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

这项工作得到了国家重点研究发展计划（2016YFD0102103）和国家自然科学基金（第31701394号）的支持。

1. 准备工作
<ol><li>
β射线诱变种群的发展
	<ol>
<li>处理约20，000个干水稻种子（含水量约14%）在 β辐照设施（例如伽马细胞）中，在100 Gy（1 Gy/min）处的137克伽马射线的japonica水稻线（例如DS552）。 注：用于治疗的种子应具有高活力（例如，发芽率 >85%）。稻谷的辐照剂量可增加到150Gy。</li>
		<li>在幼苗床上发芽后，将辐照种子播种，将幼苗单独移植到稻田中，?...

<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. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+and+type+of+inheritable+mutations+induced+by+&#947;+rays+in+rice+as+revealed+by+whole+genome+sequencing.">Frequency and type of inheritable mutations induced by &#947; rays in rice as revealed by whole genome sequencing.</a> Journal of Zhejiang University-Science B. 17 (12), 905 (2016).</li></ol>

TILLING已被证明是一个强大的反向遗传工具，用于识别基因功能分析和作物育种的诱导突变。对于一些不易观察或确定的特征，使用高通量PCR突变检测的TILLING是获得不同基因突变体的有用方法。在番茄12、小麦11和葡萄藤20的EMS诱变种群中，已采用HRM-TILLING方法进行突变筛选。本文演示了一种更简单、更强大的人力资源管理-TILLING，适用于Indel和SB...

突变是植物功能基因组学研究和新品种育种的重要遗传资源。前向遗传学方法（即从突变选择到基因克隆或品种发育）是大约20年前使用诱导突变的主要和唯一方法。McCallum等人1日开发一种新的反向遗传学方法，即"在基因组中定位诱导局部病变"，开创了一种新的范式，并自二十年代以来已应用于大量动植物物种2。TILLING特别适用于培育技术上难以确定或昂贵的特性（例如，抗病性、矿物质含量）。
TILLING最初是为筛选由化学诱变引起的点突变（例如EMS1，3）开发的。它包括以下步骤：建立一个TILLING人口;单个植物的DNA制备和汇集;靶DNA片段的PCR扩增;通过减少PCR增生和裂解的PCR增生和裂解通过CEL I核酸酶形成异质双相的形成;和识别突变个体及其特定的分子病变3，4。但是，此方法仍然相对复杂、耗时且吞吐量低。为了使它更有效率和更高的吞吐量，已经开发了许多经过修改的 TILLING 方法，例如删除 TILLING （de-TILLING） 1、3、5、6、 7，8，9，10，11，12。
HRM曲线分析基于DNA双工熔化过程中的荧光变化，是一种简单、经济、高通量的突变筛选和基因分型方法。HRM已广泛应用于植物研究，包括基于人力资源管理的TILLING（HRM-TILLING），用于筛选EMS诱变14诱导的SBS突变。在这里，我们提出了详细的HRM-TILLING协议，用于筛选水稻中伽马（α）射线引起的突变（Indel和SBS）。

<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

靶向诱导局部病变在基因组（TILLING）是一种反向遗传学的策略，用于诱导突变的高通量筛选。然而，TILLING 系统对于插入/删除 （Indel） 检测的适用性较低，传统的 TILLING 需要更复杂的步骤，如 CEL I 核酸酶消化和凝胶电泳。为了提高产量和选择效率，并使得Indels和单基级（SBS）的筛选成为可能，开发了一种新的基于高分辨率熔融（HRM）的TILLING系统。在这里，我们提出了详细的HRM-TILLING协议，并展示了其在突变筛选中的应用。该方法可以通过测量高温下双链DNA的变性来分析PCR增氧核糖核酸的突变。HRM 分析在 PCR 后直接执行，无需额外处理。此外，一种简单、安全、快速的DNA提取方法与人力资源管理-TILLING集成在一起，用于识别Indels和SBS。其简单性、鲁棒性和高通量使其在水稻和其他作物的突变扫描中具有潜在的用处。

