Research Collection
Doctoral Thesis
Development of molecular markers for the adult plant leaf rust
resistance genes Lr13 and Lr35 in wheat (Triticum aestivum L.)
Author(s):
Seyfarth, Ralf
Publication Date:
2000
Permanent Link:
https://doi.org/10.3929/ethz-a-003910435
Rights / License:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For more
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ETH Library
Diss. ETH No. 13533
Development of Molecular
Markers for the Adult Plant Leaf Rust
Resistance Genes Lr13 and Lr35 in Wheat
(Tnticum
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
ZÜRICH
for the
degree
of
Doctor of Natural Sciences
presented by
Ralf
Seyfarth
Dipl. Ing. Agr. University
of
Hohenheim, Germany
born October 26, 1967
accepted
on
the recommendation of
Prof. Dr. I.
Potrykus,
examiner
Prof. Dr. B. Keller, co-examiner
Prof. Dr. P.
Dr. C,
Stamp,
co-examiner
Feuillet, co-examiner
Zürich,
2000
aestivum
L.)
Table of contents
1.
Summary
1
2.
Zusammenfassung
3
3.
General Introduction
5
3.1
Wheat and leaf rust
5
3.2
Adult
3.3
Molecular markers and marker-assisted selection
3.4
Breeding for
3.5
The
4.
plant
resistance
6
resistance and
7
of resistance genes
sources
10
objectives of the study
Molecular
mapping of
16
the adult leaf rust resistance gene 17
Lr13 in wheat
Development
5.
of
a
molecular marker for the
adult-plant
28
leaf rust resistance gene Lr35 in wheat
General discussion
46
6.1.
Marker types and marker-assisted selection
46
6.2.
Allelism
6.
of
resistance
genes
on
chromosome
2BS
and
49
cloning of these genes
6.3.
Breeding
for resistance
6.4.
Breeding
for resistance
49
or
tolerance?
55
7.
Outlook
57
8.
References
58
1
1.
Summary
Susceptible
suffer
wheat cultivars which
severe
losses when attacked
f.sp. tritici, the
Rob. Ex Desm.
have
can
a
negative impact
the most economical and
on
the
used in marker-assisted selection
Puccinia recondita
rust. As chemical
fungicides
ecosystem, breeding for resistance
ways of
to
was
fungicides
chemical
fungal pathogen
the
agent of leaf
causal
ecological
investigation
The aim of this
by
protected by
not
are
one
molecular markers which
leaf rust resistance genes Lr13 and Lr35. Both genes
be
can
support the introgression of the
to
of
plant protection.
develop
(MAS)
is
two
acting
at the adult
stage in wheat. The adult plant growth stage is very vulnerable
to leaf rust
attack
the
in reduced floret set and
resulting
the inoculum
growth
density
conditions for the
developing
considered to be
one
is very
fungus.
yield
high
loss.
During
due to the
the adult
period
optimal growing
of
and
The adult leaf rust resistance gene Lr13 is
widely distributed
of the most
are
leaf rust resistance genes
of the world. In combination with other resistance genes it increases resistance.
Lr13 is located
from
the short
wheat.
common
crosses
on
We
of chromosome 2B of wheat and
arm
developed
We established
to detect the adult resistance at the
and 14 microsatellites that were
tested for
ThLr13
were
x
mapped
one
microsatellite
the
closest
no
linked
were
the
in the
against
growth chamber
(GWM630)
x
RFLP
probes
was
Thatcher)
(10.3 cM). Although
by using
resistance gene Lr35 gene is
many
were
and four
derived from the
located at 10.7 cM
one
RFLP
linked with the resistance gene. GWM630
marker
probes
chromosome 2B
segregating population
population (JhLr13
relative of wheat. As this gene is not
effective
on
the parents. Three RFLP
closer marker could be found
origin of
infection test in the
lines Frisai and
seedling stage. Eighty-two
Frisai. The closest marker
from Lr13. In the second
detected,
an
susceptible
previously located
polymorphism between
microsatellites
The
F2 populations derived from the
between the resistant line ThLrf3and the
Thatcher, respectively.
cross
two
originated
probe
and
again
was
polymorphisms
the AFLP
technique.
Triticum
speltoides,
were
a
wild
yet exploited in wheat breeding it is still
leaf rust worldwide. The resistance gene Lr35 is also located
2
on
wheat chromosome 2B. An F2
was
developed.
chamber to
We established
detect
resistance
segregating population
RFLP
probes
between the
were
cross
adult
l\\Lr35
x
located
on
can
Lr35 into elite
Fifty-one
chromosome
were
breeding
of
the
of the tested 80
were
polymorphic
mapped. We developed
marker from the
be used
2B
Frisai
plants in the growth
susceptibility. Ninety-six F2 plants
(sequence-tagged-site)
introgress
on
tested at the adult stage.
BCD260. This STS marker
programs to
infection test
an
or
from the
parents. Fourteen of these probes
dominant STS
probe
which
were
population
completely linked RFLP
efficiently
material.
a
in resistance
breeding
3
2.
Zusammenfassung
Weizenbraunrost, hervorgerufen durch das Pathogen Puccinia recondita Rob.
Ex
Desm.
f.sp. tritici,
eine
ist
der weltweit
Weizen. Im Falle einer Infektion
Der
Ertragseinbussen.
reagieren anfällige
Einsatz
nachteiligen
Pflanzen kann einen
wichtigsten Pilzkrankheiten
chemischer
Sorten mit
Fungizide
empfindlichen
zum
Effekt auf die Umwelt ausüben.
Schutze
der
Züchtung
auf
ökologisch bevorzugte Strategie
Resistenz ist somit eine ökonomisch und
im
des
Pflanzenschutzes.
Ziel dieser Arbeit
Lr13 und Lr35
zu
entwickeln. Beide
Infektion
Eine
effektiv.
Resistenzgene sind
während
diesem
Molekulare Marker würden
Braunrostresistenzgene.
erhöht
es
eines
für Lr13
14
zu
von
einen
verringerten Ertrag
hervor.
einzusetzen.
weltweit
am
aus
dem
meisten
verbreiteten
Braunrostresistenzgenen
hexaploiden Weizengenpool. Es
Chromosom 2B lokalisiert. In dieser Arbeit wurden
ThLr13 und die
entwickelt:
anfällige Sorte
Dafür wurden für Lr13 die
Frisai bzw.
Um die adulte Resistenz im
ThLr13 und die
Keimlingsstadium
können, wurde in der Klimakammer ein spezifischer Infektionstest
durchgeführt. Zwischen
Mikrosatelliten
Mikrosatelliten
RFLP Sonden
waren
für
einen Abstand
den
Kreuzungseltern wurden
Polymorphismen getestet.
und vier Mikrosatelliten
(ThLr13x Thatcher)
am
10,7 cM
von
waren
GWM630
82 RFLP Sonden
Diese
Sonden
und
bereits früher auf Chromosom 2B kartiert worden. Drei
Frisai kartiert werden. Der
gekoppelt.
ruft
In Kombination mit weiteren
anfällige Linie Thatcher gekreuzt.
erkennen
Züchtung
der
segregierende F2 Populationen
resistente Linie
und
in der
die Resistenz. Lr13 stammt
ist auf dem kurzen Arm
zwei
ist
Lr13
in adulten Pflanzen
erlauben, diese beiden Resistenzgene durch
es
markergestützte Selektion gezielt
Resistenzgen
nur
Entwicklungsstadium
verminderten Blütenansatz und somit einen deutlich
Das
Braunrostresistenzgene
es, molekulare Marker für die
war
war
konnten
in
der
Population ThLr13
nähesten lokalisierte Marker
zum
Resistenzgen.
In
x
(GWM630) zeigte
der zweiten
Population
eine RFLP Sonde und ein Mikrosatellit mit dem Gen
wiederum der
am
engsten gekoppelte Marker (10,7
4
cM).
Mit der AFLP Technik konnten viele
Allerdings
kein
konnte
Polymorphismen entdeckt werden.
gekoppelter Marker
mit
dieser
Technik
entwickelt
werden.
Triticum
Lr35
speltoides,
ein
diploider Verwandter
des
Weizens, ist der Donor des
Resistenzgenes. Dieses Gen ist weltweit noch wirksam, da
es
noch nicht
kommerziell ausgenutzt worden ist. Auch dieses Gen ist auf dem kurzen Arm
von
Chromosom 2B lokalisiert. Aus den zwei
Frisai wurde eine
spaltende F2 Population
Kreuzungseltern ThLr35
und
entwickelt. 96 F2 Pflanzen wurden
auf Resistenz im adulten Stadium in der Klimakammer getestet. 51
der 80
getesteten RFLP Sonden zeigten Polymorphismen zwischen den elterlichen
Linien.
14 dieser Sonden
gekoppelte
tagged-site)
die
konnten kartiert werden.
Die mit Lr35
RFLP Sonde BCD260 wurde in einen dominanten STS
vollständig
(sequence-
Marker umgewandelt. Dieser auf PCR basierende Marker erlaubt
effiziente
Zuchtmaterial.
Einführung
des
Lr35
Resistenzgenes
in
fortgeschrittenes
5
3. General Introduction
3.1 Wheat and leaf rust
Wheat is the most
widely grown
and consumed food crop in the world. It is the
staple
food of
wheat
occupies approximately 20% of
most
important agricultural commodity
Triticum
(Knott, 1989).
diploid, tetraploid
and
tetraploid
durum
(Triticum
aestivum
or
hexaploid
wide basis it
probably
Puccinia
be
It
more
belongs
(f.sp. tritici)
are
and rye
L.) and hexaploid
recondita Rob. Ex Desm.
important fungal
damage than
(crown
cycle
(Roelfs
order
(f.sp. secalis).
and
and
Wittmack),
are
wheat
f.sp.
diseases
but
uredinales
are
Puccinia
of minor
whereas
T.
(leaf
(Triticum
monococcum
importance (Roelfs
et al.
a
or
the
world
wheat
fungus,
class
are
of
specific
rust
graminis, (stem
or
rust of
As Puccinia recondita is
wheat
wheat
agronomically important
includes two different hosts:
hexaploid
of
on
the
speciales, which
Puccinia hordei
oats).
fungus
are
of
triticale), Puccinia stiiformis (stripe
rust of
tritici is
1992).
yellow
barley)
or
rust
and
macrocyclic,
major primary
aestivum L.em.
spelt (Triticum spelta L.), tetraploid wheat (Triticum durum)
speltoides
includes
it
wheat stem rust
et al.
Other
basidiomycetes
triticale),
of the
hosts for wheat leaf rust
Triticosecale
the genus
common
spectacular yield losses,
the
to
to the
barley
barley, rye
the sexual life
The
the cultivation is restricted to the
be subdivided into formae
belonging
Puccinia coronata
and
most
cause
causes
can
black rust of wheat,
of wheat,
durum
and reach up to 30%
recondita
fungi which
the
does not
severe
basidiomycetes.
for wheat
Today
(Samborski, 1985). Under favorable conditions for
rust
can
species of
Triticum is very diverse and
by Puccinia
of
one
usually
losses
(Wiese, 1991).
L.).
be
to
1998). Today
et al.
the world's cultivated land and is the
in international trade
wheat.
{Triticum
wheat
worldwide. It
yellow
The genus
brown rust caused
considered
population (Braun
used to refer to the cultivated
normally
term wheat is
Leaf
35% of the world
nearly
Thell)
and triticale
(X
L., T. dicoccum L. and 7*.
1992).
The asexual life
cycle
6
can
be
in the cereals. On these hosts, the
completed
On the alternate hosts, such
phase.
Isopyrum fumarioides
the
between
alternate
of the
races
host
generating changes
in
again dicaryotic
host. The
cropping
1992).
do not have
host is not
As the
is
pathogen
large distances.
wheat
large distances,
and
(Roelfs
et al.
3.2 Adult
plant
resistance
Seedling,
adult
plant
expressed
at
is
the
for
1992).
regions
The
than
epidemics
cycle
primary
or
wheat
(Roelfs
pathogen
et al.
is able to
where the alternate
areas
spores
are
are
spread
1992).
After the
(Saari
fully expressed.
seedling stage
from
of North America to the northern
area
rust
in the field and
transported annually
dispersal
and Prescott,
plant stages.
it
is
over
the
spring-
pathogen
reasons
over
of the
1985).
plant stage
resistance
Seedling
and
of the
of the
one
and overall resistance describe the
resistance in the other
aeciospores which
in the world
and the
occur
the spores remain viable. This is
importance of leaf
resistance
pathogen population
Prescott, 1985).
example,
economic
the
of
events like sexual
and
windborn, urediniospores
For
area
areas
severe
southern autumn-sown wheat
sown
some
genetic
1992). The importance
the
et al.
alternate hosts
more
growing (Saari
of
frequency
regions where
The
develop
and
may vary among
seasons
(Roelfs
sexual
a
role for
important
an
et al.
in
undergoes
and
released from the alternate host to infect the
are
importance
play
fungi (Roelfs
virulence combinations is unknown
are
monocaryotic
is
dicaryotic
Thalictrum, Anchusa, Clematis and
as
the alternate hosts may
cycle. Therefore,
exchange
fungus
the
is in the
fungus
at which
resistance
means
associated
nearly always
with
It is therefore almost identical to overall
resistance, which is expressed in all plant stages (Parlevliet, 1997). Resistance
against
of
a
a
plant
plant
pathogen
but not at the
resistance
resistance
which is
the
(APR).
only effective
seedling stage
in the advanced
was
The onset of APR
resistance
is
active
when
defined
can
by
plant growth stage
Zadoks
vary. In the
infection
is
(1961)
case
most
as
adult
of leaf rust
likely
to
occur
7
because of
infection
high inoculum density
(Roelfs
1992).
et al.
grain yield
can cause severe
photosynthesis, increased
photosynthates
Therefore, resistance
Until
now
described
(Mcintosh
(Nelson
rust
inherited in
as
well
et al.
genes, such
hypersensitive
as
decreased translocation of
al.
et
quantitative
as
all of them
cycle
of the
stage (Mcintosh
infection
a
et al.
has been found
attempt has been made
and
for leaf
are
A few of these
1995).
