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The reference genome of
Macropodus opercularis (the
paradise sh)
Erika Fodordo
Czimer
LowLiew
Rhie
✉
✉
Danio rerio
(Macropodus opercularis
M. opercularis
During the 20th century experimental biology gained increased inuence over descriptive biology and concom-
itantly most research eorts began to narrow into a small number of “model” species. ese organisms were not
only selected because they were considered to be representative models for the examined phenomena but were
also easy and cheap to maintain in laboratory conditions1,2. Working with these convenient experimental models
had several advantages and made a rapid accumulation of knowledge possible. It enabled scientists to compare
and build on each other’s ndings eciently as well as to share valuable data and resources that accelerated dis-
covery. As a result of this, a handful of model species have dominated the eld of biomedical studies.
Despite their broad success, these models also brought limitations. As Bolker pointed out: “e extraordinary
resolving power of core models comes with the same trade-o as a high-magnication lens: a much-reduced
eld of view”3. In the case of zebrash research this trade-o has been perhaps most apparent for behavioral
studies. Zebrash are an inherently social (shoaling) species, but most behavioral studies use them in solitary
settings, which arguably is a non-natural environment for them. erefore, the use of other teleost species with
more solitary behavioral proles is warranted for studies of individual behaviors.
Paradise sh (Macropodus opercularis Linnaeus, 1758) are a relatively small (8–11 cm long) freshwater
sh native to East Asia, Southern China, Northern Vietnam, and Laos where they are commonly found in
shallow waters with dense vegetation and reduced dissolved oxygen4. Similar to all other members of the sub-
order Anabantoidae, they are characterized by the capacity to take up oxygen directly from the air through
a highly vascularized structure covered with respiratory epithelium, the labyrinth organ (LO)5. e ability
to “air-breathe” allows anabantoids to inhabit swamps and small ponds with low levels of dissolved oxygen
that would be impossible for other sh species, therefore the LO can be considered an adaptation to hypoxic
1Department of Genetics, ELTE Eötvös Loránd University, Budapest, Hungary. 2Translational and Functional
Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA. 3Frontline Fish Genomics
Research Group, Department of Applied Fish Biology, Institute of Aquaculture and Environmental Safety, Hungarian
University of Agriculture and Life Sciences, Georgikon Campus, Keszthely, Hungary. 4Science Unit, Lingnan
University, Hong Kong, China. 5Computational and Statistical Genomics Branch, National Human Genome Research
Institute, Bethesda, MD, USA. 6 Department of Ethology, ELTE Eötvös Loránd University, Budapest, Hungary. 7These
authors contributed equally: Erika Fodor, Javan Okendo. ✉e-mail: mvarga@ttk.elte.hu; burgess@mail.nih.gov
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conditions6. e evolution of the LO has also improved hearing in some species7,8, and may have led to the
emergence of novel and elaborate mating behaviors, including courtship, territorial display, and parental care6,9.
Another interesting behavior that these sh possess is they build egg “nests” by blowing bubbles on the sur-
face of the water6,10. ese types of intricate and complex behaviors sh made them an important ethological
model during the 1970–80 s, which resulted in a detailed ethogram of the species11,12.
We propose that with recently developed husbandry protocols13 and the advent of novel molecular tech-
niques for genome editing and transgenesis, paradise sh could become an important complementary model
species for neurogenetic studies14. Furthermore, several genomes are now available for the Siamese ghting sh
(Betta splendens), a closely related species to the paradise sh15–17, so a good quality genome sequence of para-
dise sh would enable comparative ecological and evolutionary (eco-evo) studies.
