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>
	<li>McCallum, C. M., Comai, L., Green, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiology. 123, 439-442 (2000).</li><li>Taheri, S., Abdullah, T. L., Jain, S. M., Sahebi, M., Azizi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING,+high-resolution+melting+(HRM),+and+next-generation+sequencing+(NGS)+techniques+in+plant+mutation+breeding.">TILLING, high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding.</a> Molecular Breeding. 37 (3), 40 (2017).</li><li>Till, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+discovery+of+induced+point+mutations+with+high+throughput+TILLING.">Large-scale discovery of induced point mutations with high throughput TILLING.</a> Genome Research. 13 (3), 524-530 (2003).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single+nucleotide+mutation+discovery.">TILLING: practical single nucleotide mutation discovery.</a> The Plant Journal. 45 (4), 684-694 (2006).</li><li>Comai, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+discovery+of+DNA+polymorphisms+in+natural+populations+by+Ecotilling.">Efficient discovery of DNA polymorphisms in natural populations by Ecotilling.</a> The Plant Journal. 37, 778-786 (2004).</li><li>Rogers, C., Wen, J., Chen, R., Oldroyd, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion-based+reverse+genetics+in+Medicagotruncatula.">Deletion-based reverse genetics in Medicagotruncatula.</a> Plant Physiology. 151 (3), 1077 (2009).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+arabidopsis.">ITILLING: a personalized approach to the identification of induced mutations in arabidopsis.</a> Physiology. 154 (1), 25-35 (2010).</li><li>Colasuonno, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DHPLC+technology+for+high-throughput+detection+of+mutations+in+a+durum+wheat+TILLING+population.">DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.</a> BMC Genetics. 17 (1), 43 (2016).</li><li>Tsai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+rare+mutations+in+populations:+TILLING+by+sequencing.">Discovery of rare mutations in populations: TILLING by sequencing.</a> Plant Physiology. 156, 1257-1268 (2011).</li><li>Kumar, A. P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING+by+Sequencing+(TbyS)+for+targeted+genome+mutagenesis+in+crops.">TILLING by Sequencing (TbyS) for targeted genome mutagenesis in crops.</a> Molecular Breeding. 37, 14 (2017).</li><li>Dong, C., Vincent, K., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+mutation+detection+of+three+homoeologous+genes+in+wheat+by+High+Resolution+Melting+analysis+and+Mutation+Surveyor.">Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor.</a> BMC Plant Biology. 9, 143 (2009).</li><li>Gady, A. L., Herman, F. W., Wal, M. H. V. D., Loo, E. N. V., Visser, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+two+high+through-put+techniques+in+a+novel+application:+detecting+point+mutations+in+large+EMS+mutated+plant+populations.">Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.</a> Plant Methods. 5 (41), 6974-6977 (2009).</li><li>Ririe, K. M., Rasmussen, R. P., Wittwer, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+differentiation+by+analysis+of+DNA+melting+curves+during+the+polymerase+chain+reaction.">Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.</a> Analytical Biochemistry. 245, 154-160 (1997).</li><li>Lochlainn, S. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melt+(HRM)+analysis+is+an+efficient+tool+to+genotype+EMS+mutants+in+complex+crop+genomes.">High resolution melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.</a> Plant Methods. 7, 43 (2011).</li><li>Yoshida, S., Forno, D. A., Cock, J. H., Gomez, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+manual+for+physiological+rice.">Laboratory manual for physiological rice.</a> The International Rice Research Institute. , (1976).</li><li>Peng, S. T., Zhuang, J. Y., Yan, Q. C., Zheng, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSR+markers+selection+and+purity+detection+of+major+hybrid+rice+combinations+and+their+parents+in+China.">SSR markers selection and purity detection of major hybrid rice combinations and their parents in China.</a> Chinese Journal of Rice Science. 17, 1-5 (2003).</li><li>Allen, G. C., Flores-Vergara, M. A., Krasynanski, S., Kumar, S., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+protocol+for+rapid+DNA+isolation+from+plant+tissues+using+cetyltrimethymmonium+bromide.">A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethymmonium bromide.</a> Nature. 1 (5), 2320-2325 (2006).</li><li>Fu, H. W., Li, Y. F., Shu, Q. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revisit+of+mutation+induction+by+gamma+rays+in+rice+(Oryza+sativa+L.):+implications+of+microsatellite+markers+for+quality+control.">A revisit of mutation induction by gamma rays in rice (Oryza sativa L.): implications of microsatellite markers for quality control.</a> Molecular Breeding. 22 (2), 281-288 (2008).</li><li>Li, S., Liu, S. M., Fu, H. W., Huang, J. Z., Shu, Q. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+melting-based+tilling+of+&#947;+ray-induced+mutations+in+rice.">High-resolution melting-based tilling of &#947; ray-induced mutations in rice.</a> Journal of Zhejiang University-Science B. 19 (8), 620-629 (2018).</li><li>Acanda, Y., &#211;scar, M., Prado, M. J., Gonz&#225;lez, M. V., Rey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMS+mutagenesis+and+qPCR-HRM+prescreening+for+point+mutations+in+an+embryogenic+cell+suspension+of+grapevine.">EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine.</a> Cell Reports. 33 (3), 471-481 (2014).</li><li>Si, H. J., Wang, Q., Liu, Y. Y., Huang, J. Z., Shu, Q. Y., Tan, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+an+HRM-based,+safe+and+high-throughput+genotyping+system+for+photoperiod+sensitive+genic+male+sterility+gene+in+rice.">Development and application of an HRM-based, safe and high-throughput genotyping system for photoperiod sensitive genic male sterility gene in rice.</a> Journal of Nuclear Agricultural Sciences. 31 (11), 2081-2086 (2017).</li><li>Li, S., Zheng, Y. C., Cui, H. R., Fu, H. W., Shu, Q. Y., Huang, J. 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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 ...

