Original article
Genetic markers for Prunus avium L:
inheritance and linkage of isozyme loci
F Santi* M Lemoine
B Bruant
INRA, Station d’Amélioration des arbres forestiers, Centre de recherche d’Orléans, Ardon, 45160 Olivet, France
(Received
14February
1989;accepted
1January 1990)
Summary -
Thepolymorphism
of 10 enzymesystems
in wildcherry (Prunus
aviumL.)
wasanalysed using
verticalpolyacrylamide gel electrophoresis (AMY, GOT, ME)
and isoelectro-focusing (ACP, IDH,
LAP, MDH, PGM,SDH, TO)
on 286 wild cherries. Theproducts
of around41 loci could be
distinguished
in thesesystems,
13 of whichdisplayed polymorphism.
Thegenetics
of 7isozyme
loci have been studiedusing
8 fullsib families:acp1, lap1,
sdh1 andmdh1 functioned as monomers, and
got1,
idh1 and me1 were active as dimers. Two otherisozymes (pgm1
andgot2 ) appeared
to havesimple
inheritance, but it was notpossible
toverify
this. Jointsegregation
of 13 locuspairs
showedlinkage
betweenlap1
andgot1 (r
=0.03 ±
0.02)
and betweenlap1
and me1(r
= 0.05 ±0.07).
ldh1,sdh1, acp1
and mdh1 arenot linked to these loci. No
linkage
has been detected betweenacp1
and mdh1.Prunus avium L. / wild
cherry
/isozyme
/variability
/ inheritance /linkage
Résumé -
Marqueurs génétiques
pour Prunus avium L : déterminismegénétique
etgroupes de liaisons entre loci
enzymatiques. L’espèce
Prunus aviumcomprend
les cerisiers,variétés améliorées pour la
production
defruits,
et les merisiers, arbres forestiers dequalité
sur
lesquels portent
aussi des programmes d’amélioration. Il serait utile pour ces programmes dedisposer
de marqueursgénétiques,
pour les identifications clonales etinterspecifiques, l’analyse
de la variabilité naturelle et dusystème
dereproduction
et le contrôle desproduits
de croisements
dirigés.
Comme peu de marqueursgénétiques
avaient été étudiés chez P avium, nos efforts ontporté
sur la recherche et la caractérisation de locienzymatiques.
Lepolymorphisme enzymatique
a été étudiégrâce
à 286 merisiersprovenant
de France(216), d’Allemagne (14)
et deBelgique (6).
Le déterminisme et les liaisonsgénétiques
ont ététestés avec 8 descendances d’un demi-diallèle 14 x 14. Les extraits de
bourgeons prélevés
en hiver ont été
analysés
parelectrophorèse
surgel d’acrylamide (AMY, GOT, ME)
ou par isoélectrofocalisation(ACP, IDH,
LAP, MDH,PGM, SDH, TO).
Dans les zymogrammes obtenus, schématisés enfigure 1,
13 locienzymatiques polymorphes
et 28 bandesmonomorphes
sont
observés.
Leshypothèses
de déterminismegénétique (fig 1)
ont été testées par un χ2 dans les descendances(tableau II).
Deux écartssignificatifs (au
niveau 5%)
auxproportions
* Correspondence and reprints
génotypiques
attendues parségrégation
mendélienne ont été notés, mais le test deχ
2 global
est nonsignificatif.
Le déterminisme deacp1, got1,
idh1,lap1,
mdh1, me1 et sdh1 a ainsi été établi. Un seultype
de test de déterminisme étaitpossible
pouramy1, mdh2, got2, pgm1
et to 1, car les 14 parents du demi-diallèle ont les mêmesgénotypes supposés (homozygotes
pour les 4 derniersloci).
Cela n’a paspermis
d’établir avec certitude le déterminismeproposé. Cependant,
en comparant letype
de zymogramme obtenu par d’autres auteurs sur les mêmes enzymes, il semblerait que le déterminisme leplus probable
pour
got2
etpgm
1 soit celuiproposé.
