Exploration and DNA analysis of Local Citrus Genetic ...

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).


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


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

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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

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25 2627

28


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


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).


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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