Molecular and cytological characterization of the global Musa germplasm collection provides insights into the treasure of banana diversity

Most of the plant material analyzed in this study (Online Resource 1) came from the International Transit Centre (ITC, Leuven, Belgium) and was delivered in batches of in vitro rooted plantlets of about 50 accessions (five plantlets per accession), or as lyophilized leaf tissues. If fresh plant material was obtained, leaf tissues were lyophilized after ploidy level analysis and kept for further use. In particular situations—if ploidy analysis resulted in ambiguous results (1.2% of measured accessions), the plants were transferred to soil and maintained in a greenhouse for chromosome analysis. Altogether, 695 accessions were genotyped, including 327 diploids, 363 triploids and 5 tetraploids. Apart from the accessions received from ITC and from the Musa Reference DNA collection (http://​www.​musanet.​org/​Musagenotypingce​ntre/​genomicDNA), 27 accessions from Hawaii were provided by Dr. Angela Kay Kepler (Banana Specialist, Hawaii) to enlarge the representation of individual Musa subgroups, and 38 accessions were obtained during two Musa germplasm collecting expeditions in Indonesia (Sutanto et al. 2016), that are currently being introduced into the ITC collection. The exploration expedition was confined to the East-Indonesian triangle (formed by Halmahera Islands, Sulawesi and Lesser Sunda Islands) and is henceforth referred to as “Indonesian Triangle”. The samples from Hawaii and from the “Indonesian Triangle” expedition were collected as fresh leaves from young banana plants for ploidy level estimation and subsequently lyophilized for further use.

Ploidy level of each accession was estimated by flow cytometry according to Doležel et al. (1997, 2007). About 30 mg of a young leaf tissue was chopped with a razor blade in a glass Petri dish containing 500 µl Otto I solution (0.1 M citric acid, 0.5% v/v Tween 20). Crude homogenate was filtered through a 50 µm nylon mesh. Chicken red blood cell nuclei (CRBC), prepared according to Galbraith et al. (1998), were added to the suspension of Musa nuclei as an internal reference standard. After 30 min incubation at room temperature, 1 ml Otto II solution (0.4 M Na₂HPO₄) (Otto 1990) supplemented with 5 µM DAPI and 3 µl/ml of 2-mercaptoethanol were added. The samples were analyzed using Partec PAS or Sysmex-Partec CyFlow flow cytometers equipped with UV excitation and detectors for DAPI fluorescence. The gain of the instruments was adjusted so that the peak of the CRBC nuclei was positioned approximately at channel 100 on a 512 channel scale. Relative nuclear DNA content of Musa accessions was then determined by comparing peak positions of CRBC nuclei and nuclei of the sample (Fig. 1). Every accession was represented by five individual plants and ploidy was estimated in each of them.

Genomic DNA was extracted from lyophilized leaf tissues of young banana plants using a NucleoSpin Plant II kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s recommendations. Each of the accessions received from ITC collection was represented by five individual plantlets. If there were no differences in ploidy among individual plants, genomic DNA was extracted from a pooled sample containing lyophilized tissue from all of them. If plants from the same accession displayed inconsistent results during ploidy analysis, genomic DNA was extracted separately from each plant and individual plants were treated as separate accessions. For the SSR analysis, the pipeline established by Christelová et al. (2011) was used and is illustrated in Fig. 2. Briefly, 19 SSR loci (Lagoda et al. 1998; Crouch et al. 1998; Hippolyte et al. 2010) that are well distributed across ten of the eleven Musa genetic linkage groups (Hippolyte et al. 2010) were amplified using a set of M13–tailed specific primers to allow universal labelling with fluorescent dyes. Although new SSR markers are accessible thanks to the Musa genome sequence assembly completion (D’Hont et al. 2012; Martin et al. 2016), the hitherto used marker set was not enlarged, as the reproducibility and comparability of results gathered until now would then not be assured and a re-start of the whole genotyping effort would be inefficient. The PCR reaction mix contained (in the final volume of 20 μl): 10 ng of template genomic DNA, reaction buffer (consisting of 10 mM Tris–HCl (pH 8), KCl 50 mM, 0.1% Triton-X100 and 1.5 mM MgCl₂), 200 μM dNTPs (each), 1 U of Taq polymerase, 8 pmol of the M13–tailed locus specific forward primer, 6 pmol of the fluorescently labeled universal M13 forward primer, 10 pmol of the locus specific reverse primer. The cycling conditions were set as follows; initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation (94 °C/45 s), annealing at temperature corresponding to the locus–specific primer (1 min) and extension (72 °C/1 min). The final extension was allowed for 5 min at 72 °C. Purification of the PCR products was performed by ethanol/sodium acetate precipitation. Two independent PCR reactions were performed in each accession to avoid random errors of allele binning.

