C Plant
Resume:
Theor Appl Genet (****) ***: *** ***
O R I GI N A L P A P E R
G. M. Rauscher C. D. Smart I. Simko
M. Bonierbale H. Mayton A. Greenland W. E. Fry
Characterization and mapping of RPi-ber, a novel potato late blight
resistance gene from Solanum berthaultii
Received: 5 January 2005 / Accepted: 30 November 2005 / Published online: 10 January 2006
Springer-Verlag 2006
Abstract Phytophthora infestans, the causal agent of late mapping technique, named MASP-map, which located
blight, threatens potato production worldwide. An im- RPi-ber in a 3.9 cM interval between markers CT240 and
portant tool in the management of the disease is the use TG63 on potato chromosome X. The location of RPi-ber
of resistant varieties. Eleven major resistance genes have coincides with an area involved in resistance to di erent
been identi ed and introgressed from Solanum pathogens of potato and tomato.
demissum. However, new sources of resistance are con-
tinually sought. Here, we report the characterization and
re ned genetic localization of a resistance gene pre-
viously identi ed as Rber in a backcross progeny of Abbreviations AUDPC: Area under the disease progress
Solanum tuberosum and Solanum berthaultii. In order to curve BCT: Backcross to S. tuberosum progeny
further characterize Rber, we developed a set of BSA: Bulk segregant analysis
P. infestans isolates capable of identifying each of the 11 CAPS: Cleaved ampli ed polymorphic sequences
R-genes known to confer resistance to late blight in MASP-map: Multiplex allele-speci c PCR mapping
potato. Our results indicate that Rber is a new resistance R-gene: Resistance gene chr: Chromosome
gene, di erent from those recognized in S. demissum, RFLP: Restriction fragment length polymorphisms
and therefore, it has been named RPi-ber according to the
current system of nomenclature. In order to add new
molecular markers around RPi-ber, we used a PCR-based
Introduction
Potato is the fourth most important crop in the world.
Communicated by G. Wenzel
In 2004, more than 328 million metric tons of potatoes
G. M. Rauscher C. D. Smart H. Mayton W. E. Fry were produced worldwide (FAOSTAT 2005). Potato
Department of Plant Pathology,
production is threatened by diverse pathogens, the worst
Cornell University, Ithaca, NY, USA
of which is the oomycete Phytophthora infestans, the
E-mail: ****@*******.***
Tel.: +1-607-***-**** causal agent of late blight. Late blight epidemics can be
devastating, sometimes causing total crop losses (Fry
I. Simko
and Goodwin 1997). It has been estimated that the
Department of Horticulture, Cornell University, Ithaca, NY, USA
management of the disease costs $3.5 billion annually in
M. Bonierbale developing countries alone (GILB 2004).
Centro Internacional de la Papa, Lima, Peru
Management strategies for late blight include the
application of fungicides, the use of healthy seed tubers
A. Greenland
for planting and host resistance. However, the occur-
Syngenta Jealott s Hill, Bracknell, UK
rence of isolates resistant to some modern fungicides
Present address: I. Simko emphasizes the need for host resistance (Deahl et al.
USDA-ARS, Beltsville, MD, USA
1993; Goodwin et al. 1996; Grunwald et al. 2001). Ad-
ditionally, the high cost of fungicide applications, in-
Present address: C. D. Smart
creasing awareness of health and environmental risks
Department of Plant Pathology, Cornell University,
Geneva, NY, USA and world-wide pressures to minimize the use of che-
mical sprays (Fry and Goodwin 1997) also make the use
Present address: A. Greenland
of host resistance a priority.
National Institute for Agricultural Botany, Cambridge, UK
675
identi cation of Rber was a complex race, compatible
Two types of resistance to late blight have been de-
with (i.e., not recognized by) R-genes: R1, R2, R3, R4,
scribed in potato. First, general resistance slows the
R5, R6, R7, and R10. Therefore, we inferred that Rber
spread of the disease and it is understood to be often
might be a new R-gene (Ewing et al. 2000).
polygenic (Leonards-Schippers et al. 1994; Umaerus and
The goal of the research reported here was to de-
Umaerus 1994), and strongly correlated with maturity
termine if Rber was di erent from R8, R9 or R11, and to
type (Bormann et al. 2004; Simko 2002) which makes it
provide a ner map location of this R-gene. We de-
di cult to breed into new varieties (Wastie 1991).
termined the identity of Rber using a set of P. infestans
Second, speci c resistance confers immunity or near
isolates known as a tester set, since they allowed po-
immunity to the plant through a hypersensitive response
tato plants to be screened for the 11 known R-genes and
and is thought to be monogenic. Genes governing such
therefore the identi cation of new R-genes. We also
resistance have been termed R-genes, and are thought to
investigated a series of markers from diverse sources to
produce proteins involved in pathogen recognition and
provide a ner map location of this R-gene.
