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Melody S. Clark (Ed.)
i Plant Molecular Biology
I A Laboratory Manual
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With 37 Figures, Including a Color Plate
1
Technische Universitat Darmstadt
FACHBEREICH 10 — B1OLOGIE
Hi
— Bibliothek
—
SchnittspahnstraBe 10
D-6 4 2 8 7 D a r m s t a d t
Inv.-Nr.
Springer
Contents
Part I
Basic Molecular Techniques
1
1
Genomic DNA Isolation, Southern Blotting
and Hybridization
3
Isolation of Total Genomic DNA
S. WlLKIE
3
1.1
1.1.1
1.1.2
1.1.3
Introduction
Theory Underlying Extraction Procedures
Extraction of Whole Genomic DNA
by the CTAB Method
1.1.4
Micro-Isolation of DNA by the CTAB Method
1.1.5
Extraction of Whole Genomic DNA
by the Method of Dellaporta,
Wood and Hicks (1983)
1.1.6 " Extraction of DNA from Isolated Nuclei
1.1.7
Purification of DNA Extracts by Differential Density
Centrifugation on a Caesium Chloride Gradient . . . .
1.1.8
Estimation of DNA Concentration,
Purity and Quality
References
1.2
3
4
5
8
9
11
12
14
14
Southern Blotting
M.S. CLARK
1.2.1
Introduction
1.2.2 ' Isolation of Total Genomic DNA
1.2.3
Restriction Enzyme Digestion of Genomic DNA
and Check Mini-Gel
1.2.4
Agarose Gel Electrophoresis and Transfer
of DNA to Nylon Membrane
References
15
15
15
16
20
25
X
Contents
1.3
Hybridization with Radioactive Probes
M.S. CLARK
1.3.1
1.3.2
1.3.2.1
1.3.2.2
1.3.2.3
1.3.3
1.3.3.1
1.3.3.2
1.3.4
1.3.5
Introduction
Probe Labelling
PCR Labelling of Probes
Oligolabelling
Isolation of Fragments from Gels for Labelling . . . .
Purification of Probe
CL6B Spin Columns . .
Sephadex G-50 Columns
Hybridization Protocol
Filter Stripping
References
~
1.4
Non-Radioactive Methods of Detection
on Southern Blots
26
26
27
27
29,
30J
31|
31
3|
P. LEROY, M. MERLINO, S. NEGRE,
J.C. CAISSARD, P. SOURDILLE, Y.H. LU,
and M. BERNARD
1.4.1
1.4.2
1.4.3
1.4.4
1.4.5
Introduction
Extraction of Total Genomic DNA
Restriction Enzyme Digestion of the DNA
and Southern Blotting
Probe Labelling by PCR-DIG
Hybridization, Washing and Development
Plus Probe Removal
References
2
Cloning from Genomic DNA
and Production of Libraries
2.1
Use of PCRs in Plant Molecular Biology
41
4!
4v
A. GUEVARA-GARCIA, L. HERRERA-ESTRELLA,
and G. OLMEDO-ALVAREZ
2.1.1
2.1.1.1
2.1.1.2
2.1.1.3
2.1.2
Introduction
Primer Design
Defining the Right Annealing Temperature
Fidelity of PCR Products
Procedure for DNA Amplification
yj
5X1
Contents
2.1.3
2.1.4
2.1.4.1
2.1.4.2
2.1.4.3
2.1.4A2.1.4.5
2.1.5
2.1.5.1
2.1.5.2
2.1.5.3
2.2
2.2.4
2.2.5
2.2.5.1
2.2.5.2
2.2.6
2.2.7
2.2.7.1
2.2.7.2
2.2.8
2.3
2.3.1
2.3.1.1
'
2.3.1.3
PCR Optimization
Cloning
T-A System
,
Preparation of T-Vectors
Cleaning of PCR Products
Removal of Taq DNA Polymerase
by Double Phenol Extraction
Ligation and Transformation
Specific Applications for PCR Other Than Cloning . .
