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Introduction
Ambon Island is part of the Maluku Archipelago of Indonesia. This island has an area of 775 km2, is located approximately 3° S and 128° E, and has a tropical rainfor-est climate (Fig. 1). The main crops grown in the region include coconut, clove, nutmeg, sago, cacao, cassava, sweet potato, pepper, and various vegetables. Durian, langsat, breadfruit, rambutan, pineapple, papaya, banana, and plantain are among popular fruit crops grown on Ambon Island (Girsang and Kastanya, 2016). Although citrus is not an important industrial crop, citrus fruits are commonly utilized for cooking, fresh fruit, and juice. Historically, diversity and utilization of citrus on Ambon were described quite extensively by Rumphius (“The Ambonese Herbal”), a naturalist who lived on the island in the 17th century (Rumphius and Beekman, 2011). Some local citrus accessions that are grown in home gardens show characteristic traits. However, the details of their characteristics remain unknown. For precise elucidation of the traits of each accession, investigations of morphological traits and DNA analysis have been
very effective (Ueda and Higuchi, 2012). New findings of several local citrus genetic resources were reported using both morphological and molecular markers (Nasir et al., 2017; Sharafi et al., 2016; Tshering Penjor et al., 2014a, b).
Among the various types of DNA analyses, the cleaved amplified polymorphic sequence (CAPS) is a simple and reliable method for DNA analysis (Koniec-zny and Ausubel, 1993). It is easy to assay, requires only a few nanograms of DNA for the PCR amplification, and does not require the use of a DNA sequencer. CAPS markers utilize amplified DNA fragments digested with a restriction endonuclease to reveal restriction-site polymorphisms. It has several advantages for cultivar identification because it is inherited in a codominant manner. Recently, Shimada et al. (2014) developed 708 CAPS markers from sequenced-tagged-site (STS) prim-ers with designs based on cDNA. Ninomiya et al. (2015) and Nonaka et al. (2017) reported cultivar identification and parental analysis of new citrus cultivars in Japan us-ing these markers. These results indicate the usefulness of the CAPS markers for phylogenetic studies on citrus.
In the present study, first, we investigated the morphological characteristics of local citrus genetic resources grown on Ambon Island. Then, their CAPS analyses were carried out. Based on the combined results of morphological and molecular markers, the ge-
Communicated by H. GemmaReceived Jan. 11, 2018Accepted Mar. 22, 2018* Corresponding author [email protected]
Exploration and DNA analysis of Local Citrus Genetic Resources on Ambon Island of Indonesia
Simon RAHARJO1, Yuta NATORI 2, Atsu YAMASAKI 3, Akira KITAJIMA4, and Masashi YAMAMOTO2, *
1 Faculty of Agriculture, Pattimura University, Jl. M. Putuhena, Poka, Ambon 97233, Maluku, Indonesia2 Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan3 Institute of Fruit Tree and Tea Science, NARO, Akitsu-Cho, Higashi-Hiroshima, Hiroshima 739-2494, Japan4 Experimental Farm, Graduate School of Agriculture, Kyoto University, Kizugawa, Kyoto 619-0218, Japan
Abstract Exploration of local citrus genetic resources grown on Ambon Island, Maluku, Indonesia was conducted in June and July 2014. Among the 28 accessions investigated, three accessions purchased at a local market were from Seram Island close to Ambon Island. The remaining 25 accessions were local citrus genetic resources grown on Ambon Island. According to the morphological traits, the citrus collected could be grouped into lime (Citrus aurantifolia), purut (C. hystrix), calamondin (C. madurensis), pummelo (C. maxima), sweet orange (C. sinensis), and mandarin (C. reticulata). Cleaved amplified polymorphic sequence (CAPS) analysis of the accessions was conducted, and precise accession identification was possible based on the results of CAPS analysis in conjunction with the morphological traits. The results of CAPS analysis revealed that genetic diversity exists in accessions with the same name on Ambon Island. The genotypes of some accessions resembled to lime, purut, calamondin, sweet orange, and mandarin were differed from those of control species. In contrast, genotypes of all four “Lemon Bali” accessions were identical to those of pummelo. These results indicate the individuality and diversity of citrus genetic resources grown on Ambon Island. Key words: CAPS, Lime, Maluku, Pummelo, Purut
Trop. Agr. Develop. 62(3):115- 123,2018
Trop. Agr. Develop. 62(3)2018116
netic characteristics of these accessions were discussed.
Materials and Methods
Plant materials and morphological characteristicsExploration of local citrus genetic resources on
Ambon Island was conducted in June and July 2014. Although almost all samples were collected from trees grown in backyards, home gardens, or a campus field, some were purchased at a local market (Fig. 1). Leaf and fruit characteristics of each accession were recorded on site just after their collection. For DNA analysis, the eleven accessions shown in Table 1 were used as control materials. All of the control materials were preserved at the Faculty of Agriculture of Kagoshima University,
Japan.
