# Sequencing the genome of the French wheat variety Renan

Frédéric Choulet

► To cite this version:

Frédéric Choulet. Sequencing the genome of the French wheat variety Renan. IWGSC Webinar, IWGSC - International Wheat Genome Sequencing Consortium, Sep 2022, Clermont-Ferrand, France. hal-04090088

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# Sequencing the genome of the French wheat variety Renan

• Frédéric CHOULET

GDEC, INRAE-Univ Clermont-Ferrand
<figure><img src="image_2.png" /></figure>

RENAN

Pch1

<figure><img src="image_3.png" /></figure>

Ae. ventricosa

<figure><img src="image_4.png" /></figure>
□ Genome assembly: steps and techno

<figure><img src="image_5.png" /></figure>
Triticeae refseq current status (09/2022)

Reference-quality genome assemblies

* T. aestivum

* Chinese Spring (NRgene)

* Arina Lr For Jagger Julius CDC_Lancer CDC_Landmark Mace-
Norin61 Stanley SY-Mattis Spelta (NRGene)

* Zang1817 (NRGene)

* Fielder (Pacbio HiFi)

* Kariega (Pacbio HiFi)

* Renan (ONT)

* Attraktion (Pacbio HiFi)

* T. urartu AA

* T. durum CABB

* T. dicoccoides CABB

* Ae. tauschii DD

* Ae. searsii DD

* Ae. bicornis DD

* Ae. longissima DD

* Ae. sharonensis DD

* Ae. speltoides SS

Article | Open Access | Published: 08 October 2020

Origin and adaptation to high altitude of Tibetan semi-wild wheat

Weilong Guo, Mingming Xin, Zihao Wang, Yingyin Yao, Zhaorong Hu, Wanjun Song, Kuohai Yu, Yongming Chen, Xiaobo Wang, Panfeng Guan, Rudi Appels, Huiru Peng, Zhongfu Ni & Qixin Sun

Nature Communications 11, Article number: 5085 (2020) | Cite this article

DNA Research, 2021, 00001, 1-7

doi: 10.1093/dnares/dsab008

Advance Access Publication Date: 12 July 2021

Resource Article: Genomes Explored

Resource Article: Genomes Explored

Chromosome-scale genome assembly of the
transformation-amenable common wheat
cultivar 'Fielder'

Kazuhiro Sato Ⓓ ¹*, Fumitaka Abe Ⓓ ², Martin Mascher Ⓓ ³, ⁴,
Georg Haberer Ⓓ ⁵, Heidrun Gundlach Ⓓ ⁵, Manuel Spannagl Ⓓ ⁵,
Kenta Shirasawa Ⓓ ⁶, and Sachiko Isobe Ⓓ ⁶

nature
genetics

LETTERS

http://mol.org/10.1038/s41588-022-01022-4

Check for updates

OPEN

Long-read genome sequencing of bread wheat facilitates disease resistance gene cloning

Abrouk Ⓓ ¹, Willem H. P. Boshoff Ⓓ ², Stéphane Cauet Ⓓ ³,
hed Mohammed Ⓓ ¹, Jan Bettgenhaeuser Ⓓ ¹, Kirsty S. Botha Ⓓ ⁵,
⁴, Renée Prins Ⓓ ⁵, ⁶ and Simon G. Krattinger Ⓓ ¹

CellPress
Partner Journal

Molecular Plant
Research Article

Genome sequences of five Sitopsis species of
Aegilops and the origin of polyploid wheat B
subgenome

Lin-Feng Li¹,²,⁵,*, Zhi-Bin Zhang¹,³,⁵, Zhen-Hui Wang⁴, Ning Li¹, Yan Sha¹, Xin-Feng Wang²,
Ning Ding³, Yang Li¹, Jing Zhao¹, Ying Wu¹, Lei Gong¹, Fabrizio Mafessoni¹, Avraham A. Levy¹,³,
and Bao Liu¹,∗

<figure><img src="image_6.png" /></figure>

doi: 10.1111/jpx.13584

gene mapping in winter wheat

Sandip M. Kale¹,¹, Albert W. Schulthess¹,¹, Sudharsan Padmarasu¹, Philipp H. G. Boewen², Johannes Schacht²,
Axel Himmelbach¹, Burkhard Steuernagel², Brande B. H. Wulff¹,⁴, Jochen C. Reif¹, Nils Stein¹,⁵,∗ and
Martin Mascher¹,⁶,∗
# Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves high resolution for research and breeding

