Theobroma cacao is one of the main crops of economic importance in the world as the source of raw material for producing chocolate and derivatives. The crop is the main source of income for thousands of small farmers, who produce more than 80% of the world’s cocoa supply. However, the emergence, re-emergence and proliferation of pathogens, such as Ceratocystis spp., the causative agent of Ceratocystis wilt disease and canker disease, have been affecting the sustainability of many crops. Fungal control is laborious, often depending on fungicides that are expensive and/or toxic to humans, prompting researchers to look for new solutions to counteract the proliferation of these pathogens, including the use of biological agents such as mycoviruses. In this study, we investigated the diversity of microorganisms associated with the T. cacao pathogens Ceratocystis cacaofunesta and Ceratocystis fimbriata with a focus on the virome using RNA sequencing data available in public databases. We used a comprehensive bioinformatics pipeline containing several steps for viral sequence enrichment and took advantage of an integrated assembly step composed of different assemblers followed by sequence similarity searches using NCBI nonredundant databases. Our strategy was able to identify four putative C. cacaofunesta viruses (hypovirus, sclerotimonavirus, alphapartitivirus and narnavirus) and six C. fimbriata viruses (three alphaendornaviruses, one victorivirus and two mitoviruses). All the viral sequences identified showed similarity to viral genomes in public databases only at the amino acid level, likely representing new viral species. Of note, we present the first report of viruses associated with the cacao pathogens C. cacaofunesta and C. fimbriata and the second report of viral species infecting members of the Ceratocystidaceae family. Our findings highlight the need for further prospective studies to uncover the real diversity of fungus-infecting viruses that can contribute to the development of new management strategies.

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Open reading frame 8 (ORF8) shows one of the highest levels of variability among accessory proteins in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits the presentation of viral antigens by the major histocompatibility complex class I (MHC-I), which interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 in evading immunity and plays a role in SARS-CoV-2 replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein, defines the B.1.1.7 lineage of SARS-CoV-2, engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) with the Q27STOP mutations were identified among 49,055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents, which include Africa, Asia, Europe and South America. Based on various quantitative features, such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, nine possible T-ORF8 unique variants were defined. The question as to whether T-ORF8 variants function similarly to the wild type ORF8 is yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

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.

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.