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New Phytol.
1997), 137,
1-8
Genetic approaches in plant physiology
BY
M. KO OR NN EE F* , C. AL ON SO - BL AN CO AND
A. J. M. PEETERS
Department ofGenetics, Wageningen Agricultural University, Dreijenlaan2 6703HA
ageningen
The Netherlands
{Received3April 1997;accepted 24 June 1997
SUMMARY
The use ofgeneticsin plant biology aimsat thephysiologicalandmolecular genetical characterizationof the
phenotypic
variation for the trait under s tudy. Efficient mutant and gene isolation procedures have been developed
for
a
number
of
plant models such
asArabidopsis thaliana.
For
this,
the map position
of
the genes and insertion
mutagenesisare used. The latter also allows the characterization
of
genes that are not easily recognized in mutant
approaches,by using enhancerorgene-trapping procedures and reverse genetics.Inaddition to mutants, natural
variationpresent among wild and cultivated varieties ofa species provides an important source ofgenetic variation.
The
use of molecular markers, advanced mapping populations and specific cytogenetic stocks in case of polyploids,
enablesa detailed characterization
of
such natural variation even when
it
is
of
a quantitative and polygenic nature.
Examplesof the various genetic approachesare given.
Key words: Arabidopsis thaliana, abscisic acid ABA), gene isolationandmutagenesis, photomorphogenesis,
quantitative trait locus QTL).
INTRODUCTION
The use of genetics as a tool to dissect complex
biological processesinplants hasalong history.For
instance, a tobacco mutant called MarylandMam-
moth and different soybean varieties led to the
discover} of photoperiodism by Garner Allard
1920). Late r, during the fifties, genetic dwarfs in
peaandmaize convinced manyofthe importanceof
gibberellins asplant growth hormones. Why there-
after geneticshas notbeen used very muchinplant
physiology is less clear, despite that mutants were
shown to be crucial toolsfor theunderstandingof
biosynthetic pathways inmicro-organisms and de-
velopmental processes, e.g. inDrosophila. Towards
the end of the seventies, students of
E.
coliand
Drosophila
genetics such asSomerville andMeyer-
owitz became convinced that genetics was the wayto
go ahead in plant science. These authors,aswellas
Laibach, Redei and Feenstra before them, had
realized that
Arabidopsis thaliana
L.) Heynh. wasa
model speciesfor plant genetics, because this small
self-fertilizing crucifer has avery short generation
time. Genetics becomes even more powerful whenit
can
be
combined with molecular genetics , which
links DNAwith thephenotype. Its small genome
and ease of transformat ion were additional factors.
which established Arabidopsis as the general model
organism
in
higher plant molecular genetics.
Cur-
rently, the whole genome of this plant is being
sequenced Bevanetal.. 1997), and the impact of this
information is rapidly increasingas can beseen,for
example, from thefrequent use thatisalready made
of the partly sequenced cDNAs called expressed
sequence tags ESTs).
Besides Arabidopsis, other plant species suchas
petunia.
Antirrhinum,
maize, pea, tomato, barley and
rice have been studied for a long time, resultingin
large collectionsofgenetic stocksandgenetic maps.
They became very interesting genetic models for
specific developmental processes because of the
particular characteristics of these species, whichled
to the characterizationofsomeoftheir mutants.For
instance, for
Petunia
and
Antirrhinum,
mutations
affecting flower colour and morphology were
analysed in detail. With the advent of molecular
biology. Antirrhinum, maize and, somewhat later.
Petunia could beused for gene cloning, since well
characterized transposable elements were available.
Unfortunately, these species, except Petunia, are
difficult orimpossibleto transform, which limitsthe
complementation proof
of
having cloned
the
gene
and the further characterizat ion of those genes.
Tomato and, even more so, rice, have relatively
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M. Koornneef C. Alonso-Blanco and A.J. M. Peeters
resulting in the isolation of several disease-resistance
genes (Jones Jones , 1997) . T he la rger genom es of
barley and pea make these classic model species less
amenabletosome molecular approach es . H owev er ,
the va lue of these species sho uld not be u n d e r -
estimated, given their suitability not only for genetics
bu t a lso for plant physiology and plant biochem is try.