Lacoségrégation
de 13paires
de loci a été testée parrapport
à celle attendue en casd’indépendance (table III),
montrant une liaisonsignifi-
cative entre
got
1 etlap 1 (r
= 0.03± 0.02),
et entrelap
1 et me1 (r = 0.05-0.07).
Ce groupe de liaison estindépendant
deidh
1, sdh1, acp
1 et mdh 1 et aucune liaison ne semble exister entre ces 2 derniers loci. Toute autre relation entre loci n’a pu être testée avec les famillesanalysées.
Prunus avium L / merisier /
isozyme
/ déterminismegénétique
/ liaisonINTRODUCTION
The sweet
cherry,
Prunus aviumL,
iswidely cultivated for
its fruitcrop, for
which substantialimprovements have
been made
for
some time. The samespecies,
named the wildcherry,
grow-ing naturally
inEurope
andWest-Asia, produces
a very valuablewood, justi- fying the forest breeding
programmes whichbegan recently.
Genetic
markers would beuseful for both fruit
and woodbreeding
pro-grammes.
Theidentification
of varie-ties,
control ofbreeding material,
andthe
assesmentof specific purity
wouldbe
possible
with a series of en-vironmental influence-free traits. Co-
dominant, simply-inherited
andmapped
traits are useful
for
various purposes, such as theanalysis
of natural varia-bility
and ofmating systems,
the control of man-made crosses and ofproducts
of
forestry
clonal seed orchards.Very
fewmonogenic, simply-inherit-
ed
morphological
traits have been de- scribed for Prunus avium:fruit-juice
colour and
albinism, which
are control- ledby dominant-recessive pairs of
al-leles
(Watkin
andBrown, 1956).
The
gametophytic incompatibility S
locus is
polymorphic
with at least 6 al-leles, among
sweetcherry
cultivars(Crane
andBrown, 1937),
as well asamong
wildcherry (Berger, 1963, Santi, unpublished data),
but it isnecessary
to make crosses for thegenotype
de-termination.
Treutter and Feucht
(1985)
showeddifferences
inphenolic composition among
P aviumclones. Phenolic
com-pounds
arepotentially
valuablegenetic
markers for
breeding programmes
andpopulation genetic studies, since they
may
be linked directly
toeconomically important
traits(Doumanjou
andMarigo, 1978; Friend, 1985)
and involveregulator genes (Vernet
etal, 1986).
Unfortunately, phenolics
are often af-fected
by
environmental factors and their inheritance iscomplex.
Such
difficulties
do notusually
arisewhile
using isozymes,
the mostwidely
used
genetic markers,
which have al-ready proved
useful for numerousplants
as reviewedby Tanksley
andOrton (1983). Some knowledge
is nowavailable
on P avium enzymevariability.
Feucht and Schmid
(1985)
showed pro- tein andperoxidase banding pattern differences among
P avium clones.Kaurisch et al
(1988) pointed
out vari-ability
for 3isozyme
loci(aconitase-2,
6
phosphoglucodeshydrogenase-1,
phosphoglucoisomerase-2).
Neverthe-less,
the number of enzymes studied is still limited and no inheritance has been established. Inaddition
nolinkage
mapof
P avium isavailable.
The purpose of this
study therefore
was to increase the number of
isozyme
loci available for P avium and to test their inheritance and
linkage
relation-ships.
MATERIAL AND
METHODS
Plant material
Enzyme variability
was studied with 286 wild cherriessampled throughout
most of France(186)
and in 4populations
in:Normandy (Northwest
France, 61trees),
the Ardennes(Northern
France 19trees),
Southern Germa- ny(14 trees)
and SouthernBelgium (6 trees).
