A T2T genome assembly of subtropic maize inbred line CML312 with Nanopore simplex reads

Tropical and subtropical maize germplasm harbor stress-tolerance alleles that are critical for breeding resilient cultivars capable of withstanding increasingly extreme weather conditions driven by climate change. However, high-quality reference genomes (e.g., T2T genomes) remain unavailable. Here, we report a near-complete assembly of the CML312 maize genome, a subtropical maize line, generated using only three ONT PromethION R10.4 flow cells with standard simplex reads, without dedicated ultralong ONT sequencing or PacBio HiFi data. De novo assembly with hifiasm (ONT), short-read polishing, and Hi-C scaffolding produced a 2.312 Gb genome. The assembly reached a consensus quality value of 42.46 (99.994% accuracy) and 98.3% BUSCO completeness. Integrating ab initio, homology-based, and ONT direct RNA sequencing evidence, we annotated 44,758 protein-coding genes and 74,408 transcripts with more complete gene structures supported by native full-length transcripts from ONT DRS, and 85.87% of the genome was classified as repetitive sequence. The CML312 genome carried a higher 45S ribosomal DNA copy number comparing to the temperate reference genomes, and megabase scale structural variation was strongly associated with knob heterochromatin. Knob-embedded genes showed attenuated expression. Genome assembly further showed that the white seed coat phenotype in CML312 may be caused by elevated Ccd1 copy number and structural variation in the y1 promoter sequence. This cost-effective, near-complete, and comprehensively annotated CML312 genome assembly establishes a scalable paradigm for assembling complex plant genomes with standard nanopore simplex reads and ONT DRS data, while providing a critical community resource for mining adaptive alleles to accelerate maize germplasm improvement.