APR
can act as a
quantitative way. Seedlings
frequency
to the
(Boyle
to determine the
with
1997).
a
of the
increased
sporulation
bacteria and
and Aust,
show
same race
or
and reduced
against viruses,
known about the mechanism of APR
(Qayoum
(QTL)
effective at the
are
plant.
(hypersensitive response, HR)
death
latency period, decreased
Although APR
trait loci
Lr12, Lr13, Lr22a, Lr22b, Lr34, Lr35 and Lr37, however,
at the adult
cell
breeding.
2000). The major resistance genes
the whole life
resistance but also in
with
in wheat
1997).
rust resistance genes have been
susceptible infection type whereas adult plants respond
pathogen
Agrios
1992;
important
is very
stage
specific leaf
1995)
throughout
or
only effective
are
(Roelfs
simple Mendelian fashion. Almost
seedling stage
major
race
respiration and
tissue
1997; Messmer
et al.
a
rate of
et al.
during heading
losses due to reduced floret set, reduced rate of
at the adult
than 45
more
An attack of Puccinia recondita
infected
from
and suitable environmental conditions for
rate.
fungi, little
is
No concerted
factors which affect its expression
Line, 1985).
3.3 Molecular markers and marker-assisted selection
Until
recently, morphological
markers
linkage
to desired traits and to select
(Weber
and
a
time and
species. Often
subvital
used in
indirectly
plant breeding
much
maize
patience, however,
genes with
mutations
as
had
negative effects
to
be
used
to
to define
for these traits in crop plants
Wricke, 1994). Classical phenotypic markers
few well characterized species such
long
were
(Rafalski
were
et al.
plentiful
1996).
in
It took
obtain several markers for
such
as
dwarfism, albinism
only
or
a
one
other
(Weber and Wricke, 1994). Moreover,
8
morphological
reasons, the
markers often
use
of
only
can
be detected in adult
morphological markers
Biochemical markers
(protein markers)
amino acids of enzymes with little
or no
in
plant breeding
based
are
effect
on
on
the
polymorphisms
the
on
techniques, however, only
example, in maize less
small to find close
wheat, the important leaf
Molecular
sequence
(DNA)
are
few
than 40
linkage
biochemical marker
protein
enzymatic activity.
markers
being applied
to
a
-
-
-
-
-
-
-
creation of
simple
mapping
of
quantitative trait loci (QTL)
traits
mapping of mutations
transgenics
genetic diagnostics
for
plant breeding
population genetics
identification of individuals
taxonomy
and evolution
germplasm characterization
identification of
easily distinguishable.
Winzeleret al.
For
In
polymorphisms
proprietary germplasm
estimation of genome size
(1995).
in number and
central to
the
in
biochemical markers.
of
-
-
and
as:
molecular
-
are
variety of problems
-
-
on
mapping
or
present
verifiable and this number is to
genetic maps
identification
the
proteins
based
morphological
such
With
means
are
theoretically almost unlimited
1996)
frequent.
high, which
In this
rust resistance gene Lr19 can be detected with the
of
et al.
charged
isoenzymes
Ep-DIc developed by
wide
are
has been limited.
agronomically important genes (Becker, 1993).
to
disadvantage
(Rafalski
level
For these
differences in
case, the variation between different varieties and lines is
that
plants.
are
nucleic
overcoming
DNA markers
plant
acid
genome
the
are
analysis
9
According
to Winter and Kahl
markers focuses
single
of
into
on
three
in
plant breeding
major issues: (I)
resistance
resistance and
for
breeding
into
genes
of molecular
the acceleration of the
introgression
the accumulation
cultivars
the
(III)
to
quantitatively
species
cultivated donor lines
or
(pyramiding)
the
of
multiple
generate
improvement of
the
use
disease resistance genes from wild
superior cultivars, (II)
drought
(1995),
major and/or
and
yield, protein
as
now a
large
number of research
projects that
content or
considerable number of molecular markers have been
quantitative characters. Particularly for disease
effort
was
and is made to
main
staple crops
(reviewed by
such
Mohan
of MAS
application
Frisch et al.
(1999)
markers in the
1997).
published
Numerous
for
form. A
qualitative
pest resistance much
molecular makers for the
addressed
papers
in the last years. For
(1998), Knapp (1998),
breeding
applied
and
wheat, maize, rice, barley, potato and sugarbeet
al.
et
were
Xie and Xu
(1997),
as
or
developed
and
develop different types of
questions
address
prospects of marker-assisted selection (MAS) in theoretical
al.
by
and cold tolerance.
There is
and
durable
more
value of crops
agronomic
inherited traits such
minor
questions
example, Hospital
where to
as
theoretical
Hoisington (1998)
Ribaut and
such
the
on
integrate
et
and
molecular
program, the number of markers which should be used,
the number of progeny which should be tested and whether molecular markers
could
actually
few reports
have
et al.
(Ribaut
al.
developed
One
a
applied plant breeding.
1995; Reddy
1997;
Stuber,
rice lines with
et al.
strategies
Han et al.
rice
variety
to increase
(1997).
1997), barley (Han
1997).
Yoshimura
major resistance
major resistance gene against
popular
and
in
There are,
however,
MAS experiments. Successful work has been done in
on
(Yoshimura
et
impact
an
gene
bacterial
of eastern India
malting quality
traits in
in rice
et al.
was
(1997).
barley by
MAS
bacterial
is
more
effective than
phenotypic
blight.
introgressed
Different
were
into
breeding
compared by
These authors conclude that the combination of
genotypic selection
and maize
(1995) successfully
pairs against
blight
by Reddy
al.
et
1997)
et al.
rice
phenotypic
selection alone. In
maize, Stuber (1997) found that identifying and mapping of QTLs is effective.
10
Furthermore, the transfer of
was
optimal. By increasing
genome
breeding
segments with
deleterious
the fast and reliable MAS Ribaut et al.
marker(s) which co-segregate
an
introgressed QTLs, the recipient
chromosomal
requirements for MAS
sufficient for
-
application of
by MAS
(1997)
program.
The essential
-
donor
line to another
one
increase the selection pressure in backcrosses in their maize
able to
were
the number of
replaced by
was
effects. Due to the
-
two to four QTLs from
MAS)
or are
closely
linked
screening large populations
technique should
economical and
plant breeding programs
(1
cM
or
are:
less is
probably
with the desired trait
efficient way for
the marker
in
be
highly reproducible
breeder-friendly (Mohan
At present, the two last
points
PCR-based markers such
as
can
with the molecular markers
et al.
be achieved
sequence-tagged
across
laboratories,
1997)
relatively
sites
well with the
(STS)
use
of
and microsatellites.
These marker types allow:
-
-
an
a
earlier
plant sampling
faster DNA
because of the small amount of tissue
preparation
because of the small amount of
required
template
DNA
required
-
a more
PCR
-
the
efficient
handling
of
large sample size,
because of the
efficiency
of
technology
possibility
amplification
of
automation
reactions
can
be
as
the
extraction
performed
in the 96
of
genomic
or
384 microtiter
DNA
and
plate-
format
3.4
Breeding for resistance
Breeding
for resistance
occurrence
breed for
of
new
new
and
requires
virulent
a
sources
of resistance genes
constant inflow of
pathogen
races
new
resistance genes. The
makes it necessary to
permanently
resistant elite lines. Over the years, effective resistance
genes
11
are
often
recombination
have
mixtures
of
Mutations
Zadoks
the
years
as a source
isolated
from
urediospores of
1
x
10~6
and
where the
and
the
ineffective after
two
races
ha of
1
variety
a
few
of various
occurs
single
seasons.
yield,
the farm level and
the
a
regional level,
use
a
mutation
of the
different
to
day. Depending
on
a
and
of
of
conventional,
to become virulent on
agricultural strategies
development
of the
level. On the farm
region
an
adequate application of nitrogen, the
time to escape the
farmers
and
epidemiological
zone,
agencies
cooperate. In addition, seed distribution
good
resistances must be available
called
practice, however,
government
level,
epidemic
one
use
of
variety,
containing different resistance genes. On
of different combinations of
In
are
pathogen
pathogen,
an
less
was
early seeding
promising strategy (Knott, 1989).
breeders,
become
can
genes
longevity
pathogen
reduce the
of seed mixtures with lines
areas
of
dikaryotic pathogen,
an
use
with
frequency
be
the
new
with 1% infection of
multilines which is the combination of several resistance genes in
or
fact,
(Samborski, 1985).
throughout the world
breeding
several
could be realized
can
in rust
in leaf rust. Parlevliet and
resistance
To prevent the
measures
of the
to occur. In
rusts
The average
existing. Agricultural
agricultural methods
The fusion
with the resistance gene is grown, the number of
(Kilpatrick, 1975).
on
Asexual
readily
occurs
susceptible wheat
resistance to wheat rusts
resistant lines and reduce
races.
of many mutant spores each
conditions,
growing
host.
susceptible wheat plants inoculated
assuming heterozygosity
climatic
monogenic inherited
occurrence
virulent
new
produce 1011 uredospores per day. With
area
than 10 years
for
sexual
reasons:
intermediate
expect mitotic recombination
potential for production
a
the
on
provide the basic variation that
to
is due to several
which is called anastomosis,
(1977) estimated that
10"5
there is
occur
well
hyphae
been
leaf rust could
on
to
and it is reasonable to
strains
x
pathogens. This
can serve as
of germ tubes and
1
the
known
is
recombination
fungi
by
overcome
in
a
resistance
gene-deployment,
et al.
1992).
is
in
a
this is difficult unless all
given
zone
must be controlled and
(Roelfs
genes
to
agree
adequate
and
12
for durable resistance is the most ambitious
Breeding
from the attack of
The
fact that these resistances
are
genetically complex.
individual effects
the
on
plant phenotype.
must be combined at the
(QTLs)
same
and that the effect of individual
does
resistance
supposed
confer
not
also called
pyramidisation,
against
same
to the
the
to the
one
individual genes
the
however, do
that several
regions
When
each
only
a
single
invariably
give higher
act
but
pathogen
is
considerably.
combination,
resistance genes and/or QTLs
the
more
the
e.
genes.
independently.
effective gene; the
more
effective gene is
Furthermore,
be resistant to the rust
some cases
act
different level of resistance,
a
phenotype of
resistance
This kind of
cultivar has several genes for resistance
determining
effective but in
individual
significant impact
a
the
to
attack
that confers lower resistance.
are
not
may interact to
a
fungal
the least resistance is masked, i.
conferring
only
smaller
easily identified.
resistance
complete
rust reaction
two or more genes will not
have
is not
are
have
to breed for durable resistance is the
pathotype.
usually exhibits the
overcome
implies
This
to the
Because several genes
disease, it is generally assumed that the genes
same
epistatic
regions
of different
A cultivar with two genes,
gene
challenging, due
is
goal.
to achieve this
time in order to have
to be durable and to reduce the
promising approach
Another
protect plants
to
expression of polygenic traits, they generally
in the
involved
existing
are
polygenic inherited resistance in breeding
of
use
pathogen. Several ways
a
project
also
against
Genes
for
independently. Thus,
levels of resistance. A
races
cultivar with
a
against
races
disease
which the
which have
resistance,
several resistance genes
single
gene
by
itself
small effect, but in combination with other genes the effects
might
can
be
greater (Knott and Yadav, 1993). Extreme forms of complementary resistance
require
There
give
the presence of two
are numerous
an
or more
examples
enhanced level
expressed.
of genes for disease resistance that interact to
resistance
or
genes for the resistance to be
(Samborski
and
Dyck, 1982;
Ezzahiri and
Roelfs, 1989). This complementary interaction, which may be additive, results
in
a
higher
durable
level of resistance than that conferred
resistance to leaf
(Roelfs, 1988).
Lr13 and
rust
by
is associated with
perhaps Lr12,
both adult
the
a
single genes. The
most
few gene combinations
plant resistance genes,
in
13
combination with Lr34
of
the basis of most of this resistance. The combination
are
(adult resistance) and Lr16 (seedling resistance)
Lr13
enhanced resistance: rust
interaction but
produced
virulent to Lr16 did not result in
races
an
also
a
results
in
compatible
incompatible infection type (Samborski
and
Dyck,
1982).
In order to
necessary
stay ahead of constantly changing
breeders
wheat
for
resistance genes from
pools of
to their
wheat
sources
the
are
crossability
other than
of not
sources
with
maintain
to
hexaploid
rust
pathogens
genetic
it has been
diversity by seeking
wheat. The different gene
common
yet exploited resistance genes. According
wheat the
species
are
divided into three gene
pools.
The
primary gene pool
Genes of
species belonging
to the
transferred to wheat with almost
group
includes
modern
genome, old land
with lower
races
ploidy levels
primary gene pool
problems (Dyck
no
and
as
such
to this group. The
genome donor T
belong
to the
well
wheat
occurs
in
normal
and
resistance genes is
1985).
The direct
diploid (AA
pairing
crosses
and
Wheat
between
one
between
some
are
genetic
of
are
ploidy
the solution in such
tetraploid
level
and
cases.
then
to
Genes
level to
wheat, the transfer of
and
Kerber,
genotypes of hexaploid and diploid
can
hexaploid
possible
wheat and the
wheat, however, is sometimes difficult and the hybrid is sterile. Bridge
are
also
recombination
possible without problems of Fi sterility (Dyck
cross
with the
donor, T. tauschii, also
hexaploid
DD) progenitors
species
related to the A
sexual crosses
and
be
AABBDD
(T, durum)
another, from
to
chromosome
hybrids produced from
tetraploid (AABB)
variety
the
(T. dicoccoides)
D genome
diploid
primary gene pool. Within this group
Because
with
diploid Einkorn (T. monococcum),
urartu and the
can
Kerber, 1985). This
hexaploid spelt (T spelta).
tetraploid durum
as
to transfer desired traits from one
another.
and
hexaploid wheat (T, aestivum)
AABB genome and wild relatives of durum wheat
belonging
of common wheat
be transferred first from
wheat.
