通过高分辨率熔解分析在水稻TILLING群体中鉴定突变位点

摘要

本文介绍了基于高分辨率熔解分析（HRM）的靶向诱导基因组局部突变（TILLING）技术方案。该方法利用DNA双链熔解过程中荧光信号的变化，适用于对插入/缺失（Indel）和单碱基替换（SBS）进行高通量筛选。

摘要

靶标诱导基因组局部突变检测技术（TILLING）是一种用于高通量筛选诱导突变的反向遗传学策略。然而，传统的TILLING体系在插入/缺失（Indel）检测方面适用性较低，且需要更为复杂的步骤，例如CEL I核酸酶消化和凝胶电泳。为了提高通量和筛选效率，并实现对插入/缺失突变和单碱基替换（SBSs）的同步检测，已开发出一种基于高分辨率熔解曲线（HRM）的新型TILLING系统。本文详细介绍了一种HRM-TILLING实验方案，并展示了其在突变筛选中的应用。该方法通过检测双链DNA在高温下的变性过程，分析PCR扩增子中的突变情况。HRM分析可在PCR完成后直接进行，无需额外处理步骤。此外，本方法整合了一种简单、安全、快速（SSF）的DNA提取技术，可同时用于识别插入/缺失突变和单碱基替换突变。由于其操作简便、结果稳定且通量高，该技术在水稻及其他作物的突变扫描中具有广泛的应用前景。

引言

突变体是植物功能基因组学研究和新品种育种的重要遗传资源。大约20年前，正向遗传学方法（即从突变体筛选到基因克隆或品种培育）曾是利用人工诱变的主要且唯一手段。McCallum 等人¹开发的一种新型反向遗传学方法——TILLING（Targeting Induced Local Lesions In Genomes，基因组中诱导局部突变的靶向技术），开创了一种新范式，并已被广泛应用于大量动物和植物物种中²。TILLING 特别适用于那些在技术上难以或成本较高的性状育种（例如抗病性、矿物质含量）。

TILLING 最初用于筛选化学诱变剂（如 EMS^1,3）诱导的点突变，其流程包括以下步骤：构建 TILLING 群体；提取并混合个体植株的 DNA；靶 DNA 片段的 PCR 扩增；通过 PCR 产物的变性与复性形成异源双链，并利用 CEL I 核酸酶进行切割；最后鉴定突变个体及其特异性的分子变异位点^3,4。然而，该方法仍相对复杂、耗时较长且通量较低。为了提高效率和通量，已发展出多种改进的 TILLING 方法，例如缺失 TILLING（De-TILLING）（表 1）^{1,3,5,6,7,8,9,10,11,12}。

高分辨率熔解曲线分析（HRM）基于DNA双链解链过程中荧光信号的变化，是一种简单、经济且高通量的突变筛查与基因分型方法¹³。HRM已广泛应用于植物研究中，包括基于HRM的TILLING技术（HRM-TILLING），用于筛选由EMS诱变诱导的单碱基替换（SBS）突变¹⁴。本文提供了利用HRM-TILLING技术在水稻中筛选γ射线诱导的突变（包括插入缺失突变和单碱基替换突变）的详细实验方案。

方案

1. Preparations

1. Development of γ-rays mutagenized populations
   1. 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 γ irradiation facility (e.g., gamma cell).
      NOTE: Seeds used for treatment should have a high viability (e.g., with a germination rate >85%). The irradiation dose for indica rice could be increased to 150 Gy.
   2. 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.
   3. 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.
   4. 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 °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.
   5. 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±2) °C and night (24±2) °C].
2. Sampling of leaf tissues: Cut one disk (Φ ~2 mm) from the fully extended leaf of each seeding at the same position using a hole puncher.
3. Preparation of DNA extraction solutions
   1. 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.
   2. Buffer B: Add 20 mL of 1 M Tris-HCl (pH 8.0) and 80 μL of 0.5 M EDTA to make a final volume of 100 mL.
4. PCR primers
   1. 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 (>75%) or too low (<25%) GC content are also not good for HRM analysis.
   2. Primers for quality control: Use the 24 SSR markers distributed on the 12 rice chromosomes from Peng et al.¹⁶.

2. DNA Extraction

1. 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 °C freezer for 10 min.
2. Defreeze the plate at room temperature, and then incubate at 95 °C for 10 min.
3. Add 50 μL of buffer B to each well and mix well by vortexing.
4. Centrifuge the plate for 1 min at 1,500 x g. The supernatant is ready for PCR.

