Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure

doi:10.21203/rs.3.rs-3222040/v1

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Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure

https://doi.org/10.21203/rs.3.rs-3222040/v1

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published 20 Mar, 2024

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Black flounder (Paralichthys orbignyanus, Pleuronectiformes) is an economically important marine fish with aquaculture potential in Argentina. In this study, we sequenced the whole genome of this species using an Illumina sequencing technology. We started with two independent libraries (from one female and one male; each with 150 bp paired-end reads, a mean insert length of 350 bp, and > 35 X-fold coverage). The assemblies yielded a genome size of ~ 538 Mbp. Analysis of the assemblies at the core gene level revealed that more than 98% of the core genes were present, with more than 78% of them having more than 50% coverage. This indicates a somehow complete and accurate genome at the coding sequence level. This genome contains 25,231 protein-coding genes, 445 tRNAs, 3 rRNAs, and more than 1,500 non-coding RNAs of other types. On the other hand, in vertebrates the number of transposable elements (TEs) is related to genome size, but it remains unclear whether the size of introns and exons also plays a role. Therefore, the main objective of the present study was to determine whether the small genome size of Black flounder and other Pleuronectiformes is related to the size of their introns and exons. Indeed, Black flounder, along with pufferfishes, seahorses, pipefishes and anabantid fish appear to have smaller genomes than most other teleost groups. We performed a comparative genomic analysis between Black flounder and other teleost order, in order to determine if the small genomic size could be explained by gene features, including the whole genome genes and introns sizes. We show that the smaller genome size of flounders can be attributed to several factors, including changes in the number of repetitive elements, and decreased gene size, particularly due to lower amount of very large and small introns. Thus, these components appear to be involved in the genome reduction in Black flounder.

Pleuronectiformes

Paralichthys orbignyanus

Black flounder

genome

intron size

Ensembl

evolution

The Pleuronectiforms are a fish order with remarkable biological interest due to their adaptations to demersal life and the presence of a drastic metamorphosis from bilateral pelagic larval symmetry to flatfish symmetry in the adult stage (Robledo et al., 2017; Lü et al., 2021). This order consists of a total of 772 species distributed among 129 genera and 14 families (Nelson & Wilson., 2016). Moreover, some of the species are of great economic importance due to their importance to fisheries and aquaculture (Seitz, 2008). Within this order, the genus Paralichthys is one of the most relevant species for aquaculture. For example, in Asia, aquaculture of the Japanese flounder (Paralichthys olivaceus) is well established (Seikai, 2002). In Latin America, other species belonging to this genus have also been identified with potential for aquaculture. The Black flounder (Paralichthys orbignyanus) in Brazil, Argentina, and Uruguay (Díaz de Astarloa, 2002) and the Chilean flounder (Paralichthys adspersus), endemic to Peru and Chile, are worth mentioning because of their importance to fisheries and aquaculture. Black flounder inhabits shallow estuaries and coastal waters from Rio de Janeiro (Díaz de Astarloa, 2002) to the Gulf of San Matías in northern Patagonia (Díaz de Astarloa & Munroe, 1998). Because this species is a promising candidate for aquaculture, numerous zootechnical studies have been conducted over the last two decades (Bambill et al., 2006; Boccanfuso et al., 2019; López et al., 2009; Magnone et al., 2015; Sampaio & Bianchini, 2002; Sampaio et al., 2007; Radonic et al., 2007; Radonic & Macchi, 2009). However, there is a knowledge gap regarding the genetic resources of this species. For example, only 12 protein sequences are deposited in the GenBank® sequence database (http://www.ncbi.nlm.nih.gov/). This is even more remarkable considering that genetics and genomics are among the most important information for the development of the aquaculture and fisheries industry (Cerdà et al., 2010; Robledo et al., 2017). Due to market demand and its high nutritional value, Black flounder aquaculture has great potential for marine farming in South America. However, there is a bottleneck in the production of Black flounder. One of the first obstacles to the development of Black flounder farming is its slow growth rate, which contrast to the rapid growth observed in nature (Haimovici et al., 2022). Another important problem to solve is its nutrition. In this sense, the development of economically and environmentally sustainable fish food for this species is needed. Finally, the development of Black flounder farming in South America, and especially in Argentina, requires market analysis and bioeconomic models to develop sustainability strategies for this product. From this point of view, and from other aspects such as the study of sex determination and differentiation, sex ratio, nutrition, reproduction, genetic population structure and other important biological traits, obtaining a reference genome is a starting point for the study of all these processes and mechanisms, and consequently it would help for the promotion of Black flounder aquaculture in South America. In the last decade, next-generation sequencing (NGS) technology has gained acceptance, and has played a critical role in obtaining whole genome sequences from different non-model fish species. According to the Ensembl Genome Browser (https://www.ensembl.org), hundreds of fish species with whole genome sequences (WGS) are available to date, and the number of species with sequenced genomes is continuously increasing. Among Pleuronectiformes, thirteen species have their genome sequenced: Tongue sole (Cynoglossus semilaevis) (Chen et al., 2014), Turbot (Scophthalmus maximus) (Maroso et al., 2018), Senegalese sole (Solea senegalensis) (Guerrero-Cózar, et al., 2021), Japanese flounder (Paralichthys olivaceus) (Shao et al., 2017), Hogchoker (Trinectes maculatus), Pelican flounder (Chascanopsetta lugubris), Oriental sole (Brachirus orientalis), Bloch's tongue sole (Paraplagusia blochii), New Zealand turbot (Colistium nudipinnis), Ocellated flounder (Pseudorhombus dupliocellatus), Starry flounder (Platichthys stellatus), and Indian halibut Psettodes erumei) (Lü et al., 2021). In addition, recent studies have compiled the genomes at chromosome level of Turbot (Martínez et al., 2021) and Spotted halibut (Verasper variegatus) (Zhao et al., 2021). A recent study also analyzed the origin of the specialized body structure of flatfishes, the genomes of 11 flatfish species representing 9 of the 14 Pleuronectiformes families were sequenced (Lü et al., 2021). In this scenario, sequencing and annotation of the Black flounder genome would expand the knowledge of this species so that alternative genomic methods could be used to improve fish breeding and to conduct comparative and evolutionary studies with other flatfish species. Comparative genomics has revealed striking differences in genome size even in closely related species. In teleosts, this variation is remarkable, with enormous variance not only in genome size but also in chromosome number (Mank & Avise, 2006; Sarropoulou & Fernandes, 2011; Yuan et al., 2018). Previous studies have clearly shown that Pleuronectiformes, along with pufferfishes, seahorses, pipefishes, and Anabantiformes have the smallest genome compared to other teleost groups (Hinegardner, 1968; Hinegardner & Rosen, 1972). Some authors have associated the reduction of the genome size in amniotes with a decrease in intron size (Zhang & Edwards, 2012). Alternatively, the frequency and size of repetitive elements was also correlated with genome size (Yuan et al., 2018). Pleuronectiform species have only less than 9.0% repetitive elements (Aparicio et al., 2002), compared to ~ 60% in sea lamprey (Petromyzon marinus) (Smith et al., 2018), coelacanth (Latimeria chalumnae) (Nikaido et al., 2013), and salmon (Salmo salar) genomes (Lien et al., 2016). However, the causes and consequences of this putative reduction in the genomes of these fish remain unclear. In general, the variations in genome size are explained by the balance between several genome-level mechanisms that lead to genome enlargement (duplication, transposable elements, and polyploidy) and others that lead to genome reduction (deletions and DNA repair mechanisms) (Brainerd et al., 2001).

