title: GBS of Facchinia cherlerioides subsp. cherlerioides Fcc1-4
design: Total genomic DNA was extracted and fragmented using the restriction enzyme BamHI. BamHI is insensitive to methylation and has a 6 bp recognition site. In general, the protocol is almost identical to Elshire et al. (2011). Restriction of the DNA was performed in 20 ľL reactions with 1× NEBbuffer 4, 10 U BamHI (New England Biolabs, Ipswich, MA, USA), 5 ľL of DNA and with an incubation of 2 h at 37 °C. For ligation the complete restriction reaction stock (20 ľL) was used in 50 ľL reactions together with 1× T4 DNA Ligase Buffer with 10 mM ATP, 40 U T4 DNA Ligase (New England Biolabs, Ipswich, MA, USA) and 3.6 ng adapters. Ligation was carried out at 22 °C for 60 min and denaturation of the enzyme at 65 °C for 30 min. After ligation of adapters containing barcodes for each sample (Elshire et al. 2011), the product was cleaned up with the Macherey-Nagel PCR purification kit (Macherey-Nagel, Düren, Germany). 25 ligation products (i.e. the maximum number of different barcodes used) were combined in one column and eluted in 2× 15 ľL elution buffer. PCR were carried out in 50 ľL reactions with 25 ľL (i.e. 1×) NEB Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA), 200 ľmol of each primer and 3 ľL of the combined and cleaned ligation products of 25 samples. For PCR, the following program was used: 5 min at 72 °C, 30 s at 98 °C, followed by 35 cycles of 98 °C for 10 s, 65 °C for 30 s and 72 °C for 90 s followed by a final elongation step at 72 °C for 5 min. PCR primers were the TruSeq Universal Adapter primer (Illumina, Inc., San Diego, CA, USA), which was complementary to the 3 strand of the barcode adapters and four different TruSeq Adapter Index primers that bind to the 3 strand of the common adapter. PCR products were loaded on a 1.4% agarose gel and fragments between 200 and 600 bp were cut out in order to remove remaining primers and adapters. Clean-up and extraction from the gel was carried out with the Macherey-Nagel PCR purification kit (Macherey-Nagel, Düren, Germany) with one change to the manufacturers protocol. The PCR product was eluted with 2× 15 ľL ddH2O instead of with the elution buffer provided. Libraries were checked for quality on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For paired-end sequencing of the libraries an Illumina HiSeq 2500 system (Illumina, Inc., San Diego, CA, USA).Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS ONE, 6, e19379. doi: http://dx.doi.org/10.1371/journal.pone.0019379