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>
		<li>Sow the irradiated seeds after germination on a seedling bed and transplant seedlings individually to a paddy field and grow into the M1 population. 
		NOTE: Direct seeding of M1 plants sparsely could also be applied to save labor cost. Prevent outcrossing of M1 plants with other rice varieties using physical or biological isolation means.</li>
		<li>Bulk-harvest M2 seeds from the M1 plants, with 1-2 seeds from each M1 panic, to form an M2 population. 
		NOTE: In practice and for simplicity, harvest all seeds of M1 plants and after fully mixing, a portion is sampled to form an M2 population.</li>
		<li>Soak about 5,000 M2 seeds in water for 24 (indica rice)-36 (japonica rice) h at room temperature. Then let the seeds to germinate at 37 &#176;C for 2 days on moist filter paper in Petri dishes. 
		NOTE: More M2 seeds can be germinated for analysis to increase the probability of identifying mutants.</li>
		<li>Place the germinated seeds to seeding panels with small holes and grow them hydroponically for 3-4 weeks in a culture solution modified from Yoshida et al.15 in a glasshouse with a 12 h photoperiod [daytime (30&#177;2) &#176;C and night (24&#177;2) &#176;C].</li>
	</ol>
	</li>
	<li>Sampling of leaf tissues: Cut one disk (&#934; ~2 mm) from the fully extended leaf of each seeding at the same position using a hole puncher.</li>
	<li>Preparation of DNA extraction solutions
	<ol>
		<li>Buffer A: Add 2 mL of 5 M NaOH and 10 mL of 20% Tween 20 to make the final volume of 50 mL. Freshly prepare buffer A before DNA extraction.</li>
		<li>Buffer B: Add 20 mL of 1 M Tris-HCl (pH 8.0) and 80 &#956;L of 0.5 M EDTA to make a final volume of 100 mL.</li>
	</ol>
	</li>
	<li>PCR primers
	<ol>
		<li>Primers for HRM analysis: Design primers for amplification of the target sequence using software (e.g., Primer Premier5) and synthesize by a commercial company. 
		NOTE: Because HRM is less applicable to the analysis of long fragments, and hence, the amplicons should be less than 400 bp. Fragments with too high (&#62;75%) or too low (&#60;25%) GC content are also not good for HRM analysis.</li>
		<li>Primers for quality control: Use the 24 SSR markers distributed on the 12 rice chromosomes from Peng et al.16.</li>
	</ol>
	</li>
</ol>
2. DNA Extraction
<ol>
	<li>Place 4 leaf discs into each well of a 96-well PCR plate, add buffer A solution (50 mL/well). Freeze the plate in a -80 &#176;C freezer for 10 min.</li>
	<li>Defreeze the plate at room temperature, and then incubate at 95 &#176;C for 10 min.</li>
	<li>Add 50 &#956;L of buffer B to each well and mix well by vortexing.</li>
	<li>Centrifuge the plate for 1 min at 1,500 x g. The supernatant is ready for PCR.</li>
</ol>
3. PCR Amplification
<ol>
	<li>PCR Optimization
	<ol>
		<li>Add the following reagents to each PCR well: 1 &#956;L of DNA (the supernatant in step 2.4), 5 &#956;L of 2x Master Mix (containing 2x PCR buffer, 4 mmol/L MgCl2, 0.4 mmol/L 2&#39;-deoxyribonucleoside triphosphates (dNTPs), and 50 U/mL Taq DNA polymerase, 0.2 &#956;L each of 10 &#956;mol/L primers, and make a final volume up to 10 &#956;L using nuclease free water.</li>
		<li>Use a gradient-capable thermal block to determine the optimal annealing temperature for each target fragment by using the following PCR program: 5 min at 94 &#176;C, followed by 40 cycles of 30 s at 94 &#176;C, 30 s at 52-62 &#176;C (gradient temperature), and 30 s at 72 &#176;C, with a final extension at 72 &#176;C for 8 min and a hold at 16 &#176;C.</li>
		<li>Examine amplicons on 1% agarose gels for determination of the optimal annealing temperature. 
		NOTE: An optimal annealing temperature should enable specific amplification of the target fragment, without nonspecific amplification and primer dimerization.</li>
	</ol>
	</li>
	<li>PCR for HRM analysis 
	NOTE: HRM compatible plates and DNA of M2 seedlings extracted as described in step 2.4 are used for PCR.
	<ol>
		<li>Perform PCRs in a final volume of 10 &#956;L, with 1 &#956;L of DNA (supernatant), 5 &#956;L of 2x Master Mix, 0.2 &#956;L each of 10 &#956;mol/L primers, and 1 &#956;L of 10x fluorescence dye. In each plate include one wild type (WT) parented sample and one negative (without DNA) control.</li>
		<li>Add a drop of mineral oil to each well to prevent evaporation.</li>
		<li>Seal the plate with adhesive film and centrifuge at 1,000 x g for 1 min.</li>
		<li>Run the PCR using the optimized annealing temperature. 
		NOTE: In a few cases, fluorescence dye may affect PCR amplification, hence the dye is added after completion of PCR. In such cases, the dye is incorporated into DNA strands by post-PCR denaturing and annealing.</li>
	</ol>
	</li>
</ol>
4. HRM Scanning and Mutation Confirmation
<ol>
	<li>Remove the adhesive film from plate and insert the plate into an HRM machine.</li>
	<li>Select New Run from the file menu or press the Run button at the top of the screen.</li>
	<li>Specify the starting and ending temperatures for the melt from 55 &#176;C to 95 &#176;C. 
	NOTE: After the first run, the range of melt temperature can be determined for a particular fragment; hence, the melt temperature can be adjusted for subsequent analysis of the same fragment to save time.</li>
	<li>Select samples for high resolution melting analysis, exclude samples similar to the negative control.</li>
	<li>Normalize the melting curves to have the same beginning and ending fluorescence. Visually confirm that the Lower Min and Lower Max temperature cursors are in a region of the curves.</li>
	<li>Keep the &#8710;F (difference of fluorescence) level at the default setting of 0.05.</li>
	<li>Select the Common versus Variant from the Standards selection list and choose the Normal sensitivity.</li>
	<li>Use the WT as the control; samples with a &#8710;F value of &#8805;0.05 from WT are considered to contain mutant plant.</li>
	<li>Identification and confirmation of mutant plants.
	<ol>
		<li>Identify the four plants, of which each mutant pool was made.</li>
		<li>Extract DNA from each of these plants using a CTAB method according to Allen et al.17 with some modifications. 
		NOTE: DNase-free RNase and NaAc are not used when extracting DNA using the CTAB method described by Allen et al.17, as the DNA quality is good enough for further PCR amplification. Adjust the DNA to a final concentration of ~25 ng/&#956;L after quantification using a spectrophotometer.</li>
		<li>Amplify the target fragment using PCR primers and program the same as for HRM analysis.</li>
		<li>Identify specific molecular lesions by Sanger sequencing of the amplicons. 
		NOTE: Once the completion of PCR amplification, send the amplicons to a company for Sanger sequencing. The molecular lesion can be identified by comparing the sequences between the M2 plant and the WT.</li>
	</ol>
	</li>
</ol>
5. Quality Control of Selected Mutants with Molecular Markers
<ol>
	<li>Perform PCR for the 24 SSR markers in a final volume of 10 &#956;L with 25 ng of genomic DNA (extracted using the CTAB protocol), 5 &#181;L of 2x master mix, 0.2 &#181;L of each of 10 &#956;M SSR primers.</li>
	<li>Run PCR using the following program: 5 min at 94 &#176;C, followed by 30 cycles of 30 s at 94 &#176;C, 30 s at 55 &#176;C and 30 s at 72 &#176;C, with a final extension at 72 &#176;C for 7 min.</li>
	<li>Separate the amplicons on 8% polyacrylamide gels and reveal polymorphism of amplified fragments by silver staining18.</li>
	<li>Compare the SSR haplotypes of selected variants and the WT. 
	NOTE: Induced mutants often have SSR haplotypes identical to their WT, if more than one SSR markers are different between a variant and the WT, the variant is high likely to be a genetic contaminant (e.g., a mixture or outcrossed plant) rather than an induced mutant18.</li>
</ol>

