AUS DEM INSTITUT FÜR
PFLANZENZÜCHTUNG, SAATGUTFORSCHUNG UND POPULATIONSGENETIK
DER UNIVERSITÄT HOHENHEIM
FACHGEBIET: ANGEWANDTE GENETIK UND PFLANZENZÜCHTUNG
PROF. DR. A. E. MELCHINGER
QTL MAPPING OF RESISTANCE TO SCLEROTINIA
SCLEROTIORUM (LIB.) DE BARY IN SUNFLOWER
(HELIANTHUS ANNUUS L.)
ZUR ERLANGUNG DES GRADES EINES DOKTORS
DER AGRARWISSENSCHAFTEN VORGELEGT
DER FAKULTÄT AGRARWISSENSCHAFTEN
DER UNIVERSITÄT HOHENHEIM
DIPL.-ING. SC. AGR.
(BOSNIEN UND HERZEGOWINA)
STUTTGART - HOHENHEIM
Die vorliegende Arbeit wurde am 29. April 2005 von der Fakultät Agrarwissenschaften der
Universität Hohenheim als “Dissertation zur Erlangung des Grades eines Doktors der
Agrarwissenschaften (Dr. sc. agr.)” angenommen
Tag der mündlichen Prüfung: 13. Juni 2005
Prodekan: Prof. Dr. Stahr
Berichterstatter, 1. Prüfer: Prof. Dr. Melchinger
Mitberichterstatter, 2. Prüfer: Prof. Dr. Spring
3. Prüfer: Prof. Dr. Blaich
To my parents
1 General Introduction 1
2 QTL mapping of Sclerotinia midstalk rot resistance in sunflower1 17
3 QTL mapping of resistance to Sclerotinia midstalk rot in RIL of sunflower population
NDBLOS × CM6252 28
4 Identification and validation of QTL for Sclerotinia midstalk rot resistance in sunflower
by selective genotyping3 37
5 General Discussion 47
6 Summary 59
7 Zusammenfassung 62
8 Acknowledgements 65
9 Curriculum vitae 66
1 Z. Micic, V. Hahn, E. Bauer, C.C. Schön, S. Tang, S.J. Knapp, A.E. Melchinger (2004) QTL
mapping of Sclerotinia midstalk rot resistance in sunflower. Theor. Appl. Genet.
2 Z. Micic, V. Hahn, E. Bauer, C.C. Schön, A.E. Melchinger (2004) QTL mapping of
resistance to Sclerotinia midstalk rot in RIL of sunflower population
NDBLOSsel × CM625. Theor. Appl. Genet. 110:1490-1498.
3 Z. Micic, V. Hahn, E. Bauer, C.C. Schön, S. Tang, S.J. Knapp, A.E. Melchinger (2004)
Identification and validation of QTL for Sclerotinia midstalk rot resistance in
sunflower by selective genotyping. Theor. Appl. Genet. 110:233-242.
analysis of variance
CV cross validation
CPS conventional phenotypic selection
LG linkage group
LOD log 10 odds ratio
MP parental mean
MAS marker-assisted selection
QTL quantitative trait locus or loci, depending on the context
proportion of genotypic variance explained by QTL
proportion of genotypic variance explained by QTL in the data set
proportion of genotypic variance explained by QTL in a test set
RE relative efficiency of MAS
RIL recombinant inbred lines
SG selective genotyping
SSR simple sequence repeat
1. General Introduction
The cultivated sunflower (Helianthus annuus L.) (Figure 1) ranks with soybean
[Glycine max (L.) Merr.], rapeseed (Brassica rapa L., and B. napus L.), and peanut (Arachis
hypogaea L.) among the four most important annual crops in the world grown for edible oil.
In recent years, the sunflower oil has been increasingly used for industrial purposes.
Figure1: Cultivated sunflower (Helianthus annuus)
Diseases represent the major limiting factors of sunflower production worldwide.
