全 文 ：利用流式细胞仪测定荚蒾属植物的基因组大小
*
张宁宁1，2，杨 静1，孙卫邦1＊＊
(1 中国科学院昆明植物研究所昆明植物园，云南 昆明 650201;2 中国科学院大学，北京 100049)
摘要:荚蒾属 (Viburnum)植物在园林中广泛用作观赏灌木，并且其具有优良园艺性状的杂交种在世界范
围内越来越受欢迎。本研究利用流式细胞仪测定了 14种荚蒾属植物的基因组大小。二倍体中基因组大小
变化范围是 2. 55 pg (陕西荚蒾)到 4. 26 pg (琼花)。同时，琼花的核型也较不对称，这可能反应了它的
育种历史。四倍体物种珊瑚树的基因组大小 (7. 62 pg)是其他二倍体物种的两倍还多，这揭示了该属的
多倍化在进化中可能并不遥远。该研究为荚蒾属细胞遗传学和分类学的深入研究奠定了基础，并为该属杂
交育种提供有用的信息。
关键词:荚蒾属;流式细胞仪;基因组大小;变异;染色体
中图分类号:Q 75 文献标识码:A 文章编号:2095－0845(2014)06－730－07
Genome Size Estimation of Viburnum (Adoxaceae)
Species by Using Flow Cytometry*
ZHANG Ning-Ning1，2，YANG Jing1，SUN Wei-Bang1＊＊
(1 Kunming Botanical Garden，Kunming Institute of Botany，Chinese Academy of Sciences，Kunming 650201，China;
2 University of Chinese Academy of Sciences，Beijing 100049，China)
Abstract:Viburnum species are widely used as ornamental shrubs in gardens and their hybrids with brilliant horti-
cultural traits are increasingly popular worldwide． In this study，genome sizes of fourteen Viburnum species were esti-
mated by using flow cytometry． Genome size for diploids ranged from 2. 55 pg in V. schensianum to 4. 26 pg in
V. macrocephalum form． keteleeri． And V. macrocephalum form． keteleeri also had a more asymmetrical karyotype
which may reflect its breeding history． The genome size of tetraploid species V. odoratissimum (7. 62 pg)was unusu-
ally more than twice as big as those of other diploid species，which revealed that polyploidization in this genus may
be not remote in its evolution． These results lay a foundation for further research in the cytogenetics and taxonomy of
Viburnum and provide useful insight into future hybridization efforts for this genus．
Key words:Viburnum;Flow cytometry;Genome size;Variation;Chromosome
Viburnum，a genus of Adoxaceae，containing
approximately 175 species of shrubs and small trees，
distributes most widely in Northern Hemisphere with
major centers of species diversity in eastern Asia
and Latin America (Ｒehder， 1908; Killip and
Smith，1931;Morton，1933;Kern，1951;Dono-
ghue，1983;Hara，1983)． Ｒemarkable variation in
features such as endocarp shape，fruit color，leaf
morphology， bud morphology， and inflorescence
form distinguish Viburnum into ten traditionally rec-
ognized sections，namely sections Lentago，Mega-
lotinus，Odontotinus，Opulus，Oreinotinus，Pseudot-
inus，Solenotinus，Tinus，Tomentosa and Viburnum
(Oersted，1861;Hara，1983)．
植 物 分 类 与 资 源 学 报 2014，36 (6):730～736
Plant Diversity and Ｒesources DOI:10．7677 /ynzwyj201414022
＊
＊＊
Funding:中国科学院知识创新工程重要方向项目“西南地区重要植物类群分类、评价与挖掘”(KSCX2-EW-J-24)
Author for correspondence;E-mail:wbsun@mail. kib. ac. cn
Ｒeceived date:2014－02－07，Accepted date:2014－04－24
作者简介:张宁宁 (1990－)硕士生，研究方向:植物引种驯化与保护生物学。E-mail:zhangningning@mail. kib. ac. cn
Viburnum species are widely used as ornamental
shrubs in gardens and hybrids with brilliant horticul-
tural traits are increasingly popular worldwide．
Though viburnums are varied in form，the ideal or-
namental that combines fragrance，colored flowers，
small stature，evergreen or brilliantly colored foli-
age，and luxuriant fruit does not exist (Egolf，
1962)． Hybridization between related species could
achieve features in needs，so it is important to obtain
more genetic and taxonomic information about the
genus．
DNA C-value，the haploid nuclear DNA con-
tent，is correlated with many biological characteris-
tics，such as cell and nuclear volume，size of the
chromosomes，and duration of male meiosis (Ben-
nett，1987)． Moreover，variation in genome size is
commonly observed in species within the same genus
in various groups of plants (Wong and Murray，
2012;Ｒussell et al．，2013)． The study of genome
size is a powerful tool for understanding taxonomy
and evolutionary history within a genus． To date，on-
ly three species of Viburnum have been investigated
for genome size． Haploid(1C)values of V. bitchiuense，
V. lantana and V. opulus were reported as 3. 80 pg，