Purified PCR products were diluted 40-fold in Hi-Di formamide containing the internal size standard (GeneScan™-500 LIZ size standard; Applied Biosystems, Foster City, CA, USA) and loaded onto a capillary electrophoresis DNA analyzer (ABI 3730xl, Applied Biosystems) after 5 min denaturation (95 °C). Electrophoretic separation and signal detection were carried out with default module settings. In order to reduce the cost and increase throughput of the genotyping platform, samples were multiplexed for electrophoretic separation. Up to fourfold multiplexing was applied by combining four PCR products, labelled with four different fluorescent dyes (6-FAM, VIC, NED and PET) into a single sample for loading. The resulting data were analyzed and called for alleles using GeneMarker^® v1.75 (Softgenetics, State College, PA, USA), manually checked and implemented into marker panels (Christelová et al. 2011).

The extent of genetic diversity among all samples was evaluated using the Nei´s genetic distance coefficient calculation (Nei 1973) and subsequent cluster analysis was done using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA; Michener and Sokal 1957). To enable joint analysis of all ploidy levels (2×, 3× and 4×) the genotypic data was converted into binary (coded by 1/0 = presence/absence) and analyzed as a dominant marker’s record (Weising et al. 2005). Dendrograms were constructed based on the results of UPGMA analysis and visualized in FigTree v1.4.0 (http://​tree.​bio.​ed.​ac.​uk/​software/​figtree/​). The first dendrogram that comprised all analyzed samples (not shown) was used to identify problematic accessions whose position in the dendrogram did not agree with their current classification and were therefore not used to build the “core subset”. The core dataset comprised reliable accessions only, for which the clustering pattern agreed with the classification based on morphological descriptors. The genotyping and evaluating pipeline is shown in Fig. 2. The dissimilarity index threshold of 0.25 was used to assess the grouping of the accessions on the dendrogram. Bootstrap support for individual branches was calculated on 1000 replicates and values above 35% (0.35) were used to confirm the fundamental subclustering pattern. To evaluate the informative power of individual SSR loci, several characteristics were calculated, such as Polymorphism information content (PIC; Botstein et al. 1980), allele number, observed heterozygosity (H _o, in the diploid dataset) and major allele frequency using Powermarker v3.25 (Liu and Muse 2005).

Flow cytometry was used to estimate the ploidy in all 495 accessions that were received as fresh leaf tissues. The remaining accessions were received either as lyophilized leaf tissues (151) or DNA samples from the Reference DNA collection (49; http://​www.​musanet.​org/​Musagenotyping centre/​genomicDNA). Ploidy of these later accessions was retrieved from the Musa germplasm information system database (MGIS; http://​www.​crop-diversity.​org/​mgis/​). For a majority of the samples, the expected ploidy was confirmed (Online Resource 1). However, some accessions e.g. Gebi (ITC0877) and Mwitu Pemba (ITC1545) reported to be triploid AAB, were found to be diploid. Moreover, accessions Tongkat Langit Papua (ITC1716) and M. borneensis (ITC1531) showed ambiguous results with DNA content between diploid and triploid, or between triploid and tetraploid for accession Sar (ITC0898) and SUP 1 (AAB, Indonesian Triangle mission 2). SSR analysis indicated triploid status by the occurrence of three alleles at some loci. However, this observation does not guarantee three complete chromosome sets, nor it excludes the possibility of tetraploids status. Only chromosome counting would give a definite answer.

