Variation in Genome Size, Ploidy, Stomata, and rDNA Signals in Althea

in Journal of the American Society for Horticultural Science

Althea (Hibiscus syriacus) is a shrub prized for its winterhardiness and colorful summer flowers. Altheas are tetraploids (2n = 4x = 80); however, breeders have developed hexaploids and octoploids. Previous studies report anatomical variation among polyploids, including stomata size. The purpose of this study was 4-fold. First, identify genome size and ploidy variation in cultivars via flow cytometry and chromosome counts. Second, create a ploidy series consisting of 4x, 5x, 6x, and 8x cytotypes. Third, investigate the ploidy series for variation in stomatal guard cell lengths, stomatal density, and copy number of fluorescent ribosomal DNA (rDNA) signals. Fourth, investigate segregation patterns of rDNA signals in a subset of pentaploid seedlings. Flow cytometry revealed most cultivars to be tetraploid with holoploid 2C genome sizes from 4.55 ± 0.02 to 4.78 ± 0.06 pg. Five taxa (‘Aphrodite’, ‘Pink Giant’, ‘Minerva’, Azurri Satin®, and Raspberry Smoothie™) were hexaploids (6.68 ± 0.13 to 7.05 ± 0.18 pg). Peppermint Smoothie™ was a cytochimera with tetraploid cells (4.61 ± 0.06 pg) and octoploid cells (8.98 ± 0.13 pg). To create pentaploids, reciprocal combinations were made between hexaploid ‘Pink Giant’ and tetraploid cultivars. To create octoploids, seedlings were treated with agar solutions containing 0.2% colchicine or 125 μM oryzalin. Guard cell lengths were significantly different among the four cytotypes: 4x (27.36 ± 0.04 μm), 5x (30.35 ± 1.28 μm), 6x (35.59 ± 0.63 μm), and 8x (40.48 ± 1.05 μm). Measurements of stomatal density revealed a precipitous decline in average density from the 4x cytotype (398.22 ± 15.43 stomata/mm2) to 5x cytotype (194.06 ± 38.69 stomata/mm2) but no significant difference among 5x, 6x, and 8x cytotypes. Fluorescent in situ hybridization (FISH) revealed an increase in 5S and 45S rDNA signals that scaled with ploidy: 4x (two 5S + four 45S), 6x (three 5S + six 45S), and 8x (four 5S + eight 45S). However, pentaploid (5x) seedlings exhibited random segregation of rDNA signals between the 4x and 6x cytotypes, including all six possible combinations (two 5S, three 5S) × (four 45S, five 45S, six 45S).

Contributor Notes

This study was funded by the Oregon Department of Agriculture and Oregon State University.

Corresponding author. E-mail:

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Article Figures

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    Photomicrograph of metaphase chromosomes from root tip cells of Hibiscus syriacus ‘Diana’ (2n = 4x = 80) viewed at ×1000; scale bar = 10 μm.

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    Flow cytometry estimates of average holoploid 2C genome sizes in a subset interploid crosses between tetraploid Hibiscus syriacus cultivars and hexaploid H. syriacus ‘Pink Giant’. Reciprocal combinations indicated by “|” symbol, whereas “x” represents a unidirectional cross. Vertical bars represent mean ± se for each cross. White letters within vertical bars represent the number of seedlings measured for each cross. Horizontal 4x bar represents the range of tetraploid genome sizes found among cultivars of H. syriacus. Horizontal 6x bar represents the range of hexaploid genome sizes found among cultivars of H. syriacus. Horizontal 5x line represents the theoretical pentaploid genome size based on an average of 4x and 6x genome sizes.

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    Stomata length and density in a ploidy series in Hibiscus syriacus. Photomicrographs above bar graph are representative cuticle peels for each ploidy level viewed at ×630 magnification (scale bar = 10 μm). Ploidy series included tetraploid H. syriacus Bali™ (12-0023), pentaploid hybrid H. syriacus ‘Pink Giant’ × Bali™ (H2013-078-01), hexaploid H. syriacus ‘Pink Giant’ (11-0217), and an octoploid, oryzalin-treated, open-pollinated seedling from H. syriacus Bali™ (OP2014-19).

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    Fluorescent in situ hybridization (FISH) analysis of metaphase, root tip chromosomes in a ploidy series of Hibiscus syriacus. Chromosomes displaying variation in 5S (red) and 45S (green) rDNA loci (scale bar = 10 μm). (A) tetraploid H. syriacus Bali™ (two 5S signals + four 45 signals). (B) hexaploid H. syriacus ‘Pink Giant’ (three 5S signals + six 45S signals). (C) oryzalin-treated (125 μM for 5 d), octoploid seedling from H. syriacus Bali™ (four 5S signals + eight 45S signals).

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    Fluorescent in situ hybridization (FISH) analysis of metaphase, root tip chromosomes in pentaploid seedlings of Hibiscus syriacus. Chromosomes displaying variation in 5S (red) and 45S (green) rDNA loci (scale bar = 10 μm). (A) H2013-078-01 ‘Pink Giant’ × Bali™. (B) H2013-131-06 ‘Pink Giant’ × ‘Blushing Bride’ (C) H2013-124-19 ‘Helene’ × ‘Pink Giant’. (D) H2013-017-21 ‘Aphrodite’ × ‘Pink Giant’. (E) H2013-085-02 ‘Pink Giant’ × ‘Red Heart’. (F) H2013-049-01 ‘Diana’ × ‘Pink Giant’.

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