2.1 Sample collection, sequencing, and karyotype analysis
One adult maleO. bidens collected from Hengyuan farm in Dongyang City, Zhejiang Province, was used for genome sequencing and assembly. Physiological sex was identified using the squeezing method to produce seminal fluid from the fish abdomen. Muscle tissue was collected and treated following the QIAamp DNA Mini Kit (QIAGEN, Germany) protocol and was quickly frozen in liquid nitrogen for genomic DNA (gDNA) sequencing. Tissues including the testis, ovary, brain, heart, liver, kidney, muscle, eyes, and skin of the male individual were subjected to transcriptome sequencing using TRIzol-extracted total RNA from the pooled samples.
The gDNA extracted from the muscle tissue was randomly sheared to 300-500 bp fragments and amplified using PCR. A DNA library with an insert size of 350 bp was prepared and constructed using DNA library prep kits (TruSeq, Illumina, U.S.). The short insert library was sequenced using the Illumina Hiseq 2500 platform in 150 PE mode to estimate genome size and heterozygosity by k-mer frequency distribution using Genomescope (Ranallo-Benavidez et al. 2020). Meanwhile, the gDNA extracted from fresh muscle was used to construct the DNA Nanopore library for long-read sequencing using the PromethION platform (Oxford Nanopore, Oxford, UK), according to the manufacturer’s instructions.
To prepare the Hi-C library for chromosome-level genome assembly, 1 g of muscle tissue from the same male individual was fixed with formaldehyde, digested using HindIII (restriction enzyme), biotin marking, physical shearing, and DNA amplification, and finally used to construct the Hi-C library with insert sizes of 350 bp. As previously described, the Hi-C library was sequenced using the Illumina HiSeq 2500 platform with PE 150 mode (Rao et al., 2014).
Transcriptome sequencing was conducted to estimate the coverage rate of the assembled genome over gene regions and predict gene models. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from nine tissue pools collected from the same male individual mentioned above. RNA quality was assessed using a Nanodrop spectrophotometer (Labtech, Ringmer, UK). The RNA was used to construct the Illumina RNA-seq library and sequenced using an Illumina NovaSeq 6000 in PE150 mode (Illumina Inc., San Diego, CA, USA), as previously described (Liu et al., 2021).
The karyotype of the hooks out carp was examined by metaphase spread from the head kidney cells of six-month-old fish artificially bred from the Hengyuan farm. Chromosome samples were obtained by phytohemagglutinin injection, colchicine-air drying, and Giemsa staining, as previously described(Wang et al., 2008).