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Genetic Mapping of the Or
5
Gene for Resistance to Orobanche Race E in Sunflower
Shunxue Tang, Adam Heesacker, Venkata K. Kishore, Alberto Fernandez, El Sayed Sadik,
Glenn Cole, and Steven J. Knapp*
ABSTRACT
and widely used are single dominant genes (Burlov and
Kostyuk, 1976; Pogorletsky and Geshele, 1976; Vran-
Orobanche cumana Wallr. (⫽ O. cernua Loefl., broomrape), a
ceanu et al., 1980; Burlov and Artemenko, 1983; Ish-
weedy parasitic plant, is a serious pest of cultivated sunflower (Helian-
thus annuus L.). Breeding for resistance has been crucial for protecting Shalom-Gordon et al., 1993; Sukno et al., 1998, 1999; Lu
sunflowers from broomrape damage, a challenging task because new
et al., 2000). The development of Orobanche-resistant
races of the pathogen continually emerge and ultimately defeat known
inbred lines is complicated by the weedy and noxious
resistance genes. Despite several attempts to identify DNA markers
characteristics of the parasite, the need for geographic
tightly linked to Orobanche resistance genes, the closest reported thus
containment, susceptible escapes and other screening
far is 5.6 centimorgans (cM) downstream of Or
5
, a gene for resistance
variability, the genetic complexity of physiological races
to Race E. The Or
5
locus was placed on the simple sequence repeat
of the pathogen, genetic background effects, and geno-
(SSR) map of sunflower by genotyping and phenotyping 262 recom-
type ⫻ environment interactions; hence, Orobanche re-
binant inbred lines (RILs) from a cross between elite inbred lines (PHC ⫻
sistance is an ideal target for molecular breeding. De-
PHD) segregating for resistance to Orobanche Race E. Polymerase
spite the complexities underlying Orobanche resistance
chain reaction (PCR) multiplexes were used to screen 78 SSR marker
loci, strategically positioned throughout the genome, for polymor- breeding in sunflower, race-specific dominant genes seem
phisms between resistant and susceptible bulks of PHC ⫻ PHD RILs.
to protect the crop and are ideal sources of resistance
The bulks were polymorphic for three of five Linkage Group 3 (LG3)
for single-cross hybrid breeding because they only need
SSR marker loci amplified by the PCR multiplexes. The RILs were
be incorporated into one parent or the other. Moreover,
phenotyped for resistance to Race E and genotyped for 13 SSR mark-
allelic and nonallelic resistance genes can be pyramided
ers from the upper end of LG3. The Or
5
locus mapped to the end of
by working opposite sides of a hybrid pedigree.
LG3 distal to the SSR marker loci (the closest SSR marker locus
The first Orobanche-resistant sunflowers were devel-
was 6.2 cM downstream of Or
5
. The terminal and perhaps telomeric
oped by introgressing resistance genes from Jerusalem
location of Or
5
on LG3 sheds light on difficulties, past and present,
artichoke (H. tuberosus L.) to cultivated sunflower (Vran-
of identifying flanking DNA markers tightly linked to Or
5
.
ceanu et al., 1980). The first (Race A) resistant cultivars
(Kruglik A-41 and Saratovsky 169) were developed by
1916 (Pustovoit, 1976; Parker and Riches, 1993). Resis-
B
roomrape is a weedy parasitic plant and serious pest
tance to Race A was overcome by the emergence of the
of cultivated sunflower in Europe, especially south-
more virulent Race B by 1928 (Pustovoit, 1976; Parker
ern Europe, the Balkans, and the Mediterranean (Par-
and Riches, 1993). By 1935, open-pollinated cultivars
ker and Riches, 1993). Seed yield losses from broomrape
(e.g., Jdanovsky 8281 and 8885) resistant to Race B had
infestations in susceptible sunflower genotypes can be
been developed. Resistance to Race B was ultimately
substantial (Bulbul et al., 1991; Parker and Riches, 1993;
transferred to Peredovik and VNIIMK1646 (Melero-
Shindrova, 1994; Dominguez, 1996b; Blamey et al., 1997).
Vara et al., 1989; Fernandez-Martinez et al., 2000). By
Because O. cumana has a broad host range and produces
the early 1960s, Race A and B resistance genes were
an extraordinarily large number of small, long-lived,
defeated by the emergence of Race M, purportedly, a
facilely dispersed seeds, control through crop manage-
complex of 17 to 22 highly virulent subraces (Petrov,
ment has been difficult (Ish-Shalom-Gordon et al., 1993;
1968; Melero-Vara et al., 1989).
