advanced backcross QTL analysis: a method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theoretical and Applied Genetics 92: 191-203.

Thabuis, A., Palloix, A., Servin, B., Daubèze, A.M., Signoret, P., Hospital, F., Lefebvre, V. 2004. Marker assisted introgression of 4 Phytophthora capsici resistance QTL alleles into a bell pepper line: validation of additive and epistatic effects. Molecular Breeding 14: 9-20.

Tuberosa, R., Salvi, S., Sanguienti, M.C., Landi, P., Maccaferri, M., Conti, S. 2002a. Mapping QTLs regulating morpho-physiological traits and yield in drought-stressed maize: case studies, shortcomings and perspectives. Annals of Botany 89: 941-963.

Tuberosa, R., Salvi, S., Sanguineti, M.C., Landi, P., MacCaferri, M., Conti, S. 2002b. Mapping QTLs regulating morpho-physiological traits and yield: case studies, shortcomings and perspectives in drought-stressed maize. Annals of Botany 89: 941-963.

63

Tuinstra, M.R., Ejeta, G., Goldsbrough, P.B. 1997. Heterogeneous inbred family (HIF) analysis: a method for developing near-isogenic lines that differ at quantitative trait loci. Theoretical and Applied Genetics 95: 1005-1011.

Van Berloo, R., Aalbers, H., Werkman, A., Niks, R.E. 2001. Resistance QTL confirmed through development of QTL-NILs for barley leaf rust resistance. Molecular Breeding 8: 187-195.

Visscher, P.M., Haley, C.S., Thompson, R. 1996. Marker-assisted introgression in backcross breeding programs. Genetics 144: 1923-1932.

Vladutu, C., McLaughlin, J., Phillips, R.L. 1999. Fine mapping and characterization of linked quantitative trait loci involved in the transition of the maize apical meristem from vegetative to generative structures. Genetics 153: 993-1007.

Wang, G.L., Paterson, A.H. 1994. Assessment of DNA pooling strategies for mapping of QTLs. Theoretical and Applied Genetics 88: 355-361.

Yano, M. 2001. Genetic and molecular dissection of naturally occurring variation. Current Opinion in Plant Biology 4: 130-135.

Ying, J., Lee, E.A., Tollenaar, M. 2000. Response of maize leaf photosynthesis to low temperature during the grain-filling period. Field Crops Research 68: 87-96.

Zeng, Z.-B. 1994. Precision mapping of quantitative trait loci. Genetics 136: 1457-1468.

Zhang, Q., Shen, B.Z., Dai, X.K., Mei, M.H., Saghai Maroof, M.A., Li, Z.B. 1994. Using bulked extremes and recessive class to map genes for photoperiod-sensitive genic male sterility in rice. Proceedings of the National Academy of Sciences of the USA 91: 8675-8679.

Zheng, K., Subudhi, P., Domingo, J., Magpantay, G., Huang, N. 1995. Rapid DNA isolation for marker assisted selection in rice breeding. Rice Genetics Newsletter 12: 255-258.

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9 Appendix

Table A.1: Statistical analysis of the light response curves of carbon exchange rate (CER), operating quantum efficiency of PS II (ΦPSII), efficiency of open PS II reaction centers (Fv'/Fm'), photochemical quenching factor (qP), quantum efficiency for dissipation by down regulation (ΦNPQ), and quantum efficiency of other non-photochemical losses (ΦNO) in the chilling tolerant line ETH-DH7, chilling sensitive line ETH-DL3 and the near isogenic lines NIL152 and NIL146. Significant differences (P < 0.05) for pair-wise comparisons within the photon flux density (PPFD) are indicated by different letters. Empty rows indicate no significant differences.

Comparison at 25 °C Comparison at 15 °C

Trait PPFD ETH

-DH

7

NIL

146

NIL

152

ETH

-DL3

ETH

-DH

7

NIL

146

NIL

152

ETH

-DL3

CER 50 100 200 a ab ab b 400 a ab ab b 800 a ab ab b 1200 a ab a b 1600 ab ab b a a ab a b

ΦPSII 50 a ab a b 100 a ab a b 200 a ab a b 400 a a a b 800 b ab b a a a a b 1200 ab ab b a a a a b 1600 ab ab b a a a a b

Fv'/Fm' 50 a ab a b 100 a ab a b 200 a a a b 400 a b ab c 800 b ab b a a b ab c 1200 ab ab b a a b ab c 1600 ab ab b a a b ab c

qP 50 a ab a b 100 a ab a b 200 ab a ab b a a a b 400 b a b ab a a a b 800 b ab b a a a a b 1200 ab ab b a a a a b 1600 ab ab b a a a a b

65

Table A.1: continuation.

