The genome of Citrus australasica reveals disease resistance and other species specific genes.

BACKGROUND: The finger lime (Citrus australasica), one of six Australian endemic citrus species shows a high natural phenotypic diversity and novel characteristics. The wide variation and unique horticultural features have made this lime an attractive candidate for domestication. Currently no haplotype resolved genome is available for this species. Here we present a high quality, haplotype-resolved reference genome for this species using PacBio HiFi and Hi-C sequencing. RESULTS: Hifiasm assembly and SALSA scaffolding resulted in a collapsed genome size of 344.2 Mb and 321.1 Mb and 323.2 Mb size for the two haplotypes. The nine pseudochromosomes of the collapsed genome had an N50 of 35.2 Mb, 99.1% genome assembly completeness and 98.9% gene annotation completeness (BUSCO). A total of 41,304 genes were predicted in the nuclear genome. Comparison with C. australis revealed that 13,661 genes in pseudochromosomes were unique in C. australasica. These were mainly involved in plant-pathogen interactions, stress response, cellular metabolic and developmental processes, and signal transduction. The two genomes showed a syntenic arrangement at the chromosome level with large structural rearrangements in some chromosomes. Genetic variation among five C. australasica cultivars was analysed. Genes related to defense, synthesis of volatile compounds and red/yellow coloration were identified in the genome. A major expansion of genes encoding thylakoid curvature proteins was found in the C. australasica genome. CONCLUSIONS: The genome of C. australasica present in this study is of high quality and contiguity. This genome helps deepen our understanding of citrus evolution and reveals disease resistance and quality related genes with potential to accelerate the genetic improvement of citrus.

Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes.

Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus. Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased, with the available genomes of varying accuracy and completeness. We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species; Citrus australis (round lime) using highly accurate PacBio HiFi long reads, complemented with Hi-C scaffolding. Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C. australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness (BUSCO). Repeat analysis showed that more than 50% of the genome contained interspersed repeats. Among them, LTR elements were the predominant type (21.0%), of which LTR Gypsy (9.8%) and LTR copia (7.7%) elements were the most abundant repeats. A total of 29 464 genes and 32 009 transcripts were identified in the genome. Of these, 28 222 CDS (25 753 genes) had BLAST hits and 21 401 CDS (75.8%) were annotated with at least one GO term. Citrus specific genes for antimicrobial peptides, defense, volatile compounds and acidity regulation were identified. The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2, 4, 7 and 8. This chromosome scale, and haplotype resolved C. australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.

Pathotyping Citrus Ornamental Relatives with Xanthomonas citri pv. citri and X. citri pv. aurantifolii Refines Our Understanding of Their Susceptibility to These Pathogens.

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host-pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen.

Pathogenicity of Phyllosticta citricarpa Ascospores on Citrus spp.

Citrus black spot, caused by Phyllosticta citricarpa, is one of the most important fungal diseases in many citrus-growing regions with hot and humid summers. Ascospores and conidia are known to contribute to epidemic development of the disease. However, pathogenicity testing has never been done for pure ascospores produced from fully characterized P. citricarpa isolates, due to the inability to induce the sexual state in vitro. Recently, an in vitro mating technique was developed to readily produce pure P. citricarpa ascospores for use in host inoculation studies. To test the pathogenicity of P. citricarpa ascospores, we inoculated Troyer citrange leaves and Murcott tangor fruit with ascospores produced in vitro from characterized P. citricarpa isolates. Typical symptoms of citrus black spot occurred. Recovery of P. citricarpa isolates from symptomatic lesions and their characterization using genetic markers enabled us to identify recombinant genotypes among the isolates recovered from ascospore inoculations and, as such, fulfill Koch's postulates for ascospores. We have also identified Troyer citrange seedlings as a potential model system for citrus black spot inoculation studies, because it allows typical symptoms of citrus black spot to be expressed with a much shorter latent period than on fruit. This will facilitate future studies of epidemiological aspects of P. citricarpa ascospores relative to conidia and improve our understanding of the citrus black spot pathosystem. The susceptibility of Troyer citrange seedlings will also facilitate experimenting with disease management methods, aimed at reducing the impact of citrus black spot.