Jean-Marc Aury Ⓓ1,*, Stefan Engelen Ⓓ1, Benjamin Istace Ⓓ1, Cécile Monat Ⓓ2, Pauline Lasserre-Zuber<fnref n="2" />, Caroline Belser Ⓓ1,
Corinne Cruaud Ⓓ3, Hélène Rimbert Ⓓ2, Philippe Leroy Ⓓ2, Sandrine Arribat Ⓓ4, Isabelle Dufau<fnref n="4" />, Arnaud Bellec Ⓓ4,
David Grimbichler Ⓓ5, Nathan Papon<fnref n="2" />, Etienne Paux Ⓓ2, Marion Ranoux<fnref n="2" />, Adriana Alberti Ⓓ1,<fnref n="6" />, Patrick Wincker Ⓓ1 and
Frédéric Choulet Ⓓ2,<fnref n="4" />

## Abstract

**Background:** The sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years owing to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions, but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost.

**Results:** Here, we report on an optimized procedure based on long reads produced on the Oxford Nanopore Technology PromethION device to assemble the genome of the French bread wheat cultivar Renan.

**Conclusions:** We provide the most contiguous chromosome-scale assembly of a bread wheat genome to date. Coupled with an annotation based on RNA-sequencing data, this resource will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide a framework to generate high-quality assemblies of complex genomes using ONT.

**Keywords:** wheat, hexaploid genome, long-reads, nanopore sequencing, genome assembly, haplotype characterization, introgressions

<footnote n="1">¹Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France</footnote>

<footnote n="2">²GDEC, Université Clermont Auvergne, INRAE, UMR1095, 63000 Clermont-Ferrand, France</footnote>

<footnote n="3">³Commissariat à l'Energie Atomique (CEA), Institut François Jacob, Genoscope, F-91057 Evry, France</footnote>

<footnote n="4">⁴INRAE, CNRGV French Plant Genomic Resource Center, F-31320, Castanet Tolosan, France</footnote>

<footnote n="5">⁵Mésocentre Clermont Auvergne, DOSI / Bâtiment Turing, 7 avenue Blaise Pascal, 63178 Aubière, France</footnote>

<footnote n="6">⁶Present address: Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (IBSC), 91198, Gif-sur-Yvette, France</footnote>

<footnote n="4">⁴Correspondence address. Jean-Marc Aury, Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France. E-mail: jmaury@genoscope.cns.fr; Frédéric Choulet, GDEC, Université Clermont Auvergne, INRAE, UMR1095, 63000 Clermont-Ferrand, France. E-mail: frederic.choulet@inrae.fr</footnote>
Step#1: sequencing

<figure><img src="image_7.png" /></figure>
Step#2: read assembly

https://www.youtube.com/watch?v=GUb1TZyMWsw

<figure><img src="image_8.png" /></figure>

Early stages

-> error rate in reads: 12%

-> error rate in assembly: 4%

<figure><img src="image_9.png" /></figure>

Nanopore

Renan

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

o basecallin

o polishing

<figure><img src="image_14.png" /></figure>

Illumina
30x

ACGTAGACGTACGGCTGCAATCGCATTCATCGGCGGGGGGGGGCTGCTCGTCGCTCCTCGT assembly
Step#3: scaffolding

<figure><img src="image_15.png" /></figure>
□ Assembly metrics

❖ Metrics

➢ Genome size=14.3 Gb

➢ 2904 scaffolds (=138/chr)

<figure><img src="image_16.png" /></figure>

❖ Quality Assessment

Genes: 98% CS genes found

TEs: 92% CS ISBPs found

QV: lower (99.95% accuracy) than NRGene-based assemblies (99.99%)
<figure><img src="image_17.png" /></figure>

Step#4: pseudomolecule construction

<figure><img src="image_18.png" /></figure>

2904 scaff

1. CS-guided

2. HiC

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

21 chromosomes

X not efficient enough to build a
chromosome-scale assembly

<figure><img src="image_21.png" /></figure>
□ Step#4-1: CS-guided pseudomol construction

<figure><img src="image_22.png" /></figure>
☐ Step#4-1: CS-guided pseudomol construction