Another
bridging
crosses
diploid
to
method
14
applicable
to the transfer of
genetic
material to both
wheat is with natural
amphiploids
these two wheats
the D genome in
amphiploid
hybrid
or
that have the A genome in
with
common
with
common
hexaploid
selected for resistance is crossed to wheat, and the
wheat. The
partially fertile
is then backcrossed several times to the wheat cultivar to obtain fertile
plants. Although 25,000 cultivars
to search for
of wheat
available, there is ongoing need
are
resistance genes in the other
new
sources.
leaf rust resistance in most of the cereal crops
international
scale.
expected from
Consequently,
advanced
breeding
The
secondary gene pool
Out
of
the
numbered
49
(Mcintosh
et al.
hexaploid
wheat and
gene
pool.
least
one
and
one
were
This gene
from
DDMM genome
species,
secondary
are
11
and
resistance
et al.
breeding
with
common
is
T
1998),
31
genes
originated
from
Ten leaf rust
material from the
aestivum.
secondary
T.
ventricosum with the
to be the
Stebbins, 1956),
and
at
Gene transfer from these
possible. For example,
(Sarkar
wheat
an
be
can
resistance
rust
hexaploid spelt (Triticum spelta).
into
on
Kerber, 1985).
leaf
unnumbered
speltoides, which is supposed
common
for
genes
(Dyck
being utilized
now
the donor of the Lr37 resistance gene. The
was
T.
are
The known genes for
pool includes the polyploid Triticum species that have
genome in
genome of
new
stocks
introgressed
species by recombination
genome
few
1995; Kolmer, 1996; Dubcovsky
resistance genes
that
tetraploid and hexaploid
diploid S
progenitor
also
of the B
belongs
to the
gene pool. This species has contributed several resistance genes
used
in
cultivar
improvement,
for
example:
Lr28,
Lr35,
Lr36,
Neepawa*6/7~. speltoides F-7, Manitou*6/7. speltoides E-11, Neepawa*6/7".
speltoides
S
H-9 and
genome
is
Neepawa*5/7". speltoides
related
to
the
homoeologous chromosomes
chapter
genome,
can
about the resistance gene
appear to
develop normally
degeneration. This probably
inability
B
to nourish the
for
a
occur
(Mcintosh
et al.
recombination
but the
number of
frequency
If the
a
days
have
the
of such
two
(see
crosses
signs
endosperm
developed
As the
is very low
and then show
breakdown of the
embryos
1995).
between
Lr35). Often the embryos
results from
embryo.
0-1
of
and its
for at least 8-10
15
days, they
can
often be removed, cultured
produce plants (Knott, 1989). Embryo
special
tertiary
is often the
rescue
the
to
tertiary
pool
gene
Resistance genes from 7. umbellulatum
are
to obtain
transferred to
chromosomes
recombination
methods
hexaploid wheat (Mcintosh
homoeologous
are
between
available for
are
to those of
the
related.
(Lr25, Lr26, Lr45)
cereale
et al.
1995, Knott, 1996). Their
wheat, but during meiosis there is
homoeologous
inducing
distantly
more
(Lr9), Thinopyron ponticum (Lr19, Lr24,
Lr29), Thinopyron intermedium (Lr38) and Secale
no
only choice
pool
gene
Species belonging
were
medium and induced to
hybrids.
fertile
The
on
chromosome
recombination. Radiation
pairs. Several
can cause
random
breaks in chromosomes which will reunite at random and result in translocated
chromosomes.
method.
An
Numerous
alternative
recombination is to
Sears
(1977),
method
the
use
which is
successful
According
can
to Knott
relatives of wheat is
wheat.
in
fact, in
overcome
Alien
a
by
gene
there is
or
screening
should
integration
process.
ensures
physiologically
located
that
on
pairing
this
and
developed by
the
long
arm
only homologous
cases
resistance from
into wheat
be done
different from that in cultivated
to assume that it will be more durable:
no reason
new, virulent race of a rust
integration
mutant line
by
there is little evidence that resistance in the wild
genetically
number of
a
homoeologous
phi locus,
product PH1
induced
been
and recombine.
(1989)
Consequently,
inducing
deletion line for the
a
pair
for
have
X-ray induced ph1b/ph1b
of chromosome 5B. The gene
chromosomes
transfers
is
thoroughly
a
an
alien
species has been
pathogen.
long
to
and
ensure
laborious
maximum
process
so
that
results from the
16
3.5 The
objectives of
In this work,
we
inherited adult
the
wanted to
plant leaf
markers would allow to
breeding
study
integrate
the resistance genes
efficiently
Furthermore, MAS would allow
against the
durable resistance.
molecular markers for the monogenically
rust resistance genes Lr13 and Lr35 in wheat. These
material via MAS.
resistance genes
develop
same
pathogen
in
one
into advanced
to combine several
line to achieve
a
more
17
Molecular
4.
of the adult leaf rust resistance gene
mapping
Lr13 in wheat
Seyfarth R,
(1999)
Feuillet
C, Schachermayr G, Messmer M, Winzeler M,
Submitted to Journal of Genetics and
Keller B
Breeding
Abstract
Resistance of wheat to leaf rust
or
only
more
at later
can
genes. Lr13 is effective
develop
important
linkage
a
gene. We
between
only
(Lr) genes,
at the adult
seven are
stage
at the
seedling stage
map around Lr13 and
wheat
two
lines
a
objective
and
resistance
of this work
one
of
was
to
molecular marker for this resistance
segregating populations
ThLr13
adult-plant
and is considered to be
leaf rust resistance genes. The
developed
the
expressed either
stages of plant development (adult-plant resistance). Among
than 45 leaf rust resistance
the most
be
Frisai
derived from the
and
ThLr13
crosses
Thatcher,
and
respectively. Eighty-two RFLP probes,
14 microsatellites and 226 AFLP
primer
combinations
In the first
Frisai)
three RFLP
the
were
tested for
probes and
four microsatellites
population
second
microsatellite
were
gene. In both
linkage.
(ThLr13
x
linked. No AFLP
populations
x
linked with Lr13, whereas in
were
Thatcher)
population (ThLr13
one
RFLP
amplification product
probe
was
the wheat microsatellite GWM630
and
one
linked with the
was
the closest
marker at 10 cM from Lr13.
Introduction
Wheat leaf rust caused
Henn. is
use
one
by Puccinia
of the most
devastating fungal
of resistant vaneties is
minimizing
the
losses
recondita Rob.
an
caused
leaf
Desm.
f.sp.
tritici Eriks. &
diseases in wheat worldwide. The
economical and
by
ex
rust.
environmental-friendly
Resistant
varieties,
way for
however,
18
particularly
those
resistant. The
carrying only
ability
the host resistance
Among
the
Lr13 is
probably
1995).
For
of the
pathogen
than 45 leaf rust
more
the most
example,
(Rajaram
pathotypes
1988).
As
a
of the
Trigo, Mexico)
Under normal
et
(Ezzahiri
showed
an
Roelfs,
and
1989).
increased resistance
illustrate the observation that
pathogen
to
can
confer
are
quantitative
CIMMYT
(Centra
expressing
the Lr13
conditions, this resistance
is
combination of two
or more
interactions is
parasite
lack of isolates with
has been
al.
the
longer
no
1995). However, Ne2m
the
as
the
yield
is
(Anand
can
one
et al.
line
and sometimes
morphological
not
called
be
1991)
used
as
against
Lr16 also
the
same
However,
the
traditional hostdue to the
necrosis gene
marker for Lr13
it maps at 33
examples
due
impossible
for accurate
leaf
presumably
using
hybrid
an
against
These
1995).
al.
et
addition, hybrid necrosis is
drastically
breeding process
resistance genes
(Yoshimura
a
so
effective in
Lr13 and
Dyck, 1982).
virulence genes. The
as
of
and durable resistance
time-consuming
previously defined
the resistance gene. In
trait
pyramiding
and
resistance genes in
specific
resistance gene Lr13
combination
(Samborski
complementation
et al.
al, 1995). However, in combination with
The
broad-spectrum
overcome
(Mcintosh
Lr34 it appears that Lr13 is the basis of the most durable resistance
rust
durably
breeding.
gene worldwide
single gene Lr13
(Mcintosh
not
which
plant stage which is characteristic for
adult-plant resistance (APR).
areas
frequently
resistance genes identified in wheat,
de Maiz y
et al.
acts at the adult
wheat-growing
new
of the wheat varieties
most
resistance gene
most
(Lr)
widely distributed
Mejoramiento
only
to evolve
are
constant efforts in resistance
requires
International de
gene
resistance gene
one
Ne2irt
(Mcintosh
detection
cross over
et
of the
units from
agronomically negative
reduced. The introduction of molecular markers in
to select lines which contain the desired
composition
of
resistance genes is therefore very valuable.
Here,
we
have
developed
two
where the resistance gene
linkage maps
of the
region
Lr13 is located. Furthermore,
of chromosome 2B
we
have found
a
microsatellite marker, GWM630, which segregates at 10 cM distance from the
Lr13 resistance gene.
19
Material and methods
Plant material
Studies
performed
were
Zürich-Reckenholz),
resistant
ThLr13)
Two
the
isogenic
near
which
on
the
susceptible spring
(NIL) Thatcher*7/Frontana,
developed by Dyck
was
segregating populations
population consisted
et al.
linkage analysis.
for
the resistance gene Lr13 is effective at the adult
the gene
was
seedling stage according
(1984). Infection
Plants of the
chamber.
(ThLr13
Frisai)
x
the infection
were
to as
performed
The first
between the
line Thatcher.
stage, detection of
to the method
developed by
with the leaf rust isolate 96505
avirulent for Lr13 under controlled conditions in
(FAL, Zürich-Reckenholz)
growth
was
the
F2 individuals
151
parental
Although
Pretorius et al.
cross
population comprised
derived from the NIL ThLr13 crossed with the recurrent
done at the
on
(referred
R.L.4031
of 156 F2 individuals derived from the
NIL ThLr13 and Frisai. The second
(FAL,
Frisai
(1966).
developed
were
variety
wheat line Thatcher and
susceptible spring
line
wheat
tested at the
type 50 F2 plants
of the first
resulting F2 progeny
re-tested
as
population
for resistance. To confirm
seedling stage
were
a
F3 families
at the
seedling
stage. Each F3 family consisted of 20 individuals. The second F2 population
originating from
resistance at the
Scoring
of the
urediospores
ranging
the
artificially
as a
performed
was
for
genomic
as
infected
suspension
screening
Isolation of
x
Thatcher
plants
was
digests.
labeling
kit
also infected and scored for
10
polymorphism
days
(Roelfs
probes
Graner et al.
(Amersham,
were
et al.
spraying
A scale
1992).
with molecular markers
DNA from leaf tissue of 5-week-old wheat
by
after
(Philips, Paris).
used to determine infection types
described
RFLP
done
with mineral oil "Soltrol"
(1990).
(ßamHI, BgH\, Dra\, EcoRI, EcoRV, HindWl
DNA
was
seedling stage.
from 0 to 4
Parental
ThLrl3
cross
and
labeled with
Switzerland)
seedlings
was
Seven restriction enzymes
Xba\)
were
[32P]dCTP
according
used for
using
to
the
a
genomic
random
primer
manufacturer's
20
instructions.
were
RFLP
Eighty-two
used to
screen
mapped
polymorphisms.
chromosome 2B
on
http://wheat.pw.usda.gov),
chromosome
derived from wheat
(Devos
whereas the
(PSR
and
were
The
probe GLK456
was
DNA
and oat
developed by
was
probes
used
by Plaschke
of microsatellites
analysis
was
performed
carried out
was
Switzerland)
(1995)
et al.
Sorrells
Devos et al.
(1991).
derived from the wheat
were
described
Dr.
Rôder
M.
(IPK,
(MWG, Germany)
were
(1998). PCR-amplification
by Rôder
automated sequencers
on
LiCor 4200
and
as
and Rôder et al.
were
probes
Gatersleben, Germany). The microsatellites GWM 120, 319, 388 and 630
described
the
database
M.E.
Dr.
1993;
on
The
(CDO).
mapped by
kindly provided by
were
probes
Liu and Tsunewaki
study
al.
et
Graingene
the
in
probes had
48
1993; Laurie
kindly provided by
The 14 wheat microsatellites used in this
variety Chinese Spring and
probes,
genomic
(Cornell University, USA). The probe PSR912
(1993).
susceptible parents
of the other 34
position
described
are
(http://wheat.pw.usda.gov)
al.
et
GLK), barley (BCD)
and
CD0388
and
BCD1709
Out of these
not known, The cDNA and
was
to chromosome 2B of wheat
the resistant parent ThLr13 and the
Thatcher and Frisai for
been
probes mapping
et al.
(1998). Fragment
ALFexpress (Pharmacia,
fluorescence
with
labeled
primers.
performed
described
AFLPs
were
(1995)
with minor modifications
Frisai.
Digestion
were
of
as
on
by Zabeau
the F2
plant
performed according
to HartI et al.
tested. The
volume of 25
kinase buffer
labeling
\x\ containing
(10
potassium acetate)
1 hour at
for
et al.
and 1U T4
(pH 7.5),
was
1991). Phenotypic
done
bulks
and Vos et al.
cross
and PCR
ThLr13
x
amplification
226 Sse8387l/Msel
primer
extending into genomic sequences
10 uM Sse8387l
primer,
10 mM
primers
2
MBq
was
[y-33P]
made in
50 mM
The incubation time
at 65°C for enzyme inactivation.
a
ATP, 1 X
magnesium acetate,
polynucleotide kinase.
