28. The global Musa genomic consortium: A boost for banana improvement - Frison, E.A^[47]., J.V. Escalant^[48], S. Sharrock^[49]

Abstract

Bananas (Musa spp.) are of great importance to small-scale farmers in the developing countries of the tropics and sub-tropics. The crop can be grown in a range of environments and production systems, and provides a nutritious staple food and a significant source of revenue all year round. Growing populations in many of the countries where bananas provide a vital food source mean that productivity increases are essential. However, such increases in production must be brought about in the face of growing pest and disease pressure, and constantly changing environmental and economic conditions. In order to accelerate efforts in producing improved varieties of Musa, the International Network for the Improvement of Banana and Plantain (INIBAP) was instrumental in the formation of PROMUSA, the Global Programme for Musa Improvement, in 1997. Within the framework of PROMUSA, a global Musa Genomics Consortium was launched in 2001. The consortium aims to apply the newly available genomic technologies, which cover the analysis and sequencing of all the DNA, its genes, their expression, recombination and diversity, directly to the sustainable improvement of this major crop. This paper gives details of the importance of genomic studies for the improvement of a crop such as Musa, in which all the important cultivars are highly sterile; the resources available to the members of the consortium; and the incremental strategy developed for achieving the consortium's aims. Musa provides an ideal model species for genomics studies because of the small size of its genome, the comparisons that can be made between sterile, vegetatively propagated cultivars and seed-fertile wild species, and the different levels of ploidy available among members of the genus. It is also one of the few plant species with biparental cytoplasmic inheritance: paternal inheritance of mitochondria and maternal inheritance of chloroplasts.

1. INTRODUCTION

Bananas* are the developing world's fourth most important food crop (after rice, wheat and maize) in terms of gross value of production. The crop is grown in more than 100 countries throughout the tropics and sub-tropics, with an annual world production of around 98 million tonnes, of which around a third is produced in each of the African, Asia-Pacific, and Latin American and Caribbean regions [1]. Around 87% of all the bananas grown worldwide are produced by small-scale farmers for home consumption or for sale in local and regional markets. They provide a staple food for millions of people, particularly in Africa, an area where the green revolution has had little influence. As well as providing a cheap and easily produced source of energy, bananas are also rich in certain minerals and in vitamins A, C and B6. Growing urbanisation in many developing countries means that the crop is becoming more and more important as a source of revenue, sometimes providing the main source of income for rural communities. Bananas thus play an important role in poverty alleviation.

* Bananas comprise a diverse group, including cooking types such as plantains and a wide range of dessert types, and consist of interspecific hybrids of Musa spp. of different ploidy levels.

Bananas will grow in a range of environments and produce fruit throughout the year, thus providing a source of energy during the 'hungry period' between the harvest of other crops. They are particularly suited to intercropping systems and to mixed farming with livestock. Due to their suitability for production in backyard systems, bananas are also an important component of periurban agriculture. When grown in perennial production systems, bananas maintain cover throughout the year, thus protecting the soil from rain and wind erosion. Furthermore, if their biomass is used as mulch, soil fertility and organic matter remain stable.

Approximately 13% of worldwide banana production is destined for the export market. For many countries, especially in Latin America and the Caribbean, bananas provide an essential source of foreign exchange. The value of banana exports greatly outranks that of other fruits, such as apples and oranges, as well as vegetables such as tomatoes and potatoes. Banana exports are worth nearly $1 billion annually for Ecuador, the world's largest banana exporter. The export banana industry is also the backbone of the economies of many Caribbean countries, and the crop plays a vital role in the social and political fabrics of the islands.

In many countries, bananas are more than just a food crop [2]. They provide an important source of fibre (for example Abaca/Manila hemp in the Philippines), and among other uses, can be fermented to produce alcohol. Bananas have also been considered as a useful tool to deliver edible vaccines. The fruit can be eaten uncooked, it is sterile before peeling, and it is often the first solid food eaten by babies.

Because of growing populations in many of the countries where bananas provide a vital food source, productivity increases are essential. Such increases in production must, however, be brought about in the face of growing pest and disease pressure and ever-changing environmental and economic conditions.

