The main genetic diversity observed in cultivated citrus results from a reticulate evolution involving four ancestral taxa whose radiation occurred in allopatry. In such context, GWAS analysis, genome diversity and transcriptomic studies will be significantly enhanced through pangenome approaches. We report the implementation of a super-pangenome for cultivated citrus, established with de novo assemblies of C. medica, C. reticulata and C. micrantha, released for the first time alongside a published chromosome-scale assembly of C. maxima. Repetitive element annotation revealed that half of each genome consisted of transposable elements or DNA-satellites. The new genome assemblies display strong synteny and collinearity, while discrepancies are observed with the C. maxima assembly. Resequencing information from 55 accessions helped to explore the intra- and interspecific diversity of the ancestral taxa and their relationships with horticultural groups. Diagnostic SNPs of the ancestral taxa revealed interspecific introgressions in several representative accessions of C. reticulata, C. maxima and C. medica as well as insights into the origin and phylogenomic structures of horticultural groups. PAV analysis revealed a gene whose absence or presence was specific to one of the ancestral taxa. Diagnostic PAV analysis uncovered a large chloroplastic introgression in C. medica chromosome 4. The analysis of the functional enrichment and species-specific adaptations in the citrus super-pangenome revealed distinct functional specialisations. This highlights the evolutionary paths that have shaped species, contributing to the diversity in the citrus super-pangenome while maintaining a shared foundation of essential biological processes. We established a Genome Hub, offering a platform for continuous genomic research.

Oceanian citrus species, including wild taxa native to Australia and Papua New Guinea, form a genetically distinct clade within the Citrus L. (1753) genus. These species remain largely underexplored, despite their adaptation to diverse environments and relevance for citrus improvement. To support their use in breeding and evolutionary studies, we generated a high-quality, Chromosome-scale genome assembly of an Australian finger lime accession (SRA 1002), a natural interspecific hybrid. The genome was assembled using long-read sequencing, optical mapping, and a high-density genetic map, resulting in nine pseudomolecules covering over 97% of the genome. Using this reference, we analyzed whole-genome resequencing data from 132 accessions representing the diversity of Asian and Oceanian citrus. Variant calling across the dataset produced a high-resolution catalogue of single nucleotide polymorphisms (SNPs) and derived database of SNP fully discriminant of 20 Citrus species (DSNPs), enabling detailed exploration of inter- and intra-specific diversity. The data reveal a strong population structure within the group with clear heterozygosity variation between ancestral species and admixed accessions, reflecting their complex evolutionary history and hybridization patterns. This study provides the first integrated genomic framework for Oceanian citrus diversity, offering essential tools for downstream applications in citrus breeding, conservation, and evolutionary genomics. The resources generated lay the groundwork for future association studies and the targeted introgression of beneficial traits into cultivated citrus.

