E gel system

Sequencing of the two RRLs was obtained using 200 ng of DNA that was purified by agarose gel electrophoresis as described above, enzymatically sheared, end-repaired, and adapter-ligated using the Ion Xpress Plus Fragment Library Kit (Life Technologies). Obtained DNA material was size-selected using the e-gel system (Invitrogen, Carlsbad, CA, USA) and bands corresponding to 100 bp of inserts were collected and quantified by qPCR using a StepOnePlus Real-Time PCR System (Life Technologies). Selected fragments were clonally amplified, purified, and sequenced using the Ion One Touch 100 Template Kit and the Ion PGM Sequencing Kit with two Ion 318 chips (Life Technologies), for the two RRLs.

Two Ion Torrent libraries (one for each honey) were prepared following a standard protocol already applied in other experiments (i.e. [34 (link), 35 (link)]). In summary, about 200 ng of DNA from each extracted sample was enzymatically sheared, end-repaired and adapter ligated using the Ion Xpress Plus Fragment Library kit (Life Technologies). Then, resulting DNA material was size selected using the e-gel system (Invitrogen, Carlsbad, CA, USA) and bands corresponding to 200 bp of inserts were collected and quantified by qPCR using a StepOnePlus Real-Time PCR System (Life Technologies). Then, DNA was end-repaired and ligated with a specific barcode using the Ion Xpress Plus Fragment Library and Ion Xpress Barcode Adapters 1–16 kits (Thermo Fisher Scientific Inc.). Then, the two libraries were quantified with the Ion Library Quantitation kit (Thermo Fisher Scientific Inc.) by qPCR with the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific Inc.), pooled together and clonally amplified by emulsion PCR with the Ion PGM Hi-Q OT2 kit. Sequencing reactions were obtained on an Ion 318 v2 chip (Thermo Fisher Scientific Inc.) using the Ion PGM Hi-Q Sequencing kit. The same chip was used to sequence at the same time other barcoded libraries used for different purposes.

Ion Torrent compatible-NEXTflex DNA Barcodes- (Bioo Scientific, Austin, TX), were selected as a cost-effective alternative to standard IonXpress barcodes supplied by ThermoFisher Scientific. Barcode ligation was performed with the T4 DNA ligase enzyme component of the NEBNext fast DNA library prep set for Ion Torrent (New England Biolabs, Ipswich, MA). Barcoded products were then subjected to a purification process using 180μl Agencourt AMPure XP mix (Beckman Coulter, Brea, CA) and subsequent 80% (v/v) Ethanol wash. Following this, the barcoded mtDNA fragments were examined on an E-gel system (ThermoFisher Scientific, Waltham, MA) utilising an iBase unit to visualise the running gel. This allowed us to identify and recover fragments between 250–350 base pairs. Libraries were size selected at a target peak of 330bp thus maximising efficiency under the limitations of 200bp sequencing chemistry.

Young leaf tissues were collected from three-week-old plants of each accession for DNA isolation using the hexadecyltrimethylammonium bromide (CTAB) method [73 ]. The DNA was quantified and normalized to 20ng/μl. GBS was performed following the double-restriction enzyme digestion protocol for library construction and Ion Proton system sequencing [74 (link)]. Briefly, 10μl of the normalized DNA from each accession was double-digested with PstI and MspI restriction enzymes (New England BioLabs, Inc., Ipswich, MA, USA) and then ligated to two adapters with barcodes [74 (link)–76 (link)]. After the adapters were ligated, the samples were pooled together for PCR amplification and subsequently size-selected for 250-300bp amplicons using the E-gel system (Thermo Fisher Scientific, Waltham, MA, USA). Libraries (80 pM) were prepared and sequenced in an Ion Proton system using Ion PI Hi-Q Chef and Sequencing 200 kits with two Ion P1v3 chips (Thermo Fisher Scientific, Waltham, MA, USA). SNPs were called using a reference-based SNP calling pipeline of TASSEL 5 [77 (link)]. A rye custom reference genome was obtained from the Plant Genome and Systems Biology (PGSB) website (http://pgsb.helmholtz-muenchen.de/plant/rye/gz/download/) [78 ] and used as a reference to locate the SNPs within the rye genome.

We amplified purified SureSelect-enriched DNA libraries and non-template control through PCR (11 cycles) with the use of Herculase II Fusion Polymerase. Before assessing DNA quality and quantity with High Sensitivity DNA Assay on TapeStation System, we purified each sample using AMPure XP beads. Based on the evaluated concentration of SureSelect-enriched DNA libraries, we calculated the amount of each sample to be included in the pool using the following formula: volume of barcoded sample: V(f)xC(f)/nxC(i). V(f) is the final required/needed volume of the pool (20 µl), C(f) is the initial concentration of all SureSelect-enriched DNA libraries in the pool, n is the number of samples to be combined, and C(i) is the initial concentration of each barcoded sample. To avoid the presence of additional fragments in each library, we size-selected our pools by agarose gel electrophoresis using the integrated E-Gel system (Thermo Fisher Scientific), purified them using Agencourt AMPure XP beads (Beckman Coulter Genomics) and finally validated using High sensitivity DNA assay on 4200 TapeStation. The molarity of pooled libraries was 756 and 525 pmol/l, respectively (see Supplementary Fig. 1). Since the Ion Chef requires concentration of a loaded pooled library to be 50 pM, we diluted our samples using low TE buffer.