Collection of new diversity of wild and cultivated bananas (Musa spp.) in the Autonomous Region of Bougainville, Papua New Guinea

Bananas (Musa spp.), including dessert and cooking types such as Plantain, are of major importance in the tropics for both subsistence and food security (FAO 2014). Originating from the South-East Asia/West Oceania region, this crop has a complex domestication history (De Langhe et al. 2009; Perrier et al. 2011). The name “banana” corresponds to different species of the Musa genus, and to their hybrids. The genus Musa is divided into two sections corresponding to distinct phylogenetic clades, Musa (2n = 22) and Callimusa (2n = 20 or 18) (Häkkinen 2013; Janssens et al. 2016). The edible bananas from the section Musa are composed of either genome A (Musa acuminata Colla), or A in combination with B (M. balbisiana Colla) or S (M. schizocarpa N.W. Simmonds). Edible bananas from the Musa section are diploid, triploid or more rarely tetraploid, the most popular cultivars being triploid from Groups such as Cavendish (AAA) and Plantain (AAB). Edible bananas from the section Callimusa, called Fe’i bananas, are associated with T genome (M. textilis Née), but have been far less studied and their origin remains obscure. Recent results indicate that the Fe’i bananas arose from complex domestication schemes and comprise accessions with different ploidy (Christelová et al. 2017; www.crop-diversity.org/mgis/). As edible bananas bear seedless fruits, they are propagated vegetatively. This mode of propagation and extremely low level of fertility make cultivated bananas particularly vulnerable to diseases and abiotic stress and the occurrence of new, potentially better adapted genotypes is limited to rare mutation events. Therefore, the diversity of cultivated bananas was fixed over long periods of time to the existing genotypes. Even if the diverse genetic make-up of banana varieties grown worldwide exhibit a wide range of traits (Heslop-Harrison and Schwarzacher 2007), this pattern slows down cultivated bananas’ evolution and puts banana-based agrosystems at risk by hampering rapid adaptation of the crop to new threats.

Breeding programs in banana focus primarily on creating improved triploid or tetraploid varieties (Ortiz 2013; Tomekpe et al. 2004). In addition to multiple ploidy levels, the sterility associated with the production of seedless fruits is a challenge for breeders. However, the use of fully or partially fertile, parthenocarpic, edible diploids eases the process of creating progenies in a largely sterile crop (Tomekpe et al. 2004; Tenkouano et al. 2011). In this context, assessing the extent of wild and cultivated banana diversity, conserving it and making it available for further use is a priority.

Papua New Guinea (PNG), including neighbouring islands, is a recognized centre of diversity and potentially a domestication centre for banana (Christelová et al. 2017; Lebot 1999; Sardos et al. 2016b). Four banana collecting missions¹ were organized to mainland PNG and the Bismarck Archipelago in 1988–1989 which revealed high levels of diversity in the country. In total, 264 wild and cultivated accessions were collected, of which 86% appeared to be unique genotypes (Arnaud and Horry 1997). These accessions were sent to both the National Banana Germplasm Collection at Laloki, Port Moresby and to the Bioversity International Musa Germplasm Transit Centre (ITC)² in Belgium for conservation purposes. Currently, more than 25 years after the PNG missions, 230 of the accessions collected in PNG are still conserved in vitro in the ITC. Over the years, the PNG accessions have been valuable resources for breeders and researchers and have significantly improved our knowledge of banana (e.g. Ploetz et al. 1999; Geering et al. 2005; Raboin et al. 2005; Ball et al. 2006; Volkaert 2011; Till et al. 2010; Hřibová et al. 2011; Teo et al. 2011; Christelová et al. 2011, 2017; Valdez-Ojeda et al. 2014; Janssens et al. 2016; Sardos et al. 2016a, b).

Interestingly, out of the 177 cultivated accessions collected in PNG that are still conserved in the ITC, 100 are cultivated diploids with AA genome composition. These accessions are mainly used by local populations as cooking varieties and are therefore of great interest for the improvement of cooking triploids such as Plantains. Due to a civil conflict (1988–1998), the region which later became the Autonomous Region of Bougainville (AROB) was not visited by the expeditions in the 1980′s (Fig. 1).