the initiation of defense responses. Eleven resistance
genes have been introgressed into the cultivated potato
(Solanum tuberosum) from its wild relative Solanum
demissum (Van der Plank 1963; Wastie 1991), and they Materials and methods
are named R1, R2 R11. Of these, only ve have
been located on the genetic map of potato: R1 on Plant material
chromosome V (Leonards-Schippers et al. 1992); R2 on
chromosome IV (Li et al. 1998); and R3a, R3b (Huang Host di erential set
et al. 2004), R6, and R7 on chromosome XI (El-Khar-
botly et al. 1996). However, R5 R11 have recently been We used a di erential set of S. tuberosum genotypes,
identi ed as alleles of R3 (Huang 2005), which would each one containing one known resistance gene (Ta-
also locate them in chromosome XI. Of the R-genes ble 1). This di erential set was obtained from the US
from S. demissum, only R1(Ballvora et al. 2002) and R3a Department of Agriculture Potato Germplasm In-
(Huang et al. 2005) have been cloned and sequenced. troduction Station in Sturgeon Bay, WI, USA and had
R-genes from other hosts of P. infestans have been been used previously (Abu-El Samen et al. 2003; Spiel-
reported. Three resistance genes against P. infestans man et al. 1989). Historical and collection data are
have been mapped in tomato a close relative to potato: available for each accession (USDA 1999). The cultivars
Ph-1 on chromosome 7, Ph-2 on chromosome 10 Norchip or Katahdin were used as R-gene-free controls
(Moreau et al. 1998), and Ph-3 on chromosome 9 with no known R-genes, and served to indicate the
(Chunwongse et al. 2002). An R-gene from Solanum success of inoculations (Mastenbroek 1952). For all ex-
bulbocastanum on chromosome VIII (Helgeson et al. periments, plants were grown and maintained under
1998; Naess et al. 2000), named RB, has now been greenhouse conditions at the Cornell University facilities
cloned (Song et al. 2003). Rpi has been found in S. (Ithaca, NY).
pinnatisectum and mapped to chromosome VII (Kuhl
et al. 2001). We previously reported an R-gene located
chromosome X of Solanum berthaultii (Ewing et al. Backcross mapping population
2000), named Rber, since it segregates from the
S. berthaultii parent in a backcross with S. tuberosum. The backcross population was developed by Bonierbale
Unfortunately, P. infestans has been shown to rapidly et al. (1994). Brie y, an interspeci c progeny was de-
overcome the classic R-genes from S. demissum when veloped by crossing a dihaploid S. tuberosum clone
they were deployed in potato cultivars. The result is that (USW-2230 Saco, GS 193 in the GRIN database)
such speci c resistance has had short durability (Wastie (USDA 1999) as female with an individual of the ac-
1991), and the immunity hoped for in such R-genes has cession PI473331 of S. berthaultii from Cochabamba,
contributed little to practical late blight management. Bolivia (USDA 1999). One individual from the F1
However recent reports (Bormann et al. 2004; Stewart progeny (M200-30) was then backcrossed with the 2x S.
et al. 2003) suggest that there may be some residual tuberosum parent clone HH1-9 to generate the backcross
disease suppression e ect of R-genes even when inter- to S. tuberosum (BCT) progeny (Bonierbale et al. 1994).
acting with a compatible isolate. Knowledge of sig- Originally the progeny consisted of 158 diploid in-
naling pathways and downstream events may open new dividuals, of which 133 were still available for this study.
approaches to disease suppression, so it is important to A total of 665 genotypes of the BCT progeny were
learn as much as possible about such genes. produced at Centro Internacional de la Papa (CIP
Rber was mapped to a 13 cM interval between the Lima, Peru) and shipped to Cornell University as a true
restriction fragment length polymorphism (RFLP) seed to use in this study. They were tested for, and found
markers CT214b and TG63 in a diploid backcross pro- to be free of, major potato viruses and other pathogens.
geny. However, it was uncertain if Rber was a new gene For both the original and extended progenies, in vitro
plants were obtained and maintained at 15 C until
or an allele of a resistance gene previously introgressed
from S. demissum. The isolate of P. infestans used for the planting them in the greenhouse and eld in July 1999.
676
Table 1 Di erential set of
R-gene Accession no. Origin Reference
Solanum tubersoum
R1 Kennebec Wisconsin, USA
R2 PI 203905 Limburg, Netherlands Mastenbroek (1952)
R3 PI 203902 Spielman et al. (1989)
R4 PI 203900 Netherlands Mastenbroek (1952)
The accession numbers corre- R5 PI 303146 Scotland, UK Malcomson and Black (1966)
spond to the plant introduction R6 PI 587059 Netherlands
numbers assigned by USDA. R7 PI 303148 Scotland, UK Malcomson and Black (1966)
Origin stands for the country R8 PI 303149 Scotland, UK Malcomson and Black (1966)
collecting the material, which R9 Hogdson 2573 Wisconsin, USA Malcomson and Black (1966)
subsequently donated it to the R10 PI 423656 Netherlands
Potato Germplasm introduc- R11 PI 587060 Netherlands
tion station (Sturgeon Bay, WI)
tester set (Table 2). A dichotomous key (Fig. 1) was
Plants were then stored as tubers for future use. For the
then built to facilitate the identi cation of each of the 11
characterization of Rber we used the pedigree clones of
known R-genes from S. demissum.
the BCT progeny.
