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Theor Appl Genet (2004) 109:92–102
DOI 10.1007/s00122-004-1599-7
ORIGINAL PAPER
B. Prez-Vich · B. Akhtouch · S. J. Knapp · A. J. Leon ·
L. Velasco · J. M. Fernndez-Martnez · S. T. Berry
Quantitative trait loci for broomrape (
Orobanche cumana
Wallr.)
resistance in sunflower
Received: 31 October 2003 / Accepted: 5 January 2003 / Published online: 13 February 2004
Springer-Verlag 2004
Abstract Broomrape (Orobanche cumana Wallr.) is a
root parasite of sunflower that is regarded as one of the
most important constraints of sunflower production in the
Mediterranean region. Breeding for resistance is the most
effective method of control. P-96 is a sunflower line
which shows dominant resistance to broomrape race E
and recessive resistance to the very new race F. The
objective of this study was to map and characterize
quantitative trait loci (QTL) for resistance to race E and to
race F of broomrape in P-96. A population from a cross
between P-96 and the susceptible line P-21 was pheno-
typed for broomrape resistance in four experiments, two
for race E and two for race F, by measuring different
resistance parameters (resistance or susceptibility, number
of broomrape per plant, and proportion of resistant plants
per F3family). This population was also genotyped with
microsatellite and RFLP markers. A linkage map com-
prising 103 marker loci distributed on 17 linkage groups
was developed, and composite interval mapping analyses
were performed. In total, five QTL (or1.1,or3.1,or7.1
or13.1 and or13.2) for resistance to race E and six QTL
(or1.1,or4.1,or5.1,or13.1,or13.2 and or16.1) for
resistance to race F of broomrape were detected on 7 of
the 17 linkage groups. Phenotypic variance for race E
resistance was mainly explained by the major QTL or3.1
associated to the resistance or susceptibility character
(R2=59%), while race F resistance was explained by QTL
with a small to moderate effect (R2from 15.0% to
38.7%), mainly associated with the number of broomrape
per plant. Or3.1 was race E-specific, while or1.1,or13.1
and or13.2 of were non-race specific. Or13.1, and or13.2
were stable across the four experiments. Or3.1, and or7.1
were stable over the two race E experiments and or1.1
and or5.1 over the two race F experiments. The results
from this study suggest that resistance to broomrape in
sunflower is controlled by a combination of qualitative,
race-specific resistance affecting the presence or absence
of broomrape and a quantitative non-race specific resis-
tance affecting their number.
Introduction
Sunflower broomrape (Orobanche cumana Wallr.) is an
obligate, holoparasitic angiosperm that lives attached to
the roots of sunflower (Helianthus annuus L.), depleting
the plant of nutrients and water. Sunflower broomrape is
nowadays regarded as one of the most important con-
straints of sunflower production in countries surrounding
the Black Sea, as well as in central Europe, Spain and
Israel (Blbl et al. 1991; Shindrova 1994; Alonso et al.
1996; Domnguez et al. 1996). Attacks are frequently
severe and yield losses can reach 50% (Domnguez
1996a). Control of this parasite remains extremely diffi-
cult because the thousands of tiny seeds produced by a
single broomrape plant can be easily dispersed by wind,
water, animals, humans, machinery or soil attached to
agricultural products. The seeds may remain viable for
15–20 years and will germinate in the presence of the host
plant (Skoric 1988). Several control methods have been
Communicated by C. Mllers
B. Prez-Vich ()) · B. Akhtouch · L. Velasco ·
J. M. Fernndez-Martnez
Instituto de Agricultura Sostenible (CSIC),
Apartado 4084, 14080 Crdoba, Spain
e-mail: bperez@cica.es
Tel.: +34-957-499210
Fax: +34-957-499252
S. J. Knapp
Department of Crop and Soil Science,
Oregon State University,
Corvallis, OR 97331, USA
A. J. Leon
Advanta Semillas,
Ruta 226, Km 60, 7620 Balcarce, Buenos Aires, Argentina
S. T. Berry
Advanta Biotechnology Laboratory, SES-Europe,
3300 Tienen, Belgium
Present address:
S. T. Berry, Advanta Seeds UK Ltd.,
Station Road, Docking, King’s Lynn, Norfolk, PE31 8LS, UK
investigated including the use of herbicides (Garca-
Torres et al. 1994, 1995), soil solarization (Sauerborn et
al. 1989), crop rotation with pepper as a catch crop
(Hershenhorn et al. 1996), modifying sunflower planting
times (Aydin and Mutlu 1996) and the use of biological
agents (Thomas et al. 1999; Klein and Kroschel 2002;
Shabana et al. 2003).
Breeding for resistance is considered the most effec-
tive and feasible method of controlling sunflower broom-
rape. Genetic resistance to broomrape was introduced into
sunflower in the early breeding programs in the former
USSR (Pustovoit 1966). However, the widespread use of
resistant cultivars has led to the appearance of new races
of the parasite, which overcome existing resistance genes
(Skoric 1988), thus there is a continuous need for new
resistance sources. Races A to E of broomrape have been
described (reviewed in Alonso 1998), and these can be
identified using a set of sunflower differentials, each
carrying a single dominant gene (Or1 through Or5,
respectively) (Vrnceanu et al. 1980). Although a mono-
genic and dominant inheritance of resistance to races A to
E was found in most genetic studies (Pogorletsky and
Geshele 1976; Vrnceanu et al. 1980; Ish-Shalom-Gordon
et al. 1993; Sukno et al. 1999), some reports pointed to a
more complex inheritance of the trait, including two
dominant genes (Domnguez 1996b), one recessive gene
(Ramaiah 1987), double recessive epistasis (Kirichenco et
al. 1987) or even quantitative inheritance (Pustovoit
1966).
