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

Abstract

Background: The oat × maize addition (OMA) lines are used for mapping of the maize genome, the studies of centromere-specific histone (CENH3), gene expression, meiotic chromosome behavior and also for introducing maize C4 photosynthetic system to oat. The aim of our study was the identification and molecular-cytogenetic characterization of oat × maize hybrids.

Methods: Oat DH lines and oat × maize hybrids were obtained using the wide crossing of Avena sativa L. with Zea mays L. The plants identified as having a Grande-1 retrotransposon fragment, which produced seeds, were used for genomic in situ hybridization (GISH).

Results: A total of 138 oat lines obtained by crossing of 2,314 oat plants from 80 genotypes with maize cv. Waza were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated in 66 lines by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1. Genomic in situ hybridization (GISH) detected whole maize chromosomes in eight lines (40%). All of the analyzed plants possessed full complement of oat chromosomes. The number of maize chromosomes differed between the OMA lines. Four OMA lines possessed two maize chromosomes similar in size, three OMA-one maize chromosome, and one OMA-four maize chromosomes. In most of the lines, the detected chromosomes were labeled uniformly. The presence of six 45S rDNA loci was detected in oat chromosomes, 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 maize chromosomes, but the introgression of maize chromatin in the oat chromosomes. Five of 66 hybrids were shorter in height, grassy type without panicles. Twenty-seven OMA lines were fertile and produced seeds ranging in number from 1-102 (in total 613). Sixty-three fertile DH lines, out of 72 which did not have an addition of maize chromosomes or chromatin, produced seeds in the range of 1-343 (in total 3,758). Obtained DH and OMA lines were fertile and produced seeds.

Discussion: In wide hybridization of oat with maize, the complete or incomplete chromosomes elimination of maize occur. Hybrids of oat and maize had a complete set of oat chromosomes without maize chromosomes, and a complete set of oat chromosomes with one to four retained maize chromosomes.

Keywords: Doubled haploids (DH); Fertility; Genomic in situ hybridization (GISH); Oat × maize hybrids.

Figure 1. Oat haploid embryo formed after crossing with maize.

(A); germinated haploid embryo on 190-2 medium (B); haploid plant on MS medium (C); acclimatization of haploid plant in perlite (D); DH plants in the greenhouse (E); panicles of OMA line STH5.8536/1, some of immature panicles are senesced (F); grassy OMA line STH5.8429 without panicles (G). Photo credit: Edyta Skrzypek.

Figure 2. Flow cytometry histograms of oat plants; (A) control 2n, (B) doubled haploid 2n, (C) haploid 1n and (D) mixoploid.

Figure 3. Identification of oat × maize F1 plants.

PCR products of genomic DNA of oat, maize, and a selection of 22 oat ×maize F1 plants shown after electrophoresis in 1.5% (w/v) agarose gel. Bands represent 500 bp DNA fragments that were amplified with marker Grande-1. Marker leader is shown in the first line. Maize cv. Waza specificity is shown by product presence in maize DNA (positive control) and absence in oat cv. Stoper DNA (negative control). The presence of retained maize chromosomes is indicated in 14 out of the 22 F₁ plant DNAs shown. Photo credit: Tomasz Warzecha.

Figure 4. Visualization of added maize chromosomes in oat genome by genomic in situ hybridization (GISH).

(A) STH 4.4690f plant with tetrasomic addition of maize; (B) STH 5.8504b plant with monosomic addition of maize chromosome, (C) STH 5.8436b plant with disomic addition of maize chromosome. The maize chromosomes are labelled uniformly, (D) STH 4.4576 plant with disomic addition of maize chromosome. A banding pattern is visible on additional chromosomes. Maize gDNA is labeled with digoxigenin and detected anti-dig FITC (green fluorescence), rhodamine-5-dUTP—labeled 25S rDNA (red fluorescence) is used as an internal control of hybridization efficiency. Chromosomes are stained with DAPI (blue fluorescence). Scale bar: 10 µm. Photo credit: Dominika Idziak-Helmcke.

Figure 5. Visualization of added fragments of maize chromosomes in oat genome by genomic in situ hybridization (GISH).

(A) STH 4.4690d; (B) STH 4.4576; (C) STH 4.4606 F1 plants. The arrows point to maize introgressions into oat chromosomes (green fluorescence). Yellow signals result from colocalization of hybridization signals for maize gDNA (green fluorescence) and 25S rDNA (red fluorescence). Chromosomes are stained with DAPI (blue fluorescence). Scale bar: 10 µm. Photo credit: Dominika Idziak-Helmcke.