A chromosome-level, haplotype-resolved genome assembly and annotation for the Eurasian minnow (Leuciscidae: Phoxinus phoxinus) provide evidence of haplotype diversity

Abstract

Background: In this study, we present an in-depth analysis of the Eurasian minnow (Phoxinus phoxinus) genome, highlighting its genetic diversity, structural variations, and evolutionary adaptations. We generated an annotated haplotype-phased, chromosome-level genome assembly (2n = 50) by integrating high-fidelity (HiFi) long reads and chromosome conformation capture data (Hi-C).

Results: We achieved a haploid size of 940 megabase pairs (Mbp) for haplome 1 and 929 Mbp for haplome 2 with high scaffold N50 values of 36.4 Mb and 36.6 Mb and BUSCO scores of 96.9% and 97.2%, respectively, indicating a highly complete genome assembly. We detected notable heterozygosity (1.43%) and a high repeat content (approximately 54%), primarily consisting of DNA transposons, which contribute to genome rearrangements and variations. We found substantial structural variations within the genome, including insertions, deletions, inversions, and translocations. These variations affect genes enriched in functions such as dephosphorylation, developmental pigmentation, phagocytosis, immunity, and stress response. In the annotation of protein-coding genes, 30,980 messenger RNAs and 23,497 protein-coding genes were identified with a high completeness score, which further underpins the high contiguity of our genome assemblies. We performed a gene family evolution analysis by comparing our proteome to 10 other teleost species, which identified immune system gene families that prioritize histone-based disease prevention over NB-LRR-related-based immune responses. Additionally, demographic analysis indicates historical fluctuations in the effective population size of P. phoxinus, likely correlating with past climatic changes.

Conclusions: This annotated, phased reference genome provides a crucial resource for resolving the taxonomic complexity within the genus Phoxinus and highlights the importance of haplotype-phased assemblies in understanding haplotype diversity in species characterized by high heterozygosity.

Keywords: Phoxinus; annotation; comparative genomics; haplotype-phased genome assembly; immune system; structural variants; transposable elements.

Graphical Abstract

Figure 1:

K-mer spectra profile of Phoxinus phoxinus generated from raw PacBio HiFi reads with GenomeScope2. The y-axis shows the k-mer counts and the x-axis shows sequencing depth. The clear bimodal pattern observed indicates a diploid genome with high heterozygosity of 1.43%. The haploid genome size was estimated to be around 805 Mb.

Figure 2:

Repeat landscape of transposable elements (TEs) in the Phoxinus phoxinus genome across different Kimura substitution levels (in %). TEs on the left side of the histogram represent recently active TEs with low divergence from the consensus sequence of TEs, while TEs toward the right side of the histogram represent ancient TEs with higher degrees of divergence.

Figure 3:

Distribution of heterozygosity along the 25 chromosomes in P. phoxinus shown for Hap1 (upper plot) and Hap2 (lower plot). Mean heterozygosity estimates were calculated in 1-Mb bins and converted to the number of heterozygous sites per kb across all 25 chromosomes with alternating colors to differentiate adjacent chromosomes.

Figure 4:

Inferred demographic history of P. phoxinus from PSMC analysis based on Hap1. The time ranges cover the Miocene, Pliocene, Early to Late Pleistocene, and Holocene, each shown in different colors. The x-axis shows years before present on a logarithmic scale, and the y-axis shows the estimated effective population size. Bootstrap results are shown as transparent lines. Hapl2 showed a similar curve.

Figure 5:

Synteny plot of all 25 chromosomes of the P. phoxinus Hap1 (top, blue line) and Hap2 (bottom, orange line) of assemblies. Aligned regions between both haplomes are shown in blocks of gray, and unaligned regions are shown in gaps of white. Inversions are represented in orange, translocations in green, and duplications in light blue.

Figure 6:

GO network of 54 functionally annotated P. phoxinus–specific genes. Genes cluster in 8 major GO terms: “DNA replication,” “DNA repair,” “regulation of DNA-templated transcription,” “response to oxidative stress,” “defense response to virus,” “cellular response to stimulus,” “regulation of response to reactive oxygen species,” and “negative regulation of TORC1 signalling.” Bubble size indicates the frequency of the GO term in the Gene Ontology annotation database (i.e., larger bubbles correspond to more general terms). Edges on the graph connect highly similar or linked GO terms to each other.

Figure 7:

Ultrametric phylogenetic tree of selected teleost species and P. phoxinus. Numbers on the branches represent counts for expanded (+) and contracted (–) gene families. Positive numbers indicate significant gene families that have been expanded since the split from the last common ancestor and negative numbers indicate significant gene families that have been contracted since the split from the last common ancestor.