The International Network for the Improvement of Banana and Plantain (INIBAP) was created in 1985 in response to the threat posed by the spread of a devastating fungal disease, black Sigatoka, to Africa. This disease, together with a range of other fungi, bacteria, insects, nematodes and viruses, causes heavy losses to banana farmers worldwide, the majority of whom cannot afford chemical pesticides to control these parasites. INIBAP has always recognised the need for improved pest- and disease-resistant varieties as the most effective and sustainable way of increasing yields, but is also aware of the difficulty of breeding such varieties in a crop where most of the cultivated varieties are highly sterile. Indeed, despite the efforts of INIBAP and its partners, progress in the development of improved varieties suitable for small-scale farmers has been slow. Recognising this, the Musa research community agreed that a global-level initiative was required to accelerate the impact of improvement efforts. This culminated in 1997 with the formation of PROMUSA, the Global Programme for Musa Improvement. The major thrust of PROMUSA is to develop a wide range of improved Musa varieties, bringing together conventional breeding and biotechnology, supported by research that is carried out on pests and diseases within the various working groups that constitute PROMUSA [3].

The formation of PROMUSA means that a mechanism for collaboration and information exchange between researchers involved in Musa genetic improvement is now in place. This has allowed the global prioritisation of research needs, and the acceleration of progress through the formation of synergistic partnerships. More than 100 researchers worldwide participate in the Global Programme.

Given the importance of genetic information for the sustainable improvement of Musa, a meeting of researchers wishing to apply the new and rapidly developing genomics technologies to Musa was held in France in 2000. At this meeting, it was agreed that such an effort would require participation and collaboration of scientists around the world. In order to bring together and enhance the combined expertise of the different participating laboratories, it was therefore agreed to form a 'Global Musa Genomics Consortium' in the framework of PROMUSA [4]. The launch of the Global Musa Genomics Consortium took place in Arlington, Virginia in July 2001.

2. THE GLOBAL MUSA GENOMICS CONSORTIUM

2.1. Aims of the consortium

The Consortium aims to apply genomics to the sustainable improvement of Musa (banana and plantain), a crop of world importance. The consortium believes that newly available genomic technologies, which include the analysis and sequencing of all the DNA, its genes, their expression, recombination and diversity, can now be applied directly to the sustainable improvement of this major crop. The consortium aims to develop freely accessible resources for Musa genomics, and use the new knowledge and tools to enable both targeted conventional breeding and transgenic strategies. The genomics strategy will also allow better utilisation and maintenance of Musa biodiversity. Furthermore, knowledge of Musa genomics, a monocotyledon cultivated as a polyploid, will provide a model resource for the exploitation of the genomes of other important species, increasing the usefulness of all genomic information.

The consortium will benefit, in the development of new varieties, from the successes of current genome and genomics programmes and enabling technologies in delivering new genetic knowledge and tools. Information will be obtained from the complete genomic sequences of Arabidopsis and rice, as well as the extensive sequence tags of other species, and these will be applied to Musa improvement. High throughput technologies, developed primarily for human genome analysis but now widely available, will be accessed to put in place rapidly the resources and tools needed.

The overall aims of the Global Musa Genomics Consortium lie within the context of improvement of the world's prosperity through fighting poverty and food insecurity in developing countries. The consortium will achieve this aim by using the tools and expertise at its disposal to increase the productivity of the developing world's fourth most important crop, a staple food and key cash crop for nearly a billion people. As a result of genomics research, banana and plantain productivity can be increased in ways which will remain sustainable, particularly in the face of changing economic, social and environmental conditions. The consortium believes that such increases in productivity gained through fundamental knowledge and application of genomics will help to ensure future food and income security for millions of men, women and children in the developing world.

2.2. How genomics can be applied for Musa improvement

In most crops, improvement and breeding methods are based on crossing elites and selecting the best performing progeny, a strategy impossible in the sterile Musa cultivars. Nevertheless, by the end of the twentieth century, efforts to improve Musa using diploid and tetraploid lines were beginning to produce results, and the first improved cultivars began to be distributed on a wide scale. However, progress in genetic improvement remains slow, and the potential for new genomic methodologies to make a significant difference in banana improvement is clear.

In the short term, the development of molecular markers and marker-assisted selection (MAS) methods will without doubt improve the efficiency of selection of improved cultivars for defined traits such as pest and disease resistance, abiotic stress tolerance, quality and post-harvest fruit characteristics [5-8]. The genetic maps now under construction will improve selectability of quantitative traits such as yield, and also allow better selection of parents for breeding programmes. However, it is generally agreed that more DNA markers are needed in order to accelerate the screening process. Because it is not known how the environment affects the pattern of the expression of the genome, the development of new and more markers should also be a way to predict how a particular genotype could respond, and therefore to predict if a particular genotype is more or less adapted to a specific situation.

In the medium term (5-10 years), breeding should provide new varieties. Gene isolation and the analysis of mutants will allow characterisation of critical alleles of genes of agronomic value and the relevant regulatory sequences. Transgenic approaches will offer the potential for direct transfer of these genes into current triploid cultivars, avoiding the difficulty of reconstructing already satisfactory varieties from crosses between unimproved and minimally selected lines.