Parker and Riches, 1993; Ruso et al., 1996; Sukno et al.,
Vranceanu et al. (1980, 1986), in a classic study, identi-
1999; Roman et al., 2001). The primary line of defense
fied five physiological races (A to E) of Orobanche by
against broomrape, other than quarantine, has been ge-
using five dominant genes (Or
1
, Or
2
, Or
3
, Or
4
, and Or
5
)
netic resistance (Sackston, 1992; Ruso et al., 1996; Sukno
resistant to Race A, A ⫹ B, A ⫹ B ⫹ C, A ⫹ B ⫹ C ⫹
et al., 1999; Lu et al., 2000).
D, and A ⫹ B ⫹ C ⫹ D ⫹ E, respectively. The five races
While various genetically simple and complex sources
were subsequently identified and substantiated in other
of Orobanche resistance have been described in sun-
analyses and geographic regions (Melero-Vara et al.,
flower (Pustovoit, 1976; Russell, 1981; Krokhin, 1983;
1989; Bulbul et al., 1991; Saaverdra del Rio et al., 1994a;
Kirichenko et al., 1987; Ramaiah, 1987; Saaverdra del Rio
Shindrova, 1994). Vranceanu et al. (1980, 1986) identi-
et al., 1994b; Dominguez, 1996a), the most important
fied homozygous differentials for Or
1
, Or
2
, Or
3
, Or
4
, and
Or
5
for discriminating between the races and resistance
S. Tang, A. Heesacker, V.K. Kishore, and S.J. Knapp, Dep. of Crop
and Soil Sci., Oregon State Univ., Corvallis, OR 97331-3002, USA;
A. Fernandez, Pioneer Hi-Bred Int., Avda. Reino Unido s/n, Edificio
Abbreviations: AFLP, amplified fragment length polymorphism; BSA,
Aditec 2a Planta, 41012 Sevilla, Spain; E.S. Sadik, Pioneer Hi-Bred
bulked segregant analysis; cM, centimorgan; CMS, cytoplasmic-genic
Int., Belediye Binasi Kat 3, Ahmetbey-Kirklareli, Turkey; and Glenn
male sterility; LOD, likelihood odds; MAS, marker-assisted selection;
Cole, Pioneer Hi-Bred Int., 18285 County Road 96, Woodland, CA
PCR, polymerase chain reaction; RAPD, random amplified polymor-
95695-9340, USA. Received 12 Sept. 2002. *Corresponding author
phic DNA; RIL, recombinant inbred line; RFLP, restriction fragment
(steven.j.knapp@orst.edu).
length polymorphism; SCAR, sequence characterized amplified re-
gion; SSR, simple sequence repeat.Published in Crop Sci. 43:1021–1028 (2003).
1021
1022 CROP SCIENCE, VOL. 43, MAY–JUNE 2003
lines (PHC and PHD) developed by Pioneer Hi-Bred Interna-
genes (Kruglik A-41 for Race A, Jdanovsky 8281 for
tional (Johnston, IA). PHC is a cytoplasmic-genic male sterile
Race B, Record for Race C, S-1358 for Race D, and
(CMS) maintainer susceptible to Orobanche RaceE(or
5
or
5
).
P-1380 for Race E). Similar to many of the downy mil-
PHD is a CMS fertility restorer resistant to Orobanche Race
dew [Plasmopara halstedii (Farl.) Berl. & de Toni in
E(Or
5
Or
5
). Seed was produced in Woodland, CA, by bagging
Sacc.] resistance genes described in sunflower (Mouze-
and separately harvesting individuals from each of 262 F
2
yar et al., 1995; Roeckel-Drevet et al., 1996, Vear et al.,
lineages for four generations (F
5
seed was produced by bulking
1997), new Orobanche resistance genes often confer
five individuals per RIL). Leaves were harvested from ten
resistance to earlier races. Several analyses in segregat-
3-wk-old greenhouse-grown F
5
seedlings from each RIL. DNA
ing populations have shown that Or
1
to Or
5
are either
was isolated from bulked fresh leaf samples using a modified
allelic or tightly linked (Vranceanu et al., 1980, Ish-
CTAB method (Webb and Knapp, 1990).
Shalom-Gordon et al., 1993; Sukno et al., 1998, 1999;
Fernandez-Martinez et al., 2000).
