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Practical guidelines for the early screening and field evaluation of banana (Musa spp.) for resistance to three major traits: Fusarium wilt (Fusarium oxysporum f. sp. cubense), leaf spot diseases (Pseudocercospora spp.) and drought. The guidelines have been produced by experts from the Evaluation Thematic Group of MusaNet, the global network for Musa genetic resources (www.musanet.org) coordinated by the Alliance of Bioversity and CIAT.

Summary Chromosome rearrangements and the way that they impact genetic differentiation and speciation have long raised questions from evolutionary biologists. They are also a major concern for breeders because of their bearing on chromosome recombination. Banana is a major crop that derives from inter(sub)specific hybridizations between various once geographically isolated Musa species and subspecies. We sequenced 155 accessions, including banana cultivars and representatives of Musa diversity, and genotyped‐by‐sequencing 1059 individuals from 11 progenies. We precisely characterized six large reciprocal translocations and showed that they emerged in different (sub)species of Musa acuminata, the main contributor to currently cultivated bananas. Most diploid and triploid cultivars analyzed were structurally heterozygous for 1 to 4 M. acuminata translocations, highlighting their complex origin. We showed that all translocations induced a recombination reduction of variable intensity and extent depending on the translocations, involving only the breakpoint regions, a chromosome arm, or an entire chromosome. The translocated chromosomes were found preferentially transmitted in many cases. We explore and discuss the possible mechanisms involved in this preferential transmission and its impact on translocation colonization., Significance Statement Chromosome rearrangements, the way that they emerged and their potential impact on speciation and breeding raise important questions. We report on the characterization of several large reciprocal translocations in banana, Musa acuminata, identify the genetic group in which they emerged, characterize their impacts on chromosome recombination, and explore possible mechanisms allowing their colonization.

ost banana cultivars are triploid derived from Musa acuminata (2n=2x=22), sometimes combined with Musa balbisiana (2n=2x=22). These species and subspecies diverged following geographical isolation in distinct Southeast Asian continental regions and islands. Contact between them was made possible by human migration and led to the selection of seedless parthenocarpic hybrids. M. acuminata subspecies were suggested to differ by a few large chromosomal rearrangements based on chromosome pairing configurations in inter-subspecies hybrids. The precise nature of these rearrangements, their distribution in the diversity and their consequence on chromosomal segregation in structural heterozygote remained to be documented. 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 Mb and 10 Mb segments from chromosomes 01 and 04, respectively, and showed that it locally generated high segregation distortions and reduced recombinations 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 within the wild accessions, it was only found within malaccensis sub-species accessions, thus suggesting that this rearrangement occurred in this sub-species. We propose mechanisms for the spread of this rearrangement in Musa diversity and propose that this structure may have played a role in the emergence of triploid cultivars. Knowledge on these structures and their impact on chromosomal recombination and segregation will now allow to take them into account for the genetic analysis of traits of agronomic interest (QTL, GWAS) and for the choice of crosses in breeding programs. (Texte intégral)

Bananas cultivars are triploid or diploid seedless parthenocarpic clones derived from the hybridization between Musa acuminata subspecies and, for some of them, with the species M. balbisiana. Based on chromosome pairing configuration in hybrids, M. acuminata subspecies were reported to differ by a few large chromosomal structural rearrangements which may have occurred since their divergence following geographical isolation. We analyzed with DArTseq, chromosome segregation in the progenies of a seedy M. acuminata banana accession and discovered strong segregation distortion, reduced recombination and abnormal linkage in two regions of references chromosomes 1 and 4. Pair-end resequencing of the parental accession and comparison to the Musa acuminata references sequence, showed evidence of a heterozygous reciprocal translocation involving two distal segments of 3 Mb and 10 Mb from chromosome 1 and 4. This translocation was then confirmed through BAC-FISH and PCR experiments. The two chromosome structures were found mutually exclusives in gametes and the rearranged structure was transmitted preferentially to the progeny. Analysis of the distribution of this distinct chromosome structures in Musa diversity allowed to make hypotheses about its origin. (Texte intégral)

Detailed description of tools and processes used to improve the Musa acuminata reference sequence and additional figures. (PDF 1604 kb)

