# Sequencing complex genomes of Triticeae

Frédéric Choulet

► To cite this version:

Frédéric Choulet. Sequencing complex genomes of Triticeae. Pacbio PRISM 2024, Pacbio, Apr 2024, Lisbonne, Portugal. hal-04584678

HAL Id: hal-04584678

https://hal.science/hal-04584678

Submitted on 23 May 2024

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<figure><img src="image_1.png" /></figure>
# Sequencing complex genomes
of *Triticeae*
# Sequencing complex genomes
of *Triticeae*

PacBio PRISM 2024
April 24th 2024 – Lisbon
❖ Triticum/Aegilops evolution

<figure><img src="image_1.png" /></figure>

wheat wild relatives

durum wheat

bread wheat
❖ Triticum/Aegilops evolution

<figure><img src="image_2.png" /></figure>

wheat wild relatives

durum wheat

bread wheat
❖ Triticum/Aegilops evolution

<figure><img src="image_2.png" /><figcaption>Bread wheat - T. aestivum</figcaption></figure>

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

15 Gb

- Wild diploids (AA, BB, DD, SS, UU, NN, GG...)

- Wild and cultivated tetraploids (AABB, DDNN, ...)

- Cultivated hexaploids (AABBDD)

<figure><img src="image_4.png" /></figure>
❖ Triticum/Aegilops evolution

<figure><img src="image_3.png" /><figcaption>Bread wheat - T. aestivum</figcaption></figure>

<figure><img src="image_4.png" /></figure>

15 Gb

- Wild diploids (AA, BB, DD, SS, UU, NN, GG...)

<figure><img src="image_5.png" /></figure>

- Wild and cultivated tetraploids (AABB, DDNN, ...)

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

<figure><img src="image_7.png" /></figure>

- Cultivated hexaploids (AABBDD)

<figure><img src="image_8.png" /></figure>
<figure><img src="image_5.png" /></figure>

Triticeae genome composition

○ Transposons (85%)

○ Genes (2%)

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

1st assembled genome:
Chinese Spring (IWGSC Science 2018)

○ 3x ~1.3 million TEs

○ 3x ~35,000 genes

<figure><img src="image_7.png" /></figure>
<figure><img src="image_9.png" /></figure>

Triticeae genome composition

○ Transposons (85%)

○ Genes (2%)

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

1st assembled genome:
Chinese Spring (IWGSC Science 2018)

○ 3x ~1.3 million TEs

○ 3x ~35,000 genes

<figure><img src="image_11.png" /></figure>
<figure><img src="image_8.png" /></figure>

A-B-D comparative genomics

<figure><img src="image_9.png" /></figure>
<figure><img src="image_12.png" /></figure>

A-B-D comparative genomics

<figure><img src="image_13.png" /></figure>
History of sequencing *Triticeae* genomes

T. aestivum ABBDD

<table><thead><tr><th></th><th></th><th></th><th>N50</th></tr></thead><tbody><tr><td>• 2014</td><td>WGS (Illumina)<br/>1 chr BAC-by-BAC (Roche/454)</td><td>IWGSC 2014</td><td>90,000 contigs/chr 5 kb</td></tr><tr><td>• 2017</td><td>WGS (Illumina+Pacbio CLR)</td><td>Choulet et al. 2014</td><td>1 Mb</td></tr><tr><td>• 2018</td><td>RefSeq (NRGene Illumina)</td><td>Zimin et al. 2017</td><td></td></tr><tr><td>• 2020</td><td>10 genotypes (NRGene Illumina)</td><td>IWGSC 2018</td><td>23 Mb</td></tr><tr><td>• ..</td><td>Renan (ONT+Illumina)</td><td>Walkoviak et al. 2020</td><td></td></tr><tr><td>• ..</td><td>Fielder (Pacbio HiFi)</td><td>Aury et al. 2022</td><td>48 Mb</td></tr><tr><td>• ..</td><td>Kariega (Pacbio HiFi)</td><td>Sato et al. 2021</td><td>68 contigs/chr 21 Mb</td></tr><tr><td>• ..</td><td>Attraktion (Pacbio HiFi)</td><td>Athiyannan et al. 2022</td><td>15 scaff/chr 30 Mb</td></tr><tr><td>• ..</td><td>Attraktion (Pacbio HiFi)</td><td>Kale et al., 2022</td><td>74 contigs/chr 17 Mb</td></tr></tbody></table>