Once the tester set of P. infestans isolates was de-
veloped, it was used to characterize the pedigree clones
parents of the BCT progeny (USW22-30, HH1-9, and
P. infestans isolates
M200-30; the B11B clone was used as a representative
of S. berthaultii), two of which were known to carry the
A set of 30 isolates of P. infestans was characterized for
Rber gene (M200-30 and B11B). Detached lea et assays
compatibility/incompatibility with each of the 11 known
were again used to identify speci c reactions against
R-genes. The set of P. infestans isolates was obtained
each isolate. The cultivar Norchip was used as a
from the Cornell University P. infestans culture collec-
positive control in every inoculation due to its high
tion, and included strains from Ecuador, Kenya,
susceptibility and absence of known R genes to
Mexico, Uganda, United States, Peru, South Africa and
Poland (Table 2). The strains were chosen on the ex- P. infestans.
pectation that phenotypic di erences would occur
among isolates from the geographically diverse regions.
Phenotypic characterization of the BCT progeny
Speci c information regarding the isolates is available
upon request. For the assays, isolates were rst grown
The presence or absence of Rber was determined in the
on Rye B medium (Caten and Jinks 1968) in a growth
chamber at 15 C to encourage sporulation and then extended progeny and recon rmed in the original BCT
progeny. We used isolate US940480 (ATCC# 208834, a
transferred to new media every other week.
member of the US-8 clonal lineage) obtained from the
The isolates were tested on each host di erential
Cornell University P. infestans culture collection. We
plant using a detached lea et assay (Black et al. 1953;
had previously shown that this isolate can distinguish
Dorrance and Inglis 1997). Each inoculation involved
Rber from 8 out of the 11 known R-genes (Ewing et al.
recently expanded detached lea ets from 5-, 6-week-old
2000), and so it was used in both eld inoculations and
plants of each host di erential. Fifty microliters of a
detached lea et assays. Field inoculations are desirable
suspension bearing at least 15,000 zoospores/ml were
because they re ect better resistance under agricultural
deposited on the abaxial side of the lea ets using Petri
conditions. The original BCT progeny had been pre-
dishes with water agar (15 g/l) on the bottom as moist
viously exposed twice to the US8 isolate (US940480)
chambers (Tooley et al. 1986). Each inoculated lea et
was incubated for 7 days at 15 C and scored for pre- (Ewing et al. 2000). The US8-compatible individuals
from the original progeny and all individuals from the
sence/absence of infection (detected as sporulation from
extended progeny were inoculated in the eld at
the lea et) on the seventh day after inoculation
Freeville, NY, during the summer of 1999.
(Dorrance and Inglis 1997; Rivera-Pena 1990).
For eld studies in 1999, plantlets from in vitro cul-
Wastie (1991) and Dorrance et al. (1997) have sug-
tures were transplanted into Ji y7-Peat Pellets (Ji y
gested that small-scale tests, such as detached lea ets,
Products) supports and grown under greenhouse con-
should be interpreted with caution, because the condi-
ditions between May 15 and June 15; from June 15 to
tions may not reliably re ect the real resistance or sus-
June 18 they were conditioned to the eld environment
ceptibility interaction other than in extreme cases.
in cold frames and transplanted to the eld in single
Therefore, we scored as compatible only those inter-
plant plots during the week of June 18. The spacing
actions in which sporulation was evident. All cases
between plots was 0.86 m, and the space between rows
without sporulation, and where hypersensitive response
was 0.9 m. The cultivar Atlantic was included as a
was present, were scored as incompatible. Each test was
positive control because of its high general susceptibility
repeated at least ve times and only those strains that
to late blight.