Open image in new window

Located in Near Oceania, the AROB is composed of two main islands, Bougainville and Buka, and of smaller surrounding islands. While it is an autonomous region of Papua New Guinea, it belongs geographically and ecologically to the Solomon Islands Archipelago. Agricultural systems in Bougainville and Buka are typical of the West Oceania region, where people practise a rain fed and shifting agriculture with fallow periods that can reach up to 15 years. The most important staple crop all over these islands is sweet potato (Ipomoea batatas (L.) Lam.) followed by banana, coconut (Cocos nucifera L.) and a range of root and tuber crops (Bourke et al. 2002).

Organized by NARI and Bioversity International, a banana collecting mission in the AROB took place from 19 October to 31 October 2016, and explored the islands of Bougainville, Buka and Sohano. This report presents the genetic diversity collected during the prospections, which targeted new wild and cultivated germplasm for conservation purposes. When possible, the prospections were coupled with the systematic ploidy measurement and SSR genotyping of the collected accessions. These two activities took place at the Musa Genotyping Centre (MGC) held in the Institute of Experimental Botany (Olomouc, Czech Republic). The SSR profiles of the collected accessions were added to the results obtained in the study published by Christelová et al. (2017) in order to better characterize the genetic diversity of the new accessions collected in this region. We discuss here the diversity of the genotypes collected in Bougainville and show that doubling plant prospection with “real-time” ploidy measurements and SSR genotyping adds substantial value to collecting missions.

The collecting mission to AROB was a fruitful exploration. Despite the four extensive collecting missions achieved in PNG in the 1980′s, many new cultivars were discovered.

Coupling ploidy estimation and SSR genotyping using the standardized platform for molecular characterization of Musa germplasm (Christelová et al. 2011) with the field prospections was very useful for different purposes. First, the joint analysis of the CS and of the AROB accessions helped in refining or confirming the classification of the AROB accessions by complementing the observations made in the field. The most appropriate stage for banana cultivars description and identification is when the first fruits are ripe (TAG 2010) but it was not always possible on the field to find plants at this particular stage of development. Second, for the same reason and also due to G x E interactions, the formal identification of synonyms/duplicates was not always possible. The molecular characterization of the collected accessions allowed detecting potential duplicates within the newly collected accessions but also within the joint CS-AROB datasets. It also allowed identifying nearly identical genotypes deriving from clonal diversification. Due to the accumulation of mutations and epigenetic changes, strictly identical genotypes do not always have strictly identical phenotypes, and therefore do not correspond to the same varieties, but they give a good estimation of the genetic diversity that was collected.

The only limitation we found using this set of markers is for the Callimusa accessions, which appear genetically very similar with the SSR markers used. The high rates of missing data observed overall in this section correspond to non-amplifying loci. It suggests high rates of null alleles for some of the markers used or the total absence of the site for some others such as the marker mMaCIR164 which was missing in all accessions of the section Callimusa. The transferability rate of SSR markers between species within genera in monocots was estimated 60% in average, of which only 40% are expected to be polymorphic (Barbará et al. 2007). As the SSR markers used here were developed from the Musa species M. acuminata and M. balbisiana (Crouch et al. 1998; Lagoda et al. 1998; Hippolyte et al. 2010) that belong both to the Musa section, the Callimusa species may be too genetically distant. Recently, SNPs called from the mapping of Callimusa reads on the M. acuminata reference genome were not accurate (Y. Hueber and M. Rouard pers. com.) and may also reflect high levels of differentiation. A set of markers developed specifically for targeted Callimusa species would be likely to lead to different results. Therefore, the interpretation of the results on the wild and cultivated Callimusa genotypes in this study should be made cautiously.

