Genetic relationships among ginger accessions based on AFLP

Transcription

Jurnal
60 Bioteknologi Pertanian, Vol. 8, No. 2, 2003, pp. 60-68
S. Wahyuni et al.
Genetic relationships among ginger accessions
based on AFLP marker
Kekerabatan antar nomor-nomor aksesi jahe berdasarkan marka AFLP
S. Wahyuni1, D.H. Xu2, N. Bermawie1, H. Tsunematsu2, and T. Ban2
1
Indonesian Spices and Medicinal Crops Research Institute, Jalan Tentara Pelajar No. 3, Bogor 16111, Indonesia
2
Japan International Centre for Agrobiological Sciences (JIRCAS)
ABSTRAK
Jahe merupakan salah satu tanaman penting dari jenis temutemuan. Di Indonesia dikenal tiga tipe jahe, yaitu jahe merah,
jahe besar, dan jahe kecil. Ketiga tipe jahe tersebut mempunyai
bentuk, warna, aroma, dan komposisi kimia rimpang yang
berbeda. Untuk mengetahui kekerabatan antartipe dan dalam
tipe pada jahe, 28 nomor aksesi yang terdiri atas 22 aksesi
jahe Indonesia dan masing-masing 3 aksesi jahe asal Afrika
dan Jepang digunakan dalam penelitian ini. Total DNA
diekstrak dari rimpang dengan metode cetyltrimethyl ammonium bromide (CTAB) kemudian dimurnikan menggunakan
serbuk magnet. Amplified fragment length polymorphism
(AFLP) dilakukan mengikuti prosedur pada AFLP TM plant
mapping kit (PE Applied Biosystem) dan hasil akhir
polymerase chain reaction (PCR) dipisahkan pada 5% gel
poliakrilamid dalam ABI 373 sequencer. Jumlah fragmen
yang teramplifikasi pada setiap kombinasi primer AFLP ratarata mencapai 96 dengan kisaran 47-137 fragmen. Pengamatan dengan menggunakan 21 kombinasi primer menghasilkan
221 pita polimorfis. Dendrogram berdasar unweighted pair
group methods of arithmetic average (UPGMA) dari semua
nomor aksesi yang digunakan dapat diklasifikasikan menjadi
tiga kelompok utama. Jahe merah secara genetik jauh dari
jahe besar, tetapi mempunyai kekerabatan yang dekat dengan
beberapa aksesi jahe kecil. Keragaman genetik dari jahe kecil
(Ht = 0,25) lebih tinggi dari jahe besar (Ht = 0,08).
[Kata kunci: Zingiber officinale, jahe, AFLP, keragaman genetik]
ABSTRACT
Ginger (Zingiber officinale Rosc.) is a rhizomatous perennial
herb and one of the important crop of the genus Zingiber.
There are three types of ginger in Indonesia, i.e. big ginger,
small ginger, and red ginger. Their rhizomes differ in shape,
color, aroma, and chemical composition. To understand the
genetic relationships among the three types of ginger, 28
accessions consisted of 22 Indonesian cultivated ginger and
3 accessions each of African and Japanese commercial ginger
were analyzed. Total DNA was extracted from rhizome using
cetyltrimethyl ammonium bromide (CTAB) method then
purified by magnetic beads. Amplified fragment length
polymorphism (AFLP) was carried out according to the
protocol described in AFLP TM plant mapping kit (PE Applied
Biosystem) and the final polymerase chain reaction (PCR)
products were separated on 5% denatured polyacrylamide gel
on an ABI 373 sequencer. The number of fragments produced
by a primer combination of AFLP ranged from 47 to 137 with
an average of 96. A total of 221 polymorphic bands were observed by using 21 selective primer combinations. Dendrogram
based on unweighted pair group methods of arithmetic average
(UPGMA) revealed that the gingers could be classified into
three major clusters. The red ginger was genetically far from
the big ginger, but close to some accessions of small ginger.