showed consistent reactions were chosen to comprise a
677
Table 2 Set of Phytophthora infestans isolates tested and the correspondent reaction with the R-genes carried by the respective members of
the di erential host set
Isolate/plant Country of origin R0 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11
EC1 Ecuador C C I C I I I C I I I I
KE980002(KE2) Kenya C C C I I C C C I I C C
KE980004 Kenya C C C C C C C C C I C C
KE980008 (KE8) Kenya C C C C I C C C I I I I
KE980012 Kenya C C C I I C C C I I C I
KE980014 (KE14) Kenya C C I C I C I C I I C I
KE980016 Kenya C C I C I C I C I I C C
MX980002 Mexico C C C I I C C C I I C C
MX980005 Mexico C C C I I C C C I I C C
MX980006 Mexico C C C I I C C C I I C C
MX980082 Mexico C C C I I C C C I I C C
MX980083 Mexico C C C I C C I I I I C C
MX980084 Mexico C C C I I C I I I I C C
MX980085 (MX85) Mexico C C C I I C I I I I C C
MX980099 Mexico C C C I I C C I I I C C
MX980100 (MX100) Mexico C C C C C C C C I I C C
PE970002 Peru C C C C C C C C I I C C
PO149 Poland C C C I C C C C I I C C
SA960001 South Africa C C C C I C I I I I I C
UG980001 Uganda C C C C C C C C V V C I
UG980005 (UG5) Uganda C C C I I C C C I I I I
UG980006 Uganda C C C I I C C C I I I I
UG980008 Uganda C C I C I C I C I I C C
UG980010 Uganda C C C I I C C C I I I C
US980066 - US11 USA C C C C C C C C I I C C
US970001 (US17) USA C I I C C C I C I I I I
US940480 (US8) USA C C C C C C C C I C C C
US940501 US1 USA C I I I I I V I I I I I
IPO428.2 Netherlands C C I C I C C C I I C C
VK98014 Netherlands C C I C I C I C I I I C
The accession numbers are given by the year of isolation (i.e., 1998) and the consecutive number from the FryLab collection (Cornell
University), isolates IPO428.2 and VK98014 were provided by W. Flier (Plant Research International, The Netherlands). Isolates in bold
were chosen for the tester set, codes in parenthesis correspond to Fig. 1, italics represent clonal lineages. Reaction to inoculation is
recorded as C Compatible; I Incompatible and V Variable
Potato production practices typical of northeastern controlled as needed by applications of Lorox DF
USA were used to encourage plant growth. Fertilization (lineron, Dupont, Wilmington, DE, USA) and Dual II
was at the rate of 167 kg/ha of a 13-13-13 blend of (metalochlor, Syngenta, Greebsboro, NC, USA) at re-
nitrogen, phosphorous and potassium. Weeds were commended label rates. Insects were controlled with the
Fig. 1 Flow chart for
identi cation of resistance
genes to Phytophthora infestans
in potato germplasm. The
names in bold correspond to
Table 2. C/I stand for
compatible/incompatible
reactions, respectively, and the
numbers in parentheses are
the resistance genes that show
the corresponding reaction
678
applications of Provado 3.75 oz/A (imidacloprid, Bayer then ampli ed as follows: initial denaturation 5 min at
94 C, followed by 30 cycles of 94 C for 60 s, 55 C for
CropScience, Durham, NC, USA), Sevin XLR 1 qt/A
30 s, and 72 C for 90 s, and a nal extension at 72 C for
(carbaryl, Bayer CropScience), or Ambush 6.4 oz/A
(permethrin, Amvac chemical corp, Newport Beach, 5 min. The ampli cation products were visualized on
CA, USA) when necessary. 1% agarose gels and cleaned using the Wizard PCR
Inoculation with P. infestans took place during the preps DNA puri cation system (Promega, Madison,
evening of August 13 by applying a suspension (10 ml) WI, USA).
containing 150 sporangia/ml of isolate US940480 to To nd polymorphisms in the progeny we performed
each plant. Immediately prior to inoculation, the entire Southern hybridizations with each tomato probe. DNA
area had been sprinkler-irrigated to an equivalent of from each of the BCT pedigree clones was digested in
about 0.2 in. of rainfall. The inoculum was applied with separate reactions with: DraI, EcoRI, EcoRV, HaeIII
a hand-held sprayer. Sporangia had been obtained from (all from Invitrogen Corporation), and BstNI (New
sporulating lesions on lea ets of potato cv. Atlantic. England Biolabs, Beverly, MA, USA). Digestion and
The epidemic developed rapidly and rate of disease Southern blot procedures were done as described by
development on each progeny genotype was recorded. Sambrook et al. (1989) using Hybond N+ membrane
The amount of foliar disease (as a percentage of the total (Amersham Pharmacia Biotech) for capillary transfer.
tissue a ected) was evaluated every 3 days between For hybridization bu er we used Hyb-9 Hybridization
September 5 and October 2 using the methods and solution (Gentra Systems, Plymouth, MN, USA).
Probes were labeled with 32Pa-dATP with the
guidelines previously described (Ewing et al. 2000).
These data were used to calculate the area under the Random Primers DNA Labeling system (Invitrogen
disease progress curve (AUDPC) as described by Shaner Corporation) and cleaned with Bio-Spin 30 Tris columns
et al. (1977). Those plants that showed inconsistent re- (Bio Rad, Hercules, CA, USA). Hybridization took
place for at least 4 h at 65 C. Washes were performed
sults or that died in the eld for reasons other than late
with 2 SSC 0.1% SDS for 15 min, 1 SSC 0.1%
blight were subsequently tested using detached lea ets
SDS for 15 min, and 0.5 SSC 0.1% SDS for 15 min
from plants grown in the greenhouse as described be-
at 65 C. The membranes were then exposed to BioMax
fore.
MS lm (Eastman Kodak Company, Rochester, NY,
USA) overnight at 80 C and developed with a Kodak
DNA extraction X-Omat automatic developer.
DNA was obtained from each of the individuals in the
original and extended BCT progeny. Expanding lea ets Multiplex allele-speci c polymorphism mapping
were collected from 826 plants growing under green-
house conditions and were placed in liquid nitrogen. We used a mapping technique that we named Multiplex
DNA was extracted using a CTAB extraction protocol Allele-Speci c Polymorphism (MASP-map), since it al-
(Doyle and Doyle 1990). The precipitated DNA was lows the simultaneous ampli cation of one allele and a
treated with 5 ll of a solution (10 mg/ml) of RNAse A control band. It is designed to follow a single allele
for 30 min at 37 C. It was then visualized in a 0.8% through a mapping progeny, via a modi cation of the
agarose gel in TAE bu er to verify its integrity, and similar methods such as PASA (Okimoto and Dogson
concentration was measured using a Hoefer DyNA 1996) and tetra-primer ARMS-PCR (Ye et al. 2001).
