Acuminata Subspecies Research Articles

Cryopreservation of zygotic embryos derived from BBTV resistant accessions of wild banana Musa acuminata

The wild banana species Musa acuminata encompasses numerous subspecies, some of which serve as ancestors of edible bananas. Preserving these subspecies is critical for food security, especially in regions where bananas are staple crops. While cryopreservation of M. acuminata subsp. burmanicoides zygotic embryos using air dehydration have shown high success rates, this method has not been effective for other subspecies. With at least 15 subspecies displaying various morphological traits, including seed morphology, alternative cryopreservation methods warrant exploration. In a recent study, successful cryopreservation using the droplet-vitrification method was achieved for mature zygotic embryos of accession of M. acuminata subspecies tomentosa, rutilifes, and malaccensis, all of which are resistant to the Banana bunchy top virus (BBTV). Using this approach, subjecting embryos to dehydration for 20 min in PVS2 solution and employing a regeneration medium comprising MS medium supplemented with benzyladenine and thidiazuron resulted in a success rate of 77.4 % (86 % survival rate × 90 % regrowth rate) for subsp. tomentosa and 50.4 % (70 % survival rate × 72 % regrowth rate) for subsp. malaccensis. Conversely, for subsp. rutilifes, dehydration for 10 min led to a success rate of 15.6 % (30 % survival rate × 52 % regrowth rate). Subspecies emerged as the primary factor influencing embryo regrowth, suggesting the need for protocol optimizations tailored to other subspecies. These findings highlight the potential of droplet-vitrification as a long-term preservation method for certain subspecies, while underscoring the necessity for further optimization for the other subspecies of M. acuminata.

Striking variation in chromosome structure within Musa acuminata subspecies, diploid cultivars, and F1 diploid hybrids.

The majority of cultivated bananas originated from inter- and intra(sub)specific crosses between two wild diploid species, Musa acuminata and Musa balbisiana. Hybridization and polyploidization events during the evolution of bananas led to the formation of clonally propagated cultivars characterized by a high level of genome heterozygosity and reduced fertility. The combination of low fertility in edible clones and differences in the chromosome structure among M. acuminata subspecies greatly hampers the breeding of improved banana cultivars. Using comparative oligo-painting, we investigated large chromosomal rearrangements in a set of wild M. acuminata subspecies and cultivars that originated from natural and human-made crosses. Additionally, we analyzed the chromosome structure of F1 progeny that resulted from crosses between Mchare bananas and the wild M. acuminata 'Calcutta 4' genotype. Analysis of chromosome structure within M. acuminata revealed the presence of a large number of chromosomal rearrangements showing a correlation with banana speciation. Chromosome painting of F1 hybrids was complemented by Illumina resequencing to identify the contribution of parental subgenomes to the diploid hybrid clones. The balanced presence of both parental genomes was revealed in all F1 hybrids, with the exception of one clone, which contained only Mchare-specific SNPs and thus most probably originated from an unreduced diploid gamete of Mchare.

Shared pedigree relationships and transmission of unreduced gametes in cultivated banana.

Cultivated bananas resulted from inter(sub)specific hybridizations involving Musa species and subspecies (M. acuminata subspecies, M. schizocarpa, M. balbisiana) and the subsequent selection, centuries ago, of hybrids with parthenocarpic, seedless fruits. Cultivars have low fertility and are vegetatively propagated, forming groups of somaclones. Relatively few of them, mainly triploids, are grown on a large scale and characterization of their parental relationships may be useful for breeding strategies. Here we investigate parental relationships and gamete-type contributions among diploid and polyploid banana cultivars. We used SNP genotyping data from whole-genome sequencing of 178 banana individuals, including 111 cultivars, 55 wild bananas and 12 synthetic F1 hybrids. We analysed the proportion of SNP sites in accordance with direct parentage with a global statistic and along chromosomes for selected individuals. We characterized parentage relationships for 7 diploid cultivars, 11 triploid cultivars and 1 tetraploid cultivar. Results showed that both diploid and triploid cultivars could have contributed gametes to other banana cultivars. Diploids may have contributed 1x or 2x gametes and triploids 1x to 3x gametes. The Mchare diploid cultivar group, nowadays only found in East Africa, was found as parent of two diploid and eight triploid cultivars. In five of its identified triploid offspring, corresponding to main export or locally popular dessert bananas, Mchare contributed a 2x gamete with full genome restitution without recombination. Analyses of remaining haplotypes in these Mchare offspring suggested ancestral pedigree relationships between different interspecific banana cultivars. The current cultivated banana resulted from different pathways of formation, with implication of recombined or un-recombined unreduced gametes produced by diploid or triploid cultivars. Identification of dessert banana's parents and the types of gametes they contributed should support the design of breeding strategies.