There was no clear genetic differentiation between the small
and big types of ginger. The genetic diversity of small ginger
(Ht = 0.25) is higher than that of the big ginger (Ht = 0.08).
[Keywords: Zingiber officinale, ginger, AFLP, genetic variation]
INTRODUCTION
Ginger (Zingiber officinale Rosc.) is a perennial
rhizomatous herb of the family Zingiberaceae. Its
origin is unknown, probably in tropical Asia and China
(Purseglove et al. 1981). In Indonesia, gingers are
grown in 13 provinces, but the main producing areas
are Aceh, East Java, North Sumatra, West Java,
Lampung, and Central Java. Ginger cultivation in these
areas is considered to be beneficial. Ginger can be
planted at the altitude up to 1,000 m asl, but the
optimum condition for high yield is at 300-500 asl.
Ginger is used worldwide as a cooking spice,
condiment, and herbal remedy. It is the underground
roots or rhizomes that are used for culinary and
medicinal purposes. The Chinese have used ginger
for at least 2,500 years as digestive aid, antinausea and
rheumatism. In Malaysia and Indonesia, ginger is
widely used as beverage such ginger tea to warm
body. In Arabian medicine, ginger is considered as an
aphrodisiac, while some African believe that eating
ginger regularly will help repel mosquito. Nowadays,
ginger is extensively cultivated in Asia and Africa, but
61
Genetic relationships among ginger accessions based on AFLP marker
in commerce the main ginger exporting countries are
China and India, which supplied almost 75% of the
world annual requirement of over 18,000 tons of ginger.
The major ginger importing countries are USA, Japan,
Europe, and Middle East countries.
Indonesian ginger has been described as two
variants, i.e. white ginger (Zingiber officinale var.
officinale) and red ginger (var. sunti) (Rugayah 1994),
it is in agreement with Valeton classification (1918).
Based on phenotyphic characters, Rostiana et al.
(1990) distinguished ginger into three groups, i.e. big
ginger, small ginger, and red ginger. Big ginger has big
rhizome size, less pungent and less fibrous, average
plant height 68.63 + 12.75 cm. Small ginger has smaller
rhizome size, fibrous and pungent, plant height 49.16
+ 7.29 cm, while red ginger has small rhizome size with
red skin color, more pungent and more fibrous, plant
height 48.23 + 14.05 cm, and has darker green leaves
compared with two others. According to the usage,
small ginger and red ginger are generally used for
medicinal purposes and cooking spices, while big
ginger is used for food, beverage, and cooking spices.
De Guzman and Siemonsma (1999) reported that there
are three types of ginger in Indonesia, namely (1) jahe
badak = jahe gajah = jahe putih besar, (2) jahe merah
= jahe sunti, and (3) jahe putih kecil = jahe emprit.
Their rhizome differs in shape, color, aroma, and
chemical composition, and all types can be considered
as cultivars.
Genetic diversity of ginger germplasm collected from
several locations in Indonesia was low (Bermawie et
al. 2001). Although they consisted of big, small and
red gingers, morphologically they showed considerable phenotypic variations for many traits such as
rhizome size, color, flesh color, flavor/pungency, yield,
fiber, and rhizome chemical content. However, it is
difficult to distinguish accessions within the group
compared to accessions of different group. Therefore,
the relationships between and within the groups of
ginger have to be studied to pursue genetic improvement of ginger.
Polymerase chain reaction (PCR)-based methods for
genetic diversity analyses have been developed, such
as random amplified polymorphic DNA (RAPD),
random fragment length polymorphism (RFLP),
amplified fragment length polymorphism (AFLP), and
inter simple sequence repeat (ISSR/SSR). Each
technique is not only differed in principal, but also in
the type and amount of polymorphism detected. AFLP
technique is based on the selective PCR amplification
of restriction fragments from a total digest of genomic
DNA. The technique involves three steps: (1) restric-
tion of the DNA and ligation of oligonucleotide
adapters, (2) selective amplification of sets of restriction fragments, and (3) gel analysis of the
amplified fragments (Vos et al. 1995). Typically, 50-100
restriction fragments are amplified and detected on
denaturing polyacrylamide gel. AFLP has been
recognized as a reliable and efficient DNA marker
system (Vos et al. 1995). It has been proven the most
efficient for estimating diversity in barley (Russel et
al. 1997), provides detailed estimates of the genetic
variation of papaya (Kim et al. 2002), and have been
used to analyze the genetic diversity of various plants
such as tea (Lai et al. 2001), eggplant (Mace et al.
