# A new annotation effort for the T2T genome

Antonio Santiago, Camille Rustenholz

► To cite this version:

Antonio Santiago, Camille Rustenholz. A new annotation effort for the T2T genome. GRAPEDIA: the birth of a centralized, federative portal, CIG Grapedia, Sep 2023, Valencia, Spain. hal-05283624

HAL Id: hal-05283624

https://hal.inrae.fr/hal-05283624v1

Submitted on 25 Sep 2025

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# A new annotation effort for the T2T genome
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Horticulture Research, 2023, 10: uhad061

https://doi.org/10.1093/hr/uhad061

Horticulture
Research

Horticulture
Research

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Article

# The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding

Xiaoya Shi¹,²,†, Shuo Cao²,³,†, Xu Wang²,⁴,†, Siyang Huang²,⁵, Yue Wang²,⁶, Zhongjie Liu², Wenwen Liu², Xiangpeng Leng¹, Yanling Peng², Nan Wang², Yiwen Wang⁶, Zhiyao Ma², Xiaodong Xu², Fan Zhang⁴, Hui Xue², Haixia Zhong⁷, Yi Wang⁶, Kekun Zhang⁸, Amandine Velt¹⁰, Komlan Avia¹⁰, Daniela Holtgräwe¹¹, Jérôme Grimplet¹², José Tomás Matus¹³, Doreen Ware¹⁴,¹⁵, Xinyu Wu⁷, Haibo Wang¹⁶, Chonghuai Liu¹⁷, Yuling Fang⁸, Camille Rustenholz¹⁰,*, Zongming Cheng¹⁸,*, Hua Xiao²,⁷,* and Yongfeng Zhou²,¹⁹,•

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PN40024
~42X HiFi reads
assembled with hifiasm
Assembly metrics

<table><thead><tr><th></th><th>PN40024.v4</th><th>PN40024.v5 (T2T)</th></tr></thead><tbody><tr><td>Total sequence length</td><td>462.2 Mb</td><td>494.9 Mb</td></tr><tr><td>Number of scaffolds / contigs in the 19 pseudomolecules</td><td>165</td><td>20</td></tr><tr><td>Scaffold / contig N50</td><td>6.6 Mb</td><td>25.9 Mb</td></tr><tr><td>Number of Ns</td><td>1.6 Mb</td><td>0</td></tr><tr><td>BUSCO complete genes</td><td>98.2%</td><td>98.5%</td></tr></tbody></table>

More complete and
more contiguous
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<figure><img src="image_7.png" /><figcaption>PN40024.v4</figcaption></figure>
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<figure><img src="image_9.png" /><figcaption>PN40024.v4</figcaption></figure>
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# Real Telomere-to-Telomere assembly

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* 19/19 centromeres identified

* 36/38 telomeres identified but chr15 and chr17 telomeric regions are composed of 45S rDNA tandem repeats
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Eight heterozygous regions

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<figure><img src="image_15.png" /><figcaption>Pinot noir</figcaption></figure>

X

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<figure><img src="image_17.png" /><figcaption>Schiava N</figcaption></figure>

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

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9 selfings

PN40024

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<figure><img src="image_22.png" /><figcaption>PN40024.v4</figcaption></figure>
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<figure><img src="image_24.png" /><figcaption>PN40024.v5 (T2T)</figcaption></figure>

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Phasing is much improved!
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# PN40024 Genomics

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downloads 191/month bioconda-install 12k build passing stars 302

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We can use these new liftoff coordinates to generate a table of correspondences/equivalences between annotation versions or even other grapevine cultivars/species.

Apollo

Lifted genes from v4.3 annotation: 34950 out of 35386 (98,76%)

243 with more than 1 copy

389 extra gene paralogs in total

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# Short-term Scientific Missions (STSMs)

## STSM in INRAE Colmar

## June & July 2023

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Implementing the PN40024
reference genome in the
Grapedia database and portal

cost
EUROPEAN COOPERATION
IN SCIENCE & TECHNOLOGY
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# New RNA-seq samples for PN40024

<figure><img src="image_35.png" /><figcaption>Very young leaves</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>Leaves 10-15 cm</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>Young inflorescence</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>Flowering inflorescence</figcaption></figure>

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

<figure><img src="image_40.png" /><figcaption>Very young berries</figcaption></figure>

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

<figure><img src="image_42.png" /><figcaption>Green berries</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>Ripe berries</figcaption></figure>
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New RNA-seq samples for
PN40024

