Abstract
The karyotypes of 21 herbaceous peony (Paeonia) cultivars were studied using root tip squashes revealing a wide variety of ploidy levels. There were three tetraploid (2n = 4x = 20), 11 triploid (2n = 3x = 15), and one diploid (2n = 2x = 10) cultivars in the hybrid group; five triploid (2n = 3x = 15) cultivars in the Itoh group; and one diploid (2n = 2x = 10) cultivar in the lactiflora group (LG). The asymmetry index (AI) ranged from 59.61% (‘Cytherea’) to 64.03% (‘Little Red Gem’). The karyotypes of all peony cultivars were 2A with 60% metacentric (m), 20% submetacentric (sm), and 20% subtelocentric (st) chromosomes. The karyotypic background of these 21 herbaceous peony cultivars is discussed in the context of the evolution of ploidy and the three cultivar groups. These results provide cytological information that would assist in a peony hybridization program.
Herbaceous peonies are perennial flowers belonging to section Paeonia of the genus Paeonia in the Paeoniaceae. Herbaceous peonies usually blossom from April to June and have a variety of flower patterns and flower colors, making them famous traditional flowers in China and offering them an extremely important position in the global cut-flower market (Yang et al., 2015; Yu et al., 2011). According to a new classification, section Paeonia has 25 species and more than 1000 cultivars around the world (Hong et al., 2010; Sang et al., 2004). The American Peony Society has divided herbaceous peony into three groups: lactiflora, hybrid, and Itoh. The LG uses Paeonia lactiflora as the parent (2n = 2x = 10) (Hong et al., 2010), and the hybrid group (HG) uses P. lactiflora, P. officinalis, P. macrophylla, and other species as the parents, whereas members of the Itoh group (IG) are the product of distant hybridization in which peony (section Paeonia) served as the female parent and species or cultivars of tree peony (section Moutan) served as the male parent (Qin, 2004; Yang and Yu, 2016).
Herbaceous peonies have rich germplasm diversity in terms of external morphology and chromosome ploidy (Hao et al., 2016; Ji et al., 2014). Species and cultivars have been used in the hybridization of different cultivar groups resulting in complex genetic backgrounds. Members of the LG are all diploid (Hong et al., 2010). The HG consists of a variety of species involved in hybridization. There are many natural tetraploids such as P. officinalis (2n = 4x = 20) and P. macrophylla (2n = 4x = 20). Cultivars of the HG usually have either of these species as their parents, so most cultivars in this group are triploid or tetraploid (Sang et al., 2004). Cultivars of the IG have characters that are intermediate to herbaceous and tree peony (Wu et al., 2011).
Much cytological work has been carried out on Paeonia. Dark (1936) found that the basic chromosome number was x = 5. Ding and Liu (1991), Hong et al. (1988), La Cour (1952), and Yang and Zhu (1989) studied the ploidy of P. mairei and P. obovata, finding that diploid (2n = 2x = 10) and tetraploid (2n = 4x = 20) species exist in China. Hong et al. (1988) and Sang et al. (2004) found that P. veitchii and P. anomala are both diploid (2n = 2x = 10). According to Zhang and Shao (2000), P. lactiflora and P. obovata are diploid (2n = 2x = 10), and Li and Xu (1990) found that ‘Huang Jin Lun’ and ‘He Bao’, two cultivars of the LG, are diploid (2n = 2x = 10). Few researchers have focused on the study of different peony cultivar groups. Only Ma and Yu (2013) reported two triploid (2n = 3x = 15) cultivars, Buckeye Belle and Red Red Rose, which belong to the HG.
There is some research on the karyotype of peony and tree peony species or cultivars. When the arm ratio is between 0.1 and 0.5, using the ratio of the longest chromosome to the shortest one, the karyotype of a cultivar can be judged. When the ratio is less than 2:1, it is referred to as 2A, but when the ratio is between 2:1 and 4:1, it is referred to as 2B (Stebbins, 1971). The karyotype of peony is 2n = 2x = 6m + 2sm + 2st; i.e., most peonies belong to 2A (Sang et al., 2004), whereas only Hong et al. (1988) and Liu (2016) found 2B among a population of P. obovata and a hybrid of ‘Zhu Sha Pan’ and ‘Cream Delight’, respectively. All tree peony species are diploid (Zhao, 2014), but Li and Zhang (1982) found that one cultivar, Shou’An Hong, is triploid (2n = 3x = 15). The karyotype of tree peony is usually 2n = 2x = 10 = 6m + 2sm + 2st, 2n = 2x = 10 = 8m + 2st, 2n = 2x = 10 = 8m + 2sm, or 2n = 2x = 10 = 5m + 2sm + 3st but all are 2A (Hou et al., 2006).