4. 04 pg and 4. 15 pg respectively (Olszewska and
Osiecka，1984;Siljak-Yakovlev et al．，2010)．
In this investigation，we estimated the genome
sizes of fourteen Viburnum species using flow cytome-
try as basic information for further research，com-
pared the variation of genome sizes in Viburnum，
and explored the relationship between genome sizes
and chromosomes．
1 Materials and methods
1. 1 Plant materials
Fourteen Viburnum spp． from the collections of
Kunming Botanical Garden (KBG)were included in
this investigation． V. hupehense and V. odoratissimum
were cuttings introduced from Chengdu Botanical
Garden (CBG)． Wild seeds of V. congestum and
V. schensianum were collected from Kangding (Si-
chuan，China)and Yangcheng (Sichuan，China)
respectively． Seeds of Glycine max‘Williams 82’
and Zea mays‘B73’were kindly provided by Insti-
tute of Crop Science，Chinese Academy of Agricul-
tural Sciences． All seeds were germinated and grown
in KBG． The investigated species，somatic chromo-
some numbers (2n)，ploidy level and source loca-
tions are listed in Table 1． The somatic chromosome
numbers and ploidy level are based on previous cyto-
logical studies and unpublished data (Egolf，1962)．
Table 1 Somatic chromosome numbers (2n)，ploidy level
and locations of the investigated species
Section and species 2n Ploidy level Location
Section Odontotinus
V. betulifolium 18 diploid KBG
V. foetidum
var． ceanothoides 18 diploid KBG
V. hupehense 18 diploid CBG
Section Solenotinus
V. corymbiflorum 18 diploid KBG
V. odoratissimum 32 tetraploid CBG
V. odoratissimum
var． awabuki — — KBG
V. taitoense 18 diploid KBG
Section Tomentosa
V. plicatum 16 diploid KBG
Section Viburnum
V. carlesii 18 diploid KBG
V. congestum 18 diploid Kangding，Sichuan
V. macrocephalum
form． keteleeri 18 diploid KBG
V. rhytidophyllum 18 diploid KBG
V. schensianum 18 diploid Yangcheng，Sichuan
V. utile 18 diploid KBG
1. 2 Measurement of genome size by flow cy-
tometry
Nuclear DNA content estimation was performed
by flow cytometry on fresh leaf tissue，using a proto-
col developed by Doleel et al． (2007)． Young leav-
es of Viburnum samples (one to three individual plants
per species，depending on the number of available
plants)and a reference standard［To ensure the ac-
curacy of the results，Zea mays‘B73’(2. 35 pg /1C)
1376期 ZHANG Ning-Ning et al．:Genome Size Estimation of Viburnum Species by Using Flow Cytometry
(Schnable et al．，2009)was used as the reference
standard of V. odoratissimum and V. odoratissimum
var． awabuki，on account of rather great disparity of
genome size between tetraploid species and Glycine
max‘Williams 82’(1. 13 pg /1C)(Schmutz et al．，
2010)． The other species were with Glycine max
‘Williams 82’．］ were co-chopped with a razor
blade in a petri dish containing 0. 5 mL ice-cold Gal-
braith’s buffer (45 mmol·L－1 MgCl2，20 mmol·L
－1
MOPS，30 mmol·L－1 sodium citrate，0. 1% Triton
X-100，pH= 7. 0)and 0. 5 mL ice-cold 1% PVP-10
(Galbraith et al．，1983)． The nuclei suspension was
filtered through a 48 μm nylon mesh，and then
2. 5 μL ＲNase (10 mg·mL－1)and 250 μL propidi-
um iodide (0. 2 mg·mL－1)solution were added． Af-
ter incubation for 30 min at 0 ℃ in darkness，all the
homogenates were analyzed based on lightscatter and
fluorescence signals produced from 20 mW laser illu-
mination at 488 nm using a Accuri C6 flow cytome-
ter． At least 5 000 nuclei were collected in each
measurement and repeated at least three times for
each of the species．
The genome size was calculated as the ratio be-
tween sample and standard peaks multiplied by the
genome size of the standard． Differences in genome
size among species were tested by one-way analysis
of variance (ANOVA)，and multiple comparison
tests were determined by the Tukey HSD test (P≤
0. 05)with SPSS (Statistical Package for the Social
Sciences)v 16. 0 for Windows．
