Abi3730xl

For all investigated species, total RNA from a mix of embryos of different developmental stages was extracted using TRIZOL (Invitrogen), and reverse transcribed into cDNA. Fragments of candidate genes were amplified by means of RT-PCR. Gene-specific primers were designed based on published sequence information and sequenced embryonic transcriptomes of Glomeris ([37 (link)] Janssen and Posnien 2014) and Euperipatoides ([36 (link)] Janssen and Budd 2013). Nested PCRs were run with internal primers, using a first PCR as template. Primer sequences are summarized in Additional file 1: Table S1. All investigated gene fragments were cloned into the PCRII vector (Invitrogen) and sequenced on an ABI3730XL automatic sequencer (Macrogen, Seoul, South Korea). Gene identification-numbers are listed in Additional file 2: Table S2. Colorimetric in-situ hybridizations for all investigated species were performed as described in [38 (link)] Janssen et al. (2018). For confocal microscopy, embryos were stained with SIGMAFAST Fast Red TR/NaphtolAS-MX (SIGMA) instead of BM Purple (ROCHE). Cell nuclei were visualized by either incubation of the embryos in 3–5 μg/ml of 4–6-diamidino-2-phenylindole (DAPI) or SYBR Green in phosphate buffered saline with 0.1% Tween-20 (PBST-0.1%).

DNA was extracted from two samples of fresh leaf segments using a Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. We amplified two genes, i.e., the plastid gene matK and the trnL intron with the trnL-F intergenic spacer (called simply trnL-F). The gene matK was amplified using the primer combination 371-F [25 (link)] and Trnk-2R [26 ]. The gene trnL was amplified using the primer combination Trnlf-C and Trnlf-F [27 (link)]. PCR conditions were as follows: initial denaturizing at 98 °C for 30 s; 30 cycles, each cycle consisting of one step of denaturizing at 98 °C for 10 s; annealing, at 60 ºC for 20 s and extension at 72 °C for 30 s; and final extension at 72 °C for 10 min. A control including PCR mix without DNA template was included in each PCR. Success of the PCR amplifications were tested in 0.7% agarose stained with GelRed. PCR products were purified using the QIAquick protocol (Qiagen). PCR products were sequenced bi-directionally by ABI 3730xl in Macrogen using the same primers as for PCR amplification.

Primers for amplification of the ompA gene were synthesised by Macrogen Co., Ltd., Korea. The gene was amplified using Taq DNA polymerase (PrimeSTAR GXL DNA Polymerase, Takara Bio. Inc., Japan) with the forward (OMPA-F, 5′-AGGATCCATGAAAAAAACAGCAATTGCATTGA-3′) and reverse (OMPA-R, 5′-TCTCGAGTTATTTGTTACCTTTAACAGCGATTTC-3′) primers; the sequences of these primers were modified from Gao et al. [49 ]. The PCRs contained 5 μl of 50 ng/μl genomic DNA, 10 μl of 5X PCR buffer, 5 μl of 2 mM dNTPs, 1 μl (5 U) of Taq DNA polymerase, and 5 μl of each of 2 μM forward and reverse primers in a total volume of 50 μl. The reaction mixtures were incubated for 30 cycles consisting of initial denaturation at 98 °C for 3 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 15 s, extension at 68 °C for 1 min; and final extension at 68 °C for 3 min. The PCR products were purified using a GF-1 Ambiclean kit (Vivantis Technologies Sdn. Bhd., Malaysia), and the cleaned products were subjected to sequencing with both primers using an Applied Biosystems automatic sequencer (ABI 3730XL) (Macrogen Co., Ltd., Korea). The sequence chromatograms were checked for quality, and the ompA sequences were confirmed using the BLASTN program.

For the sequencing reactions of the 18S rRNA gene, DNA was harvested from monocultures cultured in GYT medium (see above). DNA was extracted using the phenol-chloroform method [81 ]. The DNA was PCR amplified using Teg polymerase (Matís ohf., Reykjavík Iceland; denaturation at 94 ${}^{°}$ C for 5 min followed by 30 cycles of denaturation at 94 ${}^{°}$ C for 45 s, annealing at 62 ${}^{°}$ C for 30 s and extension at 72 ${}^{°}$ C for 90 s with a final extension at 72 ${}^{°}$ C for 10 min) using 18S primers: 18S001F forward and 18S13R reverse [6 (link),82 (link)]. The product was cleaned using QIAquick PCR Purification Kit (Qiagen) and the sequencing carried out on ABI 3730XL capillary sequencer at Macrogen Inc. (Amsterdam, The Netherlands).