The inheritance and
linkage analyses
were made with 1
year-old plants
of 8 fullsibfamilies,
which were chosen in a 14 x 14 half-diallelaccording
to theavailability
ofmaterial and the
variability
ofparent isozyme phenotypes:
nr13(clone
108 x clone229),
nr22(109
x208),
nr27(111
x143),
nr36(111
x229),
nr71(171
x195),
nr80(195
x226),
nr81(195
x229),
nr87(208
x226).
Buds were
preferred
to leaves for enzyme extraction. Buds weresampled during
the1987-1988 winter. The
samplings
were madein the
original
stands inNormandy
andBavaria on 15 to 100
year-old
trees. Thesamplings
of other wild cherries were made on 1-7year-old vegetative copies
in a clonebank in Olivet. The
sampling period
varied from November 1987 to March 1988. The buds weresampled
from the lateral orapical position,
on short orlong
shoots. The ma-jority
of the buds werevegetative,
and ex-ceptionally
floral.Sample preparation
Immediately
aftersampling,
the buds werefrozen in
liquid nitrogen,
freeze-dried and vacuum-stored until extraction.Lyophiliza-
tion does not alter enzyme
activity,
since theextracts of fresh and
lyophilized
buds of 5clones had similar
electrophoretic patterns.
100 mg of scale-free buds were
put
into an aluminiumbag,
frozen inliquid nitrogen
and crushed with a hammer. The
powder
was soaked for 1 h in 1.2 ml of extraction buffer
(20
mM triscitricpH
= 7.5containing
12 mM
2β-mercaptoethanol,
5 mM dithi-otreithol, 2 mM
polyethylene glycol (MW
=6 000),
2% w/vPVP).
When less than 100 mg of buds were available, the buffer volume wasadjusted.
Thehomogenate
wascentrifuged
for 30 min at 14 000 g, and thesupernatant,
transferred intoplastic
vials,was stored at -60 °C until
electrophoresis.
Electrophoretic procedure
The trisboric-EDTA
polyacrylamide gel
elec-trophoresis (Page) system (Dalet
andCornu, 1989)
wasperformed
for 3 enzymes. Gluta- mate oxaloacetate transaminase(GOT,
EC2.6.1.1.)
was runthrough
a 10%acrylamide
"running gel"
and a 5%acrylamide "stacking gel".
Malic enzyme(ME,
EC1.1.1.40)
andamylase (AMY,
EC3.2.1.1)
were runthrough
7% and 10%
acrylamide "running gels",
res-pectively.
The powersupply
was set at 100V for 1 h, then
bromophenol
blue and 15μl (GOT)
or 20μl (ME
andAMY)
of extractwere loaded into each slot. The
voltage
was then increased to 250 V and maintained for 5 h(AMY)
or increased to 350 V and main- tained for 6 h(GOT, ME).
Thetemperature
of theelectrolytic
buffer waskept
at 4 to 6 °Cusing
a cooler.The other enzymes were run on 245 x 125 x 0.5 mm
isoelectrofocusing (IEF) gels, containing acrylamide
andbisacry-
lamide
(concentration
T = 5%, C =4%), Servalyt
carrierampholytes (2%) w/v
3-10
pH gradient,
0.7% w/v 4-6pH gradient;
the latter was not added when
running deshydrogenases,
1μl/ml
TEMED and1.8 mM ammonium
persulphate.
Thegels
were run on 2
"Multiphor
II"apparatus,
with thecooling plates
maintained at 2 °C. Theelectrolytes
used were those describedby
Kinzkofer and Radola
(1981).
Six toeight μl
of extract were loaded
using
a hand-madesilicon
applicator strip
with 64 holes,lying
across the
gel.
The powersupply
was setat a maximum of 1200 or 1500 V, 45 mA and
40 W for the 2
gels,
and each run lastedabout 2 h.
The
staining procedures
were those ofCardy
et al(1983)
for ME,GOT,
isocitratedeshydrogenase (IDH,
EC1.1.1.42.),
malatedeshydrogenase (MDH,
EC1.1.1.37)
andphosphoglucomutase (PGM,
EC2.7.5.1.).