Orobanche Resistance Phenotyping
Because physiological races of broomrape seem to
The parents and RILs (F
5
seedlings) were screened for
rapidly evolve or are exposed by selection pressure
resistance to broomrape Race E in a growth chamber at Pio-
stemming from the broad deployment of individual re-
neer Hi-Bred Agroservicios Spain S.L., Sevilla, Spain, in 2000
sistance (R) genes, the search is continually on for new
using seeds of the Race E broomrape population collected
resistant gene specificities (Sackston, 1992; Dominguez
from Ecija, Sevilla, Spain, in the summer of 1998. Broomrape
et al., 1996; Ruso et al., 1996; Sukno et al., 1998, 1999;
seeds were collected from plants infesting the hybrid Florasol
Fernandez-Martinez et al., 2000). During the last several
(resistant to Race D, but susceptible to Race E) in a nursery
where several hundred rows of Race E-resistant hybrids were
years, resistance to Race E (conferred by Or
5
) has been
grown and none were infected. The broomrape seeds were
defeated by the emergence of Race F in Spain. Genes
homogeneously mixed with a 1:1 mixture of sand and peat at
for resistance to the new race have been identified in
the rate of 250 mg per kg. PHC, PHD, two susceptible controls
cultivated and wild sunflowers and seem to confer resis-
(Coronil and Florasol), and the RILs were screened for resis-
tance to earlier races (A through E), as has been the
tance to Orobanche. Five seeds of each entry were planted in
historical pattern (Vranceanu et al., 1980, 1986; Melero-
6- ⫻ 10- ⫻ 10-cm plastic pots filled with the infested soil mix-
Vara et al., 1989; Dominguez, 1996a; Ruso et al., 1996;
ture. The plants were grown under a 14-h photoperiod using
Gagne et al., 1998; Sukno et al., 1998, 1999; Fernandez-
25⬚C day and 18⬚C night temperatures and constant ≈60%
Martinez et al., 2000).
humidity. Two-month-old plants were carefully removed from
The development of inbred lines resistant to Oro-
the pots to phenotype for the presence or absence of emerged
banche can be accelerated by marker-assisted selection
or underground broomrape stalks (nodules). RILs with no
infected plants were scored as resistant (R), RILs with 100%
(MAS), for example, by identifying and culling hetero-
infected plants were scored as susceptible (S), and RILs with
zygotes and susceptible escapes (phenotyping errors)
a mixture of infected and uninfected plants were scored as
and performing genotypic selection on seedlings and
segregating (H).
other preanthesis growth stages. Or
5
has not been placed
on the SSR map of sunflower, and few DNA markers are
Bulked Segregant Analysis and SSR Genotyping
presently found in the region surrounding Or
5
.Luetal.
(2000) used bulked segregant analysis (BSA) (Michel-
Bulked segregant analysis (Michelmore et al., 1991) was
more et al., 1991) to identify a random amplified poly-
performed by screening 78 SSR marker loci amplified by the
PCR-multiplexes (13 six-plexes) described by Tang et al. (2003).
morphic DNA (RAPD) marker (UBC120_660) and five
Resistant (R) and susceptible (S) bulks were produced by
DNA sequence characterized amplified region (SCAR)
pooling equal quantities of DNA from 10 putatively homozy-
markers (RTS05, RTS28, RTS40, RTS29, and RTS41)
gous resistant and 10 putatively homozygous susceptible RILs,
linked to Or
5
. The RAPD and SCAR markers and Or
5
respectively. Two independent R and S bulks were produced
were mapped in two segregating populations (Lu et al.,
and screened. The 78 SSR markers were screened for polymor-
2000) and the SCAR markers were placed on LG17 of
phisms between PHC and PHD and between the replicate
the CARTISOL restriction fragment length polymor-
bulked DNA samples using the PCR-multiplexes and genotyp-
phism (RFLP) map (Lu et al., 1999). The closest SCAR
ing methods described by Tang et al. (2003). The forward prim-
marker mapped 5.6 cM distal to Or
5
, and no DNA mark-
ers in each six-plex were labeled with different combinations
ers other than the RAPD (22.5 cM upstream) flanked Or
5
.
of fluorophores (6FAM, HEX, TET, or NED) to facilitate mul-
The goal of the present study was to identify SSR
tiplex genotyping. PHC, PHD, and the bulks were subsequently
screened for polymorphisms using 11 additional SSR markers
markers tightly linked to Or
5
and, in the process, to place
from the upper end of LG3 (CRT392, CRT314, ORS1040,
the Or
5
locus on the public molecular genetic linkage
ORS1112, ORS683, ORS372, ORS820, CRT197, ORS777, ORS-
map of sunflower (Tang et al., 2002; Yu et al., 2003).
657, and ORS1021). The RILs were genotyped for 13 SSR
Secondarily, we tested the utility and sensitivity of fluo-
markers found to be polymorphic between PHC and PHD and
rescent PCR-multiplex SSR genotyping (Tang et al.,
between R and S bulks. The genotyping assays were performed
2003) as a tool for screening bulked-segregant DNA
using post-PCR multiplexing.
samples (Michelmore et al., 1991) in sunflower.
Genetic Analyses and Map Construction
MATERIALS AND METHODS
The expected segregation ratio for Or
5
among F
5
RILs was
Plant Materials
0.4375 homozygous-resistant (Or
5
Or
5
) to 0.125 segregating
(3 Or
5
__:1 or
5
or
5
) to 0.4375 homozygous susceptible (or
5
or
5
).