1999), peach (Manubens et al. 1999), apple (Guolao et
al. 2001), rapeseed (Lombard et al. 1999), wild radish
(Man and Ohnishi 2002), and Musa sp. (Wong et al.
2001; Ude et al. 2002). Another marker system which
can be used for genetic diversity analysis is ISSR. The
use of this technique for genetic diversity analysis
has been reported on tea (Lai et al. 2001; Mondal
2002), and Botrycum pumicola (Camacho and Liston
2001)
The objective of this study was to understand the
genetic relationships between and within the types of
ginger by using AFLP marker.
MATERIALS AND METHODS
Plant materials
A total of 22 accessions of cultivated ginger grown in
Indonesia including small, red and big gingers were
used as a material for analysis. More over 3 accessions
of African ginger (Ivory Coast, Bouake) and 3 accessions of Japanese ginger were used as a test sample.
The list of materials used were shown in Table 1.
DNA extraction
Total genomic DNA was extracted from rhizomes by
using cetyltrimethyl ammonium bromide (CTAB)
methods (Doyle and Doyle 1990). The DNA was then
purified with silica beads (MagExtractor Plant Genome
Kit, Toyobo. Co.) to obtain a better quality of DNA
which appropriate for AFLP. A few sample of DNA
obtained were loaded in 0.8% agarose gel, and
λÉDNA were loaded too as a standard for estimation
of the quantity and quality of the DNA. The gel was
finally stained with ethidium bromide and viewed on
the UV transiluminator light at gel documentation
system.
62
S. Wahyuni et al.
Table 1.
Description of ginger accessions used in this study.
Accession code
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
AA
AB
Origin
Rhizome size
Ina, West Java, Sukabumi
Ina, West Java, Cianjur
Ina, West Java, Garut
Ina, West Java, Sumedang
Ina, Central Java, Salatiga
Ina, Central Java, Boyolali
Ina, West Java, Cisewu
Ina, West Java, Sumedang
Ina, West Java, Garut
Ina, West Java, Maja
Ina, West Java, Wado
Ina, Central Java, Salatiga
Ina, West Java, Wado
Ina, West Java, Bogor
Ina, Papua, Manokwari
Ina, Papua, Manokwari
Ivory Coast, Africa
Ivory Coast, Africa
Ivory Coast, Africa
Japan
Japan
Japan
Big
Big
Big
Big
Big
Big
Small
Small
Small
Small
Small
Small
Small
Small
Small
Small
Small
Small
Big
Big
Small
Small
Big
Small
Big
Small
Small
Big
Amplified Fragment Length Polymorphism (AFLP)
AFLP was carried out according to the protocol
described in AFLP TM plant mapping (PE Applied
Biosystems). Genomic DNA was digested with 5 units
EcoRI and 1 unit MseI at 37oC for 6 hours, then ligated
with 5 pmol EcoRI and 50 pmol MseI adaptor in a total
volume of 20 µl by one weiss unit of T4 DNA ligase
in a PCR core mix (dNTPs, MgCl2, PCR buffer, rTag
polymerase; TOYOBO), then incubated for overnight
at 16oC. Pre-amplification was performed in a total
volume of 20 µl containing 3 µl DNA template, 0.125
µM EcoRI +A and MseI + C primers, 0.2 mM dNTPs,
0.4 unit rTag polymerase, 1.5 mM MgCl2 and 1x PCR
buffer, and amplified on the thermocycler. The reaction
condition was as follow: 25 cycles of 94 oC for 20
seconds, 56oC for 30 seconds and 72oC for 2 minutes;
and one cycle of 60oC for 30 minutes. Pre-amplification
product was then diluted ten times as template for
selective amplification. It was carried out in a total
volume of 20 ml consisted of 3 µl diluted pre-selective
amplification product, 1 µl 10 pmol MseI and 1 µl 2
pmol EcoRI primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1x
Rhizome color
White
White
White
White
White
White
Red
Red
White
White
White
White
White
White
