Abstract: Copper (Cu) is an essential micronutrient for plants, but excess Cu is phytotoxic and poses a serious threat to crop production. However, the genetic basis of Cu tolerance in rice, particularly at the seedling stage, remains largely unexplored. In this study, we developed a high-throughput phenotyping system using a strip-shaped black mesh to efficiently and accurately quantify root and shoot growth dynamics under Cu stress. Using a recombinant inbred line population derived from a cross between Lemont (LM) and Teqing (TQ), two varieties with contrasting Cu tolerance, we performed QTL mapping based on multiple time-series traits. Six novel QTLs controlling Cu tolerance at the seedling stage were identified on chromosomes 1, 3, 5, and 12. Through combined analyses of resequencing, gene expression profiling, and knockout mutant phenotype analysis, we revealed OsTIP4;3, encoding a tonoplast intrinsic protein (TIP), as the causal gene underlying the major QTL controlling Cu tolerance on chromosome 1, qCuTR1. A single non-synonymous single nucleotide polymorphism (SNP) in OsTIP4;3 led to an Ala119Val substitution and was likely associated with differential Cu tolerance between the two haplotypes prevalent in japonica and indica subspecies. Additionally, the phytochelatin synthase gene OsPCS1 was identified as a candidate gene for qCuTR5; its knockout conferred enhanced Cu tolerance, suggesting a negative regulatory role distinct from canonical phytochelatin-mediated heavy metal detoxification. Promoter polymorphisms in OsPCS1 may contribute to natural variation in Cu tolerance. Our findings reveal new genetic components underlying Cu tolerance in rice and provide valuable targets for breeding Cu tolerant varieties, as well as a foundation for elucidating the molecular mechanisms of Cu detoxification in plants.

Key words: rice, copper tolerance, QTL mapping, OsTIP4, 3, OsPCS1, haplotype analysis