Scientific report: "Genetic diversity of seed samples collected from Vietnam wakegi pps

J. Sci. Dev. 2009, 7 (Eng.Iss.1): 22 - 29 HA NOI UNIVERSITY OF AGRICULTURE
22
Genetic Variation of Wakegi Onion (Alllium  wakegi
Araki) in Vietnam
Đa dạng di truyền các mẫu giống hành wakegi thu thập từ Việt Nam
Pham Thi Minh Phuong
1
, Shiro Isshiki
2
and Yosuke Tashiro
2
1
Department of Vegetables, Fruits, Flowers and Ornamental plants,
Faculty of Agronomy, Hanoi University of Agriculture, Gialam, Hanoi, Vietnam
2
Faculty of Agriculture, Saga University, Saga city, Japan
TÓM TẮT
Để đánh giá nguồn gen các mẫu giống hành wakegi (Alllium × wakegi Araki) trong việc lai tạo các
cây họ hành thì đa dạng di truyền trong và giữa các mẫu giống hành wakegi thu thập từ miền Bắc,
Trung và Nam của Việt Nam và hành hoa, hành củ thu thập từ Nhật, Thái Lan đã được nghiên cứu dựa
trên các đặc điểm hình thái, sinh lý và tính đa hình DNA của nhân, tế bào chất và ti thể. Kết quả
nghiên cứu tế bào và phân tích GISH chứng tỏ tất cả các mẫu giống hành wakegi từ Việt Nam là con
lai giữa hành hoa và hành củ và có tế bào chất được nhận từ hành hoa. Chúng có 8 nhiễm sắc thể
nhận từ hành hoa và 8 nhiễm sắc thể nhận từ hành củ. Kết quả phân tích RAPD cho thấy 16 trong số
40 mồi sử dụng đã tạo ra 160 đoạn DNA trong tất cả các mẫu giống, trong đó có 135 đoạn đa hình.
Trên cây di truyền được xây dựng từ kết quả của phân tích RAPD, sự đa dạng di truyền được thấy
giữa các mẫu giống hành wakegi. Một số nhóm phụ được hình thành tuy nhiên các mẫu giống được
thu từ một địa điểm không cùng vào một nhóm. Các phân tích RAPD khác loài đã thu được 145 đoạn
DNA. Trong số các đoạn DNA thu được từ các mẫu giống hành wakegi thì 85 đoạn giống hành hoa và
71 đoạn giống hành củ. Trên cây di truyền 3 nhóm này được tách biệt rõ ràng lần lượt là nhóm các
mẫu giống hành wakegi, nhóm các mẫu giống hành củ và nhóm mẫu giống hành hoa. Khoảng cách di

truyền trên cây di truyên giữa hành wakegi và hành củ là 0.3 và giữa hành wakegi và hành lá là 0.5.
Key words: Alllium × wakegi Araki, đa dạng di truyền, DNA tế bào chất, DNA ti thể, hành wakegi,
RAPD.
SUMMARY
To evaluate wakegi onion (Alllium × wakegi Araki) strains as genetic resource for breeding of
onions in Vietnam, genetic variation among and between wakegi onions collected from the North,
Central and South regions in Vietnam and Japanese bunching onion, added shallot strains from
Japan, Thailand were studied basing on morphological, physiological characters and polymorphisms
of nuclear, chloroplast and mitochondrial DNAs. The results from cytological and genomic in situ
hybridization (GISH) analyses demonstrated that all the examined wakegi onion strains from Vietnam
were hybrid between Japanese bunching onion and shallot with the cytoplasm from Japanese
bunching onion and possessed eight chromosomes from shallot and eight chromosomes from
Japanese bunching onion in the somatic root tip cells. The intraspecific analysis among wakegi onion
showed that 16 out of 40 primers used in RAPD analysis produced total 160 distinct bands in all the
strains examined and 135 bands were polymorphic. In the dendrogram of RAPD data, variation was
detected between wakegi onion strains. There were many sub- groups however all the strains from the
same region were not always formed into one sub-group. The interspecific analysis produced total
145 DNA bands and among wakegi onion band patterns detected, 58 bands were similar to those
obtained from Japanese bunching onion, 71 bands similar to those from shallot. Three distinct groups
were found as shallot group, wakegi onion group and Japanese bunching onion group. The genetic
distance value between wakegi onion and shallot in phylogenetic tree was 0.3, and that between
wakegi onion and Japanese bunching onion was 0.5.
Journal of Science and Development 2009: Tập VI, No 6: 46-55 HA NOI UNIVERSITY OF AGRICULTURE
23
Key words: Alllium × wakegi Araki, chlotoplast DNA, genetic variation, mitochondrial DNA,
RAPD, wakegi onion.
1. INTRODUCTION
Wakegi onion is an interspecific hybrid of
Japanese bunching onion × shallot (Tashiro, 1980;
Tashiro et al., 1990) and has been cultivated in