Since the release of the Banana (Musa acuminataDH Pahang) sequence in July 2012, significant progresses were made to improve the quality of this reference sequence as well as the access to this information. This presentation intends to give an update on the status of the sequence assembly and gene annotation as well as the information systems that handle those data. An improved version of the assembly was produced (Martin et al in Prep.). The total scaffold number was reduced of 79% (7513 vs 1532), with a N50 that increased from 1.3Mb (65 scaffolds) to 3.0 Mb (26 scaffolds). Unknown sites (N) were reduced from 17.3% to 10.0%. Finally 89.5% of assembly was anchored to the 11 chromosomes compared to the previous 70%. The unanchored sequences represent now 46.5 Mb compare to 140.4 Mb before. For the gene annotation, a new release of annotation is in progress that have been mapped on the assembly v2 and will take advantage of improvements coming from manual curation (~ 2,000 genes out of the 36,542) and NCBI RefSeq released in October 2014. Finally, an overview of the new version of the Banana Genome Hub (Droc et al, 2013) will be presented. It includes additional features like JBrowse, a chromosome Browser and is being linked to the resources genetics being genotyped by Next Generation Genotyping and accessible via the Musa Germplasm Information System (MGIS). (Texte intégral)

Structural heterozygosity is thought to be one of the main causes of sterility in banana (Musa spp.) hybrids. These structural variations impact chromosomal segregation and recombination, limiting crossing possibilities and complicating genetic analyses. It is thus important to better understand their nature and location in the genome. For the production of the Musa accuminata reference genome sequence (D'Hont et al., Nature, 2012), we generated a genetic map that allowed anchoring 70% of the genome assembly on the 11 Musa chromosomes. The genetic map was based on a self progeny of the wild diploid Musa acuminata 'Pahang', the parent of the sequenced doubled haploid DH-Pahang. Strongly distorted markers were found on linkage group 1 and part of linkage group 4 that might indicate a structural rearrangement affecting these two linkage groups. We have developed a bioinformatic pipeline to help characterize structural variation based on resequencing approaches. We are using this pipeline on resequencing data of DH-Pahang and its parent and are densifying the genetic map with DArTseq markers to better understand the causes of the strong markers distortions and their link with structural variation.

Mycosphaerella fijiensis is a hemibiotrophic ascomycete causing the black leaf streak disease (BLSD) of banana that is commonly called Black Sigatoka disease. BLSD is regarded as the most economically important threat to banana production and affects almost all edible banana cultivars grown worldwide. The M. fijiensis infection cycle goes through a long symptomless phase in the plant, during which the fungus grows in the mesophyll intercellular space, before the appearance of disease symptoms in the form of necrotic areas. Despite the economical importance of the BLSD disease, little is known about the physiological and molecular events occurring during the pathogen life cycle in the plant and on the genetic bases of resistance. To better characterize the interaction, we first established a bioassay based on detached leaves maintained in vitro, associated to phenotyping with an image analysis software package (Visilog® Noesis). This allows higher throughput phenotyping and more precise monitoring of the reaction of banana accessions to M. fijiensis strains. In a preliminary experiment, we also analysed transcriptome changes in three accessions showing contrasted reactions to M. fijiensis, using RNA-Seq. First results of this analysis will be presented. (Texte intégral)

Mycosphaerella fijiensis is a hemibiotrophic ascomycete causing the black leaf streak disease (BLSD) of banana that is commonly called Black Sigatoka disease. After a period of epiphytic growth on the leaf surface, fungal hyphae penetrate the leaf through the stomata. The fungus grows then in the mesophyll intercellular space, without penetrating leaf cells. This biotrophic phase can last for a few weeks before the appearance of the first symptoms of the necrotic phase. Despite the economical importance of the BLSD disease, little is known on the physiological events occurring during the pathogen life cycle in the plant . To learn more about these physiological events, we established a bioassay based on detached leaves maintained in vitro, associated to phenotyping with an image analysis software package (Visilog R°Noesis) that allows monitoring of the reaction of banana cultivars to M. fijiensis strains. In a preliminary experiment, we also analysed transcriptome changes in three accessions showing contrasted reactions to M. fijiensis, using gene expression profiling by RNASeq. First results of this analysis will be presented.

