Ascertaining maternal and paternal lineage withinMusa by chloroplast and mitochondrial DNA RFLPanalyses

F. Carreel, D. Gonzalez de Leon, P. Lagoda, C. Lanaud, C. Jenny, J.P. Horry,and H. Tezenas du Montcel

Abstract: In banana, the maternal transmission of chloroplast DNA and paternal transmission of the mitochondrialDNA provides an exceptional opportunity for studying the maternal and paternal lineage of clones. In the presentstudy, RFLP combined with hybridization of heterologous mitochondrial and chloroplastic probes have been used tocharacterize 71 wild accessions and 131 diploid and 103 triploid cultivated clones. In additon toMusa acuminataandMusa balbisiana, other species from the fourMusa sections were studied to investigate their contribution to the originof cultivated bananas. These molecular analyses enable the classification of theMusa complex to be discussed. Resultsascertain relationships among and between the wild accessions and the mono- and interspecific diploid and triploidbananas, particularly for theacuminatagenome. Parthenocarpic varieties are shown to be linked toM. acuminatabanksii and M. acuminata errans, thus suggesting that the first center of domestication was in the Philippines – NewGuinea area.

Key words: Musa, RFLP, cpDNA, mtDNA, lineage.

Résumé: Chez le bananier, la transmission maternelle de l’ADN chloroplastique et paternelle de l’ADN mitochondrialoffre la possibilité d’étudier les filiations maternelles et paternelles entre clones. Cette étude RFLP à l’aide de sondeshétérologues mitochondriales et chloroplastiques a permis de caractériser les génomes cytoplasmiques de 71 accessionssauvages, 131 bananiers cultivés diploïdes et 103 triploïdes. En plus des bananiers sauvagesMusa acuminataet M. balbisiana,d’autres espèces des 4 sections du genreMusa ont été étudiées pour évaluer leur contribution à l’origine des bananierscultivés. La classification du complexeMusa est ici discutée. Ces résultats permettent d’établir des apparentementsentre les bananiers sauvages, et les cultivars diploïdes et triploïdes mono ou inter-spécifiques en particulier au sein dugénome acuminata. Il est montré que les variétés parthénocarpiques sont apparentés àM. acuminata banksiiet M. acu-minata erranset donc suggèrent un centre de domestication primaire du bananier dans la zone Philippines – NouvelleGuinée.

Mots clés: Musa, RFLP, ADNcp, ADNmt, filiation.

Carreel et al. 692

Introduction

Bananas (Musaspp.) are native to Southeast Asia and thewestern Pacific. They are widely distributed throughout thesubtropics where man has spread them by vegetative propa-gation. Cultivated clones are parthenocarpic and often quite

sterile; cultivars produce seedless starchy fruits that developwithout fertilization. Indeed, wild types are very fertile; theirfruits, which are full of seeds, have little starch. TwoMusaspecies out of the 28 described were first noted in 1865 byKurz (as reported in Stover and Simmonds 1987) as beingthe origin of most cultivated clones. Although these twowild species are diploid (2n = 2x = 22), cultivated varietiescan be diploid, triploid (2n = 3x = 33), or, more rarely,tetraploid (2n = 4x = 44). Cheesman’s (1947) botanical stud-ies showed that once disregarding parthenocarpy and steril-ity, cultivated clones may be related toMusa acuminataonly (eumusa section, haploid A genome) or areinterspecific hybrids betweenM. acuminata and Musabalbisiana (eumusasection, haploid B genome). Based onmorphological observations of the characters that differenti-ate these two species and on the ploidy level of the differentclones, Simmonds and Shepherd (1955) recognized fivemain genomic groups of cultivated bananas designated AA,AB, AAA, AAB, and ABB. Within each group, relatedclones are associated in a subgroup. For example, the most-cultivated sweet export bananas all belong to the Cavendish

Genome45: 679–692 (2002) DOI: 10.1139/G02-033 © 2002 NRC Canada

679

Received 2 July 2001. Accepted 16 April 2002. Publishedon the Research Press Web site at http://genome.nrc.ca on20 June 2002.

Corresponding Editor: K.J. Kasha.

F. Carreel1 and C. Jenny. CIRAD Neufchateau, SainteMarie, 97 130 Capesterre BE, French West Indies.D. Gonzalez de Leon,2 P. Lagoda, C. Lanaud, J.P. Horry,3

and H. Tezenas du Montcel.3 CIRAD, Avenue Agropolis,34 398 Montpellier CEDEX 5, France.

1Corresponding author (e-mail: carreel@cirad.fr).2Present address: Paseo del Atardecer 360, Villas de Irapuato,Irapuato 36650 Guanajuato, Mexico.

3Present address: CIRAD, BP153, 97 202 Fort de France,French West Indies.

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subgroup, AAA. The West and Central African cooking ba-nanas, although highly variable for some morphologicalcharacteristics such as bunch shape and colour, are classifiedin the Plantain subgroup, AAB. Isozymes (Horry 1989;Lebot et al. 1993) and molecular surveys (Jarret et al. 1992;Gawel et al. 1992) confirmed this morphological groupingand subgrouping.

Most banana production, both of dessert and cookingtypes, is based on triploid cultivars. These cultivars are natu-ral combinations of different A and B genomes and havebeen fixed over hundreds of years of human selection. Thesevarieties are presently threatened by several pests and dis-eases. Breeding programs aim to create resistant clones witha similar fruit quality for each type of consumption. The fi-nal hybrids selected by the different institutes’ improvementprograms are either triploid or tetraploid, depending on thebreeding scheme. In all the crossing steps, the genitors arewild species and cultivated diploids that are hybridized todifferent natural triploid or autotetraploid cultivars. A goodknowledge of the relationships within and among wildclones, diploid accessions, and triploid cultivars is requiredto optimize the identification of the diploid parents to beused in creating resistant and performing hybrids (Bakry etal. 1997). Little data exists on relationships between clones.Some morphological characters and isozyme data led to theidentification of the A genome of the AAB Plantain as beingrelated toM. acuminatasubsp.banksiiand to some cultivarsfrom Papua New Guinea (Horry 1989). But for the otherclones, interspecificity or intersubspecificity and polyploidyobscure these relationships.

