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Haplotype-resolved genome assembly of the tetraploid potato cultivar Désirée
Tim Godec*
1,2
, Sebastian Beier
3
, Natalia Yaneth Rodriguez-Granados
4
, Rashmi Sasidharan
4
,
Lamis Abdelhakim
5
, Markus Teige
6
, Björn Usadel
3,7
, Kristina Gruden
1
, Marko Petek
1
1 National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana,
Slovenia
2 Jožef Stefan International Postgraduate School, Ljubljana, Slovenia
3 Institute of Bio- and Geosciences (IBG-4 Bioinformatics), Bioeconomy Science Center
(BioSC), CEPLAS, Forschungszentrum Jülich GmbH, Jülich, Germany
4 Plant Stress Resilience, Institute of Environmental Biology, Utrecht University, Utrecht, The
Netherlands
5 PSI (Photon Systems Instruments), Drásov, Czech Republic
6 Molecular Systems Biology (MOSYS), Department of Functional and Evolutionary Ecology,
University Vienna, Vienna, Austria
7 Faculty of Mathematics and Natural Sciences, Institute for Biological Data Science, Cluster
of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Düsseldorf,
Düsseldorf, Germany
* corresponding author
corresponding author email: tim.godec@nib.si
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Abstract
Cultivar Désirée is an important model for potato functional genomics studies to assist
breeding strategies. Here, we present a haplotype-resolved genome assembly of Désirée,
achieved by assembling PacBio HiFi reads and Hi-C scaffolding, resulting in a
high-contiguity chromosome-level assembly. We implemented a comprehensive annotation
pipeline incorporating gene models and functional annotations from the Solanum tuberosum
Phureja DM reference genome alongside RNA-seq reads to provide high-quality gene and
transcript annotations. Additionally, we provide a genome-wide DNA methylation profile
using Oxford Nanopore reads, enabling insights into potato epigenetics. The assembled
genome, annotations, methylation and expression data are visualised in a publicly
accessible genome browser ( https://desiree.nib.si ), providing a valuable resource for the
potato research community.
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Background & Summary
Potato ( Solanum tuberosum ) is one of the most important and widely cultivated crops
worldwide, with a significant role in global food security and agricultural research. Despite its
significance, many studies still rely on the genome of the double monoploid (DM) clone of
group Phureja DM1–3 516 R44
1,2 which lacks a substantial portion of the gene repertoire
and variability found in cultivated tetraploid potato varieties.
The potato cultivar Désirée is a red-skinned late-season potato variety, originally bred in the
Netherlands in 1962 by crossing parent cultivars Urgenta and Depesche (Potato Pedigree
Database)
3
. It is still cultivated due to its favourable agronomic traits, such as predictable
yields and high tolerance to drought and some pathogens
4
. It has also been used in
breeding programs, yet a genome assembly for the Désirée cultivar has not been available.
In research, it has been propagated in tissue cultures, and used for genetic manipulation
including gene overexpression
5
, gene silencing
6
, and Crispr-Cas gene editing
7
.
Although haplotype-resolved genome assemblies are becoming common in diploid
organisms, the high heterozygosity rate, extensive repeat content, and the autopolyploid
nature of cultivated potatoes still present significant challenges for generating high-quality
haplotype-resolved assemblies. Currently, five haplotype-resolved genomes of autotetraploid
potato cultivars are publicly available
8–12 as well as several phased diploid genomes
13–15
. The
recently published haplotype-resolved tetraploid potato assemblies rely on labour-intensive
techniques such as single-pollen sequencing
10 or the use of parental and crossing material
11
,
which may not always be available.
Adding to existing publicly available genomes, we provide a reference quality (CRAQ overall
AQI of 97.5) haplotype-resolved genome assembly of the tetraploid cultivar Désirée,
assembled using solely PacBio HiFi and Illumina Hi-C data. Our assembly is accompanied
by a comprehensive structural and functional gene annotation reaching 99.4 % BUSCO
completeness for Solanaceae, accompanied by orthology to DM genes. For the potato
research community, we provide an online resource featuring a genome browser and
downloadable genomic assembly and annotation files, providing a valuable tool for studies
involving allele-specific expression or promoter analysis.