While the mitochondrial genome was already available for this species18 a full genome sequence was lack-
ing. Here, we provide a brief description and characterization of a high quality, de novo paradise sh reference
genome and transcriptome assembly.
e paradise sh used to establish our colony and the source of
the transcriptome samples were purchased from a local pet store (Trioker Ltd., Érd, Hungary). Adult paradise
sh were kept in aerated glass aquariums in the animal facility of the Institute of Biology at ELTE Eötvös Loránd
University. Husbandry conditions were specied previously13. Embryos were raised at 28.5 °C and staged as
described before19. All experimental procedures were approved by the Hungarian National Food Chain Safety
Oce (Permit Number: PE/EA/406—7/2020). Animal experiments in Hungarian academic research centres
are regulated by decree no. 40/2013 (14.II.) issued by the Hungarian Government, which was draed based
on Directive 2010/63/EU on the protection of animals used for scientic purposes. e research on paradise
sh in Dr. Varga’s laboratory was made possible by permit no. PE/EA/406-7/2020, issued by the Pest County
Government Oce on the basis of the above-mentioned government regulation. Wild-caught adult paradise sh
were captured in the areas surrounding Hong Kong and the specimens were handled in accordance to protocols
outlines in the Research Ethics Approval Application via Lingnan University (Reference number: EC051/2021).
Permission to collect wild specimens were also granted in a permit obtained from the AFCD (Agriculture,
Fisheries, and Conservation Department). e permit number is “AF GR CON 11/17 Pt. 7”.
RNA samples were collected from a mix
of embryonic stages (stage 9 – 5 days post fertilization), from caudal tail blastema taken at 3- and 5-days post
amputation, from the kidney, heart, brain, ovaries of an adult female, and the brain and testis of an adult male
paradise sh, respectively. Total RNA was isolated using TRIzol (Invitrogen, 15596026), following the manufac-
turer’s protocol. Samples were puried twice with ethanol and eluted in water. Quality and integrity of the samples
was tested on an agarose gel, by Nanodrop, and using an Agilent 2100. Ribosomal RNA (rRNA) was removed
using the Illumina Ribo-Zero kit and paired-end (PE) libraries were prepared using standard Illumina protocols.
Samples were processed on an Illumina NovaSeq PE150 platform, and a total of 218,715,409 PE reads (2x 150 bp)
were sequenced, resulting in ~65 Gbp of raw transcriptomic data.
Genomic DNA samples were isolated from the tail n of the parental F0 male and female paradise sh using
the Qiagen DNeasy Blood and Tissue Kit (cat no: 69504). Samples were eluted in TE and sent for library prepa-
ration and sequencing. Sample quality-checks were performed using standard agarose gel electrophoresis and
with a Qubit 2.0 instrument. For Illumina short-read sequencing a size-selected 150 bp insert DNA library was
prepared and processed on the Illumina NovaSeq. 5000 platform. Approximately 100 million PE reads (2 × 150
bp) were sequenced for each parent, resulting in approximately 60X coverage for each genome. For PacBio HiFi
long-read single molecule real-time (SMRT) sequencing libraries, genomic DNA was prepared using whole
tissue from the 6 month old F1 ospring and the Circulomics Nanobind tissue kit. Sequence libraries were pre-
pared using the PacBio SMRTbell Template Preparation Kit and HiFi sequenced on a Sequel II platform. A total
of 4,885,238 reads (average length: 15.5 kbp) resulted in ~73 Gbp of raw genomic sequence data.
All software versions used are listed in Supplementary TableS4. The raw data
pre-processing was conducted by doing quality control, adapter trimming, and ltration of the low-quality reads
using trim galore wrapper around FASTQC and Cutadapt20. e genome assembly was generated with the hiasm
genome assembler21 using the High-Performance Computing facility at the National Institute of Health. For the
assembly, 32 cores processing units and 512 Gb of memory was used. e lower and the upper bound binned
K-mers was set to 25 and 75, respectively. e estimated haploid genome size used for inferring reads depth was
set to 0.5Gbp. e rest of the hiasm default settings were used to assemble the homozygous genome with the
build-in duplication purging parameter set to -l1. e primary assembly Graphical Fragment Assembly (GFA) le
was converted to FASTA le format using the awk command.
e Trinity assembler22 was used to create a set of RNA transcripts from the bulk
RNA-seq data. To aid in gene prediction, we downloaded the reviewed Swissprot/Uniprot vertebrate proteins
(Download date 12/01/2022; entries 97,804 proteins) for homology comparisons in annotation pipelines. Gene
prediction was done using the AUGUSTUS23 and GeneMark-ES24 sowares as part of the BRAKER pipeline25 to
train the AUGUSTUS parameters. Final annotation using the assembled transcripts and the vertebrate proteins
database was done using the MAKER pipeline26 with the EvidenceModeller27 tool switched-on to improve gene
structure annotation.
