Chiloglottis trapeziformis is a sexually deceptive Australian orchid that provides a valuable system for studying orchid genome evolution, structural variation, and the molecular basis of specialized pollination. However, high-quality nuclear genome resources remain scarce for most orchids, particularly Australia's diverse terrestrial lineages. To address this gap, we integrated PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C chromatin-contact data to generate the first chromosome-scale, haplotype-resolved nuclear genome assembly for an Australian terrestrial orchid, Chiloglottis trapeziformis. Hi-C guided scaffolding resolved two haplotypes into 20 chromosomes each, consistent with the reported karyotype and genome size (2n=40, haplotype sizes of 1.58 Gb and 1.91 Gb). Genome completeness was high for both haplotypes, recovering 95.1% and 95.5% complete BUSCO genes for haplotype 1 and haplotype 2, respectively. De novo repeat annotation revealed a repeat-rich genome (85.79-88.25% repetitive sequence), dominated by LTR retrotransposons. Evidence-guided annotation identified 16,287 and 16,548 protein-coding genes in Haplotype 1 and Haplotype 2, respectively. Phylogenetically informed comparisons placed C. trapeziformis as sister to Anoectochilus roxburghii among sampled Orchidoideae and showed broad gene-order conservation. Comparing the two haplotypes for structural variation, we identified large inter-haplotype inversions containing functionally annotated genes with detectable RNA expression, with focal examples further supported by local Hi-C contact patterns and breakpoint-level inspection. Inversion-overlapping genes did not show elevated dS relative to collinear background. This assembly and annotation resource provides a foundation for population and conservation genomics, structural and comparative analyses, and genome-enabled hypothesis testing of molecular traits underlying sexual deception in orchids.
Zhang, Z., Jones, A., Schwessinger, B., Peakall, R., Wong, D.
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