DNA extraction and CAPS analysisDried leaves were provided for total DNA extrac-
tion using the Nucleon Phytopure kit (GE Healthcare Life Science, NJ, USA). In control accessions, total DNA was extracted from fresh leaves using Isoplant II (Nip-pon Gene, Japan).
The CAPS genotypes were identified using four of the citrus CAPS markers (Table 2) developed by Shimada et al. (2014) that showed efficiency for cultivar identification in a previous study (Ninomiya et al., 2015). The PCR reaction mixture of 12.5 µL consisted of 10 ng of template DNA, 10 pmol of each primer, 10× reaction buffer, and 0.5 units of Prime taq DNA polymerase (GeNet Bio, Korea). PCR reactions were performed in a Veriti 200 (Applied Biosystems, ThermoFisher Scien-tific, Waltham, Massachusetts, USA) thermal cycler that was programmed as follows: initial heating at 94°C for 3 min, next two cycles each of denaturation at 94°C for 30 sec, at 62, 60, 58, and 56°C for 30 sec, extension at 72°C for 1 min, followed by 35 cycles for 30 sec at 94 °C, 30 sec at 54 °C, and 2 min at 72 °C, and final extension for 7 min at 72 °C.
The PCR products were digested with restriction enzymes (Takara Bio Inc., Shiga, Japan) under the fol-lowing conditions. Each of 4 µL of the PCR products was mixed with 1.0 µL of the reaction buffer and 2 to 3 units of the restriction enzyme, and then the final volume was adjusted to a total of 10 µL with sterile water. After diges-tion at 37°C for more than 4 h, the digested products were electrophoresed on 1.5% agarose gels (Seakem GTG Agarose, Takara Bio, Otsu, Japan), and stained with GelRed (Biotium, Hayward, California, USA). The resulting bands were detected under UV light. Based on the results of the banding pattern of gel electrophoresis, each genotype was designated as “aa”, “ab”, and “bb” according to the fragment size (Table 2).
Sumatra
Kalimantan
Java
SulawesiPapua
Ambon I.Seram I.
N128 ̊ N128 ̊15
S3 ̊30
S3 ̊45
1
234567
8910
11
INDONESIA
AMBON ISLAND
Ambon City
Table 1. Control accessions used in the DNA analysis.
No. Latin name Common nameC1 Citrus hystrix DC purutC2 C. aurantifolia (Cristm.) Swingle mexican limeC3 C. aurantium L. ‘Kaiseito’ sour orangeC4 C. limon (L.) Burm. f. ‘Allen Eureka’ lemonC5 C. maxima (Burm.) Merr. ‘Banpeiyu’ pummeloC6 C. medica L. ‘Marubusshukan’ citronC7 C. sinensis (L.) Osbeck ‘Hamlin’ sweet orangeC8 C. reticulata Blanco ‘Yoshida Ponkan’ ponkan (mandarin)C9 C. sunki (Hayata) hort. ex Tanaka sunki (mandarin)C10 C. reshni hort. ex Tanaka cleopatra (mandarin)C11 C. madurensis Lour. calamondin
Fig. 1. Map of Indonesia and citrus collection sites in Ambon Island.
Note: 1=Pasar Mardika (Market); 2=Negeri Lama; 3=Near Hunuth; 4=Balai Benih Induk Hortikultura (Telaga Kodok); 5=Univ. Pattimura; 6=Near Univ. Pattimura; 7=Taeno Bawah Village; 8=Taeno Village; 9=Taeno Atas Village; 10=Telaga Pange Village; 11=Keranjang Village.
Raharjo et al.: Exploration and DNA analysis of local Citrus in Ambon Island of Indonesia 117
Results and Discussion
Local citrus genetic resources on Ambon Island, their morphological traits, and their usage
In Ambon Island, citrus is referred to as “Lemon” in general; the common name of pummelo (C. maxima (Burm.) Merr.) is “Lemon Bali”. Thus, “Lemon” on Ambon Island does not mean common lemon (C. limon (L.) Burm. f). There is no citrus orchard on Ambon Island, thus all citrus has been cultivated in home gar-dens. However, a citrus orchard exists on Seram Island located close to Ambon Island, where sweet orange and mandarin are grown.
There are six major citrus types on Ambon Island: “Lemon Nipis”, “Lemon Purut”, “Lemon Suwanggi”, “Lemon China”, “Lemon Bali”, and “Lemon Kisar”. The meanings of these names are as follows; Nipis: no specif-ic meaning in Indonesian languages, Purut: “rough skin” in Indonesian or Malay language, Suwanggi: “witch or witchcraft” in local Ambonese language, China: a name of country, Bali: a name of the island in eastern Java, and Kisar: a name of an island in southwestern Maluku. Almost all types on the island have been propagated by seedlings, which means the existence of diversity within each type. In our investigation, all six types could be found and analyzed. The results are shown in Tables 3 and 4 and Fig. 2. Among 28 accessions investigated, three accessions (#1, 3, and 4) purchased at a local market were from Seram Island. The remaining 25 ac-cessions were local citrus genetic resources grown on Ambon Island.