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Sources for the reference data used to create Figs. 2, 3: refs. 28–34.
We performed OrthoFinder analysis35,36 with default parameters, using predicted peptides (including all alter-
native splice versions) of the zebrash genome assembly GRCz11, medaka genome assembly ASM223467v1 and
B. splendens genome assembly fBetSpl5.3. Sequences were downloaded from the ENSEMBL and NIH/NCBI
Assembly homepages, respectively.
e Illumina short read les (accession number ERR3332352) were downloaded from
the Vertebrate Genome Project (VGP) database (https://vertebrategenomesproject.org/). Illumina short read
sequencing was also performed on genomic DNA obtained from the tails of 3 wild-caught sh from the Hong
Kong region. Trim galore version 0.6.10 (https://github.com/FelixKrueger/TrimGalore), a wrapper around cut-
adapt and fastqc was then used to trim the illumina adapter sequences and to discard reads less than 25 bps.
DRAGMAP version 1.3.0 (https://github.com/Illumina/DRAGMAP) was used to map the reads to the reference
genome. e resultant sequence alignment map (SAM) le was then converted to binary alignment map (BAM),
sorted, and indexed using samtools. e Picard was then used to add the read groups information in BAM le.
e genome analysis tool kit (GATK) was then used in calling the variants by turning on the dragen mode. e
bamtools stats and the plot-vcfstats was used in the downstream analysis and visualization of the genomic vari-
ants in the variant call le (VCF).
e assembly and all DNA and RNA raw reads have been deposited in the NCBI under the BioProject study
accession PRJNA824432. Within that project there is the GenBank assembly macOpe2 (GCA_030770545.1), 9
RNA-seq raw sequence data les (SRX20729884, SRX15898419, SRX15898418, SRX15898417, SRX15898416,
SRX15898415, SRX15898414, SRX15898413, SRX15898412), one PacBio HiFi genomic raw sequence data le
(SRX15948463), one Illumina PE short read genomic raw sequence data le for the assembly (SRX15948462)
and Illumina short read genomic raw sequence data les for the 3 wild-caught samples (SAMN39260618,
SAMN39260619, SAMN39260620)37,38. e variant data for this study have been deposited in the European
Variation Archive (EVA) at EMBL-EBI under accession number PRJEB7448139.
We generated the de novo reference genome sequence for this
species using 150X coverage of PacBio SMRT HiFi long-read sequencing and the hiasm genome assembly pipe-
line21. e nal assembly consisted of 483,077,705 base pairs (bp) on 152 contigs (Supplementary TableS1). e
assembled genome demonstrated a very high contiguity with an N50 of 19.2 megabases (Mb) in 12 contigs. e
largest contig was 24,022,457 bp and the shortest contig was 14,205 bp. More than 98% of the canonical k-mers
were 1x copy number indicating that our genome assembly is of very good quality (Fig.1a). e paradise sh
genome repeat content is estimated to be ~10.4%. e “trio binning”40 mode of Hiasm was attempted using sin-
gle nucleotide variant (SNV) data collected from short read sequencing of the F0 parents, however the heterozy-
gosity rate from the lab raised sh was very low at ~0.07% making it impossible to eciently separate maternal
and paternal haplotypes. e resulting assembled reference genome is therefore a pseudohaplotype. e sequence
of the mitochondrial genome (mtDNA) was essentially identical to the previously published mtDNA sequence
for this species (16,495/16,496 identities)18. We followed the B. splendens example and numbered the M. operculis
chromosomes based on their similarity to medaka chromosomes resulting in the chromosomes being numbered
1–19, and 21–24. We performed a whole genome alignment to a recent Betta splendens assembly34 21 chromo-
somes had a 1 to 1 relationship with B. splendens chromosome 9 aligning to two separate paradise sh contigs
(Fig.1b) and M. opercularis chromosome 18 having no signicant homology to a B. splendens chromosome.
is is explained by the number of chromosomes for each species with B. splendens having 21 and M. opercularis
reportedly having 23 chromosomes41. We have not determined whether the B. splendens chromosomes fused or
if the M. opercularis chromosomes split.