Most of the rich genetic diversity observed in cultivated citrus results from a reticulate evolution involving four ancestral taxa, C. medica, C. reticulata, C. maxima and C. micrantha, whose radiation occurred in allopatry. In such an evolutive context, genome diversity studies, GWAS analysis and transcriptomic studies will be significantly enhanced through pangenome approaches. We report the implementation of a super-pangenome for cultivated citrus, established with de novo assemblies in pseudochromosomes of C. medica (346 Mb), C. reticulata (332 Mb) and C. micrantha (333 Mb), released for the first time alongside a previously published chromosome-scale assembly of C. maxima. Gene annotation of these four genome assemblies revealed 28,090, 29,477, 29,258 and 30,101 genes, respectively, with a focus on pattern recognition receptors. Repetitive element annotation revealed that nearly half of each genome consisted of transposable elements or DNA-satellites. The 3 new genome assemblies display strong synteny and collinearity, while significant discrepancies are observed with the C. maxima assembly. Resequencing information (single nucleotide polymorphisms [SNPs], small indels and gene presence-absence variation [PAV]) from 55 accessions were used to explore the intra- and interspecific diversity of the four ancestral taxa and their relationships with the main horticultural groups resulting from reticulate evolution. Diagnostic SNPs of the ancestral taxa, all over the four genomes assemblies, revealed interspecific introgressions in several accessions representative of C. reticulata, C. maxima and C. medica as well as insights into the origin and phylogenomic structures of modern horticultural groups. PAV analysis revealed a gene whose absence or presence was specific to one of the ancestral taxa (dPAV). Interestingly, diagnostic PAV (dPAV) analysis uncovered a large chloroplastic introgression in chromosome 4 of C. medica, inherited in horticultural groups and recent hybrids having this species as the male parent. Implementing the super-pangenome combined the identification of orthologous genes across the four genome assemblies and the PAV information from the resequencing data. Significant inter- and intraspecific variations were highlighted, including 25,291 core, 2,431 soft-core and 5,171 dispensable genes. The analysis of the functional enrichment and species-specific adaptations in the citrus super-pangenome revealed distinct functional specializations. This highlights the evolutionary paths that have shaped each species, contributing to the diversity in the citrus super-pangenome while maintaining a shared foundation of essential biological processes. The development of this super-pangenome is a significant milestone in citrus genomics, providing a comprehensive resource that captures the extensive genetic diversity of modern cultivated citrus. Additionally, we established a dedicated Genome Hub, offering a platform for continuous genomic research and allowing for ongoing updates and future inclusion of additional ancestral species. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionUseful germplasm for citrus breeding includes all sexually compatible species of the former genera Citrus, Clymenia, Eremocitrus, Fortunella, Microcitrus, Oxanthera, and Poncirus, now merged in the single Citrus genus. An improved knowledge on the synteny/collinearity between the genome of these different species, and on their recombination landscapes, is essential to optimize interspecific breeding schemes.MethodWe have performed a large comparative genetic mapping study including several main clades of the Citrus genus. It concerns five species (C. maxima, C. medica, C. reticulata, C. trifoliata and C. glauca), two horticultural groups resulting from interspecific admixture (clementine and lemon) and two recent interspecific hybrids (C. australis x C. australasica and C. maxima x C. reticulata). The nine individual genetic maps were established from GBS data of 1,216 hybrids.Results and discussionThe number of SNPs mapped for each parent varies from 760 for C. medica to 4,436 for the C. maxima x C. reticulata hybrid, with an average of 2,162.3 markers by map. Their comparison with C. clementina v1.0 assembly and inter-map comparisons revealed a high synteny and collinearity between the nine genetic maps. Non-Mendelian segregation was frequent and specific for each parental combination. The recombination landscape was similar for the nine mapped parents, and large genomic regions with very low recombination were identified. A consensus genetic map was successfully established. It encompasses 10,756 loci, including 7,915 gene-based markers and 2,841 non-genic SNPs. The anchoring of the consensus map on 15 published citrus chromosome-scale genome assemblies revealed a high synteny and collinearity for the most recent assemblies, whereas discrepancies were observed for some older ones. Large structural variations do not seem to have played a major role in the differentiation of the main species of the Citrus genus. The consensus genetic map is a useful tool to check the accuracy of genome assemblies, identify large structural variation and focus on analyzing potential relationships with phenotypic variations. It should also be a reference framework to integrate the positions of QTLs and useful genes identified in different analyses.

The feral cattle of the subantarctic island of Amsterdam provide an outstanding case study of a large mammalian population that was established by a handful of founders and thrived within a few generations in a seemingly inhospitable environment. Here, we investigated the genetic history and composition of this population using genotyping and sequencing data. Our inference showed an intense but brief founding bottleneck around the late 19th century and revealed contributions from European taurine and Indian Ocean Zebu in the founder ancestry. Comparative analysis of whole-genome sequences further revealed a moderate reduction in genetic diversity despite high levels of inbreeding. The brief and intense bottleneck was associated with high levels of drift, a flattening of the site frequency spectrum and a slight relaxation of purifying selection on mildly deleterious variants. Unlike some populations that have experienced prolonged reductions in effective population size, we did not observe any significant purging of highly deleterious variants. Interestingly, the population's success in the harsh environment can be attributed to preadaptation from their European taurine ancestry, suggesting no strong bioclimatic challenge, and also contradicting evidence for insular dwarfism. Genome scan for footprints of selection uncovered a majority of candidate genes related to nervous system function, likely reflecting rapid feralization driven by behavioral changes and complex social restructuring. The Amsterdam Island cattle offers valuable insights into rapid population establishment, feralization, and genetic adaptation in challenging environments. It also sheds light on the unique genetic legacies of feral populations, raising ethical questions according to conservation efforts.