White
White
White
White
White
White
Red
White
White
White
White
White
White
White
Inner part color
Yellowish cream
Yellowish cream
Yellowish cream
Grayish cream
Grayish cream
Grayish cream
Purple-red
Light purple
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Yellowish cream
Grayish cream
Light purple
Yellowish cream
Yellowish cream
Grayish yellow
Grayish cream
Yellowish cream
PCR buffer and 0.4 unit rTaq polymerase then
performed on the following program: one cycle of
denaturation at 94oC for 20 seconds, annealing at 65oC
for 30 seconds and extension at 72oC for 2 minutes,
followed by 8 cycles of a 1 oC decreasing annealing
temperature per cycle, and 23 cycles of 94oC for 20
seconds, 65oC for 30 seconds and 72oC for 2 minutes
and finally at 60 oC for 30 minutes. The final PCR
products were separated on 5% denatured polyacrylamide gel and electrophoresed on ABI 373
sequencer (Perkins Elmer/Applied Biosystem, Foster,
USA). Twenty one selective primer combinations were
used in this study.
Data scoring and analysis
A band was considered polymorphic if it was present
in at least one genotype and absent in the others. Each
accession was scored for the present (1) or absent (0)
of the polymorphic bands. Estimates of similarity were
based on simple matching (SM) coefficient (Sokal and
Michener 1958): Sij = a+d/a+b+c+d, where Sij is the
similarity between two individuals (i and j), a is the
63
number of bands present both in i and j, b is the
number of bands present in i and absent in j, c is the
number of bands present in j and absent in i, and d is
the number of bands absent both in i and j. The matrix
of similarity was then clustered using unweighted pair
group methods of arithmetic average (UPGMA) using
NTSYS-pc version 2.1 (Exeter software). Diversity
values were calculated for each locus as 1 - Σ pi 2 ,
where pi is the phenotypic frequency for each assay
unit of AFLP primer combinations (Russell et al. 1997).
RESULTS AND DISCUSSION
AFLP analysis
The bands produced by one primer combination with
three E+nnn and M+nnnn of selective bases were
ranging from 47 to 137 with an average of 96 bands
(Table 2). Primer combinations of E-ATC/M-CAGA
and AGA/CGGA perform the clearest amplified and
polymorphic bands. Figure 1 shows an example of a
gel image for the 28 accessions studied with the primer
combination E-ATC/M-CAGA.
A total of 221 polymorphic bands were obtained by
AFLP analysis with the average of 10.5 polymorphic
bands per primer combination or equal with 11.5%.
Table 2.
The amplified bands in ginger are generally not sharp
and clear. It might be due to the large genome size
of ginger (23,618 Mbp) (RBG Kew 2000). The
achievement of polymorphic bands was relatively low
(11.45%) compared with another plants such as 72.8%
on wild radish (Man and Ohnishi 2002), 57.2% on
apple (Goulao 2001), 42% on papaya (Kim et al. 2002),
and 46.8% on barley (Russel et al. 1997).
The genetic variation among 28 ginger accessions
was estimated using pair-wise comparison of genetic
similarity. The average pair-wise genetic similarity was
0.801, ranged from 0.527 to 0.992. About 37.3% of the
pair-wise comparison among the ginger accessions
exhibited genetic similarity greater than 0.90, and less
than 16% showed genetic similarity 0.70.
The most closely accessions sharing genetic
similarity were big ginger D (accession collected from
Wado, Sumedang, West Java) and E (accession
collected from Salatiga, Central Java). Both of the
places are the main production areas of ginger.