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Methods
Sample preparation and sequencing
Leaves from 4-week old S. tuberosum cv. Désirée plants were collected and flash-frozen.
High molecular weight genomic DNA (HMW gDNA) used for PacBio HiFi, Illumina and
Oxford Nanopore Technologies (ONT) sequencing was extracted from the leaf tissues using
a modified CTAB method
16
. The concentration and quality of the extracted DNA were
assessed using a NanoDrop spectrophotometer.
PacBio HiFi
HMW gDNA was sent to National Genomics Infrastructure (NGI) Sweden for library
preparation and sequencing on the PacBio Sequel II platform. We obtained 79.4 Gbp of raw
data, consisting of 4.1 million reads.
Illumina Hi-C
Leaves from 4-week old S. tuberosum cv. Désirée plants were collected, flash-frozen in
liquid nitrogen and ground using mortar and pestle. Hi-C library prep using the Omni-C kit
(Dovetail Genomics) and sequencing were performed on an Illumina NovaSeq 6000 platform
by NGI Sweden. Sequencing generated 2018.4 million paired-end (2 × 150 bp) reads.
ONT
The HMW gDNA was used for ONT DNA library prep using the SQK-LSK110 kit and
sequenced on a MinION using the FLO-MIN106 flow cell. Reads were basecalled using
Dorado (v0.7.2) with the model dna_r9.4.1_e8_sup@v3.3 which generated 5.8 Gbp. The
reads with methylation-related tags were converted to bedMethyl format using modkit
(v0.4.1).
Illumina short reads
Illumina short-read library was constructed from the HMW gDNA and sequenced on Illumina
NextSeq 2000 by ELIXIR Slovenia node to generate 150 bp paired-end reads. The
short-read sequencing generated approximately 138 Gbp of raw data, consisting of 460.1
million paired-end (2 × 150 bp) reads.
Genome size and heterozygosity estimation
The genome characteristics of S. tuberosum cv. Désirée, including genome size,
heterozygosity, and repeat content, were estimated using Illumina short-read data and a
k-mer based approach. A 21-mer frequency distribution was generated with Jellyfish
(v2.2.10), and the genome's key features were inferred using GenomeScope2 (v2.0). The
haploid genome size was estimated at 669.6 Mbp, with a heterozygosity rate estimated at
3.8–5.7%.
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De novo genome assembly, Hi-C scaffolding and quality
assessment
PacBio HiFi and Illumina Hi-C reads were initially assembled into four sets of
haplotype-resolved contigs using Hifiasm (v0.19.8-r603)
17–19
. Hifiasm primary unitigs were
searched against DM genome assembly with blastn (v2.5.0)
20 and best matches were
visualised on Graphical Fragment Assembly with Bandage (v0.8.1, Fig. 1a)
21
. We performed
quality control of the contigs using Merqury (v1.3, Fig. 1b)
22 k-mer spectra and BUSCO
completeness scores (v5.4.7, solanales_odb10 dataset)
23
. The length of haplotype draft
assemblies ranged from 761.6 Mbp to 888.4 Mbp with contig N50 sizes ranging from
7.0 Mbp to 13.7 Mbp (Table 1).
Contigs identified as contaminants were removed based on blastn (v0.8.1) searches against
a custom-built contaminant database, which includes Solanum plastid and mitochondrial
sequences and bacterial NCBI RefSeq sequences.
Decontaminated scaffolds were anchored to chromosomes by mapping Hi-C reads to each
haplotype set separately following the manufacturer’s recommended pipeline for Omni-C
data ( https://omni-c.readthedocs.io ). Briefly, Hi-C reads were mapped using BWA-MEM
(v0.7.17-r1188)
24 then the mappings were parsed with pairtools (v0.3.0)
25 followed by
samtools (v1.3.1)
26 to identify and extract valid pairs. Valid pairs were used to anchor and
orient scaffolds into chromosomes using YaHS (v1.2a.1)
27 and Juicebox Assembly Tools
(v2.17.00)
28,29
.
