September 2016. Horticultural Plant Journal, 2 (5): 279–283.

Horticultural Plant Journal

Available online at www.sciencedirect.com
The journal’s homepage: http://www.journals.elsevier.com/horticultural-plant-journal

Karyotype Analysis of Gazania rigens Varieties

ZENG Jiashi, WANG Dongxu, WU Yangqing, GUO Xiaoyu, ZHANG Yazhou, and LU Xiaoping *
Gold Mantis School of Architecture, Soochow University, Suzhou, Jiangsu 215123, China
Received 23 May 2016; Received in revised form 2 July 2016; Accepted 2 September 2016
Available online 25 November 2016

Abstract
For studying species origin, systematic evolution and phylogenetic relationship of Gazania rigens, four different G. rigens varieties, with dif-
ferent flower colors, were subjected to chromosome karyotype analysis. The somatic chromosome number in three varieties ‘Hongwen’, ‘Xingbai’
and ‘Richu’ was 2n = 10, while in ‘Zhongguo Xunzhangju’ it was 2n = 20. We speculate that the cardinal number of chromosomes in G. rigens
plants is x = 5, in which case ‘Zhongguo Xunzhangju’ is a tetraploid. The karyotype formulae of ‘Hongwen’, ‘Xingbai’ and ‘Richu’ were 2n = 8m + 2sm,
2n = 8m + 2sm and 2n = 10m respectively. The karyotype formula of ‘Zhongguo Xunzhangju’ was 2n = 18m + 2sm. The asymmetrical karyo-
type coefficients of the four G. rigens varieties ranged from 53.80% to 58.84%. Only ‘Richu’ had a ‘1A’ karyotype, while the others were relatively
symmetric ‘2A’. Karyotype analysis indicates that the three introduced varieties have a close genetic relationship.
Keywords: Gazania rigens; chromosome; karyotype analysis

1. Introduction
Gazania rigens (L.) Gaertn. is a member of the Asteraceae.
It is a herbaceous perennial named for a flower shape resem-
bling a medal. G. rigens is native to South Africa and grows
best in warm, sunny locations. Two types, tufted and decum-
bent, were introduced into China as useful groundcovers (Xie
et al., 2013). They are evergreen with colorful flowers with a
long flower life and flowering season, and have a strong
resistance to drought, heat, poor soils and moderately cold
temperatures. G. rigens is strongly agamogenic, making it easy
to propagate by cuttings, plant division, and tissue culture, and
it rapidly colonizes roadbed landscapes. Li (2011) and Wang
(2012) discussed potential landscape applications. Wang (2013)
and Zhou (2014) noted plant, leaf, flower shape, and flower
color variability of G. rigens.
The cytology of G. rigens is poorly known. Chen et al. (2003)
reported that Gazania was diploid (2n = 20), and its chromo-
some cardinal number was x = 10, but Barkley (2006) suggested
that the chromosome cardinal number was x = 9 (2n = 18). In
the present research, we analyzed chromosome karyotypes of four
Gazania varieties and found that the chromosome number in three
introduced varieties was 2n = 10, and for the variety ‘Zhongguo
Xunzhangju’, the number was 2n = 20. We propose that the chro-
mosome cardinal number in Gazania is x = 5, and that ‘Zhongguo
Xunzhangju’ is a tetraploid.
2. Materials and methods
This research was conducted from June 2013 to September
2014. ‘Zhongguo Xunzhangju’ was cultivated by the Plant Cul-
tivation and Physiology Laboratory, Soochow University, China.
‘Xingbai’, ‘Hongwen’ and ‘Richu’ varieties were introduced from
Japan and were selected for karyotype analysis.
We used the methods of Li et al. (2008) and Zhu et al. (2011).
Root samples were taken when the new root length of cuttings
from the Gazania varieties reached 1–3 cm. Roots were pre-
treated for 20–24 h in ice water, then transferred into Carnoy’s
fluid (ethyl alcohol:glacial acetic acid = 3:1), immobilized for
20–24 h at 4 °C, washed three times with 90% alcohol, then trans-
ferred into 70% alcohol, at 4 °C. Root tips were sliced and
subjected to acidolysis by immersing in 1 mol·L−1 hydrochloric

* Corresponding author.
E-mail address: longzs@suda.edu.cn
Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Ag-
ricultural Sciences (CAAS)
The Chinese version of this paper is published in Acta Horticulturae Sinica. doi:10.16420/j.issn.0513-353x.2015-0301

http://dx.doi.org/10.1016/j.hpj.2016.07.004
2468-0141/© 2016 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences
(CAAS). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
280 ZENG Jiashi et al.

