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Preparation of Rice Plant Genomic DNAfor Various Applications
Lian Zhou,
1,2,3
Rongxin Shen,
1,2,3
Xingliang Ma,
1,2,3
Heying Li,
1,2,3
Gousi Li,
1,2,3
and Yao-Guang Liu
1,2
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Guangzhou, China
2
College of Life Sciences, South China Agricultural University, Guangzhou, China
3
These authors contributed equally to this work 
Research on plant molecular biology, genetic engineering, and crop molecularbreeding involves various manipulations of plant genomic DNAs (gDNAs).These studies require preparing gDNAs from plant materials using suitablemethods according to the yield, intactness, and purity of the resultant gDNAsamples. Here we describe protocols for preparation of rice plant gDNAs forvarious applications including: (1) maxipreps and (2) minipreps of gDNAsfor restriction analysis, gene cloning, PCR amplification, genomic sequencing,and genotyping; (3) preparation of megabase-sized nuclear DNA for construc-tion of large-insert genomic libraries and long-range physical mapping; and(4) 96-well-format high-throughput gDNA preparation for PCR-based geno-typing. The methods are also suitable for other plant species including dicots.
C
 2016 by John Wiley & Sons, Inc.Keywords: DNA preparation
 
genomic DNA
 
nuclear DNA
 
plants
How to cite this article:
Zhou, L., Shen, R. Ma, X., Li, H., Li, G. and Liu, Y.-G. 2016.Preparation of rice plant genomic DNA for various applications.
Curr. Protoc. Plant Biol.
 1:29-42.doi: 10.1002/cppb.20002
INTRODUCTION
Plant molecular biology research and crop molecular breeding require analysis of ge-nomic DNAs (gDNAs). For some applications such as restriction analysis, gene cloning,PCRamplification,sequencingoflargefragments,andconstructionofgenomiclibraries,higher-quality and relatively larger-size gDNAs are required. However, for large-scalegenotyping analysis, the high throughput of the gDNA preparation method is the firstpriority. So far, many plant gDNA preparation methods have been developed for variousapplications.The CTAB (hexadecyltrimethylammonium bromide) DNA preparation method is suit-able for preparation of a large amount of high-quality gDNA (Murray and Thompson,1980; Richards et al., 1994). The resultant gDNA is suitable for Southern blot analysis,PCR amplification of large fragments, genomic library construction, and next-generationsequencing. For most applications such as restriction analysis, PCR amplification of DNA fragments, PCR-based genotyping, and next-generation sequencing, a simplerSDS (sodium dodecyl sulfate) method (Dellaporta et al., 1983) and modified CTABmethod (Doyle and Doyle, 1987) can be used for minipreps of gDNA. In the caseof large-scale genotyping with DNA markers, e.g., SSRs (simple sequence repeats),
Current Protocols in Plant Biology
 1:29-42, May 2016Published online May 2016 in Wiley Online Library (wileyonlinelibrary.com).doi: 10.1002/cppb.20002Copyright
 C
 2016 John Wiley & Sons, Inc.
Preparationof Plant gDNA
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Volume 1
 
Table 1
 A Summary of the Four Basic Protocols for Plant gDNA Preparation
Basic Protocol 1 Basic Protocol 2 Basic Protocol 3 Basic Protocol 4Time course 2-3 hr 1.5-2 hr 2-3 hr 6-8 min (96 samples)Throughput Low High Low Very highTissue amount 2-5 g
 