e genome is relatively compressed in size and has relatively small introns (mean paradise sh intron
length = 566 bp, whereas mean average teleost intron size = 1,21428) (Fig.2) and shorter intergenic regions.
e N90 for our assembly consists of 23 contigs suggesting that most chromosomes are primarily represented
by a single contig from the de novo assembly, even without any scaolding performed. Searching the contigs
with zebrash telomeric sequences revealed “telomere-to-telomere” assemblies, i.e. contigs that had telomeric
sequences at both ends in the correct orientation, for contigs ptg000004l, ptg000010l, ptg000024l, ptg000026l,
ptg000028l, and ptg000030l representing chromosomes 3, 8, 9, 15, 17 and 21, respectively (Supplementary
TableS3). ese contigs have vertebrate telomeres at both ends while the remaining contigs have one or no
stretches of telomeric sequence at the end of the contig.
Benchmarking Universal Single-Copy Orthologs (BUSCO) was used to evaluate the completeness of our
reference genome assembly with the Actinopterygii_odb10 dataset42,43. e result showed that 98.5% of the
sequence in the reference dataset had a complete ortholog in our genome including 97.3% complete and
single-copy genes and 1.2% complete and duplicate genes. Additionally, 1.2% of the genes were reported as
fragmented and 0.3% of the genes were completely missing.
Using RepeatMasker44, we ana-
lysed and characterized the repeat content in our reference genome assembly. By using a custom-built repeat
prediction library, we identied 32,955,420 bp (6.78%) in retroelements and 11,076,209 bp (2.8%) in DNA trans-
posons (Supplementary TableS2). e retroelements were further categorised into repeat families which were
made up of short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), or long
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terminal repeats (LTR) (Supplementary TableS3). e LINEs were the most abundant repetitive sequence in
the retroelement family at 3.38% (16,447,763 bp) followed by LTRs, 3.19% (15,490,642 bp), and SINEs occurred
Fig. 1 Basic genome assessment. (a) K-mer comparison plot showing copy number of the k-mers as a
stacked histogram colored by the copy numbers found in the paradise sh dra genome assembly. e y-axis
represents the number of distinct k-mers, and the x-axis shows the k-mer multiplicity (coverage). Most
k-mers are represented once (red peak) indicating high quality for the genomic assembly. (b) Whole genome
alignment of the B. splendens genome assembly to the M. opercularis assembly. B. splendens is on the X-axis
and M. opercularis on the Y-axis. B. splendens chromosomes 9 appears to map to two separate M. opercularis
chromosomes and M. opercularis chromosome 18 does not appear to have a clear homologous chromosome in
B. splendens. Chromosome numbering is based on B. splendens alignment to medaka chromosomes14,20.
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at a lowest frequency (0.21%) (Supplementary TableS2). In the LINEs sub-family, we identied L2/CR1/Rex
as the most abundant repetitive sequence (2.15%) followed closely with the retroviral (1.73%) LTR sub-family
(Supplementary TableS2). e proportion of the DNA transposons was estimated to be 11,076,209 bp (2.28%).
Overall, the proportion of retroelements (6.78%) was much higher in the genome compared to that of DNA
transposons (2.28%).
e Vertebrate Genome Project (VGP)45 had performed short read sequencing on a single paradise sh
purchased from a German pet shop (NCBI accession: PRJEB19273), and we captured 9 “wild” samples from the
New Territories in Hong Kong. We performed short read sequencing to ≥20X coverage for 3 of the wild-caught
sh and used the data in combination with the VGP eort to establish the SNP rates within the paradise sh
populations. We identied 5,867,521 variants having a quality score of greater or equal to 30 (Table1) across
4 individual sh. e transition/transversion rate was 1.41. Our analysis identied a total of 663,781 insertions
or deletions ranging from 1 to 60 bps. e rate of SNPs and the indels were 0.5% and 0.1%, respectively.
e Trinity transcriptome assembler was used to
assemble the Illumina short reads from the RNA-sequencing data22 into predicted transcripts. e transcriptome
assembly consisted of 366,029 contigs in 20,157 loci. e integrity of the transcriptome assembly was evaluated
by mapping the Illumina short reads back to the assembled transcriptome using bowtie246; a 98.4% overall align-
ment rate was achieved. e BUSCO analysis conrmed 99.6% completeness with 8.2% single copy orthologs
and 91.4% duplicated genes (i.e. multiple isoforms). A total of 0.4% of the genes were fragmented and 0.0% were
missing completely.