In Spain, broomrape has been traditionally restricted to
limited areas cropped with confectionary sunflower. From
the early 1970s onward, the parasite quickly spread to
central and southern Spain, causing serious infections in
oilseed cultivars. The first racial studies in this country
identified races overcoming Or1,Or3 and Or4, but not
Or2 or Or5 (reviewed in Melero-Vara et al. 2000). More
recent studies have shown the presence of a new race,
named race F, which overcomes all the known resistance
genes, including Or2 and Or5 (Alonso et al. 1996;
Domnguez 1999). Resistance to this new race has been
found in both cultivated and wild sunflower (Domnguez
1999; Sukno et al. 1999; Fernndez-Martnez et al. 2000;
Jan et al. 2002). Resistance to race F of broomrape in
germplasm derived from cultivated sunflower has been
reported to be recessive and controlled by alleles at two
loci (Rodrguez-Ojeda et al. 2001; Akhtouch et al. 2002).
The development of broomrape-resistant inbred lines
is not an easy task, mainly due to the difficulties in
assessing resistance under experimental conditions, where
the presence of escapes, genetic background effects, and
genotypeenvironment interactions lead to a sometimes
inefficient selection. Therefore, Orobanche resistance
genes are outstanding targets for molecular breeding
(Tang et al. 2003). DNA marker studies for broomrape
resistance in sunflower have been focused on the iden-
tification of molecular markers linked to the Or5 gene,
which confers resistance to race E of broomrape. Lu et al.
(2000), using bulked segregant analysis (BSA), developed
a linkage map containing Or5 flanked 22.5 cM by a distal
random amplified polymorphic DNA (RAPD) marker,
and from 5.6 cM to 39.4 cM by five proximal DNA
sequence characterized amplified regions (SCAR) mark-
ers. This linkage group (LG) was integrated with the
LG17 of the GIE Cartisol restriction fragment length
polymorphism (RFLP) map (Lu et al. 1999). Recently,
Tang et al. (2003), also using BSA, placed the Or5 gene
in a telomeric region of LG3 of the public simple
sequence repeat (SSR) map of sunflower (Tang et al.
2002), with the closest SSR marker mapping 6.2 cM
proximal to the Or5 locus. To date, no molecular markers
linked to genes conferring resistance to the most recent
broomrape race F have been identified.
The objective of the present research was to identify
and characterize quantitative trait loci (QTL) linked to
genes for resistance to races E and F of sunflower
broomrape.
Materials and methods
Plant materials and segregating populations
The sunflower lines used to generate the F2mapping population
were P-96, an inbred line resistant to races E and F of broomrape
developed from cultivated sunflower of Yugoslavian origin
(Akhtouch et al. 2002) and P-21, a genetic male sterile (GMS)
line of sunflower, which is highly susceptible to broomrape. P-21
was used as female for crosses with P-96. The F1together with both
parents were planted in the glasshouse during the spring of 1999. F2
seeds were produced by self-pollinating the F1plants. In the spring
of 2000, 113 F2plants were sown in the field. About 25% of the F2
plants were sterile due to the segregation of the GMS gene from
P-21. Fertile F2plants were self-pollinated to produce F3seeds.
Broomrape populations
Two different race E broomrape populations were used in this
study: SE-194, collected in southern Spain in 1994 (Sukno et al.
1999), and CU-796, collected in central Spain in 1996 (J.M.
Melero-Vara, personal communication). Both SE-194 and CU-796
were classified as race E by artificial inoculation on sunflower
differentials carrying the resistance genes Or1,Or4 and Or5. The
race F broomrape population used in this study was SE-296,
collected in southern Spain in 1999 from broomrape plants
attacking cultivars which incorporated genes of resistance to race
E. SE-296 was confirmed as race F by artificial inoculation on
sunflower differentials carrying the resistance genes Or1 and Or5
(Akhtouch et al. 2002).
Phenotypic evaluation
Four different experiments were conducted to test the reaction of
populations derived from the cross P-21P-96 to broomrape races E
and F:
Experiment 1. Evaluation of 80 F3families artificially inoculated
with broomrape population SE-194 (race E), con-
ducted in pots in the glasshouse in the winter of
2000/2001.
Experiment 2. Evaluation of 60 F3families artificially inoculated
with broomrape population CU-796 (race E), con-
ducted in pots in a mesh cage in the spring of 2001.
93
Experiment 3. Evaluation of 113 F2plants artificially inoculated
with broomrape population SE-296 (race F), con-
ducted in the field in the spring of 2000.
Experiment 4. Evaluation of 52 F3families artificially inoculated
with broomrape population SE-296 (race F), con-
ducted in the field in the spring of 2001.
F3families consisted of 15 to 30 plants. Plants of the parents as
well as F1plants from the P-21P-96 cross were also tested.
Additionally, plants of the R-5 line, which carries the resistance
gene Or5 (Sukno et al. 1999), were used as check.
In all the experiments, artificial inoculation was carried out by
planting 2-day-old sunflower seedlings in small pots (778 cm)
containing a mixture of sand and peat (1:1, v/v). Each pot
(approximately 180 g of the mixture) was carefully mixed with
50 mg of broomrape seeds to obtain a homogeneously infested
substrate. The plants were kept in a growth chamber for 15–20 days
for incubation at 25C/18C (day/night) using a 14-h photoperiod.
For experiments in the glasshouse and mesh cage, the plants were
then transplanted to larger pots containing 3 l of fertilized and
uninfected sand/silt/peat (2:1:1, v/v/v) soil mixture. For the field
experiments, the plants were transplanted into a field plot artifi-
cially infected with race F of broomrape.