RNA Amplification
Screening of Transgenic Plants
Detection of Plant Pathogens
References
61
63
63
64
65
66
66
67
68
68
L70
73
Plasmid Libraries
2:2.1
2.2.2
2.2.3
2.3.1.2
XI
M.S. CLARK
75
Introduction
DNA Isolation
Restriction Enzyme Digestion of Genomic DNA
and Plasmid
Ligation into Plasmid Vector
Preparation of Competent Cells
CaCl2 Method
Hanahan Method
Transformation Protocol
Analysis of Colonies
Colony Blotting
Mini-Prep Isolation Method
Storage of Library/Plasmids
References
75
77
77
79
81
81
82
83
85
85
89
91
91
Lambda Genomic Cloning
G. ELGAR
94
^
".'.'.'
Introduction
General Characteristics of Cloning
in Lambda Replacement Vectors
Generation of Insert DNA Representative
of the Whole Genome
Plating Out Libraries and Selecting
for Recombinants
94
.
95
96
98
XII
2.3.2
2.3.2.1
2.3.2.2
2.3.2.3
2.3.2.4
2.3.2.5
2.3.3
2.3.4
2.3.5
2.3.6
2.3.7
2.3.8
2.3.8.1
2.3.8.2
2.4
2.4.1
2.4.2
2.4.3
2.4.3.1
2.4.3.2
2.4.3.3
2.4.3.4
2.4.4
2.4.4.1
2.4.4.2
Contents
Protocol for Construction of a Lambda Genomic
Library in the Replacement Vector 22001
Preparation of Vector DNA Arms
Preparation of Insert DNA
by Partial Sau3AI Digestion
Preparation of Insert DNA by Mbol/dam
Methylase Titration
De-Phosphorylation of Insert DNA
Ligation and Packaging of Phage Libraries
Titering of Lambda 2001 Libraries.
Amplification of Lambda^2001 Genomic Libraries. .
Plating Out Lambda Libraries for Amplification
or Screening by Hybridization
Preparation of Membranes^
for Plaque Hybridizations
Isolation of Positive Phage and Generation
of High-Titer Stocks
Preparation of DNA
from Hybridization-Positive Phage
Crude Phage Mini-Preps for the Digestion
and Southern Blot Analysis of Multiple Clones . . . .
Isolation of Total Insert from Phage DNA
References
Cosmid Libraries
C.-N. Liu
Introduction
Cloning Strategy
Library Production
Preparation of Plant DNA for Cloning
Preparation of Vector DNA
Ligation and-Packaging
Amplification and Storage of a Cosmid Library. . . .
Suggestions for Screening a Cosmid Library
and Analyzing Recombinant Cosmid Clones
Screening the Cosmid Library
Restriction Mapping of Recombinant Cosmids . . . .
References
99
101
103
105
106
108
109
110
110
Ill
112
113
114
115
116
117
117
118
120
121
123
124
126
127
127
127
130
Contents
2.5
2.5.1
2.5.1.1
2.5.1.2
2.5.2
2.5.3
2.5.4
2.5.5
2.5.6
2.5.7
2.5.8
3
3.1
XIII
Yeast Artificial Chromosome (YAC) Libraries
E. MATALLANA, J. SIMPSON, and P.A. GUZMAN : . . .
131
Introduction
YAC Vectors and Cloning Strategies
Megabase DNA from Plants
Preparation of Megabase DNA from Plants
Vector Preparation
EcoRI Partial Digests of Genomic DNA
and Size Selection of EcoRI Fragments by PFGE . .
Ligation Mixtures and Preparation
x
of Sample for Transformation . T
.
Yeast Spheroplast Transformation
Preparation of Yeast Artificial Chromosome Clones
and Analysis by PFGE
Storage and Screening of YAC Libraries
References
:..
131
133
136
137
140
Extraction of RNA, Cloning and Subtractive
Hybridization
141
144
145
148
149
151
154
Isolation and Analysis of Messenger RNA
from Plant Cells: Cloning of cDNAs
P. LESSARD, V. DECROOCQ, and M. THOMAS
154
Introduction
RNA Isolation
Isolation of Total Cellular RNA
RNA Isolation from Small Amounts of Material . .