3. PCR Amplification

1. PCR Optimization
   1. Add the following reagents to each PCR well: 1 μL of DNA (the supernatant in step 2.4), 5 μL of 2x Master Mix (containing 2x PCR buffer, 4 mmol/L MgCl₂, 0.4 mmol/L 2'-deoxyribonucleoside triphosphates (dNTPs), and 50 U/mL Taq DNA polymerase, 0.2 μL each of 10 μmol/L primers, and make a final volume up to 10 μL using nuclease free water.
   2. 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 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 52-62 °C (gradient temperature), and 30 s at 72 °C, with a final extension at 72 °C for 8 min and a hold at 16 °C.
   3. 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.
2. PCR for HRM analysis
   NOTE: HRM compatible plates and DNA of M₂ seedlings extracted as described in step 2.4 are used for PCR.
   1. Perform PCRs in a final volume of 10 μL, with 1 μL of DNA (supernatant), 5 μL of 2x Master Mix, 0.2 μL each of 10 μmol/L primers, and 1 μL of 10x fluorescence dye. In each plate include one wild type (WT) parented sample and one negative (without DNA) control.
   2. Add a drop of mineral oil to each well to prevent evaporation.
   3. Seal the plate with adhesive film and centrifuge at 1,000 x g for 1 min.
   4. 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.

4. HRM Scanning and Mutation Confirmation

1. Remove the adhesive film from plate and insert the plate into an HRM machine.
2. Select New Run from the file menu or press the Run button at the top of the screen.
3. Specify the starting and ending temperatures for the melt from 55 °C to 95 °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.
4. Select samples for high resolution melting analysis, exclude samples similar to the negative control.
5. 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.
6. Keep the ∆F (difference of fluorescence) level at the default setting of 0.05.
7. Select the Common versus Variant from the Standards selection list and choose the Normal sensitivity.
8. Use the WT as the control; samples with a ∆F value of ≥0.05 from WT are considered to contain mutant plant.
9. Identification and confirmation of mutant plants.
   1. Identify the four plants, of which each mutant pool was made.
   2. 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/μL after quantification using a spectrophotometer.
   3. Amplify the target fragment using PCR primers and program the same as for HRM analysis.
   4. 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.

5. Quality Control of Selected Mutants with Molecular Markers

1. Perform PCR for the 24 SSR markers in a final volume of 10 μL with 25 ng of genomic DNA (extracted using the CTAB protocol), 5 µL of 2x master mix, 0.2 µL of each of 10 μM SSR primers.
2. Run PCR using the following program: 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C, with a final extension at 72 °C for 7 min.
3. Separate the amplicons on 8% polyacrylamide gels and reveal polymorphism of amplified fragments by silver staining¹⁸.
4. 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.

结果

HRM 扫描与分析

总共制备了来自4,560株M₂幼苗的1,140个混合DNA样本，并进行了PCR扩增。分别针对OsLCT1和SPDT扩增出大小为195 bp和259 bp的两个片段（表2）。大多数样本的熔解曲线与野生型（WT）无显著差异（ΔF < 0.05）。熔解曲线与野生型存在显著差异（ΔF > 0.05）的样本被软件以不同于野生型的颜色进行分组（图1）。

突变确认与频率

采用单株幼苗的DNA样品进行扩增，并对相应片段进行测序。通过测序色谱图可确认突变类型及其在基因上的位置（图2）。在4,560株M₂代幼苗中鉴定出3个突变位点，包括两个插入缺失突变（Indel...

讨论

TILLING 已被证明是一种强大的反向遗传学工具，可用于鉴定诱导突变，以进行基因功能分析和作物育种。对于某些不易观察或难以确定的性状，采用基于高通量 PCR 的突变检测 TILLING 方法，可有效获得不同基因的突变体。HRM-TILLING 方法已应用于番茄¹²、小麦¹¹和葡萄²⁰的 EMS 诱变群体中进行突变筛选。本文展示了一种更简便且更高效的 HRM-TILLING 方法，适用于 Indel 和 SBS 突变的筛选。

为了提高效率，采用SSF方法而非耗时耗力且需要使用有毒化学试剂的传统CTAB方法进行DNA提取。Si等人²¹比较了SSF法与CTAB法所提取DNA的质量。尽管发现SSF法提取的DNA纯度低于CTAB法，但其仍能满足水稻PCR及高分辨率熔解曲线（HRM）分析的要求。然而需要注意的是，SSF法提取的DNA不能长期保存，因此不适合用于构建永久性突变体文库。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
2× Taq plus PCR Master Mix	Tiangen, 中国	KT201	用于PCR扩增的PCR缓冲液、dNTP和聚合酶
96孔板	Bio-rad, 美国	MSP-9651	HRM分析中PCR专用板
Mastercycler nexus	Eppendorf, 德国	6333000073	用于PCR扩增
LightScanner	Idaho Technology, 美国	LCSN-ASY-0011	用于荧光信号的采集与处理
CALL-IT 2.0	Idaho Technology, 美国		用于分析荧光变化
EvaGreen	Biotium, 美国	31000-T	HRM用荧光染料
Nanodrop 2000	Thermo Scientific, 美国	ND2000	用于DNA定量