In pea , thesystem of nitro gen fixation c ouldbe
s tudied genet ica l ly, and wel l charac ter ized mutants ,
e.g. inthe ph ytoc hrom e, f lowering and gibbere l l in
pa thways , resul ted
in
excellent research, c om ple-
m e n t a r ytotha tofthe 'g ene ra l ' m ode ls .Inbarley,
s imilar approaches provided important molecular
da ta on seed germinat ion, and the recent map-based
cloning of the mildew resistance gene
Mlo
(Blischges
etal 1997) indicates that these limitations, because
of the large genome, canbeovercome . T hu s ,it is
clear that genetics nowadaysis abasic toolinplant
science. In orde r to apply this genet ic approach ,
genetic variation, which takes
a
range
of
forms,
is
essential. Thegenera t ion of mu tant va r iat ion is
genera l ly notapro blem because efficient mu tag ens
and mutagenes is procedures are available . M ore
difficult
is
the identification
of
m u t a n t p h e n o t y p e s
tha t arerelevant to theresearch topic . Form o r -
phological processes the phenot>^pe itself allows the
detec t ion of mu tan ts ; however , for some biochem ica l
and physiological traits , either thep h e n o t y p e is
relatively subtle, or the phenotype is too general, e .g.
genera l reduct ion of plan t size or vigour .An
important l imita t ion infinding mu tan ts istha tfor
many genes redundancy is present , which means tha t
muta t ions in such genes do not resul t in an obvious
vis ible phenotype s ince the redundant counterpar t
produces enough produc t to atleast partly) su b-
s t i tute for thefunction of themuta ted gene .In
addi t ion
to
induced m utan ts , another sou rce
of
genet ic var ia t ion is provided by the na tura l var iants
present withinaspecies andbycytog enetic stocks
such as chromoso mal su bs t i tut io ns . Once genet ic
variation has been identified and characterized, the
appl ica t ion of molecular genet ic techn iques to amen -
able species allows the cloning of the mutated genes.
Cloning and charac ter iza t ion
of
genes have some -
t imes given c lues about the na tureofthe observed
defec ts in the mutants . An important recent example
is the identification ofArabidopsis and tomato genes
produc ing dwar f i sm when muta ted , in whichen-
coded prote ins arerelated to steroid biosynthes is
pro te ins in mam mals (L ie t
al
1996). This prom pted
severa l authorstotes t t he effectof brass inos teroids
on these dwarf mutants , ofwhich many couldbe
rever ted to the phenotype of the wild type , whereas
others were classified as insensitive to this group of
c o m p o u n d s ( K a u s c h m a n n
et
al,\996 . Brass inos-
te roid mutants have common charac ter is t ics , such as
be ing dw arf with small round leaves and having a de-
e t io la ted phen otype when grown in da rkness. T hese
brass inos teroidsas na tura l p lan t hormones . On
othe r hand ,
it is
very im po rtan t to find an effect
the phenotypebygenes tha t h ave been c loned
sequenced but forwhich nom u t a n t p h e n o t y p
known, s ince such a phenoty pe provides key
formation about the funct ion
of
those genes . N
approaches such as reverse genetics are be
developed to genera te mutants , s ta r t ing with a DN
sequence of unknown funct ion.
THE USE OF MUTANTS TO CLONE GENES AND
THE USE OF GENES TO ISOLATE MUTANTS
A number of procedures are now available to isol
the cor re sponding DNA
of
genes only know n
the i r mutant phenotype . Themos t impor tan t
the se me thods are gene tagging and map-ba
cloning.
1. Gene tagging
Inse r t ion of D N A in a coding sequence in m
cases dis rupts the gene, resul t ing in a m u t
phenotype . Since the sequence of the inser ted DN
is known,
it is
possible
to
clone the genom ic pl
DNA f lanking the inser t ion and thereby par t of t
d i s rupted gene . In p lan t s , T-DNA and t r ansposab
elements a re most f requent ly used fortagging.
Arabidopsis, several large collections of T - D
inser t ions have been genera ted (Fe ldmann, 199
and, more recent ly, the int roduc t ion of ma
transposa ble e lements such as the A c/ D s (Bancro
et al.,1992 and the En/ I (Aa r t set al 1995) tw
element systems have been used successfully.
