GoKawiilPlant materials
For genome assembly and annotation, we used plant materials including, S. hybrid POJ2878, S. spontaneum 82-114 and S. officinarum XZ. These were cultivated in the greenhouse at Shenzhen. Tender leaves were collected for subsequent genome sequencing.
This study also resequenced 981 sugarcane accessions, including 78 S. officinarum, 290 S. spontaneum accessions and 613 cultivated varieties. Among the cultivated varieties, 246 were non-Chinese germplasms, while 327 were core domestic cultivars or breeding lines in China. The plant materials were grown at three experimental sites representing distinct climates: (1) a tropical monsoon climate at the sugarcane research base in Lingao County, Hainan Province (19° 34′–20° 02′ N, 109° 3′–109° 53′ E, altitude: 34 m); (2) a subtropical monsoon climate in Fusui County, Guangxi Zhuang Autonomous Region (22° 38′ 06″ N, 107° 54′ 15″ E; altitude 94 m); and (3) a subtropical plateau climate in Kaiyuan City, Yunnan Province (23° 30′–23° 58′ N, 103° 04′–103° 43′ E; altitude 1,051 m). Buds were first disinfected and then planted using a single-bud method within a randomized complete block design (1.2 m row spacing; 15 cm plant spacing). To ensure uniform germination, the field was subject to standardized management, including regular irrigation and pest and weed control.
DNA and RNA sequencing
DNA extraction
DNA extraction for single-molecule sequencing involved collecting samples and preparing long genomic DNA. The DNA was then purified using the QIAGEN Genomic Kit. For ONT-UL sequencing, the SDS method was used to extract DNA, omitting the purification step to preserve long DNA molecules. The DNA integrity and purity were evaluated using the NanoDrop system. Moreover, the Qubit 4.0 Fluorometer was used to measure DNA concentration.
PacBio sequencing
The library preparation process included gDNA shearing, DNA damage repair, end repair, A-tailing, hairpin adapter ligation, nuclease treatment, size selection and polymerase binding. A total of 15 μg of genomic DNA was used for each sample and sheared using g-TUBEs. We removed single-strand overhangs and performed repair, end-repair and A-tailing on the DNA fragments. Subsequently, we ligated the fragments with hairpin adapters, treated them with nuclease and purified the samples. We used the BluePippin system to select fragments with target sizes and the library size was verified in Agilent 2100. HiFi reads were sequenced on the PacBio Sequel Revio platform.
ONT-UL sequencing
Genomic DNA (8–10 μg per sample) was selected for fragments larger than 50 kb using the SageHLS HMW library system. The ONT-UL reads were sequenced on the PromethION platform.
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