Analysis of cytoplasm DNA has been thoroughly appliedto investigate phylogeny (Kim and Jansen 1998; Bukhari etal. 1999), to resolve parentage (Dally and Second 1990;Gauthier et al. 1997), or to evaluate the variability and distri-bution among and within species or a population (for exam-ple, Hong et al. 1993; Junyuan et al. 1998). The usualmonoparental transmission of chloroplast and mitochondrialDNA (cpDNA and mtDNA, respectively) provide an excep-tional opportunity for studying maternal and (or) paternallineage. Gawel and Jarret (1991a, 1991b) used cytoplasmicprobes to studyMusa classification using the hypothesis ofmaternal transmission. A major achievement was the evi-dence found for a strong bias towards maternal transmissionof cpDNA and paternal transmission of mtDNA inM. acuminata(Fauré et al. 1994a). In banana, special cyto-plasmic DNA heredity enables us to differentiate maternalfrom paternal lineage.

In the present study, we first review the structuring of alarge sample of theMusa complex according to its cyto-plasm and then focus on the relationships between andwithin wild clones and the diploid and triploid cultivars, es-pecially for the A genome, which is the most diversified.

Materials and methods

Plant materialThe cpDNA and mtDNA pattern of 305 accessions have

been characterized. Material was supplied by the Centre decoopération internationale en recherche agronomique pour ledéveloppement (CIRAD-FLHOR), Guadeloupe, theQueenslandDepartment of Primary Industries (QDPI) (Australia) for

Papua New Guinea (PNG) clones, and the Centre deRecherches Régionales sur Bananiers et Plantains (CRBP)(Cameroon). Pieces of fresh leaves were kept at 4°C duringtransport and lyophilized upon arrival. Data of 305 acces-sions, 71 wild varieties classified aseumusa and 234cultivars, are presented here and listed in Table 1 accordingto their ploidy (2n = 2x = 22, 2n = 3x = 33). Twenty-threeother accessions are wild clones belonging to the four differ-ent Musa sections. Their patterns, not discussed here, werechecked in terms of their relationship to cultivated bananas:(i) eumusa(2n = 2x = 22), M. basjoo; (ii ) rhodochlamys(2n= 2x = 22), M. velutina ‘Velutina’, M. ornata ‘Ornata’, M.laterita ‘Jamaïque’, M. sanguinea ‘Sanguinea’, andM.Mannii (H. Wendl); (iii ) callimusa (2n = 2x = 20), M.coccinea ‘Coccinea’, M. gracilis ‘Halle No. 1’, and M.beccarii ‘Beccarii’; and (iv) autralimusa(2n = 2x = 20), M.boman ‘PNG 057’, M. jackeyi ‘Jackeyi’, M. textilis acces-sions ‘Textilis’, ‘Bangulanon’ and ‘Tangonon’,M. maclayiaccessions ‘PNG 045’, ‘PNG 339’, and ‘PNG 340’,M.lolodensis ‘PNG 364’, M. angustigemmaaccessions ‘PNG150’, ‘PNG 158’, and ‘PNG 221’,M. peekelii accessions‘PNG 315’ and ‘PNG 316’.

ProbesDNA probes were first hybridized on total DNA of 26

clones selected by International Network for the Improve-ment of Banana and Platain (INIBAP) for diversity analysesand restricted with eitherEcoRI, EcoRV, DraI, or HindIII.

Four heterologous chloroplastic probes were tested: thecytochromef gene of pea (Cyt.f, 1.4 kb) (Willey et al. 1984),rubisco large subunit gene of spinach (Rub, 1.5 kb) (R.Mache, Laboratoire de Biologie Moleculaire Vegetale,Grenoble, personal communication), and twoSalI restrictionfragments of wheat cpDNA, CpIR and CpS4 (6.5 and 10 kb)(Aubry 1990). The homologous probe pMaCIRB46 (3 kb) is87% homologous to 90 bp of tobacco cpDNA and 70% ho-mologous to 138 bp of rice cpDNA (F.C. Baurens, personalcommunication). This probe is about 6 kb from theCyt.fprobe (data not shown). Polymorphism between these 26clones was only obtained with DNA restricted byDraI withprobesCyt.f, CpS4, and pMaCIRB46. These probes and en-zymes were used to check out all the clones’ patterns.

The following seven heterologous mitochondrial probeswere tested: the genes for subunitsα, 6, and 9 of sunflowerATP synthetase (atp) (Récipon 1989); genes for subunits Iand III of wheat cytochrome oxidase (cox) (Lejeune,Laboratoire de Biologie Moléculaire Végétale, Paris XI, per-sonal communication); the maize apocytochrome gene (cob)(Dawson et al. 1984); and the 18S + 5S genes of wheatrDNA (Falconet et al. 1984). Polymorphism was observedfor 26 out of 28 probe–enzyme combinations. Probesatp6andcob revealed patterns with one band giving similar infor-mation with the different enzymes. We selectedatp6–EcoRVandcob–HindIII combinations. More complex patterns wereobserved for the other probes. When only one probe wasused, the different enzymes produced no correlating infor-mative bands. We therefore chose two to three enzymes foreach probe:atpα–DraI, atpα–HindIII, atp9–EcoRV, atp9–DraI, 18S+5S–DraI, CoxI–DraI, CoxI–HindIII, and CoxIII–HindIII. All the clones’ mtDNA patterns were noted with

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Carreel et al. 681

Species subspecies Current name of wild accessions Cp pattern Mitotype

M. acuminata P. Prentel, typeII*, Pa abyssinea I χ ADM. acuminata typeX, typeXI I δ 1M. acuminatasubsp.zebrina Zebrina, Maia Oa, Monyet I ε ADM. acuminatasubsp.zebrina Buitenzorg I ΝΑM. acuminatasubsp.errans Agutay II αM. acuminata EN13(IDN075)* II βM. acuminatasubsp.burmannica Long Tavoy P1, Long Tavoy P4 II χ ADM. acuminatasubsp. burmannicoïdes Calcutta 4 II χM. acuminatasubsp.siamea Siamea (Pa Rayong) II χM. acuminatasubsp.malaccensis Malaccensis, Pahang*, Selangor* II δ ADM. acuminata indAA101, P. cici alas* II ε ADM. acuminatasubsp.microcarpa Microcarpa, Borneo* II γ ADM. acuminatasubsp.truncata Truncata II ΝΑM. acuminata THA018, THA029, THA044 II ΝΑM. acuminata Pa Songkhla II ΝΑM. acuminata IDN 113* II ΝΑM. acuminatasubsp.malaccensis P. cici* III εM. acuminatasubsp.siamea Khae (Phrae) IV χM. acuminatasubsp.banksii Madang, Higa/BS464, PNG151, PNG162, PNG176 V φ 1M. acuminatasubsp.banksii PNG 220 V φ 2M. acuminatasubsp.banksii PNG 344, PNG 363 V φ 3M. acuminatasubsp.banksii Banksii, Paliama, Hawain3, Hawain2, Waigu V φ ADM. acuminatasubsp.banksii PNG174, PNG181, PNG234, PNG255, PNG269 V φ ADM. acuminatasubsp.banksii PNG276, PNG291, PNG292, PNG343 V φ ADM. balbisiana Cameroun VII ι type 2M. balbisiana Honduras VIII ι type 1M. balbisiana Lal Velchi, Singapuri VIII ι type 3M. balbisiana Tani, P. Klutuk Wulung, P. Batu, P. Klutuk VIII ι type 4M. balbisiana Butuhan 0 ιM. schizocarpa Schizocarpa n°1*, PNG246 VI η ADM. schizocarpa Mushu, PNG172, PNG232, PNG180 VI η 1M. schizocarpa × M.a. banksii PNG 253, PNG 180 seed VI φ ADM.a. banksii × M. schizocarpa PNG235, PNG247 V η 1