Karyotype analysis, which is the comparison of chromosome measurements, is a traditional cytogenetic step to compare genomes among related species. In this study, a karyotypic analysis of the chromosomes of 21 non-Chinese cultivars of herbaceous peony is reported. The characteristics and variation in chromosome number and karyotype have also been analyzed. Cytological data of these cultivars will allow better guidance of future breeding work.
Materials and Methods
Plant materials.
The herbaceous peonies used in this study (Table 1) were planted at the Xiaotangshan cultivation base, Beijing Forestry University, National Engineer Research Center for Floriculture, Beijing, China.
Information about the group, flower color, and parents of the 21 herbaceous peony cultivars that were used in the study of variation in ploidy and karyological diversity.
Methods.
Actively growing root tips (1–3 cm) were cut and pretreated with cycloheximide for 6–8 h in the dark, fixed in Carnoy’s solution (100% ethanol:acetic acid = 3:1) for 24 h, and then stored in ethanol at 4 °C until use. Root tips were hydrolyzed in 1 M HCl at 60 °C for 10 min, washed with distilled water three times and stained by improved carbol fuchsin for 5–10 min. Karyokinetic observations were made and documented by Leica Application Suite version 3 (Leica Microsystems, Wetzlar, Germany). All chemicals (100% ethanol, acetic acid, HCl, and carbol fuchsin) were purchased from Lanyi Co. (Beijing, China). Chromosome measurements were made using five well-spread metaphase plates per population. Karyotype analysis was performed using the criteria described by Chen et al. (2003) and Li and Zhang (1982). Karyotype symmetry was classified according to Stebbins (1971) as shown in Table 2 in this article. The AI percent (long arm length/total genome length × 100%) was determined according to the calculation defined by Arano (1963). The index of relative length [IRL (chromosome length/mean genome length)] and composition of the relative length of the genome [CRL (chromosome length/genome length)] were calculated according to Kuo et al. (1972). The resulting classification is compiled in Table 2. Chromosome morphology was determined using arm ratio (long arm/short arm). Accordingly, chromosomes were 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 longest to shortest chromosome (Lt/St) and centromere index percent (short arm length/chromosome length × 100%) were also calculated. Another AI to measure karyotype asymmetry proposed by Paszko (2006) was also used in this study. The AI is defined as the product of a component expressing the relative variation in chromosome length (CVCL) and a component expressing the relative variation in centromeric index (CVCI). Relationships between these parameters are summarized by the following equation: AI = CVCL × CVCI/100.
Classification of karyotypes in relation to their degree of asymmetry according to Stebbins (1971) and relative chromosome length of 21 herbaceous peony cultivars.
Results
Chromosome number of 21 cultivars.
The metaphase chromosomes and karyotypes of each studied species are shown in Figs. 1 and 2, respectively. Karyotypes of the studied species are compared in Table 3. No aneuploidy was observed in any species. The basic chromosome number, which is reported for the first time for these 21 cultivars, is x = 5 although they have different ploidies. The cultivars in the LG were diploid with 10 chromosomes (2n = 2x = 10), the IG cultivars were triploid with 15 chromosomes (2n = 3x = 15), and the HG had diploid, triploid, and tetraploid cultivars. ‘Little Red Gem’ was diploid with 10 chromosomes (2n = 2x = 10), ‘Old Faithful’, ‘Roy Pehrson’s Best Yellow’ and ‘Scarlet O’Hara’ were tetraploids with 20 chromosomes (2n = 4x = 20), whereas the remaining HG cultivars were triploid with 15 chromosomes (2n = 3x = 15).