They
were those of Roux and Roux(1981)
for acid
phosphatase (ACP,
EC3.1.3.2),
andthose of Beckman et al
(1964)
for leucineaminopeptidase (LAP).
For AMYstaining, gels
were soaked 2:30 h in an acetate buffer 0.2 MpH
= 4.5 with 2% w/v starch and 6%w/v CaCl 2 ,
and then in the same buffer with 3% w/v Kl and 0.3 w/v l2. PAGEgels
werefixed with 7% acetic
acid, wrapped
inplastic
foil and stored at 4-6 °C. IEF
gels
were driedand stored at room
temperature.
Checking zymogram stability Zymogram stability
was testedby varying
the
sampling
conditionsapplied
to the same clones as described in table I. No difference was noticed for 9 enzymes(ME stability
wasnot
tested), ensuring
that the observed zy- mogram differences wereindependent
of thetested
sampling
conditions.Inheritance
andlinkage
testsMendelian inheritance
hypotheses
were pro-posed
afterwatching
zymogramvariability
among the 286 wild
cherries, except
forME,
for whichonly
the 14parents
of the half-dial- lel and 5 of theirprogenies
were observed.Departure
from oradequation
to the expec- tedsegregation
ratios in the observed fami- lies were testedusing χ 2 tests.
RESULTS
Scored
loci and inheritancehypo-
theses
All the observed zymograms
of
the 286wild cherries
arerepresented
schemati-cally
infigure
1. Thirteenpolymorphic isozyme
loci and 28monomorphic bands
were scored.Inheritance hypotheses (figure 1)
areeasy to propose for
acp1, got1, idh1, lap1, mdh1,
me1 andsdh1,
since atleast
3supposed genotypes (aa, bb, ab) appear directly
from the observedphenotypes.
Thepattern
of the bb zy-mogram (nr3
onfigure 1)
of thegot1 locus suggests that
amonomorphic
band is merging
into the a-band.This
band may be theproduct of
adupli-
cated
GOT-locus.
Formdh1,
one alleleis
thought
toproduce
2 bands.Con-
versely, only
2phenotypes
ofamyl, got2, mdh2, pgm1
and to1 were re-corded in observed zymograms. Two
hypotheses
areproposed for
mdh2:the product
of the morefrequent
allele isthought
to bemerged
into a monomor-phic band;
or a null allele isinvolved.
The
expected heterozygote
was ingeneral
the leastoften
observedphenotype: 1/285 for got2, 35/285 for mdh2, 9/285 for pgm1, 24/285 for to1, suggesting
that the secondhomozy- gote
was not recordedby chance.
Further
difficulties
arise wheninterpret- ing amyl phenotypes,
since thepheno- type
with the most numerous bands isthe most
frequent (241/285).
A realistichypothesis
is to involve a null allele.Sdh1’ and
lap1’
appear assecondary products
and me1’ isprobably
a het-erodimer between me1 and a mono-
morphic
locus. Nosimple genetic hypothesis
results from the examinationof the acp2 patterns.
Segregation
at individual lociThe
scoredmonomorphic
bandsamong the 286
wild cherries
werestill monomorphic
among theprogenies.
All 14
parents
of the half diallel weremonomorphic for amyl (thought
to beheterozygote
with a null allele or ho-mozygote
with activealleles),
and formdh2, got2, pgm1
and to1 (thought
tobe
homozygotes).
The observedsegre- gation
ratios in 1progeny
of the 3 latterisozyme
loci were thoseexpected,
andfor
amyl
no evidence for a null alleleappeared (table II).
Other loci were
polymorphic
among the 14parents, allowing
more diversecrossing
combinations. The results in table II show 2departures from
Men-delian
expectations, concerning
thefamily
n° 80 for the locus sdh 1 and thefamily
n° 22 for the locusacp1.