Tropical race 4 (^R4) of Fusarium oxysporum f. sp. cubense (Foc) presents a major concern for current banana production worldwide. Two strategies are critical for controlling this pathogen: a) preventing its spread to TR4-free areas and b) identifying resistant banana genotypes. The team has established and validated a reliable and rapid greenhouse bioassay, and used it to characterize ^R4 resistance in eight banana genotypes [Gros Michel (AAA); 'Silk (AAB), 'Prats (AAB), 'Pahang (AA, Musa acuminata ssp. malaccensis), 'CIRAD 930 (AA, DH Pahang), 'Matavi'a (ABB), 'Banksii (AA) and 'Tuu Gia (AA)], where 'Grande Naine (AAA) was used as a susceptible control. All genotypes were highly susceptible, but 'CIRAD 930 and 'Pahang showed partial resistance, evidenced by highly reduced rhizome discoloration. Subsequently, we evaluated a segregating population of 'Pahang (N=80) and observed three phenotypic groups a) susceptible, b) moderately resistant and c) resistant. We concluded that the bioassay is a reliable method to screen banana germplasm for ^R4 resistance under greenhouse conditions and that 'Pahang is an important source of resistance to Foc ^R4 that should be further explored.

Banana streak viruses (BSVs) are double-stranded DNA pararetroviruses causing banana streak disease. Recently, numerous outbreaks of the disease occurred in many banana-producing areas in interspecific hybrids (#Musa acuminata# × #Musa balbisiana#) originating from virus-free parents. These infections correlated with the presence of endogenous banana streak viruses (eBSVs), viral DNA sequences integrated in the #M. balbisiana# genome only. Although integration is not needed for the viral replication cycle, some viral integrants are infectious under stress conditions by reconstituting a replication-competent genome after possible homologous recombination events. Even though the wild #M. balbisiana# Pisang Klutuk Wulung (PKW) harbours infectious eBSVs, it is resistant to BSVs. We characterised the genetic and genomic endogenous viral organisation of three BSV species in PKW in order to determine the species responsible for the viral expression in the interspecific F1 progeny.

The diversity of the banana complex can be deciphered only by jointly characterizing the original wild species and their relatives, the primitive diploid forms and the triploid varieties. Sexuality, the primary source of diversity, is strongly disrupted in the cultivated varieties (sterility, parthenocarpy and vegetative propagation) and is relayed by human selection of punctuated mutations vegetatively maintained. Many biological tools illustrate peculiar facets of the diversity and their joint analysis enables an evolutionary reading of this diversity. The access to forms resulting from ancient events and vegetatively maintained is a valuable asset. We propose various assumptions in the structure of wild species, on the domestication of the edible diploids from hybrids between wild forms, on the direct ancestry of triploids from cultivated diploids, and on the ancient migrations. The comparison with data from archaeology, linguistics and human genetics will enable the validation, refinement and dating of the proposed domestication process. (Texte intégral)

We report a reference map of Musa acuminata from 180 individuals of BORLI population. This F1 population derived from the cross between M.a. "Borneo" x M.a. "Pisang Lilin" which was chosen for mapping mainly because of the great heterozygosity of both parents (76 % each). For map construction we genotyped 180 SSR markers on 180 individuals, 12 RGA-RFLP probes on 112 individuals and 380 DArT markers on 92 individuals. Using the software Joinmap 4 at LOD 5, Kosambi calculation distance and the regression mapping algorithm, we defined 11 main linkage groups, as expected, on Borneo parent and 10 on Pisang lilin due to one translocation event in the male parent. Despite structural differences between the parents, a consensus map was built. In order to check genotyping results and to analyse structural rearrangements, we developed an original method using Neighbor Joining algorithm implemented in the genetic diversity software Darwin5. It allows visualizing graphically disruptions in alignments arising from these structural rearrangements. (Texte intégral)