Sexual breeding at the tetraploid level is a promising strategy for rootstock breeding in citrus. Due to the interspecific origin of most of the conventional diploid citrus rootstocks that produced the tetraploid germplasm, the optimization of this strategy requires better knowledge of the meiotic behavior of the tetraploid parents. This work used Genotyping By Sequencing (GBS) data from 103 tetraploid hybrids to study the meiotic behavior and generate a high-density recombination landscape for their tetraploid intergenic Swingle citrumelo and interspecific Volkamer lemon progenitors. A genetic association study was performed with root architecture traits. For citrumelo, high preferential chromosome pairing was revealed and led to an intermediate inheritance with a disomic tendency. Meiosis in Volkamer lemon was more complex than that of citrumelo, with mixed segregation patterns from disomy to tetrasomy. The preferential pairing resulted in low interspecific recombination levels and high interspecific heterozygosity transmission by the diploid gametes. This meiotic behavior affected the efficiency of Quantitative Trait Loci (QTL) detection. Nevertheless, it enabled a high transmission of disease and pest resistance candidate genes from P. trifoliata that are heterozygous in the citrumelo progenitor. The tetrazyg strategy, using doubled diploids of interspecific origin as parents, appears to be efficient in transferring the dominant traits selected at the parental level to the tetraploid progenies.

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9 . This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

Long-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

Eusocial termite queens achieve nearly maximal fertility throughout their extremely long life without apparent signs of aging. Termites represent, therefore, an ideal model for aging research. To investigate the molecular mechanisms underlying their long reproductive life, we carried out transcriptomic, lipidomic and metabolomic analyses on fat bodies of sterile short lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. In mature reproductives, genes supporting a robust mitochondrial functioning or associated with genome stability were upregulated. In most organisms, insulin signaling increases fertility but decreases lifespan, often accompanied by harmful lipid signatures. Our findings suggest that an upregulation of insulin-like peptide (Ilp9) in the fat body of termite queens is accompanied by a specific lipid metabolism, limiting fat storage, thus sustaining both high fertility and maintaining extreme lifespan. Our results highlight potential molecular targets for research into aging-related metabolic diseases linked to the accumulation of excess fat.

The Seine-Morée wastewater treatment plant (SM_WWTP), with a capacity of 100,000 population-equivalents, was fed with raw domestic wastewater during all of its start-up phase. Its microbiome resulted from the spontaneous evolution of wastewater-borne microorganisms. This rare opportunity allowed us to analyze the sequential microbiota colonization and implantation follow up during the start-up phase of this WWTP by means of regular sampling carried out over 8 months until the establishment of a stable and functional ecosystem. During the study, biological nitrification–denitrification and dephosphatation occurred 68 days after the start-up of the WWTP, followed by flocs decantation 91 days later. High throughput sequencing of 18S and 16S rRNA genes was performed using Illumina's MiSeq and PGM Ion Torrent platforms respectively, generating 584,647 16S and 521,031 18S high-quality sequence rDNA reads. Analyses of 16S and 18S rDNA datasets show three colonization phases occurring concomitantly with nitrification, dephosphatation and floc development processes. Thus, we could define three microbiota profiles that sequentially colonized the SM_WWTP: the early colonizers, the late colonizers and the continuous spectrum population. Shannon and inverse Simpson diversity indices indicate that the highest microbiota diversity was reached at days 133 and 82 for prokaryotes and eukaryotes respectively; after that, the structure and complexity of the wastewater microbiome reached its functional stability. This study demonstrates that physicochemical parameters and microbial metabolic interactions are the main forces shaping microbial community structure, gradually building up and maintaining a functionally stable microbial ecosystem.

Genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200-nucleotide resolution. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

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.

Abstract Background The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus. Findings Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated ∼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes. Conclusion Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.

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.

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.

This protocol describes the sequencing library preparation for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

This protocol describes the handling of genomics samples for the Tara Oceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.