The identification of all genes and gene functions in Musa should be possible, given the availability of high-throughout technologies. While relatively expensive to set up, because all genes are characterised in parallel, the per-gene costs are substantially lower than the cost for isolation of particular targeted genes. Furthermore, there is no cost of 'failure' to isolate a particular gene, errors are less likely, work in particular genome regions is not duplicated between laboratories, and the inherent systematic approach means genes are not missed in the assay.

2.3. Resources for Musa genomics research

The wide diversity in the genus Musa is the basic element that sustains production of this important crop, a situation that remains unchanged but is made more efficient and accessible by the tools of genomics. Breeders use diversity to produce improved varieties, allowing the crop to be grown in a wide range of environments and to meet the varied needs of the millions of people who depend on it for food and income [9]. INIBAP, entrusted by FAO, aims to conserve and make available this diversity for the benefit of present and future generations. As part of this effort, a major international germplasm collection is available. This resource is almost unparalleled in any other species. More than 1100 accessions of Musa are available from the International Musa Germplasm Collection, maintained by INIBAP at the Katholieke Universiteit Leuven (K.U.Leuven), Belgium [10]. The need for continued, open access, and fully indexed (pathogen-free) status has been highlighted by the consortium, and the continuing expansion of the collection with new accessions, particularly of wild and diploid material, is strongly supported.

As well as genotype collections, all genetic and genomic work relies on access to intercrossed families (segregating populations). Key resources for genomics also include large-insert recombinant DNA libraries, of which the most appropriate for Musa is made in BAC (bacterial artificial chromosome) vectors. Complementing this, full sets of expressed gene libraries, cDNA libraries, are required to examine gene expression profiles in different tissues and conditions. Finally, mutants and transgenic plants (including plants of other species with Musa genes) are essential to define the function of many genes.

In this respect, BAC and EST (expressed sequence tag) libraries are already available for M. acuminata. A new BAC library for M. acuminata is being developed in the framework of the Agropolis Advanced Research Platform at Montpellier, France, where an INIBAP researcher seconded from Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA), Brazil is working at the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) on the mapping of Musa acuminata translocation breakpoints, using on banana molecular cytogenetic methods such as in situ hybridisation with BACs as probes. A BAC library of M. balbisiana is also being developed at the Institute of Experimental Botany (IEB) in the Czech Republic. The project aims at producing an ordered large-insert banana B-genome library, which will be highly complementary with the BAC library of the A-genome.

Furthermore, genetic and physical maps of banana are being developed at CIRAD, France and IEB, Czech Republic, respectively, which should allow the identification of specific genes of interest. The first genetic map of banana (shared within PROMUSA), based on a wild segregating population, is currently being constructed at CIRAD using molecular markers: Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP) and Sequence-Tagged Microsatellites (STMS) [11]. Complementary work is ongoing at the Queensland Department of Primary Industry (QDPI) in Australia, where the banana segregating population from CIRAD is currently used to identify genes for resistance to Fusarium wilt. A Quantitative Trait Loci (QTL) for Sigatoka resistance has been anchored on a map developed at CIRAD and could be mapped, allowing isolation of genes (or cluster of genes) involved in resistance to the disease. Segregating populations are being studied at the International Institute of Tropical Agriculture (IITA), Nigeria and Centre Africain de Recherches sur Bananiers et Plantains (CARBAP), Cameroon. Activities are also being developed at IEB to establish a physical map of banana using fluorescence in situ hybridisation (FISH), fibre-FISH, Primed in situ Synthesis (PRINS) and chromosome painting [12-15].

Genetic transformation of bananas is now routine at a number of institutes around the world including K.U.Leuven, Belgium; Queensland University of Technology (QUT), Australia; and Syngenta, UK. Two main methodologies are being used: particle bombardment and Agrobacterium-mediated transformation [16-19]. The latter technique is considered to hold the greatest potential as it allows the insertion of larger pieces of DNA and results in fewer insertion sites and fewer gene copies. Transient and stable expression of introduced genes has been observed in a range of cultivars. The sterile nature of many cultivars means that concerns over genetic manipulation in field-grown material are small; there is, in contrast to brassicas and cereals, very little chance of spreading introduced genes into wild species.

K.U.Leuven, in collaboration with the University of Queensland, Australia, has isolated promoters from various strains of banana streak badnavirus. These promoters have allowed a very high constitutive expression of reporter genes in banana, as well as other monocot and dicot species. A promoter from sugarcane bacilliform badnavirus, cloned and shown to infect banana at the University of Minnesota, was also found by the Minnesota, Belgian and Australian groups to be highly active in both monocots and dicots [20]. The Belgian and Australian groups have also shown that this promoter is active in banana. At QUT, Australia, promoter regions derived from banana bunchy top nanovirus satellite components (S1 and S2) and banana actin genes have been isolated and characterised in transgenic banana plants.

