GoKawiilPlant material
Plants of R. tenuis and R. austrobrasiliensis were collected from natural populations in Brazil under appropriate permits and cultivated under controlled greenhouse conditions. Nine geographically distinct accessions of R. tenuis were used for genome sequencing and cytological analyses. Individual flowers and pollen were staged for meiotic analyses. All experimental material was propagated clonally from field-collected individuals to ensure genetic identity across assays.
Sequencing
High-molecular-mass DNA was extracted from young leaf tissue using a modified cetyltrimethylammonium bromide protocol. PacBio HiFi libraries were prepared and sequenced on the Sequel IIe platform. Illumina short-read sequencing was used for genome polishing and transcriptome profiling. Hi-C libraries were constructed using Arima Hi-C kits and sequenced on the Illumina NovaSeq 6000 platform. RNA was extracted from young inflorescences for expression analyses.
DNA isolation
High-molecular-mass DNA from R. tenuis and R. austrobrasiliensis was isolated from 1.5 g of material using the NucleoBond HMW DNA kit (Macherey Nagel). Quality was assessed using a FEMTO-pulse device (Agilent), and the quantity was measured using the Quantus Fluorometer (Promega).
PacBio
HiFi libraries were prepared according to the ‘Preparing whole genome and metagenome libraries using SMRTbell prep kit 3.0’ manual, with an initial DNA fragmentation by Megaruptor-3 (Diagenode) and final size-selection by BluePippin (Sage Science). Size distribution was again controlled by FEMTO-pulse (Agilent). Size-selected libraries were then sequenced on a Revio device using the Revio polymerase kit and Revio chemistry for 30 h (Pacific Biosciences).
Arima Hi-C
Plant tissues were cross-linked with 1% formaldehyde for 30 min at room temperature, and the reaction was quenched with 125 mM glycine for 10 min. Subsequently, the tissues were ground using a TissueLyser at a frequency of 30 Hz for 3 min. Nucleus extraction was performed using the CelLytic PN Plant Nuclei Isolation/Extraction Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Hi-C libraries were prepared using the Arima High Coverage Hi-C Kit (Arima Genomics, A410110) according to the manufacturer’s instructions, and were then sequenced (paired-end, 2 × 150 bp) on the NextSeq 2000 instrument (Illumina).
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