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International audience; Abstract Background and Aims Modern sugarcane cultivars (Saccharum spp.) are high polyploids, aneuploids (2n = ~12x = ~120) derived from interspecific hybridizations between the domesticated sweet species Saccharum officinarum and the wild species S. spontaneum. Methods To analyse the architecture and origin of such a complex genome, we analysed the sequences of all 12 hom(oe)ologous haplotypes (BAC clones) from two distinct genomic regions of a typical modern cultivar, as well as the corresponding sequence in Miscanthus sinense and Sorghum bicolor, and monitored their distribution among representatives of the Saccharum genus. Key Results The diversity observed among haplotypes suggested the existence of three founding genomes (A, B, C) in modern cultivars, which diverged between 0.8 and 1.3 Mya. Two genomes (A, B) were contributed by S. officinarum; these were also found in its wild presumed ancestor S. robustum, and one genome (C) was contributed by S. spontaneum. These results suggest that S. officinarum and S. robustum are derived from interspecific hybridization between two unknown ancestors (A and B genomes). The A genome contributed most haplotypes (nine or ten) while the B and C genomes contributed one or two haplotypes in the regions analysed of this typical modern cultivar. Interspecific hybridizations likely involved accessions or gametes with distinct ploidy levels and/or were followed by a series of backcrosses with the A genome. The three founding genomes were found in all S. barberi, S. sinense and modern cultivars analysed. None of the analysed accessions contained only the A genome or the B genome, suggesting that representatives of these founding genomes remain to be discovered. Conclusions This evolutionary model, which combines interspecificity and high polyploidy, can explain the variable chromosome pairing affinity observed in Saccharum. It represents a major revision of the understanding of Saccharum diversity.

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.

International audience; Admixture and polyploidization are major recognized eukaryotic genome evolutionary processes. Their impacts on genome dynamics vary among systems and are still partially deciphered. Many banana cultivars are triploid (sometimes diploid) interspecific hybrids between Musa acuminata (A genome) and M. balbisiana (B genome). They have no or very low fertility, are vegetatively propagated and have been classified as "AB," "AAB," or "ABB" based on morphological characters. We used NGS sequence data to characterize the A versus B chromosome composition of nine diploid and triploid interspecific cultivars, to compare the chromosome structures of A and B genomes and analyze A/B chromosome segregations in a polyploid context. We showed that interspecific recombination occurred frequently between A and B chromosomes. We identified two large structural variations between A and B genomes, a reciprocal translocation and an inversion that locally affected recombination and led to segregation distortion and aneuploidy in a triploid progeny. Interspecific recombination and large structural variations explained the mosaic genomes observed in edible bananas. The unprecedented resolution in deciphering their genome structure allowed us to start revisiting the origins of banana cultivars and provided new information to gain insight into the impact of interspecificity on genome evolution. It will also facilitate much more effective assessment of breeding strategies.

International audience; Background and Aims Banana 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. Methods Marker (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 Results Two 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. Conclusion Two 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.

Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its genome poses challenges that have not been addressed in any prior sequencing project due to its highly polyploid, aneuploid, heterozygous and interspecific genome structure (2n ~ 12x ~ 120, ~10Gb). We exploited the colinearity with sorghum to produce a monoploid genome sequence of sugarcane. We applied whole genome profiling (WGPTM) to 20,736 sugarcane BAC clones and anchored 11,732 of them onto the sorghum genome. A minimum tiling path of 4,660 sugarcane BAC clones that best cover the gene-rich part of the sorghum genome was selected, sequenced and assembled in a 382 Mb single tiling path (STP) of high quality sequence. A total of 25,316 protein-coding gene models were predicted on the STP, 17% of which displayed no colinearity with their sorghum orthologs. We showed that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differed by their content in transposable elements (TE) explaining their distinct genome size. We also showed that they differ by a few large chromosomal rearrangements, explaining their distinct basic chromosome numbers while also suggesting that polyploidisation arose in both lineages after their divergence. This BAC-based monoploid sugarcane reference sequence represents an essential resource for genetic and genomic studies and for future whole genome sequence assembly programmes. BAC and STP sequences as well as gene annotations are available on the sugarcane genome hub (http://sugarcane-genome.cirad.fr). We acknowledge members of the International Consortium for Sugarcane Biotechnology (ICSB) and Joint BioEnergy Institute (JBEI) for financial support.

