Fastpure plant dna isolation mini kit

High conidial suspensions (1 × 10⁸ conidia/mL) of V. dahliae strains P48, P50, and Vd991 were placed on a PDA medium and incubated at 25 °C, and the mycelium was collected after 7 days. The total genomic DNA was extracted for the identification of mating types, races, and D and ND types. Genomic DNA of each isolate was extracted using a FastPure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China) following the manufacturer’s instructions and was stored at −20 °C for polymerase chain reaction (PCR) assays.
For the genotype identification assays, D/ND, race1/2, and MAT1-1/MAT1-2 were determined by PCR with previously developed primers [35 (link),73 (link),74 (link)] (Table S2). All PCR assays in this study were performed in 20 μL reaction volumes using 2×Taq Mester Mix (Dye Plus) P112-AA (Vazyme, Nan Jing, China). PCR was performed under the following conditions: an initial 94 °C denaturation step for 10 min, followed by 30 cycles at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and a final extension of 10 min at 72 °C. The PCR products were detected by 1% agarose (Sigma-Aldrich, St. Louis, MO, USA) gel dyed with GelStain (TransGen, Beijing, China) and electrophoresis for 20 min at 120 V in 1×TAE buffer. Then the image was obtained with the Bio-Rad’s ChemiDoc XRS system.

We performed bisulfite sequencing PCR (BSP) to measure the DNA methylation level. We extracted the genomic DNA from the fruit and injected it with ddH₂O and 5′-Aza using FastPure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China). We used a DNA Methylation Kit (CW2140M, CoWin Biosciences) to convert bisulfite. BSP was performed using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The BS primers are listed in Additional file 1: Table S4. All fragments generated by BSP were introduced into the pEASY-Blunt Zero vector (CB501-01, TransGen Biotech) using ClonExpress® II One Step Cloning Kit (Vazyme, Nanjing, China). For each sample, ten individual clones were sequenced. The CyMATE software (http://www.cymate.org/) was used to analyze the sequencing results and measure DNA methylation levels.

TaqMan-qPCR was used to analyze the gene copy number in P. ceruna, P. chinensis and P. turczaninovii as previous study (Maron et al., 2013 (link)) with modified. Firstly, genomic DNA was isolated from leaves of P. ceruna, P. chinensis and P. turczaninovii by FastPure^® Plant DNA Isolation Mini Kit (DC104, Vazyme). Then the primers (AS-Tq-F/R) and probes 781-TT/GG with VIC and FAM fluorescent labeling, respectively, were designed by Primer Express 3.0.1 (Supplementary Table S1). Finally, ChamQ Geno-SNP Probe Master Mix (Q811, Vazyme) and Roche LightCycler 480 were used to analyze the fluorescence of VIC and FAM to explain 781-TT and 781-GG type gene copy number. In this experiment, the standards of 781-TT and 781-GG were the same as those used in gene expression.

After germination of parental seeds, young leaves were collected and immediately flash-frozen in liquid nitrogen, and then the samples were stored at − 80℃ for future use. DNA extraction was performed using the FastPure Plant DNA Isolation Mini Kit (Vazyme, Jiangsu, China). The concentration of the extracted DNA was evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and a Qubit 3.0 fluorometer (Life Technologies, Carlsbad, CA, USA). To assess the purity and integrity of the DNA, 1% agarose gel electrophoresis was conducted. For library preparation, a short-read library with a DNA-fragment insert size of 200–400 bp was generated using 1 μg of genomic DNA. The library preparation was carried out following the manufacturer’s instructions using a library preparation kit compatible with DNBSEQ-T7 (BGI, Shenzhen, China). Subsequently, paired-end (PE) sequencing was performed on a DNBSEQ-T7 platform using the PE 150 model.

Genomic DNA was extracted from the soybean leaves using the Fastpure plant DNA isolation mini kit (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s protocol. The concentration and quality of genomic DNA samples were determined by a NanoDrop 2000 microvolume spectrophotometer (Thermal Fisher Scientific). The quality of each DNA sample was further assessed by electrophoresis on 0.8% agarose gel. Then, the resistant and susceptible pools were generated by pooling equal amounts of DNA from 50 resistant and 50 susceptible individuals, respectively. About 5 μg of DNA from the two pools or two parental lines was used to construct paired-end sequencing libraries, which were sequenced on an Illumina NovaSeq^TM 6000 platform at Hangzhou Lianchuan Biotechnology Co., Ltd. (Hangzhou, China).

A highly efficient CRISPR/Cas9 system improved for plant genome editing⁴⁶ was used in this study. CRISPR-P (http://cbi.hzau.edu.cn/crispr/), an online tool, was used to generate a pool of target sequences, and we selected two target sites for SlHY5 based on previously reported principles⁴⁶ . Once the target sequences were selected, a PCR-based method was applied to rapidly generate multiple sgRNA expression cassettes that could be easily cloned into the pYLCRISPR/Cas9Pubi-H binary vector by Golden Gate ligation. The final construct pYLCRISPR/Cas9Pubi-H-SlHY5 was sequenced and then transformed into wild-type tomato through A. tumefaciens-mediated transformation.
After regeneration, tomato seedlings were transplanted into soil and cultured in a greenhouse. Genomic DNA of regeneration seedlings was extracted from fresh leaves using the FastPure Plant DNA Isolation Mini Kit (Vazyme, DC104). The presence of the transgene was verified by PCR to confirm the insertion of T-DNA. Subsequently, to validate the mutation of the transgenic plants, second-round PCR was performed using primers flanking the target sites. The PCR products were sequenced directly, and the superimposed sequencing chromatograms were decoded manually or using the automated web tool DSDecodeM (http://skl.scau.edu.cn/dsdecode/). Subsequently, the mutated and original sequences were aligned to show the mutagenesis.

Cucumber mock-infection and M. incognita-infection roots at different developmental stages were used for total RNA extraction and cDNA synthesis (Vazyme, China). RNA quantification was done using a Nanodrop 2000 (Thermo Fisher Scientific). The RT-qPCR analysis using SYBR Green Master Mix (Vazyme, China) was performed in ABI 6500 Real-Time PCR System (Applied Biosystems). CsTUB (accession number Csa4G000580) was used as internal control. Relative expression abundance of candidate genes was calculated with the formula 2^−ΔΔCt. All reactions were performed with four biological replicates. Genomic DNA of hairy roots was extracted using the FastPure Plant DNA Isolation Mini Kit (Vazyme, China). All the primers for characterized GFP from hairy roots and RT-qPCR analysis are listed in the Table 1.