Current (+ proposed) group,subgroup when known Current name of parthenocarpic accessions Cp pattern Mitotype

AAcv P. Tongat, Akondro Mainty, Tuu Gia, Pallenbery, Mak II α 1AAcv IDN110, P. Pipit, P. Rojo Uter, Samba, Chicame II α 1AAcv M48, M53, Gu Nin Chiao, Manang, Thong Dok Mak II α 1AAcv Wudi Yali Yalua, SF215, Niyarma Yik, P. Sasi II α 2AAcv Khai Nai On, Sa, Thong Det, Odwa II α 2AAcv Katual n°2 II α 3AAcv, P. mas type P. mas, Kirun II α 4AAcv Tha052, Khi Maeo II α 5AAcv Guyod*, Colatino Ouro, IDN077, Pa Patthalong II α ADAAcv P. Jaran, M61, Senorita, Yenai II α ADAAcv, P. Jari Buaya type P. Jari Buaya BS312, Gabah Gabah, P. Sipulu II β 1AAcv, P. Jari Buaya type Hawundu Vita, Saing Todloh, P. Gigi Buaya II β 1AAcv, P. Jari Buaya type P. Jari Buaya IDN082, Niukin II β ADAAcv P. Lilin II δAAcv P. Bangkahulu II δ 2AAcv P. Sapon II εAAcv Djum Tau II φ 5AAcv Kunburgh, Mala II φ ADAAcv Sinwobogi, P. berlin III α 1AAcv P. Trimulin III α 4AAcv Tjau Lagada III α

Table 1. Cytoplasmic pattern of the banana accessions studied.

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Current (+ proposed) group,subgroup when known Current name of parthenocarpic accessions Cp pattern Mitotype

AAcv Hom IV α 1AAcv Heva, Galeo, Sowmuk, Fako Fako V α 1AAcv Dibit, Beram, Kuspaka, Kungor V α 1AAcv SN2, Pitu, P. Madu, Padri, Japaraka n°2 V α 2AAcv Bie Yeng, Tomolo, Puka, Gulum, Bebek, Taputuput V α 3AAcv Waimara, Somani, Papat, Spiral, Ivi-Ivi, Loibwa V α 3AAcv Buladou, Fu Des, Yendisi, To’o, Navaradam, Agul, Bud V α 3AAcv Maleb, Gonub, Enar, Kekiau, Manameg Red, Buka V α 3AAcv Sepi V α 8AAcv SF265, Yangun Yefan, Mpiajhap, Jaruda, Maka V α ADAAcv Tamat, Vudu Papua, Kenar V α ADAA? cv Jongo, Lagun Vunalir V α ADAAcv Ta V φ 1AAcv Lalalur, Inori, Papat Wung,Tagamor,Gorop V φ 4AA? cv Navaradam V φ 4AAcv Mapua V φ 5AAcv Gwanhour, Tangamor, Meinje, Wikago, Igua, Kwonta V φ ADAAcv Sihir, Meleng, Himone, Grupnai, Pongani V φ ADAScv (SAcv) Japaraka n°1 (PNG 065) VI α 2AScv (SAcv) Ungota VI φ 1AScv (SAcv) Wompa, Ato VI φ 4AScv (SAcv) Vunamani VI φABcv Safet Velchi, Figue Pomme Ekona, Kunnan II δ 2ABcv ?/ ABB Aoko, PNG125, Kalapua n°2, V ι ADABcv ?/ ABB Dwarf Kalapua, Tukuru n°2* V ι ADAAA, Gros Michel Gros Michel, Dougoufoui, Cocos, Highgate, Bout Rond II α 1AAA, Cavendish Lacatan, Petite Naine, Williams, Poyo II α 1AAA, Cavendish 901, Grande Naine, Hom Thong Mokho II α 1AAA, Orotova P. Kayu, P. Umbuk, Hom Sakon Nakhon II α 1AAA, Ibota Yangambi KM5, Khom Bao II α 1AAA, Rio Leite II α 1AAA, Ambon P. Ambon, Sultana, P. Bakar II α 1AAA ? Wan II α 1AAA, Red Figue Rose Naine II α 2AAA, Orotova Orotava II αAAA Ouro Mel* II αAAA? Who-gu II φ 1AAA? Pagatau (PNG 028) II φ 4AAA? Toowoolee* II φAAA, Orotova P. Sri (IDN 060) II ΝΑAAA P. Papan III αAAA Medja V α 1AAA Palang, Marau, Kokopo 1* V α ADAAA, Lujugira/Mutika Bolo Bigouyo, Foulah, N’genge, Intokatoke, Nakitengwa V ε 1AAA, Lujugira/Mutika Mbwazirume, Nshika, Bui-se-ed, Igitsiri V ε 1AAB (ABA) Tip Kum II α 1AAB (ABA), Nendra Padaththi P. Rajah II α 6AAB (ABA), Nadan Lady Finger (AA Sucrier) II α 6AAB (ABA), Pome/Prata Foconah II α 6AAB (ABA), Mysore P. Ceylan, Zabi, Gorolo II α 7AAB (ABA), Nendra Padaththi Rajapuri India* II αAAB (ABA), Pome/Prata Prata Ana* II αAAB (ABA), Silk Figue Pomme Géante II δ 2AAB (ABA), Silk Figue Pomme Naine II δAAB (ABA) P. Nangka V α 2AAB (ABA), Laknao Adimoo, Mugus V α 3AAB (ABA) Kumanamba (PNG 195) V α 3

Table 1 (continued).

