Complex characterization of oat (Avena sativa L.) lines obtained by wide crossing with maize (Zea mays L.)

Oat wide crossing with maize

The experiments were done on 80 oat genotypes (listed in Table 1) obtained from Plant Breeding Strzelce Ltd., PBAI Group, Strzelce, Łódź Voivodeship, Poland. Oat plants and maize cultivar Waza were sown and cultivated according to Noga et al. (2016). Oat haploid plants were obtained using the wide crossing method by pollination with maize cv. Waza, as described by Marcińska et al. (2013). Three weeks after floret pollination, enlarged ovaries were sterilized in 70% (v/v) ethanol (1 min), in a 2.5% (w/v) calcium hypochlorite (8 min), and washed three times with sterile water. Then, haploid embryos were isolated from caryopses and transferred to the 190-2 medium (Zhuang & Xu, 1983) with 9% (w/v) maltose, 0.6% (w/v) agar, 0.5 mg L⁻¹ kinetin (KIN) and 0.5 mg L⁻¹ α-naphthaleneacetic acid (NAA) and pH 6.0. Haploid embryos were germinated in an in vitro chamber with a 16 h photoperiod, light intensity of 100 µmol m² s⁻¹ and temperature 21 °C. The germination capacity of haploid embryos was evaluated under a stereomicroscope (SMZ 1500, Nikon, Tokyo, Japan) and photographs were taken using a digital CCD camera (DS-Ri1, Nikon, Tokyo, Japan).

The plants developed from haploid embryos were transferred to MS medium (Murashige & Skoog, 1962) with 0.6% (w/v) agar. Then, the well rooted plants were acclimated to natural conditions by transferring them subsequently to wet perlite and soil. For chromosome doubling, the roots of haploid plants were treated by colchicine solution and the ploidy level of the plants was evaluated using a MACSQuant flow cytometer (MACSQuant, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described by Noga et al. (2016).

Next, the plants were tested for the presence of the maize retrotransposon Grande-1 to indicate maize chromatin in oat. Oat cv. Stoper and maize cv. Waza were used as a negative and positive control of the presence of the maize retrotransposon Grande-1. Leaves of studied lines, oat cv. Stoper, and maize cv. Waza (about 200 mg) were lyophilized in high vacuum at 40 µbar, coil temperature −52 °C (lyophiliser FreeZone 6L; Labconco, Kansas City, MO, USA). Then, the tissue was homogenized using ball mill (MM400; Retsch, Haan, Germany) for 5 min at frequency 25 Hz.

DNA extraction and PCR analyses

DNA was extracted from 20 mg of lyophilized leaves, using the Genomic Mini AX Plant kit (A&A Biotechnology, Gdynia, Poland) according to the producer recommendations. After precipitation, the DNA pellet was washed twice in 70% (v/v) ethanol and dried in a concentrator (Concentrator Plus; Eppendorf, Hamburg, Germany) for 10 min. under 20 hPa. The precipitate was suspended in sterile distilled water. DNA concentration was measured at 260 nm with a spectrophotometer NanoDrop 2000c (Thermo Scientific, USA).

Two Grande 1F (5′-AAA GAC CTC ACG AAA GGC CCA AGG-3′), Grande1R (5′-AAA TGG TTC ATG CCG ATT GCA CG-3′) primers (GenBank accession number X97604; Ananiev, Phillips & Rines, 1998) were used for the PCR reaction, which in successive cycles enabled the amplification of the 500 bp retrotransposon region of Grande-1 and detection of the presence of maize chromatin in oat plants. For the PCR, the thermostable Taq polymerase (Fermentas, USA) was used.

After DNA isolation and dilution (50 µg/µl DNA), 5 µl of DNA was taken and introduced into reaction mixture using 0.5 µl of each two primers. The reaction mixture was placed in a thermocycler (2720 Thermal Cycler; Applied Biosystems, Foster City, CA, USA) and used the following thermal program: 1. Initial denaturation −94 °C, 5 min.; 2. Cyclic denaturation: 94 °C–30 s; 3. Starter connection—58 °C, 30 s; 4. Polymerization—72 °C—30 s; 5. Final Polymerization—72 °C, 5 min. Stages 2-4 were repeated cyclically 25 times. In the last stage the samples were cooled to 4 °C until removed from the amplification apparatus (Ananiev, Phillips & Rines, 1998).

The obtained products were mixed with 2.5 µL DNA Gel Loading Dye buffer (Thermo Fisher Scientific, Waltham, MA, USA) and then separated in 1.5% agarose gel with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) in TBE buffer, under 90 V for 90 min. DNA markers of 100 bp to 1000 bp and concentration of 0.5 µg/µl (GeneRuler 100bp; Fermentas, Waltham, MA, USA) were used to estimate the length of PCR products. The image of electrophoretic separation was archived using the Imagemaster VDS gel reader (Amersham, Pharmacia Biotech, Piscataway, NJ, USA) and the Liscap Capture Application ver. 1.0. Electrophoretic gel analysis was performed using GelScan ver. 1.45, (Kucharczyk Electrophoretic Techniques, Warsaw, Poland). The plants identified as having a Grande-1 retrotransposon fragment, which were also fertile and produced seeds, were used for genomic in situ hybridization (GISH).