Interspecific introgression patterns reveal the origins of worldwide cultivated bananas in New Guinea.

Hybridizations between Musa species and subspecies, enabled by their transport via human migration, were proposed to have played an important role in banana domestication. We exploited sequencing data of 226 Musaceae accessions, including wild and cultivated accessions, to characterize the inter(sub)specific hybridization pattern that gave rise to cultivated bananas. We identified 11 genetic pools that contributed to cultivars, including two contributors of unknown origin. Informative alleles for each of these genetic pools were pinpointed and used to obtain genome ancestry mosaics of accessions. Diploid and triploid cultivars had genome mosaics involving three up to possibly seven contributors. The simplest mosaics were found for some diploid cultivars from New Guinea, combining three contributors, i.e., banksii and zebrina representing Musa acuminata subspecies and, more unexpectedly, the New Guinean species Musa schizocarpa. Breakpoints of M. schizocarpa introgressions were found to be conserved between New Guinea cultivars and the other analyzed diploid and triploid cultivars. This suggests that plants bearing these M. schizocarpa introgressions were transported from New Guinea and gave rise to currently cultivated bananas. Many cultivars showed contrasted mosaics with predominant ancestry from their geographical origin across Southeast Asia to New Guinea. This revealed that further diversification occurred in different Southeast Asian regions through hybridization with other Musa (sub)species, including two unknown ancestors that we propose to be M. acuminata ssp. halabanensis and a yet to be characterized M. acuminata subspecies. These results highlighted a dynamic crop formation process that was initiated in New Guinea, with subsequent diversification throughout Southeast Asia.

Hybridization, missing wild ancestors and the domestication of cultivated diploid bananas.

Hybridization and introgressions are important evolutionary forces in plants. They contribute to the domestication of many species, including understudied clonal crops. Here, we examine their role in the domestication of a clonal crop of outmost importance, banana (Musa ssp.). We used genome-wide SNPs generated for 154 diploid banana cultivars and 68 samples of the wild M. acuminata to estimate and geo-localize the contribution of the different subspecies of M. acuminata to cultivated banana. We further investigated the wild to domesticate transition in New Guinea, an important domestication center. We found high levels of admixture in many cultivars and confirmed the existence of unknown wild ancestors with unequal contributions to cultivated diploid. In New Guinea, cultivated accessions exhibited higher diversity than their direct wild ancestor, the latter recovering from a bottleneck. Introgressions, balancing selection and positive selection were identified as important mechanisms for banana domestication. Our results shed new lights on the radiation of M. acuminata subspecies and on how they shaped banana domestication. They point candidate regions of origin for two unknown ancestors and suggest another contributor in New Guinea. This work feed research on the evolution of clonal crops and has direct implications for conservation, collection, and breeding.

