Dnaeasy plant mini kit

The R. solani AG2-2IIIB isolate BBA 69670 (DSM 101808) deriving from sugar beet grown in Bavaria, Germany, was used in the study. Fungal mycelia were grown in liquid potato dextrose broth (PDB) in darkness at 23–24 °C for 48 h, under shaking conditions (150 rpm). DNA was extracted using the QIAgen DNAeasy Plant Minikit (Qiagen). For RNA the fungus was grown in PDB or sugar beet media containing 4 g surface-sterilized sugar beet pieces in 100 ml sterile H₂0 and cultured as above. RNA was isolated by RNeasy kit (Qiagen), and cDNA was prepared at Vertis Biotech, Freising, Germany.

Cotton plants (Gossypium hirsutum) showing typical leaf curling, vein thickening and enation on the abaxial side of leaf were collected from a field in Lucknow, U. P., India (26° 50′ 49.2″ N, 80° 56′ 49.2″ E). The leaf sample of the diseased cotton plant was collected from a private land with the verbal consent of the owner to pick the leaf sample for academic purpose (Gossypium hirsutum is not an endangered or protected species), in September of 2010. Total genomic DNA from diseased cotton leaf was isolated using DNAeasy plant mini kit (QIAGEN, Germany) following the manufacturer’s instructions. Begomoviral complex was confirmed as described by Kumar et al. [30 (link)]. Full-length genome of CLCuBuV was amplified using templiPhi DNA amplification kit (GE healthcare, USA) as per manufacturer’s instructions. The RCA (rolling circle amplification) product was partially digested with 1 U of HindIII restriction enzyme (Fermentas, USA) and incubated at 37°C for 25 min. The purified DNA fragment was ligated into pBluscriptSK+ vector and transformed into Escherichia coli DH5α cells. The clone was sequenced by automated sequencer (Ocimum Biosolutions Ltd., India) and the nucleotide sequence was submitted in GenBank under the accession number KF767352. Genome organization of DNA-A component of CLCuBuV is represented in Fig. 1A.

Genomic DNA from Wassilewskija (WS), rdm1-3 and two independent lines expressing RDM1-3xFLAG was extracted using the DNAeasy Plant Mini Kit (Qiagen). DNA were sheared to 300 bp with a Covaris S2 (Covaris). Libraries, including bisulfite conversion, were made with the NuGEN Ultralow Methyl-seq kit (NuGEN) and EpiTect Bisulfite kit (Qiagen) following manufacturer’s instructions. WGBS analysis was performed similar as before^{13 (link)}. In general, raw reads were aligned to TAIR10 genome using BSMAP^{28 (link)} with –v 2 (allowing maximal two mismatches) and –n 1 (aligning to both strands). Reads were discarded if there were more than three consecutive methylated CHH sites within the reads (for 50 bp long read)^{29 (link)}. Methylation levels at each cytosine were calculated as #C/(#C+#T). DMRs between WS and rdm1 were defined using R package DMRcaller^{30 (link)}. To define CHH DMRs, parameters were applied as before^{31 (link)}. Basically, 100 bp bins with more than four cytosines and each cytosines with more than 4 read coverage as well as more than 0.1 difference between rdm1 and WS were used as cutoff. DMRs within 200 bp of each other were merged for further analysis. Cytosine methylation over rdm1 hypo CHH DMR were extracted and plotted in R. WGBS tracks displayed in Fig. 1c correspond to wild-type Col-0 samples as described in Stroud et al.^{31 (link)}.

A total of 1,218 tenera palms derived from multiple UR x AVROS crosses¹³ was selected as the study population. The population is maintained at Sime Darby R&D Centre, Malaysia and was phenotyped for the traits of M/F, S/F, K/F, F/B, O/M, O/B and O/P according to the industry standard^{29 , 30} . The phenotying of these traits, except O/P were conducted under bunch analysis to generate reliable mean values with at least 3 bunches per palm. The O/P trait was then calculated based on the multiplication between 4-year average fresh fruit bunch (FFB) and O/B.
The genomic DNA of each palm was extracted from 100 mg of dried leaf tissue and purified using the DNAeasy Plant Mini Kit (Qiagen, Germany). Genotyping of this population was done using the OP200K SNP array³¹ and 92,057 SNPs were found to be polymorphic and were used for GS purposes.

Genomic DNA was isolated from young leaves of 40 horsegram germplasm accessions using the DNAeasy Plant Mini Kit (catalog # 69104, Qiagen). The quality and quantity were checked using a NanoDrop device, a Qubit assay, and agarose gel electrophoresis. Double-digested restriction associated DNA (ddRAD) libraries were prepared. In brief, 250–1,000ng DNA was digested with two units MlucI and four units SphI restriction enzymes at 37°C overnight followed by AMPure purification. Adapters specific to MlucI and SphI were ligated to the double-digested DNA using T₄ DNA ligase at room temperature for 30min and then heat killed at 65°C for 15min. Equal volumes of five samples of ligated DNA were combined to prepare one Illumina library. (In total, eight libraries were prepared for 40 samples.) The samples were size selected (370–470bp) on a 2% SYBR safe gel and purified. The samples were enriched in nine cycles of PCR amplification, and the PCR products were purified using AMPure beads. The concentration of the ddRAD libraries was checked using Qubit, and the quality was assessed using Agilent 2200 TapeStation on a D1000 ScreenTape system. The ddRAD libraries were paired-end (2×100 ntds) sequenced using Illumina HiSeq2500.

Genomic DNA (gDNA) was isolated from horsegram variety PHG-9 (Supplementary Figure S1) using the DNAeasy Plant Mini Kit as per the manufacturer’s instructions (Catalog # 69104, Qiagen), and the DNA quality was checked using a NanoDrop device. The RNA from root and leaf tissues was isolated using TRIzol reagent (Catalog # 15596026, Invitrogen), followed by the procedure of the Direct-zol RNA MiniPrep kit (Catalog # R2050, Zymo Research). The integrity and quantity of the RNA were checked using the Agilent 4200 TapeStation system. Then, the RNA from root and leaf tissues was mixed in equimolar proportions and processed as a single sample for RNA-seq library preparation.