Two hundred and sixty-two F
5
RILs were developed by sin-
gle seed descent from a cross between two proprietary inbred The fit of the observed ratio of Orobanche resistance pheno-
TANG ET AL.: OROBANCHE RESISTANCE IN SUNFLOWER 1023
types to the expected ratio of Or
5
genotypes was checked
one or more recombinants. Predictably, the intensity of
using
2
statistics. The RIL mapping function of MAPMAKER
the susceptible allele signals in resistant bulks increased
(Lander et al., 1987) was used to construct a genetic linkage
(Fig. 2) as map distances between the SSR marker loci
map for LG3 among the PHC ⫻ PHD RILs. Loci were
and Or
5
locus increased (Fig. 3).
grouped using a likelihood odds (LOD) threshold of 10.0 and
map distances (cM) were calculated using the Kosambi (1944)
Genetic Mapping of the Or
5
Locus
mapping function.
The SSR markers identified to be polymorphic be-
tween PHC and PHD and R and S bulks were genotyped
RESULTS
on 262 PHC ⫻ PHD RILs. Eight SSR markers ampli-
Segregation of Resistance to Orobanche Race E
fied a single polymorphic locus each and five SSR mark-
The susceptible parent (PHC) and susceptible con-
ers amplified two or three polymorphic loci each. CRT-
trols (CORONIL and FLORASOL) were completely
392 amplified two loci on LG9 and one locus on LG3
infected, whereas the resistant parent (PHD) was not
(CRT392-3). ORS683 amplified two cosegregating du-
infected by Orobanche Race E. The observed segrega-
plicated loci on LG3 (Fig. 2). ORS1040 amplified two un-
tions ratio for Orobanche resistance phenotypes (54 sus-
linked polymorphic loci, one on LG3 (ORS1040-3) and
ceptible:70 segregating:138 resistant) was significantly
one on LG12 (ORS1040-12). Similarly, ORS1112 ampli-
different from the expected segregation ratio for Or
5
fied two unlinked polymorphic loci, one on LG3 (ORS-
genotypes (114.625 or
5
or
5
:32.75 Or
5
or
5
:114.625 Or
5
Or
5
)
1112-3) and one on LG10 (ORS1112-10), and CRT197
among the PHC ⫻ PHD RILs (
2
⫽ 54.826, P ⬍ 0.001).
amplified two unlinked polymorphic loci, one on LG3
We observed a deficiency of susceptible (⫺60.625) and
(CRT197-3) and one on LG17 (CRT197-17) (Tang et
excesses of segregating (⫹37.25) and resistant (⫹23.375)
al., 2002; Yu et al., 2003) (Fig. 2, 3). The SSR marker
RILs. The segregation distortion undoubtedly arose from
loci, apart from duplicate loci known to map to different
misclassifying homozygous susceptible RILs (or
5
or
5
) with
linkage groups, grouped together and none had signifi-
escapes (resistant plants) as segregating and segregat-
cantly distorted segregation ratios. Furthermore, the ob-
ing RILs (3 Or
5
__: or
5
or
5
) with escapes (no susceptible
served heterozygote frequency across SSR marker loci
plants) as resistant.
(0.112) was not significantly different from expected
heterozygote frequency (0.125).
Bulked Segregant Analysis
The locus orders for SSR markers on the PHC ⫻
PHD RIL and reference genetic linkage maps were
Two replicate samples of 10 susceptible and 10 resis-
identical (Fig. 3). Or
5
mapped distal to the SSR marker
tant RILs were selected for producing and screening
loci and was the upper terminus of LG3 (Fig. 3), as
bulked-segregant DNA samples. Forty-three out of 78
demarcated by SSR marker loci on the genetic linkage
SSR markers amplified by the PCR-multiplexes (Tang
map (Burke et al., 2002; Tang et al., 2002; Yu et al.,
et al., 2003) were polymorphic between PHC and PHD.
2003). The closest proprietary SSR marker (CRT392)
When screened on replicate R and S bulks, three SSR
was 6.2 cM downstream of Or
5
, while the closest public
markers from LG3 (ORS1222 in Set 1, ORS1036 in Set 11,
SSR marker (ORS1036) was 7.5 cM downstream of the
and ORS1114 in Set 13) were polymorphic (Fig. 1),
Or
5
locus.
suggesting that Or
5
might reside on LG3. None of the
SSR markers from other linkage groups were polymor-
phic between R and S bulks. The other two SSR marker
DISCUSSION
loci on LG3 amplified by the PCR multiplexes (ORS665
Marker-Assisted Selection for
in Set 4 and ORS949 in Set 7 of the PCR multiplexes)
Orobanche Resistance
were monomorphic between PHC and PHD.