Out of 495 accessions analyzed by flow cytometry, two cases of mixoploidy were found. The accession M. rosea-hybrid (ITC1598) comprised one mixoploid (2× + 4×) plant out of the five analyzed plantlets. Mixoploid status (3× + 6×) was detected in three out of five plants of the accession Malbhog (ITC1631). The remaining two plantlets were either triploid or hexaploid. The presence of the hexaploid plant indicates that a shift to a higher ploidy happened in vitro as hexaploid banana plants do not grow in the field and that the classification of three plants as mixoploid was not an artifact due to accumulation of cells in G2 phase of cell cycle, which is yet to be confirmed in the case of M. rosea-hybrid ITC1598. In cases where the five plantlets differed in ploidy levels without showing a mixoploid pattern (ITC1573, ITC1598), analysis of re-sent samples was carried out. No incongruent results were obtained on the re-sent samples, which indicated that the problems detected in the first batch were caused by delivery error or mislabeling. This proved the capability of the genotyping pipeline to detect mislabeled accessions and showed the robustness of the method.

In the latest systematic effort to screen ploidy levels (Doleželová et al. 2005), 1150 entries deposited in the ITC collection by that time were analyzed. Since then, 381 new accessions were introduced into the genebank, out of which 249 accessions (Online Resource 1) had their ploidy level verified by flow cytometry for the first time in this study. For the remaining accessions, repeated analyses provided a valuable feedback for the conservation strategy. As demonstrated in some accessions, e.g., mixoploid ITC1631 and 18 samples listed in Online Resource 2, the ploidy determined in the present work did not agree with previous results.

The informative power of the 19 SSR loci used in the genetic diversity study (expressed as the Polymorphism Information Content; PIC) ranged between 0.561 and 0.933 with an average value of 0.789 (Table 1). The highest observed heterozygosity within the diploid dataset was observed for the marker mMaCIR01 (H _o = 0.623), whereas the locus mMaCIR307 had the lowest H _o value (H _o = 0.250).

The UPGMA analysis of the dataset comprising all ploidy levels together grouped the accessions into clusters, which in general conformed to the morphological traits-based classification into subgroups (in 84% of accessions) as reported by germplasm donors to the collection (Musa Germplasm Information System (MGIS), Bioversity International. Accessed on February 15, 2016 at http://​www.​crop-diversity.​org/​banana/​). However, in 16% of cases, the classification reported did not agree with the clustering observed in the dendrogram. These accessions are listed in Online Resource 2 and the discrepancy between the current classification and the results obtained here are possibly caused by mislabeling or handling error, or due to misclassification before introduction into the genebank. This set of 71 “to be verified” accessions was excluded from the dataset that was used for the final dendrogram construction. Furthermore, 24 accessions for which classification was incomplete or not provided, and eight synthetic hybrid accessions were removed from the dataset, in order to increase the resolution of the “core subset” tree. The remaining 591 accessions, representing only entries with coherent classification and genotyping data, were used to construct a final SSR dendrogram. The representativeness of the subset with regard to individual species/subspecies and subgroups of Musa is summarized in Table 2. We considered this subset of accessions as the “core subset” to which any accession from the excluded ones could be added in future and its potential true/more specific classification could be proposed. However, only field verification which is carried out in parallel (Chase et al. 2016) can provide conclusive results (examples included in Online Resource 2).

Our results indicate that the SSR markers covered well the genetic diversity of the Musa genus as revealed by the clustering pattern on the dendrogram (Fig. 3, Online Resource 3). The dendrogram reveals 48 sets of accessions separated at the Nei’s dissimilarity index threshold of 0.25 (Table 3) with relatively significant bootstrap support for most of them (>35%). For the wild accessions, the sets and some singletons correspond to wild species and subspecies, while at the triploid cultivar level, the sets correspond to subgroups, of which the members are supposed to have derived as somatic mutants from an original domesticate. At the diploid cultivar level, the situation has not yet been clarified, and the sets may be composed of somatic mutants and accessions with different parentage.