Sunflower is known to be a host for almost 40 pathogenic organisms (Gulya et al., 1997). A
major fungal disease that significantly restricts the productivity of sunflower, when grown in
humid and temperate environments, is Sclerotinia sclerotiorum (Lib.) de Bary.
Sclerotinia sclerotiorum was first described in 1837 and identified as a pathogen of
sunflower by Fuckel in 1861 (Purdy, 1979). The fungus is widespread and reported in all
sunflower-growing regions of the world. The host range includes 361 plant species belonging
to 225 genera in 64 families, including Brassicaceae, Fabaceae, and Solanaceae (Purdy,
1979). Sclerotinia species belong to the class Ascomycota and are characterized by producing
mycelia and sclerotia in the asexual stage, and apothecia with asci and ascospores in the
sexual stage (Figure 2) (Gulya et al., 1997).
Figure 2: Disease cycle of Sclerotinia wilt, midstalk rot and head rot of sunflower (Source:
Mycelia from germinating sclerotia in the soil infect sunflower roots and may result in
Sclerotinia wilt. Sclerotinia wilt may occur anytime from the seedling stage until maturity.
Midstalk rot typically originates from a leaf infection of airborne ascospores landing on
wounded leaf tissue and colonizing the leaf. The infection progresses down the petiole,
Sclerotia returned to soil
during harvest and
Fungus overwinters as sclerotia
in soil and plant debris
Wilted plants with
rotting stems and
basal stem cankers
Disease = Yield loss and
increase in soil inoculum
Sclerotia in soil
With high soil moisture
sclerotia near soil surface
germinate to form apothetia
by apothecium and
Ascospores blown in
from nearby fields
Roots contact sclerotia and
sclerotia germinate and infect roots;
fungus moves from plant to plant along roots
producing a stem lesion (Figure 3) with pith degradation and sclerotia formation inside the
stem. The stalks break usually at the point of infection. S. sclerotiorum infects the midstalk
from the late vegetative stage until maturity. At the end of flowering or later, ascospores may
also infect sunflower heads. The ultimate result of head infection is the complete rot
(Masirevic and Gulya, 1992).
Figure 3: Sclerotinia sclerotiorum midstalk rot
The impact of S. sclerotiorum on yield depends on the growth stage at which plants
are infected, as well as on subsequent climatic conditions. Commonly, S. sclerotiorum
infections at the root, midstalk and head result in a total yield loss (Masirevic and Gulya,
1992). Considering the wide host range and longevity of sclerotia, S. sclerotiorum is one of
the most difficult pathogens to control. Gulya et al. (1997) proposed an integrated control
program for combating S. sclerotiorum diseases.
(1) Cultural methods to control S. sclerotiorum diseases include proper plant density and a
3- to 4- year crop rotation with non-host crops.
(2) Use of fungicides. Fungicide tests of Peres et al. (1992) in sunflower revealed that the
most consistent and highest levels of efficacy were obtained by preventive treatments before
development of the first symptoms. Curative treatments against S. sclerotiorum in sunflower
would be cheaper, however, their efficacy is highly dependent on weather conditions and the
extent of attack.
(3) Biological control agents such as adding bacteria (Expert and Digat, 1995) or soil
micoorganisms (Jones et al., 2003) to the seeds or soil reduce the disease incidence and
subsequent loss in seed yield and, therefore, represent an alternative method for controlling S.
(4) Deployment of moderate levels of resistance in the host plants.
The search for resistance to S. sclerotiorum in sunflower has been the objective of
most sunflower breeding programs worldwide (Gulya et al., 1997). Several wild Helianthus
species were described as potential sources of genes for resistance to S. sclerotiorum (Seiler
and Rieseberg, 1997) and have been used to produce interspecific hybrids (Kräuter et al.,
1991, Schnabl et al., 2002).