Thesedepartures
are notsignificant
for thetotal of 25
chi-square
tests. Neverthe-less,
alinkage
between theacp1 locus
and the
incompatibility
S-locus mayprovide
such a resultfor
thefamily 22,
if the
parents
have 1 commonS-allele,
because the male
parent
is aheterozy- gote for
theacp1
locus.Joint
segregation
atlocus pairs
Over the 13 locus
pairs tested
forjoint segregation
2pairs
hadhighly signifi-
cant
(P
<0.01) chi-square
values for 2-locussegregation
ratios. The loci in- volved are thepairs got1/lap1
andme1/lap1.
The recombination fraction between the loci is reduced in allfami-
lies for these 2pairs, although
in thecross n° 27 the
chi-square
value is notsignificant
for theme1/lap1 pair.
The estimated recombination values show a
strong linkage
betweengot1
and
lap1, and
between me1and lap 1,
but the
precise respective position of
the 3 loci is still
unknown,
since thelinkage
betweengot1
and me1 has notyet
been tested.ldh1, sdh1, acp1
andmdh1 are not linked to this 3 locus
group, according
to2, 4,
5and
2 testsrespectively.
TheAcp1/mdh1 linkage relationship
hasonly
beentested
among the
acp1, mdh1,
idh1 and sdh1loci,
and it involvedonly
14 sibs.DISCUSSION
Inheritance
A limited number
of
sibsper family
were
analysed
because buds were most oftenrestricted
in number and size.Nevertheless,
we have deter- mined that 7isozymes
detectable in Pavium buds
(acp1, got1, idh1, lap1, mdh1, me1, sdh1 )
are inherited assingle genes, since
observedsegrega-
tion ratios inseveral
full-sibfamilies
are asexpected. Acp1, lap1,
sdh1 andmdh1 seem to be monomers,
while got1,
idh1 and me1have dimer-like pat-
terns. These
patterns
are common in animals orplants (Tanksley
andOrton, 1983,
Pasteur etal, 1987), apart
fromMDH
isozymes,
which areusually
dimers.
No
moreinformation
will beforth- coming by analysing
more families of thehalf
diallel forpgm1, to1, got2
andmdh2,
since the 14parents
have thesame
homozygous genotype.
Newcrossings, involving
variable geno-types,
will be necessary toobtain
clear evidence of the allelicrelationships
among the
observed
bandsof
the pu- tative loci.Nevertheless,
one may com- pare the obtainedphenotypes
to thoseexisting
in otherorganisms. GOT isozymes
areusually dimers,
andPGM isozymes
areusually
monomerswith duplicated
bands(Pasteur et al, 1987),
so the observed
pgm
1 andgot2 pheno- types
in P avium do notdiffer from the
usualpatterns,
andtherefore
the pro-posed
inheritancehypotheses
are themost
likely.
TOisozymes
are notusually
monomers
(Pasteur
etal, 1987),
socare should be taken with the
proposed
inheritance.
Conversely, identifying
allelesof amy1
andmdh2
would bedifficult,
ifthe
proposed
inheritance iscorrect,
since thesupposed genotypes
do notalways result
in differentphenotypes.
Acp2
inheritance is notclear, though
several
progenies
have beenanalysed.
Out of
the 13isozyme loci,
9 may there-fore
beuseful
inpopulation genetic
studies. These are in addition to the 3 observed
by
Kaurischet al (1988),
whoproposed
inheritancepatterns,
whichalthough
nottested,
are the mostprobable.
Linkage relationships
P avium has 8 chromosome
pairs.
Among
the 12 variableisozyme loci
with known
inheritance,
3 are on thesame chromosome
pair,
4 are notpart of this linkage group,
but are not nec-essarily
on 4different chromosomes,
and theremaining
5 loci have unknownlinkage relationships.
Newanalyses
willtherefore
be necessary tocomplete
thelinkage map.
The sizeable
numberof polymorphic isozyme loci
in wildcherry
will proveuseful
in our researchprogramme,
in thebreeding programme
as well asfor population genetic
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