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these 14 probe–enzyme combinations. RFLP and hybridiza-tions were made according to Fauré et al. (1994b).

Statistical analysisPatterns were deduced from a presence vs. absence scor-

ing of bands in each genotype. Statistical analysis wasperformed using ADDAD software. The factorial analysisand phenograms were conducted on Dice or Jaccard dis-tances as recommended by Perrier et al. (1999). Similar re-sults were obtained for each method, thus factorial analysisof the Jaccard distances is presented.

Results

Chloroplast DNAAll the clones restricted withDraI were hybridized to the

two heterologous probes CpSal4 and Cyt.f and the homolo-gous probe pMaCIRB46. Fourteen informative levels ofbands were identified. One is specific toM. sanguinea(rhodochlamys section). Another is specific to someM. acuminataclones (pattern I, Table 2). The other 12 levelsof bands were identified in at least one wild and onecultivated banana (Fig. 1). Each wildeumusaand cultivatedvariety is characterized by one of the 10 different patternsidentified labelled 0 to IX in Table 2.

Pattern 0 (bands 3, 9, and 12 of Fig. 1) associated four Pa-pua New Guinea parthenocarpic varieties classified as AT orAAT and also to mostcallimusaandaustralimusawild spe-cies. The accession ‘Butuhan’ (usually classified as aM. balbisiana) had the same pattern.

The five patterns from I to V regroup all theM. acuminataaccessions and most of the cultivated varieties. Pattern I(bands 5, 8, and 11 of Fig. 1) associates the fourM. acuminatasubsp.zebrina accessions and five unclassi-fied wild M. acuminataaccessions. Pattern II (bands 3, 9,and 11 of Fig. 1) includes many wild accessions and culti-vated varieties of different ploidy levels, particularly clonesfrom the AAA sub-groups such as Gros Michel and Caven-dish, and the sweet bananas of the AAB subgroups such asMysore and Pome. Patterns III (bands 3, 9, and 10 of Fig. 1)and IV (bands 2, 9, and 11 of Fig. 1) were each revealed byonly one wild accession,M. acuminatasubsp.malaccensis‘P. cici’ and M. acuminatasubsp.siamea‘Khae Phrae’, re-spectively. Few cultivars revealed these patterns. Pattern V(bands 1, 7, and 11 of Fig. 1) included many clones. Thediploids are the wildM. acuminatasubsp.banksiiaccessionsand the AA cultivated varieties from Papua New Guinea.Several cultivated triploid varieties also show this patternand belong to subgroups Lujugira (AAA), Plantain (AAB),Popoulou (AAB), Laknao (AAB), and Pelipita (ABB).

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Carreel et al. 683

Current (+ proposed) group,subgroup when known Current name of parthenocarpic accessions Cp pattern Mitotype

AAB (ABA), Maia Maoli Maia Maoli V α 8AAB (ABA)? Kupulik (PNG 307) V α 8AAB (ABA)? Kofi* (PNG 310) V αAAB (ABA) Plantain 3 Vert, Nyombé n°2, n’Jock Kon V φ 1AAB (ABA), Plantain Big Ebanga, Three Hands Planty V φ 1AAB (ABA), Popoulou Popoulou, Iho u Maohi V φ 1AAB (ABA), Laknao Laknao, Kune V φ 1AAB (ABA), Iholena Luba V φ 4AAB (ABA), Popoulou Poingo V φAAB (ABA) Tomnam V φAAB (BAA), P. Rajah P. Raja Bulu (IDN 093) VIII αAAB (BAA) Muracho VIII αAAB (BAA), P. Kelat P. Pulut, P. Kelat VIII ε 2ABB, Bluggoe Poteau Geant, Poteau Nain, Dole II ι 5ABB, Ney Mannan Radjah, Ice Cream, Banane Argente II ι 7ABB, Saba Saba II ι type 4ABB Topala* V αABB, Pelipita Pelipita V ιABB, Saba IDN 107* V ιABB (BAB), P. Awak Gia Hui, Namwa Khom VIII ι 6ABB (BAB), Peyan Brazza IV, P. Kepok Bung (IDN 095) VIII ιABB (BAB), Saba Benedetta* VIII ιABB (BAB), P. Awak P. Kepok (IDN 086) IX ιABB (BAB) K. Tiparot IX ιAAT (TAA) Karoina 0 αAT (TA) Umbubu 0 αTA, TAA, TTA? Mayalopa, Sar 0 α

Note: Accessions classified in a same taxon are grouped on a same line (separated by a coma) according to their cytoplasmic patterns: chloroplasticpattern (Cp. pattern) (0 to VIII) and Mitotype (α to_ι or NA: pattern non associated to any other mitotype). Within a mitotype, clones with an identicalpattern have a same number (1 to 7) or clones may have different patterns: AD (all different). XX?: ploidy not confirmed by flow cytometry; *, missingdata for few mitochondrial RFLP probe/enzyme.

Table 1 (concluded).

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684 Genome Vol. 45, 2002

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Pattern VI (bands 1, 7, and 12 of Fig. 1) is associated toM. schizocarpa. Five cultivated varieties and one wild acces-sion from Papua New Guinea also revealed this pattern.Some of them were morphologically described as putativeAS or AAS hybrids (Sharrock 1989).

Pattern VIII (bands 4, 6, and 12 of Fig. 1) includes severalAAB and ABB cultivated varieties with all of theM. balbisiana types exceptM. balbisiana ‘Cameroun’,which presents pattern VII (bands 5, 6, and 11 of Fig. 1).This last pattern is the closest to pattern IX (bands 5, 6, and12 of Fig. 1), shown by two ABB clones.

The triploid cultivated varieties belonging to a same sub-group revealed the same pattern with the exceptions of theABB Saba subgroup and the clone ‘Pisang kepok’ in theABB Pisang awak subgroup.

Chloroplastic patterns for each individual clone are sum-marised in Table 1.

Mitochondrial DNA

PatternsMore than 100 different patterns were revealed with the

14 probe–enzyme couples studied (examples of patternsFig. 2).

The different Musa species studied have very differentpatterns with few common bands. Wild accessions within aspecies or within theM. acuminata subspecies often re-vealed different patterns.

Clones within a subgroup have identical or very similarpatterns. There are two exceptions. The AAB Laknao sub-group is subdivided in two very different patterns, and theAAA Orotova ‘P. Sri’ pattern is very different from those ofthe two other clones of the subgroup. It was not possible todraw any conclusion for some clones, because data weremissing (indicated with an asterisk in Table 1). Several AAAsubgroups show exactly the same pattern: Gros Michel,Cavendish, Orotova, Ibota, Rio, and Ambon.