Cytogenetic analysis

The seeds of oat, maize, and oat-maize chromosome addition and DH lines were germinated for 2–3 days in Petri dishes on a wet filter paper. Seedlings with the root tips of 1.5–2 cm long were immersed in ice-cold water and incubated for 24 h at 4 °C. The procedure allowed to stop the cell division at metaphase through the spindle degradation, and to increase chromosome condensation. After 24 h, the root tips were fixed in a mixture of pure methanol and glacial acetic acid at a 3:1 ratio. The material was kept at 4 °C for 24 h, and stored in −20 °C until used.

Excised root tips (about 5 mm in length) were washed in citrate buffer (pH 4.8) for 15-20 min and then digested in a solution of 20% pectinase (Sigma, St Louis, MO, USA), 1% cellulose (Calbiochem, San Diego, CA, USA) and 1% cellulase ‘Onozuka R-10′(Serva, Heidelberg, Germany) in a citrate buffer at 37 °C for 2 h. The meristems were dissected from the root tips and squashed in a drop of 45% acetic acid. Good quality preparations were frozen on dry ice. After freezing, the coverslips were removed and the slides were air-dried and stored at 4 °C until required.

In order to discriminate maize chromatin introgressions, the total genomic DNA of the maize cv. Waza was used as probe for GISH. Maize DNA was isolated from plants grown under greenhouse conditions (21 °C, photoperiod 8 h/16 h). Young leaves were frozen with liquid nitrogen and homogenized. DNA isolation was performed using the C-TAB method (Doyle & Doyle, 1987). The concentration and purity of the isolated DNA was measured by NanoDrop 2000c (Thermo Scientific, Waltham, MA, USA) and the length of the DNA sequence fragments was checked on agarose gel.

Maize genomic DNA and 25S rDNA sequences that were used as additional chromosome markers were labeled by nick-translation using the digoxigenin-11-dUTP and rhodamine-5-dUTP (Roche), respectively. The GISH procedure was performed according to the protocol of Hasterok et al. (2006). In order to verify the specificity of the gDNA probe and appropriate hybridization conditions, maize and oat root tips preparations were also used as positive and negative control in GISH, respectively. Hybridization mixture was prepared by adding appropriate volumes of 50% formamide, 10% dextran sulfate, 20 × SSC, water, SDS, and labeled DNA probes (∼500 ng/slide). Due to the high phylogenetic distance between oat and maize and related to that low degree of DNA sequence homology, the use of total genomic oat DNA to block non-specific hybridization was not necessary. Posthybridization stringent washes were carried out in 10% formamide in 0.1 × SSC at 42 °C, which is equivalent to 79% stringency. Immunodetection of digoxigenin-labeled probes was performed according to the standard protocols using FITC-conjugated anti-digoxigenin antibodies (Roche). The rhodamine probes were visualized directly. The preparations were mounted in VectaShield antifade (Vector Laboratories, Inc. Burlingame, CA, USA) containing 2.5 µm ml⁻¹ DAPI (Serva, Heidelberg, Germany). Hybridization signals were visualized and captured using fluorescence microscope Axio Imager Z2 (ZEISS, Germany) equipped with monochromatic camera AxioCam MRm (ZEISS, Oberkochen, Germany) and archived by ZEN blue program (ZEISS, Oberkochen, Germany). All images were processed uniformly and superimposed using Photoshop CS3 (Adobe).

Oat DH lines and oat × maize hybrids formation

Analysis of variance indicated no significant differences between oat genotypes in the production of embryos per emasculated florets and lines per pollinated florets (Table 2). The efficiency of oat lines production by wide crossing with maize is shown in Table 1. Total of 57,515 florets from 80 oat genotypes were pollinated with maize that resulted in development of 2,129 embryos (Fig. 1A). Average embryo formation frequency per pollinated florets was 3.58%. Six hundred and one embryos germinated within three weeks of in vitro culturing (28.23% of all embryos) (Fig. 1B). Number of embryos produced by various genotypes varied from 0 to 106 (9.69% embryos per pollinated florets). Only one genotype out of eighty examined did not formed haploid embryos (STH 5.8428). Most embryos per pollinated floret were obtained from the genotypes: STH 4.4690 (9.69%), STH 4.4606 (7.22%), STH 4.4586 (6.68%), STH 5.8425 (6.54%) and STH 4.8448 (6.37%). Even if the genotype did not have the significant effect on the haploid plants production, 15 of 80 genotypes did not develop haploid plants. Total of 210 plants survived the acclimatization to the natural condition (Figs. 1C–1E). The procedure of chromosome doubling using colchicine reduced the number of obtained lines to 138. The highest number of lines (16) was obtained from the STH 4.4690 genotype. Plants from seven genotypes completely failed this process. The ploidy of the plants was measured by flow cytometry before and after application of colchicine (Fig. 2). Most plants doubled chromosomes number, except plants STH 4.4690 n, STH 4.4690 o, STH 5.8505 b, STH 4.8417 c and STH 5.8540.

