structure, dynamics and analysis of plant genomes ii - a
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Structure, Dynamics and Analysis of Plant Genomes II
- A toolbox of molecular biology -
I. Evidence for the transgene in transformed plants
II. Natural recombination and plant breeding
III. Generation of genome maps
IV. Current status of plant genome analysis
Lecture Molecular Biology and
Biotechnology of Plants
Rüdiger Hell
Centre for Organismal Studies
Analysis of transformation events
Experiment Scutellae Regen. Plants PAT Positive GUS Positive Transform.
(R0) frequency
1 240 6 6 6 2.5%
2 300 7 ND 5 1.7
3 140 4 ND 3 2.1
4 120 0 0 0 0.0
5 150 2 1 1 0.7
• Not all regenerated plants are transgenic (‚false positive‘)
• Transgenic lines with single copy and locus insertion are wanted
Statistical analysis of transformation
Transgenic No. of No. of PPT resistant PPT sensitive % PPT
plants (R1) seeds tested plants plants plants resistant
B4 174 40 29 11 72
B6 121 33 23 9 73
Segregation analysis of transformation
How homogeneity is achieved: Haploids
• Double haploid lines are dihaploid plants and completely homozygous for each allel
• Single haploid lines are used in hybrid breeding
Anther culture of tobaco. Duchefa p27
Method 1: haploid androgenesis
• Regeneration of haploid plants from
anthers or microspores (pollen), e.g.
of barley
• Doubling of 1n chromosome set by
colchicin: dihaploid
Method 2: Fertilization with pollen
from a different species
• Wheat can be fertilized with pollen
from maize
• Double fertilization of zygote (diploid)
and endosperm takes place, but
endosperm nucleus (triploid) is unable
to develop into an endosperm
• Isolated embryo are nurtured in vitro
• Chromosomes of unrelated species
(here:maize) are gradually eliminated
during mitotic divisions
• Haploid plants result
Structure, Dynamics and Analysis of Plant Genomes II
I. Evidence for the transgene in transformed plants
II. Natural recombination and plant breeding
III. Generation of genome maps
• Genetic maps
• Molecular maps
IV. Current status of plant genome analysis
Mendel’s first law: The Principle of Segregation
• Wrinkled seed phenotype from
mutation in gene encoding starch
branching enzyme II
• Seeds carry less amylopectin but
more sucrose
• Before ripening the osmotic
potential is higher compared to
wildtype
• After ripening the water loss is more
severe: wrinkling Buchanan Box 13.3, 646
3 Spherical: 1 Wrinkled
Monohybrid cross
Mendel’s first law:
The Principle of Segregation:
The two members of a heredity factor
pair segregate from each other in the
formation of gametes.
Now:
Two members = alleles; Heredity factor = gene
Definition of a gene: DNA-fragment, that encodes
for a trait or phenotype; can exist in different forms
(wildtype/mutant, polymorphism, allele)
Polymorphism: DNA sequence variants of coding
or non-coding sectors (with or without
consequences for function)
Locus: Position in the genome with undefined size.
Two allelic genes are always at the same locus
Marker: Gene or DNA fragment, whose position in
the genome is known and defined
Monohybrid cross
Dihybrid cross
F2 generation ratio:
Mendel’s second law:
The Principle of independence:
During gamete formation, members of
one heredity factor pair segregate into
gametes independently from other
heredity factor pairs.
Recombination as basis of genetic maps
• Genetic maps maps serve for the orientation in large genomes
1. Homologous chromosomes
Each chromosome has the
same order of genes, but can
have different alleles of one locus
2.+3. Recombination (crossing-over)
Homologous chromosomes pair
(1. Meiosis)
and exchange genetic material
(DNA double strand break and
repair)
1 2 3
Terms for the description of genetic distance
• Genetic distance: The measure for distances on a chromosome is based on
the probability of recombination between two loci: the Morgan
• A distance of 1 centiMorgan (cM) between 2 loci (genes or markers) is
observed, if recombination of both loci occurs in 1% of meiosis events
= Markers separated by 1 cM have an expected rate of chromosomal
crossovers of 0.01 per generation
= A 1% probability of two markers to be separated by recombination
• As a consequence non-Mendelian inheritance ratios occur !
• A genetic distance of 1 cM is equivalent to a physical distance of app.
1.000.000 base pairs (1 Mb) in human genome and app. 203.000 base
pairs for Arabidopsis thaliana
Thomas Hunt Morgan (1886-1945). Nobel prize for medicine 1933.