Inter(sub)specific hybridizations are frequent events in plants and represent key factors in the evolution and domestication of many crops. Banana is one of such crop plants where domesticated cultivars result from hybridization events between Musa acuminata (2n=2x=22, A genome) subspecies and sometimes with Musa balbisiana (2n=2x=22, B genome). These species and subspecies diverged following geographical isolation in Southeast Asian continental regions and islands. Plant movement fostered by human migrations lead to inter(sub)specific hybridization that generated diploid and triploid hybrids with low fertility. Large chromosomal rearrangements between the species and subspecies are suspected to be involved in this low fertility. Seedless parthenocarpic hybrids were then selected by early farmers and since then dispersed by centuries of vegetative propagation. So far, two large structural variations were observed between Musa acuminata and Musa balbisiana and three large structural variations were identified within Musa acuminata subspecies using a combination of genetic and physical mapping, large insert paired read sequencing and BAC-FISH. Using genetic mapping data and FISH, a fourth structural variation was identified at heterozygous state in several cultivated accessions. However, while we know which chromosomes are implicated in this genome rearrangement, previous methods failed to clarify its exact nature. We will discuss present development based on genetic mapping and partial sequence assembly of an accession having this SV at heterozygous state to reconstruct both haplotypes and access to this new structure.

Figure S3. Chromosome numbers in Aeschynomene species. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 135 kb)

Almost 40% of the world banana production relies on triploid interspecific cultivars deriving from hybridization between Musa. acuminata (Genome A, 2n=22) and M. balbisiana (Genome B, 2n=22). These cultivars were classified based on morphological characteristics in genomic groups (AAB and ABB) subdivided into various subgroups. They include the cooking banana Plantain group (classified as AAB) that represents 18% of the banana production worldwide. The origin of these cultivars, their chromosome structure as well as its impact on chromosome recombination and segregation are still poorly known. We analyzed using Genotyping By Sequencing (GBS), the A/B chromosomes composition of a few cultivars and showed that it deviates in several regions from the conventional genomic classification. For example Plantain, classified as 'AAB,' has six genomic regions with an AAA chromosome composition and one entire chromosome set with an ABB composition. We compared the global chromosome structure of A and B genomes through the construction a high density SNP genetic map of M. balbisiana and its comparison with the M. acuminata reference sequence assembly. We identified a large reciprocal translocation between chromosome 1 and chromosome 3 and a large inversion on chromosome 5. We analyzed the A/B chromosomes recombination and segregation in a progeny from an 'AAAB' Plantain-derived tetraploid breeding accession. This revealed frequent recombination between A and B chromosomes all along the genome to the main exception of the inverted segment on chromosome 5. We observed 62% of aneuploids in the progeny that mainly involved the three chromosomes displaying large structural variations between A and B genomes. Implication of these results will be discussed.

Table S1. Accessions used for the phylogeny of the genus Aeschynomene and related genera, their origin and characteristics. (PPTX 143 kb)

Figure S4. Chromosome numbers in species of Aeschynomene related genera. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome counts are indicated in brackets. Scale bars: 5 μm. (PPTX 57 kb)

Figure S5. Phylogenetic trees based on nuclear low-copy genes. Bayesian phylogenetic reconstructions obtained for the CYP1, eif1a, SuSy and TIP1;1 genes. Diploid species (2n = 20) are in blue, polyploid species (2n ≥ 28) in black excepted A. afraspera for which the A and B gene copies are distinguished in red and green respectively. -A, −A1, −A2, -B, -B1 and -B2 indicated the different copies found. Putative A and B subgenomes of the polyploid taxa are delineated by red and green boxes in dashed lines, respectively. Numbers at branches represent posterior probability. (PPTX 56 kb)

Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (PPTX 149 kb)

Table S2. Primers used for gene amplification and sequencing. (PPTX 134 kb)

