Dneasy plant pro kit

DNeasy Plant Pro Kit (Qiagen, Hilden, Germany) was used according to manufacturer’s instructions with modification. A total of 0.3 g wood powder was first mixed with 0.1 g of PVPP and incubated with 1.8 mL of CD1 solution and 200 µL of PS solution for 1 h at 65 °C. After incubation, mixtures were centrifuged at 15,000 rpm for 20 min. Clear supernatant was collected and mixed with same volume of solution CD2 followed by centrifugation at 15,000 rpm for 20 min. Clear supernatant was collected and mixed with 1000 µL of APP solution. Mixtures were applied to QIAquick Spin Columns followed by centrifugation at 5000 rpm for 5 min. After all mixtures were applied, column was washed with 650 µL of AW1 solution followed by centrifugation at 5000 rpm at 5 min and 650 µL of AW2 solution followed by centrifugation at 5000 rpm at 5 min. Ethanol residue was removed by centrifugation at 15,000 rpm for 5 min. Genomic DNA were eluted with 50 µL of pre-warmed EB solution with centrifugation at 15,000 rpm for 5 min. Time consumed in this method was approximately within 3.5 h for 24 samples and each sample costed USD 17.

After removal from soil, seedlings were first washed with filter-sterilized 0.1% Triton-X for 10 min, and then 3% sodium hypochlorite for 20 min. Samples were drained and rinsed with autoclaved, deionized water, followed by treatment with 70% ethanol for 10 minutes. Samples were rinsed 5 times to remove ethanol before being frozen for DNA isolation. An aliquot of the final rinse was plated on R2A medium to test sterility. DNA from whole 7-day old seedlings (including both roots and shoots) was isolated using the Qiagen DNeasy Plant Pro kit according to the manufacturer’s instructions and using a Powerlyser24 homogenizer at 1500 rpm for 1 min, then 2000 rpm for 1 min.

Nodules of two independent plant roots were collected from randomized plots in three developmental stages (R2, R4 and R7; Prasad, 1999 ). Whole peanut plants with roots and nodules were transported overnight on ice from the field site to the laboratory. Roots and nodules were washed thoroughly with tap water and wiped with a paper towel before collecting two types of nodules (big and small) from roots for surface sterilization. Ten big nodules (~ 1.5–2.0 mm), five from each plant, and 20 small nodules (<1.5 mm), 10 from each plant, were selected and immersed in 70% (v/v) ethanol for 2 min, washed with sterile H₂O three times, and then soaked in 10% (v/v) commercial bleach for 5 min. After bleach treatment, nodules were carefully washed 5–6 times with sterile H₂O and rinsed of any trace elements of bleach. A total of 12 big and 12 small nodule samples (3 growth stages x 4 plots) were independently collected for total DNA isolation.
Total DNA was extracted from the nodules using DNeasy Plant Pro Kit (Qiagen), according to the manufacturer’s instructions. DNA quality and concentration were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, United States).

For metagenomic analysis, from 50 mL of each fresh sample, a cell pellet was collected by centrifugation at 4500 rpm (2000× g) for 10 min and frozen at −80 °C immediately. Samples were stored for 3 months at −80 °C. For environmental DNA (eDNA) extraction, cell pellets were frozen in liquid nitrogen and grinded into a fine powder with homogenizing pestles (SSIBio Corp., Lodi, CA, USA) in a 1.5 mL microcentrifuge tube. The freezing-homogenization procedure was conducted repetitively three times for each sample. The eDNA was extracted from the obtained homogenates using a DNeasy Plant Pro kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A purified eDNA was used as a template for polymerase chain reaction (PCR) with universal primers for amplification of the V4 region of the 16SrRNA gene [58 (link)], F515 (GTGCCAGCMGCCGCGGTAA) and R806 (GGACTACVSGGGTATCTAAT), from Bates et al. [59 (link)]. Sequencing was performed on a MiSeq platform (Illumina, Inc., San Diego, CA, USA) using a MiSeq Reagent Kit v3 (600-cycle) (MS-102-3003, Illumina, Inc., San Diego, CA, USA) for paired-end sequencing (2 × 300 bp).

Individuals of the studied species were collected from the Chinese University of Hong Kong (Table 1 and Fig 1). Fresh and healthy cladodes were stored at −80°C in a freezer immediately after collection. Voucher specimens were deposited at the Shiu-Ying Hu Herbarium (herbarium code: CUHK).
Total genomic DNA was extracted from 0.2 g of frozen cladode using the DNeasy Plant Pro Kit (Qiagen Co., Hilden, Germany) according to the manufacturer’s instructions. Prior to the sequencing conducted by Novogene Bioinformatic Technology Co. Ltd. (http://en.novogene.com/, Beijing, China), DNA quantity and quality were assessed using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, MA, USA) and 1% agarose gel electrophoresis, respectively.

For testing primer efficiency, DNA was extracted from J2 CN individuals following the lysis method [37 (link)]. For screening experiments and standard curves, solutions containing a known number of posterior ends of CN females were placed in bead-beating tubes and extracted using a DNeasy Plant Pro Kit (Qiagen, Hilden, Germany), as specified by the most recent available manufacturer instructions (August 2019), with the following modifications: 50 µL of solution PS was added together with solution CD1, and homogenization was performed at 6.0 m/s for 180 s on a Fast-Prep-24 bead beater (MP Biomedicals, CA, USA), followed by 90 min digestion at 55 °C with 16 U of proteinase K (New England Biolabs Inc., Beverly, MA, USA). A subsequential 60 s bead-beating step was performed before the first centrifugation step (12,000× g for 2 min). DNA was eluted in 100 µL of nuclease-free water. Samples from the host-screening experiment were diluted 1:10. DNA quality and concentration were assessed in an ND-1000 full spectrum UV–Vis spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).