A specific application of tagging is the use
enhance rorpro mo tor t raps (Sun daresan, 1996) .
this system
the
T - D N A
or
transposab le e lem
carriesareporter gene witho ut a promo tor or wit
min imal prom otor . W hen such a constru c t is inser t
near to a gene the express ion pa t te rn of the repor t
gene might reflect theexpress ion pa t te rn of t
endogenous gene , whose promotor
or
enhancer w
used todrive the express ion ofthe rep or ter ge
Because of r e d u n d a n c y , andalso beca use cert
funct ions might not be essent ia l , such inser t ionsd
not a lways resul t in physiologica l and morp hologic
mutant phenotypes . Very sophis t ica ted sys temso
this type , which inc lude gene t raps , have bee
developed recent ly (Sundaresan
et al
1995) . Su
trap l ines have been shown to bevery usefulf
marking specific tissues (Scheres et al 1994)o
processes . An important novel appl ica t ion of taggin
is in reverse genetics (Koes etal 1995). Plants wi
inser t ions in c loned genes , for which some sequen
information
is
available,
can be
identified
by t
ability to amplify D N A fragm ents m polymer
chain reac t ion (PCR). One PCR pr imer is based o
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Genetics in plant physiology
sought. Again a mutant phenotype wil l not always be
observed when therespective gene is disrupted ,
becauseofredundancy .
2. Map-based cloning
Map-based c loning requi resadetailed genetic m ap -
posit ion ofthe locus a nd the availability of easily
screenable l ibrar ies harbour ing la rge p lant DNA
fragments , e.g. in yeast and bac teria l artificial
chrom osom es (YA Cs and BAC s) . Th e avai labi li ty of
complete physical maps, where such clones have
been grouped and sorted into contigs that are related
to the genetic map are now available for most of the
rabidopsis genome (Schmid t et al., 1995, Zachgo et
al., 1996). This enormously facil i tates map-based
loning since it rest r ic t s chrom osom e walkingto
M ar t in ,
1995). Final proof that theright gene hasbeen
the
complementa t ion
of
by D N A
. O ther gene-cloning strategies based on mutants
Ausubel ,
is
performed compa r ing wi ld type
and
et al.,1990).
SE OF MUTANTS TO DISSECT TRAITS
for
of the m uta ted genes and pro-
Clon ing of the corre spon ding genes has often
provided impor tant ,
and
somet imes uniqu e ,
in-
formation on the mode of action of these genes and,
fur thermore , it offers the possibility of modifying
these processes,
e.g. by
reint roducing
the
cloned
genes into plants, thereby over-expressing or under-
expressing the genes.
Three different pathways, namely those affecting
abscisic acid (ABA), phytochrome and the transi t ion
to fiowering can serve as illustrations for the genetic
dissection of physiological pathways.
1. Abscisic acid
T he p lant ho rm one abscisic acid (ABA) affects man y
processes in plants . Its role in contro lling seed
dormancy
and
stomatal closure, toge ther with
its
germination and growth-inhibit ing effect ledtothe
isolation ofm ut an ts affected inABA biosynthesis
and action (Table 1) . The aba mutants , which are
defective in epoxy -carotenoid levels and ABA
provided conclusive proof for the carotenoid path-
way of ABA biosyn thesis in higher plants (Du ckha m,
Linfo rth Tay lor, 199 1; Rock Zeevaart , 1991).
