Agarose

DNA in fresh rhododendron leaves was extracted using Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China). A Kaiao K5500 spectrophotometer (Kaiao, Beijing, China) was employed to measure the OD value of DNA at 260 nm, and calculated its purity and concentration. The quality was checked by 1% agarose (Biowest, Nuaillé, France) gel electrophoresis. The gel was observed and photographed with a gel imaging system Quantum CX5 Edge 18.02a (Vilber Lourmat, Marne-la-Vallée, France). Qualified DNA samples were stored at −20 °C for later use.

Total RNA was extracted from duck spleen via TransZol up (Transgen). Reverse transcription of RNA into cDNA used a HiScriptRII One Step RT-PCR kit (Vazyme, Nanjing, China). To clone the duck HMGB1 (duHMGB1), primers (Additional file 1) were designed based on the predicated gene in the GenBank (Accession Number, XM_027469875.1) (Additional file 2).
All PCR products were analyzed using electrophoresis on a 1% agarose (Biowest, Hong Kong) gel in 1 × TAE at 120 V for 20 min. The PCR products were then cloned into a pMD19-T (TaKaRa) vector and transformed into E. coli DH5α (Vazyme, Nanjing, China). Competent cells were then sequenced.

The following reagents and instruments (with their suppliers) were used: fungal genome DNA extraction kit (TIANGEN, Beijing), Bst DNA polymerase (New England Biolabs, Beijing), betaine (Sigma Aldrich, Shanghai), agarose (BIOWEST, Hong Kong), Taq DNA polymerase (Takara, Japan), dNTPs (Beijing Zoman Biotechnology Co., Ltd., Beijing), Marker-DL2000 (Beijing Zoman Biotechnology Co., Ltd., Beijing), SYBR Green I (Biotech Co., Ltd., Beijing), isothermal water bath kettle DK-8D (Beijing Jinyi Tech. Co., Ltd., Beijing), PCR-Cycler (Biometra, Germany), electrophoresis apparatus DYY26C (Beijing Liuyi Co., Ltd., Beijing), Bio-Rad multimager (Richmond, CA), and NanoDrop 2000 (Thermo Fisher, USA).

The transcriptome of S. paramamosain was utilized to obtain the Sp-SEFIR transcript. Moreover, the AG’s total RNA was retrieved by utilizing TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the guidelines provided by the supplier. The 3′ and 5′ untranslated regions (UTR) of Sp-CFSHR were acquired through the utilization of rapid amplification of cDNA ends (RACE) technique, employing the SMART ^TM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) in accordance with the producer’s guidelines. The validation of open reading frame (ORF) was performed via synthesizing the first-strand cDNA from 1 μg of total RNA utilizing the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The ORF of Sp-SEFIR was confirmed by specific primers Sp-SEFIR-OF/OR (Table A1). Utilizing LA Taq polymerase (TaKaRa, Dalian, China), polymerase chain reaction (PCR) was conducted as per these criteria: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 100 s, and then through 72 °C for 10 min final extension. PCR outcomes were observed with 1.5% agarose (Biowest, Kansas City, MO, USA) gel electrophoresis before being connected to the pMD19-T vector (Takara, Dalian, China) for sequencing. Table A1 lists the primer sequences.

LA Taq DNA polymerase, dNTPs (TaKaRa, Japan); 5× loading buffer, DL2000, DL1500 (Dongsheng Biotech, China); agarose (BIOWEST, Hong Kong); DNA gel extraction kit (Tiangen Biotech, China); yeast extract, tryptone, NaCl, agar (Oxoid, UK); chloramphenicol (Sigma, USA); BAC/PAC DNA Isolation Kit (OMEGA, USA).