<ol><li>McCallum, C. M., Comai, L., Green, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+induced+local+lesions+IN+genomes+%28TILLING%29+for+plant+functional+genomics%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((TILLING%2C+high-resolution+melting+%28HRM%29%2C+and+next-generation+sequencing+%28NGS%29+techniques+in+plant+mutation+breeding%5BTitle%5D)+AND+%22Molecular+Breeding%22%5BJournal%5D)">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/pubmed?term=((Large-scale+discovery+of+induced+point+mutations+with+high+throughput+TILLING%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">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/pubmed?term=((TILLING%3A+practical+single+nucleotide+mutation+discovery%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">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/pubmed?term=((Efficient+discovery+of+DNA+polymorphisms+in+natural+populations+by+Ecotilling%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">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/pubmed?term=((Deletion-based+reverse+genetics+in+Medicagotruncatula%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((ITILLING%3A+a+personalized+approach+to+the+identification+of+induced+mutations+in+arabidopsis%5BTitle%5D)+AND+%22Physiology%22%5BJournal%5D)">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/pubmed?term=((DHPLC+technology+for+high-throughput+detection+of+mutations+in+a+durum+wheat+TILLING+population%5BTitle%5D)+AND+%22BMC+Genetics%22%5BJournal%5D)">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/pubmed?term=((Discovery+of+rare+mutations+in+populations%3A+TILLING+by+sequencing%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">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/pubmed?term=((TILLING+by+Sequencing+%28TbyS%29+for+targeted+genome+mutagenesis+in+crops%5BTitle%5D)+AND+%22Molecular+Breeding%22%5BJournal%5D)">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/pubmed?term=((Simultaneous+mutation+detection+of+three+homoeologous+genes+in+wheat+by+High+Resolution+Melting+analysis+and+Mutation+Surveyor%5BTitle%5D)+AND+%22BMC+Plant+Biology%22%5BJournal%5D)">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/pubmed?term=((Implementation+of+two+high+through-put+techniques+in+a+novel+application%3A+detecting+point+mutations+in+large+EMS+mutated+plant+populations%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">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/pubmed?term=((Product+differentiation+by+analysis+of+DNA+melting+curves+during+the+polymerase+chain+reaction%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((High+resolution+melt+%28HRM%29+analysis+is+an+efficient+tool+to+genotype+EMS+mutants+in+complex+crop+genomes%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">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. Laboratory manual for physiological rice. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aYoshida%2C+S+%22The+International+Rice+Research+Institute%22">The International Rice Research Institute</a>. , Manila, the Philippines. (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/pubmed?term=((SSR+markers+selection+and+purity+detection+of+major+hybrid+rice+combinations+and+their+parents+in+China%5BTitle%5D)+AND+%22Chinese+Journal+of+Rice+Science%22%5BJournal%5D)">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/pubmed?term=((A+modified+protocol+for+rapid+DNA+isolation+from+plant+tissues+using+cetyltrimethymmonium+bromide%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((A+revisit+of+mutation+induction+by+gamma+rays+in+rice+%28Oryza+sativa+L.%29%3A+implications+of+microsatellite+markers+for+quality+control%5BTitle%5D)+AND+%22Molecular+Breeding%22%5BJournal%5D)">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/pubmed?term=((High-resolution+melting-based+tilling+of+%CE%B3+ray-induced+mutations+in+rice%5BTitle%5D)+AND+%22Journal+of+Zhejiang+University-Science+B%22%5BJournal%5D)">High-resolution melting-based tilling of γ ray-induced mutations in rice.</a> Journal of Zhejiang University-Science B. 19 (8), 620-629 (2018).</li>
<li>Acanda, Y., Óscar, M., Prado, M. J., González, M. V., Rey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EMS+mutagenesis+and+qPCR-HRM+prescreening+for+point+mutations+in+an+embryogenic+cell+suspension+of+grapevine%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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/pubmed?term=((Development+and+application+of+an+HRM-based%2C+safe+and+high-throughput+genotyping+system+for+photoperiod+sensitive+genic+male+sterility+gene+in+rice%5BTitle%5D)+AND+%22Journal+of+Nuclear+Agricultural+Sciences%22%5BJournal%5D)">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. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency+and+type+of+inheritable+mutations+induced+by+%CE%B3+rays+in+rice+as+revealed+by+whole+genome+sequencing%5BTitle%5D)+AND+%22Journal+of+Zhejiang+University-Science+B%22%5BJournal%5D)">Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.</a> Journal of Zhejiang University-Science B. 17 (12), 905(2016).</li>
</ol>

