Cross-compatibility of the parents as the main factor for successful olive breeding crosses

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. 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