Musa has been the subject of diverse studies at CIRAD since the mid 1980s with the advent of biochemical and molecular markers. In the context of the Generation Challenge Programme, these have been pursued with heightened intensity through a network of international interactions. Here we report surveys of SSR markers for germplasm diversity analysis and genome mapping. A set of 500 accessions chosen to represent the species was characterized with 12 SSRs. Among them, 182 were also genotyped with DArT markers (see E. Huttner et al., speaker in this workshop). This contributes to a comprehensive understanding of Musa diversity. Simultaneously, the progeny of a cross between two heterozygous parents (Borneo x Pisang liling) are being genotyped using 150 SSR markers; the preliminary map will be presented. More recently, the production of two BAC libraries has been undertaken using a doubled haploid regenerated from anther culture of the clone Pahang. Altogether, these genomic resources will contribute to the development of a coordinated initiative of fine genome analysis to support Musa breeding. (Texte intégral)

As several other plants, the genome of banana and plantain contains integrations of Banana streak virus (BSV) sequences even though integration is not an essential step in the replication cycle of this virus. In banana two types of BSV integrants exist. Ones are non functional sequences present in both common Musa species, Musa acuminata (denoted A) and Musa balbisiana (denoted B) and it is now assumed that the integrants of the other type, containing the complete viral genome and restricted to M. balbisiana genome, become infectious by reconstituting a complete replication-competent viral genome. Thereby, an increasing record of BSV outbreaks was observed fifteen years ago among banana breeding lines and micro propagated inter-specific Musa hybrids, worldwide. Today, three widespread BSV species, Banana streak Obino l'Ewai virus (BSOIV), Banana streak Imové virus (BSImV) and Banana streak Golfinger virus (BSGfV) are known to occur as infectious integrants in the M. balbisiana genome. However, even though such integrations are known to be infectious, their presence is not sufficient to induce infection. We demonstrated that the process of genetic hybridization and abiotic stresses such as micropropagation by in vitro culture contributed in triggering episomal expression from EPRVs. Two mechanisms at least are involved in the BSV expression: the ploidy of the M. balbisiana in Musa genotypes and an additional genetic factor called BEL for BSV expressed locus concerning the triploids (Musa AAB) resulting from inter-species genetic crosses between virus-free diploid M. balbisiana (BB) and tetraploid M acuminata (AAAA) parents. Then, diploids M. balbisiana such as PKW and Pisang Batu harboring pathogenic BSV EPRVs are resistant to any multiplication of BSV while haploid genotypes such as triploids (AAB, French clair) or tetraploids (AAAB, FHIA 21) expressed BSV. Thereby, we characterized the segregation of three BSV species appearance among the AAB F1 progeny as a monogenic allelic system conferring the role of carrier to the M. balbisiana diploid parent. BSOIV and BSImV appeared in almost all infected hybrids (50% of the progeny) depending of BEL regulation while BSGfV are restricted in only half of these hybrids and subordinated by BEL. Three BAC libraries from accession of M.acuminata Cavendish subgroup cv petite naine (AAA), a wild M. acuminata subsp burmannicoides Calcutta 4 (AA) and a wild M. balbisiana PKW (BB) are explored for the pattern of integration of infectious BSV EPRVs by testing a set of different viral probes representing each time the BSOIV, lm and Gf complete genome. BSV positive BAC clones are characterised by RFLP fingerprints approaches. This analysis revealed that the three BSV species represent low-copy loci and their integration is specific to the PKW Musa balbisiana genome. BSGfV EPRVs in PKW is twice and are fully annotated after sequencing. Each BSGfV is composed of back-to-back viral sequences representing more than a whole BSV genome very similar each other. We developed molecular markers (PCR, PCR-RFLP) to distinguish each others and analysed the BSGfV EPRV segregation in the AAB F1 progeny. BSGfV EPRVs are found to be allelic, located at the same locus. In theory, both allelic EPRVs could be involved in the restitution of virions through a set of recombination events. (Texte intégral)