Morphologically both accessions have similar performance. The lowest similarity was between small
ginger K (accession collected from Garut, West
Java) and Z (commercial ginger from Japan). The
similarity matrix among the accessions is presented in
Table 3.
Number of polymorphic bands of ginger in each assay unit of AFLP.
AFLP primer
combination
Number of
amplified bands
Number of
polymorphic bands
Percentage of
polymorphic bands
ACC/CAAG
ACC/CAAC
ACC/CAAT
AAC/CACG
AAC/CAAC
AAC/CACA
AAC/CAGA
AAC/CAAT
ATA/CAGA
ATC/CGGA
ATC/CGGT
ATC/CTGA
AGA/CAGA
AGA/CTGA
AGA/CGGA
AGG/CATA
AGG/CGGA
AGG/CACG
AAG/CAGA
ACG/CAGA
ACT/CAGA
96
105
83
120
92
105
123
130
80
75
79
90
135
111
92
47
86
62
106
60
137
12
4
14
12
4
23
8
11
14
10
12
16
12
17
7
5
8
8
9
9
6
12.50
3.81
16.86
10.00
4.35
21.90
6.50
8.46
17.50
13.3
15.19
17.78
8.89
15.32
7.61
10.64
9.30
12.90
8.33
15.00
4.38
96
60-137
10.523
4-23
Average
Range
11.446
3.81-21.90
64
S. Wahyuni et al.
l▼
Data point 2732
Size 285 bp
Data point 2395
Size 250 bp
l▼
Fig. 1. Gel image of AFLP of 28 ginger accessions produced by primer combination E-ATC/M-CAGA.
The dendrogram based on UPGMA produced three
major clusters (Fig. 2). The first cluster consisted of
Japanese ginger, the second cluster consisted of all
accessions of red and three accessions of small ginger,
while the third cluster consisted of small ginger and
big ginger. The correlation coefficient between the
cophenetic matrix computed from the dendrogram and
the original similarity matrix was 0.960 (t = 7.496, p =
1), suggesting very good fit of the three representations to the rough data value.
Based on the cluster analysis, among red, small, and
big types of ginger are not clearly defined. It is not in
agreement with the common classification that distinguishes ginger into three types (big, small and red).
In the second cluster, red ginger and some accessions
of small ginger clustered together, while in the third
cluster the small and big types of ginger are not well
separated. Rhizome size and color were conspicious
characters in ginger, but AFLP result showed that
several accessions of small and big ginger were highly
similar to each other genetically, and several accessions of small ginger genetically close to red ginger.
The similar result was obtained in Musa sp. (Wong et
al. 2001). Both Musa ssp. truncata and microcarpa
have similar character which can be seen clearly in
black pseudostem, but AFLP result showed that the
two subspecies were genetically less similar to each
other. Subspecies microcarpa (black stem with nonwaxy leaves) was genetically more similar to
subspecies malaccensis (green stem with waxy
leaves). These indicated that differences in morphological characters in ginger based on rhizome size
65
Table 3. Genetic similarity between 28 ginger accessions.