QUANT 200 uorometer (Amersham Biosciences, San MASP-map allowed us to change RFLP probes into
Francisco, CA, USA). PCR markers. The rst step is to design primers from
known RFLP probe sequences and use them to amplify
and sequence the parents of the cross (Fig. 2). These
Restriction fragment length polymorphism sequences are then used to nd single nucleotide poly-
morphisms that allow the unique identi cation of one of
Restriction fragment length polymorphism probes for the parental alleles. A new allele-speci c primer is de-
signed so that the 3 end anneals to the polymorphic site.
chromosome 10 were chosen based on the tomato ge-
netic map and obtained from Dr. Steve Tanksley s lab In addition a mismatch is added in the penultimate
nucleotide on the 3 end of the speci c primer to help
(Cornell University). To obtain su cient probe DNA
for RFLP hybridization, each cloned probe was ampli- improve allele speci city.
ed by PCR using M13 universal primers in a 25 ll nal To screen individuals of the progeny for the parental
volume. For the template, we used 1 ll of a liquid cul- polymorphism of interest, PCR is carried out with three
ture of Escherichia coli transformed with the probe primers simultaneously; two that amplify both alleles
which had been diluted to half concentration in distilled and a third that is speci c for the allele with the targeted
water and incubated in boiling water for 5 min. PCR polymorphism. Thus, individuals lacking the allele of
conditions were: 2.5 mM MgCl2, 0.2 lM of each primer, interest amplify only the control band, while in in-
dividuals containing the allele of interest, both the
200 mM dNTPs, and 1 U of Taq Polymerase (Invitro-
speci c allele and the control band are ampli ed.
gen Corporation, Carlsbad, CA, USA). Each probe was
679
The two parental sequences obtained for each probe
were compared with Sequencher (version 4.0.5, Gene
Codes Corp. MI, USA) to look for single nucleotide
polymorphisms that were speci c to the S. berthaultii
parent. Speci c primers for S. berthaultii polymorphisms
were designed from the parental sequences with a Tm
about 5 C lower than those of the initial primers
(Table 3) and included a mismatch at the penultimate
base at the 3 end of the primer to improve speci city as
described by Cha et al. (1992).
When the polymorphism was close to the 5 end of
the fragment a forward primer was designed (5/6 probes,
Table 3); when polymorphisms were closer to the 3 end,
a reverse primer was designed (1/6 probes, Table 3). For
the sake of clarity, the three primers used will henceforth
be referred to as allele-speci c (usually forward),
general-forward and general-reverse.
To facilitate mapping the region of interest, we
screened only individuals with a recombination at any
point along chromosome X as rst determined by
Bonierbale et al. (1988a). Using this method, markers
mCT11, mCD5, mCT238, mCT240, and mT1682 were
added to the genetic map of chromosome X, the m
refers to RFLP makers detected by MASP-map.
Tomato marker sequences have been published by
Fig. 2 Diagram of the MASP-map method. See text for explana-
Ganal et al. (1998). In order to assess percent similarity
tion of the methodology. FP and RP stand for forward and reverse
between potato and tomato sequences, they were
primer, respectively. SFP stands for speci c forward primer. The
last box shows results of MASP-map: S.t. and S.b. stand for aligned, visually inspected to identify sites with signal
Solanum tuberosum and Solanum berthaultii, respectively. UHMB
from more than one nucleotide (i.e., double peaks), and
are U: S. t parent USW 22-30; H: S. t. Recurrent parent HH1-9; M:
coded according to standard IUPAC degenerate code.
hybrid parent M200-30; B: S.b. representative B11B, respectively
Sequences were then trimmed to the same size using
The ampli cation products are then separated and vi- Sequencher version.4.2 (GeneCodes Corp.). Since to-
sualized in agarose gels. mato sequences were obtained from ESTs, for accurate
In order to add markers to the interval between comparison, introns were edited out of potato se-
CT214 and TG63, the sequences of six tomato RFLP quences. However, alignments among potato accessions
probes were obtained from the Solanaceae Genome were done using the whole length of the sequences. To
Network (http://www.sgn.cornell.edu). From these, nd sequence similarity, the ClustalW utility of the
forward and reverse primers were designed using Primer software package MegAlign (Lasergene Corp, Clewley
Select from the DNASTAR DNA analysis software and Arnold 1997) was used. Multiple alignments were
package (DNASTAR, Inc.), trying to localize them performed using the slow/accurate option weighted to
within the 100 initial or terminal base pairs on each account for diploidy.
