J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007.
C oss-compa ibili y o he Pa en s as he Main
Fac o o Success ul Oli e B eeding C osses
Au o a Dı
´az
1,2
and An onio Ma ı
´n
Ins i u o de Ag icul u a Sos enible-CSIC, Alameda del Obispo s/n, 14080, C ´
o doba, Spain
Pila Rallo
Depa amen o de Ciencias Ag o o es ales, Uni e si y o Se illa-EUITA, C a U e a km 1,
41013 Se illa, Spain
Rau
´l De la Rosa
IFAPA, CICE, Jun a de Andalucı
´
a. A da Men´
endez Pidal s/n, 14080 C ´
o doba, Spain
ADDITIONAL INDEX WORDS.C oss-pollina ion, in e compa ibili y, mic osa elli es, Olea eu opaea, pa e ni y analysis
ABSTRACT. P e ious analyses o oli e (Olea eu opaea L.) c osses ha e e ealed an impo an deg ee o pollen
con amina ion. Fo his eason, he au ho s es ed he pa e ni y o a se o p ogenies coming om c osses among
di e en cul i a s wi hin he oli e b eeding p og am o C´
o doba, Spain, using ou polymo phic mic osa elli es. They
ound ha he expec ed pollen si ed 141 (83.4%) o he 169 samples analyzed. The con amina ion a e was ei he
almos o al o almos null wi hin each pa icula c oss conside ed, no being compa able be ween di e en c osses.
In a second expe imen he au ho s e alua ed he in luence o se e al ac o s on he success o oli e c osses, he ype
o isola ion bag, he iming and numbe o pollina ions, and he c oss-compa ibili y o he pa en s in a mul i ac o ial
assay in 2003. They obse ed no di e ences in he ype o pollina ion bag used o he numbe and iming o pollen
addi ions when hey analyzed 145 seeds. The main ac o a ec ing he success o he c osses seems o be he
in e compa ibili y among cul i a s, because i had a signi ican in luence on he a e o con amina ion. In he ailed
c osses, he au ho s clea ly de ec ed he con ibu ion o mo e han one cul i a o he pa e ni y. The esul s ob ained
he e indica e ha some knowledge o c oss-compa ibili y ela ionships in oli e is equi ed o design c osses in oli e
b eeding p og ams e ec i ely. To achie e his objec i e, he p ogeny pa en age analysis could be o g ea help
because o he high le el o pollen con amina ion ound among hose s udied he e.
G owe s ha e ob ained mos o he cu en oli e cul i a s
by empi ic selec ion o e cen u ies (Le´on e al., 2005). Se e al
b eeding p og ams a e now de o ed o eleasing new cul i a s
be e adap ed o he ac ual ends in oli e g owing in di -
e en Medi e anean coun ies, such as Is ael (La ee, 1990),
I aly (Bellini, 1993; Fon anazza and Baldoni, 1990), o Spain
(Rallo, 1995).
Ha ing a eliable c oss me hod is one o he i s essen ial
s eps o he p og ess o any b eeding p og am. In his sense,
hose c osses whose p ogeny ha e been ac ually e ilized
by he expec ed pollen dono ( he cul i a used as he a he
du ing he c osses) a e success ul. Oli e is a wind-pollina ed
species in which adding pollen o bagged b anches o he
ma e nal pa en has been he usual c oss p o ocol in b eeding
p og ams (Bellini, 1993; Fon anazza and Baldoni, 1990;
La ee, 1990; Rallo, 1995). Se e al au ho s ha e ied clo h
and pape bags in p e ious expe imen s, ob aining highe ui
se in hose b anches isola ed wi h he la e (G iggs e al.,
1975). Howe e , in all cases, hey ha e only measu ed he
pollina ion e ec i eness o he b eeding c osses by he ui
se ob ained, wi hou conside ing ha his can be he esul o
e iliza ion by di e en cul i a s. So, we conside necessa y
he use o molecula ma ke s in pa e ni y es s o e alua e he
success o he pollina ions pe o med.
Di e en esea che s ha e unde aken simila me hodolo-
gies based on he measu e o he ui se o de e mine adequa e
pollen dono s o ce ain oli e cul i a s (Mo e ini e al., 1972;
Mou ie , 2002), and in some cases hey ha e complemen ed
he s udies wi h pollen ube g ow h es s (Cue as and
Poli o, 1997; Wu e al., 2002). Ne e heless, he esul s we e
no always consis en (La ee e al., 2002), because he da a
coming om di e en yea s allow one o classi y he same pai
o cul i a s as c oss-compa ible and c oss-incompa ible.
