GBS of medicago sativa (alfalfa)
Genomic DNA was extracted from young leaves of each plant using the DNeasy Plant Mini Kit (Qiagen, Milan, Italy). Nucleic acid was quantified by a Quant-iT PicoGreen dsDNA Assay Kit (P7589, Life Technologies, Italy), checking its quality by 1% agarose gel electrophoresis. A trial digestion was carried out on 10% of the DNA samples using the Optizyme EcoRI restriction enzyme (25,000 U, Fisher BioReagents, Rodano, MI, Italy), to compare bands of cut and uncut DNA. The reaction was performed at 37 C for one hour and the enzyme was deactivated at 65 C for 20 min. DNA samples were sent to The Elshire Group Ltd. laboratory (Palmerston North, New Zealand) for outsourced library preparation and sequencing. GBS data were generated according to Elshire et al.s method with the following changes: we used 100 ng of genomic DNA and 3.6 ng of total adapters and restricted the genomic DNA with ApeKI enzyme (NEB New England Biolabs, R0643L); then, the library was amplified with Kapa Taq polymerase Alpha (KAPA Library Amplification Readymix, Kapa Biosystems KK2611) by 14 PCR cycles. The library was sequenced at the Elshire Group Ltd. facility (New Zealand) using an Illumina X Ten platform with 150 bp paired end reads. Each sample was repeated three times on three different lanes. The raw reads were collated before demultiplexing.