Eleven additional SSR markers (CRT392, CRT314, The telomeric or near-telomeric location of Or
5
sheds
light on why several hundred DNA markers had to beORS1040, ORS1112, ORS683, ORS372, ORS820, CRT-
197, ORS777, ORS657, and ORS1021) were selected screened to identify loci linked to Or
5
, why the closest
SSR marker is 6.2 cM downstream of Or
5
, and why nofrom the upper end of LG3 (Tang et al., 2002; Yu et al.,
2003) and screened for polymorphisms between PHC DNA markers other than an unconfirmed RAPD
marker (Lu et al., 1999, 2000) flank Or
5
. The presentand PHD and between R and S bulks. Simple sequence
repeat marker loci flanking ORS1036 were selected for analysis drew on ≈700 mapped SSR marker loci (Burke
et al., 2002; Tang et al., 2002; Yu et al., 2003) and, throughscreening because polymorphisms between R and S
bulks seemed more intense for ORS1036 than ORS1222 comparisons to other maps, ≈900 mapped RFLP marker
loci (Berry et al., 1995, 1996, 1997; Gentzbittel et al.,and ORS1114, suggesting that Or
5
might be more tightly
linked to the former than the latter (Fig. 1). Other than 1995, 1999; Gedil et al., 2001). Of ⬎1600 RFLP and
SSR marker loci mapped in sunflower thus far, onlyORS820 (a dominant SSR marker in PHC ⫻ PHD), the
selected SSR markers were found to be polymorphic three are within 6 to 10 cM of the Or
5
locus. The map
distances between Or
5
and the DNA markers reportedbetween R and S bulks (Fig. 2). The susceptible bulks
only produced PHC alleles (alleles from the susceptible here (Fig. 3) and elsewhere (Lu et al., 1999, 2000) could
be upwardly biased by phenotyping errors because sus-parent), whereas some of the resistant bulks produced
faint PHC allele signals, in addition to strong PHD allele ceptible escapes misclassified as resistant introduce spu-
rious recombinants and inflate map distances.signals, as predicted for bulk DNA samples harboring
1024 CROP SCIENCE, VOL. 43, MAY–JUNE 2003
Fig. 1. Sunflower simple sequence repeat (SSR) marker genotypes
amplified by polymerase chain recation (PCR) multiplex Sets 1
(Lanes 1–6), 11 (Lanes 7–12), and 13 (Lanes 13–18) on PHC, two
Orobanche resistant PHC ⫻ PHD recombinant inbred line (RIL)
bulks, two Orobanche susceptible PHC ⫻ PHD RIL bulks, and
Fig. 2. Genotypes for eight sunflower simple sequence repeat markers
PHD (shown in order for each PCR-multiplex set). The white ar-
screened on PHC (Lane 1), an Orobanche resistant PHC ⫻ PHD
rows highlight polymorphisms for three SSR markers on Linkage
recombinant inbred line (RIL) bulk (Lane 2), an Orobanche sus-
Group 3 (ORS1222 in Set 1, ORS1036 in Set 11, and ORS1114 in
ceptible PHC ⫻ PHD RIL bulk (Lane 3), and PHD (Lane 4).
Set 13).
TANG ET AL.: OROBANCHE RESISTANCE IN SUNFLOWER 1025
SSR (Burke et al., 2002; Tang et al., 2002; Yu et al.,
2003) maps of sunflower are missing 20 or more cM on
the upper end of LG3 (Fig. 3), and none of the ⬎100
SSR and RFLP markers mapped to LG3 reside in the
interval between Or
5
and UBC120_660.
The goal of identifying flanking DNA markers tightly
linked to Or
5
has not been met, partly because the Or
5
locus resides in a telomeric or near-telomeric region of
apparently high recombination. Because none of the
DNA markers described thus far are tightly linked to
or flank Or
5
, MAS is presently limited to the centro-
meric side of the Or
5
locus, and Or
5
genotypes cannot
be unequivocally identified from SCAR or SSR marker
genotypes. However, less than one in 12 individuals
selected for ORS1036 SSR marker genotypes should be
recombinant for Or
5
. The location of Or
5
in a region
of apparently high recombination is advantageous for
breeding and potentially advantageous for map-based
cloning (Zhang et al., 1994; Tanksley et al., 1995; Qi and
Gill, 2001), and has almost certainly played an important
role in the recovery of Or
5
recombinants in H. tuberosus
(hexaploid) ⫻ H. annuus (diploid) and other interspe-
cific segregating populations (Vranceanu et al., 1980; Ruso
et al., 1996; Sukno et al., 1999). The scarcity of DNA
markers near Or
5
could stem from a scarcity of DNA
polymorphisms; however, because Or
5
and other Oro-
banche resistance genes have been introgressed from
wild sunflowers, a scarcity of DNA polymorphisms in
the dragged DNA segments seems improbable. Quite the
contrary, DNA sequences flanking Orobanche resis-
tance genes from wild sunflowers should be extraordi-
narily polymorphic when compared with DNA sequences
commonly found in elite inbred lines of cultivated sun-
flower (Tang and Knapp, 2003).
