全 文 ：RESEARCH ART ICLE
研究论文
The O rigin of Garden Chrysanthemums and Molecular Phylogeny of Den-
dranthema in China based on Nucleotide Sequences of nrDNA ITS ,
trnT-trn L and trnL-trnF Intergenic Spacer Regions in cpDNA
Zhao Huien1　Wang Xiaoquan2＊　Chen Junyu1＊　Hong Deyuan2
1 College of Landscape Archi tecture , Beijing Forestry University , Beijing , 100083
2 Laboratory of S ystematic and Evolutionary Botany , Inst itute of Botany , Chinese Academy of S cience , Beijing , 100086
＊Corresponding author , xiaoq.w ang@ns.ibcas.ac.cn and chenjymc@public.bta.cn.net
基于核糖体 DNA 的 ITS 序列和叶绿体 t rnT-trnL 及 trnL-trnF 基因
间区的菊花起源与中国菊属植物分子系统学研究
赵惠恩1　汪小全2＊　陈俊愉1＊　洪德元2
1北京林业大学园林学院 , 北京 , 100083
2中国科学院植物研究所系统与进化植物学室 , 北京 , 100086
＊通讯作者 , xiaoq.w ang@ns.ibcas.ac.cn and chenjymc@public.bta.cn.net
ABSTRACT
Several sequences were applied to resolve phylogenet ic relationships wi thin Dendranthema and clarify the
origin of garden chry santhemums including sequences of nrDNA ITS , trnT-trn L and trn L-t rn F intergenic
spacer regions in cpDNA.The relationships among the species are so close that the three sequences could only
provide limited info rmation.From the evidence presented , we suggest that:① D.rhmobifolium be the
chlo roplast dono r of D.vest itum (HN)with the resembling morphology and the same trn T/L IGS sequence.
② D.vestitum , a putative ancestor , may be no t the chloroplast dono r of garden chrysanthemums.D.lavand-
uli folium might be the chloroplast dono r of the type population of D.indicum (HN)or the direct chloroplast
donor of the ancient g arden chry santhemum cultivar.③ D.zawaskii might be not the ancestor of garden
chry santhemums.
KEYWORDS
Origin , Garden chrysanthemums , Phylogenetic relationship , Dendranthema , ITS , trn T-L and trnL-F IGSs
1 INTRODUCT ION
Chrysanthemums have been cultivated for at least
1600 years(chen , 1985).During this time , more than
7000 cultivars have been developed , displaying a diverse
range of flower form , flower color , and plant habit
(Heywood et al., 1977;Anderson et al., 1990).
Chrysanthemums today are among the most popular
flower crops in the world and are used as cut flowers ,
potted plants , greenhouse and garden plants , especially in
North America , West Europe , Japan and China(Boase
et al., 1997).
Chrysanthemums are part of a hexaploid species
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Molecular P lant Breeding , 2003 , Vol.1 , No.5/6 , 597—604　
complex and have on average 54 chromosomes
(Dow rick et al., 1966), although counts of 36 ～ 75
are the possible(Langton , 1989).The exact o rigin
of the genomes in this hexaploid species is unknown
(Wolf f et al., 1994), although much w ork has been
done on the origin of garden chry santhemums includ-
ing morphology , crossing experiments , cy tology ,
RAPD and PCR-RFLP (Staf f , 1933;Dow rick ,
1953;Chen , 1957;Chen et al., 1964;Ling et al.,
1983;Li et al., 1983;Chen , 1985;Dai , 1994;Dai
et al., 1998;Kishimoto et al., 1999).
Stapf(1933)and Dow rick (1953)list the an-
cest ral species involved in the development of the gar-
den chrysanthemum in China as D.indicum , D.z-
awadsk ii , D.vesti tum and in Japan D.indicum ,
D.ornatum , D.japonense (Boase et al., 1997).
Chen(1957)and Chen(1985)proposed chry santhe-
mum originated in the middle of China f rom
D.indicum , D.zawadskii and D.vest itum.Some
Japanese scientists have proposed that chry santhe-
mum comes f rom a hybrid between D.indicum var.
procumbens and D.zawadskii var. lati lobum (Fu-
kai et al., 1998).Dai(1998)insisted that it might
derive f rom D.indicum , D.vest itum and D.nan-
kingense.
