Australian wild limes occur in highly diverse range of environments and are a unique genetic resource within the genus Citrus. Here we compare the haplotype-resolved genome assemblies of six Australian native limes, including four new assemblies generated using PacBio HiFi and Hi-C sequencing data. The size of the genomes was between 315 and 391 Mb with contig N50s from 29.5 to 35 Mb. Gene completeness of the assemblies was estimated to be from 98.4 to 99.3% and the annotations from 97.7 to 98.9% based upon BUSCO, confirming the high contiguity and completeness of the assembled genomes. High collinearity was observed among the genomes and the two haplotype assemblies for each species. Gene duplication and evolutionary analysis demonstrated that the Australian citrus have undergone only one ancient whole-genome triplication event during evolution. The highest number of species-specific and expanded gene families were found in C. glauca and they were primarily enriched in purine, thiamine metabolism, amino acids and aromatic amino acids metabolism which might help C. glauca to mitigate drought, salinity, and pathogen attacks in the drier environments in which this species is found. Unique genes related to terpene biosynthesis, glutathione metabolism, and toll-like receptors in C. australasica, and starch and sucrose metabolism genes in both C. australis and C. australasica might be important candidate genes for HLB tolerance in these species. Expanded gene families were not lineage specific, however, a greater number of genes related to plant-pathogen interactions, predominantly disease resistant protein, was found in C. australasica and C. australis.
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