Simple sequence epea s (SSRs), o mic osa elli es, a e
inc easingly becoming he ma ke s o choice o pa e ni y
analysis in plan s, because hey now o e an easy and eliable
way o es he pa e ni y o seeds and seedlings by checking
he p esence o pa en al alleles in he descendan s (Mo and-
P ieu e al., 2003; Nishizawa e al., 2005). Thei codominan
inhe i ance and high polymo phism make i possible o ob ain
high combined pa en age exclusion p obabili y alues wi h
a ela i ely low numbe o hype a iable loci (Dı´az e al.,
2006; Isagi e al., 2004). Se e al au ho s ha e used his ech-
nique in oli e o examine he pa e ni y o seedlings (De la
Rosa e al., 2004) and e en o unge mina ed seeds (Dı´az e al.,
2006; Mooke jee e al., 2005). In ac , some o hese s udies
ha e e ealed a high occu ence o con amina ion wi h undesi -
able pollen among he p ogeny o bo h con olled sel - and
c oss-pollina ions in oli e (De la Rosa e al., 2004; Dı´az e al.,
Recei ed o publica ion 11 Ma . 2007. Accep ed o publica ion 12 July 2007.
We a e g a e ul o Jun a de Andalucı´a and he CAO00-018-C7-1 p ojec o he
Spanish Minis y o Ag icul u e, Food and Fishe ies o he unding o his
esea ch.
We hank he Wo ld Oli e Ge mplasm Bank o IFAPA ‘‘Alameda del Obispo’’
(C´o doba) and he Cul i a Collec ion o IFAPA ‘‘Ven a del Llano’’ (Ja´en), bo h
in Spain, o he acili ies p o ided. We hank D . Ha a Rapopo o he help ul
sugges ions and c i ical e ision o he manusc ip .
1
P esen add ess: Di e si y Genomics G oup, NIAB, Hun ingdon Road,
Camb idge CB3 0LE (UK).
2
Co esponding au ho . E-mail: [email p o ec ed].
830 J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007.
2006). Fu he mo e, hose indings ha e a oided coming o
w ong conclusions abou he male pa en e ec on he leng h
o he ju enile pe iod (San os-An unes e al., 2005) and on
se e al ag onomic cha ac e is ics (Le´on e al., 2004a, b) in
s udies pe o med in he oli e b eeding p og am o C´o doba,
Spain, because hey ha e e ealed ha in some cases he eal
pa e ni y is no he expec ed one.
The pu pose o his esea ch was o de e mine he impac
o he ype o pollina ion bag, he numbe and iming o pollen
applica ions, and he cul i a s used as pa en s on e iliza ion
success in oli e. Fo his, we es ed he pa e ni y o oli e seeds
using mic osa elli e ma ke s.
Ma e ials and Me hods
PATERNITY TESTING IN THE ROUTINE BREEDING PROGRAM OF
C´
oRDOBA.We es ed he pa e ni y o he o sp ing o eigh
oli e c osses made using six di e en cul i a s om he b eed-
ing p og am o C´o doba (Table 1). We made he c osses in
wo di e en loca ions in sou he n Spain: a he Wo ld Oli e
Ge mplasm Bank o he Ins i u o Andaluz de In es igaci´on y
Fo maci´on Ag a ia, Pesque a, Alimen a ia y de la P oducci´on
Ecol´ogica (IFAPA) ‘‘Alameda del Obispo’’ (C´o doba) du ing
2000 and 2002, and a he cul i a collec ion o IFAPA ‘‘Ven a
del Llano’’ (Mengı´ba , Ja´en) in 2001.
We ca ied ou he c osses using he wo ypes o bags
desc ibed la e . We collec ed he pollen om pa e nal ees
loca ed in he Wo ld Oli e Ge mplasm Bank and s o ed i a
–20 C un il i s u iliza ion. We de e mined pollen iabili y
be o e use by op ical mic oscopy, using ace ic ca mine and also
by obse ing he pollen ube leng h a e i s ge mina ion du ing
24 o 48 h a 25 C in a solu ion consis ing o 10% suc ose, 0.01%
bo ic acid, and 10 mgL
–1
e acycline (Fe na´ndez-Escoba e al.,
1983). We mixed he pollen wi h six pa s o alc o inc ease i s
olume and hen we sp ayed i h ee imes e e y2don o he
ma e nal bagged b anches, as done in p e ious expe imen s (De
la Rosa e al., 2004; Dı´az, 2005). In all cases, we pe o med wo
eplica es, choosing wo ma e nal ees pe cul i a in each c oss.
STUDY OF FACTORS AFFECTING CROSS-POLLINATION SUCCESS.
To es he in luence o di e en ac o s on he success o
oli e c osses, we pe o med a pollina ion assay in he Wo ld
Oli e Ge mplasm Bank du ing 2003. We bag isola ed he
lowe ing b anches o he ma e nal ee well be o e an hesis
[wi h buds in DII phenological s age: he co olla size becoming
la ge han he calyx size (Tous and Rome o, 1993)] o a oid
any con ac wi h ai bo ne pollen. The bagged shoo s bo e 144
in lo escences on a e age. We de ised a mul i ac o ial design
o s udying he e ec o he ollowing h ee ac o s on he
same bagged shoo (Table 2) and, as done p e iously, wo
eplica es (in wo di e en mo he ees o he same cul i a )
we e made:
1. Pollina ion bag ma e ial. We in es iga ed he in luence o
wo ypes o b anch isola ion bagging ma e ial: double-
plas ic pe o a ed (1-mm-diame e holes) and Ty ek (DuPon ,
Wilming on, DE) (Del Rı´o and Caballe o, 1999; Smi h and
Mehlenbache , 1994). Resea che s ha e p e iously used bo h o
hem in oli e con olled pollina ions wi hin he a o emen ioned
b eeding p og am in C´o doba.