There is still much work to be undertaken on this
species-rich and horticultural interesting genus.There are
many poorly understood species and the taxonomic studies
are still underw ay(Boase et al., 1997).The phylogeny
of Dendranthema is the premise for probing the origin of
garden chrysanthemum.
Sequences of nrDNA ITS have been almost rou-
tinely applied to phylogenetic studies of angiosperms
at specific and generic levels(Baldw in et al., 1995;
Oberprieler , 2001), and cpDNA sequence variations
are now w idely used to invest ig ate interspecif ic rela-
tionships among angiosperms and other plants , and
the non-coding regions display the highest f requency
of mutations (Taberlet et al., 1991;Gielly et al.,
1994;Oberprieler et al., 2000).
GISH between D.vest itum , D.indicum and
D.×grandi fol ium could not resolve the o rigin of
chry santhemum by Wang et al.(2000).
In the case of chrysanthemum , nrDNA ITS and
cpDNA sequences may be a useful technology since
the three original genomes that compose the present
genome may be homologous to such an extent that a
probe originating from one contributing progenitor
species may hybridize to homologous sequences of
two or three genomes contributing to the total
chrysanthemum genome(Wolf f et al., 1994).
Comparison of data for 20 species in Argyran-
themum and Chrysantheminae indicates that the
cpDNA restriction site approach provided much more
phylogentic information than I TS sequences (Fran-
cisco-Ortega et al., 1997).Therefore an at tractive
opt ion is to apply the sequences of nrDNA ITS and
t rn T-trnL and trn L-trnF IGSs in cpDNA to ascer-
tain the o rigin of g arden chrysanthemums.
2 MATERIALS AND METHODS
2.1 Plant material
Sequences of the ITS region w ere determined
fo r 14 taxa of Dendranthema.This included all five
series in section Dendranthema and section
Chlorochlamys in China , as well as the oldest culti-
vars of f lorists chrysanthemum that could be found.
Sequences of D.coreanum and 4 species f rom other
Anthemideae genera were used as ingroup and out-
group respectively , fo r which I TS sequences were
obtained from Francisco-Ortega et al.(1997)and
Torrell et al.(1999).Outg roup selection w as based
on Bremer &Humphries(1993)and Franciscor-Or-
tega et al.(1997).
Sequences of trn T-t rn L and t rn L-trnF cp
DNA IGSs w ere determined.The 20 accessions used
are detailed in Table 1.
2.2 DNA isolation , PCR amplification , and
Sequencing
Most DNA isolations were f rom leaves collected
f rom plants cultivated in the Mei &Chry santhemum
Garden at Beijing Forest ry University .Plants sam-
pled are in Table 1.The CTAB technique of Rogers
and Bendich(1988)was used fo r DNA isolation from
the material.DNA fragments of the ITS , trnT-trn L
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Molecular Plant Breeding
and trnT-trnF IGS regions were amplified by PCR
using Universal primers for ITS(P2 (primers I TS4)
of White et al.(1990)and P1 (according to Oryza
sativa), trnT-trn L and trn T-t rn F spacer regions
(“a” , “b” , “ f” of Taberlet et al., 1991).
Table 1 List of tax a and source of plant materials
No. taxon 2n collection Locality genBank number
ITS t rnT-tr nL t rnF-L
Sect.Dendranthema
Ser.Indica Tzvel.