Hitherto, no complete resistance to S. sclerotiorum in cultivated sunflower could be
achieved, but lines derived from interspecific crosses between wild species and cultivated
sunflower showed improved resistance when infected with S. sclerotiorum (Degener et al.,
1999; Köhler and Friedt, 1999; Rönicke et al., 2004). Inheritance of resistance to S.
sclerotiorum in sunflower was generally found to be quantitative for all three forms of
infection (root, stalk, head) with different genes controlling the resistance in different organs
(Robert et al., 1987; Castaño et al., 1993; Bert et al., 2002) and no race specificity (Thuault
and Tourvieille de Labrouhe, 1988). Additive gene action prevailed over dominance or
epistasis (Robert et al., 1987; Vear and Tourvieille, 1988; Genzbittel et al., 1998; Bert et al.,
Genetic analysis of complex traits has been amended by the application of molecular
marker technologies. Molecular markers help to construct high-resolution genetic maps that
can be used for the mapping and estimation of genomic positions and genetic effects of
quantitative trait loci (QTL) involved in quantitatively inherited traits. During the last decade,
several genetic linkage maps of cultivated sunflower were published based on Restriction
Fragment Length Polymorphisms (RFLPs)(Berry et al., 1995; Gentzbittel et al., 1995; Jan et
al., 1998), Simple Sequence Repeat (SSRs) markers (Bert et al., 2002; Tang et al., 2002;
Burke et al., 2002; Yu et al., 2003), Amplified Fragment Length Polymorphism (AFLPs)
(Gedil et al., 2001), Direct Amplification of Length Polymorphisms markers (DALPs) (Langar
et al., 2003), and Target Region Amplification Polymorphism (TRAPs) (Hu and Vick, 2003).
Thus, molecular tools are available in sunflower to efficiently map QTL for agriculturally
important traits such as resistance to midstalk rot caused by S. sclerotiorum. Selecting for
favorable QTL effects based on marker data (marker-assisted selection, MAS) has great
potential for improving quantitative traits.
Concerning S. sclerotiorum resistance of sunflower, several QTL studies were
published up to now (Mestries et al., 1998; Bert et al., 2002, 2004). The authors used different
F3 populations to study the resistance to S. sclerotiorum leaf and capitulum attack. QTL
reported in these studies were based on the nomenclature defined by Gentzbittel et al. (1995),
so that they can be compared. On 14 of the 17 sunflower linkage groups (LG) QTL have been
found. In general, each of them explained less than 20% of phenotypic variance. Some of
them appeared to be specific only for one cross. One particularly strong QTL was reported on
LG1 linked to a protein-kinase gene (Genzbittel et al., 1998), but while it explained 50% of
the variation in one cross, in other crosses it explained only 15% or was absent. The most
frequent LG that carried QTL for resistance to S. sclerotiorum was LG7, probably related to
the branching genotype (Bert et al., 2004).
During the last years, attempts have been made to establish resistance against S.
sclerotiorum by genetic engineering (Lu et al., 2000, Scleonge et al., 2000, Hu et al., 2003).
These studies are based on a gene controlling the production of an enzyme oxalate oxidase
(OXO). Oxalate is a phytotoxin secreted by S. sclerotiorum. It weakens the plant tissue and
plays a key role in the pathogenicity of S. sclerotiorum. Crops with natural resistance to S.
sclerotiorum such as wheat, barley, maize, or rice, produce OXO, which breaks down and
detoxifies the phytotoxin produced by S. sclerotiorum. Contrary to such crops, sunflower has a
very low OXO activity. An OXO gene from wheat was isolated and inserted into sunflower
plants via Agrobacterium–mediated transformation. The Sclerotinia-induced lesions in
transgenic sunflower were significantly smaller than those in the control leaves (Hu et al.,
2003). Compared with the original line, this gene increased resistance but in generally, the
level was not better than in lines obtained by conventional breeding. Therefore, it should be
possible to combine the transgenic lines with natural resistance to provide a level of resistance
higher than in the currently available commercial hybrids (Bazzalo et al., 2000).