The 48 sets of accessions formed 13 clusters as denoted on Fig. 3 and in Table 3. The diversity as revealed by the UPGMA clustering is further discussed by individual groups, according to their classification as wild and cultivated diploids and edible triploid cultivars, respectively.

For the ease and clarity of the discussion regarding the diversity within the genus and to illustrate the discussed accessions in all figures, we decided to retain the former classification of genus Musa into sections Callimusa, Australimusa, Rhodochlamys and Eumusa. The accessions formerly classified under section Rhodochlamys (cluster II) are now considered as forming one assembly with the ex-section Eumusa, and entitled “Musa” (Häkkinen 2013). It may be worthwhile to revisit this naming as it creates confusion with the name of the genus ‘Musa’. Similarly, the merger between the former sections Callimusa and Australimusa (cluster IV) in the complex named Callimusa needs further study based on our results. The UPGMA tree (Online Resource 3) illustrates a distinct (and rather geographical) order in the complex with four divisions along the range of accessions: (1) Callimusa in Mainland South East Asia [from ITC1683 to ITC1532]; (2) Australimusa and some Fe’i accessions in Southeast Indonesia [from AMB006 to Fe’i Aiuri S8]; (3) Callimusa in Borneo [from ITC1568 to ITC1516B]; (4) Australimusa in the New Guinea region and the Pacific [other Fe’i, from ITC0614 to ITC0917].

Representatives of M. ornata formed a distinct separate block (cluster VI) distantly positioned from the other ex-Rhodochlamys entries (all in cluster II: M. velutina, M. mannii, M. rosea, M. siamensis, M. rubra, M. laterita, M. sanguinea). This result may indicate that M. ornata is a hybrid species, which was also suggested by Shepherd (1999), who formulated his opinion on M. ornata being a naturalized hybrid of M. velutina (Rhodochlamys) and M. flaviflora (a small species closely resembling M. acuminata).

Separate location of three well-represented subspecies of M. acuminata (burmannica, malaccensis and banksii) on the SSR tree may have a genetically indicative value. The 31 accessions of ssp. banksii form a coherent block in the middle of the cluster XI (Fig. 3) with more or less evident links to diploid AA cultivars. A comparable link for the block of 12 accessions of ssp. malaccensis in cluster III can be drawn. The block of seven accessions of ssp. burmannica is grouped with a sole and morphologically quite different AA cv. block (´Pisang Jari Buaya´, cluster I). This observation may indicate participation of this subspecies in the origin of AA cultivars to a much lesser extent, and conforms to Simmond’s (1962) findings that the subspecies does not seem to possess parthenocarpic genes.

The weakly represented East-Indonesian acuminata taxa, var. tomentosa and var. acuminata (Nasution 1991), as well as ssp. errans are spread over the “banksii sensu lato”-block in cluster XI, and are discussed further below.

Proportion of shared SSR-alleles and the proximity between ssp. zebrina, and African “AAA Mutika-Lujugira block” within the cluster X supports the previous reports on the contribution of zebrina and banksii subspecies to the formation of this triploid subgroup (Carreel et al. 2002; Boonruangrod et al. 2008; Risterucci et al. 2009; Hippolyte et al. 2012) and based on our results, the zebrina contribution is predominant.

The position of the singletons ssp. microcarpa, and ssp. sumatrana (Fig. 3, white and grey triangles) in the dendrogram seems unreliable with rather low bootstrap values for corresponding branches (0.04 and 0.12, respectively). Increasing the sample representation for each of the subspecies might be needed to produce a more robust position on the tree.