Morphological traits including the flavor of “Lemon Nipis” were similar to those of lime (C. aurantifolia (Cristm.) Swingle). The main uses of the fruits of “Lemon Nipis” are for cooking and juice, the same as for normal lime, because of the low pH of its juice (very sour) and excellent flavor. Another minor use of “Lemon Nipis” is for medicinal purposes. Both “Lemon Teh” and “Lemon Papaya” were also similar to lime. The fruit shape of both
accessions was spheroid, and the former had seedless fruit. The meaning of Teh is “tea” in Indonesian as well as Ambonese language.
There were two types similar to purut (C. hystrix DC.): “Lemon Purut” and “Lemon Suwanggi”. Although the fruit of “Lemon Suwanggi” seemed to be more sour and more juicy than that of “Lemon Purut” according to the interview, distinctive morphological differences be-tween both types were not clear. The leaves have been used for specific flavors of spicy food. All accessions investigated in the present study possessed a distinctive aroma of their leaves. Both “Lemon Purut” and “Lemon Suwanggi” could be classified into two separate types according to the leaf shape: large petiole wing and small petiole wing. In “Lemon Purut”, #4 and #13 had a large petiole wing whereas #7, #18, and #27 had a small petiole wing. In “Lemon Suwanggi”, #12 and #16 showed small and large petiole wings, respectively. In general, the petiole wing of typical C. hystrix is very large (Swingle, 1967), those accessions with a small petiole wing might have arisen as chance seedlings derived from accessions with a large petiole wing.
Morphological traits of “Lemon China” were similar to those of calamondin (C. madurensis Lour.). It is also an important seasoning on Ambon Island the same as in the Philippines (Coronel, 2011). Diversity of morphological traits in four accessions investigated in the present study was not clear. However, the fruit of #5 and #17 is small whereas that of #8 is large according to the interview. All accessions had small, round, and seedy fruits with a low pH of juice.
“Lemon Bali” was a kind of pummelo (C. maxima). All four accessions showed typical pummelo character-istics: large fruit, hard skin, characteristic aroma, and cream monoembryonic seed (Hodgson, 1967). Each accession showed a characteristic flesh color: cream to red. The number of seeds of #24 was fewer than that of other “Lemon Bali” accessions.
Although the morphological traits, including the flavor of “Lemon Kisar”, were similar to those of sweet orange (C. sinensis (L.) Osbeck), the taste of its fruits was bitter. This is different from the characteristics of sweet orange. Thus, “Lemon Kisar” is considered to be a relative of sweet orange.
Among three mandarin accessions (#1, 15, and 22), only fruits of #1 “Lemon Manis Masohi” were mature. Thus, the traits of the remaining two accessions (#15 “Lemon Siem” and #22) at the mature stage could not be determined. However, fruits of all three accessions pos-sessed the same characteristic flavor. Those mandarins
Table 2. Characteristics of the CAPS markersz used in this study.
Marker name
PCR product size (bp)
Restriction enzyme
Polymorphic allele size (bp)
a bTf0235 700 HaeIII 700 450Tf0420 400 HaeIII 400 200Tf0419 700 PvuII 700 400Gn0029 450 HinfI 450 250z Shimada et al. (2014).
Trop. Agr. Develop. 62(3)2018118
Tab
le 3
. L
ist o
f inv
estig
ated
loca
l citr
us g
enet
ic r
esou
rces
on
Am
bon
Isla
nd a
nd th
eir
tree
and
leaf
cha
ract
eris
tics.
No.
Date
Acce
ssio
n
Type
Colle
cted
site
Orig
inAl
titud
eLa
titud
eLo
ngitu
deTr
eeLe
af
(y/m
/d)
(m)
Leaf
blad
eW
ing
Heig
htW
idth
Vigo
rHa
bit
Spin
eLe
ngth
Wid
thLe
ngth
Wid
th
(m
)(m
)(m
m)
(mm
)(m
m)
(mm
)
320
14/6
/24
Lem
on N
ipis
Lim
ePa
sar M
ardi
ka (M
arke
t)Se
ram
Islan
d-
--
--
--
-
--
-
-11
2014
/6/2
5Le
mon
Nip
isLi
me
near
Hum
ut12
0 S0
3.379
42E1
28.11
921
33
Med
ium
Slig
htly
uprig
htSh
ort
65
.6 36
.5
12.9
5.4
2020
14/6
/26
Lem
on N
ipis
Lim
eTa
eno V
illag
e17
4S0
3.335
26E1
28.10
502
55
Med
ium
Slig
htly
spre
adin
gSh
ort
55.1
30.9
7.5
3.9
2120
14/6
/26
Lem
on N
ipis
Lim
e
Ambo
n Is
land
--
--
--
-Sh
ort
70.6
42.5
9.3
3.8
6
2014
/6/2
5Le
mon
Teh
Lim
eNe
grila
ma
0
S03.3
7658
E128
.1404
82.5
3M
ediu
mSp
read
ing
Shor
t
91.9
52.9
4.4
2.5
28
2014
/7/1
4Le
mon
Pap
aya
Lim
eSe
ram
Islan
dca
. 20
--
2.52.5
Vigo
rSp
read
ing
Med
ium
--
-
-4
2014
/6/2
4Le
mon
Pur
utPu
rut
Pasa
r Mar
dika
(Mar
ket)
Sera
m Is
land
--
--
--
--
40
.9 22
.8
37.2
20.8
720
14/6
/25
Lem
on P
urut
Puru
tNe
grila
ma
0
S03.3
7658
E128
.1404
8-
--
--
45
.9 27
.0
9.9
4.4
1320
14/6
/25
Lem
on P
urut
Puru
tNe
ar H
umut
131
S03.3
7888
E128
.1203
92.5
3Sl
ight
ly we
akSp
read
ing
None
36.7
27.1
30.0
23.1
1820
14/6
/25
Lem
on P
urut
Puru
tNe
ar U
niv.