M ar inet al.(1996) cloned this gene using transp oson
tagging. The ABA mutants showed the impor tance
of ABA in stress resistance (reviewed by Giraudat et
al., 1994). Three different genes of ABA-insensit ive
{abi)mu tants have been c loned. ABIl (Leung et al.,
1994; Meyer , Leube Grill, 1994) and ABI2
(Leu ng, M erlot Gira uda t , 1997) were both shown
to encode protein phosphatase 2Cs, which apparently
play
a
role
in
ABA s ignal t ransduct ion . T he ABI3
gene encodes a seed-specific transcription factor that
t ransmi ts
the
ABA signal,
as
well
as
u n k n o w n
developmental s ignals, to a number of seed-develop-
ment-specif ic genes (Parcy Gira uda t , 1997). T he
relevanceofABAinseed germ inationisshownby
the lack of seed dormancy, characteristic of almost all
ABA related mutants identif ied sofar. Recip rocal
Loci involved in the biosynthesis or mode of action of ABA , and phenotype of the mutants at these loci
Seed*
dormancy
Stomatal
closure*
ABA sensitivity
growth inhibition*
Gene function
References
BB
-t
t
Zeaxanthin epoxydase
Conversion of xanthoxin to ABA
aldehyde
Addition of sulphur to Moco of
ABA aldehyde oxidase
Protein phosphatase 2C
Protein phosphatase 2C
Seed-specific transcription factor
Mar in et al. (1996)
Schu^artz et al. (1997)
Schwartz et al. (1997)
Leung et al. (1994);
Meyer et al. (1994)
Leung et al. (1997)
Giraudat et al. (1992)
Finkelstein (1994)
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M.
Koornneef
C. Alonso-Blanco and A.J. M. Peeters
Table 2 Arabidops i s
mutan ts affecting photorecep tive pigmen ts
Sensitivity to *
Gene
Bf
R
F R
Gene function
References
HYl
HY2
HY3 PHYB)
HY4
HY5
PHYA +
Heme oxygenase J
synthase I
Phytochrome B
Cryptochrome
Transcription factor
Phytochrome A
Parks Quail (1991); Ter ry
(1997)
Parks Quail (1991); Terry
(1997)
Reed
et al.
(1993)
Ahmad Cashmore (1993)
Oyama
et al.
(1996)
Whitelam
et al.
(1993)
* The sensitivity of mutants at these loci to light of specific wavelengths is less than ( ), slightly less than (+
the same as
(
+
)
wild type.
t B, Blue light; R, Red light; FR , Far Red light.
X
Heme oxygenase and phytochrom obilin synthase control the two last steps of phytochrom e chromo ph
biosynthesis.
c rosses revea led tha t ABA produced by the embryo
controls germinat ion (Karssen
et al.,
1983) . Wi thout
ABA, seeds do not require gibbere l l in (GA) for
germinat ion as shown by the ir res is tance to the
gibbere l l in biosynthes is inhibi tors te tcyc lac is and
paclobutrazol (Leon-Kloosterz ie l
et al., 1996a, b).
In addi t ion, the insens i t ivi ty to such inhibi tors of
seed germination led to the isolation of
aba2
and
aba3
mutants and to the isolation of mutants affected
specifically in seed dormancy, which probably rep-
resent genes tha t control one of the downstream
processes affected by ABA.
phytochrome-B-de f ic ien t
hy3
mutant sugges ted
several authors that screening for insensitivity to F
might yie ld phytochrome-A-def ic ient mutants ,
indeed was la te r proven. Since
phyA
mu tants h
no obvious phenotype in white light, this specif
screen was required to f ind them. From the mome
these well defined mutants were available they ha
been used to specify the modes of action of the
dif ferent phytochromes , e .g. in seed germinat i
(Botto et al., 1995) , anthocy anin format
(Kerckhoffs
et al.,
1997) and fiowering (Bagnall
al.,
1995).
2.