人力资源管理扫描与分析
总共从4，560 M2幼苗中合成了1，140个DNA样本，并进行PCR扩增。OsLCT1和SPDT分别放大了两个大小分别为195 bp和259 bp的碎片（表2）。大多数样品的熔融曲线与 WT（μF < 0.05）没有显著差异。与 WT 显著不同的 HRM 曲线（#F > 0.05）按软件与 WT 不同的颜色分组（图 1）。
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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

在本文中，我们提出了被描述为基于高分辨率熔融分析（HRM）的基因组中靶向诱导局部病变（TILLING）的协议。该方法利用DNA双工熔化过程中的荧光变化，适用于插入/删除（Indel）和单基位（SBS）的高通量筛选。

通过高分辨率熔融在水稻种群中识别突变

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

对于技术上难以确定或成本高昂的繁殖特性（如抗病性、矿物质含量）来说，这非常有用。这是一种非常简单、经济高效、高通量的技术。首先，将四个叶盘放入 96 井 PCR 板的每个井中。在每个井中加入50毫升缓冲液 A 溶液。将盘子冷冻在零下80摄氏度的冰柜中10分钟。然后，在室温下解冻板。在95摄氏度下孵育10分钟。在每个井中加入50微升的缓冲B，通过涡旋混合。在 1，500 倍 g 下将板离心一分钟。收集作为PCRDNA的上一液。首先，使用一个和PCR模板的DNA上因，以优化退火温度。在每个PCR井中加入试剂和一微升DNA上流剂。将 PCR 管放在一个能够梯度的热块中，根据手稿调整加热程序，以确定每个目标片段的最佳退火温度。在 1%的加糖凝胶上对 PCR 产品执行电泳，以检查安胶。确定可实现目标片段特定放大的最佳退火温度。要执行 HRM 分析，请将试剂组合在一起，包括一个 10 倍荧光染料的微升和 HRM 兼容板的每个孔中的 1 微升 DNA 上流剂。在每个板块中，包括一个野生型父性样本和一个没有DNA的阴性对照。在每一个井中加入一滴矿物油，以防止蒸发。然后，用胶粘膜密封板，以1000倍g离心1分钟。使用优化的退火温度运行 PCR。现在，从板中去除粘合膜，并将板插入 HRM 机器。在软件中，从文件菜单中选择"新运行"，或按屏幕顶部的"运行"按钮。指定熔体从 55 到 95 摄氏度的起始和结束温度。选择用于高分辨率熔化分析的样本，并排除类似于负对的样本。使熔化曲线正常化，使其具有相同的开始和结束荧光。直观地确认较低的最小和较低的最高温度光标位于曲线区域中。保持三角洲 F，荧光差，在默认设置 05 的水平。从"标准"选择列表中，选择公共与变体，然后选择正常灵敏度。选择 H12 以使用野生类型作为控件。增量 F 值大于 05 的样品被视为含有突变植物。然后，使用CTAB方法，从突变池中提取四种植物的DNA，其融化曲线与野生类型明显不同。使用分光光度计进行定量后，使用 NanoDrop 2000 将 DNA 调整为每微升约 25 纳米的最终浓度。将 0.4 微升 PCR 底板添加到 PCR 管中的 DNA 样品中，将它们放入热循环器中，并调整程序以放大目标片段。然后，将 PCR 产品发送给公司进行测序。桑格测序后，通过比较M2植物和野生类型之间的序列来比较分子病变。在这项研究中，HRM扫描和分析M2幼苗在OsLCT1或SPDT基因突变显示，大多数样本的融化曲线没有显著差异，与野生类型与三角洲F小于05。突变体在温度为90至92摄氏度和81.5至83.5摄氏度时，与三角洲F大于05的HRM曲线明显不同。4，560M2幼苗的基因突变类型和位置通过测序色谱得到确认。在OsLCT1的4，304个碱基对下，一个G到A突变的异质位点被识别出来。在OsLCT1的4，240个碱基对上出现了一个单核苷酸 A 插入，在 SPDT 的 5，948 到 5，950 基对处观察到 TTC 三核苷酸缺失。我在 HRM 分析之前优化 PCR。凝胶中的染料有点有害。运行凝胶时，记得戴上手套。

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

高分辨率融合PCR用于补体受体1长度多态性基因分型：阿尔茨海默病基因易感性评估的创新工具

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

使用 Ever 设计自动化、高通量、连续细胞生长实验

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...