As shown in a number of molecular studies on bananas (e.g. Hippolyte et al. 2012; de Jesus et al. 2013), the intraspecific diversity of M. balbisiana is low compared to M. acuminata. This is reflected in our work by clustering all balbisiana accessions in a single coherent block (Fig. 3, cluster VII) that contains morphologically distinct BB forms from Mainland and Island SE Asia (‘Butuhan’, ‘P. Klutuk’ and ‘Pisang batu’) intermingled with BBs of South Asian origin. Although genetic diversity within M. balbisiana has been reported (Ude et al. 2002; Ge et al. 2005; Swangpol et al. 2007; Wang et al. 2007), it is believed that the ITC collection does not cover enough balbisiana diversity to trace the ancestral lineages of triploid cultivars comprising the B-genome (Hippolyte et al. 2012).

Another wild species, M. itinerans, ubiquitous from NE India in the west to Taiwan in the east (Häkkinen et al. 2008) is morphologically a very distinct species with characteristic long rhizomes, which was, however, not reflected by the cluster analysis. Indeed, the three representatives of this species were scattered over the dendrogram instead of being grouped in one block (black boxes on Fig. 3), which could be attributed to underrepresentation of the sample in the present dataset or potential mislabeling. On the other hand, it may also indicate the limitations of the 19 SSR marker set used. The selected 19 SSR markers proved to work very well for verifying classification of diploid and triploid cultivars subgroups (as described further below), and for some M. acuminata subspecies (see above). However, there seem to be limitations when it comes to certain species/subspecies, such as M. itinerans, M. acuminata ssp. errans and others (see Table 2), which are rather underrepresented in the dataset. As compared to the result of the first study where the platform was applied (Christelová et al. 2011), the resolution has greatly improved by enlarging the dataset, which is clearly demonstrated by the AA cv. clusters, or triploid subgroups diversification (see further below). Only future results comprising the whole diversity of the germplasm available for genotyping would give clear answer and show the borders for the application of the platform.

SSR analysis classified the large group of edible AA accessions into ten sets (highlighted in Table 3) with distinct positions on the SSR tree (Fig. 3). This not only helps in classification of these accessions, but also provides insights into the origin and putative progenitors of some triploid cultivars. The clustering reflects geographical origins as well. While the set of Island SE-Asian accessions (AA cv. ISEA 1) is connected to the malaccensis subgroup and the dessert AAA ‘Ibota’ subgroup in cluster III, the second ISEA block (AA cv. ISEA 2) co-localizes with a large spectrum of dessert AAAs within the cluster IX, suggesting that some of its members may have played a role in the constitution of cultivated AAAs. The great majority of these AA cvs. have sweet taste and are usually eaten raw.

The AA cv. African set in cluster IX contains AA cultivars found in East Africa and nearby islands of the Indian Ocean. It has been proposed that progenitors of this set (which most probably originated in the area between New Guinea, Borneo and Java and was brought to the East African coast through human migration; Perrier et al. 2009) were the diploid parents of the subgroups AAA ‘Cavendish’ and AAA ‘Gros Michel’ (Hippolyte et al. 2012). Our results support this notion. Similarly, the small group of Tongat AA cv. may be the source of another diploid ancestor of AAA ‘Cavendish’ or ‘Gros Michel’ (Hippolyte et al. 2012).

The AA cv. IndonTriNG group of cultivars, in cluster IX, originated in New Guinea and within the Triangle shaped by the islands in Eastern Indonesia Halmahera (NE), Sulawesi (NW) and Sunda (South), that was recently explored for wild and edible bananas (Sutanto et al. 2016). The set contains a number of new accessions, of which some could have played a role in the formation of less known AAA cultivars. Three specimens from the Indonesian Triangle expedition clustered in a block of AA cultivars, which contains three Philippine cultivars (therefore called AA cv. IndonTriPh block in the cluster X). Its members produce bunches that vaguely resemble those of the AAB ‘Plantain’ (e.g. Roa Cakalang, strongly resembling the French plantain bunch) and ‘Plantain-linked’ (strikingly for Guyod and GabaGaba Putih; Sutanto et al. 2016).

