Natural variation in CTF1 conferring cold tolerance at the flowering stage in rice.

Improving cold tolerance at the flowering stage (CTF) in rice is crucial for minimising yield loss, making the identification and application of cold-tolerant genes and QTLs imperative for effective molecular breeding. The long lead time, dependence on cold treatment conditions, and the inherent complexity of the trait make studying the genetic basis of CTF in rice challenging. To date, the fine-mapping or cloning of QTLs specific to CTF has not yet been achieved. In this study, single segment substitution lines (SSSLs) were constructed using HJX74 as the recipient and IR58025B, known for good CTF, as the donor. This approach led to the identification of two cold tolerance QTLs, qCTF3 and qCTF6, in rice. qCTF6 has promising breeding potential. Further, we identified the causal gene CTF1 underlying qCTF6 through map-based cloning. CTF1 which encodes a conserved putative protein, has two SNPs within its coding sequence that influence CTF in rice. Additionally, genetic variations in the promoter of CTF1 also contributes to CTF. Thirteen variant sites of CTF1 in the four cold tolerance SSSLs are consistent with the IR58025B. Moreover, we analysed 307 accessions to characterise haplotypes based on the 13 variation sites, identifying five distinct haplotypes. The selection and evolutionary analysis indicate that the cold-tolerant haplotype of CTF1 is a newly generated mutation that has undergone selection in japonica during domestication. This study not only provides a novel favourable gene for molecular breeding of CTF but also highlights the potential of CTF1 in advancing rice breeding.