Isolation of RNA from Recalcitrant Plant Tissue . .
Isolation of RNA from Organelles
Purification of mRNAs
*
RNA Analysis
:
Northern Blotting: RNA Electrophoresis
and Transfer
3.1.4.2 Northern Hybridization
3.1.4.3 Dot/Slot Blotting
3.1.5 "• Run-Off Transcription
3.1.5.1 Isolation of Nuclei
3.1.5.2 Transcription of Isolated Nuclei
.
3.1.5.3 Hybridization of Cloned Probes
to In Vitro-Labelled Transcripts
154
158
158
162
163
164
164
167
3.1.1
3.1.2
3.1.2.1
3.1.2.2
3.1.2.3
3< 1.2.4
3.1.3
3.1.4
3.1.4.1
167
169
170
172
173
174
176
XIV
3.1.6
3.1.7
3.1.7.1
3.1.7.2
3.1.8
3.1.9
3.1.9.1
3.1.9.2
3.1.10
3.2
Contents
Mapping of the 5' and 3' Termini
of Specific mRNA
Production of cDNA
Reverse Transcription
Association of Reverse Transcription with PCR . . .
Quantification of Specific mRNA
(Quantitative RT-PCR)
Cloning of cDNAs
Construction of a cDNA Library: Introduction. . . .
Construction of cDNA Libraries
Following In Vitro Amplification of cDNA
A New" Way of Cloning Differentially Expressed
cDNAs: Differential-Display Reverse TranscriptasePolymerase Chain Reaction (DDRT-PCR)
References
191
198
20j
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.2.7
3.2.8
3.2.9
3.2.10
3.2.11
Introduction
RNA Preparation
Production of Tailed First Strand cDNA C
Amplification of Representative cDNA Pools
Hybridization
Subtraction
Re-Amplification
Further Hybridization/Subtraction Cycles
Analysis of Subtracted cDNA
Cloning to Form the Subtracted Library
Concluding Comments
References
4
Characterization of Clones
201
20
201
2J
2||
2J
2j
2p
Ik
p
2J
21
:
DNA Sequencing
J.R. CLARK
4.1.1
4.1.2
182
18J!
Subtractive Hybridization
of Different mRNA Populations
H.C.C. FOOTE, G. BRADY, G.J. THORLBY,
and C.H. FRANKLIN
4.1 ^
1771
179'
179?
180J
Introduction
Generation of Nested Unidirectional Deletions . . . .
221
22jj
22"
Contents
4. 1.2.3
4. 1.2.4
4. 1.2.5
4. [.2.6
4. 1.3
4. 1.3.1
4. [.3.2
4. [.3.3
4. [.3.4
4. [.3.5
4.1.3.6
4. [.3.7
4.2 ,
Selection of Cloning Vector for ExoIII Deletion
Preparation of Closed Circular DNA
Restriction Endonuclease Digestion . ; .
ExoIII Digestion
Religation and Transformation of E. coli
Screening Transformants
Plasmid DNA Sequencing
Plasmid DNA Preparation
Template Denaturation and Primer Annealing .
Sequencing with T7 DNA Polymerase
Sequencing Gel Casting. . . .^
Sample Preparation and Electrophoresis
Gel Fixing, Drying and Autoradiography
Reading and Recording the Sequence
References
XV
227
229
231
233
235
235
236
237
238
239
241
246
247
249
255
Expression of Cloned Genes
T. PEHU
257
4.2.1
4.2.2
4.2.3
4.2.3.1
4.2.3.2
4.2.4
4.2.4.1
4.2.4.2
Introduction
Vector Construction and Expression of Protein
Protein Purification
Purification of Inclusion Bodies
Gel Electrophoresis
Western Blotting
Electroblotting
Immunological Visualization of Proteins
References
257
260
262
264
265
270
271
272
274
Part-II
Characterization of Plant DNA
279
5
5.1
5.2
5.2.1
5.2.2
Organelle DNA Isolation
M. LANDGREN and K. GLIMELIUS
281
Introduction
Isolation of Chloroplast and Mitochondrial DNA
CpDNA Isolation . . ,
MtDNA Isolation
References
281
288
290
293
298
;
I
XVI
6
Contents
RAPD Analysis: Use for Genome Characterization,
Tagging Traits and Mapping
R. WAUGH.
305
6.1
6.2
6.2.1
6.2.1.1
6.2.1.2
6.2.2
6.3
6.3.1
6.3.2 "
6.3.2.1
6.3.2.2
6.3.2.3
6.4
Introduction
Genetic Fingerprinting . . .