The SSR markers described here complement the
RAPD and SCAR markers developed by Lu et al. (2000)
Fig. 3. Composite (left) and PHC ⫻ PHD recombinant inbred line
for MAS of broomrape resistance genes in sunflowers.
(right) maps for Linkage Group 3 of sunflower. Simple sequence
Two proprietary (CRT392 and CRT314) and two public
repeat marker loci amplified by the polymerase chain reaction
(ORS1036 and ORS1040) SSR markers are located within
(PCR) multiplexes (ORS1036, ORS665, ORS1222, ORS949, and
6.2 to 11.2 cM of Or
5
. The heterozygosities of the four
ORS1114) are shown in underlined boldface type.
SSR markers among elite inbred lines fall in the range
of 0.41 (ORS1036) to 0.73 (CRT392) (Tang et al., 2003;
Of the two SCAR markers closest to Or
5
(Lu et al.,
unpublished data) and supply much needed molecular
2000), RTS05 failed to amplify bands from PHC and
marker diversity and polymorphic DNA sequences in
PHD, whereas RTS28 amplified monomorphic bands
the Or
5
region. DNA sequences for the SSR markers
from PHC and PHD (data not shown). RTS05 was 5.6
have been deposited in public databases (Tang et al.,
and RTS28 was 13.6 cM downstream of Or
5
on the map
2002; Yu et al., 2002). ORS1036 was mapped for the
of Lu et al. (2000), while the closest RFLP marker was
first time in PHC ⫻ PHD, whereas the other SSR mark-
7.1 cM downstream of Or
5
(Gentzbittel et al., 1995, 1999;
ers had been mapped in other populations (Tang et al.,
Lu et al., 1999). None of the SCAR or RFLP markers
2002; Yu et al., 2003). ORS1036, the closest public SSR
mapped upstream of Or
5
; however, using a LOD thresh-
marker to Or
5
, amplified 245- and 255-base-pair-long
old of 1.4, Lu et al. (2000) placed a RAPD marker lo-
alleles from PHC and PHD, respectively, an allele length
cus (UBC120_660) 22.5 cM upstream of Or
5
on LG3.
difference long enough for genotyping on agarose.
Because of the low LOD threshold (high probability of
ORS1036 and CRT314 seem to be universally codom-
Type I error), the placement of UCB120_660 is tenuous
inant, whereas CRT392 and ORS1040 are mixed domi-
and must be rechecked. UBC120_660 RAPD primers
nant-codominant (Tang et al., 2003; unpublished data).
failed to amplify alleles from PHC and PHD; thus, we
CRT392 and ORS1040 were dominant in PHC ⫻ PHD.
could not substantiate or refute the location of UBC-
Similar to many other SSR markers in sunflower (Tang
120_660 upstream of Or
5
. If the reported position of the
et al., 2002, 2003), CRT392 and ORS1040 produce three
RAPD marker locus is correct (Lu et al., 2000), then
or more alleles (one being null) among diverse germplasm
the RFLP (Berry et al., 1995, 1996, 1997; Gentzbittel et
al., 1995, 1999; Jan et al., 1998; Gedil et al., 2001) and accessions and, consequently, are dominant in some
1026 CROP SCIENCE, VOL. 43, MAY–JUNE 2003
crosses and codominant in others. Presumably, the null (Powell et al., 1996), they can be greatly increased by
pre- and post-PCR multiplexing (Tang et al., 2003). Typ-alleles (PCR amplification failures) were caused by sin-
gle nucleotide polymorphisms in DNA sequences tar- ically, the multiplex ratios for multiplexed SSR markers
range from six to 14, and are on par with or greatergeted by the SSR primers (Mogg et al., 2002).