Japan, China, Korea and South East Asia (Tashiro
et al.,1982; Tashiro, 1984; Inden and Asahira,
1990, Buijisen, 1990; Brewter, 1994; Tashiro et al
2006). It has the cytoplasm derived from Japanese
bunching onion (Tashiro,1980).
Wakegi onion is another small-bulb onion
with slender leaves and divides freely to form
many side shoots. Unlike Japanese bunching
onion, it forms bulbs under long day and enters
dormancy during summer. It is not very cold –
hardy and it is grown in the warmer regions of
Japan (Brewster, 1994). Arifin (Arifin and Okubo,
1996; Arifin et al., 2000) reported that there was
high genetic variation exiting among wakegi
onion strains collected from Indonesia as revealed
by isozyme, RAPD and PCR-RFLP analyses.
Wakegi onion is widely cultivated in
Vietnam as a winter crop. The bulbs are planted in
the autumn field then the shoots grown quickly
and form many tillers. However, the long dormant
stage during summer and growth retardation in
winter prevents this crop from year round
harvesting and shipping. So far, Tashiro et al.
(Tashiro et al. 2006c) had succeed to overcome
the obstacles to the harvesting and shipping of
wakegi onion through the plant breeding method.
By doubling chromosomes of wakegi onion in
callus culture, they got amphidiploid plants
(2n=32), which then were crossed with Japanese
bunching onion (2n=16) to raise new cultivar

named “Saga –otome”. This new cultivar is
triploid plant (2n=24) and propagated by bulblets.
It shows light dormancy and heat tolerance, so
suits to summer cultivation.
In Vietnam, all the parts of wakegi onion are
used as green salad, herb or to flavour the dishes
(Pham, 2002). However, wakegi onion has not
been distinguished from shallot in the market
(Pham, 2006a) though the farmers planted them
separately in the fileds.
In order to clarify the origin of wakegi
onion collection from Vietnam and to evaluate
Vietnamese wakegi onion strains as genetic
resource for future breeding of onions, genetic
variation among wakegi onion strains collected
from the North, Central and South regions in
Vietnam was studied basing on morphological
and physiological characters and
polymorphisms of nuclear, chloroplast and
mitochondrial DNAs.
2. MATERIALS AND METHODS
2.1. Plant materials
Twenty two strains of wakegi onion were
collected from the North, Central and South regions
in Vietnam during trip in December 2001. The
detail information on cultivated place and condition
of each strain was collected by an interviewing. All
the strains collected had been cultivated in the
collection sites or the nearby places. These strains
were cultivated in a plastic house of Saga

University from September 15, 2002 to May 1,
2003 (The roof of the plastic house was covered
with plastic film to protect from rainfall and frost,
but both sides and doors were opened). Ten plants
of each strain were used for morphological and
physiological observations. 20 strains of wakegi
onion from Vietnam and one additional strain from
Tiengiang province (South region) were used for
analysing interspecific variation via RAPD method.
All the wakegi onion and two addition strains
(one shallot and one Japanese bunching onion)
were used in chloroplast and mitochondrial DNA
analyses.
Furthermore, 10 strains of Japanese bunching
onion, 7 strains of shallot and 6 strains of wakegi
onion were used for inter-specific analysing via
RAPD method among them, Kujyo, 18-5 and XY3-
1 were from Japan, Thailand and Japan,
respectively and the rest was Vietnam.
2.2. Cytological analysis
Method of cytological analysis was as the
same as described by Pham T.M.P (Pham, T. M. P
et al. 2006).
2.3. GISH analysis
Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro
24
Nuclear genome of wakegi onion accessions
had been analyzed by using genomic in situ
hybridization technique with a little modification.
Root tips of the plants material were collected and