Disease reactions were assessed at physiological maturity by
counting the number of emerged broomrape shoots around each
sunflower plant. In order to minimize escapes, resistant plants were
carefully uprooted to observe any non-emerged broomrape and
nodules or stalks. Phenotypic characterization of the F2plants was
made by classifying them as susceptible (S), when plants showed
emerged or underground broomrape, or resistant (R), when they
showed no infection (RS trait) and by considering the total number
of broomrape per F2plant (NBr trait). Phenotypic characterization
of F3families was made by considering both the average number of
broomrape per F3family (NBr trait), as well as the proportion of
resistant sunflower plants within each F3family (PR trait). F3
families were also classified as segregating (i.e. those with both
resistant and susceptible plants, scored as ‘H’), susceptible (all
plants susceptible, scored as ‘A’), or resistant (all plants resistant,
scored as ‘B’).
Molecular data collection
For the molecular markers analyses, three fully expanded leaves
were cut from each of the 113 F2plants grown in the spring of 2000
and frozen at 70C. The leaf tissue was then lyophilised and
ground to a fine powder in a laboratory mill. RFLP marker analysis
was carried out as described by Berry et al. (1995), using the RFLP
probes developed and mapped by Berry et al. (1994, 1995, 1996).
Microsatellite SSR-marker data collection was performed using
primers from various sources including those described by Paniego
et al. (2002), Tang et al. (2002) and Yu et al. (2003). PCRs were
performed by using 30 ml of reaction mixture containing 1 PCR
buffer, 1.5 mM MgCl2, 0.2 mM each of dNTPs, 0.3 mM each of 30-
and 50-end primers, 0.7 U of Platinum Taq DNA polymerase
(Invitrogen, Carlsbad, Calif., USA), and 50 ng of genomic DNA.
To reduce non-specific amplification, touchdown PCR was used
with an initial denaturation at 94C for 2 min, followed by one
cycle at 94C for 30 s, final annealing temperature (TA)+10 for
30 s and 72C for 30 s. The annealing temperature was decreased
1C per cycle during each of the nine following cycles, at which
time the products were amplified for 32 cycles at 94C for 30 s, TA
for 30 s and 72C for 30 s with a final extension of 20 min at 72C.
Final annealing temperatures varied from 52C to 60C. After the
PCR reaction, the amplification products were resolved by elec-
trophoresis on denaturing polyacrylamide gels (4% acrylamide/
bisacrylamide, 19:1, 7 M urea in TBE). Bands were visualised
using a silver staining kit (Promega, Madison, Wis., USA).
Linkage map construction
Chi-square analyses were carried out on each locus to detect
deviations from the expected Mendelian ratios for codominant
(1:2:1) or dominant (3:1) markers. RFLP-SSR linkage maps were
constructed using the software MAPMAKER/EXP version 3.0b
(Whitehead Institute, Cambridge, Mass., USA) (Lander et al.
1987). Two-point analysis was used to identify LGs at an LOD
score of 3 and a maximum recombination frequency of 0.40. Three-
point and multi-point analyses were used to determine the order and
interval distances between the markers in each LG. The Haldane
mapping function was used to compute the map distances in
centiMorgans from the recombination fractions. The LG nomen-
clature follows Berry et al. (1997) and Tang et al. (2002). Multiple
loci detected by a single probe were coded with the probe name
plus the suffix ‘A’, ‘B’, or ‘C’. Based on clearly visible differences
in signal intensity on Southern blots, A was the suffix given to the
primary locus (strong hybridisation signal), and B and C to the
secondary loci (fainter hybridisation signal). LG maps were drawn
using the MapChart software (Voorrips 2002).
QTL analyses
For mapping of QTL and estimation of their effects, the method of
composite interval mapping (CIM) (Jansen and Stam 1994; Zeng
1994) was used. Computations were carried out using the software
PLABQTL version 1.1 (Utz and Melchinger 1996), which com-
bines interval mapping by the regression approach (Haley and
Knott 1992) with the use of selected markers as cofactors. The
phenotypic data consisted on trait values for each F2plant (tested
for race F, broomrape population SE-296) and for each F3family
(tested for race E, broomrape populations SE-194 and CU-796 and
race F, broomrape population SE-296). Trait values for the QTL
analysis were: (1) NBr: total number of broomrape per F2plant or
F3family averaged (2) RS: F2plants scored as resistant (scale 0 =
no broomrape per plant) or susceptible (scale 1 = one or more
broomrape per plant), and F3families scored as resistant (scale 0 =
family with all plants resistant), heterozygous (scale 1 = family
with both resistant and susceptible plants) or susceptible (scale 2 =
family with all plants susceptible) and (3) PR: proportion of
resistant plants for each F3family (= number of resistant plants per
F3family/total number of plants evaluated per F3family). Analyses
were made initially with the first statement to check the database
for errors and outliers. Next, CIM analysis was done with cofactors
chosen for each trait by a stepwise regression procedure (Fto enter:
3.5, Fto drop: 3.5) with the procedure ‘cov select’. Genome-wide
threshold values (a=0.05) for declaring the presence of QTL were
estimated from 1,000 permutations of each phenotypic trait
(Churchill and Doerge 1994; Doerge and Churchill 1996). The
threshold of the LOD score was 2.6. Estimates of QTL positions
were obtained at the point where the LOD score reaches its
maximum in the region under consideration. One-LOD support
limits for the position of each QTL were also calculated (Bohn et
al. 1996).
