Combining QTL-seq and linkage mapping to fine map a candidate gene in qCTS6 for cold tolerance at the seedling stage in rice

Cold stress caused by low temperatures is an important factor restricting rice production. Identification of cold-tolerance genes that can stably express in cold environments is crucial for molecular rice breeding. In this study, we employed high-throughput quantitative trait locus sequencing (QTL-seq) analyses in a 460-individual F2:3 mapping population to identify major QTL genomic regions governing cold tolerance at the seedling stage in rice. A novel major QTL (qCTS6) controlling the survival rate (SR) under low-temperature conditions of 9°C/10 days was mapped on the 2.60-Mb interval on chromosome 6. Twenty-seven single-nucleotide polymorphism (SNP) markers were designed for the qCST6 region based on re-sequencing data, and local QTL mapping was conducted using traditional linkage analysis. Eventually, we mapped qCTS6 to a 96.6-kb region containing 13 annotated genes, of which seven predicted genes contained 13 non-synonymous SNP loci. Quantitative reverse transcription PCR analysis revealed that only Os06g0719500, an OsbZIP54 transcription factor, was strongly induced by cold stress. Haplotype analysis confirmed that +376 bp (T>A) in the OsbZIP54 coding region played a key role in regulating cold tolerance in rice. We identified OsbZIP54 as a novel regulatory gene associated with rice cold-responsive traits, with its Dongfu-104 allele showing specific cold-induction expression serving as an important molecular variation for rice improvement. This result is expected to further exploration of the genetic mechanism of rice cold tolerance at the seedling stage and improve cold tolerance in rice varieties by marker-assisted selection.