Chromosome karyotypes of 21 peony cultivars that were used in a study of variation in ploidy and karyological diversity. The ploidy of 21 herbaceous peony cultivars can be clearly seen in each individual figure. The order of letters correspond to the letters in Table 1 (A–U); scale = 10 μm; (A) karyotype of the lactiflora group cultivars; (B–F) karyotype of the itoh group cultivars; (G–U) karyotype of the hybrid group cultivars; (A, P) diploid karyotype; (B–O, Q, S) triploid karyotype; and (R, T, U) tetraploid karyotype.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04015-17
Chromosome idiograms of 21 peony cultivars that were used in the study of variation in ploidy and karyological diversity. The relative length of the short and long arm of the chromosome and classification of karyotypes in relation to their degree of asymmetry according to Stebbins (1971) are clearly shown. The order of letters corresponds to the letters in Table 1 (A–U); scale = 10 μm. x-axis: Five pairs of chromosome idiograms of 21 peony cultivars. The first, second, and third pairs of chromosome idiograms of each peony cultivar are metacentric (m), and the arm ratio of each chromosome is from 1.01 to 1.70; the fourth pair of chromosome idiograms of each peony cultivar is submetacentric (sm), and the arm ratio of each chromosome is from 1.71 to 3.00; the fifth pair of chromosome idiograms of each peony cultivar is subtelocentric (st), and the arm ratio of each chromosomes is from 3.01 to 7.00. y-axis: chromosome relative length (%) of 21 peony cultivars. Zero on the y-axis represents the location of the centromere, with zero as the boundary. Greater than zero on the y-axis is the relative length (%) of the short arm of the chromosome. Less than zero on the y-axis is the relative length (%) of the long arm of the chromosome.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04015-17
Comparison of the range of six indexes of chromosomes parameters, and three classifications according to ploidy, index of relative length (IRL), and karyotype among 21 herbaceous peony cultivars numbered from A to U (Table 1) that were used in a study of variation in ploidy and karyological diversity.
Karyotype parameters of 21 cultivars.
The karyotypes of the studied taxa are reported here for the first time. The chromosomes of all cultivars were composed of three sections of m chromosomes, one section of sm chromosomes, and one section of st chromosomes. As Figs. 1 and 2 and Table 2 show, the IRL of the studied cultivars varied considerably. The first pair of chromosomes from ‘Cytherea’ (HG) had the longest relative total length of 26.29%, while that of ‘Lovely Rose’ (HG) had the shortest relative total length of 23.00%. With a relative total length of 16.98%, the last pair of chromosomes from ‘Henry Sass’ (HG) was the longest and that from ‘Cytherea’ (HG) was the shortest with a relative total length of 14.13% among all the studied species. The genomes of ‘Henry Sass’ (HG), ‘Border Charm’ (IG), ‘Going Bananas’ (IG), ‘Old Rose Dandy’ (IG), ‘Lemon Dream’ (IG), ‘Chalice’ (HG), ‘Etched Salmon’ (HG), ‘Fairy Princess’ (HG), ‘Henry Bockstoce’ (HG), ‘Joker’ (HG), ‘Roy Pehrson’s Best Yellow’ (HG), and ‘Scarlet O’Hara’ (HG) were one of two chromosome types, a medium long chromosome (M1) or a medium short chromosome (M2). The genomes of ‘Prairie Charm’ (IG), ‘Carina’ (HG), ‘Command Performance’ (HG), ‘Little Red Gem’ (HG), ‘Many Happy Returns’ (HG), and ‘Prairie Moon’ (HG) were composed of three chromosome types: long chromosomes (L), M1, and M2. The genomes of ‘Cytherea’ (HG), ‘Lovely Rose’ (HG), and ‘Old Faithful’ (HG) were composed of four types of chromosomes: L, M1, M2, and short (S).
Only 2A symmetry was found, and the karyotypes of the studied species were somewhat symmetric. The AI varied from 59.61% [‘Cytherea’ (HG)] to 64.03% [‘Little Red Gem’ (HG)]. The mean arm ratio ranged from 1.71 [‘Old Rose Dandy’ (IG)] to 2.17 [‘Lemon Dream’ (IG)]. The AI values varied from 3.06 [‘Henry Sass’ (HG)] to 6.14 [‘Cytherea’ (HG)].