Chromosomes 11 and 12 of haplotype 4 lacked ~20 Mbp and ~30 Mbp part of the
pericentromeric region, respectively, and haplotype 1 contained two additional unplaced
scaffolds (scaffold_22 and scaffold_23). Alignment of these scaffolds to reference genome
(DM v6.1) and inspection of Hi-C contacts suggested that these scaffolds are the missing
regions of chromosomes 11 and 12 in haplotype 4. Therefore, we remapped Hi-C reads and
incorporated these two scaffolds in haplotype 4 using Juicebox Assembly Tools (v2.17.00).
The final scaffolded assembly size amounts to 3.3 Gbp, with individual haplotypes ranging
between 762 and 888 Mb. As expected, one haplotype is highly similar to the DM haplotype,
whereas other haplotypes can be more dissimilar (Fig. 1c). A comparison of Merqury k-mer
spectra between the initial contigs and the scaffolded chromosomes (Fig. 1a) reveals that
many apparent duplications in the contigs are resolved during scaffolding. A small proportion
of sequences remains missing from the chromosomes and those can be found in the whole
genome FASTA.
The haplotype assemblies were sequentially aligned using minimap2 (v2.28) and analyzed
with SyRi (1.7.0) to identify syntenic regions and structural rearrangements which were
visualized using plotsr (v1.1.1, Fig. 1d).
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haplotype 1
haplotype 2
haplotype 3
haplotype 4
all haplotypes
Genome length (Mb)
888.4
862.7
761.6
858.5
3371.2
GC content (%)
35.31
35.27
35.12
35.47
35.3
Contig N50 (Mb)
11.5
13.7
11.7
7.0
10.8
Number of contigs
1126
867
1048
2695
5736
Chromosome length
(Mb)
721.9
729.9
698.5
709.4
2859.6
Scaffold N50 (Mb)
56.9
61.4
60.1
57.1
58.0
Number of scaffolds
705
496
523
1350
3074
Complete BUSCO (%)
96.2%
96.1%
96.6%
95.7%
99.6%
Size of repeat
sequences (Mb)
514.2
534.1
489.3
503.6
2041.2
Total gene number
76903
81184
75816
75550
309453
Table 1. Summary of the four haplotypes of the Désirée genome assembly.
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Fig. 1 General characteristics of Désirée genome assembly a) Assembly graph of primary
unitigs coloured by best match to DM chromosomes (also designated with numbers on the
graph). b) Merqury k-mer spectra for initial contigs and scaffolded chromosomes. The k = 21
was used. K-mers are categorized as read-only (grey), unique (red), and shared (blue,
green, purple, orange). Peaks corresponding to higher multiplicities indicate the presence of
highly repeated k-mers. c) Dot plot comparing cv. Désirée chromosome-anchored contigs
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with DM v8.1 chromosomes. The colour designates contig identity. d) Genomic synteny of
cv. Désirée haplotype-resolved assembly.
Genome annotation
Repeat elements in the S. tuberosum cv. Désirée genome were identified using the
Extensive de novo TE Annotator (EDTA, v2.2.1)
30
. Repetitive sequences cover 489 - 534
Mbp per haplotype, representing more than 70% of the genome (Table 2).
The prediction of protein-coding genes in the assembled S. tuberosum cv. Désirée was
determined using five complementary approaches: de novo , homology-based,
transcriptome-based, deep-learning, and reference-based predictions (Fig. 2).
Fig. 2 Workflow overview of S. tuberosum cv. Désirée genome annotation.