probe. Finally, sequences were compared to previously re-
These primers were used to amplify alleles in each ported sequences at GenBank using the BLAST algo-
individual parent from 50 ng of template DNA in a re- rithm. ESTs were compared to the dbEST database,
action containing 0.8 U of Taq DNA Polymerase which contains GenBank, EMBL, and DDBJ sequences
(Invitrogen Corporation), 1 PCR bu er (10 stock: from ESTs. Sequences for the genomic marker TG63
200 mM Tris HCl pH 8.4, 500 mM KCl), 2.5 3.0 mM were compared both with the nonredundant and the
MgCl2, 200 nM of forward and reverse primers, 200 nM EST databases.
of dNTPs, and double distilled sterile water. The reac-
tions were carried out in a nal volume of 50 ll set on ice
to minimize nonspeci c ampli cation. Prior to sequen- Cleaved ampli ed polymorphic sequences (CAPS)
cing, 1 ll of the PCR product was visualized to verify
ampli cation and size of the products on a 1% agarose The sequence of tomato probe TG63 was used to design
gel, and the remaining 49 ll were puri ed using the primers to amplify homologous sequences in potato.
Wizard PCR PrepDNA puri cation system (Promega). However, due to multiple insertions and deletions in the
Ampli cation products were sequenced by the Cornell sequence, it was not possible to design MASP-map
Biotechnology Sequencing Facility using only the for- primers. Therefore, we decided to use CAPS to trans-
ward primer for short products (around 500 bp) or both form this RFLP probe into a PCR marker. After
forward and reverse primers for longer products. screening the PCR products of all the pedigree
680
Table 3 Primers used for MASP-map
Sequence 5 3 Tm ( C) C/S
Name Prim. size (bp) Di . (bp)
CT214F GAA CGC GAA AGA GTG CTG ATA G 57.1
GAT TCC AAC ATT CAC AAG GGT
CT214BF1 54.5 63/45 618/537 81
CT214R2 CCC GCT GCC TAT GGA GAG T 59.7
CT238F2 GGA TAA GGC GGT TCT GTC 50.9
TTC GAT GCC AAT CTC CTA
CT238BF1 51.0 53/30 300/225 75
CT238R1 AAT TTC TCC ATG TTT TTC AG 47.6
CD5F TTG AGG CTA TTG TAC GAG TGT GCG 60.6
TGA GCA ACG TAA TGT GGA AAA
CD5BF1 54.3 57/30 464/270 194
CD5R AAA GCC TCT TAG GTA CAT TAT GTC G 56.2
CT11F1 AGA TTG CTT GTT TGG TGG TT 54.3
TCT CAA AAG GAA TCT TGA CAC AG
CT11BR1 54.4 61/20 316/250 66
CT11R1 TGG AGC AGT CAA CAG AGG 54.7
CT240F1 CCA AAG CCC AGG CTG TCA AG 59.7
GGT TCT AAA ATG TCC TCT TAA A
CT240BF1 49.5 55/20 890/718 172
CT240R1 AGT CGG GTG TCA CAA TAA 59.9
T1682F2 CGG AAG AAC ATG GAT TTG AAG C 55.8
CAT CTC CCA GCT CAT CAT
T1682BF4 51.9 56/10 523/325 198
T1682R2 CGT CAT TTT CCG ACG AGG ATT T 56.9
Sequences of primers used to develop and validate the MASP-map technique as well as their melting temperatures and speci c ampli-
cation conditions. The speci c primers are shown in italics; the Solanum berthaultii speci c nucleotide is underlined and mismatches are
noted in bold. The third column shows each one of the primer melting temperatures (Tm). The next column shows the anneal temperature
( C) and the extension time (s) for each PCR reaction. The product sizes (Prim. Size) di erence in size (Di .) between the products of
general and allele speci c products are illustrated in base pairs (bp)
accessions (USW22-30, HH1-9, M200-30, and B11B) exposed to Kodak BioMaxMR lm (Eastman Kodak
with multiple restriction enzymes, a polymorphism un- Company) overnight at room temperature.
ique to the S. berthaultii allele was discovered using
EcoRV. This polymorphism could then be used as a
cleaved ampli ed polymorphic sequence (CAPS, Ko- Search for new markers with bulk segregant analysis
nieczny and Ausubel 1993) marker for mapping. For the and resistance gene analogs (RGA)
nal screening, 50 ng of genomic DNA were ampli ed
using primers TG63F1 and TG63R (Table 3), and a To increase the chances of nding bands linked to Rber
master mix identical to that described for ampli cation using RGA mapping (see subsequently), we used bulk
of RFLP probes, except that the MgCl2 concentration segregant analysis (Michelmore et al. 1991). DNA of
selected BCT progeny was bulked in two groups, one
was 3 mM. The PCR conditions for this marker were:
initial denaturation 95 C for 2 min; 35 cycles of 95 C for with 10 incompatible and the other with 10 compatible
30 s; 50 C for 30 s; 72 C for 90 s; and nal extension at individuals. The respective bulks were made based on
72 C for 7 min. the genetic data (Bonierbale et al. 1994) and phenotypic
(Ewing et al. 2000) information con rmed for the ori-
The PCR product was digested with 2.5 U of EcoRV
(Invitrogen Corporation) at 37 C for 3 h and visualized ginal BCT progeny in chromosome X. The resistant and
with ethidium bromide on 1.5% agarose gels in 1 TAE the susceptible bulks each comprised individuals with
recombination events similar in the area of interest be-
bu er. The gels were examined with a FotoAnalyst In-
tween CT240 and TG63, but di ering for the rest of the
vestigation Column Mount System (Fotodyne Inc.