Thus, the main goal of the present study was to perform the first sequence and characterization of the whole genome of Black flounder and consequently, to provide an important genomic tool for further research on this species. In addition, comparative genome analysis was used to determine whether Black flounder, among other Pleuronectiformes, present a small genome size compared to other teleost species. The analysis compares exon and intron size of genes and content of transposable and other repetitive elements in different Pleuronectiformes relative to the genomes of other teleost species.

Fish sampling and DNA extraction

Live adult Black flounder, Paralichthys orbignyanus were obtained from the Estación Experimental de Maricultura (INIDEP, Argentina). Prior to dissection, fish were anesthetized with tricaine methanesulfonate (MS-222, Sigma-Aldrich). Tissue samples were then excised from the caudal fin of live specimens and preserved in 96% ethanol. Immediately before dissection, the fins were treated with iodine, and the fish were immediately transferred to a recovery tank with oxygen. The fish were then observed for an appropriate period of time to ensure that they fully recovered from anesthesia. No mortalities were recorded either during sampling or in the days that followed. Genomic DNA was extracted from one adult female and one male. Samples were lysed in 300 µl SSTNE extraction buffer (Blanquer, 1990) with SDS (0.1%) and 5 µl proteinase K (20 mg/ml) for 3 h at 55°C. After 20 min at 70°C, samples were treated for RNA digestion with 7.5 µl RNAse (10 mg/ml) for 1 h at 37°C. Total DNA was purified with cold absolute ethanol (1 ml) after protein precipitation with 5 M NaCl. DNA quality (high molecular weight > 20 kb) was first assessed on agarose gels and DNA quantity was measured using NanoDrop® ND-1000 spectrophotometer (NanoDrop® Technologies Inc). Finally, DNA concentration was accurately measured using a Qubit fluorometer (Life Technologies). Fish were handled in accordance with international animal welfare regulations.

Genome sequencing and genome assemblies

All samples were adjusted to 300 ng/uL and fragmented by ultrasonication on Covaris, aiming for a fragment size of 300 bp. Barcoded libraries were prepared using Wafergen's PrepX ILM DNA Library Kit on Apollo and beads size selected before being pooled and sequenced at 150 bp at the paired end on HiSeq-4000. All procedures followed the manufacturer's instructions. We performed multiple QC sets during library preparation using 300 ng of human gDNA samples as internal control and all QC passed for this project.

Two Illumina paired-end libraries were constructed (from one female and one male) and sequenced with a read length of 150 bp and an average fragment size of ~ 350 bp. Reads were first filtered using Trimmomatic (Bolger et al., 2014) and PrinSeq (Bolger et al., 2014; Schmieder & Edwards, 2011) to remove adapters and low-quality reads, and then sequencing errors were corrected using Quake (Kelley et al., 2010). The libraries were assembled individually using SOAPdenovo 2.04 (Luo et al., 2012) with a kmer size of 29, gap filled with GapCloser (Boetzer & Pirovano, 2012) and finally scaffolded with SSPACE3 (Boetzer et al., 2011) with default parameters.

Genome annotation

First, genomes were masked with Repeatmasker (Hancock, 2004) using the Repbase (Bao et al., 2009) and DFam (Hubley et al., 2016) databases. Using the masked scaffolds, we searched for t-RNAs, r-RNAs and nc-RNAs using tRNAScan-SE (Zou et al., 2015), RNAmmer (Lagesen et al., 2007), and Infernal (Nawrocki & Eddy, 2013) + RFAM (Griffiths-Jones 2003), respectively. Then, we performed gene prediction using GeneId (Blanco & Abril 2009) and Exonerate (Slater & Birney, 2005) trained with Uniprot/Swissprot (Pundir et al., 2016) and the Japanese flounder (Paralichthys olivaceus) genome (Shao et al., 2017). All proteins were annotated using InterProScan (Hancock & Bishop, 2004).

