Fig 1.
Induction and confirmation of tetraploidy in perennial rye.
(A) Schematic workflow illustrating the generation of perennial rye from a Secale cereale × S. strictum cross, selection of the diploid perennial line (‘Keleti1’, 2x), colchicine-induced chromosome doubling, and subsequent analyses of the tetraploid derivative (‘Keleti1T’, 4x). Colours indicate parental lines, F1 hybrid, diploid ‘Keleti1’, and tetraploid ‘Keleti1T’, as shown in the key. (B) Flow cytometry histograms showing relative nuclear DNA content of diploid (2x) and tetraploid (4x) plants. (C) Representative chromosome spreads of diploid (‘Keleti1’, 2x) and tetraploid (‘Keleti1T’, 4x) plants counterstained with DAPI. Scale bar = 10 µm.
Fig 2.
Agronomic traits of diploid and tetraploid perennial rye.
(A) Representative images of fully developed diploid (‘Keleti1’, 2x) and tetraploid (‘Keleti1T’, 4x) plants. The image shows plants prior to the late harvest, which was preceded by an early harvest (S2 Fig), together resulting in the total number of fertile tillers per plant. (B) Boxplot showing thousand-grain weight (TGW) per plant. (C) Representative seed samples of ‘Keleti1’ (2x) and ‘Keleti1T’ (4x). (D) Representative spikes of diploid and tetraploid plants. (E) Reproductive efficiency expressed as seeds per floret for individual plants; ellipses indicate individuals with moderate to high values. (F) Boxplot showing the number of seeds per spike. (G) Boxplot showing the number of fertile tillers (spikes) per plant. Data include spikes from both early and late harvests, representing the total number of fertile tillers per plant. (H) Scatterplot showing the total number of seeds per plant; dots represent individual plants, and ellipses indicate individuals with moderate to high values. Boxplots show medians, interquartile ranges, and whiskers extending to 1.5 × the interquartile range. Statistical significance is indicated as (**: P < 0.01; ***: P < 0.001). Data underlying the presented analyses are available in S1 Table.
Fig 3.
Regrowth performance of diploid perennial rye compared with Secale strictum.
(A) Representative field images showing regrowth and development of the diploid perennial rye line ‘Keleti1’ and the perennial parent S. strictum during the second year following establishment. Images were taken in April and June of the 2025 growing season. (B) Statistical comparison of regrowth performance expressed as the proportion of plants showing successful regrowth. Pearson’s chi-square test indicates that the regrowth percentage of diploid ‘Keleti1’ does not differ significantly from that of S. strictum.
Fig 4.
Molecular cytogenetic identification of chromosomes in parental, diploid, and tetraploid rye lines.
Fluorescence in situ hybridisation (FISH) analysis of mitotic chromosomes from Secale cereale, Secale strictum, the diploid perennial line ‘Keleti1’ (2x), and its tetraploid derivative ‘Keleti1T’ (4x). Chromosomes were counterstained with DAPI (blue) and hybridised with 5S rDNA (green) and the subtelomeric repeat pSc119.2 (red). The rightmost column shows merged images with chromosome identification (1R–7R) based on signal distribution patterns. Scale bars = 5 μm.
Fig 5.
Karyotypic layout and chromosome origin in parental, diploid, and tetraploid rye lines.
(A) Karyotypes of Secale cereale, Secale strictum, and the diploid perennial line ‘Keleti1’ (2x), arranged by homologous chromosome groups from 1R to 7R. For each chromosome group, representative homologous chromosomes are shown together with schematic summaries indicating inferred parental origin based on fluorescence in situ hybridisation (FISH) signal patterns. Chromosomes are shown with merged DAPI (blue), 5S rDNA (green), and pSc119.2 (red) signals. (B) Karyotype of the tetraploid derivative ‘Keleti1T’ (4x), with chromosomes arranged by homologous groups (1R–7R) and displayed as sets of four homologues. Representative merged images and corresponding schematic summaries of parental origin are shown for each chromosome group. Schematic colour coding indicates chromosome segments assigned to S. cereale, S. strictum, or regions of undetermined parental origin, as indicated in the key.