For transcriptome-based prediction, two methods were applied for short reads and Iso-Seq
reads, respectively. Short reads from multiple tissues were aligned to each haplotype using
STAR (2.7.10a)
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, and transcripts were assembled with StringTie2 (v2.2.1)
32
, followed by
Portcullis (v1.2.4)
33 for junction validation. Iso-Seq reads from five S. tuberosum cultivars
were mapped to both haplotypes using minimap2 (v2.28)
34
, and transcripts were generated
using IsoQuant (v3.3.1)
35 and TAMA Collapse (tc_version_date_2023_03_28) 36
.
BRAKER3 (v3.0.8)
37 was used in ETP mode to predict gene models by integrating de novo ,
homology-based, and transcriptome-based predictions. Repeat masking of the assembly
was performed with RepeatMasker (v4.1.2), using EDTA annotations. Protein sequences
from OrthoDB (green plant orthologs) were provided as evidence, and short-read STAR
alignments with invalid junctions removed were included.
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Helixer (v0.3.3)
38,39 was used for deep-learning-based gene prediction via its web interface
( https://www.plabipd.de/helixer_main.html ). Gene models from the S. tuberosum reference
genome (DM v6.1, UniTato annotation) were transferred to the Désirée assembly using
Liftoff (v1.6.3)
40
. All five transcript or gene model sets were consolidated using Mikado
(v2.3.4)
41 to generate a non-redundant set of transcripts. Protein-coding gene completeness
was assessed using BUSCO (Table 2, v5.4.7, solanales_odb10 dataset) and OMArk (v0.3.0,
omamer v2.0.2)
42
.
The predicted protein-coding genes were functionally annotated using EggNOG Mapper
(v2.1.11)
43 with the EggNOG database (version 5.0.2)
44 for the Viridiplantae subset. This
included categories such as gene names, Gene Ontologies (GOs), enzyme functions (EC),
and KEGG pathways, reactions, and modules, along with CAZy families, PFAM domains,
and more. Additionally, functional land-plant protein annotations were predicted using
Mercator4 (v7)
45 via the web platform ( https://www.plabipd.de/mercator_main.html ).
Annotations from EggNOG and Mercator4 were combined into the final GFF3 annotation file.
Orthologous groups between haplotypes and UniTato genes were identified using
OrthoFinder (v2.5.5)
46
. Across haplotypes, 55.3% of orthogroups contained genes from all
four haplotypes, 22.9% from three haplotypes, 19.2% from two haplotypes, and 2.7% from a
single haplotype. When comparing the Désirée annotation to UniTato, 17.24% of genes were
specific to the Désirée annotation.
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Type
haplotype 1
haplotype 2
haplotype 3
haplotype 4
Repeat elements
DNA
46.6 Mbp (6.4%)
54.1 Mbp (7.4%)
44.6 Mbp (6.4%)
45.9 Mbp (6.5%)
Helitron
36.1 Mbp (5.0%)
38.0 Mbp (5.2%)
33.6 Mbp (4.8%)
42.3 Mbp (6.0%)
LINE
12.4 Mbp (1.7%)
8.1 Mbp (1.1%)
7.5 Mbp (1.1%)
8.1 Mbp (1.1%)
LTR
176.5 Mbp (24.4%)
188.0 Mbp (25.8%)
165.9 Mbp (23.7%)
193.6 Mbp (27.3%)
LTR/Copia
16.8 Mbp (2.3%)
19.3 Mbp (2.6%)
20.3 Mbp (2.9%)
23.9 Mbp (3.4%)
LTR/Gypsy
136.2 Mbp (18.9%)
133.6 Mbp (18.3%)
130.0 Mbp (18.6%)
102.8 Mbp (14.5%)
MITE
11.9 Mbp (1.6%)
10.2 Mbp (1.4%)
13.0 Mbp (1.9%)
10.6 Mbp (1.5%)
Other