American Geneticist, worked mainly with Drosophila
Determination of a gene position by recombination
From: Chrispeels&Sadava (1999) Plants, Genes and Crop Biotechnology
• The probability for a recombination event correlates with the distance between
two loci on a chromsome
• Closely positioned loci are linked and more likely inherited together than distant
loci
• Co-segregation: Gene of the trait and the gene of the marker are inherited
together because they are closely located (linked alleles)
• Analysis of progeny of crosses for joint presence (or absence) of trait and
marker allows to determine the distance between them on a chromosome
• Crossing-over frequency can be scored: e.g. dark coloured alleles are dominant
or molecular markers to distinguish dark/light alleles are available
Crossing-over
Genetic map of loci of nitrogen assimilation in maize
From: Hirel et al. (2001) Plant Physiol. 125: 1258
NTR1
NiR
AS1
GOGAT
gln1
gln2
gln5
AS2
NR1 gln3
gln4
1 2 3 4 5 6 7 8 9 10
AS = Aspartate-Synthase
Gln = Glutamine-Synthetase
NR = Nitrate-Reductase
NiR = Nitrite-Reductase
GOGAT = Glutamat/oxo-Glutarate-
Aminotransferase
• Genetic maps form a reliable, but not very precise basis of genome analysis
Structure, Dynamics and Analysis of Plant Genomes
I. Evidence for the transgene in transformed plants
II. Natural recombination and plant breeding
III. Generation of genome maps
• Genetic maps
• Molecular maps
IV. Current status of plant genome analysis
Hierarchy and resolution of genome maps
• The physical map reflects the precise locations of genes based on DNA
basepair distance
Physical map Molecular map
Physical maps using molecular markers
• Problem of genetic mapping:
insufficient number of genes with
defined chromosome positions
available for use as markers for
mapping of trait genes
• Solution: in principle all DNA
fragments can serve as molecular
markers
• Prerequisites:
(1) Detectable difference between
alleles of different genotypes
(2) Sequence information available
(restriction site, probe)
• Example: Restriction Fragment
Length Polymorphisms (RFLP)
Detection of polymorphisms
From: K. Eimert, FG Botanik, FA Geisenheim, Website
Restriction site
Labeled probe
Genotype 1
Genotype 1
Genotype 2
Genotype 2
Genotype 1
Genotype 2
RFLP based profile of genotypes
Modified after Weising et al. (1994) CRC Press
• Advantages: High precision and
reproducibility. Disadvantage:
Sequence information required and
often radioactive labeling
• Determination of relative positions of
DNA loci on a chromosome after
crossing of differing parental
genotypes
• RFLP markers are used to document
recombination events relative to the
gene-of-interest (trait gene)
• Other physical markers: Small
nucleotide polymorphisms (SNPs),
microsatellites, AFLP
• Assistance for breeding process and
mapping of genes (‚Smart breeding‘)
Syntheny: Colinearity of genomes of different species
• Basis: RFLP markers work often between closely related species
• Successful examples: Cereals (maize/rice/millet), Brassicaceae
(Arabidopsis/rapeseed/cabbage), Solanaceae (tomato/paprica/potato)
• Same set of molecular markers (A to P) in different species
• Detection of colinearity and inversions/deletion/additions
Sch
mid
t (2
00
0)
Curr
.Op
.Pla
nt B
iol. 3
:97
A. Complete
Colinearity of 2
chromosomes of
species I and 1
B. Chromosome of species I
shows colinearity to different
chromosomes of other species
(1, 2, 3)
C. Comparison of chromosome 1
and 2 of a tetrapoid species with
chromosome I of a diploid species
1B 1A 0 1B 0 0
0
0
0
Comparative map of cereal genomes
From: Gale&Devos (1998) PNAS 95: 1971
Applications of
maps:
• breeding
(marker
assisted
breeding)
• Gene
isolation
(mapping)
• Genome
research
• Evolution,
biodiversity
Structure, Dynamics and Analysis of Plant Genomes
I. Evidence for the transgene in transformed plants
II. Natural recombination and plant breeding
III. Generation of genome maps
• Genetic maps
• Molecular maps
IV. Current status of plant genome analysis
• Sequencing of genomes
• Functional genome analysis
3764 Complete Microbial Genome Projects on 18. Oct. 2012
Procaryotes: Archaea (Pyrococcus, Halobacter, ...), Eubacteria (Clostridium, Bacillus,
Escherichia (4.100kb), Bradyrhizobium, Vibrio, ...)
