Genome size estimation and RADseq library preparation
For three species (Adelobotrys adscendens , Axinaea
costaricensis , Meriania phlomoides ), we estimated genome sizes
to select appropriate restriction enzymes for RADseq. We prepared fresh
leaf material for three individuals per species in Otto’s buffer
(Otto et al. 1981) for propidium
iodide flow cytometry (CyFlow ML flow cytometer, Partec, Münster,
Germany; 532nm/100mW laser, Cobolt Samba, Cobolt AB, Solna, Sweden)
following Temsch et al. (2010). We
used Solanum pseudocapsicum (1C = 1.29 pg DNA) as internal
standard to calculate 1C values for each run (three runs per sample) and
calculated the mean 1C per species over all runs and individuals.
Average genome sizes (1C value) for the three species were estimated as
follows: Ad. adscendens 0.344 pg, sd 0.003; A.
costaricensis 0.587 pg, sd 0.01; M. phlomoides 0.639 pg, sd
0.006. According to the Kew C-value database, these genome sizes are the
second, third and fourth estimates for the family
(Hanson et al. 2001).
In accordance with the relatively small genome sizes, we selected the
restriction enzyme PstI (New England Biolabs, Ipswich, MA, USA) for
single-digest RADseq (protocol modified from
Paun et al. (2016)). We prepared
eight RADseq libraries starting with 240 ng DNA per sample, pooling 72
samples per library, including also some repeats (using 120 ng DNA per
sample). We ligated 300 nM P1 barcoded adapters (150 nM if 120 ng input
DNA) to the restricted samples at 16°C overnight. The P1 adapters
included both index and inline barcodes, that were different from each
other by at least 3 nucleotide positions. After P1 ligation, we sheared
the DNA by sonication in a Bioruptor Pico (Diagenode, Seraing, Belgium)
using two cycles (45 seconds ‘on’, 30 seconds ‘off’; at 4°C) to obtain
DNA fragments of 400 bp on average. After P2 adapter ligation, PCR
amplification and clean-up steps (using the MiniElute PCR purification
Kit, Qiagen), we performed a final size selection for the range 220 to
850 bp using a 1.5% precasted dye-free cassette and a Pippin Prep (Sage
Science). All libraries were sequenced on an Illumina HiSeq 2500 machine
at Vienna BioCenter Core Facilities (VBCF)
(http://www.viennabiocenter.org/vbcf/next-generation-sequencing/)
as 100 bp single-end reads.