Gene family clustering and validation

To find orthologous proteins in other fish species, we downloaded the genome data of D. rerio (Howe et al., 2013), C. semilaevis (Chen et al., 2014), S. maximus (Xu et al., 2020), P. olivaceus (Shao et al., 2017) from the Zebrafish Genome Project and Ensembl. All protein sequences were aligned using BLASTP (Altschul et al., 1990) with an E value cutoff lower than 1e − 5. BLASTP results were parsed and imported into a MySQL database. Tables within were created by OrthoMCL (Li et al., 2003) to identify homologues with thresholds of percentMatchCutoff = 50 and evalueExponentCutoff = 1e − 6. To evaluate our annotation, we used Benchmarking Universal Single-Copy Orthologs (BUSCO, v3.0.237) (Waterhouse et al., 2018) to assess the assembled genome sequences. We used BUSCO with single copy orthologues from actinopterygii_odb10 to assess the completeness of the genome assembly and annotation. GO terms lists were simplified by means of semantic similarity using REVIGO (Supek et al., 2011), with a cutoff value C = 0.5 and normalized Resnik similarity measure.

Identification and comparison of Repetitive elements (REs)

Repetitive elements (REs), among them Transposable elements (TEs) were quantified for each genome applying a homology-based approach (Tarailo-Graovac et al., 2009) using RepeatMasker with Actinopterygii species repeats and default parameters. Relationship between genome size and TEs percentage in the genome were plotted using ggplot2 R package, and correlations were calculated by Spearman method.

Sex determination genes search in the Black flounder

The BLAST 2.2.29 algorithm was used to retrieve the genomic sequences of SRY-Box Transcription Factor 2 (Sox2), follicle stimulating hormone receptor (fshr), bone morpho - genetic protein receptor type-1B (bmpr1ba), forkhead box L2 (Foxl2), doublesex, mab-3 related transcription factor 1 (Dmrt1), gonadal soma-derived factor (gsdf), and a male-specific duplication of anti-Müllerian hormone (amh) from the Black flounder genome database using other Pleuronectiforms orthologous.

Analysis of C-value in teleosts

All fish C-values (in pg) available were retrieved from the Animal Genome Size Database (http://www.genomesize.com) (Gregory, 2023). As of July 2023, the website contained data for more than 1800 fish species from more than 70 orders (48 entries correspond to Pleuronectiformes species). When necessary, the average C-value per species was calculated. The C-value for P. orbignyanus was estimated assuming that 1 pg = 978 Mb (Doležel et al., 2003). Boxplots were generated using Plotly (Plotly Technologies Inc. Collaborative data science. Montréal, QC). Because most data sets were not normally distributed (according to the Shapiro-Wilk test), C-value data were analyzed with the Mann-Whitney test, comparing Pleuronectiformes with each order using JASP (version 0.16, JASP Team, 2021).

Gene features size analysis at whole genome scale in teleost orders

Complete genome annotations were retrieved from ENSEMBL in gff3 format. We selected species in orders with diverse phylogenetic relationship to Pleuronectiformes. A total of twenty seven species representatives from ten fish orders were used: Anabantiformes (Anabas testudineus, Betta splendens, Mastacembelus armatus), Carangiformes (Echeneis naucrates, Seriola dumerili, Seriola lalandi dorsalis), Cichliformes (Astatotilapia calliptera, Oreochromis niloticus, Pundamilia nyererei), Cypriniformes (Cyprinus carpio, Danio rerio, Sinocyclocheilus rhinocerous), Cyprinodontiformes (Fundulus heteroclitus, Kryptolebias marmoratus, Poecilia formosa), Perciformes (Cyclopterus lumpus, Gasterosteus aculeatus, Sander lucioperca), Pleuronectiformes (Cynoglossus semilaevis, Paralichthys orbignyanus female, Scophthalmus maximus), Salmoniformes (Hucho hucho, Oncorhynchus mykiss, Salmo trutta), Labriformes (Labrus bergylta) and Tetraodontiformes (Takifugu rubripes, Tetraodon nigroviridis). We chose to use the assembly from Black flounder's female because of the higher coverage achieved. See Supplementary Table S1 for more details. We retrieved the size of each gene model (genomic locus) by calculating the difference between the final and initial base coordinates. For each model at a given locus, we calculated the number and size of each exon. Finally, intron sizes were inferred by calculating the difference between the first base coordinate of an exon and the last base coordinate of the preceding exon.

The size distribution of exons and introns at the genome level were plotted using the kernel density estimation of the Seaborn package (parameters: gaussian kernel, band width 0.1 for exons and 0.01 for introns, and grid size of 1000 for exons and 5000 for introns). Boxplots for the size distributions were plotted using Plotly.

Genome annotation

After filtering, the remaining reads accounted for a coverage of more than 35 X-fold on each genome and were retained for assembly (Table 1).

Table 1

Genome coverage for each library. Genome sequencing (post-filtering)
Sample	Library	Read Length (bp)	Insert Size (bp)	Coverage
Female	pair-end	150	~ 350	38.7X
Male	pair-end	150	~ 350	35.8X

Insert here Table 1

The final assemblies were around 524–538 Mb, with the female being the less fragmented (Table 2).