Cultivar specific gene pool may play an important role in Musa acuminata Colla evolution

Edible bananas are hybrids of various inter- and intraspecific combinations of Musa acuminata Colla and M. balbisiana Colla at different ploidy levels brought up by coexisting selection by farmers and nature. Our current study, based on a 461-bp-long sequence of the 5′ETS rDNA of 35 diploids and 19 cultivated varieties harboring 87 informative SNPs, revealed five groups of M. acuminata subspecies; A1: zebrina, A2: malaccensis, A3: burmannica/burmanicoides/siamea, A4: banksii/errans/microcarpa, A5: truncata, and a single cluster of B: M. balbisiana. We identified a new 5′ETS rDNA allele unidentified gene pool (UGP) absent in wild type accessions but present in diploid and triploid cultivars. This new allele is probably the result of recombination event in ancient ‘cultiwild’ populations. In the present study we observed a putative crossover event re-establishing a conventional allele. Genotyping triploid cultivars revealed that the Hom group cultivars are composed of two different ancestors while Cavendish Grande Naine, Cavendish Petit Naine, Cavendish Poyo and Gros Michel have three different ancestors. Hom Thong and Hom Taiwan were shown to have different ancestors compared to Gros Michel, unexpectedly even including subspecies truncata rDNA. We further improved our earlier established 5′ETS rDNA SCAR marker system by adding two new markers identifying M. acuminata subsp. truncata (Ridl.) Kiew and the newly identified UGP allele. We suggest that for breeding purposes, involvement of partially fertile diploid cultivar/cultiwild carrying the UGP gene pool may be an approach for developing new breeding programs and improving genetic studies of edible banana.

Genome ancestry mosaics reveal multiple and cryptic contributors to cultivated banana.

SummaryHybridizations between closely related species commonly occur in the domestication process of many crops. Banana cultivars are derived from such hybridizations between species and subspecies of the Musa genus that have diverged in various tropical Southeast Asian regions and archipelagos. Among the diploid and triploid hybrids generated, those with seedless parthenocarpic fruits were selected by humans and thereafter dispersed through vegetative propagation. Musa acuminata subspecies contribute to most of these cultivars. We analyzed sequence data from 14 M. acuminata wild accessions and 10 M. acuminata‐based cultivars, including diploids and one triploid, to characterize the ancestral origins along their chromosomes. We used multivariate analysis and single nucleotide polymorphism clustering and identified five ancestral groups as contributors to these cultivars. Four of these corresponded to known M. acuminata subspecies. A fifth group, found only in cultivars, was defined based on the ‘Pisang Madu’ cultivar and represented two uncharacterized genetic pools. Diverse ancestral contributions along cultivar chromosomes were found, resulting in mosaics with at least three and up to five ancestries. The commercially important triploid Cavendish banana cultivar had contributions from at least one of the uncharacterized genetic pools and three known M. acuminata subspecies. Our results highlighted that cultivated banana origins are more complex than expected – involving multiple hybridization steps – and also that major wild banana ancestors have yet to be identified. This study revealed the extent to which admixture has framed the evolution and domestication of a crop plant.

Two large reciprocal translocations characterized in the disease resistance-rich burmannica genetic group of Musa acuminata

Background and AimsBanana cultivars are derived from hybridizations involving Musa acuminata subspecies. The latter diverged following geographical isolation in distinct South-east Asian continental regions and islands. Observation of chromosome pairing irregularities in meiosis of hybrids between these subspecies suggested the presence of large chromosomal structural variations. The aim of this study was to characterize such rearrangements.MethodsMarker (single nucleotide polymorphism) segregation in a self-progeny of the ‘Calcutta 4’ accession and mate-pair sequencing were used to search for chromosomal rearrangements in comparison with the M. acuminata ssp. malaccensis genome reference sequence. Signature segment junctions of the revealed chromosome structures were identified and searched in whole-genome sequencing data from 123 wild and cultivated Musa accessions.Key ResultsTwo large reciprocal translocations were characterized in the seedy banana M. acuminata ssp. burmannicoides ‘Calcutta 4’ accession. One consisted of an exchange of a 240 kb distal region of chromosome 2 with a 7.2 Mb distal region of chromosome 8. The other involved an exchange of a 20.8 Mb distal region of chromosome 1 with a 11.6 Mb distal region of chromosome 9. Both translocations were found only in wild accessions belonging to the burmannicoides/burmannica/siamea subspecies. Only two of the 87 cultivars analysed displayed the 2/8 translocation, while none displayed the 1/9 translocation.ConclusionTwo large reciprocal translocations were identified that probably originated in the burmannica genetic group. Accurate characterization of these translocations should enhance the use of this disease resistance-rich burmannica group in breeding programmes.

