通过高分辨率熔融在水稻种群中识别突变
1National Key Laboratory of Rice Biology, Zhejiang Provincial Key Laboratory of Crop Genetic Resources, Institute of Crop Science,Zhejiang University, 2Jiaxing Academy of Agricultural Sciences, 3Institute of Nuclear Agricultural Sciences,Zhejiang University, 4The New Countryside Development Institute at Zhejiang University
Summary
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。
Abstract
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。
Introduction
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）。
Protocol
1. 准备工作 β射线诱变种群的发展 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。 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，?…
Representative Results
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）按软件与 WT 不同的颜色分组（图 1）。 <str…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 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 |
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Cite This Article
Li, S., Yu, Y., Liu, S., Fu, H., Huang, J., Shu, Q., Tan, Y. Identifying Mutations by High Resolution Melting in a TILLING Population of Rice. J. Vis. Exp. (151), e59960, doi:10.3791/59960 (2019).