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Institute of Experimental Botany, Centre of the Region Haná
for Biotechnological and Agricultural Research, Olomouc, Czech Republic
Jaroslav Doležel
Contribution of flow cytogenetics to plant genome sequencing, gene cloning
and understanding mitotic chromosome structure
Mitoticchromosomes
Chromosome flow sorting
Genome sequencing
Gene cloning
Spatial DNA organization
Chromosomalproteins
Mitoticchromosomes
Contribution of flow cytogenetics to plant genome sequencing, gene cloning
and understanding mitotic chromosome structure
Sheath fluid
Deflection
plates
Excitation
light
Waste
Right
collector
Left
collector
Laser
Scattered
light
Fluorescence
emission
Relative fluorescence intensity
Chromosomes in
suspension
Flow karyotype
Flow
chamber
Num
ber
of
events
L
R
Flow cytogenetics
•Chromosome analysis rate :
~1000 chromosomes / sec
http://olomouc.ueb.cas.cz/Doležel et al., Crit. Rev. Plant Sci. 13: 275, 1994
Prerequisites
• Suspension
of intact mitotic
chromosomes
• Differences in
chromosome fluorescence
• Intact chromosomal DNA
(or proteins)
<Stained DNA
<Labeled DNA sequences
Chromosome analysis and sorting by flow cytometry
Sheath fluid
Deflection
plates
Waste
Right
collector
Laser
Scattered
light
Relative fluorescence intensity
Num
ber
of
events
Purified
chromosome fraction
Purified
chromosome fraction
L
R
•Chromosome analysis rate :
~1000 chromosomes / sec
•Chromosome yield :
2 – 5 x 105 chromosomes / day
Excitation
light
Left
collector
Flow karyotype
Flow
chamber
http://olomouc.ueb.cas.cz/
Chromosomes in
suspension
Waste
Fluorescence
emission
Doležel et al., Crit. Rev. Plant Sci. 13: 275, 1994
<Stained DNA
<Labeled DNA sequences
Flow cytogenetics
Chromosome analysis and sorting by flow cytometry
http://olomouc.ueb.cas.cz/
Marker-assisted selection
New breeding techniques (targeted genome modification)
The knowledge of genes and other DNA sequences is needed
Production of reference genomes
Genome sequencing and gene cloning
Reference genome sequence of a plant
species
Genome sequencesof cultivars and
landraces
Genome sequences of wild related
species
Many different genomes
Intr
ogre
ssio
nsMining useful
genes and alleles
(they may not be enough)
Genomes of many crops are complex
Hexaploid bread wheat (2n=6x=42, genome size ~17 Gb)
http://olomouc.ueb.cas.cz/
Sub
gen
om
es
Chromosomes
Highly redundant (80-90% repetitive DNA)
Doležel et al., Chromosome Res. 15:51, 2007http://olomouc.ueb.cas.cz/
Dissecting genomes to chromosomes
Offers a massive and lossless reduction of DNA sample complexity
Avoids problems due to sequence redundancy (incl. polyploidy)
Hexaploid wheat
genome
Triticum aestivum
(2n=6x=42)
1C ~ 17,000 Mbp
AA BB
DD
Chromosome arms: 225 - 585 Mbp
(1.3 – 3.4% of the genome)
D
B
;
A
Chromosomes: 605 - 995 Mbp
(3.6 – 5.9% of the genome)
Dissection to 21 chromosomes
Simplifying genome analysis
Chromosome genomics and proteomics
http://olomouc.ueb.cas.cz/Doležel et al., Biotech. Adv., 32:122, 2014
The toolbox keeps on expanding
http://olomouc.ueb.cas.cz/
Rye (Secale cereale, 2n=14)
Genome size ~8 Gb (~3x human)
Sequencing DNA amplified from
flow- sorted chromosomes
22,426 non-redundant
genes identified
Mayer et al., Plant Cell 25: 3685, 2013
Dissecting the complex genomes
Synteny between rye
(1R – 7R), barley (1H –
7H) and Brachypodium
(Bd1 - Bd5)
Major evolutionary
translocations revealed
The results indicate
reticulate evolution
of the rye genome
http://olomouc.ueb.cas.cz/
Mayer et al., Science, 2014
Towards the reference genomes
Chromosome genomics unlocked the
genomes of important crops
− Draft assemblies of complex
genomes of barley, rye and wheat
Recent advances in DNA sequencing
technologies and assemblers permit
whole genome shotgun approaches
Chromosome genomics does
continue to be very useful
