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