Methods
DNA Isolation
Basic RAPD Procedure
Analysis of Genetic Relationships
Development of Genetic Linkage Maps
CrloBal Mapping Strategies
Targeted Approaches
Near-Isogenic Lines (NILs)
Pooling Strategies
Genetic Stocks
Future Applications
References
305
306
308
308
308
314
316
316
318
318
322
324
326
328
7
RFLP Mapping of Plant Nuclear Genomes:
Planning of Experiments, Linkage Map Construction,
and QTL Mapping
j
J.H. VAN DEN BERG, S.D. CH AS ALOW,
and R. WAUGH
7.1
7.2
7.2.1
7.2.2
7.2.2.1
7.2.2.2
7.3
7.3.1
7.3.2
7.3.3
7.4
7.5
7.5.1
7.5.2
7.5.2.1
7.5.2.2
Introduction
Planning of Experiments
Homozygous Versus Heterozygous Parents
Selecting a Segregating Population
Population Type
Parent Selection and Population Size
Generating and Selecting RFLP Probes
cDNA Probes
Genomic DNA.Probes
Probes from Other Mapping Populations
Laboratory Procedures for RFLP Analysis
Linkage Map Construction
Single-Locus Segregation Analysis
Detecting Linkage Between Loci
Tests with Fixed Expected Single-Locus
Segregation Ratios
Tests for Association
334
334
337
337
338
338
341
343
343
344
344
344
345
347
350
351
352
Contents
7.5.2.3
7.5.2.4
7.5.3
7.5.3.1
7.5.3.2
7.5.4
7.5.5
7.5.5.1
7.5.5.2
7.5.6
7.5.6.1
7.5.6.2
7.5.6.3
7.5.6.4
7.6
7.6.1
7.6.2
7.6.3
7.6.4
7.6.5
7.7
The LOD Score Test
.
Analysis Strategies for Detecting Linkage
Estimating Recombination Frequencies
and Map Distances
Pairwise Estimation
Multipoint Estimation
Assembling Linkage Groups
Ordering Loci
Criteria for Comparing Locus Orders
Methods for Searching Possible Orders
Sensitivity of Map Construction to Departures
from Common Assumptions . .
Heterogeneity of Recombination Frequencies
Genotype Misclassification
Differential Viability
Crossover Interference
Mapping Quantitative Trait Loci
Analysis of Variance on Marker Classes . . . . : . . . .
Interval Mapping
Multiple-QTL Methods
Epistasis
;
Pleiotropic Effects
Conclusion
References
Part III Genetic Engineering: Methodology and Analysis
Plant Gene Transfer
R.H. POTTER and M.G.K. JONES
Introduction
Agrobacterium-Mediated Gene Transfer
8.2
Transformation of Tobacco
^
8.2.1
with Agrobacterium tumefaciens
8.2.2
Transformation of Tomato
Using Agrobacterium tumefaciens
' Direct Gene Transfer
8.3
8.3.1
Direct Gene Transfer to Rice Protoplasts .
8.3.2
Particle Bombardment
8.3.2. 1 Transformation of Barley Immature Embryos
by Particle Bombardment
XVII
354
356
357359
360
361
362
362
363
365
365
366
367
367
368
370
375
383
384
385
386
387
397
8
399
399
402
403
406
409
409
414
416
XVIII
Contents
8.4
8.4.1
8.4.2
Protoplast Fusion
Chemical Fusion.
Electrofusion . . .
References
Molecular Characterization of Transformed Plants
J.F. TOPPING and K. LINDSEY
9.1
9.2
9.3
9.3.1
9.4
9.4.1
9.4.1.1
9.4.1.2
9.4.2
9.4.3
10
Introduction
Selectable Marker Genes
Molecular Characterization of Transformants . . .