2. Numbe and iming o pollen addi ions. We ca ied ou h ee
di e en pollina ion me hodologies: a single pollina ion jus
be o e an hesis (P1); h ee pollina ions, one e e y 2 d, s a ing
Table 1. Pollen con amina ion de ec ed in oli e c osses ca ied ou om 2000 o 2002 in C´o doba and Ja´en (Andalusia, Spain).
Yea Loca ion C oss
z
Analyzed
seeds (n)
Con amina ed
seeds (n)
Pu a i e a he
(no. o seeds)
z,y
Pa e nal alleles (n)
ss OeUA-DCA3 ss OeUA-DCA9 ss OeUA-DCA18 EMO3
2000 C´o doba P ·LS 11 0 LS (11) 2 2 2 2
LS ·P 18 0 P (18) 2 2 2 2
P·A 15 0 A (15) 2 2 2 1
x
2001 Ja´en P ·A 20 0 A (20) 2 2 2 1
x
MS ·A 45 0 A (45) 2 2 2 1
x
A·Me 20 20 P (10), H (1), U (9) 5 6 5 4
2002 C´o doba A ·K 10 8 P (4–6), LS (1–3)
w
,
K (2), U (1)
34 34
A·MS 10 1 MS (9), P (1) 3 3 3 2
MS ·K 10 0 K (10) 1
21
2
MS ·A 10 0 A (10) 2 2 2 1
x
z
A, ‘A bequina’; H, ‘Hojiblanca’; K, ‘Ko oneiki’; LS, ‘Lechı´n de Se illa’; Me, ‘Memecxik’; P, ‘Picual’; MS, ‘Manzanilla de Se illa’; U = unknown.
y
No sel ing p oduc s ound, sugges ing he possible sel -incompa ibili y o some o hese cul i a s, as p e iously epo ed in he case o ‘A bequina’ (Dı´az e al., 2006) and ‘Picual’ (De la Rosa
e al., 2004; Dı´az e al., 2006).
x
‘A bequina’ is homozygous o he mic osa elli e EMO3.
w
‘Lechı´n de Se illa’ and ‘Picual’ sha e a common allele a h ee o he ou simple sequence epea loci ampli ied, and one allele o he emaining locus ma ches in bo h o hem wi h he
ma e nal ones, making i impossible o assign a single a he o wo o he seeds analyzed.
‘Ko oneiki’ oli e is homozygous o he mic osa elli es ss OeUA-DCA3 and ss OeUA-DCA18.
The o al numbe o seeds analyzed, he numbe o con amina ed seeds, he mos p obable a he o e e y seed, and he numbe o pa e nal alleles iden i ied pe c oss a e indica ed.
J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007. 831
jus be o e an hesis (P2); and h ee pollina ions, one e e y 2 d,
s a ing 3 d a e an hesis (P3).
3. C oss-compa ibili y be ween cul i a s. We ca ied ou i e di -
e en c oss combina ions o ‘A bequina’, ‘F an oio’, ‘Ko oneiki’,
and ‘Manzanilla de Se illa’ oli e. We we e in e es ed in es ing
one c oss ha had p e iously wo ked well (‘A bequina’ ·
‘Manzanilla de Se illa’), and ano he in which we had de ec ed
a high pollen con amina ion a e (‘A bequina’ ·‘Ko oneiki’).
Addi ionally, all hese cul i a s show e y in e es ing cha ac e -
is ics om a b eeding poin o iew.
We selec ed a o al o 30 seeds pe c oss o he pa e ni y
es s, 10 pe ea men , i e om e e y one o he wo ypes o
bags (Table 2), when a ailable.
DNA ISOLATION FROM OLIVE OFFSPRING AND MICROSATELLITE
AMPLIFICATION.We used wo di e en DNA ex ac ion
app oaches. Fo he p ogenies om he C´o doba ou ine
b eeding p og am, we ge mina ed he seeds and isola ed he
DNA om lea es o he oli e seedlings acco ding o Mu ay
and Thompson (1980) wi h sligh modi ica ions (De la Rosa
e al., 2002). In he pollina ion expe imen , we ex ac ed he
DNA di ec ly om uncoa ed seeds as desc ibed by Dı´az
e al. (2006). We pe o med he DNA ex ac ions using he
DNeasy Plan Mini ki (Qiagen, Valencia, CA) and ollowed
he supplie ’s ins uc ions. We collec ed a o al o 314 samples
(one seed pe ui ) o DNA isola ion and ampli ica ion
(Tables 1 and 2).
We chose a se o ou oli e mic osa elli es wi h g ea
in o ma i e po en ial (high polymo phism) and p o ed ampli-
ica ion e iciency (De la Rosa e al., 2004; Dı´az e al.,
2006)—ss OeUA-DCA3, ss OeUA-DCA9, ss OeUA-DCA18
(Se c e al., 2000), and EMO3 (De la Rosa e al., 2002)— o
he pa e ni y analyses.