Comparison at 25 °C Comparison at 15 °C

Trait PPFD ETH

-DH

7

NIL

146

NIL

152

ETH

-DL3

ETH

-DH

7

NIL

146

NIL

152

ETH

-DL3

ΦNPQ 50 ab ab b a 100 200 ab ab b a b ab ab a 400 800 1200 b ab ab a 1600 b ab ab a

ΦNO 50 a ab a b b b b a 100 ab ab a b c ab bc a 200 ab ab a b b ab b a 400 ab ab a b b ab b a 800 ab ab a b b ab b a 1200 ab b a b b ab b a 1600 ab b a b b ab b a

66

Acknowledgements

I very much appreciate Prof. Dr. Peter Stamp for his guidance, constant support and

encouragement while carrying out the research work. Without his unflinching support and

guidance, this thesis would not have been possible.

I express my heartfelt indebtedness to my supervisor, Dr. Jörg Leipner. He has guided me like

a beacon of light and has been a persistent source of encouragement throughout the entire

investigation. His suggestions in the planning and executing of the experiments have been

most helpful. His supervision gave me a sense of freedom, has broadened my scientific

outlook and inculcated a positive attitude in me. I would also like to thank him for his great

support during the writing of this thesis.

I would like thank my co-examiner Dr. Christof Sautter for his willingness to read the thesis

and to deliver his comments in a short period of time.

I would like to thank Dr. Yvan Fracheboud and Dr. Choosak Jompuk for providing valuable

basic data.

The support and help of Ernst Merz in the field and Brigitta Pichler in the glasshouse related

to the research study was indeed significant and is gratefully acknowledged.

I acknowledge with gratitude the active support and help given by Marianne Wettstein-Bättig

and Susanne Hochmann. They offered useful suggestions and active help in carrying out the

experiments in the laboratory. Without their help, it would have been very difficult for me to

bring this study to a logical conclusion.

I would also like to place on record my gratitude to all the members of the Group of

Agronomy and Plant Breeding for their encouragement and support. Special thanks go to

Sami for his help in solving statistical problems and Rainer for his suggestions.

I am thankful to the Swiss Federal Commission of Scholarship for Foreign Students and the

Swiss Federal Institute of Technology for their financial support.

I would like to place on record my sincerest gratitude to Chiesa Verena, Fredy Hubmann, and

Elisabeth Schniderlin for their support and encouragement.

The support received from my parents, Mr. Mahadevapa Biradar and Mrs. Ratnamma Biradar

has been significant and I would like to thank them for understanding the nature of my

research work, bearing with me through all these years and for motivating me to achieve my

objectives.

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I consider myself fortunate to have Shashirekha as my partner in life. Words cannot express

the depth of my affection and gratitude to her; she has displayed enormous strength, patience,

courage and perseverance in supporting me during my studies. Without her love and support I

would not have completed my PhD work. She deserves credit for all my success. During the

final phases of the research work, we were blessed with the birth of our daughter Ananya,

who will motivate both of us to achieve very best in the future.

Last but not least; I thank the Almighty for all his blessings and for helping me to achieve the

satisfactory completion of my research.

Sunil

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Curriculum Vitae

Name: Biradar, Sunil Kumar

Date of birth: 01 June 1976

Place of birth: Bidar, India

Citizen of: India

Civil status: Married

1981-1989 Primary school 1989-1992 Secondary school 1992-1994 Pre-university 1994-1998 B. Sc. in Agricultural Sciences. University of Agricultural Sciences, Dharwad,

India 1998-2000 M. Sc. in Agricultural Sciences (Genetics and Plant Breeding). University of

Agricultural Sciences, Dharwad, India. 2000-2001 Apprenticeship on molecular markers studies at ICRISAT, India 2001-2004 Research Associate at Directorate of Rice Research, India 2004-2008 Doctoral student at Swiss Federal Institute of Technology Zurich, Switzerland

Documents

Molecular and physiological analysis of chilling tolerance in maize