Diploid AA cultivars that share the banskii background appear into two sets on the tree (Fig. 3, cluster XI). The first of them called the AA cv. banksii derivatives is on the same clade as the wild M. acuminata ssp. banksii and seems to contain direct/pure derivatives of the subspecies, all coming from New Guinea. The second block is termed AA cv. ‘banksii sensu lato’ and contains several wild AAs which are geographically proximal to ssp. banksii and ssp. errans in the Philippines, var. acuminata in Western New Guinea and Maluku and var. tomentosa in Sulawesi. The possibility that AA cultivars from this set are domesticated products/hybrids of these wild acuminatas with banksii deserves to be investigated. The AA cv. ‘Pisang Jari Buaya’ set (in cluster I) is separated from other edible AAs. This cultivar is used in breeding as a well-known source of resistance to pests i.e. burrowing nematodes (Wehunt et al. 1978; Marin et al. 1998; Ray 2002), resistance to Fusarium tropical race 4 disease and tolerance to black Sigatoka (Rowe 1998). However, based on our SSR analysis, its genetic affinity to wild and edible AA is unclear and deserves special attention. The AA cv. ‘Pisang Sapon’ singleton appears to be remotely linked to subspecies zebrina only, which could point to its being an edible derivative of zebrina rather than an inter-subspecies hybrid. However, low statistical support for this particular clade does not allow for conclusive remarks. The interspecific AS (acuminata x schizocarpa) hybrids are grouped on a separate clade within cluster X, closely linked to the banksii-dominant cluster XI (Fig. 3). M. schizocarpa (Cluster V) is sympatric with M. acuminata ssp. banksii (Cluster XI) in New Guinea and hybrids have been observed, but these are probably transient F1’s, except if maintained by vegetative propagation. Indeed, several members of this subcluster show the banksii morphology except for the split fruit peels typical for schizocarpa (Daniells et al. 2001).

Indian AB cultivars ‘Ney Poovan’ and ‘Kunnan’ were clearly differentiated in this work by SSR analysis, but no conclusion could be reached on the contribution of particular A genomes to the origin of this subgroup. On the other hand, three AB cultivars, ‘Muku Bugis’, ‘Mu’u Seribu’ and ‘Mu’u Pundi’ (collected in the Indonesian Triangle expedition; Sutanto et al. 2016) were located in the ‘Plantain-linked’ set of cluster XIII (see Online Resource 3), hence at a remote distance from the previous ABs ‘Ney Poovan’ and ‘Kunnan’. Therefore we wonder whether they may have delivered the B-genome to AA cultivars to form AABs such as ‘Laknau’ and ‘Iholena’.

The UPGMA clustering analysis separated the commonly recognized accessions into distinct clusters scattered over the tree with blocks of triploid edible accessions located among clusters of wild and cultivated diploids (Fig. 3, Online Resource 3). The clustering pattern reflects the proportion of shared SSR-alleles between the accessions. Thus, the probable ancestral relationships among diploid progenitors and edible triploids can be hypothesized. Using a similar set of SSR markers, Hippolyte et al. (2012) showed the African ‘Mlali’ subgroup to be the closest 2n donor for ‘Cavendish’ and ‘Gros Michel’ subgroups. Our results confirm this concept as illustrated by the position of the block of African AA cv. (Fig. 3, cluster IX) within the clade of ‘Cavendish’ and ‘Gros Michel’ subgroups. At a broader scope, this clade is loosely linked with the AAB ‘Pome’ subgroup that was also proposed to share the eastern ancestry with the diploid African cv. ‘Mlali’ (Perrier et al. 2011). Moreover, M. acuminata ‘Pisang Pipit’, that was proposed to be related with the n-gamete donor of Cavendish bananas (Hippolyte et al. 2012) is part of the cluster, supporting its potential role in the ancestral relationships.