We ampli ied he DNA by polyme ase chain eac ion
(PCR) in 15-mL olume solu ions wi h 15 mMT is-HCl (pH
8.0), 50 mMKCl, 2.5 mMMgCl
2
, 0.25 mMo each deoxy ibo-
nucleo ide iphospha e (dNTP), 0.2 mM o o wa d ( luo es-
cen ly labeled) and e e se p ime s, 1 U AmpliTaq-Gold
polyme ase (Applied Biosys ems, Fos e Ci y, CA), and
15 ng genomic DNA empla e. We pe o med all he eac ions
in a Gene Amp PCR sys em 9600 (Applied Biosys ems) unde
he ollowing p o ile: 11-min ini ial dena u a ion s ep a 94 C,
35 cycles o 30 s a 94 C, 45 s a he annealing empe a u e
epo ed by he p e ious au ho s (De la Rosa e al., 2002; Se c
e al., 2000), 2 min a 72 C, wi h a inal ex ension s ep o 7 min
a 72 C. We dilu ed he amplicons 5- o 20- old be o e analysis
on an ABI 310 (Applied Biosys ems) au oma ic sequence
unning GeneScan so wa e ( e sion 3.7; Applied Biosys ems).
We always con i med he ma e nal allelic con ibu ion
and, when pollen con amina ion was de ec ed, we in e ed he
pu a i e male pa en o each p ogeny by emo ing he ma e nal
alleles om he comple e geno ype a he ou mic osa elli es
used, hanks o he a ailabili y o p e ious geno yping da a
(De la Rosa e al., 2004; Dı´az, 2005; Rallo, 2001). The pa e ni y
assignmen was made by eye and only when all pa e nal alleles
o he ou ma ke s ma ched he pu a i e a he .
Resul s and Discussion
CONTROLLED CROSS-POLLINATION WITHIN A ROUTINE
BREEDING PROGRAM.We we e able o disc imina e be ween
seeds p oduced by he hand pollina ion pe o med and hose
coming om he e iliza ion by undesi able pollen hanks
Table 2. Pollen con amina ion o oli e c osses, ca ied ou in 2003, as in luenced by he c oss ( emale ·male pa en ), bagging ma e ial (plas ic o Ty ek), and pollina ion ea men [only one
pollina ion jus be o e an hesis (P1); h ee pollina ions, one e e y 2 d, s a ing jus be o e an hesis (P2); h ee pollina ions, one e e y 2 d, s a ing 3 d a e an hesis (P3)].
C oss
z
Analyzed seeds
(n)
Pu a i e a he (no. o seeds)
z,y
P1 P2 P3
Pe c oss (N)
Plas ic Ty ek To al Plas ic Ty ek To al Plas ic Ty ek To al
A·K 10 (10) — 10 (10) 7 (7) 3 (3) 10 (10) 5 (4) 5 (5) 10 (9) 30 (29) P (3), GS (2), LS (2), K (1), MS (1), U (21)
A·MS 5 (2) 5 (0) 10 (2) 4 (0) 6 (0) 10 (0) 5 (0) 5 (0) 10 (0) 30 (2) MS (26), GS (2), md (2)
K·A 5 (4) 5 (5) 10 (9) 6 (5) 4 (4) 10 (9) 5 (4) 5 (5) 10 (9) 30 (27) H (9), A (3), P (3), MS (1), U (14)
K·MS 6 (0) 4 (1) 10 (1) 5 (0) 5 (0) 10 (0) 5 (0) 5 (0) 10 (0) 30 (1) MS (29), U (1)
F·K 5 (4) — 5 (4) 10 (10) — 10 (10) 5 (5) 5 (3) 10 (8) 25 (22) P (7), H (2), K (2), A (1), LS (1), MS (1), U (10), md (1)
z
A, ‘A bequina’; F, ‘F an oio’; GS, ‘Go dal Se illana’; H, ‘Hojiblanca’; K, ‘Ko oneiki’; LS, ‘Lechı´n de Se illa’; md, missing da a; MS, ‘Manzanilla de Se illa’; P, ‘Picual’; U, unknown.
y
No sel ing p oduc s ound, sugges ing he possible sel -incompa ibili y o some o hese cul i a s, as p e iously epo ed in he case o ‘A bequina’ oli e (Dı´az e al., 2006).
Fo each case, he o al numbe o seeds analyzed and he numbe o con amina ed seeds (in pa en heses) a e gi en. The o al numbe o analyzed seeds and he con amina ed ones ound
(in pa en heses) pe c oss, as well as he mos p obable a he o e e y seed a e also indica ed.
