Parthenogenesis

As training-dataset to derive the Epi-Pluri-Score we used 258 DNAm profiles, which were retrieved from the NCBI Gene Expression Omnibus (GEO) database (series numbers: GSE29290, GSE30870, GSE31848, GSE37066, and GSE40909; Supplemental Table S1). All of these DNAm profiles were generated on the Illumina HumanMethylation450 BeadChip22 (link). Raw data were transformed into β-values, and manually classified as pluripotent, somatic, or in vitro differentiated based on the sample description on the GEO website as well as additional information in accompanying publications (pESCs and ECs were excluded for derivation of the Epi-Pluri-Score). Cells considered as pluripotent are ESCs or iPSCs and were reported to be tested for their pluripotency by teratoma formation or PluriTest analysis as well as by staining of typical pluripotency markers in the respective publications.
For rigorous validation of the Epi-Pluri-Score we used an independent dataset with 2,216 samples, which were analyzed on the Illumina HumanMethylation27 BeadChip (GEO series numbers: GSE24676, GSE25047, GSE25083, GSE25089, GSE25538, GSE26033, GSE26126, GSE26519, GSE26543, GSE26683, GSE27130, GSE29661, GSE29871, GSE30090, GSE30456, GSE30601, GSE30653, GSE30759, GSE32393, GSE32861, GSE32866, GSE34035, GSE34869, GSE36812, GSE36829, GSE40097, and GSE42646; Supplemental Table S2). DNAm profiles of parthenogenic ESCs, embryonic carcinomas, and teratocarcinomas were initially excluded.

In vitro fertilizations were performed using a modified version of that described previously[27] (link). In vitro fertilization culture medium, Mouse Vitro Fert (Cook Medical; Brisbane, Australia), was used for sperm incubation, IVF and zygote culture. The components of MVF are the same as those listed for modified human tubal fluid[28] (link), except for the substitution of gentamicin for penicillin and streptomycin (personal communication; Cook Medical; Brisbane, Australia). The IVF dishes contained one 500 µL fertilization drop.
Sperm samples were thawed in a 37°C water bath for ∼30 sec. The CPM+sperm was pushed out of the straw into the IVF drop and incubated for 1 hr. The number of sperm within an IVF drop (mean±standard deviation; 6.1±2.5×10⁵) varied depending on the sperm count of the males. Sperm count variation was controlled among treatments by applying treatments to be compared to a single pool of sperm. This resulted in the same sperm concentration being utilized across treatments. Superovulated 17- to 27-day-old female mice were used as oocyte donors. After 4 hrs of co-incubation, the presumptive zygotes were washed, and only those appearing normal were cultured overnight in a 150 µL MVF drop. Approximately 18 hrs after washing, the proportion of oocytes fertilized was calculated by dividing the number of 2-cell embryos by the sum of 2-cell embryos and normally appearing presumptive 1-cell oocytes. Polyspermy and parthenogenesis are negligible in mouse IVF systems[15] (link) (personal unpublished data) and assumed consistent across treatments.

The circular or linearized transgene vector plasmids p2IS-UBC-eGFP used for embryo microinjection were treated and purified in the same way as that for in vitro transcription templates. For microinjection, the purified p2IS-UBC-eGFP plasmids were mixed with different concentrations of NLS-I-SceI mRNA or included in the digestive reaction system of I-SceI endonuclease (NEB) as the substrate as previously described for fish transgenesis [31] (link). The I-SceI nuclease was stored at −80°C in 2 µL aliquots and added into the reaction system prior to microinjection as described [31] (link), and its activity was confirmed by digestion of the plasmid p2IS-UBC-eGFP. To observe the localization of the injected DNA, two completely complementary 130 bp-long Cy3-labeled single strand DNA fragments containing two inversely flanking I-SceI recognition sequences at both ends were synthesized, denatured and annealed to be double-stranded, and then used for embryo cytoplasmic microinjection with NLS-I-SceI mRNA in the same way as transgene vector plasmids.
Microinjection was performed as described [33] , except that the materials were injected into cytoplasm instead of pronuclear in this study. The mouse or porcine embryos subjected to microinjection were collected from mated female individuals and cultured as described [33] , [21] . The porcine oocytes were collected from ovaries and subjected to in vitro maturation (IVM) as described [34] . The matured oocytes at metaphase of meiosis II (MII phase) with extruded first polar body were selected and subjected to microinjection post parthenogenetic activation by direct current electrical pulses (1.2 KV/cm, 30 µs, two times, 1 sec interval) as described [34] . The parthenogentically activated porcine oocytes (parthenogenetic embryos) were cultured as that for the collected porcine embryos.
The cultured embryos were observed under fluorescence microscopy or laser scanning confocal microscopy (LSCM, Zeiss LSM 780) to examine transgene expression or the localization of injected Cy3-labeled DNA fragments. To stain chromosomal DNAs, embryos were incubated in culture media containing 15 µg/mL Hoechst 33342(Sigma) for 30 min prior to microinjection and washed thoroughly in fresh media. To obtain transgenic founders, injected embryos were surgically transferred into oviducts of synchronized recipient female mice or sows as described [33] , [21] .

