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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)

International audience; 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.

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.

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.

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)

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)

Genetic information of plant cytoplasmic DNA presents a great interest for phylogenic studies. Recently, in Citrus, a new interest for cytoplasmic genome analysis has resulted from the impediment of somatic hybridization. Indeed this technique allows new patterns of nuclear, chloroplastic, and mitochondrial genomes that must be characterized. Cytoplasmic genome analysis among Citrus and related genera was generally done by Southern blots. This method is powerful but not applicable to in vitro plants because it requires too much fresh material. A few years ago we published a preliminary work that demonstrated the successful amplification of Citrus cytoplasmic DNA by using mitochondrial and chloroplastic universal primers and the possibility in some cases to display polymorphism by combining these amplifications with an enzymatic restriction. In the present work, the usefulness of such CAPS analysis, for cytoplasmic genome differentiation at the infra-generic level (Citrus) and intergeneric level inside of the Aurantioideae sub family, was studied on 44 genotypes from 31 species. With the combinations of primers and enzymes tested, the CAPS Technique appears more powerful for chloroplast than for mitochondria to display polymorphisms, even at the infra-generic level. Phylogenic inference are made from a cluster analysis of chloroplastic data obtained with 4 primers coupled each with 2 to 4 restriction enzymes. An example of application for somatic hybrid cytoplasmic genome characterization is presented.

Cultivated bananas have first been susPected to originate from four different wild species : Musa acuminata (A), M. balbisiana (B), M. schizocarpa (S) and the Australimusa (T) species. Banana germplasm includes di, tri and tetraploid clones. Cultivated clones have been classified in genomic groups by Simmonds and Shepherd according to their chromosome number and morphological traits; the main groups are AA, AAA, AAB, ABB, AS and AAT. Biotechnology now allows a precise and early characterisation of the genomic nature of the accessions. Flow cytometry allows a rapid determination of ploidy level of a clone and in some extend of its genomic group. Analysis of the nuclear genome using only 8 STMS allows the classification of an accession in a genomic group and subgroup. Recently, genomic in situ hybridisation has permitted to determine the specific origin - A, B, S or T- of the chromosomes of some interspecific clones. Molecular markers have also led to a better understanding of the origin of cultivated bananas, particularly through the analysis of the A genome variability. The maternal and paternal origin of the clones may be inferred respectively from the RFLP analysis of the chloroplast and mitochondria genomes. RFLP analysis of the nuclear genome of cultivated bananas reveals all the wild acuminata subspecies at their origin. For instance, this study has shown that fruit quality is related to the nature of the A genome: cooking types with a M a. bankii / errans origin, and dessert types with a M a. malaccensis / zebrina origin

Banana production is severely threatened by the Black Leaf Streak (BLS) disease caused by Mycosphaerella fijiensis. Numerous chemical applications are needed to control the fungus's impact on leaves which reduces yields and causes premature ripening of fruits. Genetic improvement for resistance to BLS disease appears as the most durable, cost and environmentally efficient control. Screening for resistance has led to the identification of two forms of resistance reported as High Resistance (HR, hypersensitive response) and Partial Resistance (PR). In connection with the breeding program, a genome mapping approach for genetic and QTL analyses was developed to characterise the HR form. An F2 segregating population of 153 plants was obtained from a cross between two wild seeded banana accessions, Musa acuminata burmannicoides type Calcutta 4 and M. a. banksii type Madang. A linkage map was constructed with 110 AFLP markers in association with 39 codominant RFLP and SSR markers to anchor the linkage groups to the banana genetic core map. Results showed significant allelic distorted segregations in the F2 progeny for 58 markers which highlight the use of molecular studies for inheritance analyses. A first mapping concluded to join the markers in 11 linkage groups. Correlations made with field observations, ranked into six classes of infection severity, led to the identification of one RFLP marker strongly associated to the resistance and a second QTL mapped onto a different linkage group with lower significance level

Banana production is severely threatened by the Black Leaf Streak Disease (BLS) caused by Mycosphaerella fijiensis. Numerous chemical applications are needed to control the fungus' impact on leaves which reduces yields and causes premature ripening of fruits. Genetic improvement for resistance to BLS appears as the most durable, cost and environmentally efficient control. Screening for resistance has led to the identification of two forms of resistance reported as High Resistance (HR, hypersensitive response) and Partial Resistance (PR). In connection with the breeding program, a genome mapping approach for genetic and QTL analyses was developed to characterise the HR form. An F2 segregating population of 175 plants was obtained from a cross between two wild seeded banana accessions, Musa acuminata burmannicoides type Calcutta 4 and M. a. banksii type Madang. A linkage map was constructed with AFLP markers in association with RFLP and SSR markers to anchor the linkage groups to the core banana genetic map. Resistance is being evaluated by field observations in two locations at CRBP in Cameroon and also by controlled inoculations. The results will serve to characterise the genetic determinism of BLSD resistance with the objective to develop marker assisted selection. (Texte intégral)

Bananas belong to the #Musaceae# family and are among the tallest monocotyledons. Main dessert banana cultivars are triploid, highly sterile, parthenocarpic and atonally propagated. They are susceptible to several diseases that seriously threaten plantations. In order to better understand banana genetics, more knowledge must be gained about their genomes. This would help to develop new improvement strategies. Major aims of the molecular studies, in progress at CIRAD at present, are the construction of genetic maps, a better understanding of chromosome structural differentiation in #Musa#, the analysis of quantitative characters as well as a comprehensive study of molecular genetic diversity. A first genetic linkage map based on RFLP markers has already been published by our lab (Fauré et al., 1993, TAG 87 : 517-526). In order to saturate this map for QTL analysis, we checked the banana genome for VNTR markers. We report here preliminary results on dinucleotide microsatellite sequences in the banana genome. Microsatellite gene tagging and targeting are discussed.

Bananas belong to the #Musaceae# family and are among the tallest monocotyledons. Main dessert banana cultivars are triploid, highly sterile, parthenocarpic and atonally propagated. They are susceptible to several diseases that seriously threaten plantations. In order to better understand banana genetics, more knowledge must be gained about their genomes. This would help to develop new improvement strategies. Major aims of the molecular studies, in progress at CIRAD at present, are the construction of genetic maps, a better understanding of chromosome structural differentiation in #Musa#, the analysis of quantitative characters as well as a comprehensive study of molecular genetic diversity. A first genetic linkage map based on RFLP markers has already been published by our lab (Fauré et al., 1993, TAG 87 : 517-526). In order to saturate this map for QTL analysis, we checked the banana genome for VNTR markers. We report here preliminary results on dinucleotide microsatellite sequences in the banana genome. Microsatellite gene tagging and targeting are discussed. (Texte intégral)