Chromosome pairing studies at meiosis have revealed that translocations are frequent in banana genomes. In Musa acuminata, seven groups of translocation have been identified (Standard, North Malaysia, Malaysia Mountains, North A, North B, Indonesia, and East Africa). Each group comprises accessions having the same chromosome structure. Translocations increase the difficulty of constructing a genetic map, studying the inheritance of agronomical characters and breeding in general. The objectives of our study were to develop a tool to characterize translocations on chromosomes based on fluorescent in situ hybridization of BAC clones (BAC-FISH) and to use this tool to characterize the translocations in the accessions 'Calcutta 4' (2n=2x=22, translocation group North A) and 'Madang' (2n=2x=22, translocation group Standard). To achieve this, a genetic map and a 'Calcutta 4' BAC library were built, the BAC-FISH technology was adapted to bananas and a banana cytogenetic map was initiated. The genetic map was based on a F2 population from a 'Calcutta 4' x 'Madang' cross. It encompasses 120 markers (20 RFLPs, 81 AFLPs and 19 SSR markers) distributed over 14 linkage groups and covering 597 cM. The 'Calcutta 4' BAC library comprises 55 152 clones with an average insert size of 100 Kb. About 1.5% of these clones present chloroplast and mitochondrial DNA inserts. This library covers 9 to 10 times the banana genome. Linkage group Il (LGII) was chosen to search for translocations since some of its characteristic (such as a high number of distorted markers) suggested that this group contained chromosomes with translocations. Four BAC clones corresponding to 3 RFLPs and 1 SSR loci distributed on LG II were localized on the 'Calcutta 4' and 'Madang' chromosomes. The results showed that the markers involved in the LG II were localized on three chromosomes, whose structure was different in 'Calcutta 4' and 'Madang', with two linked translocations observed in 'Calcutta 4'. In 'Madang', the markers mMaCIR161-rMaCIR 560, rMaCIR 1125 and rMaCIR 36 were localized on three chromosomes (A, B and C). In 'Calcutta 4' these markers were localized on only two chromosomes (A and B). The marker rMaCIR 1125 localized on the 'Madang' C chromosome, was localized in the proximal position of the 'Calcutta 4' B chromosome. We also initiated a global cytogenetic map of 'Calcutta 4' that comprises 16 loci (14 BAC clones selected by RFLP and SSR markers and the ribosome probes 45S and 5S). The 14 linkage groups of the 'Calcutta 4' x 'Madang' genetic map are anchored to this cytogenetic map. (Texte intégral)

Le complexe d'espèces des bananiers présente des originalités qui entraînent une vision particulière de la gestion de ses ressources génétiques. La multiplication clonale, associée à de fréquentes stérilités ou incompatibilités, et la coexistence de plusieurs niveaux de ploïdie rendent indispensables une connaissance approfondie des potentialités de chaque clone et des relations de phylogénie entre les différents types reconnus, pour exploiter les ressources en amélioration variétale. Les principaux programmes d'amélioration des bananiers à travers le monde possèdent des collections #in situ#. Au cours des vingt dernières années, de nombreux marqueurs de la diversité ont été mis successivement au point et appliqués au bananier. Un ensemble de 119 descripteurs agromorphotaxonomiques a été défini comme norme de description des bananiers. Des outils informatiques d'aide à l'identification fondés sur ces descripteurs ont été développés. Depuis dix ans, différents types de marqueurs moléculaires ont été utilisés sur le bananier : polyphénols, isoenzymes, RFLP et microsatellites. D'autres outils telles la cytométrie en flux et l'hybridation #in situ# ont également contribué à la caractérisation des ressources génétiques en permettant une détermination plus fine de la nature génomique des accessions étudiées : ploïdie, aneuploïdie et nombre de chromosomes affiliés à chacune des espèces d'origine. Les résultats les plus complets concernent l'étude des génomes cytoplasmiques et nucléaire à l'aide des marqueurs RFLP. Les deux génomes cytoplasmiques se caractérisent par une hérédité monoparentale. Ces apports successifs ont permis de structurer les ressources génétiques des bananiers et d'en affiner la caractérisation. Ce chapitre présente une synthèse de ces travaux