832 J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007.
o he high combined pa en age exclusion p obabili y alue
(0.92) p o ided by he ou mic osa elli es used he e and in a
p e ious s udy (Dı´az e al., 2006). We analyzed he pa e ni y o
169 seedlings coming om eigh di e en c osses pe o med
in 3 di e en yea s wi hin he C´o doba b eeding p o-
g am (Table 1). We ound ha he expec ed pollen si ed
141 (83.4%) o he 169 samples analyzed. The con amina ion
a e was no compa able be ween all c osses. Qui e he
opposi e, he con amina ion was ei he almos o al o almos
null wi hin e e y c oss (Table 1). We con i med he expec ed
pa en s o seedlings coming om he c osses ‘Manzanilla de
Se illa’ ·‘A bequina’, ‘Picual’ ·‘Lechı´n de Se illa’ and hei
ecip ocal ones, and ‘Picual’ ·‘A bequina’ and ‘Manzanilla de
Se illa’ ·‘Ko oneiki’. In he case o he c osses ‘A bequina’ ·
‘Ko oneiki’ and ‘A bequina’ ·‘Memecxik’, he le el o con-
amina ion wi h o eign pollen was nea ly o al. We could
iden i y ‘Picual’ oli e as being he p ima y pollen dono in
hese con amina ed c osses, he e o e con i ming p e iously
epo ed esul s (De la Rosa e al., 2004; Dı´az e al., 2006). We
could no de e mine he pollen dono in some cases (5.9%).
This is no unusual because he geno ypic p o ile o he ou
SSRs used he e was no a ailable o many cul i a s g own in
he wo p e iously men ioned ge mplasm collec ions. In ligh
o hese esul s, we designed an expe imen aimed a igu ing
ou he de e mining ac o s o ob aining pu e o sp ing om
oli e c osses.
STUDY OF FACTORS AFFECTING CROSS-POLLINATION SUCCESS.
Pollen om a di e en sou ce o ha added o he bagged
shoo s si ed a o al o 81 (55.9%) o 145 seeds analyzed in
his expe imen (Table 2). As no ed p e iously, we could no
de e mine he pollen dono in some cases (22.1%), al hough
he pa e nal alleles iden i ied in hose seeds (mo e han wo pe
ma ke ) clea ly sugges he con ibu ion o mo e han one
cul i a o hei pa e ni y. The dis ibu ion o hose con ami-
na ed seeds among c osses is indica i e o he main ac o
a ec ing he success o he e iliza ions, as shown he e:
1. Pollina ion bag ma e ial. Al hough we expec ed he amoun o
pollen able o gain access o bagged lowe s o be highe when
we used pe o a ed plas ic bags, nei he he con amina ion a io
wi h undesi able pollen wi hin each c oss (Table 2) no he ui
se measu ed inside bo h ypes o bags (da a no shown) we e
ela ed o he ype o bag used in pollina ion es s. So, he
pa e ni y analyses ha e demons a ed he pe meabili y o bo h
ma e ials o ai bo ne pollen. In e es ingly, his lack o isola ion
did no hampe success ul e iliza ions in ce ain cul i a
combina ions. T anspa en plas ic bags o e he possibili y o
moni o ing lo al and ui de elopmen and, he e o e, should be
selec ed i his in o ma ion is conside ed o in e es .
2. Numbe and iming o he pollen addi ions. We obse ed no
di e ences in he con amina ion a e be ween he ui
ob ained in he h ee ea men s ca ied ou du ing 2003
(Table 2), al hough in he i s wo (P1 and P2) we s a ed
pollina ion jus be o e an hesis, and in he las one (P3) we
s a ed pollina ion 3 d a e an hesis. Simila ly, we ha e no
de ec ed any possible in luence o he numbe o pollina ions,
one in ea men P1 and h ee in he emaining ea men s P2 and
P3, on he e ec i eness o c oss me hods. This indica es ha
only one pollina ion jus be o e an hesis could be su icien o
ha e a pu e p ogeny. Because pollen is qui e labo ious o collec
and i is ob ained in small quan i ies, p o ocol P1 could ep esen
impo an ime and e o sa ings.
3. C oss-compa ibili y be ween cul i a s. We obse ed no iceable
di e ences in e iliza ion success by he pollen supplied. The
high deg ee o con amina ion o some c osses and he pu i y o
o he s indica e a s ong in luence o he cul i a combina ion on
he e ec i eness o he pollina ions independen o he pollina-
ion bag ma e ial used o he numbe and iming o he pollen
addi ions (Table 2). In e es ingly, wo o he combina ions wi h
he highes le els o con amina ion wi h undesi able pollen a e
ecip ocal (‘A bequina’ ·‘Ko oneiki’ and ice e sa). De la
Rosa e al. (2004) ob ained simila esul s o he cul i a
combina ion ‘A bequina’–‘Zai y’ in p e ious expe imen s.
These esul s a e consis en wi h hose ob ained in he p e-
limina y assays (Table 1). As in ha case, we iden i ied p e-
dominan pollen dono s when we used pa icula cul i a s as he
ma e nal pa en .