Table 1 Chromosome parameters of 4 G. rigens varieties

Chromosome Hongwen Xingbai Richu Zhongguo Xunzhangju
No. Relative Arm Type Relative Arm Type Relative Arm Type Relative Arm Type
length/% ratio length/% ratio length/% ratio length/% ratio
1 12.24 1.34 m 11.51 1.98 sm 11.40 1.33 m 5.86 1.03 m
2 11.74 1.07 m 11.06 2.14 sm 11.29 1.05 m 5.79 1.06 m
3 10.74 1.19 m 11.48 1.28 m 11.00 1.21 m 5.69 1.09 m
4 10.55 1.42 m 11.41 1.18 m 10.50 1.11 m 5.61 1.27 m
5 10.54 1.27 m 10.30 1.36 m 10.04 1.15 m 5.61 1.25 m
6 9.67 1.39 m 10.01 1.37 m 9.90 1.27 m 5.46 1.07 m
7 9.55 2.05 sm 9.77 1.34 m 9.53 1.11 m 5.43 1.05 m
8 9.51 2.68 sm 9.06 1.17 m 9.17 1.18 m 5.38 1.08 m
9 8.27 1.53 m 7.91 1.17 m 9.01 1.21 m 5.33 1.62 m
10 7.19 1.15 m 7.47 1.57 m 8.15 1.04 m 5.23 1.65 m
11 5.10 1.44 m
12 5.01 1.16 m
13 5.02 2.40 sm
14 4.21 2.62 sm
15 4.74 1.02 m
16 4.62 1.19 m
17 4.50 1.15 m
18 4.16 1.32 m
19 3.77 1.07 m
20 3.49 1.13 m

acid for 8–10 min using a Thermostatic Water Bath at 60 °C,
removed and washed three times with pure water (with each
washing lasting 1–2 min). Roots were then dyed with aceto-
carmine for 5–10 min, and 1–2 mm slices of root tip were placed
on microscope slides and covered with coverslips. The bubble
inside was extruded by tapping slightly. It was then dried with
an alcohol lamp 1–2 times, and then observed and photo-
graphed using a Leica DM300 microscope.
About 40–50 clearly viewable cells of each variety were ob-
served at the metaphase. We selected cells whose number of
consistent chromosomes exceeded 85%. We photographed root
tip cells, at mitosis metaphase, that had well-separated chromo-
somes and a clear centromere. Chromosomes were measured and
matched using Photoshop CS 5.0 software and their relative
length, arm ratio, chromosome length ratio were calculated (Zhou
et al., 2009; Lu et al., 2013). The Li and Chen (1985) method
was used for karyotype analysis and the Stebbins (1971) method
was used for karyotype classification. The karyotype asymme-
try coefficient (As. K, %) was defined as (length of long arm/
length of the entire chromosome) × 100 (Arano, 1963).
3. Results
Metaphase karyotype analysis of root tip cells showed that
centromeres were mainly located at the middle (m) or near the
middle (sm) of chromosomes. There was little variation of chro-
mosome length. The number of chromosomes with length ratios
of 1.40–1.70, and arm ratios >2:1, accounted for 0–20.00% of
the total and their asymmetrical karyotype coefficients were
53.80%–58.84%. In the karyotypes of the four G. rigens vari-
eties, the chromosomes are arranged in descending order (Fig. 1,
Table 1 and Table 2).
The chromosome number of ‘Hongwen’ was 2n = 10. The
karyotype formula was 2n = 8m + 2sm, of which the fourth pair
of chromosomes was the sm type (mean arm ratio = 2.34) (Fig. 1,
Table 1). The ratio of the longest and shortest chromosome was
1.70, frequency of chromosomes with arm ratio >2 was 20.00%,
and the karyotype was ‘2A’ (Table 2).
The chromosome number of ‘Xingbai’ was 2n = 10. The karyo-
type formula was 2n = 8m + 2sm. The first pairs of chromosomes
were type sm (mean arm ratio = 2.06) (Fig. 1, Table 1). The asym-
metrical karyotype coefficient was the maximum (58.84%) and
the karyotype was ‘2A’ (Table 2).
The chromosome number of ‘Richu’ was 2n = 10. All chro-
mosomes were type m, and the karyotype formula was 2n = 10m
(Fig. 1, Table 1). The ratio of the longest and shortest chromo-
some, asymmetrical karyotype coefficient, and percentage of
chromosomes exceeding 2:1 of the arm ratio were respectively
1.40%, 53.80% and 0. The karyotype was ‘1A’ (Table 2).