Related species

T. durum ABB

T. dicoccoides ABB

T. urartu AA

Ae. speltoides SS

Ae. tauschii DD

Ae. searsii DD

Ae. bicornis DD

Ae. longissima DD

Ae. sharonensis DD

Ae. umbellulata UU
History of sequencing *Triticeae* genomes

T. aestivum ABBDD

<table><thead><tr><th></th><th></th><th></th><th>N50</th></tr></thead><tbody><tr><td>• 2014</td><td>WGS (Illumina)<br/>1 chr BAC-by-BAC (Roche/454)</td><td>IWGSC 2014</td><td>90,000 contigs/chr 5 kb</td></tr><tr><td>• 2017</td><td>WGS (Illumina+Pacbio CLR)</td><td>Choulet et al. 2014</td><td>1 Mb</td></tr><tr><td>• 2018</td><td>RefSeq (NRGene Illumina)</td><td>Zimin et al. 2017</td><td></td></tr><tr><td>• 2020</td><td>10 genotypes (NRGene Illumina)</td><td>IWGSC 2018</td><td>23 Mb</td></tr><tr><td>• ..</td><td>Renan (ONT+Illumina)</td><td>Walkoviak et al. 2020</td><td></td></tr><tr><td>• ..</td><td>Fielder (Pacbio HiFi)</td><td>Aury et al. 2022</td><td>48 Mb</td></tr><tr><td>• ..</td><td>Kariega (Pacbio HiFi)</td><td>Sato et al. 2021</td><td>68 contigs/chr 21 Mb</td></tr><tr><td>• ..</td><td>Attraktion (Pacbio HiFi)</td><td>Athiyannan et al. 2022</td><td>15 scaff/chr 30 Mb</td></tr><tr><td></td><td></td><td>Kale et al., 2022</td><td>74 contigs/chr 17 Mb</td></tr></tbody></table>

Related species

T. durum ABB

T. dicoccoides ABB

T. urartu AA

Ae. speltoides SS

Ae. tauschii DD

Ae. searsii DD

Ae. bicornis DD

Ae. longissima DD

Ae. sharonensis DD

Ae. umbellulata UU
❖ Sequencing wheat wild relatives

Wild relatives: source of genetic diversity of major interest for future wheat improvement

o T. timopheevii AAGG
France2030 PEPR A&N AgroDiv
(Salse J, Pont C et al.)

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

o Ae. ventricosa NNDD
ANR 2023-26 DeFI-Wheat (P. Sourdille et al.)
“Decipher the Fate of Introgressions from
wild relatives in the wheat genome”

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

RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Pratique

anr

agence nationale
de la recherche

o T. aestivum Courtot AABBDD
❖ Sequencing wheat wild relatives

Wild relatives: source of genetic diversity of major interest for future wheat improvement

o T. timopheevii AAGG
France2030 PEPR A&N AgroDiv
(Salse J, Pont C et al.)

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

o Ae. ventricosa NNDD
ANR 2023-26 DeFI-Wheat (P. Sourdille et al.)
“Decipher the Fate of Introgressions from
wild relatives in the wheat genome”

<figure><img src="image_15.png" /></figure>

RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Pratique

anr

agence nationale
de la recherche

o T. aestivum Courtot AABBDD
Triticum timopheevii

<figure><img src="image_12.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>T. durum
AABB</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

HiFi read production:
2 smrtcells on REVIO (Gentyane)

#ccs
Gbps
depth
avg

4.8 M
73 Gb
8x
15 kb

5.5 M
83 Gb
9x
15 kb

(17x)

<figure><img src="image_15.png" /></figure>

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

<figure><img src="image_16.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>T. durum
AABB</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

HiFi read production:
2 smrtcells on REVIO (Gentyane)

#ccs
Gbps
depth
avg

4.8 M
73 Gb
8x
15 kb

5.5 M
83 Gb
9x
15 kb

(17x)

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

<figure><img src="image_20.png" /></figure>
❖ *Triticum timopheevii*

<figure><img src="image_17.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>T. durum
AABB</figcaption></figure>

Assembly: hifiasm-0.19.8-r603

* 9.4 Gb

* 3101 contigs (max=142 Mb)

* N50=19 Mb (119 contigs)

Building 14 pseudomolecules

* 97% of bps anchored

* 41 contigs / chr[1-7]A

* 89 contigs / chr[1-7]G

<figure><img src="image_19.png" /></figure>
❖ *Triticum timopheevii*

<figure><img src="image_21.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>T. durum
AABB</figcaption></figure>

Assembly: hifiasm-0.19.8-r603

* 9.4 Gb

* 3101 contigs (max=142 Mb)

* N50=19 Mb (119 contigs)

Building 14 pseudomolecules

* 97% of bps anchored

* 41 contigs / chr[1-7]A

* 89 contigs / chr[1-7]G

<figure><img src="image_23.png" /></figure>
❖ Triticum timopheevii

<figure><img src="image_20.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>T. durum
AABB</figcaption></figure>

