Tianamp marine animals dna kit

Total genomic DNA was extracted from dissected somatic tissues using TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions.
Polymerase chain reaction (PCR) amplification of the COI gene with a 680-base pair fragment was performed using a primer pair consisting of (LCO22me2 + HCO700dy2) (Walker et al. 2007 (link)). Thermal cycling conditions were 98 °C for 10 s, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1–2 min, and a final extension of 72 °C for 7 min, following the TaKaRa Ex manufacturer’s protocol. The amplified PCR products were purified and sequenced by Sangon Biotech (Shanghai). The sequences obtained in this study have been uploaded to GenBank (OR297986–OR297991).

A total of 24 tail fins of 12 brown-marbled groupers (E. fuscoguttatus) and 12 giant groupers (E. lanceolatus) were used for genomic DNA isolation. Total DNA was extracted using a TIANamp Marine Animals DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The quality and quantity of the total DNA were determined using a NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Paired-end sequencing libraries with an insert length of 350 bp were constructed using the Illumina Truseq DNA PCR free Library Preparation Kit (Illumina, San Diego, CA, USA). The obtained libraries were then sequenced using the Illumina HiSeq X Ten platform.
The raw data were filtered and trimmed using Trimmomatic [25 (link)]. Clean reads from each sample were aligned to the assembled genome using BWA. SNPs were called using BCFtools (version 1.10.2) [65 (link)] with mpileup, call and filter programs (https://github.com/samtools/bcftools). SNPs with a depth of <8, minor allele frequency (MAF) of <0.05, >20% missing positions, a minimum quality of <30 and a number of alleles of >2 were removed using VCFtools (version 0.1.16) [66 (link)]. The F_ST values were calculated using VCFtools with parameters including a window size of 100 Kb and an F_ST window step of 10 Kb.

Forty ark clams were randomly selected, 20 mg mantles from four random individuals were collected and mixed as a sample. Ten samples were parallelly prepared for detection. Total DNA of each sample was extracted using the TIANamp Marine Animals DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Detection of OsHV-1 was performed according to a previously described protocol based on the Taqman probe method using a pair of primers (BF and B4) and the TaqMan probe BP [43 (link)]. The reaction was performed based on Bio-Rad CFX Connect Real-Time system (Hercules, CA, USA) following 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 10 s, 60 °C for 30 s. The quantification of OsHV-1 was calculated from the standard curve, which was generated from a 10-fold dilution series (10⁸–10¹ copies/mL) of the plasmid containing the target sequence.

Genomic DNA was extracted from the gills of M. lateralis by the TIANamp Marine Animals DNA Kit (No. DP324, TIANGEN, China) following the manufacturer's protocol. Subsequently, DNA was purified using the Column DNA purification kit (No. B610367, Sangon Biotech, China). The quality, purity, and concentration of DNA were assessed through electrophoresis on 1.5% agarose gels and NanoDrop Lite (Thermo Fisher, Waltham, MA, USA). IsoRAD sequencing libraries were then constructed using the method developed by Wang et al. (2016 (link)). Sequencing was performed on the Illumina Hiseq2000 platform (Novogene, China), generating 150 bp paired‐end reads for subsequent analyses.

All progenies and two parents of the mapping family were gathered for further high throughput sequencing. The strategy chosen in this study was a simple and flexible method known as 2b-RAD. Genomic DNA was extracted using TIANamp Marine Animals DNA Kit (TianGen, BeiJing). We followed the protocol of Wang et al.^{10 (link)} to construct standard 2b-RAD libraries by adopting the Type IIB restriction enzymes BsaXI and original adaptors without any selective base in the terminal 3-bp positions. Therefore, the libraries of 33 bp tags contained almost all recognition sites of BsaXI in F. chinensis genome, which can develop markers that evenly covered the whole genome as much as possible. Each library contained an individual-specific barcode incorporated during the library preparation to facilitate the pooling of all samples. All libraries were then pooled together as planed and applied to an Illumina Hiseq2500-V2 for single-end sequencing (1 × 50 bp). In consideration of the de novo genotyping strategy and requirement of establishing high quality reference, libraries of two parents were given extra sequencing depth (1.5-fold higher than progenies).

Specimens of three lampriform species were collected from three sites throughout the East China Sea between 2018 and 2021 via bottom trawling carried out by local fishermen. Muscle samples were dissected from the dorsal part of each specimen, which were then separately stored at −80 °C. We amplified the mitochondrial COI gene to identify each species. A TIANamp Marine Animals DNA Kit (TIANGEN, Guangzhou, China) was used to extract DNA from muscle samples. The mitochondrial COI gene was amplified by polymerase chain reaction (PCR) using the following primers: FishF-COI 5′ TCG ACT AAT CAT AAA GAT ATC GGC AC 3′ and FishR-COI 5′ TAG ACT TCT GGG TGG CCA AAG AAT CA 3′.

All fish samples used in this experiment have been approved by the Care and Use of Laboratory Animals of the Chinese Academy of Fishery Sciences. Before sampling, genomic DNA was extracted from cut fins by TIANamp Marine Animals DNA Kit (TIANGEN, Beijing, China) for genetic sex identification by using PCR amplification with primers sex-F and sex-R (Table 1) [31 (link)]. After anesthesia with MS-222 (20 mg/L) [29 (link)], gonads were dissected from different developmental stages of C. semilaevis, including 90-day post hatching (90 d), 6-month post hatching (6 m), and 1.5-year post hatching (1.5 y). Six females and males were sampled at each stage. Brain, gonad, liver, spleen, heart, kidney, intestine and muscle were collected from three 3 y females and males. Tissues were put into RNAwait RNAlater solution (Solarbio, Beijing, China) quickly and stored in a refrigerator at −80 °C for subsequent RNA extraction.