☐ Step#4-2: HiC corrections

Renan

<figure><img src="image_23.png" /></figure>
Step#4-2: HiC corrections

<figure><img src="image_24.png" /></figure>

* **18** chimeric scaffold

* **198** scaffold with correction of location and/or orientation

<figure><img src="image_25.png" /></figure>

21 chr + chrUn 61 Mb (<1%)
<figure><img src="image_26.png" /><figcaption>Chinese Spring</figcaption></figure>

<figure><img src="image_27.png" /></figure>

After HiC-based corrections

<figure><img src="image_28.png" /><figcaption>Chinese Spring</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>Renan Refseq_v2 versus 10 wheat genomes</figcaption></figure>
o Predicting genes is still NOT routine

**GDEC** Clermont, France – *Rimbert Leroy Choulet et al.*

**PGSB** Munich, Germany – *Spannagl Twardziok et al.*

**EI** Norwich, UK – *Swarbreck Venturini et al.*

<figure><img src="image_30.png" /></figure>

IWGSC RefSeq (Chinese Spring)

• 107,891 genes

<figure><img src="image_31.png" /></figure>
HighConfidenceGenesv1.1 (GOMAP annotation available)

Sorghum bicolor

Hordeum vulgare var. distichum

Aegilops tauschii

Triticum urartu

Brachypodium distachyon

Oryza barthii

Zea mays

Triticum aestivum RNA-Seq (Pingault et al. 2014)

<figure><img src="image_32.png" /></figure>
* HighConfidenceGenesv1.1 (GOMAP annotation available)

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

* Sorghum bicolor

<figure><img src="image_35.png" /></figure>

* Hordeum vulgare var. distichum

<figure><img src="image_36.png" /></figure>

* Aegilops tauschii

* Triticum urartu

* Brachypodium distachyon

<figure><img src="image_37.png" /></figure>

* Oryza barthii

<figure><img src="image_38.png" /></figure>

* Zea mays

<figure><img src="image_39.png" /></figure>

* Triticum aestivum RNA-Seq (Pingault et al. 2014)

<figure><img src="image_40.png" /></figure>

* EMBL EST of Triticeae

<figure><img src="image_41.png" /></figure>

* FgeneSH predictions
<figure><img src="image_42.png" /><figcaption>chr3B BAC-by-BAC</figcaption></figure>

Choulet et al. 2014

<figure><img src="image_43.png" /><figcaption>TGAC_v1</figcaption></figure>

Clavijo et al. 2017

<figure><img src="image_44.png" /><figcaption>Chr. Survey Seq</figcaption></figure>

IWGSC 2014

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

denovo gene annotation not adapted to pangenomics
o Gene projections / Annotation transfer

<figure><img src="image_47.png" /></figure>

~12%
ambiguous
mapping

> denovo gene annotation not adapted to pangenomics

> Gmap vs whole genome introduces mis-alignment errors

> Develop **MAGATT** pipeline
M

magatt

Project ID: 1089

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

120 Commits

<figure><img src="image_50.png" /></figure>

1 Branch

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5 Tags

<figure><img src="image_52.png" /></figure>

5.5 MB Project Storage

Pipeline used to transfert gene annotation (GFF3) between different versions of assemblies.

master

<figure><img src="image_53.png" /></figure>

magatt

Find file

<figure><img src="image_54.png" /></figure>

Clone

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

Upload New environment file for Singularity build

Helene Rimbert authored 10 months ago

4df3e943

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

README

<figure><img src="image_59.png" /></figure>

No license. All rights reserved

<table><thead><tr><th>Name</th><th>Last commit</th><th>Last update</th></tr></thead><tbody><tr><td>bin</td><td>NEW single-isoform mode for transfert b...</td><td>1 year ago</td></tr><tr><td>report</td><td>bug: if cds and mapping attributes existed...</td><td>1 year ago</td></tr><tr><td>rules</td><td>NEW single-isoform mode for transfert b...</td><td>1 year ago</td></tr><tr><td>.gitignore</td><td>ignore slurm log file</td><td>1 year ago</td></tr><tr><td>README.md</td><td>Update README.md</td><td>1 year ago</td></tr><tr><td>Singularity</td><td>Upload New Singularity definition file</td><td>10 months ago</td></tr><tr><td>Snakefile</td><td>bug: if cds and mapping attributes existed...</td><td>1 year ago</td></tr><tr><td>{} cluster-hpc2.json</td><td>NEW single-isoform mode for transfert b...</td><td>1 year ago</td></tr><tr><td>{} cluster-sibi.json</td><td>remove temporary fasta files to loawer di...</td><td>1 year ago</td></tr><tr><td>{} config.yaml</td><td>NEW single-isoform mode for transfert b...</td><td>1 year ago</td></tr><tr><td>{} env.yaml</td><td>Upload New environment file for Singular...</td><td>10 months ago</td></tr><tr><td>{} environment.yml</td><td>Update environment.yml with new depend...</td><td>1 year ago</td></tr></tbody></table>