• 41 contigs / chr[1-7]A

• 89 contigs / chr[1-7]G

<figure><img src="image_22.png" /></figure>
❖ Triticum timopheevii

<figure><img src="image_24.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>T. durum
AABB</figcaption></figure>

• 41 contigs / chr[1-7]A

• 89 contigs / chr[1-7]G

<figure><img src="image_26.png" /></figure>
❖ Triticum timopheevii

<figure><img src="image_23.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>T. durum
AABB</figcaption></figure>

o Comparative genomics:

BLAST search: **440,253** exons (T. durum **AABB**)
=> **98%** conserved in T. timopheevii

• T. durum AA vs. T. timopheevii AA: 99.0%id

• T. durum BB vs. T. timopheevii GG: 98.5%id
❖ Triticum timopheevii

<figure><img src="image_27.png" /><figcaption>T. timopheevii
AAGG</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>T. durum
AABB</figcaption></figure>

o Comparative genomics:

BLAST search: **440,253** exons (T. durum **AABB**)
=> **98%** conserved in T. timopheevii

• T. durum AA vs. T. timopheevii AA: 99.0%id

• T. durum BB vs. T. timopheevii GG: 98.5%id
## ❖ Aegilops ventricosa

<figure><img src="image_25.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

HiFi read production:
3 smrtcells on REVIO (Gentyane)

#ccs
Gbps
depth
avg

7.1 M
84 Gb
8x
12 kb

5.8 M
94 Gb
9x
16 kb

4.0 M
62 Gb
6x
15 kb

(23x)

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

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>
## ❖ Aegilops ventricosa

<figure><img src="image_29.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

HiFi read production:
3 smrtcells on REVIO (Gentyane)

#ccs
Gbps
depth
avg

7.1 M
84 Gb
8x
12 kb

5.8 M
94 Gb
9x
16 kb

4.0 M
62 Gb
6x
15 kb

(23x)

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>
❖ *Aegilops ventricosa*

<figure><img src="image_30.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

Assembly: hifiasm-0.19.8-r603

* 8.6 Gb

* 2887 contigs (max=225 Mb)

* N50=39 Mb (63 contigs)

Building chr1-7D pseudomolecules

* 121 contigs ordered

15:chr1D

19:chr2D

16:chr3D

17:chr4D

13:chr5D

24:chr6D

17:chr7D

<figure><img src="image_32.png" /></figure>
❖ *Aegilops ventricosa*

<figure><img src="image_34.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

Assembly: hifiasm-0.19.8-r603

* 8.6 Gb

* 2887 contigs (max=225 Mb)

* N50=39 Mb (63 contigs)

Building chr1-7D pseudomolecules

* 121 contigs ordered

15:chr1D

19:chr2D

16:chr3D

17:chr4D

13:chr5D

24:chr6D

17:chr7D

<figure><img src="image_36.png" /></figure>
❖ *Aegilops ventricosa*

<figure><img src="image_33.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

4.0 Gb

- 121 contigs ordered

15:chr1D

19:chr2D

16:chr3D

17:chr4D

13:chr5D

24:chr6D

17:chr7D

4.0 Gb

<figure><img src="image_35.png" /></figure>
❖ *Aegilops ventricosa*

<figure><img src="image_37.png" /><figcaption>Ae. ventricosa
NNDD</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>T. aestivum
AABBDD</figcaption></figure>

4.0 Gb

- 121 contigs ordered

15:chr1D

19:chr2D

16:chr3D

17:chr4D

13:chr5D

24:chr6D

17:chr7D

4.0 Gb

<figure><img src="image_39.png" /></figure>
<figure><img src="image_36.png" /><figcaption>*T. aestivum*
AABBDD</figcaption></figure>

HiFi read production:
4 smrtcells on REVIO (Gentyane)

<table><tr><td>#ccs</td><td>3.5 M</td><td>3.3 M</td><td>5.3 M</td><td>5.2 M</td><td rowspan="4">(24x)</td></tr><tr><td>Gbps</td><td>78 Gb</td><td>73 Gb</td><td>100 Gb</td><td>98 Gb</td></tr><tr><td>depth</td><td>5x</td><td>5x</td><td>7x</td><td>7x</td></tr><tr><td>avg</td><td>22 kb</td><td>22 kb</td><td>19 kb</td><td>19 kb</td></tr></table>

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

Assembly: hifiasm-0.19.8-r603

- 1744 contigs (max=278 Mb)

- N50=41 Mb
<figure><img src="image_40.png" /><figcaption>*T. aestivum*
AABBDD</figcaption></figure>