The proportion of phenotypic variance explained by each
individual QTL was calculated as the square of the partial
correlation coefficient (R2). Estimates of the additive (ai) and
dominance (di) effects, as defined by Falconer (1989), for the ith
putative QTL, the total LOD score, as well as the total proportion of
the phenotypic variance explained by all QTL, were obtained by
fitting a multiple regression model including all putative QTL for
the respective trait simultaneously (Bohn et al. 1996). Following
Bohn et al. (1996), the ratio DR ¼di
jj=ai
jj
ðÞwas used to describe
the type of gene action at each QTL: additive for DR<0.2, partial
dominance for 0.2DR<0.8, dominance for 0.8DR<1.2, and
overdominance for DR1.2. The occurrence of QTLQTL inter-
actions was tested by adding digenic epistatic effects to the model
(addadd, adddom, domadd and domdom).
94
Results
Phenotypic segregations
All the plants of the resistant parent P-96 were resistant to
all broomrape populations. Similarly, all the plants of the
susceptible parent P-21 were totally susceptible to the
three broomrape populations (Table 1). Additionally, the
differential line R-5 was resistant to the race E broomrape
populations SE-194 and CU-796, and susceptible to the
race F population SE-296 (Table 1). F1plants were
resistant to race E populations and susceptible to the race
F population (Table 1).
Resistance to race E of broomrape followed a 1:2:1
(R:H:S) ratio for broomrape population CU-726
(18R:32H:10S, c2=2.4, P=0.30). The observed segrega-
tion ratio for the race E SE-194 population (40R:32H:8S)
was significantly different from the expected segregation
ratio 1:2:1 (20R:40H:20S). There was a deficiency in the
number of both susceptible (12) and segregating (8) F3
families and a corresponding excess of resistant (+20) F3
families. Resistance to race F fitted a 1:15 (R:S) ratio for
the F2generation (10R:103S, c2=1.30, P=0.25) and a
1:8:7 ratio for the F3generation (4R:27H:21S, c2=0.35,
P=0.84).
The number of broomrape per plant showed continu-
ous distributions for both race E and F populations. For
race E, the average number of broomrape per plant was
similar with the two broomrape populations used, 0.7 for
SE-194 and 0.8 for CU-796 (Table 2). For race F, the
average number of broomrape per plant was 13.2 in the F2
and 8.6 in the F3evaluation (Table 2).
The genetic map
The parents of the mapping population were screened for
RFLPs using 772 probe-enzyme combinations (193 RFLP
probesEcoRI, EcoRV, HindIII and DraI restriction
enzyme digests) and for SSRs polymorphisms using 82
primer pairs. Sixty codominant and 19 dominant RFLP,
and 35 codominant and 4 dominant SSR-marker loci were
polymorphic between P-21 and P-96. SCAR markers and
SSR markers closest to the Or5 gene conferring resistance
to race E developed by Lu et al. (2000) and Tang et al.
(2003), respectively, were not polymorphic in the P-
21P-96 population.
After the removal of closely linked marker loci
(<1 cM), the RFLP-SSR linkage map used for QTL
mapping comprised 103 marker loci (66 codominant
RFLP + 11 dominant RFLP + 24 codominant SSR + 2
dominant SSR-marker loci). The linkage map spanned a
distance of 1,144.4 cM with an average marker interval of
13.3 cM. There were 17 LGs, corresponding to the
haploid chromosome number in sunflower. Marker cov-
erage by LG is shown in Table 3. There were a number of
regions exceeding 20 cM that were devoid of markers;
however, 97.4% of the mapped genome was within 20 cM
to the nearest marker. None of the RFLP or SSR-marker
loci deviated significantly from the expected segregation
ratios (P<0.001).
The codominant phenotypic score of race E for both
the SE-194 and the CU-796 broomrape populations
mapped on top of LG3. The map distances in centiMor-
gans are shown in Fig. 1. Both scores mapped distal to the
markers of this LG. The closest marker (ZVG406) was
4.2 cM downstream from the score of the CU-796
population and 22.8 cM downstream from the score of the
SE-194 population (Fig. 1). The map distance in the SE-
Table 1 Segregation of resis-
tance and susceptibility in the
parents, F1,F
2and F3genera-
tions of the cross P-21P-96.
The R-5 line, which carries the
resistance gene Or5, is also
included as a check
Generation Number of F2plants or F3familiesa
Race E Race F
SE-194 CU-796 SE-296
Winter-00/01 Spring-01 Spring-00 Spring-01
Res Seg Sus Res Seg Sus Res Seg Sus Res Seg Sus
P-21 0 - 15 0 - 11 0 - 10 0 - 15
P-96 10 - 0 15 - 0 10 - 0 15 - 0
F1(P-21P-96) 10 - 0 - - - 0 - 10 - - -
F2(P-21P-96) - - - - - - 10 - 103 - - -
F3(P-21P-96) 40 32 8 18 32 10 - - - 4 27 21
R-5 10 - 0 7 - 0 0 - 10 0 - 15
aRes Resistant, Seg segregating, Sus susceptible
Table 2 Mean€standard devia-
tion (SD) and range for the total
number of broomrape per F2
plant or F3family from the
cross between P-21 and P-96
Generation Broomrape race and
population No. of F2plants or
F3families No. of broomrape
Mean€SD Range
F2(P-21P-96) Race F (SE-296) 113 13.22€11.93 0.0–47.0
F3(P-21P-96) Race F (SE-296) 52 8.57€7.72 0.0–28.8
F3(P-21P-96) Race E (SE-194) 80 0.70€1.31 0.0–5.7
F3(P-21P-96) Race E (CU-796) 60 0.80€1.14 0.0–4.4
95
194 experiment could be upwardly biased by phenotyping
errors that inflate map distances (Tang et al. 2003). The
phenotypic scores for race F (dominant from the F2data
and codominant from the F3data) were not linked to any
of the 17 LGs of the P-21P-96 map.