Figure S6. Ancestral state reconstruction of ploidy levels in the genus Aeschynomene and allied genera. Ancestral state reconstruction was estimated in SIMMAP software using the 50% majority-rule topology obtained by Bayesian analysis of the combined ITS + matK sequences. Ploidy levels are indicated by different colors. Unknown ploidy levels are denoted by a dash. (PPTX 3568 kb)

Banana cultivars are derived from hybridization between Musa acuminata subspecies. Theses subspecies diverged following geographical isolation in distinct Southeast Asian continental regions and islands. Observation of chromosome pairing irregularities in meiosis of hybrids between theses subspecies suggested the presence of large chromosomal structural variations (LSV). We recently described a reciprocal translocation compare to the sequence M. acuminata reference in a subgroup of malaccensis accessions and another one in M. balbisiana. Using a self-progeny of Calcutta4 genotyped by GBS and large insert paired reads mapped on the reference genome, we characterized two large structural variations in the accession “Calcutta4” from the subspecies burmannicoides relative to the reference sequence assembly obtained with a M. a. ssp malaccensis accession. These large structural variations consist in a reciprocal translocation involving a 240 kb distal region of acrocentric chromosome 2 and a 7.2 Mb distal region of chromosome 8 and a second reciprocal translocation involving a 20.8Mb distal region of acrocentric chromosome 1 and an 11.6Mb distal region of chromosome 9. At the breakpoints, intricate events of duplication and deletion/rearrangements involving small fragments were detected. These rearrangement were validated using a preliminary assembly of “Calcutta 4” accession obtained from Nanopore Reads. We developed a methodology to exploit short-insert paired reads resequencing data to analyze the distribution of these two LSV in Musa germplasm. To do so, we designed a pipeline looking for read pair overlapping rearrangement breakpoints for the two structures. First results suggested micro-variations at rearrangement breakpoints for several representatives of Musa acuminata diversity. We thus used PacBio and Nanopore sequencing data from 2 additional representatives of Musa acuminata subspecies (ssp banksii and ssp zebrina) to design additional rearrangement breakpoints for these accessions. All breakpoints presence/absence where then tested for the 159 accessions for which paired reads were available. Results showed that both translocations are shared by all wild accessions belonging to the burmannicoides/burmannica/siamea subspecies. The chromosome 2/8 translocation is also found at the heterozygous state in two cultivars. The relatively low frequency in cultivated accessions of these alternate structures confirmed that burmannicoides/burmannica/siamea subspecies had a low contribution to cultivated bananas. Those two M. a. burmannica specific large structural variations correspond to the “Northern” group suggested by Shepherd (1999) based on meiosis observations.

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8+ T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8+ T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8+ T cell expression of CXCR3+, CD103+, CD49a+, CD69+, CD127+ homing, retention and survival markers. Furthermore, memory CD8+ T cells generated by MA immunisation significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8+ T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces.

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)

A few Musa acuminata subspecies are involved in banana cultivars. Theses subspecies diverged following geographical isolation in distinct Southeast Asian continental regions and islands. observation of chromosome pairing irregularities in meiosis of hybrids between theses subspecies suggested the presence of large chromosomal structural variations. We analyzed accession “Calcutta4” from the subspecies burmannicoides to search for chromosomal structural variations relative to the reference sequence assembly obtained with a M. a. ssp malaccensis accession. A self progeny of Calcutta4 was genotyped by GBS (genotyping by sequencing). We observed linkage between markers from reference chromosome 2 and 8 and reference chromosome 1 and 9 suggesting the presence of two reciprocal translocations involving these two pairs of chromosomes. Large insert size paired reads (5 and 8kb) from “Calcutta4” were mapped on the reference sequence to confirm the presence of these translocations and precisely locate the translocation breakpoints. Analysis of discordant read mapping suggested a first reciprocal translocation involving a 240Kb distal region of acrocentric chromosome 2 and a 7.2 Mb distal region of chromosome 8. A second reciprocal translocation involves a 20.8Mb distal region of acrocentric chromosome 1 and a 11.6Mb distal region of chromosome 9, with intricate events of duplication and deletion at the breakpoint. We are currently using BAC-­‐FISH to validate these structural variations. Perspectives are to develop PCR markers at the breakpoint to analyze the presence of these rearrangements in Musa germplasm and to analyze of the impact of these rearrangements in heterozygous accessions.