The authors declare that they have no conflict of interest.

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&amp;times; 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 ...

identifying-mutations-high-resolution-melting-tilling-population

Research

JoVE Journal

Genetics

7.3K Views.  Zhejiang University. Het zou heel nuttig zijn voor fokeigenschappen die technisch moeilijk of kostbaar zijn om te bepalen, zoals ziekteresistentie, mineraalgehalte. Het is een vrij eenvoudige, kosteneffectieve en hoge doorvoertechniek. Om te beginnen, plaats vier bladschijven in elke put van een 96-well PCR-plaat.Voeg 50 milliliter buffer een oplossing in elke put. Vries de plaat in een min 80-graden Celsius vriezer voor 10 minuten. Bevries vervolgens de plaat op kamertemperatuur.Incubeer op 95 grad...

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

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome&#8211;negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Biochemistry

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in target genes. TILLING is a highly valuable functional genomics tool for gene validation, especially in small grains in which transformation-based approaches hold serious limitations. Developing a robust mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened to find desired mutations, whereas a high mutagen concentration leads to high mortality in the population, leading to an insufficient number of mutagenized individuals. Once an effective population is developed, there are multiple ways to detect mutations in a gene of interest, and the choice of platform depends upon the experimental scale and availability of resources. The Cel-1 assay and agarose gel-based approach for mutant identification is convenient, reproducible, and a less resource-intensive platform. It is advantageous in that it is simple, requiring no computational knowledge, and it is especially suitable for validation of a small number of genes with basic lab equipment. In the present article, described are the methods for development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay.

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

Developmental Biology

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Immunology and Infection

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Biology

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

CRISPR-Based Efficient Genome Editing for Resistant Starch in Rice Breeding

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

Author Spotlight: Streamlining Rice Breeding with CRISPR/Cas for Obtaining Optimal Phenotypic and Agronomic Traits 

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Bioengineering

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

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

Samenvatting

Meer video's verkennen