1 D.vesti tum (Hem sl.) Ling 6x Moun t Tianzhushan , Anhui province AF314602 AF314621 AF314605
2 D.vesti tum 6x Moun t Funiushan , Henan province AF314622
3 D.rhombifolium Ling et Shih 2x ? Moun t Wushan , Henan province AF314603 AF314623
4 D.indicum (L.)Des Moul 4x Moun t Tianzhushan , Anhui province AF314601 AF314625 AF314606
5 D.indicum 4x Moun t Funiushan , Henan province AF314617
6 D.naktongense (Nakai)Tzvel. 8x ? 2x ? Weichang county , Hebei province AF314595
7 D.chanet ii (Levl.)Shih 4x Moun t Xiaohong , Beijing AF314596
8 D.nankingense 2x Nanjing city , Jiangsu povince AF314604 AF314627
9 D.×grandi f lorum Jinbeidahong Nanjing city , Jiangsu povince AF314598 AF314613
10 D.×grandi f lorum Huanghelou Beihan Park , Bei jing AF314599 AF314614
11 D.×grand if lorum Hangbai Tongxiang county , Zhejiang province AF314597 AF314612
12 D.×grandi f lorum Hanghuang Tongxiang county , Zhejiang province AF314618
13 D.×grand if lorum Lumudan Beihan Park , Beijing AF314615
14 D.×grandi f lorum Yinpantuogui Beihan Park , Beijing AF314619
15 D.×grand if lorum Chuju Nanjing city , Jiangsu povince AF314620
Ser.Oreast ra Shih
16 D.oreastrun (Hance)Ling 6x Moun t C hangbai , Ji lin province AF314591 AF314628 AF314608
Ser.Lavanduli folia S hih
17 D.lavandili foliun (Fisch.ex Trautv.)Ling et Shih 2x Moun t Funiushan , Henan province AF314600 AF314616 AF314607
Ser.Zaw adskiana Tzvel
18 D.zawadskii (Herb.)T zvel. 6x Moun t Huangshan , Anhui province AF314594 AF314626 AF314609
Ser.Glab riuscula Shih
19 D.dichrum Shih 2x Neiqiu count ry , Henbei p rovince AF314592 AF314630 AF314610
Sect.C hlorochlamys S hih
20 D.mongol icum (Ling)Tzvel. 2x Moun t Wulashan , Inn Mongolica AF314593 AF314629 AF314611
Others
21 D.coreanum Francisco-Ortega et al., 1997 L77802
22 Arctanthem um arcticum Francisco-Ortega et al., 1997 L77756
23 Nipponan them um nip ponicum Francisco-Ortega et al., 1997 L77772
24 Ajania paci f ica Francisco-Ortega et al., 1997 L77787
25 Artemisia canariensis Francisco-Ortega et al., 1997 L77740
26 Artemisia scopar ia Torrell et al., 1999 AF045402/AF079953
27 Artemisia scopar ia Beijing AF314624
28 Chrysanthem um segetum Beijing AF314631
The Origin of Garden Chrysanthemums and Molecular Phylog eny of Dendranthema in China
菊花起源与中国菊属植物分子系统学研究　 599　
Amplified products were purified by Geneclean
Kit following the manufacturers specif ications o r
wi th g lass milk after electrophoresis through 1.5%
agarose gel in 1×TAE buffer.
The sequencing primers used are the same as
those used as PCR primers.DNA sequencing w as
performed using an Amplied Biosystems 377 auto-
mated sequencing Ready Reaction Kit follow ing the
suppliers inst ructions.
2.3 Phylogenetic analyses
Published sequences of nrDNA of species of An-
themideae were used to determine the boundaries of the
ITS1 and ITS2 regions.Alignments of obtained se-
quences were performed by Clustal W1.4 (Thompson et
al., 1994).Only minor corrections were necessary to
adjust the output alignment of Clustal W1.4.
Fitch parsimony were performed on sequence data of
each region using PAUP Version 3.1.1 (Swofford ,
1993).Parsimony analyses(branch and bound search)
were also conducted on a combined data matrix of ITS
sequences and trn T-trnL sequences.For this analysis ,
gaps were treated as new states.
3 RESULTS
Leng th variation and base composit ion Aligned
I TS sequences f rom the taxa studied are 480bp long
(256 bp for I TS1 and 224 for I TS2).
The leng ths of ITS sequences of the taxa exam-
ined in Dendranthema are all the same in leng th
(.255 bp for I TS1 and 222bp for ITS2 , see
Figure 1).
A total of 13 variable si tes w as detected within
Dendranthema , only 4 fo r w hich are phylogeneti-
cally info rmative wi thin Dendranthema.So un-
weighted parsimony analysis of the tax a did no t gen-
erate an ideal distinguished consensus tree.