<figure><img src="image_60.png" /></figure>

README.md

## MAGATT pipeline

Marker Assisted Gene Annotation Transfert for Triticeae.

Snakemake pipeline used to transfert GFF annotation on a new assembly with a fine target mapping approach.

## Install the pipeline

```
$ git clone https://forgemia.inra.fr/umr-gdec/magatt.git
```
<figure><img src="image_61.png" /></figure>

# MAGATT (Marker-Assisted Gene Annotation Transfer for *Triticeae*)

<figure><img src="image_62.png" /></figure>

* 98% of genes mapped accurately

* + 4400 (4%) Renan specific genes
□ Introgressions

<figure><img src="image_63.png" /></figure>

telomere

Renan

40 Mb

<figure><img src="image_64.png" /></figure>

telomere

Chinese Spring

35 Mb

<figure><img src="image_65.png" /></figure>
<figure><img src="image_66.png" /><figcaption>Renan - chr7D<sup>V</sup></figcaption></figure>

telomere

<figure><img src="image_67.png" /><figcaption>Chinese Spring - chr7D</figcaption></figure>

30 Mb, ~500 genes

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>
<figure><img src="image_70.png" /><figcaption>Renan - chr7DV</figcaption></figure>

<caption>Chinese Spring - chr7D</caption>
<figure><img src="image_71.png" /></figure>

# TE dynamics

* PhD Nathan Papon
  (2019-2022)

Daron et al. 2015

Daron et al. Genome Biology 2014, 15:546
http://genomebiology.org/2014/15/12/546

<figure><img src="image_72.png" /></figure>

Genome Biology

RESEARCH

Open Access

## Organization and evolution of transposable elements along the bread wheat chromosome 3B

Josquin Daron¹,², Natasha Glover¹,², Lise Pingault¹,², Sébastien Theil¹,², Véronique Jamilloux³, Etienne Paux¹,², Valérie Barbe⁴, Sophie Mangenot⁴, Adriana Alberti⁵, Patrick Wincker⁴,⁶, Hadi Quesneville³, Catherine Feuillet¹,² and Frédéric Choulet¹,²*

Wicker et al. 2018

Wicker et al. Genome Biology (2018) 19:103
https://doi.org/10.1186/s13059-018-1479-0

Genome Biology

RESEARCH

Open Access

<figure><img src="image_73.png" /></figure>

CrossMark

## Impact of transposable elements on genome structure and evolution in bread wheat

Thomas Wicker¹¹, Heidrun Gundlach²², Manuel Spannagl³, Cristobal Uauy³, Philippa Bornl³, Ricardo H. Ramírez-González², Romain De Oliveira¹, International Wheat Genome Sequencing Consortium¹, Klaus F. X. Mayer²,³, Etienne Paux⁴ and Frédéric Choulet*

De Oliveira et al. 2020

Frontiers in Genetics | www.frontiersin.org

August 2020 | Volume 11 | Article 891

## Structural Variations Affecting Genes and Transposable Elements of Chromosome 3B in Wheats

Romain De Oliveira¹, Hélène Rimbert¹, François Balfourier¹, Jonathan Kitt¹, Emeric Dynomant¹, Jan Vrána², Jaroslav Doležel³, Federica Cattonaro³, Etienne Paux¹ and Frédéric Choulet*
<figure><img src="image_74.png" /></figure>

~1 Mb
○ TE modeling with CLARITE and ClariTeRep

https://github.com/jdaron/CLARI-TE

<figure><img src="image_75.png" /></figure>

<figure><img src="image_76.png" /></figure>

TREP++
5000 known wheat TEs

<figure><img src="image_77.png" /></figure>

Classification

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

ClariTeRep
~500 families

<figure><img src="image_80.png" /></figure>
3A

3B

3D

<figure><img src="image_81.png" /><figcaption>► Near-complete **TE turnover** since A-B-D divergence</figcaption></figure>
<figure><img src="image_82.png" /><figcaption>Families</figcaption></figure>