− Validation of whole-genome
shotgun assemblies
− Chickpea, garden pea, barley,
bread wheat, rye, …
IWGSC, Science, 2018
Post reference genome era
http://olomouc.ueb.cas.cz/
A need to obtain sequences from particular chromosomes
from particular accessions
‐ Targeted saturation of genetic maps, marker development,
characterization of alien introgressions, gene cloning, …
Sequencing large and polyploid genomes is expensive
… and requires the analysis of huge volumes of data
Chromosome-centric approach
simplifies these tasks
M2 plants
Seed
M3 seedM1 plants
EMS
treatment
M3 plants
Confirm
http://olomouc.ueb.cas.cz/
Mutagenesis and progeny screening (loss of function)
Sánchez-Martín et al., Genome Biol. 17:221, 2016
Rapid gene cloning (MutChromSeq)
M2 plants
Seed
M3 seedM1 plants
EMS
treatment
M3 plants
Confirm
http://olomouc.ueb.cas.cz/
Mutagenesis and progeny screening (loss of function)
Chromosome sorting and DNA amplification
− Wild type
− Mutants
Sánchez-Martín et al., Genome Biol. 17:221, 2016
Rapid gene cloning (MutChromSeq)
1. Assemble wild type chromosome
2. Map mutant reads
3. Call variants
4. Determine mutation overlap in contigs
M2 plants
Seed
M3 seedM1 plants
EMS
treatment
M3 plants
Confirm
http://olomouc.ueb.cas.cz/
Mutagenesis and progeny screening (loss of function)
Chromosome sorting and DNA amplification
− Wild type
− Mutants
Next-gen sequencing and data analysis
Candidate genes:
• Barley
Eceriferum-q
• Wheat Pm2
Sánchez-Martín et al., Genome Biol. 17:221, 2016
Rapid gene cloning (MutChromSeq)
Mitotic chromosome condensation
http://olomouc.ueb.cas.cz/
Organization of DNA in mitotic metaphase chromosomes?
Protein composition of mitotic metaphase chromosomes?
Analysis of isolated mitotic chromosomes is a suitable approach
How they do it?
https://www.shmoop.com/dna/dna-packaging.html
Hope you finished condensing your
DNA, we're dividing in 10 minutes!
Awwww …
Intl Rev Cell Mol Biol, First Edition, 183, 2015
Chromosome Conformation Capture
(3C) technique and its derivatives
(4C, 5C, Hi-C)
Hi-C analyses interactions of DNA loci
across the whole genome using next
generation sequencing
Spatial DNA organization in chromosomes
http://olomouc.ueb.cas.cz/
Hi-C
interaction
matrix
Slice 6
Separation by reversed-phase nanoLC
(separation of peptide mixture)
1
2
3
4
567
8 910
1112
13
14
NanoLC-ESI-MS
and MS/MS
NanoLC-MALDI-MS
and MS/MS
Petrovská et al., Cytogenet. Genome Res. 143:78-86, 2014 http://olomouc.ueb.cas.cz/
Bulk flow cytometric purification of barley mitotic chromosomes
(7 – 11 x 106 copies)
Which proteins make plant chromosomes?
Summary
http://olomouc.ueb.cas.cz/
Mitoticchromosomes
Chromosome flow sorting
Genome sequencing
Gene cloning
Spatial DNA organization
Chromosomalproteins
Mitoticchromosomes
Our team
http://olomouc.ueb.cas.cz/
Tomáš Beseda
Petr Cápal
Jarmila Číhalíková
Zdeňka Dubská
Hana Jeřábková
Miroslava Karafiátová
Marie Kubaláková
Aleš Pečinka
Beáta Petrovská
Hana Šimková
Romana Šperková
Jan Vrána
Our team
http://olomouc.ueb.cas.cz/
Tomáš Beseda
Petr Cápal
Jarmila Číhalíková
Zdeňka Dubská
Hana Jeřábková
Miroslava Karafiátová
Marie Kubaláková
Aleš Pečinka
Beáta Petrovská
Hana Šimková
Romana Šperková
Jan Vrána
Acknowledgements
Kansas State University
Bikram Gill
Bernd Friebe
Vijay Tiwari
John Innes Centre
Brande Wulff
Burkhard Steuernagel
Nikolai Adamski
Palacký University
Marek Šebela
René Lenobel
Jana Uřínovská
Ivo Chamrád
University of Zurich
Simon Krattinger
Anupriya Thind
Thomas Wicker
Dovetail Genomics
Brandon Rice
Mike Reardon
International Wheat
Genome Sequencing
Consortium
Kellye Eversole
Munich Information Center
for Protein Sequences Munich
Klaus Mayer
Mihaela Martis
IPK Gatersleben
Nils Stein
Martin Mascher
Axel Himmelbach
Ines Walde
Many thanks for your attention!
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