37°C, followed by 10 min
polymorphisms
(1999).
reaction of the Sse8387l
mM Tris-acetate
(1993)
material of the
genomic DNA, ligation of adapters
combinations with two selective nucleotides
were
and Vos
was
Screening
by bulked segregant analysis (BSA) (Michelmore
were
composed by sampling
an
equal
amount of
21
DNA
(250 ng) from
10 F2 individuals
10 F2 individuals
showing
showing
resistant infection
a
Polymorphisms found between
rust.
tested
type after inoculation with leaf
parental
the
infection type and
lines and the
pools
were
F2 individuals different from those used for the BSA. The
30
on
susceptible
a
amplification products
Electrophoresis
acrylamide gel
5%
on a
denaturing Polyacrylamide gel.
done for 105 min at constant power
was
was
separated
were
exposed
for 2-3
days
(60 W).
to Biomax MR films
The dried
(Kodak).
Linkage analysis
Linkage estimation
(Lander
MAPMAKER
to
based
was
et al.
1987).
centiMorgan (cM) according
the
on
maximum
likelihood
The recombination fraction
to Kosambi
method
was
using
transformed
(1944).
Results
Segregation analysis
populations
of the F2
Two different F2
populations
gene. The first
population
34
plants
a
resistant
a
(X2
=
1.05) and
population. Genetic mapping
were
retested
The second
as
F3 families
days
is
was
at the
mapping population
83
=
for
used for
mapping
an
on a
F2
phenotype and
intermediate
subset of 50 F2
derived from the
50 showed
a
cross
a
gene
1:2:1
this
plants which
reaction. The 1:2:1
inherited
subpopulation
the resistance gene Lr13.
between the NILs
resistant reaction, 67
susceptible
monogenically
confirmed the intermediate action of Lr13. A
was
intermediate
39
seedling stage.
was
a
as
carried out
phenotype
5.22)
seedlings,
in
and 34
(X2
Frisai and
inherited
showed
segregation
an
x
a
plants,
intermediate
ThLr13
This indicated that the gene followed
ThLr/3and Thatcher. Out of 151 F2
an
cross
of the Lr13 resistance
after infection of the
phenotype,
susceptible phenotype.
segregation
mapping
derived from the
was
consisted of 156 F2 individuals. Ten
individuals showed
for
developed
were
resistance
gene
of 89 F2 individuals
22
Genetic
mapping of
Linkage
studies
populations.
the Lr13 gene
performed
were
RFLP
probes
tested
polymorphic
for
a).
Two
region of
between
F2
previously
parental
lines,
five
were
probes BCD1709,
the
linked with the resistance gene. The closest RFLP
were
arm
the
(Tab. 4/1). Only
a
genetic distance of 16.9
polymorphic probes mapped by
the short
segregating
chromosome 2B of wheat. Out of the 82 RFLP
between ThLr13 and Frisai
probe, BCD1709, showed
4/1
on
polymorphism
CD0388 and GLK456
different
two
and microsatellites used in this work had
been shown to be located
probes
the
on
of chromosome 2B
cM to the Lr13 gene
Laurie et al.
were
(1993)
(Fig.
in the distal
not linked with the resistance
gene.
The microsatellites
were more
polymorphic
than the RFLP
probes
between the
parents ThLr13 and Frisai. Indeed, five of the 14 microsatellites tested
polymorphic
them
between the two parents ThLr13 and Frisai
(GWM120, GWM319, GWM388
resistance gene
(Fig. 4/1 a).
and
The closest
GWM630)
were
(Tab. 4/1).
proximal
to Lr13
on
microsatellite, GWM630,
We used the AFLP
ThLr13
226
x
technique in
Frisai to find
new
primer combinations
between 70 and 100
was
lines
polymorphism
when the
and
was
the F2
high,
Each
bulks
no
(data
linkage
not
was
were
mapping population
polymorphisms
one
RFLP
was
primer
combination
cross
was
and
one
generated
In total 65
shown). Although
primer
between the
the
degree
of
found with the resistance gene Lr13
tested
on
the F2
population.
derived from the
analysis
lower than in the first
probe (PSR912)
found to be
was
polymorphic fragments differing
and the recurrent parent Thatcher. Marker
of
probes mapped
population generated by the
amplification products per genotype.
the
linkage
(Fig. 4/1 a).
used.
were
polymorphic fragments
The second
All
located
markers at the Lr13 resistance gene locus. In total,
combinations revealed three to six
parental
mapping population.
chromosome 2B. No molecular marker
linked distal to the resistance gene 2B
Four of
linked with the Lr13
at 10.7 cM from Lr13. and thus is the marker which showed the closest
with the Lr13 resistance gene in this
were
cross
between ThLr13
showed that the
population ThLr13
microsatellite
(GWM630)
x
were
frequency
Frisai.
Only
found to be
23
polymorphic
between the NILs ThLr13 and Thatcher. Both markers
with the
resistance
inherited
whereas
gene Lr13. The
the
microsatellite
Therefore, the mapping
GWM630 showed
mapped
were
at 22 cM
a
with
PSR912
was
was
dominantly
codominantly
combined data.
(Fig. 4/1 b).
As in the first
population,
the
mapped probes
to the resistance gene.
techniques in
Cross
Polymorphic
Linked RFLP
probes
RFLP
probes
probes
Tested microsatellites
Polymorphic
microsatellites
Linked microsatellites
Tested AFLP combinations
Polymorphic
Linked AFLP
inherited.
Microsatellite
polymorphic
markers and linked markers found
two different crosses, ThLr13
x
Frisai and ThLr13
Thatcher.
Tested RFLP
linked
of 10.3 cM with the resistance gene while PSR912
Table 4/1: Number of tested markers,
with different marker
probe
GWM630
performed
was
linkage
proximal
located
RFLP
were
combinations
ThLr13X Frisai
ThLr73x Thatcher
82
82
5
1
3
1
14
14
5
1
4
1
226
65
0
x
24
2B
-
2B
Lr13
cM
-Lr13
10.7
cM
-
Xgwm630
103
6.2
-
Xbcd1709
—
Xgwm630
3.3
-Xgwm319
2.2
-
Xcdo388
-
Xglk456
11,7
3.8
40
-Xpsr912
—
3.2
Xgwm388
-Xgwm120
(A)
Figure
1:
Linkage map
of
RFLP
probes and microsatellites on chromosome 2B of wheat. The
is indicated by an arrowhead. (A) Map of chromosome 2B in
ThLr13x Fnsal. (B) Map of chromosome 2B in the cross ThLr?3xThatcher. RFLP probes:
approximate position of the centromere
the
cross
(B)
BCD, CDO, GLK and PSR; microsatellites: GWM.
25
Discussion
In this
study,
two different
F2 populations
resistance gene. The level of
population originating from
probes tested
for
mapping
polymorphism detected by RFLP
the parents ThLr13 and Frisai.
polymorphic
were
developed
were
between these two
was
Only 6%
parental lines.
the Lr13
low in the
of the RFLP
In
hexaploid
wheat, the lack of polymorphism between lines revealed by RFLP is well known
(Liu
and
Tsunewaki, 1991).
population
showed
with Rôder et al.
of
(1998)
polymorphism
Despite the
material for
are
successfully
is
which
possibly
of
level
which is in agreement
a
much
were
cross
relationship
theoretically
higher level
wheat than any other
microsatellite
due to the close
polymorphic
between ThLr13
of the
crossing
share 99% of the genome.
polymorphism, NILs represent
likely
some
to be linked to the gene of interest.
develop
of
the
best
for the
Pm1,
NILs have been
molecular markers for disease resistance genes in
wheat, e.g. for the Lr9 and Lr24 leaf
or
one
segregating
molecular markers linked to desired traits because any
used to
1994, 1995)
hexaploid
derived from the
was
NILs and therefore
finding
polymorphism
RFLP probe and
population
low
polymorphism (35%),
who found that microsatellites had
one
and Thatcher. This is
partners which
rate of
and information content in
Only
marker system.
in the second
higher
a
The microsatellites tested in the first
rust resistance genes
Pm2 and Pm3
powdery
(Schachermayr
et al.
mildew resistance genes
(Hartletal. 1993, 1995).
In
both
populations
all
molecular
markers
mapped proximal
were
resistance gene and the microsatellite GWM630
was
marker at 10 cM from Lr13. All the tested RFLP
located distal to the Lr13 gene
which
were
et al.
1993, Laurie
were
1993; Rôder
et al.
region
region
of chromosome 2B
were
around the centromere showed
observed
a
complete absence
resistance gene. The
mapping of
of
a
previous
RFLP
polymorphic
maps
(Devos
polymorphism
probes
probes located
in the
but not linked. Thus, the
normal level of
the RFLP
and microsatellites
1998; http://wheat.pw.usda.gov)
monomorphic between the parental lines. Two
telomeric
we
et al.
to
the
the closest molecular
probes
according
to
polymorphism
in the
region
whereas
distal to the
and microsatellites in this
26
region
has been
performed
synthetic wheat) (Liu
1993; Rôder
far in wide
so
Tsunewaki, 1991;
and
1998). Therefore,
et al.
found here could be due to the
use
Devos et al.
the lack of
of two
(wheat
crosses
spelt
x
wheat
1993, Laurie
polymorphism
hexaploid
or
that
wheat parents
as
x
et al.
we
have
crossing
partners. New segregating F2 populations with different parents would possibly
increase the chance to find
precisely determine
available RFLP
polymorphism in
this
region
and therefore
the location of the resistance gene. So far
probes
and microsatellites in the
probes and microsatellites
are
we
more
used all
region of Lr13. Therefore,
needed in the future to find
the resistance gene Lr13. Other molecular marker
new
closer marker for
a
could also be
techniques
used.
We have used the AFLP
Unfortunately,
226 different
was
none
primer
of the
marker
the ThLr73x Frisai
was
developed (HartI
et al.
1997).
In
et al.
1999),
markers
desired traits.
Only
our
population
found which
were
can
also
serve as
the
linked to
disease
tip
yellow
the
necrosis and
rust
case
(Yr18)
of
necrosis
Ne2ITI
barley yellow
the
dwarf virus
gene
microsatellite
completely linked
closely
linked marker.
a
more
the
markers have been
useful for
were
was
(Bdv1)
on
Because of the
shown to be linked to
GWM630 which is
a
(Anand
can
not
et
al.
NeZ!,
for
frequent recombination
gene
(Lr34),
chromosome 7DS. In
pair (Net, Ne2) responsible
resistance
primer
genetic linkage between
Lr13, however, this morphological marker
of
than
of
resistance genes and
resistance gene
complementary
detection
cases more
number of resistance genes against leaf rust
(Mcintosh, 1995).
and
accurate
and
Lr13,
member of the
a
it
(96) completely
testing
morphological
practical breeding. Singh (1992, 1993) demonstrated
leaf
a
a
Although
tools for the indirect selection of the
very limited number of
a
amplified by using
in other
to be tested to find
combinations could also increase the chance to obtain
Morphological
was
linked to the resistance gene.
primer combinations had
(Büschges
which
mapping population.
low number of primer combinations
a
linked markers could be
on
polymorphic fragments
combinations
shown that with
1900 different
technique
be
hybrid
between
used
1991).
PCR-based marker and shows
a
a
for
The
closer
27
linkage represents
Nevertheless,
into MAS
markers
the
seems
should
a
genetic
density mapping
of
or
successful
genetic analysis of
Lr13
this
linked
be
application
resistance
large scale EST sequencing
future
and
of cloned sequences such
development
such
and
integration
to Mohan et al.
with
less
1
molecular
cM
with
the
in MAS.
locus.
new
The
basis for future
development
of
high-
additional
probes from coding sequences in
mapping projects
as
of GWM630
(1997)
than
presented here provides the
microsatellite markers, the derivation of
sources
quite big
yet. According
co-segregate
a
better marker for the resistance gene Lr13.
distance is still
not feasible
resistance gene for
The
potentially
BAC clones will
as
well
provide
closely linked markers for Lr13.
as
other
new
the basis for the
28
5.
Development of
molecular marker for the
a
adult-plant
leaf
rust resistance gene Lr35 in wheat
Seyfarth R,
Appl
Feuillet
C, Schachermayr G, Winzeler M, Keller B (1999) Theor
Genet 99: 554-560
Abstract
The
objective
resistance
of this work
Lr35.
gene
chromosome 2B from T.
of 96 F2
population
probes
completely
BCD260
programs
Lr35 in its
on
diploid relative of
wheat
parents
a
a
cross
was
the
races
introgressed
wheat. A
on
to
segregating
analyzed. Out
2B,
51
Three
cross.
Lr35. The
of 80 RFLP
detected
of
them
a
were
cosegregating probe
sequence-tagged-site (STS) marker.
lines derived from several
and all of them reacted
virulent
leaf rust
between the resistant line
chromosome
of
PCR-based
breeding
originally
plant
European breeding
tested with the STS marker. None of these lines has
pedigree
leaf rust
a
was
resistance gene
converted into
was
gene
derived from
the
linked with the
was
marker for the adult
a
susceptible variety Frisai
between
A set of 48 different
no
speltoides,
previously mapped
polymorphism
develop
Lr35
The
plants
ThatcherLr35 and the
to
was
negatively
a
donor for
with the STS marker. As
Lr35 have been found in different
areas
of the
world, the STS marker for the Lr35 resistance gene is of great value
to
the
genes into
introgression
of this gene in combination with other leaf rust
(Lr)
support
breeding material by marker-assisted selection.
Introduction
Leaf rust caused
the most
by
Puccinia recondita f. sp. tritici is considered to be
important fungal diseases of wheat.
resistance genes have been characterized
To date,
more
one
of
than 40 leaf rust
(Knott 1989; Mcintosh
et al.
1995).
29
Most of them
are
whereas
plant,
a
effective from the
few of them
during this period
(1961)
but not at the
stage)
adult
(Roelfs
significance
in the adult
Although they
inherited in
are
between
the
adult
resistance in
and decreased infection
upon infection with
was
an
a
resistance
of
one
(Mcintosh
et al.
the
1995)
other resistance
with
Until now,
quantitative
genes.