Table 2

Black flounder assembly statistics
Assembly	Female	Male
# Contigs (≥ 1000 bp) Scaffold N50 Size (bp) Longest Scaffold (bp)	78,084 11,125 194,365	75,834 11,694 185,663
Total Scaffold Length (bp)	538,587,025	540,288,577
GC Content (%)	41.39	41.38
%N	0.13	0.09

Insert here Table 2

To verify annotation completeness, we checked for BUSCO orthologues in Actinopterygii DB, where 3459 (3252 complete and 198 fragmented, 94,7% of the selected database) were identified in the combined assemblies (for more information see Table 3).

Table 3

Gene product annotations results, GeneId, Exonerate, Infernal (with RFAM, DB, tRNAScan-SE and RNAmmer)
	Female	Male
Proteins KOG Uncharacterized	25,262 3541 ~ 4%	25,231 3670 ~ 4%
tRNAs (all types)	509	524
rRNAs	11	9
snRNAs	49	57
Others ncRNAs	971	929

Insert here Table 3

Transposable elements (TEs) identification and quantification

RepeatMasker showed large differences in the frequency of repetitive elements (REs) between fish species. Figure 1 compares percentage and proportion of TEs and other REs among different fish genomes. In general, lineages with larger genome sizes had a higher frequency of REs (Fig. 1). In the same line, small genomes such as T. nigroviridis and Pleuronectiformes (especially P. orbignyanus) presented a very low frequency of REs in general. Among the Pleuronectiformes, the genome of P. orbignyanus has a lower proportion of DNA transposons and a higher proportion of long (LINEs) and short (SINEs) interspersed nuclear elements. We also studied the relationship between genome size with different genome elements, showing strong correlation with TEs percentage (r = 0.847; p < 0.001), total intron size (r = 0.939; p < 0.001) and total exon size (r = 0.807; p < 0.001) (Fig. 2).

Gene family clustering

Gene families were clustered according to their similar structures into three different flatfish families: Scophthalmidae (S. maximus), Paralichthyidae (P. orbignyanus and P. olivaceus), Cynoglossidae (C. semilaevis), and D. rerio using OrthoMCL. A total of 10,769 putative specific genes among the five species and 1234 putative specific gene families were identified among the four flatfish species included in the analysis (Fig. 3). Among the GO terms describing the gene families shared by the flatfishes, we identified key biological features associated to flatfish’s evolution (Supplementary Table S2). In this line, we identified GO terms shared by all flatfishes that have been recently associated to genes under positive selection or rapidly evolving (Lü et al., 2021), corresponding to biological process classes such as phosphatidylinositol dephosphorylation (GO: 0046856) or protein transport (GO:0015031). Also, a number of features involved in adaptation to benthic lifestyle are also represented in the GO terms shared by all flatfishes, such as adaptation to lower light intensity, asymmetric pigmentation and body plan, and muscle development (Supplementary Table S2), which agrees with previous observations (Lü et al., 2021; Shao et al., 2017).

Black flounder sex determination genes

The presence of sox2, fshr, bmpr1ba, foxl2, dmrt1, and gsdf ORF in the Black flounder genome database was detected using the orthologous gene of Paralichthys olivaceus (Japanese flounder) (PRJNA369269) as a template, suggesting the presence of conserved genes related to sex determination between these two flounder species, indicating possible shared biological functions or evolutionary relationships.

C-value comparison between Pleuronectiformes and other fish orders

Using publicly available C-value data, we performed a comparative analysis of haploid genome sizes in fish (Fig. 4). We used data from 1504 species belonging to 73 orders (according to the NCBI taxonomy database). The estimated C-value of P. orbignyanus is 0.56 pg, which corresponds to a small fish genome size (5th percentile = 0.61 pg). The average C-value for Pleuronectiformes is 0.734 pg, and the only order with a significantly lower average C-value is Tetraodontiformes (0.618 pg, P < 0.001). Gerreiformes, Ophidiiformes, Uranoscopiformes, Osmeriformes, and Chaetodontiformes also have lower average C-value than Pleuronectiformes, but statistical analysis did not reveal a significant difference. Eight of 49 species (16.3%) of Pleuronectiformes had C-values in the 5th percentile range. In addition, 51.5% of Tetraodontiformes species (34/66) had C-values in the 5th percentile, consistent with the fact that this order had the lowest mean C-value. Other orders represented in this 5th percentile range are: Gerreiformes 2/4 (50%), Uranoscopiformes 2/4 (50%), Syngnathiformes 14/32 (43.75%), Ophidiiformes 2/5 (40%), Anabantiformes 5/28 (17.86%), Chaetodontiformes 2/15 (13.3%), Spariformes 5/39 (12.82%), Istiophoriformes 1/8 (12.5%), Carangiformes 3/26 (11.54%), Blenniiformes 5/44 (11.36%), Perciformes 8/95 (8.42%), Gobiiformes 3/40 (7.5%), Gadiformes 1/20 (5%), Acanthuriformes 1/23 (4.35%), Siluriformes 3/111 (2.70%), Lutjaniformes 1/37 (2.70%), Cypriniformes 8/394 (2.03%) and Centrarchiformes 1/51 (1.96%).

We additionally analyzed the C-values of all Pleuronectiformes species (Fig. 5). The C-value of P. orbignyanus is one of the smallest in the order (Pleuronectiformes 10th percentile value = 0.55 pg), with three members of the family Paralichthyidae having lower C-values (Pseudorhombus jenynsii 0.54 pg, Paralichthys dentatus 0.53 pg, and Pseudorhombus arsius 0.49 pg). Finally, two other species from different families had smaller C-values: Rhombosolea tapirina 0.55 pg (Rhombosoleidae family), and Pleuronectes platessa 0.39 pg (Pleuronectidae family).

Taken together, these results indicate that low genome size is a trait of Pleuronectiformes, and particularly in species from Paralichthyidae (including P. orbignyanus).

