Name: identifying mutations by high resolution melting in a tilling population of rice
Uploaded: 2019-09-02T13:00:00
Description: Watch this Scientific Journal Video about Identifying Mutations by High Resolution Melting in a TILLING Population of Rice at JoVE.com

This work was supported by the National Key Research and Development Program of China (No. 2016YFD0102103) and the National Natural Science Foundation of China (No.31701394).

1. Preparations
<ol>
	<li>Development of &#947;-rays mutagenized populations
	<ol>
		<li>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 &#947; irradiation facility (e.g., gamma cell). 
		NOTE: Seeds used for treatment should have a high viability (e.g., with a germination rate &#62;85%). The irradiation dose for indica rice could be increased to 150 Gy.</li>
		<...

<ol>
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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...

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 (e.g., disease resistance, mineral content).
TILLING was initially developed for screening point mutations induced by chemical mutagens (e.g., EMS1,3). It includes the following steps: the establishment of a TILLING population(s); DNA preparation and pooling of individual plants; PCR amplification of target DNA fragment; heteroduplexes formation by denaturation and annealing of PCR amplicons and cleavage by CEL I nuclease; and identification of mutant individuals and their specific molecular lesions3,4. However, this method is still relatively complex, time consuming, and low-throughput. To make it more efficient and with higher throughput, many modified TILLING methods have been developed, such as deletion TILLING (De-TILLING) (Table 1)1,3,5,6,7,8,9,10,11,12.
HRM curve analysis, which is based on fluorescence changes during the melting of the DNA duplex, is a simple, cost-effective, and high-throughput method for mutation screening and genotyping13. HRM has already been widely used in plant research including HRM based TILLING (HRM-TILLING) for screening SBS mutations induced by EMS mutagenesis14. Here, we presented detailed HRM-TILLING protocols for screening of mutations (both Indel and SBS) induced by gamma (&#947;) rays in rice.

<table>
	<tbody>
		<tr>
			<td>2&#215; Taq plus PCR Master Mix</td>
			<td>Tiangen, China</td>
			<td>KT201</td>
			<td>PCR buffer, dNTP and polymerase for PCR amplification</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Bio-rad, America</td>
			<td>MSP-9651</td>
			<td>Specific plate for PCR in HRM analysis</td>
		</tr>
		<tr>
			<td>Mastercycler nexus</td>
			<td>Eppendorf, German</td>
			<td>6333000073</td>
			<td>PCR amplification</td>
		</tr>
		<tr>
			<td>LightScanner</td>
			<td>Idaho Technology, USA</td>
			<td>LCSN-ASY-0011</td>
			<td>For fluorescence sampling and processing</td>
		</tr>
		<tr>
			<td>CALL-IT 2.0</td>
			<td>Idaho Technology, USA</td>
			<td></td>
			<td>For analysis of the fluorescence change</td>
		</tr>
		<tr>
			<td>EvaGreen</td>
			<td>Biotium, USA</td>
			<td>31000-T</td>
			<td>Fluorescence dye of HRM</td>
		</tr>
		<tr>
			<td>Nanodrop 2000</td>
			<td>Thermo Scientific, USA</td>
			<td>ND2000</td>
			<td>For DNA quantification</td>
		</tr>
	</tbody>
</table>

identifying mutations by high resolution melting in a tilling population of rice

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.

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 (&#916;F &#60; 0.05). HRM curves significantly different from the WT (&#916;F &#62; 0.05) were grouped with color(s) di...

Watch this Scientific Journal Video about Identifying Mutations by High Resolution Melting in a TILLING Population of Rice at JoVE.com

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

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).

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

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