D.oreast rum T T T C G A A C C T G T T
D.d ichrum G C A T A G A C C A C N C
D.mongolicum G T A T A A A C C A C T C
D.zawadskii G T A T A A T T T A C T C
D.naktongense G T A T A A A C C A C T C
D.chanet ii G T A C A G A C C A C T C
D.×grandi folium `Hangbai ju G C A C A A A C C A C N C
D.×grandi folium `Jinbeidahong G N A C A A A C C A C T C
D.×grandi folium `Huanghelou G C A C A A A C C A C T C
D.lavan dul i folium G T A C A A A C C A C T C
D.indicum G C A C A A A C C A C T C
D.vesti tum (Anhui) G T A C A A A C C A C T C
D.rhombi folium G C A C A G A C C A C C C
D.nankingense G N A C A N A C C A C T C
D.coreanum G N A C A A A C C A C T C
Figure 1 The thi rteen variable posi tions in aligned I TS(ITS1 (255bp)and ITS2 (222bp)) sequences f rom some Dendranthema species
The four informative posi tions in D.are in blod
The posi tions w here the species have the same nucleot ides are not show n
Leng th for trnT-trn L IGS region in this study
ranged from a low of 471bp in Dendranthema
dichrum to a high of 487bp in D.vest itum (Anhui
province)(Figure 2).D.dichrum has a unique 8 bp
deletion from 74 ～ 81.Dendranthema vestitum (AH)
has a unique 6 bp insertion because of repeats of TAA
from 116.D.vestitum (Henan province)and D.rh-
ombifolium have a 4 bp insertion because of 3 repreats of
TTTA from 137.The other varietion is the number of
polyA from 227 ～ 241 in the spacer region.The
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Molecular Plant Breeding
number of Dendranthema oreastrum is 15 , Those of
D.vestitum , D.lavandulifolium D.indicum (Henan)
and four cultivars of D.×grandi folium (`Jinbeida-
hong , `Hangbaiju , `Huanghelou , `Lumudan )is
14 , three cultivars of D.×grandi folium (`Hang-
huangju , `Yingpantuogui , `Chuju )13 , D.zawa-
dskii , D.dichrum , D.mongolicum 12 , D.vestitum ,
D.rhombi folium and D.indicum (Anhui)11.No
other variation was observed among the taxa within Den-
dranthema for the trnT-L ICS w ith the exception of a
single point mutation(T-G)at the 269 position.
A total of 2 info rmative sites w ere detected in
partial trn L-trnF sequences f rom Dendranthema
(see Figure 3).
D.×grandi folium `Hangbaiju TATTAACT ------ ---- G (14A) T
D.×grandi folium `Jinbeidahong TATTAACT ------ ---- G (14A) T
D.×grandi folium `Huanghelou TATTAACT ------ ---- G (14A) T
D.×grandi folium `Lumudan TATTAACT ------ ---- G (14A) T
D.lava ndu li folium TATTAACT ------ ---- G (14A) T
D.indicum (Henan) TATTAACT ------ ---- G (14A) T
D.×grandi folium `Hanghuang TATTAACT ------ ---- G (13A) T
D.×grandi folium `Yinpantuogui TATTAACT ------ ---- G (13A) T
D.×grandi folium `C huju TATTAACT ------ ---- G (13A) T
D.vesti tum (Anhui) TATTAACT TAATAA 　 ---- G (14A) T
D.vesti tum (Henan) TATTAACT ------ T TTA　 G (11A) T
D.rhombi folium TATTAACT ------ T TTA　 G (11A) T
D.indicum (Anhui) TATTAACT ------ ---- G (11A) T
D.zawadskii TATTAACT ------ ---- G (12A) T
D.nankingese TATTAACT ------ ---- G (12A) T
D.oreast rum TATTAACT ------ ---- G (15A)G
D.mongolicum TATTAACT ------ ---- N (12A) T
D.d ichrum ------ ------ ---- G (12A) T
Figure 2 The variable positi ons in aligned tr nT-t rnL sequences from some Dendra nthema species (492)
Gaps are indicated by “-”
D.vesti tum (Henan) G　　A
D.indicum G A
D.lava ndu li folium G A
D.zawadskii T G
D.oreast rum G G
D.Mongol icm ????...???? T G
D.d ichrum T G
Figure 3 The informat ive positions in aligned tr nL-tr nF (partial)se-
quences f rom som e Dend ranthema species (257bp for D.mongolicm
and 359bp for others)
Uncertain sites are show n as “ ?”