Whole genome scale analysis of gene features size in teleost fish

To find other possible factors affecting the relatively small size of the P. orbignyanus genome, we investigated whether this might be related to genetic traits, such as smaller intron sizes. To this end, we first identified the coordinates of all exons and introns for each gene model in the genome of P. orbignyanus and other twenty-six fish species. Using these data, we determined the size of all exons and introns and inferred the size of the genes (exons + introns sizes) (Fig. 6). The estimated and measured C-values for these species show a partial correlation with the size of genes and introns, but not with exon size (Supplementary Fig. S1). In this sense, the total gene size is reduced in P. orbignyanus compared to each species analyzed here (Fig. 6A). Similarly, G. aculeatus (Perciformes) and T. nigroviridis (Tetraodontiformes), species with small genomes, also show a trend toward smaller gene size, which is also confirmed in the percentile analysis of gene size distribution (Supplementary Fig. S2 and Supplementary Table S3). Note, however, that this is not a general trend, as other species with small genomes (B. splendens or C. semilaevis) do not show a general reduction in gene size (Fig. 6A), or that the distribution of gene size in species with larger genomes does not necessarily show a trend toward longer genes, as in the case of Cypriniformes genomes (Fig. 6A). Note that the total number of genes in P. orbignyanus (21531) is comparable to that of other Pleuronectiformes species (21811 bp in C. semilaevis and 21447 in S. maximus) (Fig. 6B and Supplementary Tables S3), which falls within the range of 20–30 thousand genes observed in most species. Interestingly, the genome of T. nigroviridis has the lowest number of genes (14075), whereas other species with large genomes, such as Cypriniformes (~ 32–52 thousand) and, especially, Salmoniformes (~ 51–180 thousand), typically have higher numbers of genes. These results suggest that major genomic rearrangements such as duplication/deletion within coding gene regions, may also be a feature that explains genome size in teleost fishes.

Next, we examined the introns and exons size distribution in fish genomes at the whole genome level. In general, we found that the differences between average and median are smaller for exon sizes when than for intron sizes, suggesting a more compact distribution of introns. It should also be noted that tails towards higher feature size are larger for introns, suggesting that variability in intron size leads to larger introns rather than larger exons (Fig. 7).

We found no major differences in the distribution of exon sizes between species. The median distribution ranged from 119 bp (T. nigroviridis) to 132 bp (F. heteroclitus), while interquantile values 75th -25th difference (IQR) span from 84 bp to 105 bp (S. rhinocerous and P. formosa, respectively). Among Pleuronectiformes, P. orbignyanus (median size value = 127 bp, IQR = 95 bp) has a smaller number of large exons, with the shift occurring more evidently at the 80th quantile (Fig. 7, Supplementary Fig. S3 and Supplementary Table S4). This shift in large exon size is also observed in species with small genome size, such as T. nigroviridis and G. aculeatus. This could have a differential effect on protein size, as we observed a shift toward smaller predicted protein size in Black flounder compared with other fish species, including Pleuronectiformes, T. nigroviridis and D. rerio (Supplementary Fig. S4).

Larger differences between species are observed in intron sizes (Fig. 7, Supplementary Fig. S3 and Supplementary Table S5). Median size values range from 121 bp (T. nigroviridis) to 1139 bp (D. rerio), and IQR values are highly variable, ranging from 286 in T. nigroviridis to 2602 in D. rerio. However, in P. orbignyanus, we did not detect a significant reduction in intron size at the genomic level. The largest difference in Pleuronectiformes is observed for large introns, with very large introns (> 10000 bp) being extremely rare in P. orbignyanus (Fig. 7, see insert). The 99th quantile for this species is 7311 bp, whereas in other Pleuronectiformes it is 16966 bp (C. semilaevis) and 19470 bp (S. maximus) (Supplementary Fig. S3 and Supplementary Table S5). The 99th quantile in other orders is highly variable: in T. nigroviridis, the value of the 99th quantile is 6002 bp (which is the lowest value) and increasing to 40237 bp in D. rerio. These observations are consistent with the intron size distributions for these two species, where there is a shift towards lower sizes in T. nigroviridis, and a shift towards larger intron sizes in D. rerio, including an evident shoulder at ~ 1000 bp (Fig. 7). Finally, P. orbignyanus genes have few small introns, starting at the 1th quantile (73 bp). In summary, intron size may help explain genome shrinkage (as in T. nigroviridis) or expansion (as in D. rerio), and to some extent, our results suggest that this may have an impact to explain P. orbignyanus genome shrinkage compared to or other Pleuronectiformes. In this sense, the lower amount of very large and small intron contributes to reduce the overall coding genes size in P. orbignyanus.

Teleost is the largest group of vertebrates, with more than 35,000 species described (Fricke et al., 2020) with and undoubtably associated disparity of forms and functions. About 20 years ago, the first complete genome of a teleost, the Japanese pufferfish (Takifugu rubripes), was sequenced (Aparicio et al., 2002). Since then, the number of fish species sequences has continuously increased, largely because of advances in sequencing technologies and assembly algorithms (Ravi & Venkatesh, 2018). According to NCBI and as of August 2023, the genome of only about 3% (1071) of all fishes has been sequenced. However, these numbers will soon be outdated due to large-scale sequencing of fish species genomes. For example, the 10,000 Fish Genomes Project (Fish10K) (Fan et al., 2020) aims to sequence and obtain reference genomes from representative fish species and has recently begun using long-read sequencing and Hi-C technology for at least one representative species from all families to obtain better quality reference genomes. All the information obtained from Fish10K, together with genome sequences from other laboratories (such as the one presented in this study), will help improve breeding programs and promote sustainable aquaculture in the future, as well as perform genome editing and genomic selection (Lu & Luo, 2020).