Singh 1992),
frequency
breeding
most
It
The
et al.
are
1995).
race
and
distributed
(Kolmer 1992).
and Pretorius
a
hypersensitive
1995).
et al.
as
reaction
So far,
it confers durable
and Kolmer
resistance
The
1989;
(Drijepondt
world,
(German
Pretorius
latency period
(Mcintosh
programs in the
Lr34
gene
increased
causes an
and uredium size
widely
resistance
but it shows enhanced effectiveness
genes
important
genes which
seven
(Drijepondt
way
resistance in combination with other genes
probably
is of very
stage
monogenic fashion, the type of resistance
a
avirulent leaf rust
used in many
plant growth stage
losses due to reduced floret set
at the adult
breeding.
by Zadoks
pathogen during heading (the
In contrast, the resistance gene Lr13 induces
1989).
defined
was
stage have been described (Mcintosh
German and Kolmer 1992;
Lr34
grain yield
in wheat
and
stage. Resistance
in the advanced
An attack of the
severe
the whole life of the
at the adult
plant resistance
1992). Therefore, resistance
only effective
expresses
cause
only effective
only effective
seedling stage.
can
et al.
economical
differs
is called adult
resistance that is
as a
are
seedling stage through
1992). Lr13
genes
only
is
worldwide
in combination
combination
of the
adult
resistance genes Lr13 and Lr34 appears to be the basis of most of the durable
leaf rust resistance
APR genes is
confers
1997).
from
an
(Roelfs 1988).
important factor
wheat, the
resistance gene Lr35
from chromosome 2S of the
to chromosome 2B of
hexaploid
diploid
wheat. To
for Lr35 have been found until know
Pretorius
data).
infection
by
an
breeding.
avirulent
our
was
transferred
were
knowledge
no
(Kolmer
derived
by Kerber
wild relative Triticum
of
Lr35 also
race
In contrast to the resistance genes Lr13 and Lr34 which
Dyck (1990)
and
example shows that combination
for successful resistance
hypersensitive reaction upon
common
races
This
and
speltoides
virulent leaf rust
(Kerber and Dyck 1990; Kloppers
1995; Kolmer 1997, RF Park, personal communication,
The Lr35 gene has not yet been used in modern varieties
own
(Mcintosh
et
30
al.
1995).
To maintain
a
wide spectrum of resistance
against pathogens
in cultivated
wheat, the introgression of resistance genes derived from hexaploid wheat and
wild relatives is very valuable. The
hexaploid
al.
wheat
1989).
been
can
be
efficiency
improved by
the
of
use
introgression
of alien genes to
of molecular markers
Molecular markers for translocated resistance genes have
developed (Schachermayr
Procunier et al. 1995;
Dedryver
al.
et
et al.
1994,
1995; Autrique
1996; Naik
et al.
can
was
necrosis
described
was
knowledge
no
as a
practically
morphological
In this
study,
technologies
we
developed
for the adult
completely
(Anand
or
et al.
1991)
pleiotropism
marker for Lr34
plant
necrosis
in wheat
with leaf
tip
(Singh 1992). To
plant leaf
rust
far.
molecular markers based
RFLP and
on
PCR
leaf rust resistance gene Lr35. These markers
linked with the Lr35 resistance gene and will be
to combine Lr35 with other effective
durability
hybrid
useful molecular markers for adult
so
1995;
Gupta 1991). However, Ne2m
and
programs. A strong genetic association
resistance genes have been reported
are
(Singh
not be used for accurate detection of Lr13
breeding
our
found to be linked to Lr13
already
al.
et
et
1998). Morphological
markers for Lr13 and Lr34 have been described. A gene for
(Ne2m)
(Asiedu
a
valuable tool
resistance genes and thus
improve the
of leaf rust resistance.
Material and Methods
Plant material
The resistant line R.L.6082
spring
(Tc*6/R.L.5711,
wheat line Thatcher with the
Winnipeg, Canada)
and
the
the
conditions in
resulting F2 progeny
a
growth
near
Lr35 gene,
isogenic
line
[NIL]
of the
developed by Dr P. Dyck,
susceptible spring wheat variety Frisai (FAL-
Reckenholz, Zurich, Switzerland)
plants of
a
were
were
chamber. The
crossed.
Hundred
and
thirty-seven
tested for resistance under controlled
linkage analysis
was
performed
subset of 96 F3 families also evaluated for resistance in the field.
with
a
Forty-eight
31
European
wheat and
spelt breeding
lines
do not contain the Lr35 resistance gene
(described by
Siedler et al.
used to validate the
were
1994)
that
specificity
of
the molecular marker for Lr35.
Artificial infection of adult
Seeds
germinated
were
plants
under controlled conditions
Whatman
wet
on
germination. Rooting seedlings
were
sand. The
a
plants
grown in
were
transferred into
light intensity
in the
continuously
was
with
16h,
permanently
1982)
was
a
was
following
pathogens.
two weeks up to 450
day/night temperature
of
given
F2 individuals
twice
were
|imol/m2-s.
according
artificially infected
at
growth stage DC
Park,
University of Sydney, Australia).
with mineral oil
«Soltrol
plant. After inoculation, plants
24 h in the dark. Ten
1984)
(small
uredinia
considered
chlorosis)
170»
as
and
with
photoperiod
Humidity
to
Hoagland (Jones,
(Zadoks
51-55
sprayed
were
kept
at 16°C and 100%
et al.
Dr R.
as
on
the
days after inoculation (growth conditions: photoperiod
the
on
flag
90 % relative
leaves. A scale
necrosis),
and
2
(IT):
humidity) plants
ranging
ITs 0
(small uredinia
were
from 0 to 4
(immune),
with
(large
uredinia
without
chlorosis)
were
a
humidity for
;
(Roelfs
(fleck),
or
16
scored
chlorosis)
resistant, and ITs 3 (medium-size uredinia with
4
set
was
(Philips Petroleum, Paris)
used to describe infection type
was
increased
was
The
growth,
(kindly provided by
Urediospores
were
h, 19°C/15°C day/night temperature,
for the infection type
hygienic regime
daily.
with leaf rust isolate 95502 avirulent for Lr35
entire
filled with
In the first week of
1974)
suspension
plastic tubes
19°C/15°C.
to 50%. 80 ml of a nutrition solution
regular
ensure
|imol/m2'S. Light intensity
150
set to
to
chamber under strict
growth
to avoid contamination with other wheat
the
paper 3MM
1
were
without
considered
as
susceptible.
Scoring
for leaf rust resistance in
A field trial
was
carried out in
Eleven seeds of each F3
m
rows.
a
family
The field rust nursery
a
field trial
field rust nursery
chosen for
was
(Haag, Switzerland)
linkage analysis
artificially
were
inoculated with
a
in 1998.
planted
in 1.5
mixture of 16
32
Puccinia
f.sp. tritici
recondita
ThLr35 and Frisai
response data
classified
as
(resistant
and
recorded three times
susceptible individuals in
isolation, plant
harvested and
described
Thatcher
on
the
flag
pooled.
was
a
family)
parents
segregating
or
analysis
material from
al.
et
F3 families consisting of
(1990). Genomic
with
digested
were
relative to ThLr35and Frisai.
DNA isolation and Southern
by Graner
The
leaf. The F3 families
homozygous resistant, homozygous susceptible
DNA isolation and RFLP
For DNA
Switzerland.
in
also evaluated for resistance in the rust nursery. Host
were
were
prevalent
races
five
blotting
of
DNA
restriction enzymes
11
plants
was
performed
were
as
Frisai, ThLr35 and
(Dra\, EcoR\, EcoRV,
H/ndlll, and Xba\).
Southern
on
hybridization
(BCD)
(FBA, FBB, PSR,
and oat
Heun et al.
(CDO).
(1991).
Graingene
described
al.
performed
chromosome 2B. The cDNA and
from wheat
the
was
by
(1993).
The
The
data base
P.
genomic
TAM and
DNA
probes previously mapped
probes
WG), Triticum
probes CDO370
used
tauschii
and WG996
(http://wheat.pw.usda.gov).
(1991).
The
The
probe PSR540
Leroy (INRA, Clermont-Ferrand
was
and
were
derived
(KSU), barley
were
probes BCD260, BCD1119, CDO405
Graner et al.
Dr
with 80 RFLP
described
described in
are
probe MWG950
GIS
Genoble
the
probes FBA199, FBA374, FBB4, FBB47
FBB75.
and
TAM18
KSUF11
(Kansas State University, USA)
were
kindly provided by
and Dr G.E. Hart
(Texas
was
mapped by Devos
France) kindly provided
Probe
by
A&M
et
Club,
Dr B.S.
and
Gill
University, USA),
respectively.
Probe
labeling
with
Switzerland) according
PCR
amplification
Polymerase
32P
was
a
labeling
kit
(Amersham,
to the manufacturer's instructions.
of wheat
chain reaction
contained 0.625 units of
genomic DNA with specific primers
(PCR)
was
performed
in
a
25liI reaction volume. It
Taq DNA polymerase (Sigma, Switzerland),
buffer
(10mM Tris-HCI pH 8.3,
1mM
of
each
performed with
50mM KCl 1.5mM
MgCI2,
and 0.001%
1
x
PCR
gelatin),
dNTP, 10|iM primers and 50ng of genomic DNA template.
33
Amplification
performed
was
Bioconcept, Switzerland)
reaction
was
subjected
in
PTC-200
a
follows: after
as
to 30
one
thermocycler (MJ-Research,
cycle
at 94°C for 3
at 94°C for 45 sec, 59°C for 45
cycles
72°C for 1 min. The extension of the
amplified product
min, the
and
sec
achieved at 72°C
was
for 5 min.
Conversion of
RFLP
an
A BamH\ I Hind\\\
probe
fragment of
resulting clone BCD260/0.9
This
fragment
GAG GTC TTG AC
3')
designed
were
Switzerland). They
from
3')
ThLr35,
of
units
restriction
2% agarose
The PCR
was
the PCR
was
done
marker
enzyme
2h of
(5' GAA GTA GTC
the
digestion
at
CGC TAC CAC AG
fragment
cloned
(Microsynth,
amplification of genomic DNA
population.
were
mapping analysis.
primers BCD260F1 (5' GAA GTT AAA
of
was
in
subcloned. The
CAPS
A
isolated
(Cleaved Amplified
developed by digesting the amplified
(4bp recognition site, CTNAG).
added
directly
the
to
37°C, the fragments
were
Ten
reaction
after
separated
on a
gel.
subcloned into the
3')
F2
was
probe
fragments amplified between BCD260F1
sequenced.
AC
the
RFLP
a
with the restriction enzyme Dde\
amplification. After
were
end
used for PCR
and
probe BCD260
as
and the
each
at
Polymorphic Sequence)
fragments
used
was
and BCD260R2
were
Frisai
STS marker
a
0.9kb of the
sequenced
was
to
The
specific
designed in
«pGEM-T Easy»
STS
primer
(5'
(1.5 kb)
(Promega, Switzerland)
and
TTT TGA GAA TCA GTC ATC
the insertion found in the ThLr35 allele. Visualization of
product amplified by
on a
35R2
vector
and BCD260R2
1% agarose
gel
the
primer
combination BCD260F1
and 35R2
stained with ethidium bromide.
Linkage analysis
Linkage estimation
summed
based
on
the recombination
frequency
frequency of recombination types among the
recombination
using
was
frequency
was
transformed to map units
the Kosambi function. The recombination
r
total
(defined
progeny).
(centiMorgan)
frequency
was so
the
as
The
without
low that
we
34
estimated
the
recombination
probability
frequencies
double
of
as
crossing
overs
as
and
zero
the
additive.
Results
Segregation analysis
of the F2
population derived from the
cross
ThLr35x
Frisai
To map the Lr35 resistance gene,
between the line ThLr35 and the
Evaluation of resistance
either 0
was
ThLr35
(large
4
or
-
uredinia without
uredinia)
small
chlorosis)
reaction
distinguished (data
shown). To confirm the
plants,
additional resistance tests
of 137 F2
individuals,
103
F2 individuals
were
monogenically
inherited
cross
scored for resistance.
of
types
were
F2 individuals showed
susceptible. The
gene
3:1
confirmed
susceptible parent Frisai
heterozygous plants
performed
a
could not be
results obtained with the F2
in
F3 and F4 generations. Out
resistant reaction whereas 34
segregation
the
of the F2
like the resistant parent
like the
(Fig. 5/1). Intermediate
not
were
growth chamber. The infection type
a
(immune
1
-
variety Frisai
a
done after artificial infection at the adult stage
was
under controlled conditions in
plants
F2 individuals derived from
137
dominant
(X2: 0.0024)
action
of
for
the
a
Lr35
resistance gene.
A subset of 96
randomly
F3 families (74 resistant plants, 22 susceptible plants)
chosen and scored for
susceptibility
progeny of the susceptible F2 plants
whereas
resistant
the
the
or
progeny
of
the
were
resistant
segregating. Ninety F3
or
resistance in
field trial. The
homozygous susceptible
F2 plants
were
families showed the
either
in the
field,
homozygous
infection type
same
F2 individuals in the growth chamber. For six F3 families
were
a
was
as
no
phenotypic data
obtained in the field. These results demonstrated that the
phenotypic data
generated
conditions.
in the
growth chamber
Genetic
mapping
heterozygous F2 individuals
resistant
plants by segregation
identical to those obtained under field
are
was
could
performed
be
in
distinguished
in the F3 families.
this
from
subpopulation
the
as
homozygous
of wheat
grown in the
(B),
10
days
D
B
(C)
and
susceptible F2 individual (D).
after artificial infection with the leaf rust isolate
95502.
resistant F2 individual
growth chamber
Resistant parent ThLr35 (A),
susceptible parent Frisai
Figure 4.2/1: Flag leaves
%^MW^A^'ui:î^^^0Si^^^^'f^^^^^^&
36
Genetic
Eighty
mapping
RFLP
of the Lr35 gene
probes
chromosome 2B
which
chosen for
were
showed
polymorphism
probes
which
gave
hybridization.
shown
be
to
located
Out of this set,
51
probes
hybridization pattern
clear
were
mapped
in
and
inherited
were
segregating population consisting
a
group.
