Ploidy and karyotype analysis of two Begonia species

Abstract

Begonia cucullata (Begoniaceae) is an ornamental plant used in gardening and landscaping that is capable of flowering in four seasons and possesses strong resistance to abiotic stresses, giving it considerable commercial value. Research on B. cucullata currently focuses mainly on cultivation-related physiology, while less attention is paid to ploidy and karyotype identification. In this study, one important wild Begonia species, and five horticultural varieties of B. cucullata were used as experimental materials for karyotype analysis. The results indicate that the ploidy of wild species is 2n=34, while the variety ‘Big Series’ is a tetraploid (2n=68). These findings are of considerable importance for breeding varieties of Begonia with excellent horticultural traits.

Begonia cucullata (Begoniaceae) is a perennial evergreen herb (Lim 2014). Due to its attractive flowers and leaves, and strong resistance to abiotic stresses, it is widely used in landscaping. Early provenances (B. subvillosa, B. cucullata var. cucullata) as well as late provenances (B. roezlii and B. foliosa var. miniata) (Tebbitt 2005) have allowed the expansion of varieties after repeated hybridization. B. cucullata is a commercial variety that is capable of blooming in four seasons. After continuous hybridization with other germplasm, it gradually formed a horticultural variety group of B. cucullata that is widely cultivated and used as an ornamental commodity. At present, research on B. cucullata mainly focuses on reproduction and cultivation-related physiology (Ma et al. 2020; Li et al. 2021), while less attention is paid to the ploidy and karyotype identification of B. cucullata.

Since the chromosomes of Begonia are small, the centromere is difficult to observe, so most cytological studies to date only reported the number of chromosomes (Legro and Doorenbos 1971; Nakata et al. 2007). Chromosome counting and ploidy determination of 62 varieties of the B. cucullata variety group were carried out by Zeiling (1962), who found diploid, triploid, and tetraploid varieties. Only a few species with relatively long chromosomes, such as B. wuisenioris (2n=14), have been reported, following a karyotype analysis (Peng et al. 2014). In addition, the number of chromosomes in Begonia is controversial.

There are no studies on the karyotype of B. cucullata. A clearer understanding of the ploidy and karyotype of existing high-quality varieties, which is the main objective of this study, would be a valuable contribution to the breeding of Begonia varieties with superior horticultural traits. In this paper, we provide the first report of the karyotype of B. cucullata and B. subvillosa, which is one of the early provenances.

Materials and methods

Plant materials

B. subvillosa, B. cucullata ‘Bada’, and B. cucullata ‘Big Series’, ‘D21’, ‘106’, ‘18vs79’ were purchased from the Beijing Tianhuiyuan Green Technology Research Institute Co., Ltd. (Beijing, China).

Experimental method

Actively growing root tips (1–3 cm) were pretreated with saturated 0.002 mol L⁻¹ 8-hydroxyquinoline solution for 3 h in the dark at 4°C, fixed in Carnoyʼs solution (ethanol : acetic acid=3 : 1, v/v) for 24 h, then stored in 70% ethanol at 4°C until use. The mixed enzyme solution (2.7% cellulase +0.3% pectinase, pH=4.8) was dissociated at 37°C for 15–35 min, then root tips were stained with 5 mg mL⁻¹ DAPI.

Karyotype analysis

For each taxon, 30 cells were observed with an epifluorescence microscope (Olympus BX51, Tokyo, Japan) when chromosomes could be clearly identified, and photos were processed with Cytovision software (Leica, Wetzlar, Germany). Chromosome (short and long arms) measurements were made using five well-spread metaphase plates per species using Image Pro Plus 6.0 (Media Cybernetics Inc., Rockville, MD, U.S.A). Depending on the length of the chromosome, the length of the short arm, and the position of the constriction, paired chromosomes were arranged in order from short to long, and the chromosomes were numbered. Chromosome data were imported into Excel to create idiograms (i.e., karyotype patterns). Adobe Photoshop 2019 was used to process images.

Chromosome morphology was determined using an arm ratio (long arm/short arm) and classified as either m (1.01–1.70), sm (1.71–3.00), or st (3.01–7.00) (Levan et al. 1964). The ratio of the longest to the shortest chromosome and average centromere index percentage (short arm length/chromosome length×100) were also calculated.