Identification of oat × maize hybrids

A total of 138 oat lines obtained by crossing with maize were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1 (GenBank accession number X97604; Ananiev, Phillips & Rines, 1998). Copies of the Grande-1 are densely dispersed and located on each of the maize chromosomes. The 500 bp PCR product was identified in 66 oat plants. Figure 3 shows the exemplary agarose gel with 500 bp product corresponding to the maize retrotransposon Grande-1. Out of the 66 OMA lines containing maize chromatin, only 27 were fertile and produced seeds (Table 1). The 20 OMA lines that produced the highest number of seeds were then selected for cytogenetic analysis.

The seeds of two out of 20 OMA lines did not germinate. Among the remaining 20 lines, 8 lines (40%) had the character of additive lines, since GISH has detected one or more maize chromosomes in their cells. All of the analyzed plants possessed full complement of oat chromosomes. The number of detected maize chromosomes differed between the OMA lines (Table 1). They were usually smaller than the oat chromosomes and easy to discriminate (Fig. 4). In the plant STH 4.4690f, two pairs of chromosomes of varying size were found (Fig. 4A), whereas in STH 5.8504b only one maize chromosome was present (Fig. 4B). The majority of OMA lines possessed two maize chromosomes similar in size. In most of the lines, the detected chromosomes were labeled uniformly (Fig. 4C); however, in some, a banding pattern was visible that can potentially be used for the identification of maize addition (Fig. 4D). The banding pattern on both chromosomes in a given line was similar, which might indicate that the added chromosomes are homologs. The presence of six 45S rDNA loci was detected in oat chromosomes (red hybridization signals, Fig. 4), but none of the added maize chromosomes in any of the lines carried 45S rDNA locus.

Twenty of the analyzed lines did not possess whole chromosomes of maize, but the introgression of maize chromatin was found in the oat chromosomes. A total of 12–14 hybridization signals for the maize gDNA probe were detected (Fig. 5). In all these lines, six of the maize gDNA hybridization signals colocalized with 25S rDNA sequence hybridization signals. In another pair of chromosomes, two strong signals were present in the pericentromeric positions on each of the chromosomes. Additionally, weaker maize gDNA signals were observed in one or two pairs of chromosomes of the lines STH 4.4690d (Fig. 5A), and STH 4.4606a (Fig. 5C), depending on the metaphase plate, and in one chromosome pair in the case of the line STH 4.4576 (Fig. 5B).

The colocalization of the gDNA and 25S rDNA signals may be related to the hybridization of the 25S rDNA sequence present in the maize genomic DNA to the same sequence in the oat genome. These sequences are highly conservative, hence the presence of these signals in the chromosomes of the analyzed lines. Additional hybridization signals found in 2–3 pairs of chromosomes, depending on the analyzed line, are presumably maize DNA sequences that have been incorporated into the oat chromosomes. Accurate determination of the origin of these sequences requires further analysis.

Vigor of DH and OMA lines in their effectiveness in seeds production

The progeny of oat-maize hybrids showed some differences in morphology. Five of 66 hybrids were shorter in height, grassy type without panicles (Fig. 1G). Three of them had only a few short shoots with small panicles (Fig. 1F). Leaves’ blades color and panicle shape were similar to their oat parent.

Analysis of variance showed significant variation in fertility in dependence on the addition of maize chromosomes to the oat genome (Table 1). OMA plants produced significantly less seeds than DH lines. Negative correlation coefficient (−0.279) for seeds production dependently on the maize chromosomes/chromatin added to the oat genome was observed.

Sixty-three fertile DH lines out of 72 which did not have an addition of maize chromosomes or chromatin produced seeds (Table 2). Nine DH lines belonging to various genotypes did not produce seeds. The number of seeds was from 1 to 343, dependently on the genotype. In total, 3,758 seeds were produced. The highest number of seeds (343 in total) was produced by the STH 5.8429 genotype.

Twenty-seven OMA lines (from 66 identified) were fertile and produced seeds ranging in number from 1–102 (Table 2). In total, 613 seeds were produced by OMA lines. As for DH lines, the number of seeds produced by OMA lines depended on genotype, however nearly 41% of OMA plants were sterile. The differences in the seed production were not dependent on the number of chromosomes added to the oat genome or introgression of chromosome fragments to the oat chromosomes. OMA lines with the addition of whole chromosomes produced seeds as well as the ones with the introgression of fragments of maize chromosomes. All DH and OMA lines were fertile and produced seeds in the subsequent generation.