Using next-generation sequencing (NGS) technologies, we were able to sequence, assemble, and annotate the genome of non-model species, the Black flounder. Genome sequencing identified 25,231 protein-coding genes, which is higher than the 21,787 protein-coding genes described in Japanese flounder (Shao et al., 2017). The estimated genome size of Black flounder is ~ 538 Mbp, similar to the genome size of other flatfish species: Japanese flounder (534 Mb) (Shao et al., 2017), Turbot (568 Mb) (Figueras et al., 2016), Spotted halibut (556 Mb) (Zhao et al., 2021), ~ 15% bigger than the tongue sole genome (477 Mb) (Chen et al., 2014) and ~ 11% smaller compared to the Senegalese sole genome (~ 612 Mb) (Manchado et al., 2016). In addition, another 8 flatfish species have genome sizes ranging from 399.64 Mb in Ocellated flounder to 643.91 Mb in Japanese flounder (Lü et al., 2021). Before to fully determine the genome size, it is recommended to use multiple complementary methods to assess the genome quality (Gurevich et al., 2013). In this study, we assessed the N50 sizes of contigs and scaffolds and the completeness of the genome using KOG and BUSCO. The results of quantitative measurements to determine assembly completeness using BUSCO showed a high percentage of conserved orthologs in turbot (Xu et al., 2020). High BUSCO values (94.7%) were observed in Actinopterygii using the combined assemblies, to compensate for the lack of long reads. Future transcriptome and long reads sequencing studies are therefore required to provide a comparative, more comprehensive and quantitative overview of the level of completeness achieved.

The comparative analysis of homologous genes reveal that 1234 putative orthologous groups were exclusive for the three different flatfish families (Scophthalmidae, Cynoglossidae, and Paralichthyidae) (Fig. 3). Within clusters several semantically similar GO terms correspond to features that are key for adaptation to benthic lifestyle (Supplementary Table S2). It is important to note that a recent comparative genome analysis has examined the origins of flatfish body structure, among other important features, from an evolutionary perspective using the genomes of eight new species from fourteen families of Pleuronectiformes (Lü et al., 2021), and we identified some gene clusters shared by Pleuronectiformes associated to these features. However, more comprehensive comparative analyses including the South American black flounder can be performed once those annotated genomes become publicly available.

In this study, we performed a comparative analysis of 27 flatfish species from 10 orders to understand why Black flounder have a relatively small genome size. Genome size varies from very small genomes as in Tetraodon nigroviridis (~ 350 Mb) (Neafsey & Palumbi, 2003) to large genomes as in Salmo salar (2967 Mb) (Yuan et al., 2018). As expected, the comparative analysis revealed that Black flounder has one of the smallest genomes among the compared species, which is consistent with the results of previous studies in which flatfish genome sizes were among the smallest of all teleosts (Figueras et al., 2016; Xu et al., 2020). This observation is also consistent with the analysis of fish C-values (Figs. 4 and 5). Indeed, the genomes of B. splendens (Anabantiformes), G. aculeatus (Perciformes, suborder Gasterosteiformes), C. semilaevis (Pleuronectiformes), T. rubripes, and T. nigroviridis (Tetraodontiformes) in the selected data set belong to groups with typically small genome size according to the average C-value (Fig. 4). In addition, several C-values of Pleuronectiformes are within the 10th percentile of the distribution, especially in the families Paralichthyidae (including Black flounder), Rhombosoleidae and Pleuronectidae. The large genome size in salmonids could be explained by a specific round (called 4R) whole-gene duplication event in this lineage (Lien et al., 2016). Although genome size diversity in teleosts could be the cause of the tremendous diversity of morphology, ecology, and behavior in this group (Volff, 2005), the origin of this dispersion is still unknown.

Genome size is not only affected by the teleost-specific rounds of whole-genome duplications (3R and 4R) (Meyer & Van de Peer, 2005). There are also other genomic structures that has been related to the variation of genome size. It is well known, the changes of proportion of REs (repetitive elements) can modify the size of the genome (Yuan et al., 2018; Canapa et al., 2015). Complementary to the changes of REs, the numbers of exons and introns could also explain the changes in genome size in teleost fish (Jakt & Johansen., 2022).

Most of these REs come from specific sequences that replicate and move through the genome, called transposable elements (TEs) (Bourque, 2009). Thus, Black flounder is consistent with the compact genomes observed in other flatfish genomes, with ∼5% TEs, slightly more than the 3% in pufferfish another species of extremely small genome (Aparicio et al., 2002). Consequently, the results of this study are consistent with the generally accepted fact that variation in the genome is related to the amount of repetitive DNA in eukaryotic species (Kidwell, 2002), which has also been demonstrated in teleost fishes (Chalopin et al., 2015). The abundance of TEs varies with genome size and position in the fish tree of life (Shao et al., 2019). In this work, we compare the comparison proportion of REs (Fig. 1): DNA transposons, long and short interspersed nuclear elements (LINEs and SINEs, respectively), and long terminal repeats (LTRs) in 27 fish species. In Black flounder and the order of Pleuronectiformes in general, the proportion of all REs was low, which is probably one of the reasons for the small size of the Black flounder genome. This study (Fig. 2) revealed an extraordinary abundance of transposable elements (TEs) in zebrafish. However, it is particularly surprising that despite the large number of TEs, the zebrafish genome itself is not exceptionally large (Fig. 2). However, this does not provide a clear explanation for the lack of correspondence between the proportion of TEs and the total genome size in this particular species, and further studies are needed to shed light on this intriguing phenomenon.