Photo receptive pigmen ts
The control of growth and development by the
qual i ty , qua nt i ty and dura t ion of l ight is descr ibed as
photomorphogenes is . Plants perce ive information
from l ight through pigment sys tems such as phyto-
chrome and c ryptochrome . The complexi ty of the
regula t ion of photomorphogenes i s by phytochrome
comes from the fact that different types of phyto-
chr om e enco ded by at least 45 different genes exist
(Pra t t , 1995) . Th ese phy toch rom es differ in the ir
photo-s tabi l i ty and the ir tempora l and develop-
mental expression. For some processes these dif-
fe rent types of phytochrome might have dif fe rent
modes of ac t ion. Mutants a t the
hyl-hy5
loci, wh ich
are defective in specific aspects of photomorpho-
genes is and which are recognized by the ir e longated
hypocotyls in white light, were first described by
Koornneef, Rolff Sprui t (1980) . Subsequent ly , the
molecular nature of all f ive mutants was elucidated
(Table 2) . The ident i f ica t ion of the blue l ight
receptor depended ful ly on the c loning of the
HY4
gene (Ahm ad Cash mo re , 1993) , and the sub -
sequent charac ter iza t ion of the c loned gene (Lin
et
al.,
1995) . The compar i son of the phytochrome
3. Floral initiation
The t rans i t ion f rom the vegeta t ive to the repr
duct ive meris tem which produces f iowers , is poor
unders tood a t the molecular leve l . To increase o
unders tanding of this important process in high
plants a genet ic approach has been sugges te
Genet ic var ia t ion for this t ra i t i s abundant . F
ins tance , dozens of mutants have been found
Arabidopsis
that either delay or advance the tr
s i t ion to f lowering (Hau ghn , Schul tz M art ine
Zapater , 1995
;
Peete rs Ko orn neef 1996), as wel
in other spec ies , but in none of these mutants h
flowering been completely abolished. In contrast
mutant s w i thout a r eproduc t ive phase , em f m u t a
( S u n g
et al.,
1992) lacking the vegetative phase h
been isolated. The hypothesis that fiowering is t
default state in
A rabidopsis,
which is repressed by
E M F produ c t s , was then e s tab l i shed (Sung
et a
1992;
M a r t i n e z - Z a p a t er et al., 1994; Weigel , 199
The effect of these product(s) can be modified b
var ious processes control led by the f lowering- t im
genes and also by environmental factors such as lig
and tempera ture . A number of the f lowering t im
genes ,
inc luding LD (Lee et al., 1994a ) , C
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Genetics in plant physiology
{G Oand
LD
and a
{FGA . Using
a
system with
GO gene productisswitched on,a careful
of
the floral initiation proce ss
in
relat ion
to
by the develop men tal switch
be performed (Simon, Igeno Coup land ,
The
fact tha t
the
genetics
of the
floral
a comp lex regulat ion
com-
hard ly accessible for experim ental analysis .
URAL GENETIC VARIATION
ofa species prese nt in natu re contains
at
m any different g enes.
In
contras t
onedoes not expect variants
are
stron gly affected
in
vigo ur, since selection
of
variat ion
for
basic research, al though
is thetype that has been,and
is,
exploited
for
plant breeding. Fu r therm ore ,
ofna tur al se lection has ledtogenotypes
to specific enviro nm ents. Th is a daptat ion
in
ecological/
the
species, w hich
is
very obvious
in
of cult ivated pla nts . Th is source of
to
physiological
has not bee n v ery accessible for genetic
and
even less
so for
mo lecular genetic
by quanti tat ive
on theexpression of the
A
n u m b e r
of
developments
in
gene tics, such
as
in m arker technology (Rafalski
and in
the improvemen t
of
statistical
the
genetic detection
of
single
the genetic variation for such
are
identified,
be
similar
to
The analys is
of
Q T L s
is
based
on
the association
at
specific positions
on the
thema p posit ion ofthe
The
availability
of
efficient m ark er
in a
s laborious way as com pared with other m arker
such as R FL Ps and i sozymes . Th e problem
can be
solved
by
using
zygous mapping populat ions such as setsofdoubled
haploids (DHs), recombinant backcross l ines
(RBLs), also called backcross inbred l ines (BILs)
(Ramsey et
al,
1996), introgression lines (Us) (Fshed
Zam ir, 1995) or substi tut ion l ines. M ore advanced
material
of
this kind are near-isogenic l ines ( N IL s) ,
differing in a small introg ression from a corre-
sponding genotype .
