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).