High-resolution melting-based TILLING of γ ray-induced mutations in rice
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Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.
Key wordsMutation screening High-resolution melting (HRM) analysis Targeting Induced Local Lesions IN Genomes (TILLING) Mutant Indel γ ray Rice
建立适用于筛选伽马射线诱发突变的、基于高分辨率熔解曲线(high-resolution melting,HRM)技术的高通量定向诱导基因组局部突变技术 (Targeting Induced Local Lesions IN Genomes, TILLING)体系。
关键词突变筛选 高分辨率熔解曲线(HRM) 定向诱导基因组局部突变技术(TILLING) 突变体 插入缺失 伽玛射线 水稻
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