The existence of several somatic mutants in Musa, a result of vegetative propagation, but mainly as mutants obtained after irradiation or using chemical mutagens, creates a resource of potential value for functional genomics (relating gene sequence to function), and also for the study of evolutionary mechanisms [21]. Haploid plants of M. acuminata have been produced through anther culture, and perfectly homozygous diploid plants have been obtained through colchicine treatment. Tetraploid plants can be easily obtained through doubling of diploids, allowing a comparison of plants with the same genetic composition, but with different ploidy levels [22].

Research is still required to develop efficient strategies for using information from Arabidopsis and rice genomics research. A full-normalised cDNA from different banana tissue could be isolated and made as a microarray for high-throughput genomic screening. Chips could be created (one chip may contain 15,000 elements) and used on Arabidopsis or/and rice maps to find any similarity, and therefore to identify new QTLs [23].

3. A STRATEGY FOR THE MUSA GENOMICS PROGRAMME

The consortium reviewed the current resources available in Musa genomics, the types of resources, and information available in other plant species and examples of the utilisation of these resources. Based on this, an incremental strategy for Musa genomics was defined to develop and utilise genomic tools for rapid improvement of the crop, including the discovery of genes of agronomic and economic importance, and the measurement and utilisation of the biodiversity in the genus.

The following deliverables of the Musa genomic programme have been identified:

(a) The complete sequence of the Musa genome,

(b) The identification of each gene in the genome,

(c) The definition of the function of each gene in the genome,

(d) The complete map of gene expression - the transcriptome - during development and under various biotic and abiotic stresses,

(e) The definition of all the alleles of each gene present in all genotypes of the species,

(f) Development of usable genetic markers for major traits,

(g) The measurement of variation and diversity between all accessions.

These deliverables will permit the directed breeding of improved varieties of banana and plantain, allowing environmentally friendly and sustainable production. The technology will allow transfer of genes freely between different accessions, maintaining their regulation, and also give the ability to transfer genes between other species and banana. The ability to manipulate bananas at the genome level has considerable potential for the improvement of the sterile, mostly triploid crop. A secondary aim, one which is in line with the goals of some funding agencies, is the provision of both genomic information and technology that would assist exploitation of genomes of other agronomically and medically important species.

Genomics is already a key to plant breeding and biodiversity utilisation strategies, and there is little doubt that the goals will be reached in due course. However, members of the consortium recognise that funding on the scale required might not be available over the next five years, so an incremental strategy was developed. The coordinated programme will permit access and application of markers and genes in the short term, while in the longer term driving towards the complete genomic information required, with minimal duplication of work.

3.1. Components of the incremental strategy for genomics

In the incremental strategy, a series of parallel projects are proposed, involving genomic DNA and cDNA in parallel, with additional work leading to the application of these results by breeding teams. These projects will focus on the following areas:

3.1.1. Physical maps

Following creation of the BAC library resources, physical maps require three different types of data: fingerprints, hybridisation of data using anchored probes, and cytogenetic data. The physical organisation data can be obtained by individual groups working in partnership, and leads on to the sequencing programme.

3.1.2. Sequencing

The consortium strongly recommended that a start be made as soon as possible on sequencing complete BACs (80-125 kb each) so that some information about the structure of large genomic tracts becomes available. Already, considerable information about other genomes is coming from such a strategy, and the results will feed into the complete and systematic Musa genomics project.

3.1.3. cDNA characterisation

EST sequencing - the sequencing of the ends of expressed genes cloned as cDNA - has been used in many species, but new information is showing these data are not as valuable as full-length cDNA sequences. The data is, however, useful to search for genes, so it was concluded that a framework with relatively low coverage of the genome would be useful, which would be integrated with the BAC sequencing data.

3.1.4. Functional genomics/transcriptomics

Leading on from the cDNA characterisation, the consortium felt that production of a cDNA microarray would be very valuable and should be given high priority, so that the characterisation of gene expression profiles could begin quickly. Once available, the microarray would be exploited to reveal genes involved in disease resistance, in the first instance. These would then be sequenced, and genomic clones obtained from the BAC libraries.

3.1.5. Mutagenesis

The consortium recognised that approaches to gene discovery and functional analysis through mutation analysis (both insertion and deletion) are important components of the consortium. A detailed proposal for the numbers of plants and types of neutron bombardment felt to be optimum was also prepared.

