Yu Jianghui, Suo Shaoran, Zhou Huang, Li Wenyu, Zhang Di, Li Lingling, Wang Zhijun, Ding Xiaoping, Yuan Dingyang, Liu Citao, Duan Meijuan
College of Agriculture, Hunan Agricultural University, Changsha, Hunan 410128, China; Yuelu Shan Laboratory, Changsha, Hunan 410128, China; Beijing Golden Seed Agricultural Science and Technology Co., Ltd, Beijing 100194, China.
College of Agriculture, Hunan Agricultural University, Changsha, Hunan 410128, China; Yuelu Shan Laboratory, Changsha, Hunan 410128, China.
Gene. 2025 Jun 2;964:149600. doi: 10.1016/j.gene.2025.149600.
Cold tolerance during the germination stage is a critical agronomic trait that influences the geographic adaptation and yield stability of rice. In this study, we systematically analyzed genetic variations of the COLD1 gene using the 3 K Rice Core Collection, revealing significant associations between haplotypes and cold tolerance at the germination stage. We identified two key SNPs (SNP145938700 and SNP145938916) in the upstream regulatory region of COLD1, which stratified the population into three distinct haplotypes: Hap1, Hap2, and Hap3. An association analysis of 493 germplasms subjected to cold stress demonstrated that Hap2 (SNP145938700-AA/SNP145938916-TT) exhibited superior cold tolerance, with a seedling survival rate of 62.76 %, significantly higher than that of Hap1 (55.88 %) and Hap3 (48.25 %) (P < 0.01). Haplotype distribution analysis across 2,840 germplasms revealed that Hap2, predominantly found in japonica subspecies (TEJ/TRJ), has a latitudinal adaptation advantage, suggesting its genomic architecture harbors unique cold-adaptive modules. We developed two kompetitive allele-specific PCR (KASP) markers, Cold1-1-kasp and Cold1-2-kasp, to differentiate between Hap1 (TT/GG), Hap2 (AA/TT), and Hap3 (AA/GG) with 100 % accuracy. Validation using 35 rice germplasms for production applications confirmed that carriers of Hap2 exhibited significantly higher seedling survival rates than those of Hap3. Furthermore, molecular marker-assisted selection (MAS) was employed to introgress Hap2 from the cold-tolerant donor Meixiangzhan 2 into the cold-sensitive cultivar R9311, resulting in the novel cold-tolerant germplasm RY218. In BC2F5 generations, RY218 achieved a seedling survival rate of 69.17 %, representing a seven-fold improvement over the recurrent parent. This study establishes COLD1 Hap2 as a key genetic determinant of germination stage cold tolerance and provides robust KASP markers for precision breeding in rice.
发芽期的耐冷性是影响水稻地理适应性和产量稳定性的关键农艺性状。在本研究中，我们利用3K水稻核心种质系统分析了COLD1基因的遗传变异，揭示了单倍型与发芽期耐冷性之间的显著关联。我们在COLD1上游调控区鉴定出两个关键单核苷酸多态性位点（SNP145938700和SNP145938916），这两个位点将群体分为三种不同的单倍型：Hap1、Hap2和Hap3。对493份遭受冷胁迫的种质进行关联分析表明，Hap2（SNP145938700-AA/SNP145938916-TT）表现出优异的耐冷性，幼苗存活率为62.76%，显著高于Hap1（55.88%）和Hap3（48.25%）（P < 0.01）。对2840份种质的单倍型分布分析表明，主要存在于粳稻亚种（TEJ/TRJ）中的Hap2具有纬度适应优势，这表明其基因组结构包含独特的冷适应模块。我们开发了两种竞争性等位基因特异性PCR（KASP）标记，Cold1-1-kasp和Cold1-2-kasp，以100%的准确率区分Hap1（TT/GG）、Hap2（AA/TT）和Hap3（AA/GG）。使用35份用于生产应用的水稻种质进行验证，结果证实Hap2的携带者比Hap3的携带者表现出显著更高的幼苗存活率。此外，采用分子标记辅助选择（MAS）将耐冷供体美香占2中的Hap2导入冷敏感品种R9311中，获得了新型耐冷种质RY218。在BC2F5代中，RY218的幼苗存活率达到69.17%，比轮回亲本提高了7倍。本研究确定COLD1 Hap2是发芽期耐冷性的关键遗传决定因素，并为水稻精准育种提供了可靠的KASP标记。