Genome annotation. We analyzed the predicted genes using OrthoFinder35 compared to the Betta splendens17,
medaka47 and zebrash48 genomes (Fig.3). Our analysis shows that 89.6% of the predicted genes (18,057/20,157)
of paradise fish could be assigned to orthogroups (Fig.3b), of which only a very low percentage – 2.5%
(511/20,517) – were present in species-specic orthogroups (Fig.3c). A vast majority of the annotated genes
Daniorerio
Gasterosteus aculeatus
Oryzias latipes
Takifugu rubripres
Te traodon nigroviridis
Cyprinus carpio
Macropodus opercularis
Polyodon spathula
Ictaluruspunctatus
Latescalcarifer
Oreochromisniloticus
Kryptolebiasmarmoratus
Mola mola
Poecilia mexicana
Xiphophorusmaculatus
Fundulus heteroclitus
Astyanax mexicanus
Paedocypris micromegethes
Betta
splendens
500
1000
3000
5000
0.3 0.6 0.9 1.2 1.5
(Estimated) genomesize(Gb)
Mean intron size (bp)
Gene count
20,000 30,000 40,000 50,000
Fig. 2 Distribution of intron sizes for various species of sh. M. opercularis is at the low end of the spectrum
for sh genome size, similar to puer sh species. e line denotes the linear regression line tted over the
data. Grey areas denote the 0.95 condence interval of the t. e diameter of the circles correlates to the
approximate gene count (20 to 50 thousand).
SNV*Indels***
n ts/tv** n
5,867,521 (0.3%) 1.35 633,781 (0.03%)
Tab le 1. Paradise sh variants call summary statistics from four sh compared to the reference assembly. *SNV
- single nucleotide variants. **ts/tv - transitions to transversions ratio. ***Indels - insertions/deletions.
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(17,546/20,517) had orthologs in at least one of the analyzed species, with 70% (14,067/20,517) having orthologs
in all the other species (Fig.3d). e ratio of shared orthogroups also supports the expected phylogeny (Fig.3e).
No custom code was generated for this project. All software with parameters are listed in Supplementary
information.
Received: 29 August 2023; Accepted: 18 April 2024;
Published: xx xx xxxx
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0255075 100 0 1000 2000 010203040
Genesin
orthogroups
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(%)
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Shared:
orthogroups
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orthologs
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e authors thank Lars Martin Jakt and his team for early access to their unpublished data. is work utilized
the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We would like to thank Adam
Phillippy and Brandon Pickett for helpful discussions. e research project was part of the ELTE ematic
Excellence Programme 2020 supported by the National Research, Development, and Innovation Office
(TKP2020-IKA-05) and by the ÚNKP-22-5 New National Excellence Program of the Ministry of Culture and
Innovation from the source of the National Research, Development and Innovation Fund. is research was
supported in part by the Intramural Research Program of the National Human Genome Research Institute
(ZIAHG000183-22) for SB. LO and ISz were supported by the Frontline Research Excellence Grant of the NRDI
(KKP 140353). MV is a János Bolyai fellow of the Hungarian Academy of Sciences.
Conceptualization: Á.M., L.O., S.B., M.V. Data curation: J.O., M.V., S.B. Funding acquisition: Á.M., S.B., M.V.
Investigation: E.F., J.O., N.S., K.S., D.C., A.R., S.K., A.R. Methodology: E.F., J.O., N.S., D.C., I.S.z., L.O., M.V., S.K.,
A.R. Project administration and supervision: S.B., M.V. Writing – original and revised text: E.F., J.O., S.B., M.V.
Open access funding provided by the National Institutes of Health.
e authors declare no competing interests.
Supplementary information e online version contains supplementary material available at https://doi.org/
10.1038/s41597-024-03277-1.
Correspondence and requests for materials should be addressed to M.V. or S.M.B.
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