Favorprep plant genomic dna extraction mini kit

To evaluate the resistance trait, segregating plants were phenotyped in a greenhouse after infection with E. necator. Then, naturally infected plants showing white-grayish powder on the adaxial surface of the leaves were identified as susceptible. Later, this result was confirmed by in vitro inoculation with the fungus and phenotypic evaluation.
Subsequently, genomic DNA extraction of the segregating resistant plants was performed using the FavorPrep^TM Plant Genomic DNA Extraction Mini Kit (Favorgen Biotech Co., Taiwan). Positive controls to RUN1 (02-2/81), REN1 (91-4/27), and RUN1REN1 (P09-105/34) were included.
Phenotypically resistant plants were screened using two simple sequence repeat (SSR) markers for each resistance locus. To identify RUN1 genotypes, plants were screened with VMC4f3.1 and VMC8g9 markers (Barker et al., 2005 (link)). REN1 genotypes were identified using Sc47_20 and UDV020 markers (Hoffmann et al., 2008 (link); Coleman et al., 2009 (link)). Susceptible V. vinifera ‘Sauvignon Blanc’ and ‘Melissa’ were included as RUN1 and REN1 negative controls, respectively.
Additionally, RUN1 genotypes were screened using a set of primers designed to specifically amplify a 190 bp fragment of the MrRUN1 gene (GenBank ID: JQ904636): RUN1MG: F5′-ATAAAGCTCTTCGTATAAAT-3′ and R5′-CGATATGTGCTGACCCACA-3′. Susceptible V. vinifera ′Red Globe′ was included as MrRUN1 negative control.

For each genotype, approximately 5 g of seeds were sown and grown in a greenhouse at Rimba Ilmu, Universiti Malaya (3.1311° N, 101.6578° E), and leaves of 4-week-old seedlings were harvested for DNA isolation. Total genomic DNA was extracted using a FavorPrepTM Plant Genomic DNA Extraction Mini Kit following the manufacturer’s protocol (Favorgen, Ping Tung, Taiwan). DNA quality was assessed using 1% SYBR® Safe (ThermoFisher Scientific, Waltham, Massachusetts, USA) DNA-stained agarose gel electrophoresed in 1xTris-acetate-ethylenediaminetetraacetic acid (TAE) buffer for 60 min and was visualized under the AlphaImager Mini Imaging System (ProteinSimple, Santa Clara, California, USA). The purity of the samples was also determined using a NanoDrop 2000 spectrophotometer (Thermofisher Scientific, Waltham, Massachusetts, USA). To prepare both uniplex and multiplex PCR samples, 2XGoTaq Green PCR Mastermix (Promega, Madison, WI, USA) was used, which contains optimal concentrations of bacterial-derived Taq DNA polymerase, MgCl₂, dNTP, and reaction buffers.

Genomic DNA was extracted using the Favorprep^TM Plant Genomic DNA Extraction Mini Kit (Favorgen, Taiwan) according the manufacturer’s suggested protocol. The isolated DNA was quantified using a Nanophotometer (Implen, Germany). A total of five chloroplast DNA (cpDNA) regions—two coding cpDNA loci, matK and rbcL; the trnL intron; and two non-coding cpDNA intergenic spacer loci, trnL-trnF and psbC-trnS—and an nrDNA ITS region were amplified (Table 2). PCR was conducted in a final reaction volume of 25 μL, containing 12.5 μL of 2x PCRBIO Taq Mix Red (PCRBiosystems, United Kingdom), 10 mM of each primer, and 20 ng of genomic DNA as a template. PCR amplification was conducted in a MyCycler^TM thermal cycler system (Bio-Rad, United States). PCR conditions (Lee et al., 2016 (link)) and annealing temperatures for each primer set are shown in Supplementary Table S1. PCR products were visualized in 1% agarose gel prior to direct DNA sequencing (ABI PRISM 3730xl Genetic Analyzer, Applied Biosystems, United States), performed by the First Base Laboratory Sdn. Bhd., Malaysia. In the case where targeted sequences were available in the GenBank database through previous studies of the same plant specimen, the records were included in this study without performing additional PCR and sequencing (Table 3).