Utilization of natural alleles and haplotypes of Ctb1 for rice cold adaptability.

Abstract

Cold stress during the booting stage of rice (Oryza sativa) significantly reduces yields, particularly in temperate and high-altitude regions. This study investigates the Ctb1 gene, critical for booting-stage cold tolerance, to improve breeding of resilient rice varieties. Re-sequencing the Ctb1 promoter in 202 accessions identified six Insertions and/or deletions (InDels) and four Single nucleotide polymorphisms (SNPs), with an InDel at -1,302 bp significantly boosting Ctb1 expression and cold tolerance. Accessions carrying this InDel (Haplotype I) exhibited the highest tolerance. Near-isogenic lines (NIL-Ctb1^HapI) introduced Haplotype I into the cold-sensitive Huazhan (HZ) variety, resulting in a 5.9-fold increase in Ctb1 expression, higher seedling survival, improved pollen fertility, a 64.2 % increase in seed setting rate, and a 12 g per plant yield boost under cold stress. These findings confirm the critical role of the -1,302 InDel in cold tolerance and establish NIL-Ctb1^HapI as a valuable breeding tool for cold-resilient rice.