On the basis of the distribution of gene-rich regions than the multiplex ratios for RAPDs, but less than the
multiplex ratios for AFLPs (Powell et al., 1996).and ratios of physical-to-genetic distance in centromeric
and telomeric regions in other taxa (Ganal et al., 1989; We screened independent replicates of the R and S
bulks to check the sensitivity of the PCR- multiplexesSegal et al., 1992; Zhang et al., 1994; Tanksley et al.,
1995; Umehara et al., 1995; Gill et al., 1996a,b; Ku
¨
nzel for identifying SSR marker polymorphisms in bulked
DNA samples. The mapped markers (a mixture of dom-et al., 2000; Qi and Gill, 2001), we speculate that the
region flanked by Or
5
and the next closest DNA markers inant and codominant SSR markers) permitted a retro-
spective analysis of the sensitivity of fluorescent SSR(RTS05, CRT392, and ORS1036) may be physically
shorter than randomly selected and centromeric regions marker assays for BSA. The codominant SSR markers
were sensitive to allele dosage differences. When recom-in the sunflower genome. The density of SSR marker
loci was lowest in the distalmost and highest in the binants were present in bulks screened with codominant
SSR markers, differences in DNA template concentra-centermost regions of the genetic linkage map for LG3
(Tang et al., 2002; Yu et al., 2003) (Fig. 3), the classic tions (allele dosages) between bulks yielded progres-
sively different band intensities as a function of the num-pattern for a metacentric chromosome (Gill et al.,
1996a,b; Ku
¨
nzel et al., 2000). The density of SSR mark- ber of recombinants between Or
5
and the SSR marker
loci (Fig. 2, 3). Dominant SSR polymorphisms can onlyers was 2.54 cM per locus in the upper 17.8 cM (the
region distal to ORS1112-3), 4.78 cM per locus in the be observed between R and S bulks when recombinants
are absent in both bulks or when recombinants are ab-lower 33.5 cM (the region distal to ORS149), and 1.05
cM per locus in the centermost 35.8 cM region between sent in null allele bulks only, for example, when the sus-
ceptible parent is homozygous for the null allele andORS1112-3 and ORS149 on LG3 (Fig. 3). DNA markers
densities are typically lower in telomeric than centro- the susceptible bulk lacks recombinants or when the
resistant parent is homozygous for the null allele andmeric regions, recombination tends to be greater in
gene-rich than gene-poor regions, and ratios of physical- the resistant bulk lacks recombinants. ORS1112-3 and
ORS1040-3 fell in this category (Fig. 3). Conversely,to-genetic distance tend to be less in telomeric than
centromeric regions because of suppressed recombina- when the susceptible parent is homozygous for the dom-
inant allele and recombinants are present in the resistanttion in the latter (Ganal et al., 1989; Brown and Sundare-
san, 1991; Segal et al., 1992; Zhang et al., 1994; Tanksley bulk or when the resistant parent is homozygous for the
dominant allele and recombinants are present in theet al., 1995; Pedersen et al., 1995; Gill et al., 1996a,b;
Schnable et al., 1998; Ku
¨
nzel et al., 2000; Sandhu et al., susceptible bulk, then bands of equal intensity are am-
plified from both bulks and the dominant SSR polymor-2001; Qi and Gill, 2001).
phism is not observed between R and S bulks. ORS820
fell in this category and, as previously noted, was poly-
The Utility and Sensitivity of PCR-Multiplexes
morphic between PHC and PHD but not between R
for Identifying SSR Polymorphisms in
and S bulks.
Mixed DNA Samples
The number of recombinants between Or
5
and the
The present study was our initial test of the utility
SSR marker loci in bulks was ascertained from the SSR
and sensitivity of the PCR-multiplexed SSR markers
genotypes of CRT392, ORS1036, CRT314, ORS1040, and
(Tang et al., 2003) for bulked-segregant analysis in sun-
ORS1222 among the RILs pooled to create the bulks.
flower. The PCR-multiplexes facilitated a rapid scan of
CRT392, ORS1036, CRT314, and ORS1040 mapped 6.2
the sunflower genome for SSR markers linked to Or
5
.
to 11.2 cM downstream of Or
5
, had up to three recombi-
The analysis was completed by performing 78 PCRs (six
nant alleles out of 20 alleles per bulk, and sharply dis-
DNA samples ⫻ 13 six-plexes), but could have been
criminated between R and S bulks (Fig. 1, 2). ORS1036
completed by performing as few as 26 PCRs (screening
and ORS1222 are codominant SSR markers from oppo-
one set of R and S bulks) (Fig. 1). Historically, bulked
site ends of the region on LG3 mapped in PHC ⫻ PHD
segregant analyses have been performed, out of neces-
(Fig. 3). One out of 20 ORS1036 (7.5 cM) alleles were
sity, by screening randomly selected, unmapped RAPD
recombinant, while 7 out of 20 ORS1222 (29.5 cM) al-
leles were recombinant in the bulks. Naturally, the in-or amplified fragment length polymorphism (AFLP)
markers (Mouzeyar et al., 1995; Brahm et al., 2000; Lu tensity of the ORS1036 polymorphisms were greater
than the ORS1222 polymorphisms between R and S bulks,et al., 2000). With the emergence of dense SSR genetic
linkage maps for sunflower (Tang et al., 2002; Yu et al., but both were clearly polymorphic. ORS1222 demon-
strated that codominant SSR markers ≈30 cM away2003), BSA can be systematically performed by screen-
ing strategically positioned SSR marker loci, instead of could be identified by allele intensity differences. The
0- to 30-cM sensitivity range for SSR markers is similarrandomly selected RAPD or AFLP markers. Moreover,
SSR markers immediately supply DNA sequence-tagged- to that found for RAPD markers (Michelmore et al.,
1991). Occasionally, of course, DNA marker loci sepa-sites for subsequent analyses and DNA marker develop-
ment. While the multiplex ratios of individually typed rated from the target locus by distances greater than
≈30 cM can be identified by BSA, as was found forSSR markers are less than RAPD and AFLP markers
TANG ET AL.: OROBANCHE RESISTANCE IN SUNFLOWER 1027
local race of broomrape (Orobanche cumana Wallr.) in the sun-
ORS1114 (74.3 cM from CRT392) in the present study
flower. Genetica (The Hague) 12:151–155.