A
1.000
0.954
0.946
0.925
0.934
0.946
0.701
0.698
0.929
0.693
0.693
0.905
0.917
0.909
0.781
0.905
B
C
D
E
F
G
H
I
J
K
L
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
1.000
0.950
0.963
0.971
0.967
0.722
0.736
0.934
0.739
0.714
0.950
0.946
0.954
0.824
0.934
1.000
0.954
0.963
0.950
0.722
0.728
0.909
0.730
0.722
0.917
0.938
0.946
0.820
0.909
1.000
0.992
0.963
0.726
0.732
0.913
0.743
0.718
0.946
0.942
0.950
0.820
0.929
1.000
0.971
0.726
0.740
0.921
0.751
0.726
0.946
0.942
0.959
0.828
0.921
1.000
0.722
0.745
0.925
0.747
0.730
0.950
0.938
0.963
0.828
0.934
1.000
0.940
0.705
0.925
0.917
0.722
0.718
0.726
0.601
0.722
1.000
0.702
0.940
0.923
0.728
0.740
0.749
0.621
0.715
1.000
0.722
0.705
0.909
0.929
0.946
0.811
0.950
1.000
0.959
0.730
0.743
0.759
0.627
0.730
1.000
0.714
0.734
0.751
0.601
0.722
1.000
0.954
0.963
0.825
0.934
Q
R
S
T
U
V
W
X
Y
Z
AA
AB
A
0.913
0.871
0.921
0.913
0.672
0.697
0.876
0.867
0.880
0.643
0.676
0.871
B
0.917
0.909
0.943
0.950
0.701
0.718
0.916
0.896
0.884
0.680
0.722
0.909
C
0.884
0.884
0.943
0.917
0.710
0.710
0.903
0.896
0.834
0.656
0.689
0.892
D
0.913
0.913
0.947
0.929
0.714
0.722
0.898
0.892
0.855
0.676
0.718
0.913
E
0.913
0.913
0.947
0.929
0.714
0.722
0.898
0.892
0.855
0.676
0.718
0.913
F
0.925
0.917
0.961
0.942
0.710
0.726
0.898
0.896
0.867
0.680
0.714
0.917
G
0.705
0.714
0.719
0.714
0.913
0.913
0.673
0.701
0.647
0.544
0.552
0.680
H
0.719
0.719
0.730
0.719
0.885
0.902
0.677
0.698
0.643
0.562
0.570
0.694
I
0.942
0.892
0.943
0.934
0.693
0.693
0.898
0.896
0.900
0.656
0.705
0.884
J
0.739
0.730
0.732
0.722
0.896
0.896
0.690
0.710
0.656
0.535
0.560
0.705
K
0.739
0.730
0.728
0.714
0.905
0.896
0.673
0.701
0.647
0.527
0.535
0.680
L
0.925
0.934
0.965
0.950
0.710
0.718
0.912
0.905
0.876
0.689
0.722
0.925
M
1.000
0.975
0.820
0.946
0.929
0.929
0.961
0.963
0.722
0.730
0.929
0.917
0.880
0.685
0.718
0.921
N
O
P
Q
R
S
T
U
V
W
X
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
AA
AB
1.000
0.845
0.954
0.938
0.938
0.978
0.963
0.730
0.730
0.929
0.925
0.880
0.693
0.734
0.929
1.000
0.820
0.785
0.833
0.832
0.811
0.588
0.579
0.826
0.798
0.725
0.584
0.605
0.790
1.000
0.934
0.917
0.952
0.959
0.710
0.710
0.898
0.905
0.884
0.656
0.705
0.900
1.000
0.942
0.943
0.925
0.685
0.710
0.876
0.880
0.900
0.656
0.705
0.892
1.000
0.939
0.917
0.685
0.701
0.876
0.888
0.851
0.672
0.697
0.909
1.000
0.961
0.715
0.711
0.934
0.921
0.890
0.693
0.719
0.939
1.000
0.718
0.718
0.929
0.921
0.892
0.672
0.714
0.925
1.000
0.934
0.690
0.730
0.643
0.556
0.564
0.701
1.000
0.690
0.705
0.668
0.548
0.581
0.710
1.000
0.942
0.881
0.681
0.717
0.934
1.000
0.871
0.710
0.734
0.938
Y
1.000
0.697
0.730
0.892
Z
AA
AB
Y
Z
AA
AB
1.000
0.859
0.722
1.000
0.763
1.000
66
S. Wahyuni et al.
B. Sukabumi
B. Cianjur
B. Boyolali
B. Sumedang
B. Salatiga
B. Garut
S. Majalengka
S. Cianjur
S. Boyolali
B. Wado
B. Bogor
S. Cisewu
S. Sukabumi
S. Boyolali
S. Salatiga
B. Africa
S. Africa
B. Japan
S. Africa
S. Wado
R. Sukabumi
R. Cianjur
S. Sumedang
S. Garut
S. Papua
R. Papua
S. Japan
S. Japan
0.66
0.74
0.83
0.91
0.99
Coefficient
Fig. 2. Dendrogram of 28 ginger accessions generated by AFLP analysis; S = small ginger, B = big ginger, R = red ginger.
and color are not always an indication of the similarity
or difference in DNA marker (AFLP).