Three
complete linkage
5/2).
probes BCD260/0.9, WG996
to the Lr35 resistance gene in the
probes FBB4 and MWG950
The
and FBA199
FBB47
was
probes
cosegregated
located
used
on
have
0.5 cM
at
distance of
at 1.1 cM from the resistance gene while the
already
spanned only
which
been
mapped
in
the
cross
Synthetic
39 cM
(Fig. 5/2).
covered
was
on
chromosome 2B
Conversion of the
RFLP
marker
BCD260
into
x
and
In the cross Th/_r35
x
Opata
covered
Frisai, the
by these markers
cM, suggesting that recombination frequency
3.3
probe
the other site of the Lr35 gene at 1.1 cM. Thirteen of the
approximately
genetic region
the
on
probes BCD1119, CDO370, CDO405
(http://wheat.pw.usda.gov/ggpages/linemaps/Wheat/SxO_2.html)
a
showed
located at 0.5 cM from the gene
were
side of the resistance gene. The
one
mapping population (Fig.
Lr35, whereas the probes FBA374 and TAM18 mapped
opposite
PSR540
and
as
of 90
F3 families grown in the field. The 14 codominant RFLP probes formed
linkage
on
between the two parents ThZ_r35 and Frisai. Fourteen
a
codominant markers,
previously
were
reduced.
was
sequence-tagged-site
a
(STS)
Hybridization with
pattern
with
the
only three fragments
polymorphic fragment
showed
The
the
in
resulted in
each
parental
line
BCD 260/0.9
was
sequenced.
of 1.5 kb. The
genomic DNA is due
barley
A
primer
larger
was
size of the
to the presence of
cDNA BCD 260/0.9
The
Frisai, respectively,
designed
(data
not
an
at each end of
with these
DNA of both parents ThLr35 and Frisai resulted in
amplification products
(Fig. 5/3).
the Lr35 resistance gene.
fragment (BCD260F1/BCD260R2). PCR amplification
wheat
simple hybridization
a
of 4.3 kb and 4.5 kb in ThLr35 and
complete linkage with
a
probe
genomic
the
probe BCD 260/0.9
non
primers
on
polymorphic
fragment amplified from
intron of 900
shown). Digestion
bp compared
of the
amplification
to
-
-
110~
100-
90
80
70
60
cM
Xbcd260
Xcdo370
Xksufl 1
XtamW
Xfba374
Xfbb47
Xcdo405
Xmv/g950
2BS
2BL
>Xfba199
'Xbcd1119
Xwg996
\Xfbb4
h Xfbb75
I
(B)
0.6 cM
0.5 cM
0.6 cM
0.5 cM
0.5 cM
0.6 cM
\
Xksufl 1
Xfbb75
Xfba199
Xbcd1119
Xcdo370
Xcdo405
Xfbb4
Xmwg950
Xbcd260
Xwg996
Xpsr540
XtamW
Xfba374
-Xfbb47
Lr35
chromosome 2B in the
cross
ThLr35
x
Frisai is not known.
Figure 4.2/2: Genetic map of chromosome 2 B in a cross between Synthetic x Opata (A) and a cross between ThLr35
x Frisai (B). The arrowhead indicates the centromere of the chromosome 2B of Synthetic x Opata. The orientation of
(A)
2B
CO
38
H
il
MSRRSRRRRSRRR
S
kb
23
9.4
6.5
—
—
—
Hâ«ftAAâ*
_^#
43__
2.3
Figure
the
4.2/3: Southern
hybridization pattern
probe BCD260/0.9.
pooled
F3
progeny
population (R
weight
of
resistant
DNA
13
F2
digested genomic
extracted from the
individuals
phenotype,
marker M is A, DNA
polymorphic
was
of Xba\
digested
S
from
the
ThLr35, Frisai and the
segregating
mapping
susceptible phenotype). The molecular
with H/ndlll. The arrowheads indicate the
bands of 4.3 kb and 4.5 kb from the resistant and the
alleles, respectively.
DNA with
susceptible
39
products
with the restriction enzyme Dde\ resulted in
bp in ThLr35 (Fig. 5/4). This CAPS
450
mapping population
the
The
complete linkage
a
parents ThLr35
and
independent clones
Frisai
were
likely correspond
to two
subcloned
and
of sequences
homoeologous
position
906
compared
we
generated
insertion
A
from
to the second
genomic
DNA
were
obtained for ThLr35 which
an
additional insertion of 29
bp
type (Fig. 5/5). This insertion includes
development
of the CAPS marker.
corresponds
of
Frisai
but
showed the
none
the
to
were
29
bp
(Fig. 5/5).
In combination with
fragment
of
amplified
0.9
in
phenotypically
kb
no
in
Frisai.
was
designed
from the
29bp
insertion in ThLr35
primer BCD260F1
it led to the
the
Th/_r35 whereas
The
resistant
0,9
kb
line
fragment
resistant F3 families whereas
obtained from the
showed
from Frisai
as
chromosome 2B. Two different classes of sequences
on
specific primer (35R2)
5/5).
well
as
conclude that the sequence with the insertion
resistance locus
also
sequenced (Fig. 5/5), Five
chromosomal locations of BCD260/0.9.
the Dde\ restriction site which allowed the
Thus
BCD260R2 from the
and
were
One type of sequence derived from ThLr35 had
at
of
to the Lr35 resistance gene.
from the resistant line ThLr35
analyzed. Two different types
fragment
marker showed dominant inheritance in
fragments amplified with BCD260F1
kb
1.5
and
additional
one
was
amplification
no
amplified
of
single
product
from
amplification product
no
a
(Fig.
all
was
the
could be
susceptible F3 families (Fig. 5/6). Thus, this STS marker
recombination in the F2 progeny and
was
completely linked
with the
resistance gene Lr35.
Validation of the CAPS and STS markers in wheat
A set of 48
(Siedler
European breeding
et al.
1994)
was
lines derived from different
used to test the
for the resistance gene Lr35. These
Lr35,
as
breeding
specificity
breeding
lines
this resistance gene has not yet been
susceptible variety
Frisai
material
breeding
of the markers
are
programs
developed
known not to contain
exploited
programs. With the CAPS marker all the
characteristic pattern of the
breeding
until
breeding
(data
not
now
in wheat
lines showed the
shown).
The
40
Figure 4.2/4:
on
genomic
A CAPS marker for the Lr35 resistance gene. PCR
DNA of Th Lr35, Frisai, five resistant
individuals with the
primers
(R)
fragment
resistant parent and the resistant F2 individuals. The
agarose
gel.
one
of
performed
susceptible (S) F2
BCD260F1 and BCD260R2 followed
with Dde I. The arrowhead shows the additional
on an
and
was
450bp
fragments
by digestion
found in the
were
separated
41
RCn2 60Fl
Fr I
t-
Fill
lhLr35\
A 3511
Thl
Frl
Frll
GAAGTTAAAGAGGTcnTUACCTACVVITGAGAAGGCGGATGTTTTGCCTCC
50
GAAGTTAAA0AGGT(TTGAC(TA(\rrTGAGAAGGCGGATGTTTT0CCTCC
50
GAAGTTAAAGA<;GTCT1GA(VTA(xrrTGAGAAAG(\;GATGTTTTGCCTCC
SO
GAATOACATGCTTTTGATACATTAAGTTTGATCATTCCAATAAAATTGGT
894
ThLr35l
GAATCACATGCTTTTi ;ATATATTAAGTTTGATCATTCCAATAJVAATTG0T
887
lhU3S\\
GAATCACATGCTTTTGATATATTAAGTTTGATCGTTCCAATAAAATTGGT
89 4
Frl
gacccagtcata
+
f
Frll
-
GACCCAGTCATA
Th La 3511
gacccagtcataKjttag
k**-k*-k*-hl
Frl
Frll
* * *
-
tttagtgat
<hs
---
TTTAGTGAT
908
4ATGACTGATTC TCAAAAAAATATTTAGTGAT
9 4 4
---
-
ThU35\
-
-
-
^—-
--
-
.
ÎSR,'
******** +
gacggcaattttcagattcttgctaaagggaacgattgttataaaagatt
9 6 s
T h/.A 351
GACGGCAATTTTCAGATTCTTGCTAAAGGGAACGATTGTTATAAAAGATT
95 8
1M/A351I
GATOGCAATTTTCAGATrCTTGCTAAAGGGAAGGATTGTTATAAAAGATC
994
Figure
+
4.2/5: Nucleotide sequence comparison of the
genomic
DNA of Frisai
Sequences
additional
were
29bp
(Fr
I and Fr
amplified with
was
the
II)
and ThLr35
(ThLr35
I and ThLr35
II).
primers BCD260F1 and BCD260R2. An
to
develop
the
dominant STS
nucleotides indicate the additional Ddo\ restriction site
chromosome 2B of ThLr35.
are
from
found in the Thi/35 II allele. The arrowheads indicate the
primer sequences used
sequences
products amplified
marker.
(CTNAG)
The boxed
in the allele
on
Difforoncos between the two classes of Frisai
outside the DNA
region shown in the Figure.
42
additional 450
bp fragment
these lines. Moreover, the
result in any
the two
in lines
obtained with ThLr35
use
containing
not detected in any of
of the STS marker in the
amplification product (Fig. 5/6,
primers BCD260F1
was
and 35R2
data not
shown).
amplifying
are
breeding
a
lines did not
We conclude that
specific sequence only
the Lr35 resistance gene.
Discussion
Development
of molecular markers for the detection of Lr35
In this work, the
goal
to
was
develop
a
molecular marker for the adult
rust resistance gene Lr35 which is derived from
speltoides. Linkage analysis showed
(BCD260,
PSR540 and
gene Lr35. A
WG996)
tight linkage
complete lack of pairing
of
were
probes
Lr24
et al.
(Autrique
Lr25 and Lr29
et al.
completely
was
1995; Schachermayr
was
et al.
1995)
close
(Autrique
linkage
and FBB47
was
a
et al.
absence of recombination
or
was
1995).
genetic distance
cross are
introgressed
already found for Lr9
et al.
1995),
et al.
et al.
1996),
1995; Dedryver
(Naik
et al.
1998).
In the
case
For Lr35, eleven
Interestingly,
of
was
RFLP markers showed
a
Between the most distant probes KSUF11
of 3,3 cM
the
two RFLP markers
as
probes
covering approximately
was
well
found. This indicated that there
as
between these loci and the
which span 3.3 cM in the ThLr35
39 cM in the
cross
Synthetic
This shows that recombination is about 10 fold lower than in
hexaploid
leaf rust
1995), Lr19 (Autrique
and Lr28
recombination between these loci
Frisai
and Kerber
transferred from T tauschii, the D Genome donor of
to the resistance gene,
resistance gene.
probes
due to the low level or
wheat, recombination between the resistance gene and
observed
RFLP
(Zeller and Hsam 1983; Dyck
et al.
wheat, T.
linked with the resistance
expected
species into wheat
1994; Autrique
(Procunier
the Lr32 gene which
codominant
between molecular markers and
resistance genes from alien
(Schachermayr
three
and the reduced rate
between wheat and alien chromatin
1985). Complete linkage
that
wild relative of
a
plant leaf
wheat. These results suggest that
pairing
a cross
x
x
Opata.
between
and crossovers between
43
Figure
4 2/6
STS
products from
was
parent
variety Oberkulmer
Lr35. The
of 931
size
gene
amplification
The
bp,
isolated from the resistant parent ThLr35
Fnsal,
susceptible individual),
fragment
Lr35 resistance
genomic DNA with the primer combination BCD260F1/35R2
shown. The DNA
susceptible
marker for the
nine
F2
(R
resistant
L
6082), the
individual,
two winter wheat varieties Anna and Forno and the
The arrowhead
which showed
marker
individuals
(R
(M)
is
a
indicates the
dominantly
complete linkage
the 1kb ladder
are
inherited
S
spelt
DNA
with the resistance gene
44
chromosome 2B and
possible
but at
a
S genome of
fragment
With
detected
signals
a
T
T.
a
signal
Daud and Gustafson
(1996)
speltoides specific probe,
in the genome of
showed that
Triticum aestivum and T
in Th/_r35
hypothesis
hexaploid
that the
wheat
and Maestra and
Daud
(1996)
sitopsis species,
wheat,
were
which
have been
barely detected.
proposed
Maestra and
homoeologous pairing between chromosomes of
speltoides
occurred. A
pattern of preferential pairing of
types, A-D and B-S, confirmed that the S genome is very closely related
the B genome of wheat. When
banding analysis,
as
Naranjo
Gustafson
and
are
tetraploid and hexaploid wheat. In contrast,
B genome donor of
as
Naranjo (1998)
two
is the B genome donor of
speltoides
in the genome of the
previously
introgressed
reduced rate. This is in agreement with the
previously suggested by
(1998).
of chromosome 2S
characterizing
Friebe et al.
(1996)
wheat-alien translocations
to
by C-
could not determine the translocation
breakpoint
of chromosome 2B in the Lr35 donor line R.L. 5711. The authors
suggested
that the translocation chromosome 2B
fragments
derived
from
recombination could
relative
the
position
cross
recombination
occur
of the 13
Synthetic
Opata
x
2S
of several
of
T.
speltoides
and
that
between the translocated parts. We found that the
probes
frequency
transferred from T
chromosome
composed
was
used in
and
cross
different
and
speltoides
the
could
analysis
our
not identical between
was
ThLr35
Frisai.
x
arrangements
explain
of
A
decreased
the
fragments
the different order of the markers in
the two maps.
Marker assisted selection for Lr35
RFLP markers
are
very reliable markers for
intensive and expensive
has to be
analyzed
assay. PCR
in
provides
a
(Mohan
et al.
