Purelink plant total dna purification kit

Total DNA was extracted using a PureLink™ Plant total DNA purification kit (Invitrogen, USA) according to the manufacturer’s instructions. Samples were extracted after 1, 4, 7, and 14 days of treatment in 75 µM 5-Az, and the samples without treatment were used as the control group. The DNA quality was measured based on the A260/280 and A260/230 parameters using NanoVue (GE Healthcare).

The genomic DNA was extracted from the fresh algal isolated using the PureLink^® Plant Total DNA Purification Kit (Invitrogen, Waltham, MA, USA). Master Mix PCR Taq with dye (amb^®) was used to PCR amplification, using the forward primer p23SrV-f1 (50-GGA CAG AAA GAC CCT ATG AA-30) and reverse primer p23Sr-r1 (50-TCA GCC TGT TAT CCC TAG AG30) [16 (link)]. Additionally, other segments were amplified using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [17 (link)]. Electrophoresis was performed to analyze PCR product on (1.5%) agarose gels adding 5 µL Sybr safe (Thermo Fisher Scientific, Waltham, MA, USA) in 80 mL of TBE 1x buffer. Lastly, the genetic identification was carried out using Basic Local Alignment Search Tool (BLAST) of National Center for Biotechnology Information (NCBI). The isolated strain was identified at the species level, and the corresponding sequences were deposited in GenBank. To confirm the results obtained, molecular identification was performed twice

The DNA was extracted from 100 mg of A. flavus-infected groundnut seeds at 72 hpi with the PureLink Plant Total DNA Purification kit (Invitrogen, Waltham, MA, USA). The isolated DNA was evaluated for purity on 0.8% (w/v) agarose gel, and the concentration was determined using a Qubit Fluorometer 2.0 and stored at −20 °C for further use. The fungal load in the A. flavus-infected groundnut samples of the WT control and 4RNAi lines was determined using quantitative PCR (qPCR) with a relative quantification method [75 (link)]. The DNA concentration of each sample was normalized to 50 ng/µL. Following the test for DNA amplification using groundnut ADH 3 (EG529529) gene-specific primers, quantitative real-time PCR (qPCR) was performed to amplify the A. flavus ITS2 region, using FLAV as the target gene and ADH3 as the housekeeping gene (primer sequences shown in Supplementary Table S1). The qPCR reaction (10 µL) included 1 µL of template DNA, 0.4 mM of each primer, and 5 µL SYBR Green mix (Bioline, London, UK). The qPCR reactions were performed in biological and technical triplicates, and the Ct values for the FLAV gene were normalized using the groundnut ADH3 gene. The relative gene expression of FLAV was calculated using the 2^−ΔΔCt method [71 (link)].

Total gDNA from Phytophthora infected plant samples was isolated using PureLink Plant Total DNA Purification kit (Invitrogen, USA) as per manufacturer’s protocol. About 100 mg of root tissue was ground in liquid N₂ and resuspended in 250 μL Resuspension Buffer (supplied in the kit). The tissues were homogenized with vigorous vortexing until sample was completely resuspended. About 15 μL 20% SDS and 15 μL RNase A (20 mg/mL) were added to the tissue resuspension and incubated at 55°C for 15 minutes to complete lysis of tissues. Total gDNA was eluted in 50 μL of Elution Buffer and stored at −20°C for further downstream application. The purified DNA was evaluated in 0.8% agarose gel as well as by UV spectrophotometry. The extracted DNA was stored at −20°C.

Plants of the genus Iris distributed across the broader Alpine-Dinaric region were collected either in their natural habitats during the vegetation seasons 2016–2018, retrieved from botanical collections of the National Botanical gardens in Zagreb (Croatia) and Ljubljana (Slovenia) (Supplementary Table S2). Most vouchers are live specimens deposited within the Iris collections of the mentioned Botanical Gardens in Zagreb and Ljubljana, and one in the private garden of the corresponding author. Herbarium voucher specimens are deposited in the herbarium of the Istrian Botanical Society, Vodnjan, Croatia (not yet registered in the Index Herbariorum). Total genomic DNA was isolated from 25–100 mg dried or fresh leaves, depending on the sample, using the commercial kit PureLink^® Plant Total DNA Purification Kit (Invitrogen^TM; Waltham, Massachusetts, USA), in accordance with the manufacturer’s instructions. One sample (I24; I. sibirica subsp. sibirica; Supplementary Table S2) was excluded from SSR analysis due to poor imaging signals.