Two patterns associated with wild parthenocarpic clones.One is shared byM. acuminatasubsp.banksiiand the AABPlantain, Popoulou, and 2 Laknao and the other is shared bythe ABB Saba ‘Saba’ and some wildM. balbisianaacces-sions. Several cultivated varieties share the same pattern.However, they may have a different ploidy level and be clas-sified within different groups. Four patterns associate diploidand triploid cultivars. For example, the commercial ‘GrosMichel’ and ‘Williams’ and several AA cultivars such as‘Akondro Mainty’ or ‘Pisang Madu’.

Similarity of pattern between accessions belonging tonon-eumusasection and cultivated varieties was only re-vealed for four accessions classified as AT or AAT. They allhave the specificaustralimusaband revealed with 18S–DraIand three of them have the specific band revealed by CoxI–HindIII. They also have theM. acuminatasubsp.banksiispecific bands revealed by ATPα–DraI or EcoRV and ATP9–EcoRV. All of the other cultivated varieties revealed bandsshared with the threeeumusaspeciesM. acuminata, M.balbisiana, andM. schizocarpa,or bands not associated withany of the wild accessions studied. So the only wildaccessions included in the following analyses were those belong-ing to theeumusasection. These wild accessions and the culti-vated varieties revealed 107 informative bands and 111 patterns.

MitotypesSome of these 111 patterns are more or less similar. A

Jaccard distance using 107 informative bands was calculatedbetween the 111 wild and parthenocarpic accessions show-ing different patterns. The first four axes of the factorialanalyses obtained with this distance revealed 57.6% of thetotal variability (Fig. 3). The analysis shows a structuring ofmost accessions in nine mitotypes noted fromα to ι. Each ofthe four mitotypesβ, φ, η, and ι has two specific bands is-sued from different probes. Other mitotypes are due to theassociation of several bands issued from different probes.

The first seven mitotypes fromα to γ are related toM. acuminata, mitotypeη to M. schizocarpa, and mitotype

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Carreel et al. 685

Fig. 1. Chloroplastic patterns of the three probe–enzyme combi-nations: pMaCIRB46–DraI (bands 1–5),Cyt.f–DraI (bands 6–9),and CpSal4–DraI (bands 10–12). The 10 combinations of pat-terns observed labeled 0–IX associate the bands as follows: pat-tern 0, bands 3, 9, and 12; pattern I, bands 5, 8, and 11; patternII, bands 3, 9, and 11; pattern III, bands 3, 9, and 10; pattern IV,bands 2, 9, and 11; pattern V, bands 1, 7, and 11, pattern VI,bands 1, 7, and 12; pattern VII, bands 5, 6, and 11; pattern VIII,bands 4, 6, and 12; and pattern IX, bands 5, 6, and 12. R, Raoulmolecular weight marker (Appligene).

Fig. 2. Example of mitochondrial patterns with three probe–enzymecombinations (ATP9–DraI, Cob–HindIII, and CoxI–HindIII) for fiveaccessions. Lane a, AAA Cavendish ‘Grande Naine’α1 mitotype;lane b,M. a. burmannicoides‘Calcutta 4’, a pattern of theχmitotype; lane c, AAA Lujugira–Mutika ‘Nakitengwa’ε1 mitotype;lane d, ABB Bluggoe ‘Poteau Nain’ι5 mitotype; lane e, AAB Plan-tain ‘Three Hands Planty’φ1 mitotype; R, Raoul molecular weightmarker (Appligene).

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ι to M. balbisiana. Six wild M. acuminata and AAAOrotova ‘P. Sri’ are not related to any mitotypes.

Three mitotypes only group wild accessions. Mitotypesχandγ are only shared by a fewM. acuminataaccessions, be-longing to the subspeciesburmannicaand burmannicoïdes,and to the subspeciesmicrocarpa, respectively. Mitotypeη isshared byM. schizocarpaaccessions and four wild acces-sions classified as AS.

Six of the nine mitotypes are shared by some wild acces-sions and some diploid or triploid cultivated varieties. TheM. acuminata ‘Agutay’ accession (from The Philippines)shares the same mitotypeα as 71% of the AA cultivated va-rieties (AAcv) studied, seven out of the nine AAA sub-groups, and 6 out of the 10 AAB subgroups. SomeM. acuminataaccessions share the same mitotypeβ as someAAcv as those of the P. Jari Buaya type. TheM. acuminatasubsp.malaccensisaccessions (from Malaysia) are associ-ated with some ABcv (the one with silk morphological char-acteristics) and to the AAB Silk subgroup in mitotypeδ. The

mitotypeε links M. acuminatasubsp.zebrina(from Indone-sia) with the AAA Lujugira subgroup from East Africa.Many cultivated varieties have the mitotypeφ specific to thewild M. acuminatasubsp.banksii accessions (from PNG);they are the AAcv from PNG and several cultivated bananasfrom the AAB group, such as all the clones of the Plantainsubgroup from Africa, all the Popoulou subgroup mainlyknown in the Pacific islands, and two clones of the Laknaosubgroup from Philippines. Within this mitotypeφ, there arealso two of the three AScv from PNG. TheM. balbisianaand most of the ABB are within mitotypeι.

Patterns and mitotypes for each individual clone are sum-marised in Table 2.

Discussion

Cytoplasmic variabilityTen chloroplastic patterns were identified among the ba-

nana accessions studied, whereas there were more than 100

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686 Genome Vol. 45, 2002

Fig. 3. Factorial analysis on Jaccard distances between the 111 mitochondrial patterns obtained from the RFLP study with 14 probe–enzyme combinations. Axes 1 and 2 (below) and Axes 1 and 4 (opposite) . Patterns are indicated according to their mitotype (α to ι)and correspond to the number indicated in Table 1. The first four axes shown represent 57.6% of the total variability.

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patterns for the mtDNA. These results corroborate the gen-eral agreement that the evolution rate of the cpDNA isslower than the mitochondrial and nuclear genome one(Palmer and Zamir 1982; Lumaret et al. 1989). An angio-sperm chloroplastic genome has a slow rate of sequence sub-stitution and little structural modification (Doebley et al.1987). So a small intraspecific variability is usually ob-served (Zurawski et al. 1984). Several mtDNA restrictionpatterns are usually observed inside a species and mtDNApolymorphism is usually observed within populations (Cro-zier 1990, Tomaru et al. 1998). This polymorphism is usu-ally associated with recombinations between all the differentmitochondrial small repeated regions (Palmer and Herbon1988, Palmer 1992). This explains why the mitochondrialpolymorphism revealed was different for a given probe withdifferent enzymes.

