Dneasy plant mini prep kit

Genomic DNA was extracted using the DNeasy^® Plant Mini Prep Kit (Qiagen, Crawley, UK). Genomic DNA was quantified by Nanodrop™ (Thermo Fisher Scientific) and normalized to 0.5 ng μL⁻¹ using distilled H₂O. Quantitative PCR was run on genomic DNA using SYBR^® Green Jumpstart™ Taq ReadyMix™ (Sigma Aldrich) in an Eppendorf Mastercycler^® ep realplex quantitative cycler (Eppendorf UK Ltd, Histon, UK). Copy number was calculated using a standard curve against known concentrations of the plasmid pKAN-GFP. Primers for GFP were ATC CGG ATC ACA TGA AAC GC and AAG CTA ATG GTG CGT TCC TG resulting in a 79 bp amplicon. Cycling conditions were 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s with a melting curve afterwards to ensure a single product was being measured.

Ten individuals of grass carp and rice were used for SSR validation. For carp, genomic DNA was isolated from tail fin using a salt-extraction method with slight modifications. For rice, genomic DNA was isolated from leaves using the DNeasy Plant mini prep kit (Qiagen, Hilden, Germany). DNA concentration for each sample was determined using NanoDrop (Thermo Scientific, Waltham, MA, USA). Primers were designed using Primer premier 5 software (Lalitha, 2000 ). All the PCR amplifications were carried out in a 10 µL volume containing 1 µL 10× buffer (with Mg²⁺), 100 µM dNTPs, 0.5 µL primer pairs, 1 U Taq DNA polymerase and 20 ng genomic DNA. The reaction program was 5 min at 95 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at annealing temperature 57 °C, 30 s at 72 °C and a final extension at 72 °C for 8 min; then stored at 4 °C. The PCR products were separated by size with Genescan-400 HD size ladders (Applied Biosystems, Foster City, CA, USA), by capillary gel electrophoresis using the ABI 3730 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The peak heights and fragment sizes were analyzed using GeneMarker software (Holland & Parson, 2011 (link)).

We collected material from all available trees in the Swedish Aspen (SwAsp), which consists of 116 individuals collected from 12 different locations spanning the distribution range in Sweden [12 (link)] (Fig. 1a). Leaf material was sampled from one clonal replicate of each individual growing at a common garden experiment located in Sävar, northern Sweden. Total genomic DNA for each individual was extracted from frozen leaf tissue using the DNeasy plant mini prep kit (QIAGEN, Valencia, CA, USA). Paired-end sequencing libraries with an average insert size of 650 bp were constructed for all samples according to the Illumina manufacturer’s instructions. Whole genome sequencing and base calling were performed on the Illumina HiSeq 2000 platform for all individuals to a mean, per-sample depth of approximately 30× at the Science for Life Laboratory, Stockholm, Sweden.

Genomic DNA was extracted using the Qiagen DNeasy^® plant miniprep kit. Three individual lines of NaD1-resistant strains and three lines of the no-treatment controls were sequenced. Sequencing was completed at the La Trobe Genomics Platform, using Illumina MiSeq V3 chemistry. One run was performed for all six genomes, generating 25 million 300 bp paired-end reads. The pre-processing and variant discovery steps were performed as described by the GATK best practices and are summarized in McKenna et al. (2010) (link).

Genomic DNA was extracted using the DNeasy^® Plant Mini Prep Kit (Qiagen, Crawley, UK), quantified by Nanodrop™ (Thermo Fisher Scientific) and normalized to 0.5 ng μL⁻¹ using distilled H₂O. Quantitative PCR was run on genomic DNA using KICStart^® SYBR^® Green qPCR ReadyMix™ (Sigma Aldrich) in an Eppendorf Mastercycler^® ep realplex quantitative cycler (Eppendorf UK Ltd, Histon, UK). The known 1 copy clone α-1 was used as a reference strain and all strains were compared accordingly [7 (link)]. Data was analysed using the Pfaffl method, based on ΔΔ-Ct [37 (link),38 (link)] and normalized to ACT1 as the housekeeping gene. Primers for the heavy chain were AAT CAC AAG CCC AGC AAC AC and GGG CAT GTG TGA GTT TTG TCA C resulting in a 74 bp amplicon. Primers for the light chain were TGT CTT CAT CTT CCC GCC ATC and ATT CAG CAG GCA CAC AAC AG resulting in a 70 bp amplicon. Primers for ACT1 were GCT TTG TTC CAC CCA TCT GT and TGC ATA CGC TCA GCA ATA CC resulting in a 163 bp amplicon. Cycling conditions were 95 °C for 5 min followed by 40 cycles of 95 °C for 5 s, 58 °C for 15 s and 72 °C for 10 s with a melting curve afterwards to ensure a single product was being measured. Error bars are representative of the Gaussian error propagation of the standard deviation of s[untreated control], s[treated control], s[untreated sample] and s[treated sample] [39 ].

About 100 mg of silica dried leaf tissue was used in the total genomic DNA isolation and purification. DNA extraction was carried out with DNeasy Plant Mini-prep Kit from QIAGEN (QIAGEN, Valencia, CA), according to the manufacturer's instructions. DNA was suspended in Tris-EDTA (TE) buffer; then, each sample was diluted up to 60 ng/μL before conducting a polymerase chain reaction (PCR).

The F1 mapping population consists of 105 progenies from an inter-specific cross between two heterozygous genotypes, i.e., Z. jujuba Mill. ‘JMS2’ and Z. acidojujuba Cheng et Liu ‘Xing 16’. Since pollen abortion occurred in ‘JMS2’, ‘Xing 16’ was used as the male parent. The in vitro plantlets of the two parents and their progenies were preserved in the Research Center of Chinese Jujube, Agricultural University of Hebei, China. Leaf samples were harvested from F1 individuals and the two parents and immediately treated with liquid nitrogen and then stored at −80°C until DNA isolation. All the DNA samples were extracted using the DNeasy plant mini prep kit (Qiagen). The genomic DNA was treated with RNase to remove residual RNA.