1997). The high
simple
and fast
unique sequence
diagnostic
tools.
polymorphisms
STS
at
a
known
markers
in cereals
et al.
method
been
are
as
plants
adapted
identify
reliable
successfully
well labour
that
rapid diagnostic
a
STS markers, which
genetic locus
have
(D'Ovidio
screening
but
number of
plant breeding programs requires
(marker-assisted-selection), PCR-based
and
plant breeding
and
used
to
to
MAS
a
short
efficient
detect
1990; Weining and Langridge 1991;
45
Williams
al.
et
Tragoonmng
1991;
al.
et
1992;
Talbert
al.
et
1994;
Schachermayr 1994, 1995).
One of the
probes (BCD260/0.9)
resistance gene
was
and STS markers.
which showed
with the Lr35
converted into two different PCR-based markers: CAPS
Although
the CAPS marker is
additional step for restriction enzyme
STS marker which
complete linkage
only needs
a
digestion
a
PCR-based marker,
makes it less
practical
PCR reaction. The insertion of 29
bp
an
than the
in ThLr35
with the additional Ddel restriction site allowed to convert the RFLP marker into
a
STS marker. Thus,
large populations
screened for presence
STS could be
here,
we
amplified
or
in
breeding
absence of Lr35 in
from all the
a
now
be
fast and easy way. Since
no
programs
European breeding
conclude that this STS marker is
highly specific
lines
can
we
have tested
for the Lr35 resistance
gene.
The
pyramiding
of different resistance genes
molecular markers in classical
gene Lr35,
has been
and
a
reported
communication,
would
in
own
source
overcome
marker,
breeding programs.
be
supported by
In the
case
the
use
of the resistance
Canada, South Africa, Australia and Switzerland (Kerber
and Pretorius 1995; Kolmer 1997; RF Park,
data)
would
until
certainly
the resistance
now,
The
use
of
this
gene
as
lead to the emergence of virulent
in
a
short time. With the
help
personal
a
races
single
which
of the STS
the combination of Lr35 with other leaf rust resistance
genes which
active at the
breeding for
of
combination with other genes should be achieved. No virulence
Dyck 1990; Kloppers
resistance
can
seedling and/or
durable resistance
the adult
against
stage should allow
this disease.
a
more
are
efficient
46
6. General Discussion
6.1 Marker
types and marker-assisted selection
A first map of the human genome based
1980)
the
was
catalysator for the development of genomic maps
organisms. Polymorphisms
plant varieties paved
markers in
(Botstein
molecular markers
on
that
detected in
were
the way for the
of molecular
use
their number is
nearly unlimited,
their expression is not necessary for their detection and all markers
detected with
marker
single technique (Melchinger 1990). The increasing
a
techniques
important
traits in
useful traits for
has resulted in
nearly
a
large
number of markers for
can
be
number of
agronomically
all crops. Molecular markers have been used to
and also for map based
breeding
other
in
genomic DNA of different
development and
plant breeding. Unlike isoenzymes,
et al.
cloning strategies.
tag
Restriction
Fragment Length Polymorphism (RFLP), microsatellites, Random Amplified
Polymorphic
(AFLP)
were
PCR-based
DNA
(RAPD) and Amplified Fragment Length Polymorphism
used to
generate molecular markers.
markers
like
STS
markers
or
convenient marker
types
of low amounts of
DNA, their fast application
to be used in
plant breeding
In maize the usefulness of microsatellites has
1997). Röder
are an
et al,
excellent
important genes,
over
(1998)
source
and Korzun et al.
and
already
(1999)
the genome and the
high
rate of
possibility
most
requirement
(Ribaut
et al.
claimed that microsatellites
mapping
to automate the
our
the
selection tool.
as a
been shown
polymorphism,
numbers. The wheat microsatellites used in
are
due to their
reliability
of molecular markers for the
due to their
polymorphism between
microsatellites
study
the
of
agronomically
even
analysis
showed
of
a
distribution
large plant
high
level of
the distant parents ThLr73 and Frisai. GWM630 turned
out to be the closest linked marker for the resistance
gene LrW. Another
of PCR-based marker
(STS)
has been shown to be
a
marker type to detect wheat leaf rust resistance genes
1994, 1995). The STS marker
we
have
developed
type
reliable and efficient
(Schachermayr
et al.
for the resistance gene Lr35
47
in wheat is ready to be introduced into
marker, direct staining
bromide
A
technique
promising
new
and visualization of the PCR
eliminate the need for
can
breeding schemes.
to
generate
molecular
more
(1998).
modification
technique represents
a
Amplified Polymorphic Sequence) analysis and
Amplified Polymorphic Sequence).
between two
different
parents which does
sequences
into
mutation
nucleotide
products
the
are
amplification
afterwards
not
but
so
Arabidopsis
far there
disadvantages
are
digested with
thaliana
In
a
small
An
but
of
routinely
assays
are
(Michales
digest
wheat
plants have
to be tested.
site. The
with
a
single
the
is the
1998)
et al.
The
requirement of
an
product.
which
increase their
making methodological changes.
having
unrestricted
trait loci
no
main wheat
(QTL) (Braun
program of the SARDI
to
access
where 7000
breeding
et al,
(South
new
1998).
Australian
RFLP
marker
(Dr, K. Williams, ITMI meeting 1999, personal
on
type
in
and
technique
agronomically important crops.
Development Institute, Australia),
applicable
(derived
Amasino, 1998; Neff
of the PCR
breeding
made per year
less
and
quantitative
the kind of molecular markers used in MAS,
differences between the type of
or
dCAPS
shown that this new
methodology
programs
or
communication). Depending
more
together
few research programs and
used MAS
costs exist. Each marker
(Cleaved
appropriate restriction enzyme
conservative when
are
is the wheat
Research and
CAPS
mutation has to be found
plant breeders adopt breeding methods
survey
exception
called
Neff et al.
novel restriction
a
which
already
such reports in
no
biotechnological methods,
program
the
was
of CAPS and dCAPS
breeding efficiency
the
and
was
creates a new restriction site in one of the alleles. The
additional restriction enzyme
Conventional
of
is
point
generate
products
separated by gel electrophoresis. It
works in
A
(1998)
markers
amplified by using primers which incorporate
are
polymorphism
ethidium
electrophoresis.
and Amasino
Cleaved
dominant
a
product through
developed independently by Michales
This
As it is
application,
has its
the
advantages
speed of detection and the
and drawbacks which make it
practical plant breeding
where
large
numbers of
48
The main question which has to be solved for MAS is where in the classical
breeding it
different
categories, depending
which
plants
populations
pollinated.
are
which
consisting of
of the
application
the
application
considered.
can
crosses.
are
which
which
variety
are
self
selectively
breeding scheme:
most
are
each
be used
fertile,
cross-
breeding
important step is
to
The second part consists of the selection of the
and
(III)
testing of the
the
selected lines. In each
possible
pollinating species
to be done before
as
In the second
predictive
a
such
crossing.
and useful.
as
wheat
Here, the
or
barley
is
lines with the best allelic
Molecular characterization
tool to reduce the
phase, markers
impact.
An
segregating F2 generation
markers in the first
can
early stage
be
applied
in
number of necessary
one
key selection step
of recombination which could be the
would be such
critical step. The
a
segregating population
the desired traits whereas the rest of the
genetic
would fix
application
already
in this
progenies from
crucial
space and time for field trials is often limited. In the third
plant breeding,
a cross can
already
already
in this step would
ensure
been identified. The
that the wanted
stage
be reduced in this way, which is
molecular markers could be used as
allelic value has
of
variance is not reduced. The
number of
as
clonal
the decision which lines should be crossed. Molecular markers
to maximize their
some
lines
First of all, the identification of elite
simplify
also
common
of molecular markers is
complementarity has
could
a
There
hybrids
and
is divided into four
reproduction.
phases. (I) The first and
(II)
MAS for self
of
propagated,
pollinating
three
the basic variation.
potential parents
phase
open
are
the kind of
on
vegetatively
categories have
All
method is
provide
integrated. Classical plant breeding
should be
diagnostic tools,
application
phase
of
when the
of molecular markers
genetic composition of the
new
line is
correct. Also other traits could be detected with different molecular markers to
predict
the line
the
genetic
precisely
value which
was
potential
new
as a
not selected for and therefore characterize
crossing partner.
49
6.2 Allelism
of resistance genes
chromosome 2BS and
on
cloning
of
these genes
The three leaf rust resistance genes LrW, Lr23 and Lr35
the centromere of chromosome 2B
1999a,b).
LrW and Lr23
relationship
are
very
could be allelic to LrW
pool. It
the other
With
current
our
alleles of the
The
the
cloning
based
1995, Seyfarth
(Mcintosh
still
a
between
linked
the
between Lr23 and Lr35 is not
Triticum
derived from the
tauschii
(Lr22a) (Mcintosh
not exclude that
we can
linked and
flanking markers,
missing. Lr35
highly
reduced
does not derive from
fragment
prerequisite
hexaploid
wheat but
derived from T.
speltoides
gene does not
necessarily
been constructed
(T.
are
and the D genome
for the B genome,
Breeding
for the A genome
(T. tauschii,
a
related
species
or
reflect the
libraries that have
monococcum,
Moullet et al.
linkage
Lijavetzky
1999). So far,
no
et
library
hexaploid wheat.
for resistance
ecological
and economical
emphasis from chemistry
resistance is the most
protection,
are
of map-
recombination rate. Thus, the close
and the
marker
the
recently
The
and
1995).
et al.
LrW, Lr23 and Lr35
large insert
6.3
and
wheat gene
originated from hexaploid wheat (Lr22b)
distance between them. Moreover, the
existing
speltoides
primary
physical
is
The
of the resistance gene Lr22 that two alleles
translocated chromosome
which shows
1999)
1995).
et al.
al.
et
gene.
closely
are
on a
al.
located close to
of the two genes LrW and Lr35 is not feasible at the moment. In
cloning,
is located
One
as
al.
derived from
were
diploid Triticum
knowledge,
same
of LrW
case
case
sources.
from the
one
was
et
linked
well
as
Lr23 which
or
shown in the
was
from distinct
came
closely
between LrW and Lr35
known. The resistance gene Lr35
(Mcintosh
are
research
on
to
impacts of agrochemistry
biology
in
have caused
a
shift of
plant production. Since genetic disease
effective, economical and prophylactic way of plant
breeding
for resistance and the cultivation of resistant
50
varieties is
taking
1997). Although
of
leaf
rust
develop
available. Chemical control has been
for wheat
1982). Chemicals
be
can
in the field. Resistance
pests
have
only
pathogens
is
are
where
Europe
well
yield
to
to
and
of
possible (Buchenauer,
the
fungicides. Efforts
quality (Roelfs
control
et al.
on
advantages,
et al.
1992).
susceptible
In
on
pathogen
in
fungal
breeding
1992).
In the
costs
as
the environment have
losses.
Many
fungal
other
addition,
most available
fungicides provide
cultivars when environmental conditions
disadvantages
no
to chemicals and this may occur with the
development. Although
the
is
protect susceptible cultivars against leaf
prevent yield
developed resistance
(Roelfs
prices
needed and there
are
possible negative impact
guarantee
favorable for disease
clear
they
when
Using fungicides
no
have
inadequate
used in
however, the disadvantages of applying fungicides such
become apparent,
as
successfully
not
are
would be unnecessary if the control of
been made to increase
attack
controlling
cultivars
resistant
new
population except for appearance of
breeding
for the farmer and the
rusts
and
exclusively done by application
was
recent years,
rust
applied
to monitor the rust
necessity
be the best method for
supported and where high yields
are
pesticides (Wenzel,
effective chemical control would be valuable for situations
an
races
new
chemical
on
always
resistant cultivars will
leaf rust disease,
when
to research
priority
over
make
the
application
breeding
of
fungicides
for resistance
an
are
has
attractive
alternative.
Depending
the crop, the acreage
on
different
breeding goals
breeding
to achieve durable
resistance,
focus
major
minor genes
on
single major
genes
and
or on
only
pathogen
grown
is not
so
Netherlands, flax is
specific
breeding strategies
a
basic
on
high
a
a
to
existing. Concerning
are
question rises:
should
(QTLs)? Breeding for
resistance genes has been shown in
effective, long lasting and
which is
and environmental conditions
occupied
few hectares in
one
region
new
breeding
resistance with
exceptional
prevent the appearance of
minor crop and all the cultivars carry
to be
cases
races.
In
the pressure to
to select for new, virulent races, For
the
a
a
example,
monogenic,
resistance genes to flax rust. Since 1962 these genes have
crop
biotic
in the
race-
protected
51
the flax crop
effectively.
Four cultivars
were
20 years without loss of resistance. The
non-durable in the USA,
important
crop
(Parlevliet, 1997).
to the fields. Of course, the
last from
durability
woolly aphid resistance
in
rust resistance genes in
as a
resistance
single
breeding
1995) leading
et al.
as
1995).
by
the
This
pathogen
breeding strategy
easy to handle in
approach
gene
overcome
Considering
be
it is
a
is still
question
when it is
always very
was
problem
to
of
be
case
of
The APR resistance
a
of
et al.
introgressed
as a
the
on
(Mundt
attractive
In the
plant breeding
single
use
and
gene.
single
of
Browing, 1985),
breeders
to
race-
as
the
complete resistance and
long term,
individually,
it is not
because
to
one
into
promising
a
resistance
the
monogenic
plant
to
resistance.
limited, they should
Kerber, 1985). Several possible ways to
resistance
distribution in space and/or time
(Mcintosh
in
exploited
incorporated successively
and
line from leaf
important gene in
an
that the number of useful resistance genes is
breakdown
In the
of time until this resistance
pathogen adaptation
employed carefully (Dyck
prevent the
in the last
protecting lines from leaf
elite lines confers
to use these resistance genes
the
can
contrast, the introgressed resistance
speltoides,
breeding programs.
after another has
vary. It
already
protect
however, still
resistance to the cereal rusts
incorporation of single genes into
was
It is,
recently, small-grain breeders concentrated
specific genes for
and release
can
it is often involved in gene combinations
1995). Therefore,
overcome
was an
for instance with the
as
(Kilpatrick, 1975).
to enhanced resistance. In
et al.
to be
wheat, the effectiveness of single resistance genes
Until now, this resistance gene is not
rust attack.