QTL analysis
Several QTL associated with resistance to race E and/or
race F were identified. The number, magnitude of effect
and direction of effect of QTL identified are summarised
in Table 4 and Fig. 2. Altogether, five QTL for the three
parameters evaluated for resistance to race E of broom-
rape (NBr, RS and PR) and six QTL for the same three
parameters evaluated for resistance to race F of broom-
rape were detected on 7 of the 17 LGs. Three of these
QTL were associated with resistance to both race E and
race F.
Race E resistance
One QTL associated with resistance to race E of broom-
rape (RS trait) was detected on top of LG3 (or3.1). This
QTL was consistently detected with both race E popula-
tions SE-194 and CU-796, in which it explained a
phenotypic variance of 37.0% for SE-194 and 59.4% for
CU-796.
When the number of broomrapes per plant were
considered (NBr trait), two QTL were detected for each of
the race E populations (Fig. 2 and Table 4), explaining
15.0% of the total phenotypic variation for SE-194 and
47.5% for CU-796. In both cases, the most significant
QTL was or3.1, which showed R2values of 18.8% in the
SE-194 test and 24.5% in the CU-796 test. These R2
values of or3.1 were significantly lower than those
observed for the RS trait (Table 4). The second QTL
detected was located on LG7 (or7.1) for SE-194 and on
LG13 (or13.2) for CU-796. All the resistance-enhancing
alleles at these QTL originated from the resistant parent
P-96 (Table 4). Or3.1 exhibited a partially dominant
effect for SE-194 and a dominant effect for CU-796
(Table 4). The degree of dominance was 1.47 for or7.1
(overdominant) and 0.49 for or13.2 (partially dominant).
The test for digenic epistatic interactions between or3.1
and or13.2 was significant (P<0.01, additive additive
and dominant additive) in the CU-796 test. The
inclusion of these epistatic effects into the model
increased the R2to 65.2%, compared to 47.5% for the
model without epistasis (Table 4).
Or3.1, as well as or7.1 and or13.2, were also associ-
ated with the proportion of resistant plants (PR) in both
the SE-194 and CU-796 race E tests (Table 4). Two
Table 3 Genome coverage offered by the marker set used for
quantitative trait loci (QTL) analysis in the P-21P-96 F2popu-
lation
LGaNumber of marker locibLG coverage (cM)
RFLP SSR Mean Largest
interval Total
1 3 2 16.1 29.7 64.5
2 7 0 10.2 19.2 61.1
3 6 0 22.6 38.2 112.9
4 3 2 18.0 34.9 72.0
5 4 0 13.8 25.6 41.4
6 4 0 17.8 48.3c53.3
7 4 3 12.4 41.8 74.2
8 3 4 7.9 24.2 47.5
9 5 1 16.7 60.1c83.3
10 6 3 7.3 15.5 58.4
11 4 0 25.8 39.9 77.4
12 4 1 14.3 33.8 57.3
13 8 3 11.0 30.2 110.2
14 1 5 7.7 19.5 38.7
15 3 0 6.2 9.5 12.3
16 5 2 14.3 36.2 85.8
17 7 0 15.7 37.2 94.0
Total 77 26 14.0 60.1 1144.3
aLG Linkage group. LG nomenclature is according to Berry et al.
(1997)
bRFLP Restriction fragment length polymorphism, SSR simple-
sequence repeat
cOne marker on top of LG6 and one marker on top of LG9 were
initially unlinked and they were mapped using the ‘near’ command
of MAPMAKER/EXP by increasing the recombination default Fig. 1 Molecular map of linkage group 3 (LG03) of sunflower
containing the codominant phenotypic score for race E of broom-
rape (populations SE-194 and CU-796) in F3families from the
cross P-21P-96. The scores for race E [F3-raceE (SE-194) and F3-
raceE (CU-796)] and marker names are listed to the right of the
map. The cumulative map distances in centiMorgans are shown at
the left of the map
96
additional QTL for the PR trait were detected, the first
one (or13.1) located on LG13 linked to or13.2 in both the
SE-194 and the CU-796 populations and the second one
(or1.1) located on LG1, but only detected in the CU-794
test. Or13.1 had an overdominant effect and explained
15.0% and 24.3% of the phenotypic variance for PR in the
SE-194 and the CU-794 tests, respectively (Table 4).
Or1.1 also had an overdominant effect and explained
23.5% of the phenotypic variance for PR in the CU-794
test. Simultaneous fit of the detected QTL for PR
explained 35.5% of the phenotypic variation for SE-194
and 59.0% for CU-794. The test for digenic epistatic
interactions was also significant (P<0.01, additive
additive) for or3.1 and or13.2 in the CU-796 test. The
inclusion of the epistatic effects increased the R2to 75.2%
(Table 4).
