Escherichia coli dh5α competent cells

After being crushed using an EOG-sterilized BioMasher II (Nippi, Inc., Tokyo, Japan), DNA was extracted from the whole body of each adult D. gallinae using a DNeasy Blood and Tissue Kit (Qiagen, N.V., Venlo, Netherlands) with 50 μl of EB buffer. The DNA samples were stored at −30°C until used. PCR was conducted for mitochondrial cytochrome c oxidase subunit I (COI) using KOD FX Neo (Toyobo Co. Ltd., Osaka, Japan), and the primers FCOIDG and RCOIDG designed for COI of D. gallinae (Øines and Brännström, 2011 (link)). For 16S ribosomal RNA sequencing of symbiont bacteria, PCR was conducted using Ex Taq HS (Takara Bio Inc., Kusatsu, Japan) and two sets of universal primers, 10F-1507R or 10FF-1515R. The PCR fragment was cloned using pGEM-T Easy Vector (Promega, Madison, WI, USA), Mighty mix for DNA ligation (Takara), and Escherichia coli DH5α competent cells (Takara). Plasmids were extracted using a QIAprep Spin miniprep kit (Qiagen), the purified plasmids were subjected to sequencing reactions using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) and the sequencing primers M13F or M13RV in the flanking regions of the vector along with the bacterial universal primer 16SA2. The primer sequences are shown in Supplementary Table 2.

The open reading frame (ORF) of the FIP2-like gene was obtained using the specific primers FIP2-like-ORF (Table S1) and PCR using PrimeSTAR^® GXL DNA polymerase (Takara), which was also used to compare sequences between different samples. The amplification procedure was as follows: 98 °C for 5 min; 36 cycles at 98 °C for 10 s, 53 °C for 15 s, and 68 °C for 1 min; and a final extension at 68 °C for 10 min. The PCR products were analyzed on 1.0% agarose gels, and the putative fragment was purified using a DNA purification kit (Takara). The amplified fragment was cloned into the pMDTM 19-T Vector (Takara), transformed into Escherichia coli DH5α competent cells (Tsingke, Nanjing, China), and 10 monoclones per sample were sequenced (Tsingke). The nucleotide sequence and transcriptional amino acid sequence of FIP2-like were analyzed using alignment tool DNAMAN8.0 (Lynnon, QC, Canada). The domain of FIP2-like was predicted using the CDD Tool from the NCBI databases.

A Piwi1 cDNA fragment of 311 bp was obtained from the C. farreri transcriptome (Wang et al., 2013 (link)) and compared to the National Center for Biotechnology Information (NCBI) database using BLASTX. Amplification of 5′- and 3′-RACE were conducted with scallop testis cDNA and two specific PCR primers (PR-5′: 5′-GCAACAGACATCAA CATCTGTTTCTTGG-3′, PR-3′: 5′-ATGCTGATTGGAGCAGAGATCTTCGTGG-3′) according to the SMART™ RACE cDNA Amplification Kit protocol (Clontech, Mountain View, USA). PCR products were gel-purified and cloned into the pMD18-T vector (Takara, Otsu, Japan) then transformed into Escherichia coli DH5α competent cells (Takara, Otsu, Japan). Positive clones were selected and sequenced. The full-length cDNA sequence was assembled using DNASTAR, Lasergene version 7.1.
The identity and similarity of the deduced amino acid sequence were analyzed with other known PIWI1 (Homo sapiens, Mus musculus, Sus scrofa, Gallus gallus, Caprimulgus carolinensis, Xenopus tropicalis, Danio rerio, Alitta virens, Lottia gigantea, Crossostrea gigas, Mytilus galloprovincialis, Caenorhabditis elegans) in GenBank using the online BLASTX tool. Multiple alignments were performed using the software CLUSTALX version 1.81 and DNAMAN version 8.0. We conducted a phylogenetic analysis using the neighbor-joining method in MEGA 5.0 with 1,000 bootstrap replicates (Koichiro et al., 2011 (link)).

