Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Summary

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Abstract

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

Introduction

Mutants are important genetic resources for plant functional genomics research and breeding of new varieties. A forward genetics approach (i.e. from mutant selection to gene cloning or variety development) used to be the main and sole method for the use of induced mutations about 20 years ago. The development of a novel reverse genetics method, TILLING (Targeting Induced Local Lesions In Genomes) by McCallum et al.¹ opened a new paradigm and it has since been applied in a great number of animal and plant species². TILLING is particularly useful for breeding traits that are technically difficult or costly to be determined....

Protocol

1. Preparations

1. Development of γ-rays mutagenized populations
   1. Treat about 20,000 dried rice seeds (with moisture content of ca. 14%) of a japonica rice line (e.g., DS552) with ¹³⁷Cs gamma rays at 100 Gy (1 Gy/min) in a γ irradiation facility (e.g., gamma cell).
      NOTE: Seeds used for treatment should have a high viability (e.g., with a germination rate >85%). The irradiation dose for indica rice could be increased to 150 Gy.
   2. Sow the irradiated seeds after germination on a seedling bed and transplant seedlings individually to a paddy field and grow into the M₁....

Results

HRM Scanning and Analysis

In total, 1,140 pooled DNA samples from 4,560 M₂ seedlings were produced and subjected to PCR amplification. Two fragments with the size of 195 bp and 259 bp were amplified for OsLCT1 and SPDT, respectively (Table 2). Most samples had melting curves not significantly different from the WT (ΔF < 0.05). HRM curves significantly different from the WT (ΔF > 0.05) were grouped with color(s) different from.......

Discussion

TILLING has proved to be a powerful reverse genetic tool for identifying induced mutations for gene functional analysis and crop breeding. For some traits not easily observed or determined, TILLING with high-throughput PCR-based mutation detection can be a useful method to obtain mutants for different genes. HRM-TILLING method has been used in EMS-mutagenized populations of tomato¹², wheat¹¹ and grapevine²⁰ for mutation screening. In this paper, a si.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2× Taq plus PCR Master Mix	Tiangen, China	KT201	PCR buffer, dNTP and polymerase for PCR amplification
96-well plate	Bio-rad, America	MSP-9651	Specific plate for PCR in HRM analysis
Mastercycler nexus	Eppendorf, German	6333000073	PCR amplification
LightScanner	Idaho Technology, USA	LCSN-ASY-0011	For fluorescence sampling and processing
CALL-IT 2.0	Idaho Technology, USA		For analysis of the fluorescence change
EvaGreen	Biotium, USA	31000-T	Fluorescence dye of HRM
Nanodrop 2000	Thermo Scientific, USA	ND2000	For DNA quantification