It would be quite useful for breeding traits that are technically difficult or costly to be determined, such as disease resistance, mineral content. It's a quite simple, cost-effective, and high-throughput technique. To begin, place four leaf discs into each well of a 96-well PCR plate.Add 50 milliliters of buffer A solution into each well. Freeze the plate in a minus 80-degree Celsius freezer for 10 minutes. Then, defreeze the plate at room temperature.Incubate at 95 degrees Celsius for 10 minutes. Add 50 microliters of buffer B to each well, and mix well by vortexing. Centrifuge the plate for one minute at 1, 500 times g.Collect the supernatant as DNA for PCR. First, use the DNA supernatant from one well as PCR template to optimize the annealing temperature. Add reagents and one microliter of DNA supernatant into each PCR well.Place the PCR tubes in a gradient-capable thermal block, and adjust the heating program according to the manuscript to determine the optimal annealing temperature for each target fragment. Perform electrophoresis for the PCR product on 1%agarose gels to examine amplicons. Determine the optimal annealing temperature that enables specific amplification of the target fragment.To perform HRM analysis, combine reagents, including one microliter of 10 times fluorescence dye and one microliter of the DNA supernatant in each well of HRM-compatible plates. In each plate, include one wild-type parented sample and one negative control without DNA. Add a drop of mineral oil to each well to prevent evaporation.Then, seal the plate with adhesive film, and centrifuge at 1, 000 times g for one minute. Run the PCR using the optimized annealing temperature. Now, remove the adhesive film from the plate, and insert the plate into an HRM machine.In the software, select New Run from the file menu, or press the Run button at the top of the screen. Specify the starting and ending temperatures for the melt from 55 to 95 degrees Celsius. Select samples for high-resolution melting analysis, and exclude samples similar to the negative control.Normalize the melting curves to have the same beginning and ending fluorescence. Visually confirm that the lower minimum and lower maximum temperature cursors are in a region of the curves. Keep the delta F, difference of fluorescence, level at the default setting of 05.From the Standards selection list, select the common versus variant, and choose the normal sensitivity. Select H12 to use the wild type as the control. Samples with a delta F value greater than 05 are considered to contain mutant plant.Then, using a CTAB method, extract DNA from each of the four plants from the mutant pool with the melting curve significantly different from the wild type. After quantification using a spectrophotometer, adjust the DNA with the NanoDrop 2000 to a final concentration of approximately 25 nanograms per microliter. Add 0.4 microliters of PCR primers into the DNA sample in the PCR tubes, place them in a thermal cycler, and adjust the program to amplify the target fragment.Then, send the PCR products to a company for sequencing. After Sanger sequencing, compare the molecular lesion by comparing the sequences between the M2 plant and the wild type. In this study, HRM scanning and analysis of M2 seedlings for mutations in OsLCT1 or SPDT genes showed most samples had melting curves not significantly different from the wild type with delta F less than 05.The mutants showed significantly different HRM curves with delta Fs greater than 05 at temperatures of 90 to 92 degrees Celsius and 81.5 to 83.5 degrees Celsius, respectively. The mutation type and position on the genes from 4, 560 M2 seedlings were confirmed by the sequencing chromatograms. A heterozygous site with a mutation of G to A at 4, 304 base pairs of OsLCT1 was identified.One single nucleotide A insertion appeared at the 4, 240 base pairs of OsLCT1, and a TTC trinucleotide deletion was observed at the position of 5, 948 to 5, 950 base pairs of SPDT. I optimize the PCR before HRM analysis. The dye in the gel is a bit harmful.Remember to put on gloves when running the gel.

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Genetics

7.2K visualizações.  Zhejiang University. Seria bastante útil para traços de reprodução que são tecnicamente difíceis ou caros a serem determinados, como resistência a doenças, conteúdo mineral. É uma técnica simples, econômica e de alto rendimento. Para começar, coloque quatro discos de folha em cada poço de uma placa PCR de 96 poços.Adicione 50 mililitros de tampão Uma solução em cada poço. Congele a placa em um congelador Celsius de menos 80 graus por 10 minutos. Em seguida, descongele a placa em temperat...