A mul i ac o ial analysis o a iance e ealed ha c oss-
compa ibili y be ween cul i a s was he only ac o showing
a signi ican in luence on he a e o con amina ion (F = 67.58,
P< 0.001). Acco ding o hese esul s, in e compa ibili y be-
ween cul i a s ac ing as pa en s seems o be he mos impo an
ac o among hose s udied o achie e success in ob aining
desi ed p ogeny. The ype o bag, and he iming and numbe
o pollina ions do no seem o be c ucial ac o s. Fu he mo e,
we did no obse e spon aneous sel -pollina ions, e en when
we ca ied ou he pollen addi ions a e an hesis, which is
in ag eemen wi h a p e ious s udy abou sel -incompa ibili y
in oli e (Dı´az e al., 2006).
CROSS-COMPATIBILITY RELATIONSHIPS.The cul i a s con-
side ed he e seem o exhibi ecip ocal in e compa ibili y ela-
ionships; ha is, a ce ain cul i a pai beha es in he same
way independen ly o which cul i a ac s as he male o he
emale pa en (Table 3). This sugges s ha a game ophy ic
incompa ibili y sys em migh be ac ing in oli e (Mooke jee
e al., 2005; Sedgley, 1994). In ac , oli e lowe s ha e a we
s igma (Ciampolini e al., 1983) and gene ally binuclea e pollen
(B adley and G iggs, 1963), which a e conside ed o be wo
signs o game ophy ic sel -incompa ibili y (Sedgley, 1994).
Table 3. Compa ibili y ela ionships [c oss-compa ible (c), c oss-incompa ible (i)] be ween he oli e cul i a s used in he p esen wo k,
as e ealed by pa e ni y analysis.
Female
Male
A bequina F an oio Ko oneiki Lechı´n de Se illa Manzanilla de Se illa Memecxik Picual
A bequina i c i
F an oio i
Ko oneiki i c
Lechı´n de Se illa c
Manzanilla de Se illa c c
Memecxik
Picual c c
J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007. 833
In his sense, we always ob ained pu e p ogenies om c osses
‘Picual’ ·‘Lechı´n de Se illa’, ‘Manzanilla de Se illa’ ·
‘A bequina’ and ‘Manzanilla de Se illa’ ·‘Ko oneiki’, and
he ecip ocal ones. On he con a y, we ound ha he con-
amina ion was almos o al when we es ed he p ogeny om
he c oss ‘A bequina’ ·‘Ko oneiki’ and he ecip ocal
one. Addi ionally, we can classi y he pai ‘Picual’ ·‘A be-
quina’ oli e as c oss-compa ible, whe eas ‘A bequina’ ·
‘Memecxik’ and ‘F an oio’ ·‘Ko oneiki’ oli e a e c oss-
incompa ible (da a co esponding o he ecip ocal c osses a e
no a ailable).
The cul i a associa ions ob ained (Table 3) a e in acco -
dance wi h p e ious sel - and c oss-pollina ion esul s (De la
Rosa e al., 2004; Dı´az, 2005; Dı´az e al., 2006) and wi h he
p edominan pollen dono s iden i ied in con amina ed c osses
he e and in p e ious s udies. As an example, ‘Manzanilla de
Se illa’ was one o he main sou ces o con aminan pollen
when ‘A bequina’ was used as he mo he in sel -pollina ion
es s. So, he good esul s de i ed om he c oss ‘A bequina’ ·
‘Manzanilla de Se illa’ a e no su p ising (Tables 1 and 2).
This con adic s a p e ious epo made when es ing only he
ui se , in which he au ho conside ed ‘A bequina’ oli e as
a bad pollina o o ‘Manzanilla de Se illa’ oli e (Mou ie ,
2002). Simila ly, ‘A bequina’ oli e appea s as one o he
mos equen pollen dono s in ‘Picual’ ee sel -pollina ions
in he p e ious s udies (De la Rosa e al., 2004; Dı´az e al.,
2006) and, consequen ly, he c oss ‘Picual’ ·‘A bequina’ also
wo ked co ec ly (Table 1).
The c oss-compa ibili y in o ma ion (Table 3) could also be
use ul in hose cases in which g owe s equi e he p esence o
pollen dono s (Moo ke jee e al., 2005), such as in a eas whe e
hey a e eplacing he exis ing di e si y o cul i a s by an
inc easing homogenei y o high p oduc i e ones (Jun a de
Andalucı´a, 2002) o in coun ies whe e oli e is no a na i e
ee and hey ha e impo ed a ew cul i a s om adi ional
oli e-g owing a eas. Howe e , in he cu en s udy, we es ab-
lished he c oss-compa ibili y ela ionships be ween cul i a s
unde a i icial condi ions (con olled pollina ions), so b eede s
and g owe s mus e i y he o e lapping o bo h pa en al
cul i a bloom pe iods in pa icula loca ions and en i onmen s
o gua an ee c oss-compa ibili y in si u. Acco ding o he
bloom pe iods epo ed by Ba anco and Rallo (2005) in
esea ch ca ied ou du ing 6 yea s in C´o doba (Spain), he
cul i a combina ions ‘A bequina’–‘Manzanilla de Se illa’ and
‘Picual’–‘Lechı´n de Se illa’ (in bo h ecip ocal c osses) and
‘Picual’ ·‘A bequina’ should also be compa ible in ield
condi ions because hei blooms occu simul aneously (da a
on ‘Ko oneiki’ oli e bloom pe iod a e no a ailable).
