The species Passiflora gardneri Mast. (female parent) and Passiflora gibertii NE Brown (male parent) were kept in the Active Germplasm Bank (BAG-Passifloras), located on the campus of the State University of Santa Cruz (UESC) in the city of Ilhéus, Bahia (longitude 39 10“W, latitude 14 39”-S, altitude 78 m). Both species were obtained from the Brazilian Agricultural Research Corporation (Embrapa Cerrados), Brasilia, Brazil. The genitor species were selected based on leaf and flower characteristics. P. gardneri presents characteristics, including the structure of its flowers as well as an abundant flowering period running from September to March, which elicits the interest of the ornamental plant market. Likewise, P. gibertii is attractive because it presents early growth and flowering, and produces up to 30 flowers per day under normal conditions. Additionally, P. gibertii presents resistance to premature death and fusariosis, with has caused great damage to Brazilian passion fruit culture. Finally, P. gibertii and P. gardneri belong to the same infrageneric level (subgenus Passiflora, section Granadillastrum). The interspecific crossings between P. gardneri vs. P. gibertii were performed in a greenhouse with temperature ranging from 25 to 30 °C and a relative air humidity of 70-90%. Pre-anthesis flower buds were protected with white paper bags the day prior to artificial pollination. Fruits resulting from hybridization were protected with nylon nets. After the fruits were fully mature, the seeds were propagated. Twenty-five hybrids germinated and were kept in a greenhouse. The hybrids that presented normal growth and flowering, as well as a wide segregation of colors, shapes, and sizes in their floral parts were selected. Eight F1 interspecific hybrids (HD15-101, HD15-104, HD15-106, HD15-107, HD15-108, HD15-109, HD15-110, HD15-111) were analyzed.
Root tips of approximately 1 cm in length were collected, pre-treated with 0.002 M 8-hydroxyquinoline (8-HQ; Merck) for 1 h at room temperature (RT) and a further 21 h at 8 °C to 10 °C. After being washed twice in distilled water and fixed in Carnoy (anhydrous ethanol (Merck):glacial acetic acid (Merck) [3:1], v/v; ) for 3 h at RT, the samples were stored at − 20 °C for at least 24 h. For slide preparation, root apices were washed twice in distilled water and incubated in a humidity chamber at 37 °C with 50 μl of 2% cellulase enzyme solution (Sigma) and 20% pectinase (w/v) (Sigma) for 80 min. The enzymes were then removed using a micropipette, and the root samples were washed again in distilled water and then added 10 μl of 45% acetic acid (Merck). Roots were then macerated using needles under a stereomicroscope, covered with a cover slip, pressed firmly between filter paper, frozen in liquid nitrogen for approximately 6 min to remove the cover slip, and finally air dried. Slide preparations featuring good presentation of cells in metaphase were kept at − 20 °C until the application of cytogenetic techniques.
Conventional cytogenetic staining for establishing chromosome count was performed following the protocol of Guerra and Souza  with modifications consisting of the use of 2% Giemsa solution (Merck) for 20-30 min, followed by briefly rinsing the slides in distilled water and air drying. After staining, the slides were mounted with Neo-Mount medium (Merck) and then coverslipped.
CMA3/DA/DAPI chromosome banding
In order to locate heterochromatin rich in GC and AT, slides were aged for 3 days prior to staining. We have used the fluorochromes Chromomycin A3 (CMA3; Sigma) and 4′-6-Diamidino-2-phenylindole (DAPI; Sigma) to stain GC and AT base pairs, respectively. A combination of the non-fluorescent antibiotic Distamycin (DA; Sigma) and the fluorochrome DAPI (DA/DAPI) favors differential staining by highlighting loci predominantly composed of AT bases. Coloration with CMA3/DA/DAPI was performed following the protocol used by Guerra and Souza , with an alteration in the CMA3 concentration used . Slides were treated with 15 μl CMA3 (0.25 mg/ml) for 1 h, then washed with distilled water and dried. Subsequently, 15 μl Distamycin A (0.1 mg/ml) was applied for 30 min, following which slides were washed with distilled water and dried, then treated with 15 μl DAPI (2 mg/ml) for 30 min. Finally, slides were washed with distilled water, dried, mounted using 15 μl of assembly medium glycerol (Sigma)/Mcllvaine (1:1 v/v), and coverslipped (20 × 20 mm). Slides were stored a darkened chamber for 3 days before analysis.
In situ hybridization probes
DNA from both parent species were extracted using the protocol of Doyle and Doyle  for the production of in situ hybridization probes. For GISH, P. gibertii total genomic DNA was labeled with biotin-16-dUTP (Roche Diagnostics) via nick translation, and P. gardneri total genomic DNA was used as blocking DNA. To prepare blocking DNA, genomic DNA was cleaved with a sonicator (Qsonica Q125) in order to obtain bands preferably between 100 and 800 bp. Sonication resulted in the generation of fragments predominantly between 200 and 1000 bp. In order to break the blocking DNA, about 20 μg of genomic DNA in a final volume of 200 μl was cleaved using sonicator (amplitude 40%, alternating pulses of 2 s on and 2 s off, total duration 5 min) . The sizes of the cleaved fragments was checked using electrophoresis in agarose gel (Pronadisa) 2% using a 100 bp ladder marker as a reference (New England Biolabs). Purification of the cleaved genomic DNA was accomplished through the precipitation of nucleic acids by adding 2% of the final sodium acetate volume (Sigma) to 3 M plus 200% of the final volume of anhydrous ethanol (Merck).The mixture was stored at − 20 °C overnight and then centrifuged (Novatecnica 805 NT) for 10 min at 14,000 rpm at 20 °C to isolate the pellet and eliminate the supernatant. The pellet was dried at RT for at least 1 h before being resuspended with ultrapure water to generate a final DNA concentration of 1.1 μg/μL.
