# Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae)

## TL;DR

This paper presents a complete genome assembly for the stream-dwelling fish Opsariichthys bidens, revealing genetic adaptations to high-oxygen and high-flow environments.

## Contribution

The study provides the first telomere-to-telomere genome assembly for O. bidens, enabling precise identification of centromeres and telomeres.

## Key findings

- The genome assembly includes 38 chromosomes with high completeness and continuity.
- Expanded gene families related to oxygen utilization and endurance were identified.
- Positive selection was observed in pathways linked to mitochondrial function and metabolic regulation.

## Abstract

Opsariichthys bidens is a widely distributed freshwater fish uniquely adapted to the challenging conditions of stream environments, which are typically characterized by high dissolved oxygen and sustained high-flow velocities. To thrive in such habitats, stream fishes often evolve enhanced capabilities in oxygen utilization, energy metabolism, and sustained swimming performance. In this study, we constructed the telomere-to-telomere (T2T) reference genome for O. bidens. Genomic analyses identified several expanded gene families and pathways associated with these adaptive traits. This high-quality genome assembly provides a foundational resource for further research into the genetic mechanisms underlying ecological adaptation in stream-dwelling fishes.

Stream-dwelling fishes face diverse hydrological pressures, making the broadly distributed Opsariichthys bidens an ideal model for analyzing adaptive evolution. To elucidate its adaptation to a high-dissolved-oxygen and high-flow-velocity stream environment, a high-quality genome with comprehensive annotation is essential. In this study, we present the first telomere-to-telomere (T2T) reference genome for O. bidens, constructed using PacBio HiFi, Oxford Nanopore Ultra-long, and Hi-C technologies. The assembled genome spans 841.96 Mb, comprising 38 chromosomes, each in a single contig (contig N50 = 22.42 Mb, 2.5-fold higher than the previous version), achieving a gap-free standard with 99.34% BUSCO completeness. Additionally, 38 centromeric sequences, 37 double-telomeric sequences, and 1 single-telomeric sequence were successfully identified, providing essential molecular markers. Phylogenetic analysis revealed a divergence time of 13.5 million years between O. bidens and its closely related species Z. platypus, with collinearity analysis confirming their high genomic conservation. Gene family analysis revealed 350 expanded families enriched in pathways associated with adaptation to high-dissolved-oxygen environments (e.g., antioxidant defense, oxidative phosphorylation, mitochondrial electron transport chain) and high-flow-velocity environments (e.g., exercise endurance, myocardial contraction, actin binding). Positive selection analysis further identified multiple pathways and key genes involved in mitochondrial optimization, oxygen utilization, and metabolic regulation. The T2T assembly greatly improves assembly continuity and enabling precise identification of centromeres and telomeres for O. bidens. These results provide a robust foundation for studying its adaptive evolution to stream environment.