0.5 g 20-30 g 2-5 mgDNA length
 >
50 kb
 >
20 kb
 >
2.5 Mb
 <
10 kbStorage period Long Long Long ShortYield 100-200
 µ
g/gtissue50-100
 µ
g 200-300
 µ
g 1-3 ng
µ
l (150
 µ
l)Cytoplasmic DNA Included Included Excluded IncludedApplication PCR, genomiccloning,restriction,Southern blottingPCR, DNAmarkers,restriction,genomic cloningLarge-insertcloning,DNA-fiber FISHPCR, DNA markers
indels (insertions/deletions), and SNPs (single nucleotide polymorphisms), preparationof gDNA templates from massive samples is time-consuming and tedious. Therefore,a very high-throughput gDNA preparation method has been developed to deal withlarge number of samples for PCR-based genotyping (Wang et al., 2013). Very-large-sizegDNA is necessary for large-insert genomic library construction and long-range physicalmapping. Since the chromosomal DNA molecules are extremely fragile in liquid buffer,embedding the isolated nuclei into low-melting point agarose gel plugs can providegDNA of megabase size (Liu and Whittier, 1994).Some plant cells contain high amounts of secondary metabolites, such as polyphe-nols, tannins, or polysaccharides, which are difficult to remove. Thus, some modifica-tions should be included during gDNA preparation. For example, polyvinyl pyrrolidone(PVP) helps to remove polyphenols by forming hydrogen-bonding complexes (John,1992). NaCl can be applied to remove the polysaccharides (Lodhi et al., 1994). AfterCTAB lysis, anion-exchange chromatography can also help to improve the quality of thegDNA extracted from oak, elm, pine, fir, poplar, maize, and rhododendron (Csaikl et al.,1998).Weintroducetheoriginallarge-scale(maxi)CTABpreparationmethodinBasicProtocol1 of this article, which yields high-quality DNA. In Basic Protocol 2, we describe amodified SDS and CTAB mini-extraction method. Basic Protocol 3 provides a methodfor preparation of megabase-size nuclear gDNA. In Basic Protocol 4, we describe a low-cost, very-high-throughput method for rapid preparation of gDNA and application of thetemplate DNAs to PCR reactions in a 96-well format. These protocols are summarizedin Table 1.
 BASIC  PROTOCOL 1
PREPARATION OF PLANT GDNA USING CTAB
This protocol describes a gDNA extraction method using the amine-based cationic qua-ternary surfactant CTAB in the extraction buffer for lysing cells and denaturing proteins.CTABcanformasolublecomplexwithnucleicacidsunderhigh-saltconditions(
>
0.7MNaCl), and precipitate DNA at lower salt concentrations (
<
0.5 M). This method is suit-ableforlarge-scaleDNAextractionandyieldshigh-qualitygDNA,whichcanbeusedforcloning, Southern blot hybridization, and other manipulations that require high-qualityDNA.
Preparationof Plant gDNA
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Volume 1 Current Protocols in Plant Biology
 
 Materials
Rice plant tissueLiquid nitrogenCTAB extraction buffer (see recipe)24:1 (v/v) chloroform/isoamyl alcohol10% (w/v) CTABCTAB precipitation buffer (see recipe)High-salt TE buffer, pH 8.0 (see recipe)10 mg/ml RNase A (store at
20
°
C)Isopropanol (or ethanol)70% ethanolTE buffer, pH 8.0 (no NaCl; see recipe)Lambda DNARestriction enzyme (e.g.,
 Hin
dIII,
 Eco
RI)Mortars and pestles50-ml centrifuge tubes and tube rack 55
°
 and 65
°
C water bathsShakerCentrifuge (Eppendorf 5810R) and angle rotor (F-34-6-38, with adapters) for50-ml centrifuge tubesSpectrophotometer (NanoDrop2000, Thermo Scientific)Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000)
Grinding of plant tissue
1. Grind about 4 to 5 g rice plant tissue for each sample into a fine powder with amortar and a pestle in the presence of liquid nitrogen. Transfer the frozen tissuepowder to an organic solvent–resistant 50-ml centrifuge tube and temporarily storein a freezer when grinding other samples.
Work with even numbers of samples for each round of gDNA preparation, to facilitatecentrifugation.Use young tissue (leaves) and avoid larger stems and veins to achieve higher gDNA yields with minimal contamination of polysaccharide. If samples need to be stored in a freezer, it is desirable to first freeze the samples withliquid nitrogen in advance. Avoid thawing the samples during storage and before adding extraction buffer in thegDNA preparation.
 Extraction and purification of gDNA
2. Add 20 ml (or 4 ml per g tissue) CTAB extraction buffer (preheated to 80
°
C) andmix thoroughly by inversion. Incubate the lysate mixture at 65
°
C for 15 to 20 min,with occasionally shaking.3. Add 15 ml (or 0.75 times the volume of the added extraction buffer) of 24:1 (v/v)chloroform/isoamyl alcohol to each tube and mix well by inversion. Set the tubes ina slanted position in a shaker and shake at about 50 rpm for 10 min. Centrifuge at 15min at
12,000
×
g
 (8,000 rpm in Eppendorf F-34-6-38 rotor), room temperature.
 Be sure to balance the tubes when adding chloroform/isoamyl alcohol.
4. Recover supernatant into a 50-ml centrifuge tube. Add 0.1 vol of 10% CTAB andmix thoroughly.
 Be careful not to transfer the impurities and debris.
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Current Protocols in Plant Biology Volume 1

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