Strong-positive samples from PCV2-positive samples were selected by qPCR, and the standard curve was established. The qPCR amplification of PCV2 was carried out in 20-μL reaction mixtures containing 10 μL of TB Green II (TaKaRa), 0.8 μL of Forward primer (F), 0.8 μL of Reverse primer (R), 0.4 μL of ROX and 50 ng of DNA template. A strong linear relationship was also evident, with an R2 of 0.999, and the E was 100.91%. The linear relationship between Ct and the copy number was calculated as: Ct = − 3.3X+ 31.35. Based on geographic region, prevalence time and positive rate, representative strains were selected from positive samples for genome sequencing. The selected positive samples used primers F2/R2 to amplify the genome. Subsequently, positive PCR products 1767 nts in length were purified using the DNA Glue recovery kit (TaKaRa). The purified PCV2 genomic DNA products were ligated into the PMD19-T vector (TaKaRa) and then used to transform Escherichia coli DH5α competent cells (TaKaRa). Positive bacterial suspensions were sent to Shanghai Huada Corporation for sequencing. Whole genome was sequenced twice, and the sequence was submitted to the NCBI GenBank database.

The 16 S rRNA gene and five denitrifying genes (narG, nirS, nirK, norB and nosZ) were amplified from extracted DNA using the primers following Table 1[23] (link)–[26] . We pooled the three replicates of PCR products from each of the denitrifying functional genes and 16 S rRNA gene for clone libraries analysis. Agarose gel electrophoresis of the 50 μl PCR products were performed prior to purification (QIAquick Gel Extraction Kit, Qiagen). Purified PCR products were ligated into the pMD18-vector (Takara, Japan) and transformed into Escherichia coli DH5α competent cells (Takara, Japan). Positive clones were grown in Luria-Bertan (LB) medium overnight at 37°C. Plasmids containing target gene fragments were identified by agarose gel electrophoresis and sequenced using an automatic capillary sequencer (ABI3730, USA).

One sgRNA targeting the GAPDH locus was designed using online software: CRISPR-offinder [19 (link)]. Oligonucleotides coding for the sgRNAs were annealed and assembled into a linearized px330 vector (addgene, #42230) according to the method described by Zhang at the Broad Institute of MIT [11 (link)]. The oligonucleotides coding for the sgRNAs were denatured using a thermocycler with the following program: 95 °C, 5 min; 65 °C, 30 min; and hold at 4 °C. Then, annealed oligos were ligated with BbsI-digested px330 vector, and subsequently, the ligation mixture was transformed into Escherichia coli DH5α competent cells (TakaRa, Otsu, Japan). All the primer pairs and guide RNA sequences are listed in the Supplemental Table S1.
The donor vector (pCDNA3.1-GAPDH-GFP-KI-donor) was constructed using pCDNA3.1(+) as the backbone. The GFP sequences flanking with homology arms were synthesized and inserted into pCDN3.1(+) by restriction enzymes (Genscript, Nanjing, China). Both the 5′ and 3′ homology arms for HR events at GAPDH locus were 900 bp in size. Detailed donor vector sequences are listed in the Supplementary Materials.

Total RNA from the liver of fish was determined using the Total RNA Extraction Kit (TaKaRa Code No: 9767). RNA integrity was verified via 1% (w/v) agarose gel electrophoresis, while the concentrations and purities were examined with a micro-ultraviolet spectrophotometer. Fast Quant First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China) was used to reverse-transcribe the first-strand cDNA, in accordance with the manufacturer’s instructions. The specific primers were designed using Primer Premier 5 and Oligo 6.0 software based on the sequences of the CAT gene and ECHS1 gene of Nile tilapia in the National Center for Biotechnology and Information (NCBI) (Table 2). Based on the intermediate fragments, 5′RACE- and 3′RACE-nested PCR-specific primers for CAT and ECHS1 genes were designed (Table 3). The purified DNA was cloned into the pMD18-T Vector (TaKaRa, Shiga, Japan), and then transformed into Escherichia coli DH5α-competent cells (TaKaRa, Shiga, Japan). Positive clones were selected and sent to biotechnology company (Lifei Biotechnology Co., Shanghai, China) for full-length sequencing.
The DNA copy number was calculated using the following formula: $$\mathrm{N}\mathrm{o}.\mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{p}\mathrm{i}\mathrm{e}\mathrm{s}=\frac{6.02·{10}^{23}(\mathrm{c}\mathrm{o}\mathrm{p}\mathrm{i}\mathrm{e}\mathrm{s}·{\mathrm{m}\mathrm{o}\mathrm{l}}^{-1})·\mathrm{D}\mathrm{N}\mathrm{A} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}(\mathrm{n}\mathrm{g})}{\mathrm{D}\mathrm{N}\mathrm{A} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}(\mathrm{b}\mathrm{p})·660(\mathrm{d}\mathrm{a}\mathrm{l}\mathrm{t}\mathrm{o}\mathrm{n}\mathrm{s}·\mathrm{b}\mathrm{p}-1)}$$