The a e age empe a u e and ela i e humidi y du ing
he blooming pe iod (Ap il and May) in he loca ions in which
we ca ied ou he c osses we e 17.5 C and 67.3% in 2000,
17.0 C and 60.1% in 2001, 16.7 C and 64.2% in 2002, and
18.6 C and 59.5% in 2003 espec i ely. E en hough he
clima ic condi ions a e supposed o a ec he compa ibili y
beha io o he oli e cul i a s (B adley and G iggs, 1963), we
ha e ob ained consis en esul s in all he c osses made in
wo di e en yea s (‘Picual’ ·‘A bequina’, ‘Manzanilla de
Se illa’ ·‘A bequina’, ‘A bequina’ ·‘Ko oneiki’, and ‘A be-
quina’ ·‘Manzanilla de Se illa’; Tables 1 and 2). These esul s
ag ee wi h hose ob ained in a p e ious s udy in which he sel -
incompa ibili y o ‘A bequina’ and ‘Picual’ oli e e ealed
in a iable in di e en en i onmen s (Dı´az e al., 2006).
In sho , c oss-compa ibili y ela ionships a e e ealed as
being a i al ac o in he success o oli e c osses and, he e o e,
o b eeding p og ams. This in o ma ion is also o g ea in e es
in he design o new plan a ions. Mic osa elli e ma ke s could
be e y use ul o con i m u u e esul s abou he gene ic con ol
o he compa ibili y sys em ac ing in oli e, which is s ill
unknown.
Li e a u e Ci ed
Ba anco,D.andL.Rallo.2005.E
´pocas de lo aci´on y madu aci´on,
p. 281–292. In: L. Rallo, D. Ba anco, J.M. Caballe o, C. Del Rı´o,
A. Ma ı´n, J. Tous, and I. T ujillo (eds.). Va iedades de oli o en
Espan
˜a, Vol. II. Va iabilidad y selecci´on. Jun a de Andalucı´a,
MAPA, and Ediciones Mundi-P ensa, Mad id, Spain.
Bellini, E. 1993. Va iabilidad gen´e ica y he edabilidad de algunos
ca ac e es en las plan as de semillas de oli o ob enidas po
c uzamien o. Oli ae 49:21–34.
B adley, M.V. and W.H. G iggs. 1963. Mo phological e idence o
incompa ibili y in Olea eu opaea L. Phy omo phology 13:141–156.
Ciampolini, F., M. C es i, and R.N. Kapil. 1983. Fine ul as uc u al
and cy ochemical cha ac e is ics o s yle and s igma in oli e.
Ca yologia 36:211–230.
Cue as, J. and V. Poli o. 1997. Compa ibili y ela ionships in
‘Manzanillo’ oli e. Ho Science 32:1056–1058.
De la Rosa, R., C.M. James, and K.R. Tobu . 2002. Isola ion and
cha ac e iza ion o polymo phic mic osa elli es in oli e (Olea eu o-
paea L.) and hei ans e abili y o o he gene a in he Oleaceae.
Mol. Ecol. No es 2:265–267.
De la Rosa, R., C.M. James, and K.R. Tobu . 2004. Using mic o-
sa elli es o pa e ni y es ing in oli e p ogenies. Ho Science
39:351–354.
Del Rı´o, C. and J. Caballe o. 1999. A new bag o oli e pollina ion
s udies. Ac a Ho . 474:233–235.
Dı´az, A. 2005. Desa ollo y ca ac e izaci´on de nue os mic osa ´eli es y
SNPs y aplicaci´on en la mejo a gen´e ica del oli o (Olea eu opaea
L.), C´o doba Uni , C´o doba, Spain. PhD Diss.
Dı´az, A., A. Ma ı´n, P. Rallo, D. Ba anco, and R. De la Rosa. 2006.
Sel -incompa ibili y o ‘A bequina’ and ‘Picual’ oli e assessed by
SSR ma ke s. J. Ame . Soc. Ho . Sci. 131:250–255.
Fe na´ndez-Escoba , R., G. G´omez-Valledo , and L. Rallo. 1983.
In luence o pis il ex ac and empe a u e on in i o pollen
ge mina ion and pollen ube g ow h o oli e cul i a s. J. Ho . Sci.
58:219–227.
Fon anazza, G. and L. Baldoni. 1990. P opues a de un p og ama de
mejo a gen´e ica del oli o. Oli ae 34:32–40.
G iggs, W.H., H.T. Ha mann, M.V. B adley, B.T. Iwaki i, and J.E.
Whisle . 1975. Oli e pollina ion in Cali o nia. Cali o nia Ag . Exp .
S a. Bul. 869:1–50.
Isagi, Y., T. Kanazashi, W. Suzuki, H. Tanaka, and T. Abe. 2004.
Pollina ion pa e ns in Magnolia obo a a e ealed by mic osa elli e
pa e ni y analysis. In . J. Plan Sci. 165:1047–1053.