Similarly, our results support the conclusion of Hippolyte et al. (2012), that the AAA ‘Ibota’ subgroup (cluster III) originated from malaccensis subspecies (but missing the banksii contribution; Perrier et al. 2009) and further substantiate it with AA cultivars from Island SE Asia (AA cv. ISEA 1) as potential contributors to the formation of this triploid subgroup. Another AAA subgroup ‘Lakatan’ also called ‘Pisang Berangan’ (cluster IX) suggests the contribution of ISEA 2 edible AA cultivars. The poorly defined AAA ‘Orotava’ seems to be loosely connected with Vietnamese AA cvs ‘Ayam’ and ‘Chuoi La Rung’.

While for the AAA subgroups ‘Pisang Ambon’, ‘Leite’ and ‘Rio’ (AAA Hetero on Fig. 3, cluster IX) no clear donors of the 2n and n-gamete were identified previously, our results suggest that their putative progenitors may come from the AA cvs. from the Indonesian Triangle including New Guinea (AA cv. IndonTriNG, Fig. 3). Moreover, our analysis proved that the AAA ‘Pisang Ambon’ subgroup (cluster IX; Online Resource 3 for details) is genetically distinct from the ‘Gros Michel’ subgroup, while the opposite has been suggested based on its morphological appearance and from the analysis of mitochondrial and plastid DNA (Carreel et al. 2002). Although for the ‘Red’ subgroup a malaccensis background was proposed (Perrier et al. 2009), our results (cluster IX, Fig. 3) do not support this assumption.

The four morphologically distinctive clone-sets of the African AAA ‘Lujugira/Mutika’ subgroup (Nfuuka, Musakala, Nakabululu, Nakitembe; Karamura 1998; Karamura and Pickersgill 1999) form a single coherent block in the cluster X of the SSR dendrogram. The existence of a previously assumed fifth clone set (Mbidde), containing only bananas for beer making, has recently been abandoned (Kitavi et al. 2016), its cultivars being morphologically and genetically indistinguishable from one or other of the aforementioned clone sets. Our results are in line with Kitavi et al. (2016) findings that the four clone sets are part of a single subgroup stemming from a single original AAA hybrid that has probably been formed in eastern Indonesia (Perrier et al. 2011) and diversified subsequently by multiple somatic mutations. A similar conclusion can be made for the subgroup of AAB ‘African Plantains’ that is subdivided into three clone types: Horn, False Horn and French, based on inflorescence morphology (De Langhe et al. 2005). Their phenotypic differentiation is not reflected by SSR analysis (cluster XIII, Fig. 3). The fact that these clone types cannot be separated into different subsets on the tree despite their striking morphological differentiation (Vuylsteke et al. 1996; Daniells et al. 2001) seems to confirm the fact that the differences are due to epigenetic changes, or resulted from somatic mutations occurring along the centuries of cultivation (Simmonds 1966; De Langhe et al. 1995; Crouch et al. 2000; Noyer et al. 2005).

The same cluster XIII contains subgroups ‘Iholena’ and the Philippine ‘Laknau’, with a bunch morphology reminding that of AAB Plantains, which are hence assembled under the name AAB Plantain-linked. The ‘Iholena‘originated in mainland New Guinea (Perrier et al. 2009; Kagy et al. 2016), but its place in the ‘Plantain-linked’ set with accessions from eastern Indonesia points to a broader area of origin. Because of the proved banksii genetic background of both A genomes in the plantain AAB subgroup (Lebot et al. 1993; Carreel et al. 2002; Kagy et al. 2016), it is proposed that the Plantain-linked cultivars have the same A-background.

Similarly the cluster VIII contains a heterogeneous set of different genome-combinations. Representatives of the ABB triploid subgroup ‘Pisang Awak’ and the Indian AAB ‘Silk’ subgroup were reported to share the malaccensis A-genome (Perrier et al. 2009), but this conclusion is weakly supported by our results. Interestingly, two supposedly Silk accessions—verified triploids cultivated in East Africa—are on the same branch as AB ‘Kunnan’. The possible genotypic relation between ‘Silk’ and ‘Kunnan’ warrants further examination.