HiFi read production:

4 smrtcells on REVIO (Gentyane)

<table><tr><td>#ccs</td><td>3.5 M</td><td>3.3 M</td><td>5.3 M</td><td>5.2 M</td><td rowspan="4">(24x)</td></tr><tr><td>Gbps</td><td>78 Gb</td><td>73 Gb</td><td>100 Gb</td><td>98 Gb</td></tr><tr><td>depth</td><td>5x</td><td>5x</td><td>7x</td><td>7x</td></tr><tr><td>avg</td><td>22 kb</td><td>22 kb</td><td>19 kb</td><td>19 kb</td></tr></table>

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

Assembly: hifiasm-0.19.8-r603

- 1744 contigs (max=278 Mb)

- N50=41 Mb
<figure><img src="image_38.png" /></figure>

A-B-D: complete TE turnover
TE evolution model?

▪ Wicker et al. *Genom Biol* 2018

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

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

T. aestivum

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

T. durum

T. dicoccoides

<figure><img src="image_42.png" /></figure>

T. urartu

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

Ae. tauschii
<figure><img src="image_42.png" /></figure>

A-B-D: complete TE turnover
TE evolution model?

▪ Wicker et al. *Genom Biol* 2018

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

<figure><img src="image_44.png" /></figure>

T. aestivum

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

T. durum

T. dicoccoides

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

T. urartu

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

Ae. tauschii
<figure><img src="image_44.png" /></figure>
<figure><img src="image_48.png" /></figure>
<figure><img src="image_45.png" /></figure>

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

~4,000-13,000 novel insertions / subgenome (~1%)

* Transposition rate is ~ constant

* No impact of polyploidy

* No TE burst

* All families produced novel copies recently

➤ Equilibrium model of evolution
<figure><img src="image_49.png" /></figure>

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

~4,000-13,000 novel insertions / subgenome (~1%)

* Transposition rate is ~ constant

* No impact of polyploidy

* No TE burst

* All families produced novel copies recently

➤ Equilibrium model of evolution
○ Pacbio HiFi unlocked sequencing of *Triticeae* (complex) genomes

○ Assembly stops at satellite repeats
Ultra-long reads useful for T2T gap-less assemblies

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

○ 25x ~10..30 contigs per chr

○ Bionano map still useful to reach gold standard

○ Hi-C map required to build pseudomolecules

□ Perspectives

wheat pangenome in progress

12 wheat landraces representing the worldwide diversity
=> starts in 2024 (Pacbio-Bionano-HiC + IsoSeq)

■ ERC project "Wheat3D"

- IPS2 (Benhamed, Latrasse)

- URGI (Alaux)

- INRAE GDEC

■ NSF project "WheatDiversity"

- IWGSC (J. Stein, Eversole)

- U Georgia (Buell)

- Corteva Agroscience (Johnston)

- INRAE GDEC
* Pacbio HiFi unlocked sequencing of *Triticeae* (complex) genomes

* Assembly stops at satellite repeats
* Ultra-long reads useful for T2T gap-less assemblies

<figure><img src="image_51.png" /></figure>

* 25x ~10..30 contigs per chr

* Bionano map still useful to reach gold standard

* Hi-C map required to build pseudomolecules

□ Perspectives

wheat pangenome in progress

12 wheat landraces representing the worldwide diversity
=> starts in 2024 (Pacbio-Bionano-HiC + IsoSeq)

* ERC project "Wheat3D"

- IPS2 (Benhamed, Latrasse)

- URGI (Alaux)

- INRAE GDEC

* NSF project "WheatDiversity"

- IWGSC (J. Stein, Eversole)

- U Georgia (Buell)

- Corteva Agroscience (Johnston)

- INRAE GDEC
❖ GDEC INRAE-UCA

GENTYANE

E. Belmonte

V. Gautier

C. Poncet

Team BIOINFO

V. Pailler

H. Rimbert

P. Lasserre-Zuber

F. Leloup

Team GeCO

P. Sourdille et al.

Team PALEOEVO

J. Salse

C. Pont et al.

Team DiGEN

S. Bouchet

M. Ranoux

J. Kitt et al.

<figure><img src="image_48.png" /></figure>
❖ GDEC INRAE-UCA

GENTYANE

E. Belmonte

V. Gautier

C. Poncet

Team BIOINFO

V. Pailler

H. Rimbert

P. Lasserre-Zuber

F. Leloup

Team GeCO

P. Sourdille et al.

Team PALEOEVO

J. Salse

C. Pont et al.

Team DiGEN

S. Bouchet

M. Ranoux

J. Kitt et al.

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