Ces dernières années des hybrides interspécifiques bananiers ont développé la maladie de la mosaïque en tirets, sans possibilité de contamination externe par le Banana streak badnavirus-BSV responsable. L'explication proposée fait l'hypothèse que les virions observés proviennent de séquences virales endogènes - EPRVs BSV, présentes dans le génome bananier. L'objectif de notre étude a été de tenter de trouver une explication à l'expression de la maladie au cours de l'hybridation conventionnelle pour deux croisements #Musa# interspécifiques - PKW (BB) x IDN 110 4x (AAAA) et P. batu (BB) x P. pipit 4x (AAAA). Nous avons tout d'abord montré l'existence d'une ségrégation à caractère mendélien de la maladie sur les descendances F1 et révélé la présence du BSV dans 50% des plants. L'étude de l'EPRV BSV souche Obino l'Ewaï (BSV-O1) a montré sa présence à l'état homozygote dans le génome #M. balbisiana# uniquement. Une carte génétique du locus responsable de l'expression de la maladie -BEL (BSV expressed locus) a été proposée sur la base de l'analyse de 10 marqueurs AFLP de l'expression ou non de la maladie, tous localisés chez les parents #M. balbisiana#. Les analyses génétiques révèlent chez le parent #M. balbisiana#, la présence d'un système allélique monogénique lui conférant le rôle de porteur sain. La régulation des EPRVs a été abordée au travers de l'analyse d'apparition des trois souches BSV présentes dans les hybrides malades: la souche BSV-O1 dans 98% des cas, BSV-Im dans 88% et BSVGF uniquement dans la moitié d'entre eux. Les EPRVs correspondantes sont dans les génomes #M. balbisiana# et dans la population hybride, suggérant leur caractère pathogène. L'analyse génétique a montré que les souches BSV-O1 et BSV-Im sont génétiquement liées et dépendent du facteur génétique BEL. La souche BSV-GF apparaît non liée génétiquement au locus BEL mais reste sous son contrôle. L'existence d'au moins trois EPRVs pathogènes dépendantes du facteur BEL implique que la nature de ce dernier est différente de séquences virales. Plus généralement des éléments utiles à la compréhension de ce mécanisme de régulation des EPRVs ont été recherchés en explorant le comportement "sain" du parent femelle #M. balbisiana#. Ce dernier présente une résistance tant à l'expression des EPRVs qu'à celle des souches BSV lors d'inoculations extérieures. (Texte intégral)

Recently introduced inter-specific #Musa# hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with several Banana streak badnavirus (BSV-Obino I'Ewai (BSV-OI), BSV-Imové (BSV-Im) and BSV-GoId Finger (BSV-Gf) strains), the causal agent of banana streak disease (BSD). One hypothesis suggests that BSD occurrence in these inter-specific hybrids results from activation of corresponding BSV endogenous pararetrovirus sequences (EPRVs) integrated into the Musa genome rather than from external sources of infection. The process of genetic hybridisation and abiotic stress such as micropropagation by #in vitro# culture may be responsible in triggering episomal expression of these BSV integrants. In order to test the first hypothesis we carried out a genetic analysis of banana streak disease incidence in a F1 triploid (AAB) population produced by hybridisation between virus- and disease-free #M. balbisiana# Pisang Klutug Wulung PKW (BB diploid) and #M. acuminata# IDN 110T (AAAA tetraploid) parents. Molecular AFLP markers segregating with the presence or absence of episomal viral particles were identified in the #M. balbisiana# genome only. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the #M. balbisiana# parent The three different strains were detected by IC-PCR in diseased hybrids. The BSV-OI and BSV-Im strains appeared in almost all diseased hybrids while the BSV-Gf strain appeared only in about half of these hybrids. Genetic analysis resulting from AFLP results showed that BSV-OI EPRVs' regulation depends on a genetic factor: BEL (BSV Expressed Locus) (Lheureux et al. 2003). Further experiments showed that BSV-lm EPRVs' regulation depends on the same genetic factor too. Even if BSV-Gf appears only in half of diseased hybrids, some elements suggest that BSV-Gf strain appearance and the activation of its corresponding EPRV(s) seem to be subordinated to the BEL factor. Three BAC libraries from cv Cavendish (AAA), cv Calcutta 4 (AA) and PKW (BB) were constructed in order to studies genome structure and evolution of #Musa# species. We have explored the pattern of integration for the different BSV EPRV suspected infectious by testing a set of different probes representing each time the BSV-OI, Im and Gf complete viral genome. This analysis revealed that the three EPRVs under analysis represent low-copy loci and their integration is specific to the PKW #Musa balbisiana# genome. These studies provide preliminary results towards characterisation of infectious BSV EPRV and definition of mechanism under laying their potential activation or nonactivation upon stress conditions in banana. (Texte intégral)