72.8 Mbp (10.1%)
76.2 Mbp (10.4%)
69.7 Mbp (10.0%)
71.3 Mbp (10.1%)
SINE
5.1 Mbp (0.7%)
6.6 Mbp (0.9%)
4.7 Mbp (0.7%)
4.9 Mbp (0.7%)
Total
514.2 Mbp (71.2%)
534.1 Mbp (73.2%)
489.3 Mbp (70.1%)
503.6 Mbp (71.0%)
Protein-coding genes
Total gene number
76903
81184
75816
75550
Mean gene length (bp)
1695.85
1610.97
1687.71
1677.79
Mean CDS length (bp)
1062.59
1032.74
1060.23
1061.68
Mean exon number
5.28
5.04
5.31
5.28
Mean intron number
4.28
4.04
4.31
4.28
Complete BUSCO (%)
94.1%
93.3%
95.4%
93.7%
Single Omark HOGs
82.9%
82.5%
84.3%
82.8%
Duplicated Omark HOGs
11.6%
11.6%
11.5%
11.9%
Missing Omark HOGs
5.5%
5.9%
4.2%
5.4%
Mercator4 proteins annotated
(%)
93.5%
93.5%
93.7%
93.5%
Mercator4 proteins classified
(%)
50.5%
46.5%
50.7%
50.0%
Mercator4 bins occupied (%)
94.2%
93.9%
94.6%
94.3%
Table 2. Summary of genome annotations for each haplotype.
Data Records
The raw sequencing data, including Illumina Hi-C, Illumina paired-end, PacBio HiFi, and
ONT reads, have been deposited at the National Center for Biotechnology Information
(NCBI) Sequence Read Archive (SRA) under BioProject number PRJNA1185028. Plastid,
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mitochondrial and bacterial sequences used for removal of contaminant contigs were
downloaded from NCBI RefSeq release 218. Transcriptomic data used for gene annotation
was downloaded from public repositories: SRA under accessions PRJNA1192223,
PRJNA1186376, PRJNA718240, PRJNA803222, PRJNA1209787 and PRJNA1191209; the
Gene Expression Omnibus (GEO) under accession GSE232028; and the National Genomics
Data Center (NGDC) under accession CRA006012. Existing gene models used in the gene
annotation pipeline were downloaded from https://unitato.nib.si and https://spuddb.uga.edu .
The genome assemblies of the four haplotypes have been submitted to NCBI GenBank
under the BioProject accessions PRJNA1196677, PRJNA1196678, PRJNA1196679 and
PRJNA1196680. The assembled genome, including annotations, methylation profile and
identified orthologs, is hosted in a Zenodo repository under DOI: 10.5281/zenodo.14609304
and is also accessible via an interactive genome browser at https://desiree.nib.si .
Technical Validation
We assessed the assembly quality and completeness using DNA sequencing read mapping,
CRAQ, BUSCO analysis, and Merqury k-mer based evaluation. Illumina reads were mapped
with BWA (v0.7.17), while PacBio and ONT reads were aligned using minimap2 (v2.28).
Mapping rates were 99.90%, 100.00%, and 99.74% for Illumina paired-end, PacBio, and
ONT reads, respectively. CRAQ (v1.0.9)
47 analysis of PacBio and Illumina mappings yielded
a regional AQI of 96.3 and an overall AQI of 97.5, classifying the assembly as reference
quality (AQI > 90). Assembly completeness was assessed with BUSCO (v5.4.7) using the
solanales_odb10 lineage database, identifying 5930 (99.6%) of the 5950 BUSCO
orthologous groups in both the whole genome and chromosome-only assemblies (Table 1).
Merqury (v1.3) analysis, using a Meryl (v1.3) database constructed from Illumina reads,
estimated genome completeness at 98.57% for the whole genome and 95.73% for the
chromosomes. The estimated QV values were 54.30 and 58.53 for the whole genome and
chromosomes, respectively.