(Fig. 1 and 3) and by Mouzeyar et al. (1995) for a RAPD
Dominguez, J. 1996a. R-41, a sunflower restorer inbred line, carrying
marker 43.7 cM downstream of the Pl
1
locus in sun-
two genes for resistance against a highly virulent Spanish popula-
flower.
tion of Orobanche cernua. Plant Breed. 115:203–204.
Dominguez, J. 1996b. Estimating effects on yield and other agronomic
The 78 SSR marker loci amplified by the PCR-multi-
parameters in sunflower hybrids infested with the new races of
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sunflower genome, and estimated to be within ≈6.4 cM
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of any locus in the genome, and hence should be rou-
flower Assoc., Paris.
Dominguez, J., J.M. Melero-Vara, J. Ruso, J.F. Miller, and J.M. Fer-tinely powerful for identifying SSR marker loci linked
nandez-Martinez. 1996. Screening for resistance to broomrape (Or-
to phenotypic loci. Candidates SSR marker loci can be
obanche cernua) in cultivated sunflower. Plant Breed. 115:201–202.
identified by screening the PCR-multiplexed SSR mark-
Fernandez-Martinez, J.M., J.M. Melero-Vara, J. Munoz-Ruz, J. Ruso,
ers for some phenotypic loci, as was the case for Or
5
,
and J. Dominguez. 2000. Selection of wild and cultivated sunflower
but not for others, depending on polymorphisms in the
for resistance to a new broomrape race that overcomes resistance
of the Or5 gene. Crop Sci. 40:550–555.
segregating population. We have identified another 222
Gagne, G., P. Roeckel-Drevet, B. Grezes-Besset, P. Shindrova, P.
highly polymorphic single-locus SSR markers for BSA
Ivanov, C. Grand-Ravel, F. Vear, D. Tourvielle de Labrouhe, G.
and NIL screening for cases where leads are not pro-
Charmet, and P. Nicolas. 1998. Study of variability and evolution
duced by screening the SSR marker loci amplified by
of Orobanche cumana populations infesting sunflower in different
the PCR multiplexes (Tang et al., 2003). The density,
European countries. Theor. Appl. Genet. 96:1216–1222.
Ganal, M.W., N.D. Young, and S.D. Tanksley. 1989. Pulsed field gel
distribution, and polymorphisms of the 300 single-locus
electrophoresis and physical mapping of large DNA fragments in
SSR markers should be sufficient for tracking down
the Tm-2a region of chromosome 9 in tomato. Mol. Gen. Genet.
polymorphic SSRs linked to virtually any unmapped
215:395–400.
phenotypic locus in sunflower.
Gedil, M.A., C. Wye, S. Berry, B. Segers, J. Peleman, R. Jones, A.
Leon, M.B. Slabaugh, and S.J. Knapp. 2001. An integrated re-
striction fragment length polymorphism-amplified fragment length
ACKNOWLEDGMENTS
polymorphism linkage map for cultivated sunflower. Genome 44:
Oregon Agric. Exp. Stn. Tech. Paper No. 11 938. This re-
213–221.
search was funded by grants to S.J. Knapp from Pioneer Hi-
Gentzbittel, L., E. Mestries, S. Mouzeyar, F. Mazeyrat, S. Badaour,
F. Vear, D. Tourvieille de Labrouhe, and P. Nicolas. 1999. A
Bred Intl., the USDA Nat. Res. Initiative Competitive Grants
composite map of expressed sequences and phenotypic traits of the
Program Plant Genome Program (no. 1998-35300-6166), and
sunflower (Helianthus annuus L.) genome. Theor. Appl. Genet.
USDA Coop. State Res. Educational Ext. Service Initiative
99:218–234.
for Future Agricultural and Food Systems Plant Genome Pro-
Gentzbittel, L., F. Vear, Y.X. Zhang, A. Berville, and P. Nicolas. 1995.
gram (no. 2000-04292).
Development of a consensus linkage RFLP map of cultivated sun-
flower (Helianthus annuus L.). Theor. Appl. Genet. 90:1079–1086.
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