Banana mild mosaic virus (BanMMV) is currently one of the major viral constraints for Musa germplasm exchanges, since the virus is propagated vegetatively. Current data show that synergistic infections with banana streak virus (BSV) or cucumber mosaic virus (BSV) can lead to severe necrotic symptoms, although single infections by BanMMV cause no or only very mild symptoms. Very few data are currently available on the epidemiology of BanMMV. In order to gain insights into it, we have undertaken studies on BanMMV molecular variability. An IC-RTPCR based detection method has been set up. Amplification products, located within the RdRp gene, have been cloned and sequenced for over 35 different accessions originating from CIRAD-Guadeloupe's Musa collection, with at least 3 clones sequenced for each accession. Sequence comparisons show that an important molecular variation exists between isolates originating from distinct accessions. On the other hand, sequences originating from a given accession are generally very conserved. Our results tend to indicate that BanMMV could be preferentially but not exclusively transmitted vegetatively. (Texte intégral)

Inter-specific #Musa# hybrids have been found to be infected with several Banana streak pararetrovirus strains (BSV-Obino l'Ewai (BSV-Ol), BSV-Imové (BSV-Im) and BSV-Gold Finger (BSV-GF) strains) resulting from activation of BSV endogenous pararetrovirus sequences (EPRVs) integrated into the #Musa# genome rather than from external sources of infection. The process of genetic hybridisation may be responsible in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid (AAB) population produced by hybridisation between virus- and disease-free #M. balbisiana# (BB) and #M. acuminata# (AAAA) parents. Molecular AFLP markers were identified in the #M. balbisiana# genome only. Analysis of the segregation of these markers allowed the construction of a genetic map containing the locus associated with BSV infection called BSV Express locus (BEL). The BSV-Ol and BSV-Im strains appeared in almost all diseased hybrids while the BSV-GF strain appeared only in about half of these hybrids. Genetic analysis resulting from AFLP results showed that BSV-Ol and BSV-Im EPRVs' expression depends on the same genetic factor, the BEL locus. Although the BSV-GF is not genetically linked to the BEL locus, its appearance and the activation of corresponding EPRV(s) seem to be also subordinated to the BEL locus. So, the discovery of several BSV EPRVs linked to BEL locus implicates that BEL differs in nature from BSV EPRVs.

Inter-specific #Musa# hybrids have been found to be infected with Banana streak pararetrovirus Obino I'Ewai strain (BSV-Ol) responsible of Banana Streak Disease (BSD). One hypothesis suggest (i) that BSD occurrence in these hybrids results from activation of BSV-Ol endogenous pararetrovirus sequences (EPRVs) integrated into the #Musa# genome rather than from external sources of infection and (ii) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid (AAB) population produced by hybridisation between virus- and disease-free #M. balbisiana# (BB) and #M. acuminata# (AAAA) parents. Half of the F1 progeny expressed BSV particles. Ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the #M. balbisiana# genome only. Analysis of the segregation of these markers allowed the construction of a genetic map containing the focus associated with BSV infection called BSV Express Locus (BEL). A genetic mechanism is involved in BSV appearance, conferring the role of carrier to the #M. balbisiana# parent. Two previously unidentified BSV strains in addition to BSV-Ol, have been furthermore detected in these diseased hybrids: BSV-Imové (BSV-Im) and BSV-Gold Finger (BSV-GF) strains. The BSV-Ol and BSV-Im strains appeared in almost all diseased hybrids while the BSV-GF strain appeared only in about half of these hybrids. The close relationships between the presence of these two new BSV strains and the presence of corresponding EPRVs in the #M. balbisiana# parent genome shows that the #M. balbisiana# genome contains at least two other pathogenic BSV EPRVs: BSV-Im and BSV-GF EPRVs. Genetic analysis resulting from AFLP results showed that BSV-0l and BSV-Im EPRVs' expression depends on the same genetic factor, the BEL locus. Although the BSV-GF is not genetically linked to the BEL locus, the BSV-GF strain appearance and the activation of corresponding EPRV(s) seem to be also subordinated to the BEL locus. So, the discovery of new BSV EPRVs linked to BEL locus implicates that BEL differs in nature from BSV EPRVs. Our interest is now the understanding of (i) the mechanism of activation of these strains resulting in BSV expression from EPRVs and (ii) the apparent resistance of the #M. balbisiana# parent against BSV expression and external infection.