2 Ｒesults
Flow cytometric analysis of Viburnum species
resulted in high-quality histograms． Figure 1 shows
histograms from the mixture of isolated nuclei of
V. foetidum var． ceanothoides，V. plicatum and V. cong-
estum respectively with Glycine max，and V. odorati-
ssimum var． awabuki with Zea mays． The genome si-
zes of fourteen investigated species were calculated．
The mean， standard deviation (SD)，maximum
(Max．)and minimum (Min．)of genome sizes (C
values in pg) ，the mean of nuclear DNA contents
(2C DNA content (Mbp) ) ，and the number of rep-
licates (N)for each species are shown in Table 2．
Table 2 The mean，standard deviation (SD)，maximum (Max．)and minimum (Min．)of genome size (C values;1C /pg) ，
the mean of nuclear DNA content (2C DNA content;Mbp) ，and the number of replicates (N)for each species
Section and species
C values (1C /pg)
Meana SD Max． Min．
2C DNA content
(Mbp)b
N
Section Odontotinus
V. betulifolium 3. 25b 0. 02 3. 29 3. 22 6 357. 00 5
V. foetidum var． ceanothoides 3. 24b 0. 05 3. 35 3. 20 6 337. 44 5
V. hupehense 2. 94a 0. 06 3. 03 2. 86 5 750. 64 4
Section Solenotinus
V. corymbiflorum 2. 66a 0. 01 2. 68 2. 64 5 202. 96 4
V. odoratissimum 7. 62e 0. 13 7. 80 7. 48 14 904. 72 5
V. odoratissimum var． awabuki 7. 32f 0. 14 7. 52 7. 19 14 317. 92 3
V. taitoense 3. 13b 0. 12 3. 23 2. 91 6 122. 28 5
Section Tomentosa
V. plicatum 2. 75a 0. 05 2. 83 2. 69 5 379. 00 5
Section Viburnum
V. carlesii 2. 83a 0. 04 2. 88 2. 78 5 535. 48 5
V. congestum 2. 80a 0. 06 2. 85 2. 72 5 476. 80 3
V. macrocephalum form． keteleeri 4. 26c 0. 18 4. 41 3. 89 8 332. 56 6
V. rhytidophyllum 3. 92d 0. 12 4. 13 3. 82 7 667. 52 6
V. schensianum 2. 55a 0. 08 2. 62 2. 44 4 987. 80 3
V. utile 2. 66a 0. 07 2. 77 2. 55 5 202. 96 5
a Means followed by the same letter were not statistically different according to the multiple comparison Tukey-HSD tests at P≤0. 05;
b 1 pg DNA= 978 Mbp according to Doleel et al． (2003)．
237 植 物 分 类 与 资 源 学 报 第 36卷
Fig. 1 Histograms of relative fluorescence intensity of four Viburnum species
In these species analyzed，the nuclear DNA
content in Mbp ranged from 4 987. 80 Mbp in V. schen-
sianum of section Viburnum to 14 904. 72 Mbp in
V. odoratissimum of section Solenotinus． In diploid
species (2n=16 or 18)，the genome size varied from
2. 55 pg in V. schensianum to 4. 26 pg in V. macroce-
phalum form． keteleeri． The tetraploid species V. odora-
tissimum (2n = 32)contained the highest value of
7. 62 pg，while the variety V. odoratissimum var． aw-
abuki had 1C value of 7. 32 pg． The differences in
genome sizes among these species analyzed were sta-
tistically significant (P≤0. 05)except when compa-
ring V. utile with V. hupehense， V. corymbiflorum，
V. plicatum，V. carlesii，V. congestum or V. schensian-
um，and when comparing V. taitoense with V. betuli-
folium or V. foetidum var． ceanothoides．
3 Discussion
3. 1 Genome size variation in Viburnum
Genome size was currently available for more
than 7 000 plant species，varying from 0. 006 48 pg
in the carnivorous plant Genlisea margaretae to
152. 23 pg in the monocot Paris japonica (Greilhu-
ber et al．，2006;Bennett and Leitch，2010，Pelli-
cer et al．，2010)． Compared to values reported by
Leitch et al． (1998)and Soltis et al． (2003)，the
genome size of analyzed Viburnum species fell into
the categories“small”(1C values＞1. 4－3. 5 pg)to
“medium”(＞3. 5 to ＞14. 0 pg)．
The result of multiple comparison tests showed
that the genome size of diploid species in the same
sections of Odontotinus，Solenotinus and Viburnum
varied significantly． Ｒesearches based on chloroplast
trnK intron and nuclear ribosomal ITS DNA se-
quences revealed that sections Odontotinus，Solenoti-
nus and Viburnum were non-monophyletic (Dono-
ghue et al．，2004) ，which was confirmed by the
study of duplicated nuclear gene GBSSI (Winkworth