treated with 2nM 8-hydroxyquinoline at 4
o
C for
24 hr. After fixation in the mixture of acetic acid
and ethyl alcohol (1:3), the root tips were treated
with 0.1% cellulase – onozuka RS (Yakult
Pharmaceutial Co., Ltd., Tokyo), 0.1% pectolyase
Y-23 (Sheishin Pharmaceutical Co., Ltd., Tokyo)
and 0.1% cytohelicase in 10mM citrate buffer (pH
4.5) at 37
o
C for 1hr, washed in distilled water, and
squashed in 45% acetic acid. After removal of the
coverslips by the dry-ice method, the preparations
were treated with 45% acetic acid to dehydrate the
chromosomes, and the preparations were
subsequently air-dried over night. Total DNA of
Allium fistulosum (Kujyo) extracted by CTAB
method was labeled with biotin-16-dUTP by
nicktranslation Kit (Boheringer Mannheim Co.
Ltd.) and used as a probe. Total DNA of shallot
was used as a blocking DNA. The chromosome
preparations were denaturated in 70% formamide,
2 × SSC at 70ºC for 3min, dehydrated through a
prefreezed ethanol series of 70, 90 and 100% for 5
min each, and air – dried. The hybridization
mixture for 100 µl (total volume) consisted of
50% formamide, 2 ×SSC, 10% dextran sulphate,
1µg of biotinylated A. fistulosum total DNA, and
30 µg of unlabeled shallot total DNA. The mixture

(10 µl) was applied to each slide and covered with
a plastic cover slip.
Biotin- labelled DNA was detected with
FITC- conjugated avidin. Each slide was treated
with 50 ml of a blocking buffer (4 × SSC
containing 10 mM MgSO
4,
0.1% Tween 20, 3%
bovine serum albumin) for 15 min at 37
o
C, and
incubated with 50 ml of 20 mg/ml fluorescein
avidin DN (Vector Laboratories, Burlingame,
Calf.) in the blocking buffer for 45 min at 37
o
C.
After washing in a washing buffer (4 × SSC
containing 10 mM MgSO
4,
0.1% Tween 20) five
times, 3 min each, at room temperature, the
signals from biotin were amplified. Each slide was
incubated with 50 ml of 20 mg/ml FITC-
conjugated anti - avidin D (Vector Laboratories,
Burlingame, Calf.) in the blocking buffer for 15
min at 37
o
C and then washed as described above.
Chromosomes were counterstained with 1 µg/ml
propidium iodide. Slides were mounted with

antifade (Vectashield, Vector Laboratories
Burlingame, Calf.).
The GISH image was observed on
fluorescence microscope DMRXA (Leica Co.,
LTD.) using software QFISH (Leica Co., Ltd.).
2.4. Morphological and physiological
observations
Three characters as plant height, leaf
diameter and number of tillers per plant were
recorded at 2.5 months after planting. Leaf color
was observed in all the growing cycle, and bulb
skin color was noted after harvest. Number of
days from planting to bolting was counted, and
flower morphology was observed at full bloom.
2.5. RAPD analysis
Method of RAPD analysis was the same as
described by Pham T.M.P (Pham, T. M. P et al.
2006).
2.6. Mitochondrial DNA analysis
Method of mitochondrial analysis was as the
same as describled by Pham T.M.P (Pham, T. M. P
et al. 2006).
3. RESULTS
3.1. Cytological analysis
The results from root tip cell observations by
squash and GISH methods showed that all the
strains collected possessed 16 chromosomes in the
root tip cell. Two satellite chromosomes were
distinguished clearly; one was typical type of
shallot and the other was typical type of Japanese

bunching onion. In GISH analysis, 8
chromosomes from shallot were observed clearly
in yellow green color and 8 chromosomes from
Japanese bunching onion in orange red color
(Figure 1). Among the wakegi onion examined,
there was no variation in the size and shape of the
chromosome except the satellite size and position.
3.2. Morphological and physiological
observations
Morphological and physiological characters
of all the strains observed were given in table 1.
All the wakegi onion strains collected had
dark green and erect leaves. There were variations
in all the characters observed: time for sprouting
Genetic variation of wakegi onion (Alllium × wakegi Araki) in Vietnam
25
arranged from 3.7 to 9.6 days, plant height from 4.2
to 58.8 cm, leaf diameter from 7.6 to 10.6 mm and
the number of tillers per plant from 8.0 to 18.2.
All the strains collected produced flower
stalks in Dec.2003. The flower bud and the flower
morphology were intermediate between that of
shallot and Japanese bunching onion.

