High-resolution melting-based TILLING of γ ray-induced mutations in rice

Abstract

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.

中文概要

目的

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。

创新点

建立起了基于HRM技术、适用于伽玛射线诱发的小片段插入/缺失突变的高通量TILLING体系(HRM-TILLING)。

方法

通过不同野生型/突变型比例混池DNA的HRM分析,确定HRM检测不同类型插入/缺失突变的能力,确定M₂植株突变检测的适宜混池比例,并用一个伽玛诱变M₂ 群体(n=4560)筛选OsLCT1和SPDT两个基因的突变体,确定实际效果。

结论

以4 株M₂植株混样,采用HRM可以有效检出突变。建立的基于HRM的TILLING体系适用于伽玛射线诱发突变的高通量筛选。