Three New Genome Assemblies Support a Rapid Radiation in Musa acuminata (Wild Banana).

Edible bananas result from interspecific hybridization between Musa acuminata and Musa balbisiana, as well as among subspecies in M. acuminata. Four particular M. acuminata subspecies have been proposed as the main contributors of edible bananas, all of which radiated in a short period of time in southeastern Asia. Clarifying the evolution of these lineages at a whole-genome scale is therefore an important step toward understanding the domestication and diversification of this crop. This study reports the de novo genome assembly and gene annotation of a representative genotype from three different subspecies of M. acuminata. These data are combined with the previously published genome of the fourth subspecies to investigate phylogenetic relationships. Analyses of shared and unique gene families reveal that the four subspecies are quite homogenous, with a core genome representing at least 50% of all genes and very few M. acuminata species-specific gene families. Multiple alignments indicate high sequence identity between homologous single copy-genes, supporting the close relationships of these lineages. Interestingly, phylogenomic analyses demonstrate high levels of gene tree discordance, due to both incomplete lineage sorting and introgression. This pattern suggests rapid radiation within Musa acuminata subspecies that occurred after the divergence with M. balbisiana. Introgression between M. a. ssp. malaccensis and M. a. ssp. burmannica was detected across the genome, though multiple approaches to resolve the subspecies tree converged on the same topology. To support evolutionary and functional analyses, we introduce the PanMusa database, which enables researchers to exploration of individual gene families and trees.

Evolution of the Banana Genome (Musa acuminata) Is Impacted by Large Chromosomal Translocations.

Most banana cultivars are triploid seedless parthenocarpic clones derived from hybridization between Musa acuminata subspecies and sometimes M. balbisiana. M. acuminata subspecies were suggested to differ by a few large chromosomal rearrangements based on chromosome pairing configurations in intersubspecies hybrids. We searched for large chromosomal rearrangements in a seedy M. acuminata ssp. malaccensis banana accession through mate-pair sequencing, BAC-FISH, targeted PCR and marker (DArTseq) segregation in its progeny. We identified a heterozygous reciprocal translocation involving two distal 3 and 10 Mb segments from chromosomes 01 and 04, respectively, and showed that it generated high segregation distortion, reduced recombination and linkage between chromosomes 01 and 04 in its progeny. The two chromosome structures were found to be mutually exclusive in gametes and the rearranged structure was preferentially transmitted to the progeny. The rearranged chromosome structure was frequently found in triploid cultivars but present only in wild malaccensis ssp. accessions, thus suggesting that this rearrangement occurred in M. acuminata ssp. malaccensis. We propose a mechanism for the spread of this rearrangement in Musa diversity and suggest that this rearrangement could have played a role in the emergence of triploid cultivars.

DArT whole genome profiling provides insights on the evolution and taxonomy of edible Banana (Musa spp.).

Background and Aims Dessert and cooking bananas are vegetatively propagated crops of great importance for both the subsistence and the livelihood of people in developing countries. A wide diversity of diploid and triploid cultivars including AA, AB, AS, AT, AAA, AAB, ABB, AAS and AAT genomic constitutions exists. Within each of this genome groups, cultivars are classified into subgroups that are reported to correspond to varieties clonally derived from each other after a single sexual event. The number of those founding events at the basis of the diversity of bananas is a matter of debate.Methods We analysed a large panel of 575 accessions, 94 wild relatives and 481 cultivated accessions belonging to the section Musa with a set of 498 DArT markers previously developed.Key Results DArT appeared successful and accurate to describe Musa diversity and help in the resolution of cultivated banana genome constitution and taxonomy, and highlighted discrepancies in the acknowledged classification of some accessions. This study also argues for at least two centres of domestication corresponding to South-East Asia and New Guinea, respectively. Banana domestication in New Guinea probably followed different schemes that those previously reported where hybridization underpins the emergence of edible banana. In addition, our results suggest that not all wild ancestors of bananas are known, especially in M. acuminata subspecies. We also estimate the extent of the two consecutive bottlenecks in edible bananas by evaluating the number of sexual founding events underlying our sets of edible diploids and triploids, respectively.Conclusions The attribution of clone identity to each sample of the sets allowed the detection of subgroups represented by several sets of clones. Although morphological characterization of some of the accessions is needed to correct potentially erroneous classifications, some of the subgroups seem polyclonal.