In the present study, we focused on midstalk rot due to its importance in sunflower
growing areas in Germany, and the availability of a reliable resistance test (Degener et al.,
1998). The latter determines the mycelium extension in leaves and stems as a measure for
resistance to midstalk rot caused by S. sclerotiorum. Three resistance (leaf lesion, stem lesion,
speed of fungal growth) and two morphological traits (leaf length, leaf length with petiole)
Based on observations of stem lesion, two inbred lines of different genetic origins
(NDBLOS and TUB5-3234) with high level of resistance to S. sclerotiorum (Degener et al.,
1999) were crossed with a highly susceptible line (CM625) to develop two segregating
populations for the estimation of QTL in this study.
To obtain information about the prospects of marker-assisted selection (MAS) for
increasing the level of resistance to S. sclerotiorum in sunflower, the objectives of the present
study were to:
(1) estimate the number, genomic positions, and genetic effects of QTL involved in
midstalk-rot resistance to S. sclerotiorum in two F3 populations,
(2) verify the QTL for midstalk-rot resistance in recombinant inbreed lines (RIL) of the
NDBLOSsel × CM625 population, and
(3) asses the consistency of QTL for midstalk-rot resistance across populations of different
Bazzalo, M.E., I. Bridges, T. Galella, M. Grondona, A. Leon, A. Scott, D. Bidney, G. Cole,
J.L. D’Hautefeule, G. Lu, M. Mancl, C. Scelonge, J. Soper, G. Sosa-Dominguez and L.
Wang. 2000. Sclerotinia Head rot resistance conferred by wheat oxalate Oxidase in
transgenic sunflower. Proc. 15th Int. Sunflower Conf., Toulouse, France, K 60-65.
Berry, S.T., A.J. Leon, C.C. Hanfrey, P. Challis, A. Burkholz, S. Barness, G.K. Rufener, M.
Lee and P.D.S. Caligari. 1995. Molecular marker analysis of Helianthus anuus L. 2.
Construction of an RFLP linkage map for cultivated sunflower. Theor. Appl. Genet.
Bert, P., F. Jouan, D. Tourvieille de Labrouhe, F. Seere, P. Nicolasand F. Vear. 2002.
Comparative genetic analysis of quantitative traits in sunflower (Helianthus annuus L.) 1.
QTL involved in resistance to Sclerotinia sclerotiorum and Diaporthe helianthi. Theor.
Appl. Genet. 105:985-993.
Bert, P., G. Dechamp-Guillaume, F. Seere, I. Jouan D., Tourvieille de Labrouhe, P. Nicolas
and F. Vear. 2004. Comparative genetic analysis of quantitative traits in sunflower
(Helianthus annuus L.) 3. characterization of QTL involved in resistance to Sclerotinia
sclerotiorum and Phoma macdonaldi. Theor. Appl. Genet. 109:865-874.
Burke, J.M., S. Tang, S.J. Knapp and L.H. Rieseberg. 2002. Genetic analysis of sunflower
domestication. Genetics 161:1257-1267.
Castaño, F., F. Vear, and D. Tourvieille. 1993. Resistance of sunflower inbred lines to various
forms of attack by Sclerotinia sclerotiorum and relations with some morphological
characters. Euphytica 68:85-98.
Degener, D., A.E. Melchinger, R.K. Gumber and V. Hahn. 1998. Breeding for Sclerotinia
resistance in sunflower: a modified screening test and assessment of genetic variation in
current germplasm. Plant Breed. 117:367-372.
Degener, J., A.E. Melchinger and V. Hahn. 1999. Optimal allocation of resources in
evaluating current sunflower inbred lines for resistance to Sclerotinia. Plant Breed.
Expert, J.M. and B. Digat. 1995. Biocontrol of Sclerotinia wilt of sunflower by Pseudomonas
fluorescens and Pseudomonas putida strains. Can. J. Microbiol. 41:685-691.
Gedil, M.A., C. Wye, S. Berry, B. Segers, J. Peleman, R. Lones, A. Leon, M.B. Slabaugh and
S.J. Knapp. 2001. An integrated restriction fragment length polymorphism-amplified
fragment length polymorphism linkage map for cultivated sunflower. Genome 44:213-221.
