Oligo dt 23vn primer

To detect viruses in alfalfa samples, total DNA and RNA was extracted from each of the 1,068 samples using the Plant Genomic DNA Extraction Kit (Beijing Solarbio Science and Technology Co., Beijing, China) and the TRIzol reagent (Zhonghuihecai, Shaanxi, China), respectively, according to the manufacturer’s recommendations. For RNA samples, the cDNA was synthesized using HiScript II reverse transcriptase and oligo (dT)23VN primer (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. PCR (used for DNA virus detection) and RT-PCR (used for RNA virus detection) were performed using the Taq PCR Master Mix Kit (Jiangsu CoWin Biosciences, Taizhou, China) with the following programs: predenaturation at 94°C for 2 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2 min; and final extension at 72°C for 2 min. Primers used to amplify specific sequences of AMV, pea streak virus (PeSV), lucerne transient streak virus (LTSV), ADV, MsAPV1, Medicago sativa alphapartitivirus 2 (MsAPV2), and ALCV are listed in Supplementary Table 3. The PCR products were resolved by gel electrophoresis on a 1.2% agarose gels and purified using SanPrep Column DNA Gel Extraction Kit (Sangon Biotech). The purified PCR products were verified by Sanger sequencing by Sangon Biotech.

Total RNA was extracted from the head (excluding salivary gland), salivary gland, midgut, and muscle of H. halys adults and was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). First‐strand cDNA was synthesized using HiScript^® II Q RT SuperMix with gDNA wipe and the Oligo(dT)23VN primer (Vazyme Biotech, Nanjing, China). Quantitative reverse transcription PCR (qRT‐PCR) was carried out on LightCycler^® 480 II (ROCHE, Basel, Switzerland) with a reaction volume of 10 μl mixture containing 1 μl of cDNA, 5 μl of 2×QuantiFast^® SYBR^® Green PCR Master Mix (Qiagen, Germany), 0.2 μl of forward primer, 0.2 μl of reverse primer and 3.6 μl of nuclease‐free water. The sequences of PCR primers are listed in Table S2. The PCR involved a 95℃ step for 5 min followed by 40 cycles at 95℃ for 10 s, 60℃ for 30 s. Raw data from qRT‐PCR were obtained as cycle threshold (Ct) values determined by single threshold mode. A standard curve was obtained for each set of primers to ensure 90%–100% efficiency. All reactions were run in triplicate with four independent biological replicates. The expression levels of mRNAs were normalized against H. halys 18S rRNA (Mogilicherla et al., 2018) and were calculated using the 2^‐ΔΔCt method (Livak & Schmittgen, 2001). Data on qRT‐PCR were analyzed using one‐way ANOVA (SPSS, Chicago, IL, USA) with Dunnet's Multiple Comparisons test.