Table 2 Karyotype characteristics of 4 G. rigens varieties

Variety Chromosome Centromere location Chromosome Asymmetrical Percent with Arm Karyotype
number m sm length ratio karyotype arm ratio >2 index classification
(longest/shortest) coefficient/%
Hongwen 10 8 2 1.70 58.80 20.00 20 2A
Xingbai 10 8 2 1.54 58.84 10.00 20 2A
Richu 10 10 1.40 53.80 0 20 1A
Zhongguo Xunzhangju 20 18 2 1.68 55.86 10.00 40 2A
Karyotype Analysis of Gazania rigens Varieties
Fig. 1 Metaphase chromosome maps (left), karyogram (middle) and ideogram (right) of 4 G. rigens varieties
The chromosome number of ‘Zhongguo Xunzhangju’ was
2n = 20. The seventh pair of chromosomes was type sm (mean
arm ratio = 2.50), and the karyotype formula was 2n = 18m + 2sm
(Fig. 1, Table 1). Length ratio of the chromosome was 1.68.
The number of chromosomes with more than 2:1 of arm ratio
accounted for 10.00% of the total, the asymmetrical karyotype
coefficient was 55.86%, and the karyotype was ‘2A’ (Table 2).
4. Discussion
Chen et al. (2003) suggested that the chromosome cardinal
number of Gazania was x = 10, karyotype formula was
281
2n = 2x = 18m + 2sm, and the karyotype was ‘2A’, which
corresponded to the karyotype of ‘Zhongguo Xunzhangju’ in
this study. However, our results show that somatic cells of
three G. rigens varieties, introduced from Japan, had ten
chromosomes. It is possible that the chromosome cardinal
number of Gazania was x = 5, and ‘Zhongguo Xunzhangju’ is
a tetraploid.
The chromosome morphology differed among the varieties.
The main chromosome type was m, and in addition, ‘Hongwen’,
‘Xingbai’ and ‘Zhongguo Xunzhangju’ each had a pair of sm
chromosomes. Based on karyotype characters, only ‘Richu’ was
a ‘1A’ type, while the other three varieties were the ‘2A’ type
282
with a relatively symmetrical karyotype. The diversity of karyo-
types corresponds to the phenotypic variation in these G. rigens
varieties.
We found poor germination in the filial generation of
‘Zhongguo Xunzhangju’ and the other Japanese Gazania. This
may be related to the sterility caused by a previous hybridiza-
tion event or low triploid fertility.
A major component of evolution involves the quantitative
and structural variation of chromosomes (Li et al., 1983). Li
et al. (1983) and Wang et al. (1991) suggested that the cardinal
number of chromosomes in wild species of the genus
Dendranthema was x = 9, which exhibited euploidy variation
with most species being tetraploid and hexaploid. The cultivars
of this genus mostly had aneuploid variation based on hexa-
ploidy. Li et al. (2007) researched the karyotypes of fourteen
chrysanthemum varieties. The different cultivars were all aneu-
ploids with chromosome numbers between 48 and 56 compared
to hexaploid (2n = 54). Zhu et al. (2011) found that the vast
majority of traditional Chinese chrysanthemum varieties were
hexaploid or aneuploid based on hexaploid, while a few variet-
ies had aneuploid variation based on septuploidy and octoploidy.
In addition, the ascending or descending order of aneuploid varia-
tion in plant chromosome cardinal numbers led to quantitative
and structural variation of chromosomes, and played a signifi-
cant role in intraspecific differentiation and speciation (Li, 1988).
Wang and Li (1987) found that evolution of Sanvitalia was af-
fected by ascending aneuploid variation on cardinal chromosomes.
The original cardinal number in Sanvitalia was x = 9 and there