and Donoghue，2004)． The differences of genome
sizes in these three sections might indicate the same
3376期 ZHANG Ning-Ning et al．:Genome Size Estimation of Viburnum Species by Using Flow Cytometry
phylogenetic relationships． Further researches were
needed as the absence of other more sections and
species in this study．
3. 2 Ｒelationships between genome size and chr-
omosomes in Viburnum
Among these diploid species，V. macrocephalum
form． keteleeri of section Viburnum possessed the lar-
gest genome size，which was nearly two times that of
V. schensianum in the same section，and it also had
a much more asymmetrical karyotype (based on un-
published data)． This species is a famous historical
ornamental plant in China． The unusually large ge-
nome size may be a result of its genome accumula-
tions through its history of artificial breeding，during
which repetitive sequences increased following the
structural rearrangement of chromosomes．
The genome size of V. plicatum，whose base
chromosome number was x= 8，was not the smallest
among other diploid species with x = 9． Loss of one
pair of chromosomes did not decrease the genome
size of V. plicatum correspondingly，which revealed
that robertsonian translocation of centromeres might
be the main driver of chromosome changes in Vibur-
num． In previous studies，both 2n= 16 and 18 chro-
mosome forms occurred in V. plicatum，which could
not be distinguished by vegetative characteristics，
but the 2n = 16 plants produced the most abundant
fruit． These results provided evidence from the ge-
nomic level for the view proposed by Egolf (1962)
that most of the minor morphological variations in Vi-
burnum that had been given varietal rank were the
result of genic or intrachromosomal changes，rather
than the result of changes in chromosome numbers．
Within the genus Viburnum，there was a posi-
tive relationship between nuclear DNA content and
ploidy level． For example，the genome size of tetra-
ploid V. odoratissimum in section Solenotinus was
2. 43 times that of V. taitoense from the same sec-
tion，implying that the polyploidization process dur-
ing V. odoratissimum evolution was accompanied by
genome size increase． This was in contrast to reports
for other genera where DNA content per haploid ge-
nome in polyploids was found to be less than their
diploid relatives． This phenomenon had been inter-
preted as a balance mechanism to reduce some nu-
cleotypic effects of DNA gain due to polyploidy
(Bennett，1972;Poggio et al．，1989;Murray et
al．，1992;Dimitrova and Greilhuber，2000)． After
the formation of a polyploid，its diploidization would
gradually decrease the genome size by mechanisms
such as DNA elimination or chromosomal rearrange-
ment and so on (Yang，2001)． However，in this
case of Viburnum， that the C-value of tetraploid
V. odoratissimum was more than twice that of other
diploids might indicate this polyploidization hap-
pened not far enough to undergo the process of dip-
loidization． V. odoratissimum var． awabuki had a
similar genome size to V. odoratissimum indicating
that V. odoratissimum var． awabuki was also tetra-
ploid． Prediction of ploidy level by genome sizes is
very informative for hybridization and plant develop-
ment efforts．
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Supplementary materials
5376期 ZHANG Ning-Ning et al．:Genome Size Estimation of Viburnum Species by Using Flow Cytometry
Histograms of relative fluorescence intensity of ten Viburnum species
Note:There were three apparent peaks in the histogram produced by the mixed free nuclei of V. odoratissimum and Zea mays in flow cytometry，
which were the peak of Zea mays diploid，Zea mays tetraploid and V. odoratissimum diploid respectively． This was because young
leaves of Zea mays were in mitosis period，which had two kind nuclei of diploid and tetraploid．
637 植 物 分 类 与 资 源 学 报 第 36卷