L'opposition entre la richesse de la diversité génétique naturelle et la pauvreté de la diversité exploitée en agriculture entraine des risques. Les risques pour la culture se traduisent par: - Une perte de compétitivité économique de la filière vis-à-vis du marché international et de la qualité du produit vis-à-vis de l'attente du consommateur. - Une perte de compétitivité vis-à-vis de l'environnement. Risque phytosanitaire (introduction de nouvelles maladies, absence de sources naturelles de résistance, évolution des populations pathogènes). - Pollution du milieu. Les risques pour l'environnement dus à la mauvaise utilisation d'irritants: - Apparition de résistances chez les parasites. - Parmi les outils disponibles pour contrer ces phénomènes, la création variétale joue un rôle prépondérant.

Les bananes comestibles sont issues pour l'essentiel de deux espèces sauvages diploïdes : #Musa acuminata# et #Musa balbisiana#. Il convient de distinguer les productions destinées au marché d'exportation de celles qui s'adressent aux marchés intérieurs. Les programmes d'amélioration génétique ont pour objectif la création de variétés résistantes aux maladies et aux ravageurs. La stérilité des bananiers cultivés oblige à la création de variabilité par voie sexuée (hybrides tétraploïdes et triploïdes). D'autres méthodes sont également utilisées : la mutagenèse, la variation somaclonale et la transformation génétique.

La maladie de la mosaïque en tirets des bananiers BSD frappe aujourd'hui la majorité des zones productrices de bananes. Le virus responsable BSV est un badnavirus. Ces dernières années, la véritable contrainte a résidé dans l'association étroite de l'apparition de la maladie avec l'obtention et la diffusion de nouvelles variétés de bananiers. Des cultivars sains multipliés par CIV ou des géniteurs utilisés lors de croisements génétiques restituent soit des vitroplants soit des hybrides contaminés. Un pourcentage non expliqué de plants BSD voisin de 50% a été observé dans les descendances d'hybridation de géniteurs sains. Ceci a été interprété comme le résultat d'une ségrégation à caractère mendélien de l'expression de la maladie. Au moins 4 souches BSV (Mys, Wu, Gf et Im) existent sous diverses formes : libres comme particule épisomale infectieuse, ou intégrées sous forme de séquences partielles ou totales dans le génome B. Le modèle actuel BSV-Wu suggère une double recombinaison conduisant à l'excision des deux fragments internes et à la circularisation aboutissant à la forme superenroulée transcriptionnellement active. L'analyse du motif activable n'a pas montré d'homologie avec les rétrotransposons pour leurs éléments majeurs. Afin d'identifier et d'analyser les mécanismes moléculaires induisant la multiplication de la souche BSV-Wu au cours de l'amélioration conventionnelle, nous avons tenté de définir un marqueur génétique viral utilisable par les généticiens pour identifier les bananiers sains. Des marqueurs moléculaires liés à la présence ou à l'absence de virus épisomal ont été recherchés par Bulk segregant analysis et par AFLP dans la F1 de deux croisements interspécifiques (IDN110 4A x PKW 2B). 11 marqueurs polymorphes relies au parent B ont été isolés : 7 ségrègent avec le facteur déclenchant et sont corrélés à l'expression du virus épisomal, 4 avec le phénotype 'absence de virions'. Leur analyse a permis d'établir une carte génétique du locus impliqué dans l'expression du virus. Le marqueur de la maladie le plus proche est à OcM du facteur déclenchant. L'analyse de sa séquence révèle une homologie de 86% avec le rétrotransposon 'Monkey' identifié chez #Musa Acuminata#. Ce pathosystème constitue du fait de la présence simultané de séquences intégrées et d'un système de réplication du virus, un candidat idéal pour une hypothèse forte de gene silencing.