chromosome to minimize the area of interest. The bulks
Hartland, WI, USA).
contained the same amount of DNA from each of 10
individuals in such a way that the incompatible group
had the S. berthaultii allele (segregating from M200-30
Simple sequence repeats
and in common with B11B) for the area of interest, re-
gardless of the rest of the chromosome; and the com-
Previous studies have shown that SSR marker STM1056
patible bulk contained alleles from the S. tuberosum
is located close to TG63 in potato (Milbourne et al.
progenitors in the area between CT240 and TG63 but
1998), and hence was also screened for progeny that are
could di er in the rest of the chromosome.
recombinant on chromosome X. For ampli cation we
We used resistance gene analog (RGA) ampli cation
followed the procedure and primers described by
to search for markers linked to Rber because previously
Milbourne et al. (1998). PCR products were run on a
cloned R-genes to late blight are known to code for
5.5% Long Ranger Gel (Cambrex Corporation, East
nucleotide binding sites (NBS) and leucine-rich repeats
Rutherford, NJ, USA) in a SequiGen GT Nucleic Acid
(LRR) (Ballvora et al. 2002; Song et al. 2003). Primers
Electrophoresis Cell (Bio Rad Inc.). The gels were run in
0.6 TBE bu er at 2,000 V for about 2 h, dried and designed to amplify resistance gene analogs, including
681
using a backcross linkage evaluation with P=1 10 5
regions containing NBS, LRR, and protein kinase
(Table 4) were obtained. PCR was carried out using and the Kosambi mapping function. We added the in-
50 ng of DNA and 0.8 U of Taq DNA Polymerase formation for the new markers to a di erent (new)
(Invitrogen Corporation), 1x PCR bu er (10 stock, chromosome and used the distribute command. Once
200 mM Tris HCl pH 8.4, 500 mM KCl), 2.5 mM the markers were located on chromosome X, we found
MgCl2, 100 nm of 33P-cATP labeled forward primer the best localization for them using the ripple
and 200 nM of reverse primer, 200 nM of dNTPs, and command.
bidistilled sterile water in a nal volume of 15 ll. The
thermocycler (Hybaid Touchdown TD7200, Thermo
Electron Corporation, Waltham, MA, USA) was pro- Results
grammed as follows: initial denaturation at 94 C for
2 min, followed by 35 cycles of denaturation at 94 C for Characterization of Rber
30 s; annealing of 51 56 C depending on the primer pair
for 30 s, extension at 72 C for 20 s, and a nal extension The 30 isolates of P. infestans we tested were diverse in
step of 72 C for 2 min. Each of the reactions was mixed their interaction (compatible/incompatible) with the
with 5 ll of formamide loading bu er (80% formamide, di erential plants (Table 2). Of these isolates, about half
10 mM EDTA, 1 mg/ml bromophenol blue, 1 mg/ml had unique phenotypes; 20% were compatible with R-
xylene cyanol), denatured for 3 min at 94 C and quickly genes 1, 2, 5, 6, 7, 10, and 11; 10% were compatible with
chilled. RGA products were separated and visualized 1, 2, 3, 4, 5, 6, 7, 10, and 11; 6.6% were compatible with
using the same procedure described for SSRs. 1, 3, 5, 7, 10, and 11; 6.6% with 1, 2, 5, 10, and 11; and
6.6% with 1, 2, 5, 6, and 7. In general, isolates with the
same compatibility phenotype came from diverse geo-
Data analysis graphical locations, except for some isolates from
Mexico that showed the same compatibility pattern.
