Bigdye terminator cycle sequence kit v 3

Three non-coding intergenic spacer regions of cpDNA—rps16–trnQ, atpA–rps14, and rpl16–rps3—were sequenced in all Erianthus accessions by using Sanger sequencing of each PCR product. Sequence variations in these regions were identified between E. arundinaceus accessions from Japan and Indonesia [52 (link)]. Primer pairs to amplify each region (Table S5) were designed from the chloroplast genome sequence of ‘JW630’ (GenBank accession No. LC160130). PCR was performed in a 15-μL mixture containing genomic DNA (20 ng), 5× PrimeSTAR buffer (TaKaRa, Shiga, Japan), 0.4 mM each dNTP (TaKaRa), 5 pmol of each specific forward and reverse primer, and 0.5 units PrimeSTAR HS-DNA polymerase (TaKaRa) in a GeneAmp PCR System 9700 thermal cycler (Life Technologies) as follows: 98 °C for 1 min, followed by 30 cycles of 98 °C for 15 s, 56 °C for 15 s, and 72 °C for 2.5 min. Amplification products were purified with a QuickStep2 PCR Purification Kit (Edge Biosystems, Gaithersburg, MD, USA) and were used as templates for sequencing. Cycle-sequencing was performed with a BigDye Terminator Cycle Sequence Kit v. 3.1 (Life Technologies) using specific primers (Table S5) in the same thermal cycler. Sequencing products were purified on a Sephadex G-50 column (GE Healthcare, Uppsala, Sweden) and sequenced in an ABI3500 genetic analyzer (Life Technologies).

The E. arundinaceus cp genome was sequenced by using pyrosequencing. Total E. arundinaceus genomic DNA was sheared by nebulization and then amplified by emulsion PCR. Amplification products were sequenced on a 454 GS FLX Titanium platform (Roche, Basel, Switzerland) [59 (link)]. Chloroplast sequence reads were extracted by local BLASTN searches using the cp genome of S. officinarum [31 (link)] as a reference and assembled with Newbler software (v 2.5; Roche). Homopolymer regions (poly A/T and poly G/C) and the junctions between single-copy regions (LSC and SSC) and IRs were amplified and confirmed using primers designed from the E. arundinaceus cp sequence (S1 Table) and PrimeSTAR HS DNA polymerase (TaKaRa, Shiga, Japan). PCR products were purified in a QuickStep2 PCR Purification system (Edge Biosystems, Gaithersburg, MD, USA). They were cycle-sequenced with a BigDye Terminator Cycle Sequence Kit v3.1 (Life Technologies, Foster city, CA, USA) and sequenced using an ABI3130xl genetic analyzer (Life Technologies) using primers described below (S1 Table).