Nanodrop 1000 v 3

The concentration of DNA sample was measured using the absorbance method (NanoDrop 1000 v3.7.1, Thermo Scientific). Absorbance of ultraviolet light at wavelengths of 260 nm and 280 nm were used to calculate the optical density (OD) 260/280 ratio. These values are used to compare the ratio of nucleic acid concentration in the sample (OD 260 nm) to that of protein and organic contaminants (OD 280 nm). A ratio of 1.8 was considered ideal for the OD 260/280 ratio, indicating minimal protein and organic contamination. The average DNA concentration was taken from 2 readings. All DNA samples were diluted with nuclease free water and the concentration was verified by Nanodrop.

We used PAXgene blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) as per the kit instructions for the collection of peripheral blood samples (2 × 2.5 ml) in both the sessions. Further, we used PAXgene blood miRNA Kit (PreAnalytiX GmbH, Hombrechtikon, Switzerland) as per the kit instructions for the isolation of total RNA. Next, we tested the purified RNA samples for purity and concentration using the NanoDrop 1000 v.3.7 (Thermo Fisher Scientific, USA). In addition, we used the Ambion's Human GLOBINclear^TM kit (Applied Biosystems, USA) as per the kit insert, for the depletion of globin mRNA. Further, we used the 2100 Bioanalyzer (Agilent Technologies, Germany) to measure the RNA integrity of the samples, before diluting to 50 ng/μl using RNase-free water. A total of 2 μg of RNA was assayed on the Illumina HumanHT-12 v4 bead array (Illumina Inc.; San Diego, CA, USA), which targets more than 47,000 probes.

For each plant, an equal amount of tissue from each of the four sampled culms was pooled to give a total of 60–70 mg. This tissue was ground to a powder under liquid nitrogen using a mortar and pestle. While the sample was frozen, RLY buffer from an Isolate II RNA Plant Kit (Bioline, London, UK) was added and the material was allowed to thaw to a slurry before additional grinding was performed. Total RNA was subsequently isolated following the manufacturer’s protocol which included an on-column DNA digestion using DNase I. The RNA was eluted with 60 μl RNase-free water and the flow-through reapplied to the spin column for a second round of elution. Quality and quantity was assessed by electrophoresis on a 1.5% agarose gel and spectrophotometry using a NanoDrop 1000 v 3.7 (ThermoFisher Scientific, Wilmington, DE, USA). RNA was also isolated as above for small sections of the rhizome samples.

Samples of newly expanded leaves of plants were collected and stored at – 80 °C until DNA extraction. Collected samples were ground using a TissueLyser II (Qiagen, USA). After being ground, genomic DNA was extracted using the CTAB method (Fulton et al. 1995 ). The DNA quantity and quality were determined by NanoDrop 1000 V.3.7 (Thermo, USA).
DNA from the F₁ plant used for making the F₂ population was sequenced by one lane Illumina HiSeq2500 (60 Gb, 2 × 125 nt Paired End). Reads were mapped to the reference genome C. annuum ‘CM334’ v.1.55 (Kim et al. 2014 (link)) (https://www.peppergenome.snu.ac.kr/) using BWAmem (Li 2013 ). Single nucleotide polymorphisms (SNPs) were identified using Freebayes (Garrison and Marth 2012 ). A total of 167 evenly spaced SNP markers (Table S1) were selected and named based on their physical position on the C. annuum genome sequence ‘CM334’ v.1.55 (Kim et al. 2014 (link)).
DNA solutions of F₂ plants were prepared for genotyping through the KASP™ technology (KBioscience, UK), which was carried out by the Dr. van Haeringen laboratorium B.V., Wageningen, NL. A linkage map was constructed using the JoinMap 4.1 software (Van Ooijen 2006 ). Map distances were calculated using the Kosambi mapping function.

In our research, genomic DNA isolation from biopsy samples embedded in paraffin blocks was carried out using a commercially available DNA FFPE isolation kit (GeneRead^TM FFPE kit, Qiagen, Hilden, Germany). The quality and concentration of the isolated DNA samples were assessed with a spectrophotometer (NanoDrop 1000 V3.7, Thermo Scientific, USA).