Table 4 QTL affecting broomrape race E (SE-194 and CU-796 broomrape populations) and race F (SE-296 broomrape population)
resistance in the P-21P-96 cross
Generation and
broomrape race TraitaQTL LG Position
(cM)bSupport
interval
(cM)c
Left
locusdLOD R2(%) Gene effectse
addjj=ajj
F3-E (SE-194) RS or3.1 3 0 0–11 ZVG406 7.71 37.0 0.72** 0.17 0.24
NBr or3.1 3 0 0–16 ZVG406 3.47 18.8 0.97* 0.47 0.48
or7.1 7 54 50–55 ZVG398 3.09 16.9 0.17 0.25 1.47
Total 2.72 15.0
PR or3.1 3 0 0–19 ZVG406 2.76 15.2 0.15** 0.04 0.27
or7.1 7 53 47–55 ZVG398 3.47 18.8 0.04 0.03 0.75
or13.1 13 15 6–17 ZVG59 2.72 15.0 0.07* 0.09* 1.28
or13.2 13 81 74–101 ZVG547 2.91 18.1 0.09** 0.02 0.22
Total 7.32 35.5
F3-E (CU-796) RS or3.1 3 0 0–10 ZVG406 11.1 59.4 0.88** 0.03 0.03
NBr or3.1 3 1 0–21 ZVG406 3.54 24.5 1.37** 1.47** 1.07
or13.2 13 81 80–92 ZVG547 3.09 25.2 0.49** 0.24 0.49
Total 8.12 47.5
Total epistasis 13.3 65.2
PR or.1.1 1 39 29–54 SAD17 3.32 23.5 0.07 0.12 1.71
or3.1 3 0 0–12 ZVG406 3.01 21.6 0.28** 0.17 0.61
or7.1 7 55 54–63 ZVG586 3.74 26.5 0.12* 0.07 0.58
or13.1 13 54 49–63 ZVG472 3.44 24.3 0.01 0.09 9.00
or13.2 13 87 71–96 ZVG547 4.31 33.3 0.16** 0.002 0.01
Total 8.64 59.0
Total epistasis 17.2 75.2
F2-F (SE-296) RS or1.1 1 63 46–64 ZVG1 2.63 11.3 0.11* 0.21** 1.91
NBr or1.1 1 54 39–60 SAD17 3.04 11.8 4.00** 3.00 0.75
or5.1 5 0 0–4 ZVG418 4.62 17.4 4.78** 1.51 0.32
or13.1 13 37 34–42 ZVG524 4.38 16.6 5.22** 2.17 0.42
Total 7.89 27.9
Total epistasis 10.3 34.8
F3-F (SE-296) RS or5.1 5 7 6–26 ZVG20 3.00 24.6 0.36** 0.21 0.58
or16.1 16 54 43–68 MS771 3.24 26.3 0.33** 0.09 0.27
Total 5.55 40.6
NBr or1.1 1 30 27–55 SAD17 2.80 23.4 1.91 2.47 1.29
or4.1 4 19 18–31 MS534 3.74 29.6 3.82** 1.48 0.39
or13.2 13 84 71–95 ZVG547 2.63 25.6 6.12** 2.91 0.53
or16.1 16 47 41–68 MS711 3.31 26.7 5.49** 0.33 0.06
Total 9.83 60.3
or13.1 13f42 25–51 ZVG524 2.07 17.7 1.75 3.55 2.03
PR or4.1 4 23 18–38 MS534 4.82 36.4 0.18** 0.01 0.05
or13.2 13 83 80–93 ZVG547 4.46 38.7 0.17** 0.04 0.24
or16.1 16 57 54–68 ZVG1023 4.45 34.2 0.19** 0.08 0.42
Total 10.5 62.8
aRS Resistance or susceptibility, NBr number of broomrape per F2plant or F3family, PR proportion of resistant plants within each F3
family
bAbsolute position from the top of the LG
cRegion flanking each QTL peak in which LOD scores decline by one
dLeft locus flanking the likelihood peak for a putative QTL. The MS prefix denotes SSR marker loci and the ZVG prefix denotes RFLP
marker loci
eaAdditive effect. For the traits NBr and RS, () means a decrease of the trait value due to P-96 alleles. For the trait PR, (+) means an
increase of the trait value due to P-96 alleles (an increase in the proportion of resistant plants). dDominant effect. djj=ajj= type of gene
action at each QTL. aand destimates, as well as total R2and LOD score values were obtained from a simultaneous fit of all putative QTL
using multiple regression
fQTL detected below the LOD threshold
Significance levels: **0.01 probability level, *0.05 probability level
97
Race F resistance
Three QTL associated with NBr were identified on LG1
(or1.1), LG5 (or5.1) and LG13 (or13.1) in the F2
evaluation for race F. The most significant QTL were
on LG5 (R2=17.4%) and LG13 (R2=16.6%). The other
QTL showed an R2value of 11.8% (Table 4). The three
QTL explained 27.9% of the phenotypic variation for
NBr. QTL on LG1 and LG13 were attributable to the
resistant parent (P-96) alleles, which served to decrease
the total number of broomrape (Table 4), whereas those
on LG5 was attributable to the susceptible parent (P-21).
The degree of dominance ranged from 0.75 to 0.32,
indicating that the three QTL were partially dominant.
The LOD support limit of 1.0 ranged from 21 cM for
or1.1 to 4 cM for or5.1 (Table 4). The test for digenic
Fig. 2 Quantitative trait loci
(QTL) map for broomrape re-
sistance in the P-21P-96 pop-
ulation for the traits (1) total
number of broomrape, NBr
(continuous lines), (2) resis-
tance or susceptibility, RS (da-
shed lines), and (3) proportion
of resistant plants, PR (dotted
lines). The QTL map generated
by CIM shows the likelihood of
odds (LOD) score on the y-axis
along each chromosome on the
x-axis. The axes are indepen-
dently scaled to accommodate
chromosome length and maxi-
mum LOD scores. The columns
represent results of the different
evaluations as follows: first
column F3race E evaluation
with broomrape population
SE-194, second column F3race
E evaluation with broomrape
population CU-796, third
column F2race F evaluation
with broomrape population
SE-296, and fourth column F3
race F evaluation with broom-
rape population SE-296
98
epistatic interactions was significant (P<0.05, additive
dominant) for or5.1 and or1.1 (Table 4). Additionally,
another significant QTL on LG 13 was detected when the
variation associated with or13.1 was removed in the F2
test (data not shown). According to its position and effect,
this QTL might correspond to or13.2.