Jun a de Andalucı´a. 2002. El oli a andaluz. Jun a de Andalucı´a,
Se ille, Spain.
La ee, S. 1990. Aims, me hods, and ad ances in b eeding o new oli e
(Olea eu opaea L.) cul i a s. Ac a Ho . 286:23–36.
La ee, S., J. Ta yan, J. Le in, and A. Haskal. 2002. Impo ancia de la
polinizaci´on c uzada en dis in as a iedades de oli o cul i adas en
oli a es in ensi os de egadı´o. Oli ae 91:25–36.
Le´on, L., L.M. Ma ı´n, and L. Rallo. 2004a. Pheno ypic co ela ions
among ag onomic ai s in oli e p ogenies. J. Ame . Soc. Ho . Sci.
129:271–276.
Le´on, L., L.M. Ma ı´n, and L. Rallo. 2004b. Repea abili y and
minimum selec ion ime o a y acid composi ion in oli e p oge-
nies. Ho Science 39:477–480.
Le´on, L., A. San os-An unes, L.M. Ma ı´n, A. Ga ido, and L. Rallo.
2005. Ob enci´on de nue as a iedades po c uzamien os, p. 407–420.
In: L. Rallo, D. Ba anco, J. Caballe o, A. Ma ı´n, C. Del Rı´o, J. Tous,
834 J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007.

and I. T ujillo (eds.). Va iedades de oli o en Espan
˜a, Vol. III. Mejo a
gen´e ica y bio ecnologı´a. Jun a de Andalucı´a, MAPA, and Ediciones
Mundi-P ensa, Mad id, Spain.
Mooke jee, S., J. Gue in, G. Collins, C. Fo d, and M. Sedgley. 2005.
Pa e ni y analysis using mic osa elli e ma ke s o iden i y pollen
dono s in an oli e g o e. Theo . Appl. Gene . 111:1174–1182.
Mo and-P ieu , M.E., C. Raquin, J.A. Shyko , and N. F asca ia-
Lacos e. 2003. Males ou compe e he maph odi es o seed si ing
success in con olled c osses in he polygamous F axinus excelsio
(Oleaceae). Ame . J. Bo . 90:949–953.
Mo e ini, A., G. Bini, and E. Bellini. 1972. The pe o mance o se e al
F ench and Spanish able oli e cul i a s in he Ma emma dis ic o
Tuscany. Ri is a della O o lo o u icol u a I aliana 56:3–19.
Mou ie , N. 2002. Sel - e ili y and in e -compa ibili ies o six een
oli e a ie ies. Ac a Ho . 586:209–212.
Mu ay, M.G. and W.F. Thompson. 1980. Rapid isola ion o high-
molecula -weigh DNA. Nucl. Acids Res. 8:4321–4325.
Nishizawa, T., Y. Wa ano, E. Kinoshi a, T. Waha a, and K. Ueda.
2005. Pollen mo emen in a na u al popula ion o A isaema se a um
(A aceae), a plan wi h a pi all- ap lowe pollina ion sys em. Ame .
J. Bo . 92:1114–1123.
Rallo, L. 1995. Selecci´on y mejo a gen´e ica del oli o en Espan
˜a.
Oli ae 59:46–53.
Rallo, P. 2001. Desa ollo y aplicaci´on de mic osa ´eli es en oli o (Olea
eu opaea L.), C´o doba Uni , C´o doba, Spain. PhD Diss.
San os-An unes, A., L. Le´on, R. De la Rosa, J. Al a ado, A. Mohedo,
I. T ujillo, and L. Rallo. 2005. The leng h o he ju enile pe iod in
oli e as in luenced by igo o he seedlings and he p ecoci y o he
pa en s. Ho Science 40:1213–1215.
Sedgley, M. 1994. Sel -incompa ibili y in woody ho icul u al species,
p. 141–163. In: E.G. Williams, A.E. Cla ke, and B.R. Knowx (eds.).
Gene ic con ol o sel -incompa ibili y and ep oduc i e de elop-
men in lowe ing plan s. Kluwe , Do d ech , The Ne he lands.
Se c, K.M., M.S. Lopes, D. Mendoncxa, M. Rod igues Dos San os, M.
Laime Da Caˆma a Machado, and A. Da Caˆma a Machado. 2000.
Iden i ica ion o SSR loci in oli e (Olea eu opaea)and hei
cha ac e iza ion in I alian and Ibe ian oli e ees. Mol. Ecol. No es
9:1171–1173.
Smi h, D.C. and A. Mehlenbache . 1994. Use o Ty ek housew ap o
pollina ion bags in b eeding hazelnu (Co ylus a ellana L.). Ho -
Science 29:918.
Tous, J. and A. Rome o. 1993. Va iedades del oli o. Fundaci´on ‘‘La
Caixa,’’, Ba celona, Spain.
Wu, S.B., G. Collins, and M. Sedgley. 2002. Sexual compa ibili y
wi hin and be ween oli e cul i a s. J. Ho . Sci. Bio echnol. 77:
665–673.
J. AMER.SOC.HORT.SCI. 132(6):830–835. 2007. 835