Completeness of gene annotation was assessed using OMArk (v0.3.0, omamer v2.0.2),
BUSCO (v5.4.7) and Mercator4 (v7). OMArk analysis demonstrated that our annotation
captured 94.1%-94.6% of Hierarchical Orthologous Groups (HOGs) per haplotype, with
duplication rates ranging from 11.5% to 11.9% (Fig. 3a). When combining genes from all
haplotypes, the proportion of complete HOGs reaches 99.3%, meaning that not all
conserved genes are present in all haplotypes. Similarly, BUSCO analysis reported a
haplotype completeness range of 93.3%–95.4% (Table 2), while the whole genome
annotation achieved 99.4% completeness. Protein classification via Mercator4 revealed that
93.9%–94.6% of Mercator bins were occupied per haplotype, increasing to 97.5% when
combining all proteins (Table 2). As expected, the Mercator bin with the largest proportion of
missing proteins was associated with clade-specific metabolism (Fig. 3b). Additionally, the
classified proteins showed no significant deviation from the median protein length,
confirming consistency in annotation quality (Fig. 3c).
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Fig. 3 Validation of gene annotation. a) OMArk quality assessment showing consistency,
completeness and count of proteins across all four haplotypes. b) Histogram showing the
percentage of Mercator4 functional bins occupied by the Désirée proteins. c) Histogram
displaying the distribution of proteins grouped by their percentage deviation from the median
protein length.
Usage Notes
The presented Désirée genome assembly is of high contiguity, completeness and phasing
quality and presents a valuable resource for haplotype-aware transcriptomics, proteomics
and epigenomics analyses. The transfer of UniTato annotations
48 provides translation of
gene identifiers from the DM to the Désirée genome. The RNA-seq datasets used to
supplement gene model annotation are predominantly from mature leaf and root tissue, thus
genes specifically expressed in other tissue and developmental stages may not be fully
captured in the current annotation.
The genome was produced from a plant propagated in tissue culture for over a decade. A
recent pangenome study
49 found that in vitro propagated plants of the Solanum section
Petota have greater numbers of TEs in their genomes. While this seems to hold for LTR
elements and DNA transposons in the Désirée genome, overall TE expansion is not evident.
Examining the DNA methylation profile available in the Désirée genome browser might
provide more insight into specific transposable element expansion in this cultivar.
Recently, efforts were made to generate potato pangenomes
9,49
. However, the number of
included phased tetraploid genomes is still limited. Including Désirée and more phased
tetraploid genomes will improve the completeness of potato pangenome. This will bridge
knowledge gaps in potato genomics and give potato breeders a powerful toolkit for
developing more resilient and productive cultivars.
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted January 14, 2025. ; https://doi.org/10.1101/2025.01.14.631659doi: bioRxiv preprint
Code Availability
The code, scripts and command-line tool commands used for genome assembly, annotation
and quality control are freely available in the GitHub repository
https://github.com/NIB-SI/desiree-genome .
Acknowledgement
This work benefits from resources and services provided by ELIXIR, a distributed
infrastructure for life science data, funded by national governments and the European
Commission, particularly the Elixir-SI node for performing Illumina paired-end sequencing.
Funding for this work was provided by the European Union's Horizon 2020 research and
innovation programme project ADAPT (grant agreement No GA 2020 862-858), Slovenian
Research and Innovation Agency (ARIS) project grants P4-0165, P4-0431, and J4-3089. SB
and BU are supported by the German Federal Ministry of Education and Research (BMBF)
in the frame of the German Network for Bioinformatics Infrastructure (de.NBI).
Author contributions
TG : Methodology, Data curation, Investigation, Visualization, Writing - Original Draft. SB :
Investigation, Writing - Review & Editing. BU : Writing - Review & Editing. NYRG : Resources,
Writing - Review & Editing. RS : Resources, Writing - Review & Editing. LA : Resources,
Writing - Review & Editing. MT : Funding acquisition, Writing - Review & Editing. KG : Funding
acquisition, Conceptualization, Writing - Review & Editing. MP : Conceptualization, Validation,
Resources, Supervision, Project administration, Writing - Review & Editing.
Competing interests
The author(s) declare no competing interests.
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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