English

Banana and plantain (Genus: Musa; Family: Musaceae) are giant perennial rhizomatous herbs native to South East Asia and Western Pacific. Present study analyzed the genetic population structure and diversity of South Indian Musa cultivars belonging to different genome groups using random amplified polymorphic (RAPD) markers. The total genetic diversity for all groups (Ht) was 0.22 and within groups (Hs) was 0.07 indicating high genetic variability among groups (Gst=0.69). Gene flow (Nm) was also found to be relatively very low with a value of 0.23. Genetic variability parameters and AMOVA revealed that genetic diversity is mainly present among the genome groups. Analysis of genetic diversity and structure revealed highest diversity and heterozygosity in AA group followed by AAB. Nei (1973) gene diversity was 0.12 ± 0.17 in AA group followed by 0.09 ± 0.14 in AAB group. Dendrogram derived based on UPGMA method also grouped AA cultivars into two different clusters and separated them from the wild Calcutta 4 (AA) indicating the involvement of several Musa acuminata subspecies in the origin and evolution of AA diploids. The current work revealed the status of genetic diversity and population structure among Musa genome groups implying the need of effective conservation of diploid M. acuminata (AA) group. Key words: Banana, genetic diversity, Musa, random amplified polymorphic (RAPD), population structure.

Genotype by Environment (G x E) Modeling of the Variable Initiation of Parthenocarpy sensu stricto in Musa: Elucidation of the Environmental Components of Variable Expressivity of Parthenocarpy in a Facultative Apomictic Musa acuminata Subspecies Microcarpa Model System

Genotype by Environment (G x E) Modeling of the Variable Initiation of Parthenocarpy sensu stricto in Musa: Elucidation of the Environmental Components of Variable Expressivity of Parthenocarpy in a Facultative Apomictic Musa acuminata Subspecies Microcarpa Model System

Genotype by Environment (G x E) Modeling of the Variable Initiation of Parthenocarpy sensu stricto in Musa: Elucidation of the Environmental Components of Variable Expressivity of Parthenocarpy in a Facultative Apomictic Musa acuminata Subspecies Microcarpa Model System

To better understand the genome by environment (G x E) interactions that need to be accommodated in order to better predict hybrid performance for a high breeding value

Elucidation of origin of the present day hybrid banana cultivars using the 5′ETS rDNA sequence information

In the present study, our intention was to elucidate the genetic relation of M. acuminata subspecies and analyse the diversity of the M. balbisiana gene-pool using nuclear ribosomal gene loci based marker system. Additionally the obtained information allowed elucidating the structure and ancestry of the nuclear genomes of diploid and triploid cultivars. By establishing the nucleotide sequence of the rDNA locus for M. acuminata and partially for M. balbisiana and their comparative analysis revealed that the 5′ETS region was the most divergent between acuminata and balbisiana genomes. Based on the SNP sites identified in this region a PCR based system was built, which revealed four gene-pools among M. acuminata wild types, while M. balbisiana showed no sequence divergence. The developed markers proved to be a powerful tool in the identification of the acuminata component of diploid and triploid hybrid cultivars and discovery of unexpected genotypes.

Identification of dCAPS Markers that Discriminate A and B Cytoplasms in Banana (Musa spp.)

