Nucleospin 96 plant 2 kit

The marijuana strains genotyped in this study were provided by author DH (grown by Health Canada authorized producers) and represent germplasm grown and used for breeding in the medical and recreational marijuana industries (S2 Table). Hemp strains were provided by author JV (Health Canada hemp cultivation licensee), and represent modern seed and fibre cultivars grown in Canada as well as diverse European and Asian germplasm (S3 Table). DNA was extracted from hemp leaf tissue using a Qiagen DNeasy plant mini kit, and from marijuana leaves using a Macherey-Nagel NucleoSpin 96 Plant II kit with vacuum manifold processing. Library preparation and sequencing were performed using the GBS protocol published by Sonah et al [15 (link)]. The raw sequence has been deposited in the NIH Sequence Read Archive (SRA), under BioProject PRJNA285813. SNPs with a read depth of 10 or more were called using the GBS pipeline developed by Gardner et al. [16 (link)], aligning to the canSat3 C. sativa reference genome assembly [3 (link)]. Quality filtering of genetic markers was performed in PLINK 1.07 [17 (link)] by removing SNPs with (i) greater than 20% missingness by locus (ii) a minor allele frequency less than 1% and (iii) excess heterozygosity (a Hardy-Weinberg equilibrium p-value less than 0.0001). After filtering, 14,031 SNPs remained for analysis.

Genomic DNA was extracted from lyophilized plant tissues using NucleoSpin 96 Plant II kit (Macherey-Nagel, Duren, Germany), following the manufacturer's instructions. Quality of genomic DNA was checked by agarose gel electrophoresis and quantity was estimated using Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA).
Two genomic libraries (96 + 9 including one negative control each), were prepared using the Pst1 restriction enzyme according to the procedures published in [27 (link)], with the modification that adapters were designed with a PstI overhang (S1 Table). Libraries were sequenced on the Illumina HiSeq2000, version 3 chemistry. Each library was sequenced on two flowcell lanes to achieve the equivalent of 48-plex pooling. All these steps were performed at the Cornell University Biotechnology Resource Center (BRC).

Genomic DNA was isolated from leaf tissue of 16 randomly selected individuals from each population. A set of 720 DNA samples (45 populations × 16 individuals) were isolated along with the control sample Tift 23D₂B₁ using NucleoSpin® 96 Plant II Kit (Macherey-Nagel, Germany). Electrophoresis (0.8% agarose gel) was performed to test the quality of the DNA and quantified based on lambda DNA (MBI Fermentas, USA). The final working DNA samples were normalized uniformly at a concentration of 10 ng/µl.
Twenty-nine SSR markers (Supplementary Table 2), identified as highly polymorphic and distributed over all the seven linkage groups based on earlier studies^{66 (link),67 (link)} were used. The detailed methodology followed for DNA extraction, SSR genotyping protocol and allele calling using Genemapper 4.0 (Applied Biosysterms) is explained in Patil et al.⁴⁶ .

Leaf tissue was collected using a hole punch from each accession in each cross once, and collected in duplicate from both sets of parents. Punched tissue was dried using a freeze dryer before being shipped to the AAFC Kentville Research and Development Centre in Kentville, Nova Scotia, Canada for DNA extraction. Tissue was lyophilized and then ground using a 2010 Geno/Grinder (SPEX SamplePrep, Metuchen, NJ, USA). Whole-genome DNA was extracted using a NucleoSpin 96 Plant II kit (Machery-Nagel, Düren, Germany) with the following modifications to the kit protocol: samples were incubated in lysis buffer for 60 min, and were processed using a vacuum manifold with an additional step of filtering lysate through receiver plates before proceeding to the binding step. DNA samples were quantified using the QuantiFluor dsDNA System and the GloMax-Multi+Microplate Multimode Reader with Instinct (Promega, Madison, WI, USA).
Library preparation and DNA sequencing were performed at L’Institut de Biologie Intégrative et des Systèmes (IBIS) at Université Laval, Quebec City, Québec, Canada using an Illumina HiSeq 2000 and the GBS approach,^{26 (link)} with the restriction enzyme ApeKI.^{34 (link)} Each F₁ progeny was sequenced once, while each parent in each cross was sequenced in duplicate as recommended by Gardner et al.³⁵

For the extraction of genomic DNA approximately 25 mg of dried root material of each tomato accession was weighed and inserted in a 96 deep well plate. Samples were freeze dried for 1 week using a freeze dryer (Christ Alpha 1-4 LD plus, Germany). For the extraction of DNA a Nucleospin 96 Plant II kit (Macherey-Nagel, Germany) was used and the manufacturer's protocol was followed. The quality of the gDNA was checked on a 1.5% agarose gel. The concentration of the gDNA was calculated using the Quanti-iTTM PicoGREEN dsDNA assay kit (Invitrogen). gDNA was diluted in TE buffer pH 8.0 (10 mM Tris and 1 mM EDTA) and stored at 4°C.

The DNA extraction from the pooled leaf samples of each plot was carried out using the Macherey‐Nagel NucleoSpin 96 Plant II kit (robot extraction) following the manufacturer's protocol. We used both provided lysis buffers separately (cetrimonium bromide [CTAB] lysis buffer PL1 and a sodium dodecyl sulfate [SDS]‐based lysis buffer PL2) to enhance the amount of extracted DNA. Five microliters of product was quantified using a Qubit 2.0 fluorometer (Life Technologies) and the broad‐range assay kit following the manufacturer's protocol before equally pooling the extracts from the same plot.

A mapping population consisting of 188 F₂ plants was developed from a cross between MNK-1 and Annigeri 1. MNK-1 is a kabuli type, extra-large-seeded chickpea variety having Fe and Zn concentrations of 72.88 and 30.98 mg kg⁻¹, respectively. Annigeri 1 is a desi-type, medium seed size variety having Fe and Zn concentrations of 98.67 and 37.07 mg kg⁻¹, respectively. Fresh, young leaves were collected from parental lines, and each F₂ individual and high-quality genomic DNA was extracted using high throughput NucleoSpin® 96 Plant II Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol. The DNA was quantified using a spectrophotometer (Shimadzu UV160A, Japan) and used to prepare the GBS library for SNP discovery. Individual F₂ plants were selfed, and seeds harvested were advanced to develop an F_2:3 population for phenotyping seed Fe and Zn contents.