Fig. 1. Somatic chromosomes (2n) of wakegi onion. a: Feulgen method; b: by genomic in situ
hybridization (GISH) method. The red (dark) arrowheads point the satellite carried chromosomes.
Yellowish green fluoresces: chromosomes of Japanese bunching onion, orange-red fluorescence:
chromosomes of shallot (white arrowheads)
Table 1. Morphological and physiological characteristics of wakegi onion
Strain
number
Collection market
Time for
sprouting (days)
Plant height
(cm)
Leaf diameter
(mm)
Number of
tillers per plant

W- 2 Baoyen, Laocai 5.6 ± 0.5 50.4±1.1 9.7±0.3 9.4±1.6
W- 4 " 5.2 ± 0.7 47.6±1.0 9.5±0.2 9.5±1.0
W- 5 " 7.5 ± 0.5 44.7±1.7 9.1±0.5 10.5±0.8
W- 6 Sapa, Laocai 8.1 ± 1.1 49.3±1.8 8.9±0.3 13.2±1.1
W- 7 " 9.6 ± 0.8 42.7±0.8 9.7±0.3 8.0±1.1
W- 8 " 6.0 ± 1.0 43.8±0.8 9.8±0.1 9.9±2.1
W- 24 Sapa, Laocai 4.8 ± 0.6 54.6±2.1 9.6±0.4 17.7±1.2

W- 21 Cocleu, Laocai 3.7 ± 0.2 47.9±2.4 9.0±0.3 10.3±0.1
W- 22 " 7.7 ± 0.4 42.4±0.5 10.0±0.4 10.6±0.7
W- 26 " 8.6 ± 0.5 45.0±1.8 10.1±0.3 9.2±1.6
W- 29 " 5.0 ± 0.3 48.6±4.8 9.2±0.1 17.2±1.4
W- 31 " 8.8 ± 0.3 45.4±1.9 9.3±0.5 14.7±0.3
W- 32 " 5.8 ± 0.4 48.6±1.7 9.4±0.2 18.2±1.1
W- 34 " 5.1 ± 0.4 50.7±2.6 9.4±0.1 13.6±1.0
W- 77 " 8.6 ± 0.4 47.7±1.3 9.4±0.1 12.2±0.1
W- 76 " 6.4 ± 0.4 53.1±1.3 9.0±0.2 17.8±1.6
W- 28 Huongcanh, Vinhphuc 5.5 ± 0.3 49.3±0.7 9.9±0.2 18.0±2.3
Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro
26
W- 30 " 4.8 ± 0.1 48.5±0.6 9.9±0.2 14.1±1.8
W- 44 Dongxuan, Hanoi 8.5 ± 0.6 50.7±0.7 7.6±0.4 17.6±1.0
W- 35 Trangbang,Tayninh 5.5 ± 0.5 51.0±.14 9.7±0.2 16.7±2.0
W- 43 Dongba, Hue 6.8 ± 1.8 58.8±1.5 9.2±0.1 15.4±2.1

Table 2. Number of total and polymorphic bands detected in
RAPD analysis of wakegi onion
Primer Sequence No. of total bands No. of polymorphic bands
OPA-02 5’-TGCCGAGCTG -3’ 14 13
OPA-04 AATCGGGCTG 9 7
OPA-10 GTGATCGCAG 8 5
OPA-11 CAATCGCCGT 13 10
OPA-16 AGCCAGCGAA 9 9
OPA-19 CAAACGTCGG 16 15
OPA-20 GTTGCGATCC 13 13
OPG-02 GGCACTGAGC 2 2
OPG-03 GAGCCCTCCA 11 9
OPG-04 AGCGTGTCTG 8 8

OPG-06 GTGCCTAACC 9 7
OPG-09 CTGACGTCAC 10 9
OPG-11 TGCCCGTCGT 4 4
OPG-12 CAGCTCACGA 13 10
OPG-13 CTCTCCGCCA 10 9
OPG-18 GGCTCATGTG 11 5
Total 160 135

3.3. RAPD analysis
In the intraspecific analysis, 16 out of 40
primers used in RAPD analysis produced total 160
distinct bands in all the strains examined (Table 2).
135 polymorphic bands were scored among the
strains. In the RAPD amplification of the all the
strains examined from three primers OPA-16, OPG-
02 and OPG-04, all the bands detected were
polymorphic bands. Small variations in the presence
or absence of the bands could be observed between
wakegi onion strains from both Vietnam and other
countries. Significant variation observed between
wakegi onion strains and shallot and Japanese
bunching onion. In the dendrogram of RAPD data
(Figure 2), variation was detected between wakegi
onion strains. There were many sub- groups however
all the strains from the same region were not always
formed into one sub-group. In the interspecific
analysis, total 145 DNA bands were detected.
Fig. 2. Dendrogram from RAPD data Fig. 3. Dendrogram based on RAPD data
Genetic variation of wakegi onion (Alllium × wakegi Araki) in Vietnam
27
Among wakegi onion band patterns detected, 58
bands were similar to Japanese bunching onion
while 71 of those similar to shallot (Figure 4). From
the dendrogram based on RAPD data (Figure 3),
three distinct groups were found as shallot group,
wakegi onion group and Japanese bunching onion
group. The genetic distance value between shallot
and wakegi onion was 0.3, and that between shallot

and Japanese bunching onion was 0.5. Some small
variations could be seen inside each group.