The banana RFLP cytoplasmic variability revealed is quiteconsiderable compared with other plants such as beans(Phaseolus vulgaris; Khairallah et al. 1992) and cocoa(Theobroma cacao; Laurent et al. 1993). More cytoplasmicpolymorphism was identified among the banana’s ancestralspecies than among the cultivars. Only wild clones exhibitthe chloroplastic patterns I and VII and mitotypeγ andη ora specific mitochondrial pattern. The chloroplastic patternIX is only represented by cultivars, but is closely related to

the VII ones. An even broader polymorphism may existamong wild species, but was not studied here because fewwild clones have been prospected and they were chosen fortheir value in cultivar breeding.

Nine chloroplastic patterns and nine mitotypes are specificto the A, B, and S genome of wild and (or) cultivated clones.Among the 81 possible cytotypes, wild clones are dividedinto 19 cytotypes, the diploid cultivated clones are dividedinto 12 cytotypes, and the triploid clones are divided into 14cytotypes. Quite wide general cytoplasmic variability hasbeen conserved throughout the domestication of bananas.Some cytotypes only exist in some cultivated clones so do-mestication created new associations between the cpDNApattern and mitotype that were retained until the present ow-ing to the sterility and vegetative propagation of cultivatedbananas.

ClassificationThe classification obtained from both the cpDNA and

mtDNA analyses is detailed in Table 2.

Wild accessionsEighteen species belonging to the fourMusasections have

been studied. Even more species have been described; unfor-tunately, only one representative is available for each species

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Fig. 3 (continued).

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or accessions of several of these species no longer exist incollections. Here we focused on theeumusasection, whichincludes most parthenocarpic bananas. The accessions stud-ied belong to four species. Only one accession representsM.basjoospecies that originate from Japan; it exhibits specificchloroplastic and closed mitochondrial patterns not sharedwith any other accession studied.

Musa schizocarpa(only found in PNG) is the leastvariable of the three remaining species according to its cytoplasmicDNA. Only one chloroplastic and two mitochondrial patternswere identified among the six accessions studied.

The mtDNA analyses place the nineM. balbisianaacces-sions studied into five patterns. Four are the morphotypesproposed by Horry. cpDNA isolates one of thesemorphotypes,M. balbisiana ‘Cameroun’. Little is knownabout the origin of this clone found by H. Tezenas duMontcel (personal communication) in Ekona (West Camer-oon), where it was probably introduced by the German peo-ple before the World War I. The fifth mtDNA patternbelongs to the accession ‘Butuhan’. This clone also has adifferent cpDNA pattern that it shares with theaustralimusaspecies, so an interspecific origin is suspected and discussedlater.

As in previous morphological and molecular analyses,M. acuminatais found to be the most diversified species ofthose from which cultivated bananas originated. The mor-phological variability lead to a classification into six to ninesubspecies depending on the authors. The different wildM. acuminataaccessions are distributed in five of the ninecpDNA patterns and seven of the nine mitotypes identified.

Both chloroplast and mitochondrial DNA analyses differ-entiate theM. acuminatasubsp.banksii accessions studiedfrom the otherM. acuminataclones. This subspecies is geo-graphically isolated from others in Papua New Guinea andin some North Indonesia islands. It is also, withM. acuminatasubsp.errans from Philippines, the only sub-species to be preferentially autogamous. The several mtDNApatterns observed classified this subspecies into four popula-tions that match up quite well with the region of origin ofthese four populations. MtDNA associates the clones foundin the Madang region with those of its northern islands. Italso groups the two clones from the western province at theIndonesian frontier. Most west and east Sepik clones are as-sociated.

Three of the four clones defined asM. acuminatasubsp.zebrina show a specific cytotype (I-ε). The fourth clone,‘Buitenzorg’, has a specific mitochondrial pattern.Musaacuminatasubsp.zebrina is defined as a subspecies of theIndonesian islands.

All of the other subspecies originate from the northernpart of theacuminataarea of distribution: i.e., from Thai-land, Malaysia, and the Philippines. They have the samecpDNA (II), but are dissociated into four mitotypes,α forM. acuminata subsp. errans, δ for M. acuminata subsp.malaccensis, γ for M. acuminatasubsp.microcarpa, and χfor M. acuminatasubsp.burmannicoidesand M. acuminatasubsp. burmannica. The accessionM. acuminata subsp.siamea‘Siamea’ also presents this II-χ cytotype. However,the accession ‘Khae Phrae’ also usually classified amongM. acuminatasubsp.siameahas a specific cpDNA pattern (IV).

For M. acuminatasubsp.banksii many accessions havebeen collected (Sharrock 1989), but for most subspecies fewaccessions are available. TheM. acuminataclones can inter-breed, and the classification into subspecies is mainly due tospatial and temporal isolation. Also, more accessions areneeded to differentiate intrasubspecific variability fromintersubspecific variability. However, this cytoplasmic DNAanalysis shows that for accessions available for study there isno clear difference of cytotype betweenM. acuminata subsp.burmannicaand M. acuminatasubsp.burmannicoides, theopposite of the De Langhe and Devreux (1960) propositionthat was based on morphotaxonomic data. Cytoplasmic re-sults confirm Shepherd’s (1989) cytologic translocation anal-ysis that distinguishM. acuminatasubsp.truncata clearlyfrom M. acuminatasubsp.microcarpa.

Few wild accessions have a specific mitotype (labeled nain Table 2) or a chloroplastic pattern not shared (III) by anyother wild accession. The morpho-taxonomic classificationassociates ‘Buitenzorg’ withM. acuminatasubsp.zebrinaand ‘P. cici’ with M. acuminatasubsp.malaccensis, but cy-toplasmic data differentiate those two accessions from theirusual subspecies. The accessions ‘Tha 018’, ‘Tha 029’, and‘Tha 044’, ‘IDN 113’, or ‘Pa (Songkla)’ are not commonlyclassified within any subspecies. Some of these accessionscould have originated from mutations, but, as explained be-low, the mtDNA mutation rate seems low. We believed thatmost of these clones must be the only accession studiedfrom specific populations.