A single nucleotide polymorphic sequence (SNP) in the trnL-F intergenic spacer region of chloroplast DNA that discriminate Musa acuminata (AA) cytoplasm from Musa balbisiana (BB) cytoplasm has been found. This mutation was initially amplified using a pair of universal primers and converted into dCAPS (derived cleaved amplified polymorphic sequence) markers. Using these markers in combination with flow cytometric analysis, ‘Pisang Klutuk’ (syn. ‘Pisang Awak’) was found to be a triploid ABB cultivar (not a wild BB accession as previously classified). A 15-bp long deletion was also found within the trnL-F spacer sequence of M. acuminata subspecies banksii. This mutation will be useful as a specific marker for the cytoplasm derived from this subspecies. These maternally inheritable chloroplast DNA mutations discovered in this study are vital for better understanding the origins of banana cytoplasm to clarify the lineage of banana cultivars and the contribution of wild progenitors to cultivated types.

MORPHOLOGICAL CHARACTERIZATION OF MALAYSIAN WILD BANANA MUSA ACUMINATA

Fourteen populations of Musa acuminata ranging from populations in the lowlands of northern (ssp. siamea) to central Malaysian region (ssp. malaccensis) and highland banana (ssp. truncata) were characterized based on chromosome number and 46 morphological characters. A large amount of variation was observed within the populations. However, only highland bananas appeared morphologically distinct. Lowland populations both from northern and central Malaysia were found to be overlapping and no distinguishing pattern was observed. The morphological characters found variable within these populations were related to developmental changes and mutations. The results obtained in this study were not revolutionary. However, the survey of a large number of characters treated with multivariate techniques further sharpened the existing groupings of the Musa acuminata subspecies. Key words: Musaceae/ Malaysia / ssp. malaccensis I ssp. truncata I ssp. siamea

Genetic Diversity in Musa acuminata Colla and Musa balbisiana Colla and some of their natural hybrids using AFLP Markers.

Genetic diversity and relationships were assessed in 28 accessions of Musa acuminata (AA) Colla and Musa balbisiana (BB) Colla, and some of their natural hybrids, using the amplified fragment length polymorphisms (AFLP) technique. Fifteen AFLP +3 primer pairs produced 527 polymorphic bands among the accessions. Neighbor-joining and principal co-ordinate (PCO) analyses using Jaccard's similarity coefficient produced four major clusters that closely corresponded with the genome composition of the accessions (AA, BB, AAB and ABB). The AFLP data distinguished between the wild diploid accessions and suggested new subspecies relationships in the M. acuminata complex that are different from those based on morphological data. The data suggested that there are three subspecies within the M. acuminata complex (ssp. burmannica Simmonds, malaccensis Simmonds, and microcarpa Simmonds). 'Tjau Lagada' (ssp. microcarpa), 'Truncata' [ssp truncata (Ridl.) Shepherd] and 'SF247' [ssp. banksii (F.Muell) Simmonds] clustered very closely with 'Gros Michel' and 'Km 5', indicating that more than one M. acuminata subspecies may be involved in the origin of triploid AAA bananas. 'Calcutta 4' (ssp. burmannicoides De Langhe & Devreux) and 'Long Tavoy' (ssp. burmannica) were closely related and could be together in the same subspecies. This study also showed that there is much more genetic diversity within M. balbisiana that was split into two groups: (1) 'I-63' and 'HND' and (2) 'Los Banos', 'MPL' (Montpellier), '10852', 'Singapuri', 'Etikehel', and 'Butohan 1' as the other.

Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata andM. balbisiana)

Cell nuclei were isolated from leaf tissues of wild banana (Musa balbisiana, M. acuminata ssp.banksii andM. acuminata ssp.errans) and of the two vegetative clones of diploid cultivar “Pisang Mas”. Relative fluorescence intensity was measured on propidium iodide-stained nuclei by flow cytometry. Nuclei isolated fromGlycine max with known nuclear genome size were used as internal standard to determine nuclear DNA content ofMusa in absolute units. The results of the study showed that the size of nuclear genome ofMusa is smaller than previously estimated. In general, it is smaller in comparison with many other angiosperms. Furthermore, it was found that nuclear DNA content ofM. balbisiana (genome BB) is significantly lower than that ofM. acuminata subspecies and cultivars (genome AA). This finding should permit estimation of genome composition in triploidMusa clones with expected hybrid composition. Flow cytometry is proposed as a useful technique with potential applications in taxonomy, breeding and biotechnology ofMusa.