Four QTL affecting NBr were identified on LG1
(or1.1), LG4 (or4.1), LG13 (or13.2) and LG16 (or16.1)in
the F3evaluation for race F. The four QTL jointly
explained 60.3% of the phenotypic variation. Individual
R2estimates for the four QTL were similar, ranging from
29.6% to 23.4% (Table 4). All the resistant-enhancing
alleles originated from the resistant parent P-96. A gene
action value of 1.29 was calculated for or1.1, which
indicated that this QTL might be overdominant in effect.
Partially dominant gene action was identified for or4.1
and or13.2, while or16.1 was additive in effect. The
or13.1 QTL was detected in the F3evaluation with an
LOD score below the threshold (Table 4).
Only or1.1 (R2=11.3%) was detected for the RS trait in
the F2evaluation for race F resistance (Table 4). In the F3
evaluation, or5.1 (R2=24.6%) and or16.1 (R2=26.3%)
were associated with the same trait (Table 4). Or1.1 and
or16.1 were attributable to the resistant parent (P-96)
alleles, whereas or5.1 was attributable to the susceptible
parent (P-21).
Three QTL for PR in the F3evaluation for race F
resistance were located on LG4 (or4.1,R2=36.4%), LG13
(or13.2,R2=38.7%) and LG16 (or16.1,R2=34.2%). The
positions of these QTL were similar to those of QTL
detected for the total number of broomrape. Increases in
the proportion of resistant plants were all due to P-96
alleles (Table 4). The effect of or4.1 was additive
d
jj=a
jj
¼0:05ðÞ, while a partially dominant gene action
was identified for or13.2 d
jj=a
jj
¼0:24ðÞand or16.1
d
jj=a
jj
¼0:42ðÞ.
Discussion
Phenotypic segregation for race E and race F
Phenotypic segregation for broomrape resistance indicat-
ed that resistance to race E in the P-96 line is dominant
and determined by alleles at one locus, as observed in the
CU-796 experiment, whereas resistance to race F in this
line is recessive and controlled by alleles at two loci.
These results are in agreement with previous reports for
race E (Sukno et al. 1999) and for race F (Rodrguez-
Ojeda et al. 2001; Aktouch et al. 2002).
The observed segregation ratio in the race E SE-194
experiment was significantly different from that expected
for a one-gene segregation. There was a deficiency in the
number of both susceptible and segregating F3families
and a corresponding excess of resistant F3families.
Previous studies using the same race E broomrape
population (SE-194) but infecting a different resistant
line (Sukno et al. 1999), or using a different race E
broomrape population (CU-796) in the same genetic
background (line P-96, this study), indicated a monogen-
ic, dominant inheritance to race E resistance. In addition,
none of the RFLP or SSR-marker loci in the P-21P-96
map deviated significantly from the expected one-gene
segregation ratios. In consequence, we concluded that the
segregation distortion from a 1:2:1 ratio observed in the
SE-194 experiment arose from misclassification of sus-
ceptible and segregating F3families as segregating and
resistant, respectively, caused by some susceptible plants
which escaped broomrape infection.
QTL analysis for race E resistance
One major QTL (or3.1) affecting race E resistance was
detected. This QTL was identified for all the resistance
parameters studied, and was stable over the two race E
experiments carried out in different environments and
with different race E broomrape populations. The dom-
inant gene controlling resistance to race E has been
designated Or5 in previous classical genetic studies in
which monogenic inheritance of resistance to race E of
broomrape was observed (Vrnceanu et al. 1980; Sukno et
al. 1999). This gene was mapped by Lu et al. (2000) and
Tang et al. (2003). Tang et al. (2003) placed the Or5 gene
in a telomeric region of LG3 of the public SSR linkage
map of sunflower, with the closest SSR marker mapping
6.2 cM proximal to the Or5 locus. The map position of
the QTL or3.1 detected on top of LG3 corresponds to the
region where Or5 was previously mapped. In fact, the
score of race E resistance in both the SE-194 and the CU-
796 race E tests mapped distal to the markers in LG3. The
closest marker (ZVG406) was 4.2 cM downstream from
the race E score in the F3-CU-796 test (Fig. 1). The
parental line P-96, which most likely contains the Or5
gene, contributed the positive allele at or3.1. Therefore,
we have strong evidence that the major QTL affecting
broomrape resistance to race E in the P-21P-96 popu-
lation (or3.1) represents the known qualitative Or5
resistance gene.
Three additional QTL affecting resistance to the race E
broomrape populations SE-194 and CU-796 were detect-
ed on LG7 (or7.1) and LG13 (or13.1 and or13.2). These
three QTL were associated with the proportion of resistant
plants for both race E populations, with the corresponding
disease-screening tests being performed in different
environmental conditions. This indicated that they are
stable across environments. Or7.1 also affected the total
number of broomrape in the SE-194 test, and or13.2 was
associated with the same trait in the CU-796 test. In
addition, a fourth QTL, or1.1, affecting the proportion of
resistant plants was only detected in the CU-796 test. It is
evident from these results that there are factors other than
Or5 that influence resistance to race E in the P-96 line.
Whilst the or3.1 QTL had a major effect on presence
or absence of race E broomrape (RS trait), other QTL
detected in the present research were associated with the
number of broomrape per plant. So far, broomrape
resistance to race E in sunflower has been considered a
99
qualitative trait, as simple inheritance patterns have
usually been found with clear resistant and susceptible
groups (Sukno et al. 1999; Lu et al. 2000; Tang et al.
2003). Our results suggest that resistance to race E in P-96
is not only the result of a major gene Or5, but it is also
composed of a quantitative component that influences the
number of broomrape per plant. Although the relative
weight of both mechanisms of resistance will probably be
genotype dependent, the results of the present research
revealed that the Or5 QTL accounted for a greater
proportion of the phenotypic variance than the QTL
associated with the number of broomrape per plant.