Gentzbittel, L., F. Vear, Y.X. Zhang, A. Berville and P. Nicolas. 1995. Development of a
consensus linkage RFLP map of cultivated sunflower (Helianthus annuus L.). Theor. Appl.
Gentzbittel, L., S. Mouzeyar, S. Badaoui, E. Mestries, F. Vear, D. Tourvieille de Labrouhe
and P. Nicolas. 1998. Cloning of molecular markers for disease resistance in sunflower,
Helianthus annuus L. Theor. Appl. Genet. 96:519-525.
Gulya, T. J., K. Y. Rashid, and S. N. Masirevic. 1997. Sunflower diseases. In Sunflower
technology and production, Madison, Wisconsin, USA. p. 263-379.
Hu, J. and B.A. Vick. 2003. Target region amplified polymorphism: A novel marker
technique for plant genotyping. Plant Mol. Biol. Reporter. 21: 1-6.
Hu, X., D.L. Bidney, N. Yalpani, J.P. Duvick, O. Crasta, O. Folkerts and G. Lu. 2003.
Overexpression of a hydrogen peroxide-generating oxalate oxidase gene evokes defense
responses in sunflower. Plant Physiol. 133:170-181.
Jan, C.C., B.A. Vick, J.F. Miller, A.L. Kahler and E.T. Butler. 1998. Construction of an RFLP
linkage map for cultivated sunflower. Theor. Appl. Genet. 96:15-22.
Jones, E.E., A. Mead and J. Whipps. 2003. Evaluation of different Coniothyrium minitans
inoculum sources and application rates on apothecial production and infection of
Sclerotinia sclerotiorum sclerotia. Soil Biology and Biochemistry:409-419.
Köhler, H. and W. Friedt. 1999. Genetic variability as identified by AP-PCR and reaction to
mid-stem infection of Sclerotinia sclerotiorum among interspecific sunflower (Helianthus
annuus L.) hybrid progenies. Crop Sci 39:1456-1463.
Kräuter, R., A. Steinmetz and W. Friedt. 1991. Efficient interspecific hybridization in the
genus Helianthus via "embryo rescue" and characterization of the hybrids. Theor. Appl.
Langar, K., M. Lorieux, E. Desmarais, Y. Griveau, L. Gentzbittel and A. Bervillé. 2003.
Combined mapping of DALP and AFLP markers in cultivated sunflower using F9
recombinant inbred lines. Theor. Appl. Genet. 106:1068-1074.
Lu, G., D. Bidney, Z. Bao, X. Hu, J. Wang, T. Vortherms, C. Scelonge, L. Wang, W. Bruce
and J.Duvick. 2000. Constitutive and Sclerotinia disease resistance in sunflower. K 72-77.
Proc. of 15th Int. Sunflower Conf. Toulouse, France.
Masirevic, S, and T. J. Gulya. 1992. Sclerotinia and Phomopsis – two devasting sunflower
pathogens. Field Crops. Res. 30:271-300.
Mestries, E., L. Gentzbittel, D. Tourvieille de Labrouhe, P. Nicolas and F. Vear. 1998.
Analysis of quantitative trait loci associated with resistance to Sclerotinia sclerotiorum in
sunflowers (Helianthus annuus L.) using molecular markers. Mol Breed 4:215-226.
Nelson, B. 2000. Sclerotinia diseases of sunflower
Peres, A., L. M. Allard and Y. Regnault. 1992. Sclerotinia sclerotiorum (Lib.) de Bary: A
study of fungicides to control attacks on sunflower floral buds. In Proc. 12th Int. Sunflower
Conf., Novi Sad, Yugoslavia, July 25-29. p. 808-813
Purdy, L. H. 1979. Sclerotinia sclerotiorum: History, diseases and symptomatology, host
range, geographic distribution and impact. Phytopathology 69:875-880.