Promethion sequencer

Genomic DNA and RNA were extracted using FastPureTM Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China) and RNA-easyTM Isolation Reagent (Vazyme, Nanjing, China), respectively. DNA and RNA quantification and qualification were performed using 1% agarose gel electrophoresis, a Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA), and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Nanopore libraries were prepared using SQK-LSK108 and sequenced on a Nanopore PromethION sequencer. DNA libraries for short-read whole genome sequencing (WGS) were constructed using the Illumina TruSeq DNA PCR-free library preparation kit (Illumina, CA, USA) with 300- to 500-bp fragment sizes, and sequenced on the Illumina NovaSeq 6000 platform to generate 150-bp paired-end (PE) reads. Transcriptome libraries were constructed with a TruSeq RNA Library Prep Kit v2 (Illumina, CA, USA) with an insert size of 200–400 bp, after polyA selection, and sequenced on an Illumina NovaSeq 6000 platform, and 150-bp PE reads were generated. The Hi-C library construction process includes cross-linking, restricted enzyme digestion (MboI), end repair, DNA cyclization, and purification (Yu et al., 2022 (link)). PE-150-bp reads were generated on Illumina NovaSeq 6000 platforms.

For genome sequencing, the genomic DNA was extracted by the QIAGEN Genomic kit followed the manufacturer’s instructions^{65 (link)}. Nanodrop and Qubit (Invitrogen) were used to quantify the DNA. Nanopore libraries were prepared by SQK-LSK108 and sequenced using a Nanopore PromethION sequencer. The rest of the DNA was used to generate short-read sequences using an MGI-SEQ platform, with 150-bp read length and 300–500 DNA-fragment insert size. Hi-C libraries were created from fresh megagametophyte, following a previously published method^{66 (link)}. Briefly, the tissue was fixed in formaldehyde, lysed and the cross-linked DNA was digested overnight with HindIII. Sticky ends were biotinylated and proximity-ligated to generate chimeric junctions, which were subsequently physically sheared to 500–700 bp in size. The initial cross-linked long-distance physical interactions were then represented by chimeric fragments, which were processed into paired-end sequencing libraries. Paired-end reads were produced on both the MGI-SEQ and Illumina HiSeq X platforms. See Supplementary Note 3 for details on transcriptome, organelle genome and small RNA sequencing.

The maize (Zea mays) inbred line Mo17 was grown in a greenhouse with conditions of 30°C for 16 h under light and 25°C for 8 h in the dark. After 14 d, the fresh young leaf tissue was collected and frozen immediately in liquid nitrogen for DNA extraction. High-molecular-weight genomic DNA prepared by the cetyltrimethylammonium bromide (CTAB) method and followed by purification with Qiagen genomic kit (Qiagen, 13343) was used for the construction of both PacBio HiFi sequencing libraries and ONT common sequencing libraries. High-molecular-weight genomic DNA prepared by the nuclei method^{73 (link)} was used for the construction of ONT ultralong sequencing libraries. ONT and PacBio sequencing libraries were run on the Nanopore PromethION sequencer and PacBio SequeII platform, respectively. Detail libraries construction and sequencing methods are described in the Supplementary Methods.