The clustering of some of the AROB accessions within the CS was particularly interesting with regard to the diversification history of cultivated bananas. The emergence of triploid cultivars ensued from sexual diversification through the occurrence of unbalanced meiosis leading to unreduced gametes within edible diploids (De Langhe et al. 2009; Perrier et al. 2011). For example, the AAA Cavendish clones resulted from a natural cross between two AA landraces from the Mlali Group (2n gamete donor) and “Khai Nai On” (n gamete donor) (Carreel et al. 2002; Raboin et al. 2005; Perrier et al. 2009; Hippolyte et al. 2012). In this context, the positions of the three AAA AROB015 “Laguai”, AROB017 “Banawa” and AROB058 “Korukapi” in the diversity tree are also of particular interest as they are the only polyploids observed within a wide cluster of edible AA collected in PNG and closely related to M. acuminata subsp. banksii (F.Muell.) N.W.Simmonds, the Papuan subspecies of M. acuminata. Until now, only diploids have been reported in this genetic cluster (Christelová et al. 2017; Sardos et al. 2016b) and this pattern suggests that these triploids likely resulted from natural sexual crosses within Papuan edible diploid bananas. The two tetraploid accessions collected, AROB027 “Buka” and AROB056 “Kalmagol”, are also of particular interest. Their locations in the tree and their morphologies suggest the Groups Pisang Awak (ABB) and Silk (AAB) or Kunnan (AB) as parents, respectively. None of these triploid Groups is native to the region but the Pisang Awak Group is grown worldwide. In PNG, it is notably used to produce a local alcohol named Jungle Juice. Seeds are known to be quite common in cultivars from the Pisang Awak Group in their centre of origin, Malaysia (Simmonds 1966). Given the triploid status of this Group, viable progeny would be likely resulting from the natural cross of an unreduced gamete (3n) from the mother plant with a haploid pollen grain (n gamete) from a diploid plant. The tetraploid plant obtained from such a cross could be similar in morphology to its 3x parent, such as AROB027 “Buka”, and may be wrongly classified as part of the Pisang Awak Group. This may be the origin of a debate regarding the ploidy level of the Pisang Awak Group, as some tetraploids have been reported within this Group (Pillay et al. 2006).

The occurrence of a tetraploid linked to the Silk Group (AAB) and Kunnan Group (AB), which are both from India, is more surprising. Even though AROB056 “Kalmagol” was more frequently planted than AROB027 “Buka”, we didn’t note an abundance of Silk locally, as only a single plant was observed in the town of Arawa (Sachter-Smith et al. 2017) and no diploid AB was recorded. Therefore, this tetraploid was likely introduced to the island. Allen already collected the variety under the name “Kalamagol” in the 1960′s (Rosales et al. 1999) showing it was already there 50 years ago. Given the thousands of Indian indentured labourers who were brought to the Pacific, mainly to Fiji, by the British Empire in the 19th century, AROB056 “Kalmagol” may have reached Bougainville from India via Fiji. Interestingly, “Kalamagol” sounds similar to “Kalaimagal” which is a feminine name of Indian origin.

Due to high levels of sterility, many banana varieties are clonal selections from a single clone. For example, the more than 150 known Plantain varieties are considered to be derived from the clonal diversification of a single original plant (Noyer et al. 2005). Comparing the AROB accessions and the CS, we identified six pairs and a triplet of identical genotypes. If it strongly suggests that these pairs/triplet are duplicates, it is not the case for the pair AROB023 “Morou”/AROB055 “Tambra”. AROB055 “Tambra” is indeed variegated, that is to say that its leaves and fruits exhibit white stripes, while AROB023 “Morou” is not (Sachter-Smith et al. 2017). Variegation in plants is due to the partial fixation of a mutation in the chlorophyll-synthesis pathway (Marcotrigiano 1997). It is therefore likely that AROB055 “Tambra” ensues from the clonal diversification of AROB023 “Morou” and was intentionally selected by farmers for its attractive morphology. Such a case was already documented for cassava in the Pacific archipelago of Vanuatu, located further south of the Solomon Islands, where farmers capture all the variations of an initial genotype, independently of whether these variations might be agronomically useful (Caillon and Lanouguère-Bruneau 2005; Sardos et al. 2008). Here, we therefore documented in banana the fixation of an obvious mutation followed by the intentional and likely local selection of the variant by farmers. If variegated cassava is mostly used as an ornamental plant, we observed variegated bananas sold in the market in Buka town, attesting the potential value of growing such a variety.

The results of the collecting mission presented in this paper confirmed PNG as an important hotspot for banana genetic diversity as new genotypes, and new alleles, were collected despite the extensive collecting missions performed in the country in the past. The results of the SSR genotyping achieved concomitantly with the mission allowed highlighting not only the diversity cultivated in the AROB but also the different diversification processes at work in the region despite the crop’s vegetative mode of propagation. The maintenance of crop evolution under farmer management is key to the successful establishment of in-situ and on -farm conservation initiatives (Bellon et al. 2017). Our results suggest the occurrence of gene flow, the accumulation of mutations and the introduction of new varieties in the AROB. Even though further studies should be undertaken in the future to fully characterize and understand these processes, we already have good insights that AROB and, by extension, PNG is an excellent candidate for the establishment of on -farm conservation programmes for Musa.