were secondary cardinal numbers, x = 12 and x = 13. Zhang
and Gu (2005) pointed out that there was descending aneu-
ploid variation on cardinal chromosomes of Streptopus. In addition
to two known cardinal numbers (x = 8 and x = 9), a new one,
x = 7, was found. And it seemed that the cardinal number of
x = 7 was merged by the centromeres of a pair of large m-type
chromosomes in genome of x = 8. Chu and Sun (1986) consid-
ered that the chromosome cardinal number of Morus was x = 14,
which was widely cited (Yu, 1986; Yu et al., 1996; Guo and
Yu, 2000). However, He et al. (2013), on a whole genome se-
quencing study of Morus notabilis, found that chromosome
cardinal number of this genus should be x = 7. Li et al. (2014)
showed that there were fourteen chromosomes in the somatic
cells of Morus yunnanensis. Therefore, our study suggests that
G. rigens introduced from Japan may be the original type of
the Chinese varieties, with relatively simple flower shape and
color, and lower ploidy (2n = 2x = 10). Most Chinese varieties
have large, colorful and complex flowers, and ploidy values of
2n = 4x = 20; these existing varieties were formed by artificial
selection after natural variation and cross-pollination.
It is not uncommon to see plant chromosome cardinal numbers,
ascending or descending, produced by aneuploid variation among
species within a genus. There are derivative or secondary car-
dinal numbers of aneuploid variation based on original cardinal
number in the Asteraceae. Barkley (2006) determined that the
chromosome cardinal number of G. rigens was x = 9 (2n = 18),
which may be generated by descending aneuploid variation based
on a tetraploid species. In contrast, Chen et al. (2003) sug-
gested that the chromosome cardinal number of Gazania was
ZENG Jiashi et al.
x = 10, but this might not be the original cardinal number of this
genus. Specification of the true Gazania chromosome cardinal
number clearly needs further research.
Acknowledgments
This work was financially supported by Project for Suzhou
Science and Technology Support Program (Agriculture)
(SNG201409).
References
Arano, H., 1963. Cytological studies in subfamily Carduoideae (Compositae)
of Japan. IX. Bot Mag (Tokyo), 76: 32–39.
Barkley, T.M., 2006. Flora of North America. Oxford University Press, New
York, 19: 196.
Chen, R.Y., Song, W.Q., Li, X.L., Li, M.X., Liang, G.L., Chen, C.B., 2003.
Chromosome atlas of major economic plants genome in China, vol. III.
Science Press, Beijing, pp. 207–208.
Chu, R.Y., Sun, X.X., 1986. The studies on cytogenetics of plants of the genus
Morus: I. The number of chromosome of some mulberry varieties. Acta
Sericol Sin, 12: 199–202. (in Chinese)
Guo, H.R., Yu, M.D., 2000. The study of karyotype of cell chromosome of
artificial triploid mulberry variety 9204. Acta Sericol Sin, 26: 65–70. (in
Chinese)
He, N.J., Zhang, C., Qi, X.W., Zhao, S.C., Tao, Y., Yang, G.J., Lee, T.H., Wang,
X.Y., Cai, Q.L., Dong, L., Lu, M.Z., Liao, S.T., Luo, G.Q., He, R.J., Tan,
X., Xu, Y.M., Li, T., Zhao, A.C., Jia, L., Fu, Q., Zeng, Q.W., Gao, C., Ma,
B., Liang, J.B., Wang, X.L., Shang, J.Z., Song, P.H., Wu, H.Y., Fan, L., Wang,
Q., Shuai, Q., Zhu, J.J., Wei, C.J., Zhu, S.K., Jin, D.C., Wang, J.P., Tao, L.,
Yu, M.D., Tang, C.M., Wang, Z.J., Dai, F.W., Chen, J.F., Liu, Y., Zhao, S.T.,
Lin, T.B., Zhang, S.G., Wang, J.Y., Wang, J., Yang, H.M., Yang, G.W., Wang,