The level of genetic variation among ginger
accessions tested was low, revealed by diversity index
value of only 0.22 from the AFLP data. This was
comparable with clonally propagated species such
Elymus repens, D = 0.33 (Szczepaniak et al. 2002) or
Potentilla spp., D = 0.20 (Hansen et al. 2000). The low
genetic variation of ginger was possibly caused by the
mode of ginger propagation which is mostly propagated by rhizome. Ginger sometimes produced
flower, but rarely beared fruit (Purseglove et al. 1981).
By AFLP analysis, genetic variation within small
ginger (Ht = 0.255) was broader compared with big
ginger (Ht = 0.079).
Big ginger was collected from 7 locations, while
small ginger from 9 locations and mostly from West
Java. The Sundanese (West Java tribe) call jahe badak
(means big) for their garden plantation to distinguish
it from the common ginger which run wild (Burkill
1935). Later big ginger is popular to the farmer and
widely cultivated for export purposes. On the other
hand small ginger is usually cultivated for domestic
uses such a cooking spice and medicinal purpose.
Farmer provided their own seed for small ginger, and
trade over region sometimes for big ginger. It is more
fare understandable why the diversity index of big
ginger was lower than that of small ginger.
Marker specific to accession
There is no unique molecular marker band which can
be used to distinguish red, small or big ginger. Not all
primer pairs produced specific bands. The specific
bands produced by certain primer combination were
only detected on small ginger accessions collected
from Wado (O) (Fig. 3). A specific band related to red
ginger was also observed, but only one band, one
primer combination and not a major band, so it was not
reliably used for marker yet. Mostly unique bands were
on red ginger included some accessions of small
ginger (Table 4). Both the red and small types of ginger
which have the same specific marker bands, morphologically have similar shape and size of rhizome
and smaller compared to others.
67
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Fig. 3. Specific molecular bands (arrow) of small ginger collected from Wado (15) with ATC/CAGA primer (lower) and ATC/
CGGA (upper).
Table 4. Molecular marker bands specific to certain accession of ginger.
Accession
Red ginger and 3 accessions
of small ginger
Small ginger collected
from Wado
Red ginger
Primer
combinations
Number of
specific bands
ATC/CGGA
ATC/CGGT
AGA/CTGA
AGA/CGGA
ATC/CTGA
ATC/CAGA
AAC/CACA
ACC/CAAT
ATC/CAGA
AGG/CGGA
ATC/CGGA
AAC/CACA
2
1
4
2
2
1
2
4
1
2
3
1
CONCLUSION
Based on AFLP marker, the 28 ginger accessions could
be classified into three major clusters. The red ginger
was genetically far from the big ginger, but close to
Size (kb)
154.2; 223.2
81.53
80.4; 158.2; 183.8; 225
82; 292.4
68.9; 108.4.
108.1
248.8; 46.
279.4; 329.8; 331.9; 431.5.
285.5
113.4; 202.4
105; 108; 162.4
> 500
some accessions of small ginger. There was no clear
genetic differentiation between the small and big
types of ginger. The genetic diversity of small ginger
(H = 0.25) is higher than that of the big ginger (Ht =
0.08). Specific molecular marker bands of small ginger
accessions collected from Wado can be recognized.
68
S. Wahyuni et al.
ACKNOWLEDGEMENT
The authors gratefully acknowledge the financial and
technical support by JIRCAS (Japan International
Centre for Agrobiological Sciences) for this research.
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Genetic relationships among ginger accessions based on AFLP

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