For FISH, pTa71  clones (a donation from the Biosystematics Laboratory, Institute of Biology, State University of Campinas, SP, Brazil) were used to obtain probes for 45S rDNA sites, which were labeled with biotin-16-dUTP (Roche Diagnostics). Probes for 5S rDNA sites were obtained via polymerase chain reaction (PCR) using specific primers (5′-GTGCGATCATACCAGRYTAATGCACCGG-3′ and 5′-GAGGTGCAACACGAGGACTTCCCAGGAGG -3′)  and labeled with digoxigenin-11-dUTP (Roche Diagnostics). The probes were labeled using nick translation, with a final DNA concentration of 1 μg, following the protocol proposed by the manufacturer.
The 45S and 5S rDNA probes were used for the identification of marker chromosomes, allowing for karyotype characterization and hybrid status verification.
GISH and FISH
Slides for FISH were treated in accordance with the protocol described by Schwarzacher and Heslop-Harrison  and Souza et al.  with modifications . Slides with cytological preparations were dried at 37 °C for at least 1 h. Following this, slides were treated with 50 μl of a solution containing 1 mg/ml RNase (Sigma) in 2× SSC (salt, sodium citrate) buffer (0.3 M sodium chloride [Sigma], 0.03 M sodium citrate [Sigma]) and incubated in a humidified chamber 1 h at 37 °C. The slides were the immersed in 2× SSC at RT twice for 5 min each, and then incubated with 50 μl 10 mM hydrochloric acid (HCl; Vetec) for 5 min. Following this, HCl was removed and replaced with 50 μl of pepsin (Sigma) [10 mg pepsin/ml, 10 mM HCl (1:100 v/v)] and slides were incubated in a humidified chamber for 20 min at 37 °C. The slides were then washed in 2× SSC at RT twice for 5 min each, immersed in 4% formaldehyde (Sigma) at 4% for 10 min, and then rinsed again in 2× SSC twice for 5 min each. The wash steps were carried out using a shaker platform (Biomixer Mos-1) set at 120 rpm. Cytological preparations were dehydrated in 70% and 95% ethanol for 5 min each. After drying the slides at RT for 30 min, slides were incubated with 15 μl hybridization mix, consisting of 50% formamide (Sigma), 10% dextran sulfate (Sigma), 2× SSC (Sigma), 0.13% sodium dodecyl sulfate (SDS; Bioagency), and the probes. For GISH, we used 33 ng of probe and 3.3 μg of blocking DNA (100×), while for FISH, we used 50 ng of either the 45S or the 5S probes. The hybridization mixture was heated at 75 °C for 10 min in a thermocycler (Eppendorf Mastercycler) and immediately transferred to ice for a minimum incubation of 5 min. Cytological preparations containing the hybridization mixture were denatured in a thermocycler (Techne TC-412) containing a slide adapter at 75 °C for 10 min and incubated overnight at 37 °C in a humidified chamber. After hybridization, slides were immersed in 2× SSC for 5 min at RT to facilitate coverslip removal, moved to a Dubnoff bath (Quimis Q226M2) set at 42 °C, and immersed in 2× SSC for 5 min each, twice in 0.1× SSC for 5 min each, and twice again in 2× SSC for 5 min each. Finally, slides were dipped in 4× SSC containing 0.2% Tween 20 (Sigma) at RT for 5 min and then treated with 50 μl of 5% bovine serum albumin (BSA; Sigma). Biotin-labeled probes were detected by incubating each slide with a 0.7 μl avidin-fluorescein isothiocyanate (FITC; Vector):19.3 μl 5% BSA solution. Digoxigenin-labeled probes were detected by incubating each slide with a 0.7 μl anti-digoxigenin-rhodamine (Roche):19.3 μl 5% BSA solution. All slides containing antibodies were incubated in a humidified chamber for 1 h at 37 °C. Three washes of 5 min each with 4× SSC containing 0.2% Tween 20 were conducted to remove excess antibody. Finally, the slides were briefly immersed in 2× SSC and cytological preparations were mounted and counterstained with Vectashield® Antifade Mounting Medium with DAPI (M-1200). The slides were stored at 8-10 °C until analysis.
Metaphases following fluorochrome staining and in situ hybridization were photodocumented using an epifluorescent Olympus BX41 microscope equipped with a 5 MP Olympus DP25 digital camera and DP2-BSW software. CMA3 blocks were detected with a U-MWB filter (excitation 450-480 nm/dichroic cutoff 500 nm/emission > 515 nm) and DAPI signal with a U-MWU filter (excitation 330-385 nm/dichroic cutoff 400 nm/emission > 420 nm). Hybridizations detected using avidin-FITC were visualized with a U-MWB filter (excitation 450-480 nm/dichroic cutoff 500 nm/emission > 515 nm), while hybridizations detected using anti-digoxigenin-rhodamine were visualized using a U-MWG filter (excitation 510-550 nm /dichroic cutoff 570 nm/emission > 590 nm). DAPI counterstaining was detected with a U-MWU filter (excitation 330-385 nm/dichroic cutoff 400 nm/emission > 420 nm). Slide images, karyograms, and FITC/DAPI overlays (for GISH) and FITC/rhodamine/DAPI overlays (for 45S and 5S rDNA sites) were processed using Photoshop SC5.