Patti
mur
a -
--
0.50.3
--
Shor
t
43.8
24.6
10
.0 4.0
27
2014
/6/2
6Le
mon
Pur
utPu
rut
Taen
o Baw
ah V
illag
e
161
S03.3
8683
E128
.1053
37
1M
ediu
mUp
right
Med
ium
42.5
23.2
7.4
2.9
1220
14/6
/25
Lem
on S
uwan
ggi
Puru
tne
ar H
umut
120
S03.3
7942
E128
.1192
10.8
0.5M
ediu
mUp
right
Long
102.0
44
.1 6.7
3.2
16
2014
/6/2
5Le
mon
Suw
angg
iPu
rut
Balai
Ben
ih In
duk
Hokt
ukuc
tura
200
S03.3
7590
E128
.1114
97
3Vi
gor
Uprig
htLo
ng64
.7 36
.4
49.4
31.8
1920
14/6
/25
Lem
on S
uwan
ggi
Puru
t
Sera
m Is
land
--
--
--
-No
ne
--
-
-2
2014
/6/2
4Le
mon
Chi
naCa
lamon
din
Pasa
r Mar
dika
(Mar
ket)
Ambo
n Is
land
--
--
--
--
-
-
--
520
14/6
/25
Lem
on C
hina
Calam
ondi
nNe
grila
ma
0
S03.3
7658
E128
.1404
82
2Sl
ight
ly we
akSp
read
ing
None
45
.7 24
.1
5.8
2.1
820
14/6
/25
Lem
on C
hina
Calam
ondi
nUn
iv. of
Patt
imur
a
20S0
3.391
80E1
28.11
604
11
Med
ium
Spre
adin
gNo
ne
60.2
33.7
7.7
2.1
17
2014
/6/2
5Le
mon
Chi
naCa
lamon
din
Near
Uni
v. Pa
ttim
ura
--
-2
2M
ediu
mSl
ight
ly up
right
None
60.0
33.0
7.5
2.4
920
14/6
/ 25
Lem
on B
aliPu
mm
eloNe
ar H
umut
12
0S0
3.378
88E1
28.12
127
78
Med
ium
Slig
htly
spre
adim
gNo
ne10
5.6
61.8
24
.4 14
.7
1020
14/6
/25
Lem
on B
aliPu
mm
eloNe
ar H
umut
120
S03.3
7888
E128
.1212
710
8Sl
ight
ly vig
orSl
ight
ly up
right
None
10
2.5
77.1
20
.5 12
.7
2320
14/6
/26
Lem
on B
aliPu
mm
eloTa
eno V
illag
e17
4S0
3.335
26E1
28.10
502
78
Med
ium
Spre
adin
gNo
ne91
.7 60
.4 23
.8 16
.6 24
2014
/6/2
6Le
mon
Bali
Pum
melo
Taen
o Atla
s Vill
age
213
S03.3
8124
E128
.1024
512
10M
ediu
mSl
ight
ly up
right
None
96.5
64.8
23
.0 19
.4
1420
14/6
/25
Lem
on S
unki
st (K
isar)
Swee
t ora
nge
Balai
Ben
ih In
duk
Hokt
ukuc
tura
East
Java
200
S03.3
7590
E128
.1114
95
5M
ediu
mSp
read
ing
None
79.0
36.2
6.8
2.8
2520
14/6
/26
Lem
on K
isar
Swee
t ora
nge
Tera
ga P
ange
Vill
age
92
S03.3
8679
E128
.1024
75
7M
ediu
mSp
read
ing
None
65.5
36.3
7.0
2.9
1
2014
/6/2
4Le
mon
Man
is M
asoh
iM
anda
rinPa
sar M
ardi
ka (M
arke
t)Se
ram
Islan
d-
--
--
--
-
--
-
-15
2014
/6/2
5Le
mon
Siem
Man
darin
Balai
Ben
ih In
duk
Hokt
ukuc
tura
200
S03.3
7590
E128
.1114
95
5M
ediu
mM
ediu
mNo
ne
73.4
35.7
6.8
1.6
22
2014
/6/2
6Un
know
nM
anda
rinTa
eno V
illag
e17
4S0
3.335
26E1
28.10
502
43
Slig
htly
vigor
Slig
htly
uprig
htNo
ne61
.7 35
.2 6.1
1.7
2620
14/6
/26
Unkn
own
Unid
entifi
edKe
ranj
ang
Villa
ge13
1 S0
3.392
38E1
28.09
967
68
Slig
htly
weak
Spre
adin
gM
ediu
m77
.6 39
.4 7.4
2.5
Raharjo et al.: Exploration and DNA analysis of local Citrus in Ambon Island of Indonesia 119
Tab
le 4
. F
ruit
char
acte
rist
ics
of lo
cal c
itrus
gen
etic
res
ourc
es o
n A
mbo
n Is
land
.