The apomictic capacity of the crabapple species examined was evaluated following the methods of Liu et al. (2014) (link). To determine the parthenogenesis percentage, pistils were decapitated in the spring before the flowers opened. The parthenogenesis percentage was calculated as the number of parthenocarpic fruits that could produce seeds in the absence of fertilization relative to the number of decapitated flowers. To determine the apomeiosis percentage, asexual seeds were harvested from parthenocarpic fruits. The apomeiosis percentage was calculated as the number of triploid seeds relative to the total number of seeds tested.
Ploidy was determined using flow cytometry assays, which were conducted using the Partec CyStain UV Precise T reagent kit (PARTEC, Cod. 05-5003) per the manufacturer’s instructions. First, young leaves or embryos from mature seeds were cut into smaller pieces with a razor blade in nuclei extraction buffer. Staining buffer was then added with the one-fifth volume of nuclei extraction buffer following the manufacturer’s instructions. Next, debris was removed, and nuclei were collected by filtering the nuclear suspensions two times through a 20-µm nylon mesh. An arc lamp-based flow cytometer (PARTEC PA, Germany) was used to measure the fluorescence intensity of the nuclei. The fluorescence intensity of 30,000 nuclei per cytogram was measured. At least three different plants for each line were measured, and each sample was analyzed in triplicate.

Genomic DNA was extracted from an adult male specimen of each species, except for Strongyloides ransomi, for which one parthenogenetic female was used, and Macracanthorhynchus hirudinaceus, because only females were found. The specimens selected for DNA extraction were individually transferred to microtubes and were firstly washed with sterile PBS 1X solution pH 7.4. DNA extraction was performed by using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. All the specimens obtained were submitted to the amplification of ITS, 18S e 28S rDNA regions using the set of primers presented in Table S8 [60 (link),61 (link),62 (link),63 (link),64 (link)]. The reactions were composed of 1× buffer (50 mM KCl, 200 mM TRIS-HCl, pH 8.4); 50 mM of MgCl₂; 10 mM of dNTP’s; 0.5 U of Platinum Taq DNA polymerase (Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA); 5 pmol of each Forward and Reverse primer; genomic DNA and ultrapure water to complete a final volume of 20 μL. Amplifications were performed in a Nexus thermal cycler (Eppendorf, Hamburg, Germany) programmed to perform one cycle at 95 °C for 3 min and 35 cycles at 94 °C for 40 s; each primer’s annealing temperature as shown in Table S8 was kept for 30 s, and 72 °C for 50 s, followed by a final cycle at 72 °C for 10 min. To verify amplification, the PCR products were submitted to electrophoresis in 1% agarose gel. In case of small helminths and consequently low DNA yield, the reamplification was performed using the same protocol and primers cited previously. When nonspecific bands were present, the band of interest was purified by excision of the agarose gel and purified with the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The PCR products that presented a single band were purified directly from the microtube using the same kit. The purified PCR products were submitted to PCR sequencing using the BigDye Terminator v3.1 kit (Applied Biosystems, Waltham, MA, USA), according to the manufacturer’s instructions. Sequencing was performed by capillary electrophoresis on an ABI3130 sequencer (Applied Biosystems) [65 (link)].