All of the other wild clones not associated with any sub-species have cpDNA from one subspecies and mtDNA fromanother, suggesting an inter-subspecific origin confirmed bynuclear analysis (data not shown). This is the case of‘P. prentel’, ‘Type II’, ‘Pa abyssinea’, ‘type X’ and ‘typeXI’, ‘EN13’, ‘P. cici alas’, and ‘IndAA101’.

Interspecificity of four wild accessions suspected frommorphological analysis is confirmed here. The two acces-sions PNG235 and PNG247 have aM. schizocarpaspecificmitotype (η) and a M. acuminatasubsp.banksii specificchloroplastic pattern (V). Accession PNG253 and seed ofPNG180 have aM. acuminatasubsp.banksii mitotype (φ)and aM. schizocarpachloroplastic pattern (VI). This con-firms Argent’s (1976) hypothesis that viable hybridizationsbetweenM. acuminatasubsp.banksii and M. schizocarpaoccur in Papua New Guinea where both species are endemicand grow in the same areas.

Monospecific cultivarsThere is no classification recognized among the diploid

AAcv. Few clones have strong morphological similaritiesand are noted as ‘P. Mas’ related or ‘P. Jari Buaya’ related.All accessions related to ‘P. Mas’ have the II-α cytotype ashave many other AAcv, whereas the accessions related to‘P. Jari Buaya’ clones are the only AAcv with the II-βcytotype. The others are mainly spread over three cytotypes:II-α, V-α, and V-φ. Few clones show the III-α, IV-α, II-δ, andII-φ cytotypes. These different cytotypes may be a basis forthe AAcv classification.

Morphotaxonomy classifies the triploid clones into groupsand subgroups. Among the monospecific group AAA, all theaccessions that belong to the same subgroup share the same

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cytotype. Cytoplasmic DNA analysis clearly differentiatesthe Lujugira/Mutika sub-group from the other AAA sub-groups. Cooked or made into beer, these bananas from EastAfrica have the V-ε cytotype. Mainly eaten as fresh fruit,the clones of all the other AAA subgroups have the II-αcytotype. Within this cytotype, subgroups Gros Michel,Cavendish, Ibota, Rio, and Ambon have exactly the samecytoplasmic patterns. The few AAAcv not assigned to anysubgroup have specific cytotypes, which is evidence of theirgenomic difference: II-φ for ‘WhoGu’ and ‘Toowoolee’ bothfrom PNG and III-α for ‘P. Papan’ from Indonesia.

Interspecific cultivarsChloroplastic pattern and mitotype of an interspecific

cultivar may be related to two different species. To take suchdata into account within the classification, we propose thatthe first letter of the genomic group, as indicated in Table 2,refers to the maternally inherited chloroplast genome,whereas the last letter refers to the paternally inherited mito-chondrial genome (e.g., within the current AAB genomicgroup this would enable differentiating between BAA andABA cultivars).

The three cultivars ‘Safet Velchi’, ‘Figue Pomme’, and‘Kunnan’ are classified among the AB genomic group. Noevidence ofbalbisiana cytoplasmic genome was found forthese clones.

Within the ‘AAB’ genome group, the P. Rajah and P. Kelatsubgroups have the same maternal origin of the B genome(VIII). They may be classified as BAA. The paternal originof one of their A genomes differentiates these two subgroups(α and ε respectively). All the other subgroups have cyto-plasmic DNAs related to the A genome and may be classi-fied as ABA. Cooking ABA clones, Plantain, Popoulou, andLaknao subgroups, have the V-φ cytotype. The ABA cooking‘Maia Maoli’ clone differs in its mitotype (α). Five out of sixdessert ABA subgroups have the II-α cytotype. The ABAdessert Silk subgroup differs in its mitotype (δ).

All ABB genomic groups have a paternal origin of onebalbisiana genome. Bluggoe – Ney Mannan and Pelepitasubgroups may be classified as real ABB with a cpDNA as-sociated with patterns II and V, respectively, which are spe-cific to theacuminatagenome. Peyan and P. awak subgroupsboth have their cytoplasmic DNA associated with the B ge-nome and so may be classified as BAB. The three clones as-sociated to the Saba subgroup are distributed over verydifferent cytotypes.

So most of the parthenocarpic clones are related toM. acuminataand M. balbisiana as shown botanically byCheesman in 1947. The cytoplasmic analysis also confirmsArgent’s (1976) observation of the contribution of the spe-ciesM. schizocarpa(eumusasection, 2n = 2x = 22, haploidS genome) and theaustralimusaspecies (2n = 2x = 20, hap-loid T genome) to some cultivars.

The interspecific origin is confirmed for the SA cultivarsfound in Papua New Guinea. All have the chloroplastic pat-tern specific toM. schizocarpa(VI). Four of them have theφmitotype specific toM. acuminatasubsp.banksiialso origi-nating from Papua New Guinea and one cultivar has theαmitotype.

Four cultivars are confirmed as interspecific betweenclones related to the species of theaustralimusasection andclones related toM. acuminata (genome A): ‘Karoina’,‘Umbubu’, ‘Mayalopa’, and ‘Sar’. Their cpDNA is specificto the australimusaspecies. Their mtDNA pattern has spe-cific australimusaand M. acuminatasubsp.banksii bands.A biparental origin of the mitochondrial genome is thereforesuspected for some intersection hybrids. Natural hybridiza-tions between species from different sections with differentnumbers of chromosomes exist and may lead to partheno-carpic clones.

Chloroplastic DNA analysis on yam (Dioscorea ssp.;Terauchi et al. 1992) or sorghum (Sorghum bicolor; Chen etal. 1990) or mtDNA analysis on apple (Malus domesticus;Kato et al. 1993), bean (Khairallah et al. 1992), orVicia(Van de Ven et al. 1993) gave a different, but tallying, classi-fication with the taxonomy. These results show that it is thesame for banana. The cytoplasmic analysis differentiates theA, B, S, and T genomes and agrees most of the time as de-scribed above with the main species and subspecies morpho-logical classification. The classification of the triploids intosubgroups is recognized; all clones belonging to a subgroupusually share the same cytotype. But the classification intogroups is not directly apparent from this analysis. For in-stance, different ‘AAB’ subgroups may be spread over dif-ferent mitotypes, but one AAB subgroup can share amitotype with an AAA subgroup. This structure is explainedby the origin of the clones.