No.
Acc
essio
nFr
uitSe
edNo
te
Sk
in co
lorFr
uit su
rface
Fles
h colo
rFl
avor
Peeli
ngBi
tter -
ness
Swee
t -ne
ssSo
urne
ssDi
amete
r(m
m)
Heigh
t(m
m)
D/H
index
Peel
thick
ness
No
. of
segm
ents
Brix
ofjui
cepH
ofjui
ceEm
bryo
color
Embr
yony
Num
ber
(mm
)
3Le
mon
Nipi
sYe
llowi
sh or
ange
Smoo
thYe
llowi
sh
gree
nLi
me
Diffi
cult
None
Low
High
46.7
47.1
99.2
2.1
10.4
8.0
2 .4
Crea
mPo
ly10
.4
11Le
mon
Nipi
sGr
een
Sligh
tly sm
ooth
Gree
nLi
me
Diffi
cult
None
Low
Med
ium44
.1 46
.8 94
.2 3.1
11
.2 7.8
2.5
Cr
eam
, Pale
gree
nPo
ly14
.8 20
Lem
on N
ipis
Gree
nSm
ooth
Gree
nLi
me
Diffi
cult
None
Low
High
36.9
37.5
98.4
3.9
10.3
6.9
2.3
Crea
m, P
ale gr
een
Poly
6.8
21Le
mon
Nipi
sGr
een
Sligh
tly sm
ooth
Gree
nLi
me
Diffi
cult
None
Low
High
47.8
47.6
100.4
3.7
11
.7 7.4
2.5
Cr
eam
Poly
12.0
6Le
mon
Teh
Yello
wish
gree
nSm
ooth
Crea
mLi
me
Diffi
cult
None
Low
Med
ium56
.5 68
.8 82
.1 4.4
11
.0 6.9
2.4
-
-0.0
28
Lem
on P
apay
aLi
ght y
ellow
Med
iumW
hite
-Di
fficu
ltSli
ght
-Hi
gh5.9
7.412
5.43.5
16.5
--
Crea
m, P
ale gr
een
-2.5
4Le
mon
Pur
utYe
llowi
sh gr
een
Roug
hYe
llow
Puru
tDi
fficu
ltNo
neLo
wHi
gh35
.3 30
.7 11
4.9
2.7
9.2
11.1
2.5
Pale
gree
n, Gr
een,
Crea
mPo
ly7.6
7Le
mon
Pur
ut-
--
Puru
t-
--
--
--
--
--
--
-No
fruit
13Le
mon
Pur
ut-
--
Puru
t-
--
--
--
--
--
--
-No
fruit
18Le
mon
Pur
ut-
--
Puru
t-
--
--
--
--
--
--
-No
fruit
27Le
mon
Pur
ut-
--
Puru
t-
--
--
--
--
--
--
No fr
uit12
Lem
on Su
wang
gi-
--
Puru
t-
--
--
--
--
--
--
-No
fruit
16Le
mon
Suwa
nggi
Gree
nRo
ugh
Gree
nPu
rut
Diffi
cult
--
-42
.4 57
.2 74
.1 7.0
7.3
-
-Pa
le gr
een
Poly
12.3
Imm
ature
19Le
mon
Suwa
nggi
Gree
nRo
ugh
Yello
wPu
rut
Diffi
cult
None
Low
High
41.7
55.0
75.8
4.1
8.5
9.3
2.5
Crea
m, P
ale gr
een
Poly
25.0
2Le
mon
Chi
naYe
llowi
sh gr
een
Sligh
tly sm
ooth
Oran
geCa
lamon
dinM
edium
None
Low
High
31.7
29.3
108.4
1.7
8.4
7.4
2.5
Pa
le gr
een,
Gree
nPo
ly8.4
5
Lem
on C
hina
Gree
nSli
ghtly
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ange
Calam
ondin
Diffi
cult
None
Low
High
25.8
23.9
108.3
1.4
7.5
8.2
2.4
Pa
le gr
een,
Gree
nPo
ly6.0
8
Lem
on C
hina
Gree
nSli
ghtly
smoo
thOr
ange
Calam
ondin
Diffi
cult
None
Low
High
25.4
23.4
108.8
1.6
8.4
8.5
2.5
Pa
le gr
een,
Gree
nPo
ly6.2
Im
matu
re17
Lem
on C
hina
Gree
nSm
ooth
Yello
wish
or
ange
Calam
ondin
Diffi
cult
None
Low
High
22.4
21.8
102.4
1.2
6.2
7.6
2.5
Pa
le gr
een,
Gree
nPo
ly6.2
9Le
mon
Bali
Yello
wish
gree
nM
edium
Crea
mPu
mm
eloDi
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ltM
edium
Med
iumLo
w15
1.8
143.6
10
5.7
12.6
10.5
8.5
3.7
Crea
mM
ono
50<
10Le
mon
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Yello
wSm
ooth
Red
Pum
melo
Diffi
cult
Med
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edium
Low
124.8
11
2.2
111.3
9.7
17
.0 -
