水稻苗期耐低温基因COLD1新功能标记的设计与验证

籼稻和粳稻在苗期耐低温基因COLD1的第4外显子存在1个功能性单碱基变异SNP2,粳型COLD1 ^Jap等位基因低温耐受性表现更强,具有重要的育种利用价值。通过籼粳杂交,可将粳型COLD1 ^Jap等位基因导入籼稻品种,提高其低温耐受力。为提高COLD1基因的选择效率,根据粳型COLD1 ^Jap与籼型COLD1 ^Ind基因存在的单核苷酸差异,结合扩增受阻突变体系PCR的技术原理设计功能标记。应用5个籼稻品种、5个粳稻品种、1个籼粳杂交F₁个体以及1个籼粳杂交F₂群体对功能标记进行检测验证。结果表明,所设计的功能标记可准确区分纯合粳型COLD1 ^Jap、纯合籼型COLD1 ^Ind和杂合基因型,其扩增带型与基因型完全一致,是一种鉴定COLD1基因的有效方法。该标记弥补了前人设计的衍生型酶切扩增多态性序列功能标记费用昂贵、操作复杂及费工费时等不足,可广泛应用于水稻COLD1基因的资源鉴定和分子标记辅助选择育种。

Design and Verification of New Functional Marker of Chilling-Tolerance COLD1 Gene in Rice Seedling Stage

The nucleotide variation of SNP2 in the fourth exon of COLD1 gene associated with the difference of chilling tolerance at the seedling stage between indica and japonica rice. Individuals with japonica COLD1 ^Jap allele have higher chilling tolerance. Therefore, the COLD1 ^Jap allele of japonica rice could be introduced via hybridization, thereby enhancing the cold tolerance of indica rice. In order to improve the selection efficiency of COLD1 gene, a new functional marker for detecting the single nucleotide polymorphism SNP2 between COLD1 ^Jap and COLD1 ^Indwas designed based on the technical principles of amplification refractory mutation system PCR. Furthermore, the availability of the functional marker was evaluated using five indica varieties, five japonica varieties, one F₁ individual and one F₂ from the indica × japonica cross. The results showed that the designed functional markers could accurately identify homozygous COLD1 ^Jap, homozygous COLD1 ^Ind and heterozygous genotypes, and amplified bands were identical to their corresponding genotypes, suggesting that it was an effective method for identifying COLD1 gene using this marker designed in this study. The method is simple and inexpensive and could overcome the shortcoming of the COLD1 gene functional markers previously designed based on derived cleaved amplified polymorphic sequence. In conclusion, the new functional marker could be widely used in genetic resource identification and molecular marker-assisted selection breeding of rice COLD1 gene.