Identifying crossover locations from pollen of Arabidopsis thaliana Col x Ler F1 plants (WT) using Nanopore-seq and COmapper

We sequenced pollen DNA from Col x Ler F1 hybrid plants using Nanopore long-read sequencing and identified crossover sites with COmapper. To purify pollen grains from F1 hybrid plants and extract their genomic DNA, whole inflorescences of hybrid plants were collected and transferred to ice-cold 10% (w/v) sucrose in a 50-ml tubes. Flowers were homogenized with a blender (Tefal, BL3051KR), filtered through 80-μm nylon mesh, and centrifuged at 350g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 10% (w/v) sucrose, filtered through a 40-μm cell strainer, and centrifuged at 100g for 10 min at 4°C. The purified pellet was frozen in liquid nitrogen and ground with a mortar and pestle. The pellet powder was resuspended in CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA). Proteinase K (NEB, P8107S) and RNase A (20 mg/ml, Roche, 10109142001) were added to a final concentration of 80 U/ml and 20 μg/ml, respectively. To check pollen disruption, a 1-μl sample was diluted in a 1:10 ratio with 10% (w/v) sucrose and observed under a stereomicroscope (Leica, M165 FC). Then, 500 μl of the resuspended pellet was transferred to a 1.5-ml tube, to which an equal volume of 25:24:1 (v/v/v) phenol:chloroform:isoamylalcohol (Sigma, 77617) was added, and the mixture was homogenized by gentle manual shaking. The tube was centrifuged at 15,000g for 10 min at 4°C, and 400 μl of supernatant was transferred into a new tube, to which a Nanobind disk (Nanobind® Plant Nuclei Kit RT, PacBio, 102-302-000) was added. The tube containing the Nanobind disk was gently mixed, 400 μl isopropanol was added, and the tube was slowly inverted and incubated for 2 h at room temperature on a rotator set at 7 rpm. A magnetic rack (DynaMagTM-2, Invitrogen, 12321D) was used to separate the disk and the supernatant, and the supernatant was removed. PW1 buffer in the Nanobind Plant Nuclei Kit was used for washing the disk twice and the supernatant was removed completely by centrifugation. Then, 150 μl EB+ buffer was added onto the disc and gently tabbed and incubated overnight at room temperature. The supernatant was transferred to a new 1.5-ml tube and stored at −20°C. The DNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853) and integrity checked by gel electrophoresis. Nine micrograms of gDNA from pollen was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).