-Cr
eam
Mon
o50
<23
Lem
on B
aliYe
llowi
sh gr
een
Smoo
thCr
eam
, Pa
le pin
kPu
mm
eloDi
fficu
ltM
edium
Low
Low
162.6
14
5.0
112.1
21
.0 14
.0 -
-Cr
eam
Mon
o50
<
24Le
mon
Bali
Yello
wish
gree
nSm
ooth
Pink
Pum
melo
Diffi
cult
Med
iumLo
wLo
w13
1.9
114.2
11
5.3
25.0
15.0
--
Cre
amM
ono
12.0
14Le
mon
Sunk
ist
(Kisa
r)Gr
een
Smoo
thYe
llowi
sh
oran
geSw
eet o
rang
eDi
fficu
ltLo
wLo
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edium
67.5
66.4
101.7
5.3
11
.3 9.0
3.5
Cr
eam
, Pale
gree
nM
ono?
10.7
25Le
mon
Kisa
rGr
een
Sligh
tly sm
ooth
Yello
wSw
eet o
rang
eDi
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ltLo
wLo
wM
edium
69.6
72.2
96.4
7.4
10.3
7.6
3.3
Crea
m, P
ale gr
een
Poly
10.3
Imm
ature
1Le
mon
Man
is M
asoh
iYe
llowi
sh gr
een
Smoo
thOr
ange
-Ea
syNo
neLo
wLo
w70
.1 58
.4 12
0.0
2.7
7.2
8.3
4.3
Pale
gree
n, Gr
een
Poly
10.8
15Le
mon
Siem
Gree
nSm
ooth
Gree
n-
Diffi
cult
--
-46
.3 46
.0 10
0.7
2.9
13.0
--
Pale
gree
n, Gr
een
Poly
13.0
Imm
ature
22Un
know
nYe
llow
Smoo
thYe
llow
-Di
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lt-
--
39.7
38.7
101.9
3.0
11
.3 -
-Pa
le gr
een,
Gree
nPo
ly6.8
Im
matu
re26
Unkn
own
Gree
nM
ediu
mOr
ange
-Di
fficu
ltNo
neLo
wHi
gh50
.7 53
.4 64
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9.5
8.8
2.5
Cr
eam
, Pale
gre
enM
ono
3.0
Trop. Agr. Develop. 62(3)2018120
Fig. 2. Photographs of local citrus genetic resources on Ambon Island. 1 - 28: see Table 3.
1 2 3 4
56
79
10 11
14
15
16 17
18
20
21 22
2324
25 2627
28
Raharjo et al.: Exploration and DNA analysis of local Citrus in Ambon Island of Indonesia 121
might be very close to each other. The meanings of Manis Masohi and Siem are “sweet citrus from Masohi (a name of town in central Seram) and old name for Thailand in Indonesian language, respectively. The accession #26 was an acid citrus the same as lime. However, its morphologi-cal traits were completely different from those of lime. We could not morphologically determine its position among the six major citrus types on Ambon Island.
Comprehensive information about the origins of local Ambonese citrus species is not available. Most of the local citrus species in this study have been known for a long time in Ambon, as described by Rumphius (Rumphius and Beekman, 2011). However, some of them may be brought to Ambon from other places, such as “Lemon Manis”, “Lemon Siem”, “Lemon Sunkist”, and “Lemon Kisar”. In the seventeenth century, “Lemon Bali”, which was called “The Pompelmoose Tree” (Rumphius and Beekman, 2011), was described to be present thoughout eastern Indonesia. “Lemon Papaya” was found in Ambon uplands and was called “Lemon Java” by the Malay (Rumphius and Beekman, 2011). It was abundant in Banda Island and was thought to be native of Java.