J., Paterson, A.H., Xia, Q.Y., Ji, D.F., Xiang, Z.H., 2013. Draft genome
sequence of the mulberry tree Morus notabilis. Nat Commun, 4: 2445.
http://www.nature.com/ncomms/2013/130919/ncomms3445/full/ncomms3445
.html.
Li, C., Chen, F.D., Zhao, H.B., Chen, S.M., 2008. Karyotype diversity of 17
chrysanthemum cultivars with small inflorescences. Acta Hortic Sin, 35:
71–80.
Li, C., Chen, F.D., Zhao, H.B., Fang, W.M., 2007. Karyomorphology of fourteen
cultivars in cut chrysanthemum. Acta Hortic Sin, 34: 1235–1242.
Li, M.X., 1988. Different euploidy variation and evolution of chromosome
cardinal number on plants. Bull Biol, 7: 7–12. (in Chinese)
Li, M.X., Chen, R.Y., 1985. A suggestion on the standardization of karyotype
analysis in plants. Plant Sci J, 3: 297–302. (in Chinese)
Li, M.X., Zhang, X.F., Chen, J.Y., 1983. Cytological studies on some Chinese
wild Dendranthema species and chrysanthemum cultivars. Acta Hortic Sin,
10: 199–205.
Li, Y., Xuan, Y.H., Wang, S., Zeng, Q.W., Xiang, Z.H., He, N.J., 2014. Karyotype
analysis of Morus yunnanensis. Sci Sericult, 40: 4. (in Chinese)
Li, Y.F., 2011. Study on introduction and application of ground-cover plant
Gazania rigens L. in Suzhou area [M. D. Dissertation]. Soochow University,
Suzhou. (in Chinese)
Lu, Y.F., Jiang, J.X., Yi, Z.L., 2013. The protocol for processing of the
chromosomal images of Miscanthus for karyotype analysis using Adobe
Photoshop software. Pratacult Sci, 30: 922–926. (in Chinese)
Stebbins, G.L., 1971. Chromosomal evolution in higher plants. Edward Arnold,
London, pp. 87–93.
Wang, J.W., Yang, J., Li, M.X., 1991. Karyotype study of five species of Chinese
Dendranthema. Acta Bot Yunnanica, 13: 411–416. (in Chinese)
Wang, W., 2013. Studies on sexual reproduction biology and progenies phenotype
analysis of Gazania rigens L. [M. D. Dissertation]. Soochow University,
Suzhou. (in Chinese)
Wang, X.L., Li, M.X., 1987. Observation of chromosome on 10 Compositae
species. Plant Sci J, 5: 111–118. (in Chinese)
Karyotype Analysis of Gazania rigens Varieties
Wang, Y.L., 2012. Studies of resistance physiology and landscape application
of ‘Xingbai’ Gazania [M. D. Dissertation]. Soochow University, Suzhou. (in
Chinese)
Xie, L.M., Hu, J.X., Huang, W.C., 2013. Discussion on cutting and rapid
propagation technology of Gazania rigens L. Jiangsu Agric Sci, 41: 167–168.
(in Chinese)
Yu, M.D., 1986. Study on chromosome ploidy of mulberry. Newsl Sericult Sci,
(4): 6. (in Chinese)
Yu, M.D., Jing, C.J., Chen, W., Li, Q.R., Liao, W.M., Dai, C.X., Zhao, H., Pan,
J., Liu, M.X., Li, W., 1996. Studies on chromosome ploidy of some
germplasms maintained at mulberry resources centre of Sichuan Province.
Newsl Sericult Sci, 16: 2–5. (in Chinese)
283
Zhang, T., Gu, Z.J., 2005. A new basic chromosome number of x = 7
for the genus Streptopus (Liliaceae). Acta Phytotaxon Sin, 43: 533–538. (in
Chinese)
Zhou, J.S., Su, X.B., Tang, Y.P., Luo, S.C., Zhan, F.X., Chen, G.Y., 2009. Analysis
of chromosomal karyotype of Asparagus by use of Software Photoshop. Acta
Agric Jiangxi, 21: 73–75. (in Chinese)
Zhou, X.H., 2014. Studies on new cold resistant cultivars breeding and low carbon
cultivation techniques of Gazania rigens L. [M. D. Dissertation]. Soochow
University, Suzhou. (in Chinese)
Zhu, M.L., Liu, Q.Q., Dai, S.L., 2011. Karyotype analysis of 38 large-flowered
chrysanthemum cultivars from China. Chin Bull Bot, 46: 447–455. (in
Chinese)