As mentioned before, another feature that could modify the genome size, is the number and size of exons and introns. To this end, the coordinates of all exons and introns in the genomes of twenty-seven fish species, including P. orbignyanus, were determined. Results showed that P. orbignyanus has smaller gene sizes compared to all fish species studied, including groups with small genomes such as the Tetraodontiformes (Brainerd et al., 2001) and some Perciformes (Reid et al., 2021). Flow cytometric analyses revealed that in four pufferfish species of the family Tetraodontidae, genome size varies between 0.38 and 0.82 pg, whereas the sister family Diodontidae is larger (0.8-1 pg), likely due to DNA loss while the two families diverged during evolution (Noleto et al., 2009). This is consistent with our study, which showed that all Pleuronectiformes generally have a small genome size (0.3–1.1 pg), which is in general among the smallest of all teleost fishes. Since T. nigroviridis also shows a trend towards smaller gene sizes, this can represent one of the possible explanations for genome shrinkage.

In eukaryotes, intronic DNA is the major component of genes and genomes and plays a key role in gene regulation, and intron size is important from an evolutionary perspective (Zhang & Edwards, 2012). In teleosts, genome size and intron size are closely related, with intron size reflecting genome size (Jakt et al., 2022). To further investigate the origin of the small genome size of Black flounder, we examined the distribution of total genome size of introns and exons for 27 species in 10 fish orders. Our analyses show that, as expected, the mean and median exon sizes are smaller compared to intron sizes, suggesting a more compact distribution, as observed in other Pleuronectiformes species (Robledo et al., 2017). However, we detected a difference in the distribution of the intron size in Black flounder's genome, particularly regarding the decrease of number of very large introns and small introns (Fig. 7 and Supplementary Fig. 4). Indeed, distribution of intron size in other species with small genomes (such as T. nigroviridis and T. rubripes) indicate smaller intron size, suggesting that this may be a mechanism leading to genome size reduction in those species. However, the low content of small and very long introns in P. orbignyanus could explain the gene size reduction observed in this species. Regarding exon size, as reported in previous studies, no remarkable differences in exon size distribution were detected between species (Li et al., 2017). Considering these results, we concluded that intron size may have an impact in gene size and consequently genome shrinkage in Black flounder, particularly associated to lower contents of very large and small introns. Further comparative analysis with genomes annotated at chromosome level among Pleuronectiformes and other fish species with remarkably small or large genomes (such as T. nigroviridis and salmon genomes) may help to shed light into other mechanisms involved, such as size of intergenic regions.

One of the implications of changes in the structure of genes, such as the reduction in intron size, can clearly affect the Alternative splicing (AS) of genes. AS is an essential mechanism that plays a key role in cellular differentiation and organism development (Wang et al., 2015). In teleosts, lower AS frequencies have been observed in highly duplicated genomes (e.g., zebrafish) and large occurrences in compact genomes (e.g., pufferfish). These inverse correlations between AS frequency and genome size appear to be the same across fish species (Lu et al., 2010). This study opens a new line of research with that questions whether smaller introns could affect the AS mechanism (Mechaly et al., 2009; 2011; 2018). Nowadays, we have a huge amount of data from transcriptome analysis and from fish genomes, which is constantly increasing. This resource is extremely helpful for conducting comparative genome studies and investigating the potential correlation between alternative splicing and genome size through RNA-seq analysis.

In the last decade, several genomic, proteomic, and metabolomic studies have focused on characterizing the reproduction, development, nutrition, immunity, and toxicology of flatfish (Cerdà et al., 2008; 2010; Forné et al., 2010). Flatfish genomics is important for studying the management of wild fish populations, improving fish conservation, and increasing productivity in aquaculture (Cerdà et al., 2010). In the last two years, long-read sequencing technologies have been applied to several Pleuronectiformes species, allowing the assembly of chromosomes (Guerrero-Cózar et al., 2021; Jasonowicz et al., 2022; Lü et al., 2021; Martínez et al., 2021; Zhao et al., 2021). High-quality reference genomes are important for studying evolutionary variation in fish genome structure and organization (Varadharajan et al., 2019). Moreover, P. orbignyanus is considered ‘data deficient’ in the IUCN Red List of Threatened Species (Riestra et al. 2020), and thus genetic variation at the genomic level of this species can become the start point for future studies about the population genetic structure on this specie. This technology will allow us to perform new comparative and in-depth analyzes soon. For example, one important question that remains to be addressed is whether there is a general sex-determining gene (locus) in fish and specifically in flatfish. In the recent study by Ferchaud et al., (2021), the authors suggest SRY-Box Transcription Factor 2 (Sox2) as a possible candidate not only in Greenland halibut but also in other flatfishes. Other gene candidates include follicle stimulating hormone receptor (fshr) in Senegalese sole (de la Herrán, et al., 2023), bone morpho-genetic protein receptor type-1B (bmpr1ba) in Hippoglossus stenolepi (Jasonowicz et al., 2022), Forkhead box L2 (Foxl2) and Doublesex and mab-3 related transcription factor 1 (dmrt1) in Japanese flounder (Paralichthys olivaceus) (Shu et al., 2022), and Gonadal soma-derived factor (gsdf) in Atlantic Halibut (Hippoglossus hippoglossus) (Palaiokostas et al., 2013; Einfeldt et al., 2021). More recently, other study in the Japanese flounder carried out with amh-mutant flounders created by using the CRISPR-Cas9 system technology shown that amhy is necessary for testicular formation in this species (Hattori et al., 2022). In this study, the preliminarily analysis of sex determination genes (SD) in Black flounder, we search and found in our genome all the genes that have been associated as gene markers in flatfish, as mentioned above (see Table 4). Since all genes related to sex determination in flatfish have been found in the genome of Black flounder, we suggest that the first attempt to study the mechanism of sex determination in this species should focus on the closest species, e.g., investigate the role of dmrt1 and amy, both genes that have been studied in Japanese flounder, a species of the same order.