Results and discussion

We provide the first report of the karyotypes of B. cucullata and B. subvillosa karyotypes. Chromosomes at the mitotic metaphase of B. subvillosa are 2n=34 (Figs. 1A and 2A). The chromosome numbers of B. subvillosa have been reported as 2n=29–32 (Heitz 1927), 2n=32 (Matsuura and Okuno 1943), 2n=32+2f (Legro and Doorenbos 1969), and 2n=34 (Doorenbos et al. 1998; Dewitte et al. 2009). The 2n=34 observed in this study is consistent with that reported by Doorenbos et al. (1998) and Dewitte et al. (2009). Chromosomes of the mitotic metaphase of B. cucullata ‘Bada’ (Fig. 1B), ‘D21’ (Fig. 1D), ‘106’ (Fig. 1E), and ‘18vs79’ (Figs. 1F and 2B) each show a count of 2n=34, while in the ‘Big Series’, 2n=68 (Figs. 1C and 2C). Various chromosome numbers have been reported for B. cucullata, including n=13 (Sharma 1970; Sarkar 1989), 2n=ca.30 (Pinto-Magiio et al. 1997), 2n=33, 36, 60, and 66 (Matsuura and Okuno 1936), 2n=34 (Zeiling 1962; Legro and Doorenbos 1971; Doorenbos et al. 1998; Dewitte et al. 2009), 2n=40–60 (Sharma and Bhattacharyya 1961), 2n=56 (Bowden 1945; Legro and Doorenbos 1969), 2n=64 (Legro and Doorenbos 1971), and 2n=68 (Zeiling 1962). For B. cucullata, the 2n=34 observed in our study is consistent with that reported by Zeiling (1962), Legro and Doorenbos (1971), Doorenbos et al. (1998), and Dewitte et al. (2009), and 2n=68 is consistent with that reported by Zeiling (1962).

Fig. 1. Somatic chromosomes at metaphase of six Begonia taxa.

(A) B. subvillosa (2n=34). (B) B. cucullata ‘Bada’ (2n=34), (C) B. cucullata ‘Big Series’ (2n=68). (D) B. cucullata ‘D21’ (2n=34). (E) B. cucullata ‘106’ (2n=34). (F) B. cucullata ‘18vs79’ (2n=34). Scale bars=5 µm.

Fig. 2. Chromosome arrangement of three Begonia taxa.

(A) B. subvillosa [2n=34=16m+18sm(st)]. (B) B. cucullata ‘18vs79’ [2n=34=16m+18sm(st)]. (C) B. cucullata ‘Big Series’ [2n=68=32m+36sm(st)]. Scale bar=5 µm.

The karyotypes of the studied taxa are reported here for the first time. In B. subvillosa, of the 34 chromosomes, eight pairs of chromosomes (Nos. 3, 8, 10, 12, 14–17 in Figs. 2A and 3A) have centromeres at a median position (m-chromosome), whereas the other nine pairs of chromosomes (Nos. 1, 2, 4, 5, 6, 7, 9, 11, 13 in Figs. 2A and 3A) have centromeres at a submedian or subterminal position (sm- or st-chromosome). At least two pairs of chromosomes (Nos. 1, 4 in Fig. 2A) have secondary constrictions (SCs). The B. subvillosa karyotype formula is 2n=34=16m+18sm(st). Four taxa of B. cucullata show a karyotype similar to that of B. subvillosa. B. cucullata ‘18vs79’ has 8 pairs of m-chromosomes (Nos. 3, 7, 10, 12, 14, 15, 16, 17 in Figs. 2B and 3B) and 9 pairs of sm(st)-chromosomes (Nos. 1, 2, 4, 5, 6, 8, 9, 11, 13 in Figs. 2B and 3B). At least two pairs of chromosomes (Nos. 1, 2 in Fig. 2B) have SCs. The B. cucullata ‘18vs79’ karyotype formula is 2n=34=16m+18sm(st). In contrast, chromosomes at the mitotic metaphase of one of the horticultural varieties of B. cucullata, the ‘Big Series’, is 2n=68. Of the 68 chromosomes, 32 have centromeres at a median position (Nos. 4, 7, 8, 9, 11, 14, 16, 17 in Figs. 2C and 3C), whereas the remaining 36 have centromeres at a submedian or subterminal position (Nos. 1, 2, 3, 5, 6, 10, 12, 13, 15 in Figs. 2C and 3C). At least, eight chromosomes have SCs (Nos. 1, 2 in Fig. 2C). The B. cucullata ‘Big Series’ karyotype formula is 2n=68=32m+36sm(st).

Fig. 3. Idiograms of three Begonia taxa.

(A) B. subvillosa [2n=34=16m+18sm(st)]. (B) B. cucullata ‘18vs79’ [2n=34=16m+18sm(st)]. (C) B. cucullata ‘Big Series’ [2n=68=32m+36sm(st)].

Begonia species in this study, except for B. cucullata ‘Big Series’, which is tetraploid with 68 chromosomes, are diploid with 34 chromosomes. Based on the karyotype, the 2n=68=32m+36sm(st) consists of four sets of 17 chromosomes, x=17=8m+9sm(st), and is therefore a tetraploid (4x) of 2n=34=16m+18sm(st) (2x).

Acknowledgments

This work was financially supported by the Joint Research Center for Chinese Herbal Medicine of Anhui of IHM (grant #: yjzx2023010).

Author contribution

Gang Li: Methodology, Conceptualization, Writing-original draft. Le Chen: Methodology, Formal analysis, Validation. Rui Zhao: Formal analysis, Software. Qihang Chen: Visualization, Validation. Li Wang: Validation, Writing-review. Jaime A. Teixeira da Silva: Resources, Validation, Writing-review & editing. Shaocai Zhu: Investigation, Validation. Yuxuan Wang: Conceptualization, Supervision, Validation. Xiaonan Yu: Conceptualization, Funding acquisition, Project administration, Validation.

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