Plant sample from Cypripedium calceolus 01_Oberhof
We isolated total genomic DNA from silica-dried or herbarium leaf tissue using the NucleoSpin Plant II kit: Genomic DNA from plants (Macherey-Nagel, Dren, Germany) following a modified version of the manufacturer's manual. DNA was sheared to an average fragment size of 350 bp with a Covaris M220 Focused-ultrasonicator (Covaris, Woburn, Massachusetts, USA). We prepared dual-indexed libraries according to the instruction manual using the NEBNext Ultra II DNA Library Prep Kit for Illumina and the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1, New England Biolabs, Ipswich, Massachusetts, USA). Prior to hybridization, the libraries were pooled in equal concentrations to include 250 ng of each library, with a maximum of 15 libraries per pooled library. For the hybridization enrichment reaction, we combined the pooled libraries with the custom orchid-specific bait set Orchidaceae963 (Daicel Arbor Biosciences myBaits Target Capture Kit, Ann Arbor, MI, USA) and incubated at 60 C (hybridization temperature, TH) for 16 hours overnight, following the Standard Protocol and the Blockers Mix setup designed for plants (myBaits Hybridization Capture for Targeted NGS, User Manual v. 5.02). The bead-based cleanup of the bait-target hybrids was performed at a wash temperature (TW) of 60 C, and the hybridized libraries were subsequently amplified for 14 PCR cycles at 60 C (annealing temperature, TA). Then, we purified the amplification reaction following the PCR clean-up protocol of the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Dren, Germany). The enriched pooled libraries were sequenced on an Illumina NovaSeq 6000 (SP flow cell) or NextSeq 1000 (P1.300 flow cell) sequencing systems at the Core Facility Genomics (CF-GEN) of the Helmholtz Zentrum Mnchen, Germany (Deutsches Forschungszentrum fr Gesundheit und Umwelt, GmbH).