Phy logenetic analy ses were done using sequence
data of each region , but the informative changes fo r
the three sequences are not enough to generate a
well-defined cluster.
4 DISCUSSION AND CONCLUSION
The ITS data and trnT-trn L IGS data provide
several insights into the origin ofgarden mums.The
very low divergence of the tw o sequences in Den-
dranthema suggest that we should be cautions in us-
ing them to making strong conclusions about the ori-
gin.Fortunately , our discussions can inco rporate the
resul ts f rom previous morphological , crossing experi-
ment , numerical taxonomic , cladistic taxonomic ,
molecular sy stematics (RAPD)and cultural histo ry
studies.We think that these data sets together pro-
vide a strong basis for clarifying the o rigin of garden
chrysanthemums.
The I TS sequences suggest that D.vesticum ,
D.indicum , D.lavanduli fol ium and D.coreaum
are the closest relatives to D.×grandi fol ium .In
addition , the ITS sequence is congruent w ith previ-
ous studies supporting D.vesticum and D.indicum
The Origin of Garden Chrysanthemums and Molecular Phylog eny of Dendranthema in China
菊花起源与中国菊属植物分子系统学研究　 601　
as the ancestors of g arden mums.
Although the ITS nucleotide divergence within
Dendranthema is very low , the divergence between
D.zawaskii and D.×grandi folium is higher than that
between any other wild Dendranthema species in this
study except for that between D.oreastrum and D.×
grandi folium , even higher than that between Ajania ,
Arcthanthemum and D.×grandi folium respectively.
This suggests that D.zawaskii might be not the ancestor
of garden chrysanthemums.
In M t.Huangshan , Anhui province , D.zaw-
ask ii and D.indicum were sympatric to each other
at thei r edges , and in the near Mt.Tianzhushan ,
D.vesticum , and D.indicum were also sympatric
to each o ther at thei r edges.Any hybrids with leaves
resembling D.×grandi folium betw een D.zawa-
ski i and D.indicum could no t be found in their over-
lapping edge in the many times of investigations in
M t.HuangShan ever since 1980.Although artificial
hybrid between the hexaploid(2n=54)D.zawaskii
and the tet raploid (2n=36)D.indicum was made
(Ji , 1987), the F1-hybrid has litt le resemblance to
ancient cultivars of chrysanthemums.
The hypothesis is also supported by RAPD anal-
ysis(Daiet al., 1998).
In the light of all the above evidence , we pro-
pose that D.zawaskii might be not the ancestor of
garden chry santhemums.
The use of non-coding sequences of cpDNA fo r
the establishment of plant phylogenies can lead to in-
correct phylogenetic inferences due to the presence of
numerous length mutational events (Golenberg et
al., 1993).However sequenceing provided useful
phylogenetic information(e.g.the common inserta-
tion of 4bp shared by D.rhmobi fol ium and D.ves-
t itum (Henan province population (HN)).
The phy logenies established here demonstrate
the usefulness of trn T/L IGS of cpDNA to resolve
the o rigin of g arden mums.The t rn T- trnL IGS se-
quence st rongly suggests that D.vest itum , one of
the ancestor universally recognized , may be not the
chlo roplast donor.The sequence also suggests that
D.lavanduli fol ium mightbe the chloroplast dono r
of the type population of D.indicum (HN)o r the
direct chloroplast donor of the ancient garden
chrysanthemum cultivar.
There is other evidences f rom plant geography ,
cultural history , and hybridization coinciding wi th
the hypothesis.It w as repo rted that chry santhemum
was int roduced to Luoyang city f rom Neixiang coun-
ty among the M ounts of Funiushang of Henan in the
Donghan Dynasty (107-125 A.D), and then it w as
cultivated throughout the country (Dai , 1994).Now
the three species are still distributed in M t Fu-
niushan , hybrids w ere reported in this place and
nearby (Nakata et al., 1992;Zhao et al., 1999).