QTL analysis for race F resistance
In contrast to the preponderant role of the Or5 QTL
(or3.1) in resistance to race E, no equivalent major QTL
was identified for resistance to race F in P-96. The six
QTL associated with race F resistance identified in F2
and/or F3evaluations (or1.1,or4.1,or5.1,or13.1,or13.2
and or16.1) accounted for similar and moderate percent-
ages of the phenotypic variance for broomrape race F
resistance. One possibility is that major QTL do exist, but
they were missed due to incomplete genome coverage, or
their magnitudes were reduced by increased variation due
to environmental effects. However, race F tests were
performed in two completely different environments,
which gave similar results. In addition, the F3score for
race F (i.e. considering race F resistance as a major
phenotypic locus) failed to map in any of the 17 LGs from
the P-21 P-96 linkage map. Finally, race F resistance
was mainly explained by the NBr trait, in contrast to a
predominant role of the RS trait in race E resistance. The
percentage of the phenotypic variance of individual QTL
explained by the RS trait was similar to that explained by
the NBr trait for race F, whereas the RS trait gave
significantly higher R2values for race E resistance. In
addition, isolation of the P-96 line for resistance to race F
from a germplasm accession required several cycles of
selection, which is not consistent with a resistance
controlled by a major qualitative locus. It therefore seems
likely that race F resistance is controlled by several QTL
with a small to moderate effect, despite the existence of a
major QTL can not be completely ruled out.
Or1.1,or5.1,or13.1 and or13.2 were detected in the
evaluation of both F2and F3generations, conducted under
different environments, indicating stability across envi-
ronments. By contrast, or4.1 and or16.1 were only
detected in one environment. In addition to their envi-
ronmental stability, or1.1,or13.1 and or13.2 were non-
race specific, as they were also identified in at least one of
the experiments conducted with race E. Such QTL in the
same genome region affecting both race E and race F of
broomrape may result from linkage or pleiotropy. Link-
age would be expected since the majority of plant
resistance genes appear to be organized as clusters,
conferring resistance to different races of a pathogen
(Michelmore and Meyers 1998). On the other hand, the
role of pleiotropy should also be investigated. Common
QTL affecting resistance to both race E and race F could
also result from the same components in the mechanism
of resistance to these two races. The existence of QTL
determining resistance to different races of a pathogen is
well documented, and it has been demonstrated for other
parasitic angiosperms-plant interactions such as Striga
gesnerioides attacking cowpea [Vigna unguiculata (L.)
Walp.] (Oudraogo et al. 2001) and a number of plant-
pathogens systems (Jeuken and Lindhout 2002; Chen et
al. 2003; Udupa and Baum 2003).
Disease resistance in plants can be classified into two
major types (Vanderplank 1982). Various terms have
been used to describe the two types of resistance, such as
vertical versus horizontal resistance, qualitative versus
quantitative resistance and complete versus partial resis-
tance. Qualitative or ‘vertical’ resistance is modulated by
the interaction between a disease-resistance gene in the
host plant and an avirulence gene in the pathogen
population (Flor 1971) and is specific to pathogen race.
Quantitative or ‘horizontal’ resistance, on the other hand,
is associated with numerous genes having smaller effects
but presumably acting against a broad spectrum of
pathogenic races (Nelson 1972). The results of the present
research suggest that broomrape resistance in sunflower is
composed of both qualitative and quantitative compo-
nents. Dominant resistance to race E has a major
qualitative component determined by the main race E
QTL (or3.1), which is associated with presence or
absence of broomrape. Conversely, recessive resistance
to race F is mainly conferred by QTL that jointly
contribute with a similar, small-to-moderate effect in
decreasing the number of broomrape. Some of the latter
QTL are also associated to the quantitative component of
race E resistance. Plant disease resistance to specific
pathogens has been reported previously to show both
qualitative and quantitative components (Young 1996; Li
et al. 2001; Jeuken and Lindhout 2002).
The differences among the resistance QTL detected in
this study imply that they might function in different
pathways in the sunflower defensive system. The pre-
dominant role of or3.1 in race E resistance, its association
mainly with the RS qualitative trait, and its race speci-
ficity indicate that it may play a role in an early stage of
the plant-pathogen interaction (i.e. pathogen recognition).
In contrast, the nature of or1.1,or13.1 and or13.2 and its
lack of race specificity suggest a possible regulatory role
in the more downstream mechanisms of the sunflower
defensive system. Thus, identifying and mapping candi-
date genes underlying the QTL associated with broom-
rape resistance would greatly enhance our understanding
of the defence system of sunflower.
In conclusion, this study revealed the complex nature
of sunflower resistance to broomrape and demonstrated
that identification of QTL involved in this character is
possible in cultivated sunflower genotypes. Genetic dis-
section of broomrape resistance in sunflower provided
insight into race-specific resistance and revealed that
resistance to broomrape seems to be controlled by a
100
combination of qualitative race-specific resistance affect-
ing the presence or absence of broomrape and a quanti-
tative, non-race-specific resistance affecting their number.
The consistency of the resistance QTL identified in the P-
21P-96 cross will have to be further evaluated over
years, locations, new broomrape races, genetic back-
grounds, screening conditions and evaluation criteria in
order to validate their usefulness for marked assisted
selection (MAS). The identification of new resistant loci
from other sources is also an objective which is being
currently carried out in order to accumulate multiple
resistance alleles in a genotype. An additional approach
will be to begin identifying candidate genes underlying
sunflower broomrape resistance.
Acknowledgements B.P.V. was supported first by a post-doctoral
grant from the Spanish Government (MEC, Ref. PF 98 28731590),
and thereafter by a post-doctoral contract from the ‘Ramn y Cajal’
program (MCYT).
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