http://www.genomesonline.org/
Genome sequencing in the public domain
Species Name Genome size (kb) # ORFs
A. gambiae Malaria mosquito 278.000 14.000
A. thaliana Thale cress 115.428 25.498
C. elegans Nematode 97.000 19.099
D. melanogaster Fruitfly 137.000 14.100
H. sapiens Man ~3 x 10 6 kb ~ 30.000
M. musculus Mouse ~2.5 x 10 6 kb ~ 30.000
O. sativa indica Rice 420.000 50.000
S. cerevisiae Baker‘s yeast 12.069 6.294
Incomplete projects: 14.626
Genome sequencing: The ultimate map
Shotgun method:
fast and not precise
Clone-by-clone method:
slow but exact
• Automatic sequencing of DNA fragments up to 1000 Bp
• Bioinformatics required for data mining of sequence information
• BAC: Bacterial Artificial Chromosome, around 100.000 bp
The world before genome sequencing
Sequencing of the Arabidopsis genome:
a milestone of plant biology
The Arabidopsis genome
From: The Arabidopsis Genome Initiative (2000) Nature 408:796 TAIR: http://www.arabidopsis.org/index.html
The Sequence Annotation of Arabidopsis
From: R. Joy, 2002, ABRC
Large segments of the Arabidopsis genome are
duplicated
From: The Arabidopsis Genome Initiative (2000) Nature 408:796
Identification of ancestral karyotype of Brassicaceae
Schranz et al. (2007)
Plant Physiol. 144:286
• Reduction of
originally 8
chromosomes to
5 (Arabidopsis),
7 (Boechera) or
other numbers
Structure, Dynamics and Analysis of Plant Genomes
I. Evidence for the transgene in transformed plants
II. Natural recombination and plant breeding
III. Generation of genome maps
• Genetic maps
• Molecular maps
IV. Current status of plant genome analysis
• Sequencing of genomes
• Functional genome analysis
Expression:
• Transcription
• Post-transcription
• Gene regulation
• Epigenetics
• Translation
• Post-translation
• Protein modification
Different levels of expression of genes
• Which gDNA segments encode for genes?
• How can 5‘ and 3‘ ends be correctly predicted ? (Estimate: only 40% are
correct)
• How can Exon/Intron sequences be distinguished? (Correctness at 80-
90 %)
• How precise are the generated sequences? (on average 99.8%)
• Which genes are really expressed?
• What are the changes in expression patterns in reaction to development
and environment?
Gene encoded products:
• mRNA
• tRNA
• rRNA Gene
• microRNA
• Proteins of different functions:
Structure, membrane transporter,
enzymes, allosteric, interaction
Determination of the transcriptome of plants
• Tissue-dependent origin: all cell and tissue types of
a plant have different mRNA populations;
Problem: Different representation of cell types (e.g.
stoma cell, phloem companion cell)
• Time-dependent origin: Life cycle of the plant,
ontogeny;
Problem: accessible tissue amounts (embryonic
tissues, gametophyte)
• Condition-dependent origin: reaction to stress
(e.g. light, anoxia)
Problem: Do we know all conditions?
Leaf Mimosa cross section
(Botanik Online)
• Problem: Abundancy/redundancy in mRNA populations:
Relative frequency of mRNA molecules in a cell:
Rubisco SSU (10.000) : Pyruvate dehydrogenase (100) : Transcription factor (1)
• Solution: Deep Sequencing/next generation sequencing and bioinformatics
2. Level of functional genomics : Analysis of global
expression profiles by microarrays
From: Duggan et al. (1999) Nat. Gen. 21: 10
PCR
mRNA
• Expression mapping of the genome under different physiological conditions or
between organs
Example for nylon filter with cDNAs for multiparallel
expression analysis
Control after 6 h 200 µM methyl-jasmonate for 6 h
Daten: R. Jost, R. Hell
Proteomics: two-dimensional gelelectrophoresis
Separation by
charge (pI)
Separation by
mass (Mr)
pI
Mr
Labeling for isolation and
identification by HPLC/Mass
Spectrometry
Comparison of two samples
(Wildtype/Mutant; Stress;
Disease; Cell type etc.)
Metabolomics: Metabolite profile analysis
37.5 38.0 39.0 39.5 40.0 41.0 41.5 42.0 42.5 43.0 43.5 44.0 44.5
100
0
%
37.589
38.658
38.915
44.290
42.447 41.254
40.612
39.337
39.539
40.392
41.593 39.062
38.5 40.5 min
= Wildtype = Plant overexpressing metabolic enzyme
L. Willmitzer, MPI-MOPP, Golm
Analytics: Liquid-Chromatography-Mass Spectrometry
Gas-Chromatography-Mass Spectrometry
Mutant in comparison to wildtype
Summary: Plant genomes
• Advanced molecular-genetic methods allow to determine
genetic loci in large genomes and to follow crosses during
breeding
• Genetic maps are indispensable, but sequencing represents
the ultimative genomic map
• Functional genome analysis starts with transcriptomics and
represents the task of the future
Next lecture: Gene expression
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