Thus we recommend that the most possibile of the o-
rigin of garden chrysanthemums is that w hen the
unreduced egg of the hybrid o r the egg of the doubled
hybrid betw een D.lavanduli folium (♀) and
D.indicum (♂)accepted the pollen of D.ves-ti-
tum in Henan province , the garden chry santhemum
was formed , o rthe chrysanthemum directly came
from the hybrid betw een D.indicum (♀) and
D.vest itum (♂).
Another question to be addressed in the analysis
of the relationships betw een D.lavanduli folium ,
D.indicum , and D.vesti tum .Tanaka and Tanig-
uchi(1978)proposed that D.indicum , might come
from D.lavanduli folium .Wang et al.(1993)
thought that they are sister species derived from a
common ancestor and it unreasonable to consider one
of them evolved from the other.ITS sequence data
and trn T/L and t rn L/F regions data support the
fo rmer.The number of polyA from 227 posi tion of
the t rn T/L in tw o population of D.indicum in An-
hui and Henan is 11 and 14 respectively .This result
suggests that :① there might be intraspecific vari-
able mutation among populations of the species.②
the species might be multiple origins , the Anhui
poplution (2n =36)might come from the diploid
species(subpopulation)sympatric with each other in
M t.Tianzhu.③ Ling and Shih (1983)proposed
that there ex ists natural hybrid between D.lava-
nduli fol ium and D.indicum .Gene exchanges are
f requent betw een D.lavanduli folium and the
Henan population of D.indicum based on recently
AFLP (Zhou et al., 2002).So the material usedas
602　 　分子植物育种
Molecular Plant Breeding
D.indicum (HN) might be a natural hybrid o r
D.indicum int rog ressed w ith D.lavandul ifolium .
Chen et al.(1996)proposed that the tetraploid
D.indicum might be the donor of two genomes of
D.vestitum.ITS sequence data support this hypothesis.
D.rhmobifolium , resembling D.vestitum (HN)and
the same trnT/L IGS sequence , strongly suggested that
the species was the chloroplast donor of the latter.
D.rhmobifolium and D.indicum sympatric to each
other in Mt Wushan nearby the distribution of
D.vestitum.Thus , we hypothesize that D.vestitum
might be arisen from a doubled hybrid of D.rhmobi-
folium and D.indicum.
The trnT/L sequence data help separated the
taxa in the D.vest itum complex.Dai (1994)pro-
posed that the Anhui population is an original
species , while the Henan population is a variety ;but
Zhao(1995)proposed the reversed hypothesis.The
sequence data supported the later.
Bremer and Humphries (1993)included D.nak-
tongense in D.zawadskii .Ling and Shish (1983)
thought that D.naktongense and D.zawadskii should
be maintained an independent species.Our ITS sequence
data also support the latter.
Finally , the three sequences suggested that the
relations among species of the Dendranthema are
very close , although all the above problems should be
further studied.
In conclusion , we suggest that a definit ive an-
sw er may come from the application of sequences of
ETS and N TS and other no-coding sequences , espe-
cially no-coding sequences of cpDNA , to the plants.
This approach has been successful w ith others(Lin-
der et al., 2000)and such experiments are currently
under w ay by J.Yang (personal communication ,
Peking University).
ACKNOWLEDGEMENT
We w ish to thank the staff at the Laborato ry of
Sy stemat ic and Evolutionary Botany , Inst itute of
Botany , the Chinese Academy of Science , in part icu-
lar Yingxue Sun , Yanqun Shu and Baohua Song fo r
technical assistant;Shih Chu , Yilin Chen , Jingw u
Wang and anonymous people fo r thei r help w ith the
collection of the material used in this study ;Profes-
sor Silan Dai and professor Ji Yang for their interest
and discussion;and Dr.Neil Anderson at University
of Minnesota for his review.
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604　 　分子植物育种
Molecular Plant Breeding