contribution of flow cytogenetics - uniba.sk · 2018-10-02 · istock institute of experimental...

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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


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)


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


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


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


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


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



Chromosome sorting and DNA amplification

− Wild type

− Mutants



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



Chromosome sorting and DNA amplification

− Wild type

− Mutants

Next-gen sequencing and data analysis

Candidate genes:

• Barley

Eceriferum-q

• Wheat Pm2



Mitotic chromosome condensation


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


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


Mitoticchromosomes

Chromosome flow sorting

Genome sequencing

Gene cloning

Spatial DNA organization

Chromosomalproteins

Mitoticchromosomes

Our team


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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contribution of flow cytogenetics - uniba.sk · 2018-10-02 · istock institute of experimental...

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