A super-pangenome for cultivated citrus reveals evolutive features during the allopatric phase of their reticulate evolution
Authors: Droc Gaetan , Giraud Delphine , Belser Caroline , Labadie Karine , Duprat Simone , Cruaud Corinne , Istace Benjamin , Dos Santos Menezes Fredson , Curk Franck , Costantino Gilles , Soriano Alexandre , Mournet Pierre , Dereeper Alexis , Miranda Maëva , Marchi Elodie , Santoni Sylvain , Santana Raner , Sidibe-Bocs Stéphanie , Luro François , Choisne Nathalie , Maumus Florian , Hufnagel Barbara , Micheli Fabienne , Wincker Patrick , Aury Jean-Marc , Lemainque Arnaud , Ollitrault Patrick 
Published: October 2024

ABSTRACTMost of the rich genetic diversity observed in cultivated citrus results from a reticulate evolution involving four ancestral taxa,C. medica,C. reticulata,C. maximaandC. 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 withde novoassemblies in pseudochromosomes ofC. medica(346 Mb),C. reticulata(332 Mb) andC. micrantha(333 Mb), released for the first time alongside a previously published chromosome-scale assembly ofC. 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 theC. maximaassembly. 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 ofC. reticulata,C. maximaandC. medicaas 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 ofC. 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.