<table><thead><tr><th></th><th>PacBio</th><th>Poly-A unstranded (Ubi)</th><th>RNA-depleted-stranded (Ubi)</th></tr></thead><tbody><tr><td>very young leaves from young branch</td><td></td><td></td><td></td></tr><tr><td>leaves 10-15 cm</td><td></td><td></td><td></td></tr><tr><td>young inflorescence</td><td></td><td></td><td></td></tr><tr><td>flowering inflorescence</td><td></td><td></td><td></td></tr><tr><td>ripe berries</td><td></td><td></td><td></td></tr><tr><td>tendrils</td><td></td><td></td><td></td></tr><tr><td>internode</td><td></td><td></td><td></td></tr><tr><td>very young berries</td><td></td><td></td><td></td></tr><tr><td>green berries</td><td></td><td></td><td></td></tr><tr><td>green berries before veraison</td><td></td><td></td><td></td></tr><tr><td>green berries after veraison</td><td></td><td></td><td></td></tr></tbody></table>

* Data collected -> Camille Rustenholz's lab.

* Illumina sequencing -> Daniela Holtgräwe's lab

* PacBio sequencing -> Doreen Ware's lab

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FAIR principles

- Findability
- Accessibility
- Interoperability
- Reuse

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snake make

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docker

Ab initio
predictions

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Evidence data

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Protein
data

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Protein
alignments

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RNA-seq
unstranded

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Unstranded
transcriptome
assembly

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Iso-seq
data

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Iso-seq
assembly

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RNA-seq
stranded

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Stranded
transcriptome
assembly

Pseudomolecules
Genome

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EDTA

Softmasked
Genome

Hardmasked
Genome

Evidence
Modeler

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Filtering

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Raw gene
models

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PASA

Lifted
PN40024.v4.3
gene annotation

tRNAscan-SE

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PN40024.v5.1
Gene Annotation

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Functional
annotation
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User

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Home

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Search

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Tools

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Dashboards

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Workflows

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Datasets

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Profile

## Workflows

Home | Workflows

List of available tools

nextflow

RNA-seq Alignment

nextflow

Genome annotation

# Annotation pipeline included as a Grapedia Workflow

So that it can be used on any cultivar of interest

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docker
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# PN40024
## ID proposal for v5

in red versions reference not included in previous
geneIDs

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This could be implemented in all other cultivars
when the same annotation pipeline is used
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# PN40024
## ID proposal for v5

in red versions reference not included in previous
geneIDs

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We could choose to inherit the Chr
and Gene ID through RBH and still
have annotation-identifiable gene IDs
(the inherited IDs should not change
between annotation versions to avoid
confusion)

> This could be implemented in all other cultivars
> when the same annotation pipeline is used
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Grapedia
The Grapevine Genomics Encyclopedia

News

STSMs

Training

Team

Dissemination

Genomes

Genes

Gallery

Annual Meeting

## Grapevine Genomes

### PN40024.T2T(v5)

Genome Browser

Files download

The Genome browser contains reference and alternative (heterozygous, ALT) chromosomal regions of the T2T assembly, plus a chr00 with all unanchored contigs. It also contains accurate mapping of gene annotations (Liftoff, copy mode) of all previous versions, together with mapped long-read (Iso-seq) and short-read (Illumina) sequencing data, which was generated from vegetative and reproductive organs of PN40024 at different developmental stages. This RNA-seq data was kindly provided by Daniela Holtgräwe (Bielefeld University · CeBiTec), Camille Rustenholz (University of Strasbourg · INRAE Colmar) and Doreen Ware (Cold Spring Harbour Laboratory).
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▼ annotations 7

☐ 8x_liftoff

☐ NCBI_liftoff

☐ v0_liftoff

☐ v1_liftoff

☐ v2_liftoff

☐ v3_liftoff

☐ v4_3_just_ref

▼ Reference sequence 1

☐ Reference sequence

▼ Repeat regions 1

☐ Repeats

▼ RNA-seq data 4

☐ PN40024_Illumina_Multimapping

☐ PN40024_Illumina_Unique

☐ PN40024_IsoSeq

☐ PN40024_IsoSeq_Filtered

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Acknowledgements

**TOMSBioLab**

Tomás Matus

David Navarro

Luis Orduña

**Camille Rustenholz's lab**

Camille Rustenholz

Amandine Velt

Marie Lahaye

**Daniela Holtgräwe's lab**

Daniela Holtgräwe

Bianca Frommer

**Doreen Ware's lab**

Doreen Ware

Kapeel Chougule

Marcela Tello-Ruiz

**State Key Laboratory of Tropical Crop Breeding**

Yongfeng Zhou

Hua Xiao

**College of Horticulture**

Zongming Cheng

**Cost-Action**

Anne-Marie Digby

Jérôme Grimplet

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