GoKawiilSample collection
Wild specimens of Aspiorhynchus laticeps, Gymnodiptychus pachycheilus, P. dipogon, S. younghusbandi, Chuanchia labiosa, Platypharodon extremus and Herzensteinia microcephalus were collected from Xinjiang, Gansu and Tibet (Xizang) provinces of China. Except for S. younghusbandi, the age and sex of the other sampled individuals were not determined. The captured male and female S. younghusbandi were used to obtain the F1 generation through artificial breeding. Genomic DNA from all specimens, including the F1 generation of S. younghusbandi, was extracted from the muscle tissue using the cetyltrimethylammonium bromide (CTAB) method. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Southwest University (LAC2024-2-0127).
Library construction and sequencing
For DNA sequencing, libraries were constructed using the MGIEasy Universal DNA Library Prep Kit V1.0 (1000005250, MGI) following the standard protocol. In brief, 1 μg of genomic DNA was randomly fragmented using a Covaris system. The fragmented DNA was then selected using MGIEasy DNA Clean Beads (1000005279, MGI) to achieve an average size of 200–400 bp. The selected fragments were end repaired, 3′ adenylated and ligated with adapters. The DNA samples were then amplified by PCR, and the products were purified with MGIEasy DNA Clean Beads (1000005279, MGI). The double-stranded PCR products were heat denatured and circularized using the splint oligo sequence from the MGIEasy Circularization Module (1000005260, MGI). The resulting single-stranded circular DNA was used to construct the final library and underwent quality control. The qualified libraries were sequenced on the DNBSEQ-T7RS platform.
For PacBio HiFi sequencing, genomic DNA of S. younghusbandi F1 generation was fragmented using the Megaruptor 3 system and concentrated with AMPure PB magnetic beads. SMRTbell libraries were prepared using the Pacific Biosciences SMRTbell Prep Kit 3.0. The libraries were size selected on a PippinHT system for 20-kb molecules, followed by primer annealing and the binding of SMRTbell templates to polymerases using the Revio Polymerase Kit. After primer annealing, sequencing was conducted on the Pacific Biosciences Revio platform.
For ONT ultra-long sequencing, the high-molecular-weight genomic DNA was extracted from the same F1 generation individual. The library was prepared using a Ligation Sequencing Kit (SQK-LSK110, Oxford Nanopore Technologies), and sequencing was performed on the PromethION platform with the flowcell (version r10.4.1).
For the Hi-C library, muscle tissue from the same S. younghusbandi individual was crosslinked with 40 ml of 2% formaldehyde solution at room temperature for 15 min. Next, the tissue was ground, washed and resuspended in nuclei isolation buffers to generate a nuclear suspension for further processing. The nuclear suspension was then treated with SDS and Triton X-100, and digested overnight with MboI at 37 °C. Subsequently, the cleaved ends were labelled with biotin-14-dCTP and ligated using DNA ligase to generate chimeric DNA molecules. After reverse crosslinking and biotin removal, the fragments were enriched using streptavidin beads. Finally, adapters were added, and sequencing was performed on the DNBSEQ-T7RS platform.
Genome survey and mitochondrial phylogeny
Short reads were used to count k-mer frequencies by KMC (3.1.1)48 with default parameters. The results were subsequently subjected to genomescope 2.049 and Smudgeplot to estimate genome size and ploidy with recommended parameters. We assembled and annotated the mitochondrial genomes from short reads using MitoZ (v3.4)50 with assembler megahit–kmers_megahit 59 79 99 119 141 -clade Chordata parameters. We extracted the nucleotide sequences of 13 mitochondrial protein-coding genes and aligned sequences using MAFFT (v7.526)51 with default parameters. The alignments were concatenated into a super matrix, and a phylogenetic tree was subsequently constructed using IQ-TREE (v2.0.3)52 with the parameters -m TEST, -seqtype DNA, and -bb 10000. In addition, we added the mitochondrial genome of O. macrolepis (NC_023799.1) as the outgroup.
Haplotype-resolved genome assembly
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