Cloning of Cold6 Conferring Cold Tolerance in Rice

doi:10.16819/j.1001-7216.2025.240709

Abstract

Abstract:

【Objective】 Exploring cold-tolerant germplasm and identifying cold-tolerant genes are essential strategies to mitigate cold stress in rice. 【Method】 We treated the japonica rice variety Sasanishiki with ethyl methanesulfonate (EMS) to select mutants exhibiting enhanced cold tolerance at the seed germination stage. Candidate genes underwent cloning and functional analysis using fine mapping, CRISPR/Cas9 gene editing, expression profiling, and haplotype analysis. 【Result】 We identified the mutant M34, which displayed markedly improved cold tolerance at the seed germination stage. Genetic analysis suggested that a single recessive gene governs this trait. Fine mapping pinpointed Cold6 as the target gene. CRISPR/Cas9-mediated knockout of Cold6 confirmed its role in regulating cold tolerance during germination. Cold6 expression was observed in all rice organs, peaking in the panicles, and it encodes a protein containing several functional domains, including SNF2. Evolutionary analysis indicated differentiation of Cold6 between indica and japonica varieties, with haplotype analysis revealing 10 distinct Cold6 haplotypes. The predominant haplotypes are I-III; haplotype I is prevalent in japonica rice, whereas II and III are common in indica rice. 【Conclusion】 This study identified Cold6 as a novel regulator of cold tolerance in rice at the seed germination stage, encoding an SNF2 protein. Investigating the molecular mechanism of Cold6 enhances our understanding of the SNF2 gene family's role in rice, offering a theoretical foundation and germplasm resources for breeding cold-tolerant, high-yield northern japonica rice.

Key words: rice, cold tolerance, map-based cloning, gene editing, haplotype analysis

摘要：

【目的】挖掘鉴定水稻耐冷基因，创制耐冷种质。【方法】对粳稻品种笹锦进行了甲基磺酸乙酯(EMS)诱变，筛选发芽期耐冷性显著提高的突变体，通过遗传分析、精细定位、转基因验证、表达模式分析和单倍型分析对候选基因进行克隆和功能分析。【结果】筛选到一个发芽期耐冷性显著提高的突变体M34。遗传分析表明耐冷性状由隐性单基因控制，精细定位将目标基因锁定为Cold6。通过CRISPR/Cas9基因编辑构建Cold6敲除突变体验证Cold6参与发芽期耐冷性调控。Cold6在水稻各组织器官均有表达，其中穗部表达最高，编码SNF2等多个结构域。进化树分析显示Cold6可能存在籼粳分化，单倍型分析鉴定出Cold6可分为10个单倍型，其中单倍型Ⅰ~Ⅲ为主要单倍型，单倍型Ⅰ主要为粳稻，单倍型Ⅱ和Ⅲ主要为籼稻。【结论】本研究克隆了Cold6，一个新的水稻发芽期耐冷性调控位点，编码SNF2蛋白，对Cold6分子机理的研究不但可以加深水稻中SNF2家族基因功能的认知，还可为北方粳稻耐冷分子育种提供理论线索和种质资源。

关键词: 水稻, 耐冷性, 图位克隆, 基因编辑, 单倍型分析

TAO Shibo, XU Na, XU Zhengjin, LIU Chang, XU Quan. Cloning of Cold6 Conferring Cold Tolerance in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(6): 751-759.

陶士博, 许娜, 徐正进, 刘畅, 徐铨. 水稻发芽期耐冷基因Cold6的克隆[J]. 中国水稻科学, 2025, 39(6): 751-759.

Figures/Tables 6

Fig. 1. Phenotype of WT and M34 under 28 ℃ and 15℃ A, WT and M34 5 d after sowing under 28℃ and 15 ℃, bar = 1 cm. B, Length of shoot 5 d after sowing under 28 ℃. C, Length of shoot 5 d after sowing under 15 ℃. Data are mean ± SE (n = 5).

Fig. 2. Map-based cloning of candidate gene Cold6 A, Length of shoot of WT, M34, and F1 plants 5 d after sowing under 15℃. Data are mean ± SE (n = 5). Different letters denote significant differences (P < 0.05) by Duncan’s multiple range test; B, Distribution of shoot length of F2 population under 15℃ 5 d after sowing; C, Fine mapping of candidate gene; D, Sequence difference between WT and M34 in ORF7.

Fig. 3. CRISPR/Cas9 gene editing of Cold6 and phenotypic investigation A, Target sites and mutation sequences of Cold6 gene; B, WT and gene edited plants under 28 ℃ and 15℃ 5 d after sowing, scale bar = 1 cm; C, Shoot length of WT and gene edited plants under 28 ℃ 5 d after sowing; D, Shoot length of WT and gene edited plants under 15 ℃ 5 d after sowing. Data are mean ± SE (n = 5). Different letters denote significant differences (P < 0.05) by Duncan’s multiple range test.

Fig. 4. Expression pattern and motif domain prediction of Cold6 A, Relative expression of Cold6 in different organs of plants, data are mean ± SE (n = 3); B, Prediction of motif in Cold6.

Fig. 5. Effect of Cold6 on agronomic traits in rice A, Plant architecture of WT and cold6 mutant, scale bar = 20 cm; B, Panicle architecture of WT and cold6 mutant, scale bar = 2 cm; C, Grain shape of WT and cold6 mutant, scale bar = 1 cm; D-K, Number of panicles, plant height, panicle length, grain number per panicle, seed setting rate, 1,000-grain weight, grain length, and grain width of WT and cold6 mutant. Data are mean ± SE (n = 5).

Fig. 6. Phylogenetic and haplotype analysis of Cold6 A, Distribution of SNP and InDel on Cold6. Red color indicates the SNP causing the change of protein function predicted by PloyPhen-2; B, Phylogenetic analysis of Cold6; C, Haplotype analysis of Cold6.

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