Discussion
Since the 1960s, studies related to chromosome number, karyotype, and genome composition have been widely used in biosystematics research and studies on the phylogenetic and evolutionary relationships among biological taxa (Yang et al., 2014). The basic chromosome number of Paeonia is x = 5, all of them being euploid (Wang, 2010). Polyploidy is an important aspect of evolutionary diversification and is one of the most important cytogenetic mechanisms in plant evolution and rapid speciation (Grant, 1981; Jian et al., 2013; Levin, 2002; Stebbins, 1971). The parent of the LG is P. lactiflora (2n = 2x = 10), so the cultivars of this group are usually diploid; e.g., ‘Henry Sass’. Because there is only one triploid tree peony, ‘Shou’ An Hong’ (Li and Zhang, 1982), due to its triploid status in the IG, the female parent may be tetraploid herbaceous peony, whereas the male parent may be diploid tree peony, but this should be confirmed in a future study. The ploidy range of the HG is very wide, with diploid, triploid, and tetraploid species having been found in this study. For example, ‘Little Red Gem’ is the self-cross of ‘Gwenda F2’, which itself is the cross of P. tenuifolia (2n = 2x = 10) and P. mlokosewitschii (2n = 2x = 10). In this study, ‘Little Red Gem’ was verified to be a diploid. The female parent of ‘Chalice’ is P. lactiflora (2n = 2x = 10), whereas its male parent is P. macrophylla (2n = 4x = 20), and ‘Henry Bockstoce’ is the cross of P. officinalis (2n = 4x = 20) and P. lactiflora (2n = 2x = 10), which were confirmed as triploid in this study.
Except for five cultivars of the IG, the remaining 16 cultivars are the hybrid of herbaceous peony. The parents of the IG usually belong to P. lactiflora and P. lutea (Sun, 2007). The karyotype of herbaceous peony is 2n = 2x = 10 = 6m + 2sm + 2st, and the karyotype of P. lutea is usually 2n = 2x = 10 = 6m + 2sm + 2st, but Gong found that a Ludian population is 2n = 10 = 5m + 2sm + 3st (Gong et al., 1991, 1999; Hou et al., 2006). Most herbaceous and tree peonies belong to 2A (Sang et al., 2004), whereas Hong et al. (1988) and Liu (2016) found cases of 2B among a population of P. obovata and the hybrid of ‘Zhu Sha Pan’ and ‘Cream Delight’, respectively. In this study, however, the karyotypes of all 21 herbaceous peony cultivars were 2n = 2x = 10 = 6m + 2sm + 2st, 2n = 3x = 15 = 9m + 3sm + 3st, 2n = 4x = 20 = 12m + 4sm + 4st, and 2A.
According to Stebbins (1971), the evolutionary trend of karyotypes in the plant kingdom is from symmetry to asymmetry. Thus, relatively primitive plants in systematic evolution have relatively symmetric karyotypes, whereas derivative or more advanced plants may have an asymmetric karyotype (Deng et al., 2009, 2011; Stebbins, 1971). In our study, we also used the AI index, a new AI that was developed by Paszko (2006) to give a single value that assesses karyotype asymmetry. The AI index has the advantage of allowing a high degree of precision and sensitivity to assess karyotype asymmetry, and higher values of the AI index are considered to indicate higher levels of karyotypic heterogeneity (Paszko, 2006, Zhang et al., 2013). Generally, the mean of cultivars of the HG (4.12) had higher asymmetry karyotypes than cultivars of the IG (3.97) and the LG (3.06). A cultivar of the LG, ‘Henry Sass’, had the lowest AI index (3.06). Unfortunately, only a single cultivar was used in the LG in this study, so additional studies on a larger pool of cultivars are needed to validate our findings. Only P. lactiflora is involved in the hybridization of cultivars of the LG. However, in the HG and IG, especially the former, different species or cultivars of different groups have been used in the hybridization of these cultivars, such as P. lutea, P. tenuifolia, P. mlokosewitschi, P. macrophylla, P. officinalis, and others. The AI index can indicate an evolutionary trend, and primitive species or cultivars usually have a lower AI index, a plant having a higher AI value would indicate that it is more advanced (Deng et al., 2011; Gao, et al., 2012; Zhang et al., 2013).
Differences among karyotypes and chromosomes can reflect genetic, morphological, and cytological diversity, as shown for studies on Vernonia (Angulo and Massimiliano, 2009), Rosa odorata (Jian et al., 2010), wild Rosa (Jian et al., 2013), and Forsythia (Shen et al., 2015), which have used karyotypes and chromosomes to reflect the genetic diversity of these species or cultivars. The karyotypes of all 21 herbaceous peony cultivars in this study are similar, but their ploidies differ. To establish a future study on evolutionary trends and genetic diversity, chromosome banding, in situ hybridization, and molecular markers should be combined to provide more useful information for a breeding program.
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