Total genomic DNA was extracted according to the previously published CTAB method. The rps16 chloroplast gene and ITS were selected as maternally and biparentally inherited molecular markers, respectively [35 (link), 36 (link)]. The corresponding primers were synthesized and then subjected to polymerase chain reaction (PCR) according to a previously described reaction system and program [31 (link)]. Sequencing reactions were conducted with corresponding forward and reverse primers commercially provided by Tsingke Biotechnology Co., Ltd. (Chengdu, China).
Forward and reverse primers
Manufactured by Tsingke
Sourced in China
Forward and reverse primers are short DNA sequences used in the polymerase chain reaction (PCR) process. They serve as the starting points for DNA synthesis, initiating the amplification of a specific target DNA sequence.
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Lab products found in correlation
4 protocols using forward and reverse primers
1
Chloroplast and nuclear DNA extraction
2
Gene Expression Analysis by RT-PCR
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Gene expression analysis was performed by RT-PCR using Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Wilmington, DE, USA). Primers with Tm (melting temperature) 60 °C and 18–20 bp in length were designed by Primer 3 (Supplementary Table S2 ). IpEF1A was selected as the internal reference gene. PCR reaction mix (20 μl per well) contained 10 μl TB Green Premix Ex Taq II (TliRNaseH Plus) (2X) (Takara, Tokyo, Japan), 0.8 μl forward and reverse primers (10 μM) (Tsingke, Wuhan, China), 0.4 μl ROX Reference Dye II (Takara, Tokyo, Japan), 50 ng cDNA and RNA-free water. The two-step thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 34 s. Corresponding gene expression level was analyzed with the 2-ΔΔCt method. Elongation factor 1-alpha was used as the internal control to normalize the relative amount of mRNAs for all samples.
3
Site-Specific Mutagenesis of TtAgo Proteins
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Eight individual point mutations derived from the identified 42 TtAgo mutants were selected for further analysis and the locations of the mutations are summarized in Table 1 . To generate these point mutations de novo, site-specific mutagenesis primers were synthesized with the mutated nucleotide in the middle of the forward and reverse primers (TSINGKE Company, Xi’an, China) and are listed in Supplementary Table S1 , and the re-constructed plasmids are listed in Table 2 .
4
Fungal DNA Extraction and Molecular Identification
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The genomic DNA of the fungal mycelium growing on the PDA plate was extracted using a DNA extraction kit (GeneOn BioTech, Ludwigshafen am Rhein, Germany). The ITS, LSU, TEF1α, ACT, and GPDH regions were amplified using the primer pairs and polymerase chain reaction (PCR) programs specified in Table 1 . The amplification reactions were conducted in a 20 μL reaction volume, comprising 12.5 µL of 2× Hieff Canace® Plus PCR Master Mix (Yeasen Biotechnology, Shanghai, China, Cat No. 10154ES03), with 1 µL each (10 µM) of forward and reverse primers (TsingKe, Qingdao, China) and 1 µL of template genomic DNA. The volume was adjusted to a total of 25 µL with distilled deionized water. The PCR amplification products were observed on a 2% agarose electrophoresis gel. DNA sequencing was carried out using an Eppendorf Master Thermocycler (Hamburg, Germany) at Tsingke Company Limited (Qingdao, China), bi-directionally. Consistent sequences were obtained using MEGA 7.0. All the sequences generated in this study were deposited in GenBank (Table S1 ).
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