Tracing the origin of clonesVegetative propagation of bananas allows the preservation

of a genotype arising from a cross that occurred hundreds ofyears ago. Also, since their origin, the cytoplasmic genomesof two related clones could have mutated and become differ-ent from their parents. Clones like Plantain migrated to WestAfrica more than 3000 years ago and proved to be one of theoldest triploid cultivated bananas (De Langhe 1961). ThePlantain A genome is thought to be related toM. acuminatasubsp.banksii. The Plantains studied have exactly the samecytotype pattern as someM. acuminatasubsp.banksiiacces-sions still present in PNG. Hence, few mutations of the cyto-plasmic DNA might be expected. There is no probabilitythat two unrelated clones might have similar cytoplasmicpatterns. Thus, for two clones, an identical cpDNA pattern isevidence of a similar maternal origin and the same mitotypeis evidence of a similar paternal origin. According toTable 2, each clone may be maternally related to any clonein the same row and paternally related to any clone in thesame column.

In interspecific crosses, we can determine which ancestralgenome, A, B or S, was the mother and which was the fa-ther. For example, as we saw, the BAA clones can bedistinguished from the ABA within the ‘AAB’ group. We mayalso conclude that interspecific crosses betweenM. acuminataand M. schizocarpagave viable hybrids whatever the direc-tion of the crosses. Wild hybrid accessions have been identi-fied with M. schizocarpabeing the mother or the father.

Within theacuminatagenome, the origin of the A genomeof the cultivated clones can be maternally or paternally re-

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lated to some subspecies. Many cultivated clones have oneof the three cytotypes, V-φ, V-α, or II-α.

Within the first one, V-φ, 18 AAcv clones have cpDNAand mtDNA patterns related toM. acuminatasubsp.banksii.No other subspecies is related to them. We may concludethat these clones have a maternal and paternalM. acuminatasubsp.banksii origin. The triploid ABA Plantain, Popoulouand two out of four Laknao also have this V-φ cytotype. Asexpected from previous results with morphotaxonomy orisozymes on Plantain (Horry 1989; Tézenas 1989; Lebot etal. 1993), the two A genomes of the clones of this sub-groupare also related toM. acuminatasubsp.banksii.

Cultivars with the cytotype V-α have an intersubspecificorigin. 49 AAcv and the two A genomes of the ABA MaiaMaoli can be maternally related toM. acuminatasubsp.banksii and paternally to theM. acuminatasubsp.erransclone accession ‘Agutay’.

Clones with the cytotype II-α, for example 36 AAcv andthe AAA Cavendish and ABA Mysore clones, are also pater-nally related to thisM. acuminatasubsp.errans clone. Forthese triploid clones, the A genome inherited from themother may be linked to any subspecies with a cpDNA IIpattern.

The AB Silk-type cultivars and ABA Silk clones are II-δand so can be associated paternally withM. acuminatasubsp.malaccensis. The East African AAA Lujugira Mutika(V-ε cytotype) come from a cross between clones related toM. acuminatasubsp.banksii (from PNG), with pollen re-lated toM. acuminatasubsp.zebrina(from Indonesia) con-firmed by nuclear data (not shown).

Many triploids have exactly the same cytotype as somediploid cultivars or wild clones. These diploids are preferen-tial parents and potential donors for breeding. The sweet ex-port bananas Cavendish, as well as the Gros Michel, Ambon,Rio, and Ibota, have in this experiment the same cytoplasmicpattern as 12 AAcv. This is also true for seven AAcv and theAAA Red clones and for the AB ‘Figue Pomme’ and theABA Silk clones. The plantains have the same cytoplasmicgenome as someM. acuminatasubsp.banksii.

Thus, the lineage between diploids and triploids can besuggested. Cytoplasmic patterns can be a witness of old lin-eages, but to identify the more recent parentage relationship,those lineages would have to be confirmed by nuclear analy-ses. Clones with similar cytoplasmic DNA may have littlenuclear genome in common and the classification of theclones may not agree. Many back crosses may have oc-curred, for example, because the accession ‘Butuhan’ is mor-phologically classified among theM. balbisiana. As mentionabove, it has a chloroplastic pattern similar to manyaustralimusaand somecallimusa. Its morphotype and nu-clear genome (data not shown) are characteristic ofM. balbisiana. This clone was described by Cheesman(1947) as a typical PhilippinesM. balbisiana. In that region,there are no geographic or ecological barriers betweenM. balbisianaandM. textilis (an australimusaspecies, 2n =20) (Brewbaker et al. 1956). Some natural hybrids wereidentified. All indicate that ‘Butuhan’ must be a back crosson M. balbisianaof a hybrid betweenM. textilis as motherand M. balbisiana as father. As another example, the BAcultivars do not have any cytoplasm related to thebalbisiana

genome. But morphological and nuclear analysis confirm thepresence ofM. balbisianain their genome. Analysis of someAB synthetic hybrids (data not shown) confirm Fauré’s re-sults on cpDNA and mtDNA transmission. We may con-clude that these cultivars must have arisen from a back crossof an AB hybrid with AAcv. The nuclear analysis of the ge-nome is an interesting addition to the analysis of the cyto-plasmic genome for tracing earlier crossing events.

These relationships show that most cultivars have at leasttheir cpDNA or mtDNA related toM. acuminata subsp.banksii or the M. acuminatasubsp.errans clone ‘Agutay’.The only exceptions are the AAB triploids P. kelat subgroupwith a chloroplastic B genome and the ABB triploids withboth cytoplasmic genome related to B, P. Awak and Peyansubgroups and accessions ‘Bendetta’ and ‘Klue Tiparot’. Thethird genome not accessible with the cytoplasmic study maynevertheless also be related to these subspecies. The A ge-nome of the parthenocarpic AScv is also related to thesesubspecies. Parthenocarpy and aM. acuminatasubsp.banksiior M. acuminatasubsp.errans lineage are linked. Some morestarchy than usual seed-bearing wildM. acuminatasubsp.banksii were observed by Simmonds (1962). This is evi-dence that parthenocarpy may have arisen in Papua NewGuinea or The Philippines among theM. acuminatasubsp.banksii and M. acuminatasubsp.errans.

Acknowledgements

We thank J.W. Daniells from QDPI Australia for kindlyprovinding leaf samples of the Papua New Guinea acces-sions studied. We thank Professor Quetier and his laboratoryat Orsay University (France) for kindly provinding theheterologous probes. We thank J.L. Noyer and V. Lebot fortheir critical review of the manuscript and their encourage-ment. This work was supported by CIRAD, by grants fromthe Commission of European Communities (STD3 program)and by grants from the Banana Improvement Program of theWorld Bank and Common Fund of Commodities (CFC). Wealso thank Drs. De Langhe and G.M. Getachew for theirsupport.

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