<figure><img src="image_83.png" /></figure>

<figure><img src="image_84.png" /></figure>

* A-B-D balanced proportions

<figure><img src="image_85.png" /></figure>

* 0 family specific to 1 subgenome
<figure><img src="image_86.png" /></figure>

Family #3

<figure><img src="image_87.png" /></figure>

* Independent TE evolution in the diploids AA, BB, DD

* Some rare cases of TE amplif in the tetraploid ABB
A-B-D last
common ancestor

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>

~ 3 Myrs
TE turnover

<figure><img src="image_90.png" /></figure>

dA

<figure><img src="image_91.png" /></figure>

dB

<figure><img src="image_92.png" /></figure>

dD

Most families were active since A-B-D divergence

<figure><img src="image_93.png" /></figure>

**Equilibrium**

Hypothesis of a **structural role of TEs likely under selection pressure?**
□ Genomic variability affecting **TEs** in wheat **using assembled genomes**

PhD Nathan Papon (2019-2022)

<figure><img src="image_94.png" /></figure>
○ Material = multiple RefSeq now available

T. aestivum

<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /></figure>

x13
accessions

T. dicoccoides

T. durum

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /></figure>

T. urartu

<figure><img src="image_99.png" /></figure>

<figure><img src="image_100.png" /></figure>

Ae. tauschii

<figure><img src="image_101.png" /></figure>

<figure><img src="image_102.png" /></figure>
<figure><img src="image_103.png" /></figure>
o Extent of variability affecting TEs

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

<figure><img src="image_106.png" /></figure>

• 5% .. 15%

• B > A > D

• No genome expansion/contraction

<figure><img src="image_107.png" /></figure>
<figure><img src="image_108.png" /></figure>

## Recent TE insertions

* ~4,000-13,000 transposition events / subgenome (<1% of the genome)

<figure><img src="image_109.png" /></figure>

<figure><img src="image_110.png" /></figure>

## Impact of polyploidy?

* none

## How many families were active recently?

* 346 active families (=99.7% of all TEs)
20 most abundant
families

<figure><img src="image_111.png" /></figure>

ClariTeRep TREP

\#specific TE insertions

<figure><img src="image_112.png" /></figure>

CSAABBDD

VS
**Ae. tauschii**<sup>DD</sup>

<figure><img src="image_113.png" /></figure>

CSAABBDD

VS
**T. durum**<sup>AABB</sup>
Conclusions

* No genome expansion/contraction due to TE transposition

* No impact of polyploidy

* No TE burst

* All families were active recently

* Transposition rate is stable

* Equilibrium

* Role of TEs in genome architecture under evolutionary constraints
□ Acknowledgments

○ Génoscope, CEA-CNRS-U Evry

Jean-Marc Aury
Stefan Engelen
Benjamin Istace
Caroline Belser
Corinne Cruaud
Adriana Alberti
Patrick Wincker

○ CNRGV, INRAE Toulouse

Sandrine Arribat
Isabelle Dufau
Arnaud Bellec

○ URGI, INRAE Versailles

Michael Alaux
Nicolas Francillonne

○ Mésocentre, UCA

David Grimbichler

<figure><img src="image_114.png" /></figure>

<figure><img src="image_115.png" /></figure>

UNIVERSITÉ
Clermont
Auvergne

○ GDEC, INRAE-UCA

Cécile Monat
Pauline Lasserre-Zuber
Hélène Rimbert
Philippe Leroy
Etienne Paux
Marion Ranoux
Jérôme Salse
Carline Pont
Cécile Huneau

Nathan Papon
Romain De Oliveira
Josquin Daron

Contact:
frederic.choulet@inrae.fr

○ Funding

<figure><img src="image_116.png" /></figure>

FRANCE
GENOMIQUE

SRESRI/Région ARA

<figure><img src="image_117.png" /></figure>

L'EUROPE S'ENGAGE

en région

Auvergne-Rhône-Alpes
avec le FÉDÉR

<figure><img src="image_118.png" /></figure>

La Région
Auvergne-Rhône-Alpes

<figure><img src="image_119.png" /></figure>