The BCT mapping data set was obtained directly from We designed a ow chart using the tester set of
the Solgenes database (currently available at http:// nine isolates (Table 2, Fig. 1) to identify known R-
www.grain.jouy.inra.fr/gendatabasemirror.html). The genes. The chart is used as a dichotomous key as fol-
RFLPs, MASP-map markers, RGA, and SSR gels were lows. An unknown potato genotype is inoculated with
scored visually for presence or absence of the bands each member of the tester set independently and the
originating in the S. berthaultii parent. The genotype for reactions recorded. The results are then read sequen-
each individual was coded as A (homozygous) or H tially following the chart. For example, if a plant is in-
(heterozygous) for each marker, and these data were compatible with the strain UG980005, the next step is to
combined with the previously published framework follow the right branch. If it is compatible with the strain
markers. The chromosome map was assembled with KE980002, the left branch should be followed, and so
MapManager QTX, version 0.27 (Manly et al. 2001) on, until a terminal point is reached, which will show the
Table 4 Resistance gene analogs (RGA) and cleaved ampli ed polymorphic sequences (CAPS) primers used
Sequence 5 3
Name Target Reference
S1 GGT GGC GTT GGG AAG ACA ACG P-loop motif and NBS Leister et al. (1996)
AS1 CAA CGC TAG TGG CAA TCC
AS2 IAA IGC IAG IGG IAA ICC
AS3 IAG IGC IAG IGG IAG ICC
NBS F1 GAA ATG GGN GTN GGN AAR AC NBS Yu et al. (1996)
NBS R1 YCT AGT TGT RAY DAT DAY YYT RC
LM638 GGI GGI GTI GGI AAI ACI AC P-loop and a transmembrane region Kanazin et al. (1996)
LM637 ARI GCT ARI GGI ARI CC
LRR F1 CGC AAC CAC TAG GAG TAA C Leucine-rich repeats of RPS2 (F1/R1), Chen et al. (1998)
LRR R1 ACA CTG GTC CAT GAG GTT Xa21 (F2/R2) and Cf9 (F3/R3)
LRR F2 CCG TTG GAC AGG AAG GAG
LRR R2 CCC ATA GAC CGG ACT GTT
LRR F3 TTT TCG TGT TCA ACG ACG
LRR R3 TAA CGT CTA TCG ACT TCT
PtoKIN 1 GCA TTG GAA CAA GGT GAA Protein kinase from tomato Pto Chen et al. (1998)
PtoKIN2 AGG TGG ACC ACC ACG TA
PtoKIN3 TAG TTC GGA CGT TTA CAT Zhang et al.(2002)
PtoKIN4 AGT GTC TTG TAG GGT ATC
TG63F1 CCC AGA GTC CCC CTT CCT ATT RFLP probe TG63 This study
TG63R CGA GAT GTT GAA TTT GCG TAA GA
Resistance gene analog primers used for PCR ampli cation and search of candidate sequences linked to RPi-ber. Codes are Y=C/T;
R=A/G; D=A/G/T, N=A/C/G/T. See text for details
682
R-gene that is present. All interactions are recorded and purposes of the mapping program these are considered
analyzed to determine the number and identi cation of being homozygous for the S. tuberosum allele, al-
R-genes present in the potato genotype. though the pedigree did permit heterozygosity).
The tester set was applied to three of the pedigree
genotypes of the BCT progeny and a representative in-
dividual of S. berthaultii PI 473331 (Table 5). The S.
MASP-map sequences
tuberosum grand parent USW-2230 was compatible
with all isolates but US17, indicating the presence of R1.
Given that MASP-map marker development required
We also found that the recurrent S. tuberosum parent
the characterization of nucleotide sequences in the ped-
HH1-9 was compatible with all isolates, including
igree clones of BCT (GenBank acc. AY874391 to
US970001, which is only compatible with plants lacking
AY874411), we thought it would be useful to nd the
all resistance genes; therefore, no resistance genes were
level of similarity among them and with tomato. On
detected in HH1-9. We do not have the original S.
average, potato and tomato sequences were over 90%
berthaultii donor (a genotype of PI473331), and there-
similar. The biggest di erences were found in noncoding
fore could not assess its phenotype. However, the hybrid
sequences, e.g., for TG386, where tomato was only 70.1
parent M200-30 (product of a cross between PI473331
and 69.2% similar to S. tuberosum and S. berthaultii,
and USW22-30) carries Rber and is resistant to all iso-
respectively. Regarding S. tuberosum and S. berthaultii,
lates in the tester set, suggesting that Rber is a new re-
sequences were over 97% similar on an average. Inter-
sistance gene. Field tests performed in the Toluca Valley,
estingly the same average similarity was found between
Mexico allowed the isolation of several strains from
the S. tuberosum accessions (USW22-30 and HH1-9),
plants carring RPi-ber allele showing that RPi-ber can be
most di erences were caused by heterozygosity in one of
overcome by isolates MX990005, MX010003,
the accessions, especially HH1-9.
MX010004, and Mx010008. Testing of these isolates
Blast searches con rmed previous reports by Ganal
with the di erential set also demonstrated their com-
et al. (1998) except for CT217, where we found the best
patibility with R1 R11 (unpublished results).
homology with an Avr9/Cf9 elicited protein. Also,
mCT11, mCT214, mCT238, and mCT240, each showed
homology with sequences from EST libraries made
Genetic mapping of Rber
from potato leaves challenged with P. infestans
(P value > e 111). These results, the presence of an R-
Phenotypic characterization
gene, and QTLs for resistance to other pathogens
(Gebhardt and Valkonen 2001) may suggest that this
We con rmed the phenotype of all individuals in the
area of chromosome X could be involved in resistance
original BCT progeny evaluated by Ewing et al. (2000)
responses common to other pedigrees including tomato.
and determined the phenotype of the extended BCT
For the marker TG63, forward and reverse sequences
progeny using either eld or detached lea et assays. For
from tomato, obtained from the Solanaceae Genomics
plants from the original progeny inoculated in the eld
Network, were compared to sequences in GenBank. No
with the US8 isolate US940480 during the summer of
matches were found when the search used the
1999, the AUDPC score ranged from 0 to 3,652 U. Only
nonredundant database, but the sequences share
those individuals with AUDPC less than 1 were scored
homology with one EST from tomato roots
as incompatible and thus containing RPi-ber. The average
(EST303128, 1e 85) and
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