The
mult ip le
use of
these
populat ions without having to genotype the material
again with molecular markers, makes these genetic
stocks extremely valuable. Th is can be d emo nstrated
by the analysis of traits as different as flowering
(Jansen
et al,
1995)
and
seed dorm ancy
(van der
Schaar et
al,
1997) in the sam e set of R IL s of the two
most widely used Arabidopsis ecotypes, Landsberg
erecta {her andColum bia (Col). This popula t ion
also serves
as the
standard map ping popula t ion
in
Arabidopsis
(Lister De an, 1993).Acareful choice
of pa ren ts e.g. by cho osing extrem es of the genot^^pic
variat ion within
a
species, extends these oppo r-
tuni t ies even more . Ex amplesof'imm or ta l ' m app ing
populat ions based
on
very different gen otyp es
are
t h e R I L s inrice deriv ed from acrossof anupland
japonica variety with anindicalowland variety (Wan g
etal, 1994);inbarleytheD H s have been der ived
from crosses between malt ing
and
fodder cultivars
(Kleinhofs et al, 1993),and in
Arabidopsis
crosses
between European andAfrican ecotypes (Alonso-
Blanco
et al,
unpubl ished) . Af ter
the
location,
quantification and analysisofthe interactionsofloci
controll ing
the
trait
has
been made ,
it
will
be
impor t an t to characterize the individual loci . In
order
to
obtain genotypes w ith only mon ogenic
differences, the further backcrossing with a recu rrent
parent (often one
of
the parents
of
the initial cross)
wil l be necessary, when working with RILsorD H s .
This ' Me ndel i s ing ' of aQ T Lcan befacilitatedby
markers l inked
to the
respective loci
and
also
by
selectionofthe phenot>-peinbackcross po pulat ion s.
W h e n N I L s
are
available, this process
of
' M e n d e l i s i n g ' Q T L s hasalready been perform ed.
An al ternative approach
to the
dissection
of
natura l
genetic variation is to perform a backcrosspro-
gramme with phenotype-based selection from
the
beginning (Fig. 1). After an u m b e r ofbackcrosses,
the analysis with molecular markers wil l indicate
what chromosomal regions
of
the donor parent
are
still present in theselected linesandthereby show
the
map
posit ion
of
puta tive Q TL s . O nce
a NIL
with monogenic segregation has been obtained,the
refinement
of
the m ap posit ion
can be
done
in the
progeny of thecross of such a NILcarryingthe
introgressed gene, with
the
recurrent parent .
The
selection of recombinants a round the locus of
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M.Koornneef C. Alonso-Blanco and A.J. M. Peeters
RP DP
I I
Parents
F I
o
c
x:
F2
i
3
D
o
o
c
o
CO
0
T D
X3
0
0
O
_
O5
C
F8
I^ L RIL pop ulatio n
Genotyping with molecular markers covering the genome
Phenotyping for the trait
o
interest and QTL mapping
1
1
Constructiono NILs BILs) con taining a single QTL
Physiological and genetical characterisation of the NILs
Figure 1 A
schematic o utline
of
the production and use
of
recom binan t inbred lines RI Ls) and near isogenic
lmes NIL s). RP, recurrent parent; DP , donor paren t;
S,
selected plan t; BI L, backcross inb red line.
cloning procedures. A potential problem is that it
will be difficult to distinguish if one gene or more
than one very closely linked genes determine the
traits that segregate monogenically. The determi-
nation of a very detailed map position will be
especially important in those species where the
complete physical map is available and for which in
the near future the complete sequence of the genome
will become available. When the biochemical func-
tions of the genes located in the region of the QTL
are known, one might 'guess' the candidate gene.
Knowledge of the position of open-reading frames
will also allow the selection of clones that can be used
for transformation, which will provide the proof of
the successful cloning by complementation. Al-
though in natural alleles it will not be clear whether
one is dealing with alleles that make a functional
To knock out the wild type allele, a mutatio
approach can be followed to find null mutants. T
detailed map position is also important when tra
posons are used for this, because this will permit
choice of a genotype with a transposable element
the vicinity of the target gene and thereby incre
the chance of finding insertions in the target ge
since transposable elements have a tendency to ins
predominantly to linked sites (Sundaresan, 199
Furthermore, when many ESTs are mapped in
region of interest, it will enable the use of DN
sequences in combination with transposons to p
form reverse genetics. Such EST probes can also
used for the detection of deletions caused
irradiation mutagenesis.
Examples of 'natural' monogenic traits that ha
been cloned are many disease resistance genes (Jon
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Genetics in plant physiology
and for which map-based cloning efforts have been
initiated are the flowering-time genes
FRI
(Clarke
Dean, 1993) andFLC (Lee et al 19946) in
Arabidopsis.
CONCLUDING REMARKS
The use of genetics has been successfully exploited
to dissect plant developmental processes.
In
par-
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