(Mcintosh
resistance genes
to over 130 years
gene Lr35, which derived from T.
Until
single
gene is not sufficient anymore to
(Mcintosh
rust attack
gene is
of
breeding material
apple cultivars (Parlevliet, 1997).
some
16 and
general, single race-specific resistance
the average less than 10 years
gene LrW
1930-1950 when flax
years, when the resistance is neutralized
zero
stages of the breeding program,
was on
In
periods between
type of genes appeared
period
after their introduction into
are overcome
genes
same
in the
especially
grown for
genes
exist:
(gene deployment
(I) through
at the
a
guided
regional level, gene-
52
for-gene
multilines)
use,
cultivar. Gene
or
(II)
deployment
combination of several resistance genes in
a
is not
practicable, because
it would
require good
agreement among and discipline of the farmers and breeders (Roelfs
1992). It
that
proven
was
(Fischbeck, 1997). Why did
is that the
variety
variety selected
is finished
the risk of selection
et al.
major
by
because of such
multiple
leaf
pathogen
the
durable.
role.
against all
barrier
was
several
(Samborski
and
of resistance genes
or
Brown,
rust for
yellow
obtained
by
not
always
The
use
pathogen
this
barrier that is not
a
accident
changes
of 15 years
separately
in other
(Parlevliet, 1997).
are
It
in the
longer
cultivars,
was
plays
cases
shown in
supposed
the resistance gene LrW
and
or
In all these
complexity stepwise.
Dyck, 1982; Ezzahiri
one
the
resistance became ineffective
gene combinations exist which
overcome
resistance genes in
periods
also used
to increase its
can
against
1997). Several winter wheat
gene barriers. The
were
are
three simultaneous
a
to
be
major
Roelfs, 1989). The combination
be tested via classical virulence tests with
leaf rust isolates due to the lack of virulence of
can
the resistance genes used
cultivar
same
and
Among these combinations,
selection
the time the
risk is that many genes
resistance genes
several
two
as
(Mcintosh
multiple
pathogen
that
rust
race
in the
because the resistance genes
the
possible
by
agronomic
to be another solution. It is assumed that two or three
cultivars remained resistant to
the
not be
answer
pathogen population, which probably increases
together
needed
are
enabling
Another
pyramidisation of
or
genes
overcome
virulence
develop multilines? One
to
started, but probably will
super virulent
a
great disease pressure
for the recurrent parent may be the best
to the
pathogen appears
effective
easily
try
under
et al.
1992).
The combination
same
not everyone
(Knott, 1989).
individually exposed
(Roelfs
work
when the program is
backcrossing
are
multilines
one
problem.
line could be
some
isolates. Marker-assisted
In this case, the presence of several
already
of molecular markers in wheat
detected at the molecular level.
breeding
would
help
to
prevent the
to overcome the combined resistance genes. In the case of the non-
exploited single
resistance gene Lr35, the
marker with markers for other genes would
application
of
our
new
molecular
guarantee that this effective gene is
53
not
introgressed
effective
for
breeding
with
alone in
time
long
a
elite
an
and
major resistance
Until
these genes.
today,
breeding
line. Resistance genes would remain
contribute
only
genes
to
durable
make
sense
resistance.
Therefore,
in terms of
pyramiding
17 molecular markers for different
resistance genes have been
developed (Tab. 6/1). With
major
these markers, the
effective combination of several resistance genes in advanced
should be
Where
focus
some
-
Not all
molecular markers exist
quantitative
quantitative resistances
major genes
pathosystems
major
genes that
consists of
such
with
mixture of
a
quantitative
In
the
late
pyramiding fails, breeding
should
strategy, however, there
are
are
race-specific,
non¬
the
R10
such
wheat/leaf rust, many cultivars carry
races
overcome
because the
corresponding
breeding
lines in the
as
one
or more
pathogen population
to the various resistance genes in
experimental fields
this shows up
as
resistance.
presence
of
fully effective,
(Parlevliet, 1997).
These
breeding
problems
overcome
durable. There
blight (Turkesteen, 1993).
resistance is masked
-
gene
incomplete expression
against
as
are
an
partially
are
the cultivars grown. In
-
or
resistance. Also with this
resistance gene in potato
In
lines
problems:
durable,
-
breeding
possible.
no
on
leaf rust
of
for
non-durable
major genes quantitative
horizontal, quantitative resistance
can
be
by:
A selection
strategy which is eliminating all fully resistant plants after infection.
This selection discards all
major
this way would
polygenic (Knott, 1989).
probably
be
resistance genes and resistance selected in
54
Table 6/1
and the
:
Molecular markers for different leaf rust resistance genes in wheat
source
of the resistance genes.
Gene
Source
Marker
Reference
Lr1
T. aestivum
RFLP/STS
Feuillet et al. 1995
Lr3
T aestivum
RFLP
Saccoetal. 1998
Lr9
Ae. umbellulata
RFLP/STS
Schachermayr
Autrique
et al. 1994
et al. 1995
LrW
T. aestivum
RFLP/STS
Schachermayr
LrW
T. aestivum
Microsatellite
Seyfarth
LrW
T
N band
Yamamori, 1994
LrW
Thinopyrum
isoenzyme
Winzeler et al. 1995
RFLP
Autrique étal.
RFLP
Nelson étal. 1997
RAPD/RFLP
Schachermayr
timopheevi
turgidum
Lr23
T
Lr24
A.elongatum
Autrique
et al. 1997
et al. 1999b
1995
et al. 1995
et al. 1995
et al. 1996
Dedryver
Lr25
S. céréale
RAPD
Procunier et al. 1995
Lr27
T. aestivum
RFLP
Nelson et al. 1997
Lr28
T.
speltoides
RAPD/STS
Naiketal. 1998
Lr29
A.
e Ion g at um
RAPD
Procunier et al. 1995
Lr31
T. aestivum
RFLP
Nelson et al. 1997
Lr32
T tauschii
RFLP
Autrique
Lr34
T. aestivum
RFLP
Nelson et al. 1997
Lr35
T
speltoides
RFLP/STS
Seyfarth
Lr47
T
speltoides
RFLP
Dubcovsky
et al. 1995
étal. 1999a
et al. 1998
55
-
Linkage
with
other,
easily detectable
more
stem rust resistance gene Sr2
traits should be used. E. g., the
gives incomplete
resistance and it is
effective since 60 years. It is linked with head and stem melanism
chaff)
and
be
can
Rajaram, 1993).
easily selected
Another
which shows also
at the
(false
black
phenotypic level (Van Ginkel and
example would be
incomplete resistance
partially
the leaf rust resistance gene Lr34
and is linked with
tip
leaf
necrosis
(Singh, 1992).
-
The
of molecular markers to select
application
indirectly for
the desired QTLs
via MAS.
Breeding for resistance
6.4
Resistance is
realistic
1988).
in
state of
"less disease" and "no disease" is
vertical resistance,
interaction
durability
enhanced
quality
breeding goal.
reduce
yield
mixture of the cited resistance mechanisms
or
of
these
and
yield, breeding
gene,
ability
plant
to
(1984)
yield
amount of disease
large differences
are
1997). Together with
for resistance is still the most
ways.
alternative
an
governing translocation
high
partial
or
be defined in
can
than would
for
important
strategy
to
normally
be
incomplete resistance.
plant protection
of
of
ability
are
grouped
them
the
a
number of diverse
that contribute to disease tolerance in
involve
photosynthates.
interactions
In
pustules of
an
with
some cases
to continue to "fill" the
number of
as
expected considering the
present. Under this general heading
Some
be due to the host's
incorrectly
tolerance
more
physiological phenomena
moderate to
(Parlevliet,
(Simmonds,
losses.
to Durbin
different
but there
To breed for increased tolerance is
According
a
forever,
mechanisms
The term tolerance is often used
of
distinguished: (I) Major
be
a
non-durable; (II) polygenic, race-non-specific,
No resistance mechanism lasts
the
can
rarely
resistance, durable; (III) race-non-specific, major gene resistance
horizontal
(IV)
a
tolerance?
Four kinds of resistance
objective.
race-specific,
and
simply
or
the
mechanisms
tolerance appears to
developing grain
infection type that
in
spite
ordinarily
of
a
would
56
categorize
and/or
the cultivar
awns
as
susceptible.
contribute
substantial
developing grain.
Because
of their
In
cultivars the
some
proximity
photosynthates
of
amounts
to
flag leaf, glumes
the
to
the
developing grain, they
develop very strong source-sink relationships with the grain (Durbin, 1984).
More
emphasis
tolerance. The
relationship,
tolerance
similarities
needs
any
exists,
appear to be
host-parasite
placed
genetics of tolerance
if
are
be
to
evident
to
on
exploiting
to disease is not
horizontal
independent
resistance.
this
understood; neither its
The
(Heitefuss, 1997). Tolerant plants probably
combinations
be
high yield
and
interesting goal
suboptimal growing conditions. In
become
are not
more
important,
as
of
but
exist in most
(Agrios, 1997).
or
important
mechanisms
of the mechanisms of resistance,
Thus, breeding for tolerant plants, either for biotic
an
important type of
single
to achieve
the future, this
abiotic stress, should also
and
quality under
potential breeding goal could
resistance genes in the different gene
available in unlimited numbers.
pools
57
7. Outlook
Plant
is
breeding
widely recognized
of
long-term improvement
biology
"science". It is clear that the
of MAS will not
use
engineering
plant breeding by providing
speeding
the work in the field nursery.
coming up
in
breeding programs
could be evaluated
of molecular
plant breeding
from "art" to
replace
the classical
biotechnology
and
novel
Screening
could be done
independently
from climatic
Therefore, genetic markers could potentially be
breeding programs
such
as
does not
as
genes. Once genes
of
phenotype. This,
level to
produce
at the
stop
genetic analysis
understanding
a
or
environmental conditions.
valuable tool in
first
a
are
applied
species
impact of molecular
well
as
on
structure
and
possible
function
to
develop
and
a
much greater
relate
to
genetic manipulation
this
to
alleles and allelic combinations.
used in MAS, The resistance gene Lr35 is
a
as
resistance to all leaf rust races. The
of this gene
leaf rust
careful
races
use
Therefore,
be
applied.
pyramiding
breeding
plant
at the molecular
ready
line would most
a
the
requirement for cloning economically important
in turn, will then lead to
new
already showing
level. The contribution lies
isolated it will be
gene
is
accomplishing
of traits from wild
line. But the
one
and
the molecular level. Traits
on
The STS marker BCD260F1/BCD260R2 for the APR gene Lr35 is
resistance in
help
can
large numbers of plants
of
introgression
the combination of different genes in
biology
breeding
of variation,
sources
plant breeding objectives. Marker-assisted breeding
its value in
or
help
breeding cycle, increasing the efficiency of selection
up the
simplifying
many
With the
and
new
substantial contribution to
a
to shift conventional
process but will support it. Genetic
classical
making
agricultural productivity.
possible
it became
for
use
probably
be
very valuable gene
short-lived,
as
single
because
to be
it shows
source
new
of
virulent
would evolve. As the number of resistance genes is limited, the
of Lr35 to
the
As
strategy
we
ensure
of
have
its
long effectiveness
combining
is of crucial
importance.
other resistance genes with Lr35 should
developed and published
a
STS marker for Lr35,
of different leaf rust resistance genes should be
stations that have the financial and technical
possible,
resources.
at least at
58
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26 10 1967
Born
1974-1984
Primary
1984-1987
Technical
1987-1988
Educational tour
1988-1989
Military
1989-1991
Agncultural
1991-1992
Volonteer year
1992-1995
Agncultural studies
1995-1996
Plant
1996-1999
PhD thesis
in
and
im
Breisgau, Germany
secondary
high school
school
in
in
Knchzarten
Freibug
through Argentina
service
studies at the
breeding
in
on a
farm
at the
assistant
the
University of Hohenheim
University of Hohenheim
in
potato breeding company
group of Professor Beat Keller at the
Reckenholz and University of Zurich
Since
January 2000
Employee
at VitaPlant AG Witterswil
Switzerland
FAL-
Danksagung
Ich danke
meinem
Aufnahme
in
Interesse
Prof
Dr
an
Doktorvater Herrn
seiner
Arbeitsgruppe,
Ingo Potrykus fur
unkomplizierte Betreuung
Prof
die
Dr
Beat Keller habe ich fur vieles
gesetzte
das stete
Ueberlassung des
Gesprachsbereitschaft und fur das in
Danken
zu
Fur die
Vertrauen
gebührt ein
fachliche und engagierte Betreuung
Optimismus, ihre Begeisterung, ihre
Dr
bereitwillige
und
dieser Arbeit
Themas, die stete Forderung der Arbeit, der
mich
die
Catherine Feuillet
Herrn Prof Dr
Stamp
besonders herzlichen
der
vorliegenden
Dank
Nicht
nur
fur die
Arbeit sondern auch fur ihren
Ideen und Freiheiten, die
sie mir
stets
gewahrte
danke ich fur die Uebernahme des Koreferates
Unterstützung und nicht nur fachliche erhielt ich auch von Dr Gabriele
Schachermayr Ihr sei vielmals dafür gedankt Sie hat nicht nur die Arbeit interessiert
verfolgt sondern auch mit phantasievollen Ideen angereichert
Menschliche
Besonderer Dank
geht
auch
kritischen Phase hat sehr viel
Dr
Monika
Messmer
Auswertung
der Daten
Danke auch
sei
an
Monika Clausen
Dr
zum
Gelingen
fur die
Ihr moralischer Beistand
dieser Arbeit
Einfuhrung
in
in
der
beigetragen
das Thema und fur statistische
gedankt
Philipp Streckeisen fur die vielen praktischen Vorschlage bezüglich
an
Klimakammer- und Feldversuche
Allen
Kollegen
Zurich,
die
Dankeschön
und
mir
Kolleginnen
das
gewidmet
Leben
an
und
der FAL Zurich-Reckenholz und der Universität
Arbeiten
erleichtert
haben
sei
ein
herzliches