Interpreting the Results of Sourthern Analysis . .
Analysis of Reporter Gene Expression
gusA ((3-Glucuronidase) as a Reporter Gene . . . .
Assay of (3-Glucuronidase (GUS) Activity
by Fluorimetry
Assay of Protein Content in Tissue Homogenate .
Histochemical Localization of GUS Activity . . . .
luc (Firefly Luciferase) as a Reporter Gene
References
Molecular Characterization of Somatic Hybrids
Y.-S. Xu and E. PEHU
10.1
10.2
10.3
10.3.1
10.3.1.1
10.3.1.2
10.3.1.3
10.3.2
10.3.2.1
10.3.2.2
Introduction
Genefal Characterization of Somatic Hybrids . .
Molecular Characterization of Somatic Hybrids
Nuclear Genome
RAPDs
RFLPs
Species-Specific Repetitive DNA
Organellar Genomes
cpDNA . . .•^!_.__._
mtDNA
References
11
Cytological Characterization of Transformed Plants:
Mapping of Low-Copy and Repetitive DNA
Sequences by Fluorescent In Situ Hybridization (FISH)
I.J. LEITCH, A.Y. KENTON, A.S.
and M.D. BENNETT
*•
PAROKONNY,
4M
Contents
11.1
11.2
Introduction
Optimizing the ISH Protocol
for Detecting Low-Copy Sequences
11.2.1 Chromosome Preparations
11.2.2 High Metaphase Index
11.2.3.- Probe Types
11.2.4 Choice of Labels
11.2.5 Choice of Labelling Method
11.2.6 Choice of Detection System
11.2.7 Imaging Low-Copy Signal
11.2.8 Multiple Labelling and Reprobing
11.3
Visualizing Fluorescent ISH Signal
11.4
Chromosome Preparation
11.5
Probe Preparation
, .>
.
11.5.1 Purifying DNA by Gel Extraction
11.5.2 Labelling DNA by Nick Translation
11.6
Protocol for In Situ Hybridization
References
12
461
.--
12.2.1
12.2.2
12.2.3
12.2.4
12.2.5
12.2.6
12.2.7
12.3
12.3.1
12.3.2
12.3.3
462
462
463
463
463
464
465
465
465
466
467
467
467
467
468
482
i I-
I'i
|
!
Cytological Characterization of Somatic Hybrids:
Detection of Genome Origin by Genomic In Situ
Hybridization (GISH)
A.Y. KENTON, A.S. PAROKONNY, I.J. LEITCH,
and M.D. BENNETT
12.1
12.2
XIX
Introduction
Optimizing GISH for Distinguishing
Between Genomes
Choice of Labelling Method
Total Genomic DNA as a Probe
for In Situ Hybridization
Probe Penetration/Target Accessibility . r- -,-. . . .
Stringency
Blocking DNA
Choice of Detection System
Viewing and Recording the Results
Cytological Preparations
Pretreatment and Fixation
Chromosome Preparations
from Root-Tip Squashes
Chromosome Preparations from Enzymic Digests . .
486
486
487
487
487
488
488
489
489
490
490
493
494
495
I
XX
12.3.4
12.4
12.4.1
12.4.2
12.4.3
12.5
12.6
Contents
Meiotic Chromosome Preparations
Probe Preparation
Isolation of Genomic DNA
Biotin Labelling of Genomic DNA
by Nick Translation . . . :
Testing Biotin Incorporation
by Spot Hybridization
Genomic In Situ Hybridization
Microscopy and Photography
References
Addendum to Chapters 11 and 12
In Situ Hybridization in Plant Species
with Small Chromosomes
N. LAPITAN
A.I
A.2
A.2.1
A.2.2
A.3
Introduction
Obtaining Prometaphase Chromosomes
Using Aphidicolin
Aphidicolin Treatment for Synchronizing Tomato
Root Tip Cells
*
Chromosome Squashes
In Situ Hybridization
References
Appendix Nuclear Genome Sizes
of Important Plant Species
C.-N. Liu
Subject Index