Table 4

Summary of sex determination (SD) genes described in flatfish
Species (Common names)	Gene name (Abbreviation)	Physiological actions in reproduction fish	References
Reinhardtius hippoglossoides (Greenland Halibut)	SRY-Box Transcription Factor 2 (sox2)	role in sex determination and differentiation	Ferchaud et al., 2021
Solea senegalensis (Senegalese sole)	Follicle stimulating hormone receptor (fshr)	role in folliculogenesis	De la Herrán et al., 2023
Hippoglossus stenolepis (Pacific Halibut)	bone morpho - genetic protein receptor type-1B (bmpr1ba)	potential candidate for master sex-determining	Jasonowicz et al., 2021
Paralichthys olivaceus (Japanese flounder)	Forkhead box L2 (foxl2)	role in ovarian differentiation	Shu et al., 2021
	A male-specific duplication of anti-Müllerian hormone (amhy)	role in sex determination and differentiation	Hattori et al., 2021
	Doublesex and mab-3 related transcription factor 1 (dmrt1)	roles in sex determination and neural development	Shu et al., 2021
Hippoglossus hippoglossus (Atlantic Halibut)	Gonadal soma-derived factor (gsdf)	role in testicular differentiation	Palaiokostas et al., 2013; Einfeldt et al., 2021

Insert here Table 4

Based on the analyzed features, we concluded that the most important components that could be responsible for the reduced flounder genome are (i) the low frequency of repetitive elements, (ii) the overall reduced gene size, which may be associated mainly to (iii) the lower number of both very large and small introns with possible implications to AS. The last two components (ii and iii) have a lower value than in other species with similar C-values, suggesting that this may be a novel genome reduction strategy.

In summary, in this study we generated a genome assembly of Black flounder (Paralichthys orbignyanus). The results show a reduced genome size and frequency of REs of flounder, and we have demonstrated that this is related to reduced gene locus size due to a reduction in the number of very large and small introns. Finally, our study also serves to investigate a strategy of genome reduction in teleost fishes.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Samples were collected from fish from the Estación Experimental de Maricultura (INIDEP, Argentina) in accordance with international animal welfare regulations. No ethical approval number is available for this study because sampling occurred in 2016, before the creation of the Institutional Committee of Ethics in Animal Experimentation of INIDEP (CICUAE-INIDEP acronym in Spanish) in 2017. However, it should be noted that prior to the creation of CICUAE-INIDEP, all animal experiments followed the CCAC guidelines on: the care and use of fish in research, teaching and testing (http://www.ccac.ca).

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF SUPPORTING DATA

This sequencing data has been deposited at the National Center for Biotechnology Information (NCBI) where the Bioproject Accession ID is PRJEB36690.

DISCLOSURE STATEMENT

The authors have nothing to disclose.

COMPETING INTEREST

The authors declare that they have no competing interests exist.

FUNDING INFORMATION

This study was funded by projects grants by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT, Argentina) to ASM (PICT-2017-2839 and PICT-2020 SERIE-0696) and partially funded by JV (MPCUdG2016/130).

AUTHORS’ CONTRIBUTIONS

ASM, GFB, EJS, AT and JV participated in designing the genome sequencing. ASM collected the samples. GFB and EJS performed the genome assembly. ASM, FV, JV design and performed the comparative genomic analysis. AB and EJS participated in comparative genome analysis. NS wrote and tested a custom script to parse and extract genomic features from annotated genomes. GMS and JV contributed to the writing and interpretation of the data. ASM coordinated and designed the study. FV and ASM supervised the whole study and revised the manuscript. All authors have read and approved the manuscript for submission.

ACKNOWLEDGEMENTS

We thank the Laboratorio de Maricultura del Instituto Nacional de Investigación y Desarrollo Pesquero (INIDEP), Argentina, and E. Aristizabal for kindly providing tissue samples. The authors thank J.J. Boccanfuso and E. Ricci for their help in collecting fish samples or transporting them to Europe; M.B. Gomes Pardo for DNA extraction; and Prof. P. Martínez Portela for helpful comments on this manuscript. We would like to acknowledge the support of the Centro de Supercomputación de Galicia (CESGA) in the completion of this work. We thank Centro de Cómputos de Alto Rendimiento (CeCAR) and UBA-FCEN-QB-Cluster for granting use of computational resources which allowed us to perform most of the computational analyses included in this work. Finally, this article is dedicated to our colleague and friend Nicolás Stocchi, who recently passed away.

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Genome sequencing and analysis of black flounder (Paralichthys orbignyanus) reveals new insights into Pleuronectiformes genomic size and structure

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Abstract

Figures

Introduction

Material and Methods

Fish sampling and DNA extraction

Genome sequencing and genome assemblies

Genome annotation

Gene family clustering and validation

Identification and comparison of Repetitive elements (REs)

Sex determination genes search in the Black flounder

Analysis of C-value in teleosts

Gene features size analysis at whole genome scale in teleost orders

Results

Genome annotation

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Transposable elements (TEs) identification and quantification

Gene family clustering

Black flounder sex determination genes

C-value comparison between Pleuronectiformes and other fish orders

Whole genome scale analysis of gene features size in teleost fish

Discussion

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Declarations

References

Additional Declarations

Supplementary Files

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