In Ambon Island and throughout Maluku, citrus has various uses, including for fresh fruits (“Lemon Bali”, “Lemon Manis”, “Lemon Siem”, “Lemon Sunk-ist”, and “Lemon Kisar”), squeezed for drinks (“Lemon Nipis” and “Lemon The”), for making “rujak” or mixed fruits with hot sauce (“Lemon Bali”), cake ingredient (“Lemon Manis” and “Lemon Siem”), fish and other sea-food preparation (“Lemon China”, “Lemon Suwanggi”, “Lemon Nipis”, and “Lemon Papaya”), meat preparation (“Lemon China” and “Lemon Suwanggi”), soup and vegetable preparation (“Lemon Nipis”, “Lemon China” and “Lemon Suwanggi”), chili hot sauce or “sambal” (“Lemon Nipis”, “Lemon Purut”, and “Lemon China”), as well as oral and skin application for medicinal usages. Various uses of Ambonese local citruses have been de-scribed historically by Rumphius in his “The Ambonese Herbal” (Rumphius and Beekman, 2011), including the use of bark and flower as a natural dye (“casumba”) and the wood for making tools and furniture.
“Lemon Nipis” has various medicinal uses in Am-bon Island, for example for treating and relieving fever, cold, cough, nose irritation, and also for caring skin and hair. Meanwhile, “Lemon Purut” also is used for treating fever, cold, as well as hair and skin cares, in addition to cholesterol alleviation and appetite stimulation (leaves). “Lemon Suwanggi” has similar uses as “Lemon Purut”. The medicinal usefulness of those citrus species may relate to their flavonoid contents including hesperidin,
tengeritin, naringin, eriocitrin and eriocitrocide, in addi-tion to other medicinally useful compounds in the fruits or leaves (Indonesian Spice Council, 2011).
CAPS analysis of the local citrus genetic resources on Ambon Island
Polymorphisms were observed in all four primer and restriction enzyme combinations in CAPS analysis (Table 2). We classified the local accessions based on the results of genotype combinations. Local accessions were divided into 7 types (Table 5). Genotypes of all four “Lemon Bali” were identical to those of C. maxima. #21 “Lemon Nipis”, “Lemon Teh”, “Lemon Papaya”, #18 and 27 “Lemon Purut”, #5, 8, and 17 “Lemon China”, #26 “Unknown”, C. hystrix, C. auantifolia, C. medica, and C. madurensis belonged to Type 3. Control species, C. hys-trix, C. auantifolia, C. medica, and C. madurensis, could not be distinguished from each other in the present study. The types of the remaining accessions were dif-ferent from those of the control species. As mentioned above, morphological traits of “Lemon Nipis”, “Lemon Purut”, and “Lemon China” were similar to those of C. aurantifolia, C. hytrix, and C. madurensis. Thus, genotypes of each local citrus genetic resource were compared with those of control species showed similar morphology.
Four “Lemon Nipis” were classified into three types in CAPS analysis. Recently, large genetic variations were reported in lime-type accessions in Bhutan (Tshering Penjor et al., 2014a, b) and Micronesia (Yamamoto et al., 2018). The diversity of lime-type (“Lemon Nipis”) acces-sions grown on Ambon Island was also revealed in the present study. Seedling propagation is very common on Ambon Island, and propagation by grafting and budding is sometimes practiced mainly with commercial sweet orange and mandarin (Raharjo, unpublished). Zygotic seedlings derived from lime-type accessions adapted to a tropical climate might be better. Although the genotype combinations of #21 “Lemon Nipis” were identical to those of C. aurantifolia, genotype combinations of remaining three accessions of “Lemon Nipis” were different from those of C. aurantifolia. Also the type of “Lemon Teh” and “Lemon Papaya” was identical to that of C. aurantifolia. Further analysis is needed to determine whether these accessions are true C. aurantifolia or not.
The morphological traits of Lemon Purut” and “Lemon Suwanggi” were similar to those of C. hystrix. As in “Lemon Nipis”, the type of some “Lemon Purut” and “Lemon Suwanggi” was different from that of C. hystrix. “Lemon Purut” and “Lemon Suwanggi” could not be
Trop. Agr. Develop. 62(3)2018122
clearly distinguished from each other. However, genetic diversity existed among them. The accession #4 “Lemon Purut”, #13 “Lemon Purut”, and #16 “Lemon Suwanggi” with large petiole wings showed the same genotypes. These results indicate clear genetic differences between the accessions with large and small petiole wings. Diversity within accessions with a small petiole wing was revealed, although #7, 18 and #27 showed the same genotype combinations.
The morphological traits of “Lemon China” were similar to those of C. madurensis. There were two types of genotype combinations; those of #5, 8, and 17 were identical to those of C. madurensis and the type of geno-types were different in #2. According to the interview,
#8 was a large fruit type and #5 and #17 were small fruit types. However, the genotypes of those three acces-sions were the same. CAPS analysis using more primers should be carried out to clarify the genetic differences between large and small fruit types.
As mentioned above, all “Lemon Bali” and C. maxima control belonged to type 5. They showed typical morphological traits of C. maxima. Hence, “Lemon Bali” is considered to be true C. maxima.