3.1.6. Bioinformatics

To ensure early availability of data from genomics, and exploitation of comparative data from other species, the consortium aimed to set up a common central resource as the entry point for the data. The PROMUSA website www.promusa.org could host a data repository. Specific modules are needed to host specific tasks that could be combined within and between others; several participants have major international roles in genomic databases. To ensure maximum exploitation of sequence data, state-of-the-art bioinformatics techniques will be applied and value added rapidly to all generated sequences by knowledge transfer from other species. To this end, major plant bioinformatics centres, e.g. The Institute for Genomic Research (TIGR), USA, and the Munich Information Center for Protein Sequences (MIPS), Germany have been involved in the consortium from the earliest stage, and will also contribute their experience in genomic database development. It was suggested that early work should consider database development and its nomenclature.

3.2. Musa as a model for genomics

So far, Arabidopsis and rice have been used as model species in plant genomics because of their small genome size and ease of handling, but both species have their limitations. Complementing these two species, the Musa genome provides a powerful platform for gaining a fundamental insight into the genomes of other species. Both in structural genomics and in functional genomics, Musa can be a model for several fundamental aspects for which the other two model species cannot be used.

There is a very good knowledge of the structure of the Musa species complex based on morphological descriptors, as well as molecular markers for the chloroplast, mitochondrial and nuclear genomes. Studies have revealed great diversity in Musa, providing a good model for the study of gene regulation. Musa also offers an interesting model for genetic studies, as it is one of the few plant species with biparental cytoplasmic inheritance: paternal inheritance of mitochondria and maternal inheritance of chloroplasts [5]. Moreover, as a vegetatively propagated crop, it offers a good model to study the role of somaclonal variation and phenomena such as 'imprinting'.

Because of its relatively small size - a haploid genome of 500-600 Mbp (only 25% larger than rice) divided among 11 chromosomes - the Musa genome is highly tractable to complete functional and sequence analysis, and extensive characterisation of the genes will be possible using realistic high-throughput technologies.

In the centre of origin and diversity in Southeast Asia there are many sterile banana clones that have been genomically static or fixed for thousands of years by vegetative propagation in the same environment. There are also partially fertile and highly fertile wild diploid equivalents that have been actively evolving for the same period in the same environment, making Musa one of the most perfect models to study plant evolution at a genomic level. The combination of parthenocarpy and sterility that has led to the typical edible banana is of basic interest in a wide range of other fruit crops, and is especially unusual as there are relatively few parthenocarpic monocots.

Furthermore, in Asia, bananas have co-evolved for the same period and in the same environment with most of the Musa pathogens. Both of these co-evolutions reinforce the position of Musa as a perfect model to study plant and pathogen evolution at a genomic level. In contrast, about 3000 years ago, a few plantain varieties were introduced from the centre of origin into Africa, where they underwent secondary diversification exclusively through mutations, in the absence of most of their pathogens. This also provides a valuable tool for the study of evolution in the presence and absence of pathogens.

The variability in the different ploidy levels in Musa offers a very special opportunity to gain insight into the greater-than-additive gains in crop productivity that often accompany polyploidy. Even the most well studied polyploids like cotton and sugarcane contain only one 'type' of polyploidy within the taxon, whereas Musa includes a number of autopolyploids (AAA, AAAA, AAAAAA), and different types of allopolyploid (allotriploids AAB, ABB, and allotetraploids AABB, AAAB) in addition to the diploid M. acuminata (AA) and M. balbisiana (BB) and AB hybrids. Bananas and plantains are attractive models to study the role of hybridisation and polyploidy in the evolution of cultivated crops, and to analyse the interaction of parental genomes at the chromosomal and sequence levels. Being a monocotyledon but taxonomically very distantly related to rice, Musa is an ideal candidate for studying synteny between distantly related species.

Finally, Musa was the first species where a pararetrovirus was shown to be integrated in the plant genome with the capacity to give rise to episomal banana streak badnavirus. Understanding the mechanism behind this phenomenon may lead to important applications, such as gene targeting.

The details of the state of the art and the available tools for Musa genomics are given in the strategy document [4].

4. CONCLUSION

We are convinced that genomics research will provide a major boost to genetic improvement efforts in banana. By creating the Global Musa Genomics Consortium, we are creating an ideal environment for rapid progress despite limited resources. Furthermore, the establishment of the consortium and the early results obtained by it will attract other research teams, not necessarily interested in Musa improvement, but interested in the Musa genome as a model for critical aspects of genomics research. This will in turn strengthen and speed up the overall effort for the benefit of banana growers and consumers worldwide.

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