Fig. 4. RAPD profiles generated by primer OPG -11 in Japanese bunching onion (M: Marker II;
1: Kyujo, 2 : F-25, 3: F-9, 4: F-194, 5: F-54, 6: F-126, 7: F-01, 8: F-38, 9: F-57 and 10: F-191), shallot
(11: 18-5, 12: A-13, 13: A-23, 14: A-10, 15: A-52, 16: A-42, 17: A-68) and wakegi onion (18: XY3-1,
19: W-22, 20: W-28, 21: W-43, 22: W-35, and 23: W-192)
3.4. Chloroplast DNA analysis
The sizes of the amplified regions were about
3.2 kbp and 4.5 kp for rbcL- ORF106 and rpoC1-
rpoC2, respectively in all the strains of wakegi
onion. Total 27 bands were detected by digesting
amplified region of rbcL-ORF106 with restriction
enzymes (Alu I, Taq I, Ase I, Ecor V, Bgl II and
BamH I) and 10 bands by digesting amplified
product of rpoC1 - rpoC2 with Alu I. All the
wakegi onion strains had the same band patterns
as those of Japanese bunching onion. There was
no polymorphism among the wakegi onion strains
examined. 13 and 3 polymorphic bands were
detected between wakegi onion and shallot in
rbcL-ORF106 and rpoC1- rpoC2 regions,
respectively.
3.5. Mitochondrial DNA analysis
The size of the amplified region was 550 bp.
Total 6 bands were observed in all the strains
examined. All the wakegi onion strains had the
same band patterns as those of Japanese bunching
onion. Two polymorphic bands were detected
between wakegi onion strains and shallot strains by

digesting the amplified product with Rsa I.
4. DISCUSSION
The result from cytological and GISH
analyses demonstrated that all Vietnamese wakegi
onion strains examined were possessed eight
chromosomes from shallot and eight
chromosomes from Japanese bunching onion in
the somatic root tip cells. The results agreed with
the formula as K
(2n)
=14V+J
(t)
1
+J
(T)
2
given by
Tashiro (1984). The morphological observation
showed that flower bud and flower of all the
Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro
28
strains collected were intermediate between those
of shallot and Japanese bunching. Beside that, the
results of the morphological and physiological
observations and DNA analyses showed small
variation among wakegi onion examined, but
there was no variation between the strains from
different regions as those of shallot and rakkyo
(Pham, 2006). In DNA analyses of chloroplast and
mitochondrial DNAs, all the wakegi onion had the

same band patterns as those of Japanese bunching
onion. All above results prove that wakegi onion
in Vietnam was a hybrid between shallot and
Japanese bunching onion. These results also agree
with the conclusion given by Tashiro (1984) that
the cytoplasm of wakegi onion was derived from
Japanese bunching onion. Arifin (2000) had
reported that there were shallot strains collected
from Indonesia, of which cytoplast was derived
from shallot from Indonesia. However from
cytological analysis, one of the material plants of
which cytoplasm was reported to derive from
shallot, was confirmed to be aneuploid with
number of chromosomes 25 (Endang, 2002).
So far there was no information about bolting
of wakegi onion in the South East Asia (Buijsen,
J.R.M, 1990) and Vietnam (Pham, 2003).
However, all the strains collected showed bolting
under condition in Saga, Japan. This result proves
that the wakegi onion in Vietnam has a potential
to flower and that the growing condition has a
remarkable effect to the flowering. The bolting of
wakegi onion is the same as in the case of shallot,
the short day length and/ or low temperature
during autumn and winter supposed to stimulate
the bolting of both types of wakegi onion under
condition in Saga.
Wakegi onion is a natural hybrid between
shallot and Japanese bunching onion, and it has
intermediate in morphological characters from

both. However, in Vietnam, only one name was
used for both wakegi onion and shallot (Pham,
2003; Pham, 2006b) even though the farmers
could separate them in the field. The confusion
between wakegi onion and shallot also happens in
Indonesia (Arifin et al., 2000) and it seems to
happen in the other South East Asian countries -
where both wakegi onion and shallot are
cultivated.
In Vietnam, wakegi onion is widely used
during winter when Japanese bunching onion
absent in the market. The culture of using green
plant of Japanese bunching onion (during summer
time) and wakegi onion (during winter time) since
history proves the importance of both species the
daily life in Vietnam, therefore further study is
nessesary to improve both of these species.
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Pham Thi Minh Phuong, Shiro Isshiki and Yosuke Tashiro
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