The type of genotype combinations of “Lemon Kisar” and “Lemon Sunkist” (Kisar) were the same. Both accessions possessed fruit characteristics that were similar to C. sinensis. Thus, both accessions were considered to be identical or similar to each other. Based
Table 5. The genotypes of four CAPS markers and the type of genotype combinations of local citrus genetic resources on Ambon Island.
No. Accession CAPS marker/restriction enzymez Type of genotype combinations
Tf0235 Tf0420 Tf0419 Gn0029/HaeⅢ /HaeⅢ /PvuⅡ /HinfⅠ
3 Lemon Nipis aa aa ab aa 111 Lemon Nipis aa aa ab aa 120 Lemon Nipis aa aa aa aa 221 Lemon Nipis aa aa bb aa 3
6 Lemon Teh aa aa bb aa 328 Lemon Papaya aa aa bb aa 3
4 Lemon Purut aa aa ab aa 17 Lemon Purut aa aa bb aa 3
13 Lemon Purut aa aa ab aa 118 Lemon Purut aa aa bb aa 327 Lemon Purut aa aa bb aa 312 Lemon Suwanggi aa ab ab aa 416 Lemon Suwanggi aa aa ab aa 119 Lemon Suwanggi aa aa ab aa 12 Lemon China aa aa ab aa 15 Lemon China aa aa bb aa 38 Lemon China aa aa bb aa 3
17 Lemon China aa aa bb aa 39 Lemon Bali bb aa bb bb 5
10 Lemon Bali bb aa bb bb 523 Lemon Bali bb aa bb bb 524 Lemon Bali bb aa bb bb 514 Lemon Sunkist (Kisar) ab ab bb bb 625 Lemon Kisar ab ab bb bb 6
1 Lemon Manis Masohi aa ab bb ab 715 Lemon Siem aa ab bb ab 722 Unknown aa ab bb ab 726 Unknown aa aa bb aa 3
ControlC1 Citrus hystrix aa aa bb aa 3C2 C. aurantifolia aa aa bb aa 3C3 C. aurantium ab aa bb ab 8C4 C. limon ab aa bb aa 9C5 C. maxima bb aa bb bb 5C6 C. medica aa aa bb aa 3C7 C. sinensis ab aa ab ab 10C8 C. reticulata aa ab ab ab 11C9 C. sunki aa ab ab bb 12
C10 C. reshni aa ab bb aa 13C11 C. madurensis aa aa bb aa 3
z Shimada et al. (2014).
Raharjo et al.: Exploration and DNA analysis of local Citrus in Ambon Island of Indonesia 123
on CAPS analysis, however, types of genotype combina-tions of both accessions were different from those of the C. sinensis control.
Since three mandarin accessions “Lemon Siem”, “Lemon Manis Masohi”, and #22 “Unknown”, showed the same genotypes, they may be identical or very close to each other. However, their genotypes were different from those of some mandarins, C. reticulata, C. sunki, and C. reshni controls. The accessions #26 “Unknown”, C. hystrix, C. auantifolia, C. medica, and C. madurensis showed the same genotype combinations. However, it was difficult to consider the accession #26 as these control species since their fruit traits were completely different.
Conclusion
In the present study, we identified of local citrus accessions grown on Ambon Island using morphological and molecular markers. The combined results offer valu-able information. Several local citrus accessions showed characteristic genotypes on CAPS analysis. The results of CAPS analysis revealed that genetic diversity exists in accessions with the same name on Ambon Island. In addition, the genotypes of some accessions were differ-ent from control accessions, although the morphological traits of each local citrus accession resembled those of control ones, i.e., “Lemon Nipis” and lime; “Lemon Pu-rut”, “Lemon Suwanggi”, and purut; “Lemon China” and calamondin; and “Lemon Kisar” and sweet orange. Since seedling propagation is very common on Ambon Island, the seedlings adapted to this island survived and/or were selected. Ambon Island has a tropical rainforest climate. Thus, those local accessions are considered to be very important genetic resources that may adapt well to a tropical climate. In addition, all “Lemon Bali” accessions were considered to be true pummelo (C. maxima) based on the results of morphological as well as molecular markers. They are also important genetic resources because each “Lemon Bali” accession had different characteristic fruit traits. These results indicate the individuality and diversity of citrus genetic resources grown on Ambon Island.
In the present study, valuable new genetic informa-tion was obtained based on the results of CAPS analysis, although we used only four primer/restriction enzyme combinations. A number of CAPS markers are available in citrus (Shimada et al., 2014). Hence, CAPS analysis using more markers of local citrus grown on Ambon Island is necessary to identify each accession and their phylogenetic relationships.
Acknowledgements
We gratefully acknowledge Dr. T. Shimada of the NARO Institute of Fruit Tree and Tea Science for valu-able advice. A part of this work was supported by JSPS KAKENHI, a Grant-in-Aid for Scientific Research (B), Grant Number 24405025.
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