Kagoshima University Research Center for the Pacific Islands, Occasional Papers No.34, 105-111, 2001
Part 1, Section 3, Report 4. The Progress Report of the 1999 Survey of the Research Project“Social Homeostasis of Small Islands in an Island-zone”

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RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)ANALYSIS OF WILD SWEET POTATOES AND YAMS PICKED IN
THE YAP ISLANDS
Satoru TAURA, Tadashi ABE, Michio ONJO, Yoshitaka SAKAMAKI,Maki YAMAGUCHI, Mika TAWARA, Atsushi NISHITANI and Kotaro KAWABEAbstract
Several leaves of wild sweet potatoes (Ipomoea spp.) and yams (Dioscorea spp.) were picked invarious parts of the Yap islands. After extraction of DNA from those materials, polymerase chain reactions (PCR) were done using the selected primers for the both plants classifications. Results of random amplified polymorophic DNAs (RAPD) analysis, 4 groups found in 27 wild sweet potatoes lines.
Most of lines were included in I. triloba group. In this study, existence of I. trifida group (cross compatible to sweet potato, I. batatas) recognized. In yams, total 29 lines divided into 5 clusters, consistingof 9 groups. High variability existed.
Key words: Classification, RAPD, wild sweet potato, yam, Yap islands

Introduction
Tubers (taro, yam, sweet potato etc.) were staple foods in the Yap islands. They gradually
were replaced by cereals. Still, they are important crops, especially taro and yam. Taro is cultivated at the hollow called taro patch located near farmer’s house. It takes 4 to 5 years to harvest
them. Yams are cultivated in the forest where small trees are cut off around planting sites. Yamvines are twined about a bamboo pole stood against big tree. Both crops were transmitted to theYap islands together with mankind transmission from Asia. They have a long history. While,sweet potatoes are planted at house garden and roadside. Many different kinds in leaf shape andcolor are observed in the one site. Those sweet potatoes are kept roughly as compared with taroand yam. And most of lines are introduced during the war or after the war. Sweet potato originated in middle South America spread to the world through the three major routes(Y EN, 1974). Its are Kamote, Kumara and Batata routes. Yap located on the end of Kamoteroute. This crop also has a long history. But, it is difficult to distinguish newly introduced ones
and old ones. So, we took notice of wild spices of sweet potato. Sweet potato has several wildspices. It is said that Ipomoea trifida among them is ancestor of sweet potato, Ipomoea batatas
because of cross compatibility. We interest in variation of wild sweet potato on the end of transmission route.
Yams are cultivated from temperate to tropical zone in Asia, Africa and Oceania. About 10spices have been used for starch crop (COURSEY, 1967). Classifications of the cultivated spices
have been studied. We investigated how much variations are existed in the Yap islands.
On this study, we need rapid and easy method to be known about genetic variation. Random
Amplified Polymorphic DNAs (RAPDs) analysis by the polymerase chain reaction (PCR) is106

Satoru TAURA et al.

useful method for this kind of study. RAPD analysis of wild sweet potatoes and yams in the Yapislands were done.

Materials and Methods
Leaf samples
Wild sweet potato (Ipomoea spp.): Twenty seven leaf samples were picked in various partsof the Yap islands (Fig. 1). Four typical spices of wild sweet potatoes were added as a control(Table 1.). I. purpurea group (K150 and K148), I. triloba group (K121 and 7926), I. trifidagroup (K221 and 7930) and not classified group (8203 and K4) .
Yams (Dioscorea spp.): Yam leaf samples were collected from cultivated ones by farmer
mainly. Twenty nine leaf samples including two wild types were picked in various parts of theYap islands (Fig.1). As a control, D. domitorium, D. bulbifera (2 lines), D. alata (Alata andSolo), D. opposita (Shinshu zairai and Yamanoimo), D. japonica (Jinen-jo) and Nepal wild (notFig. 1. Picking sites of wild sweet potatoes and yams in the Yap islands.

Random Amplified Polymorphic DNA (RAPD) Analysis of Wild Sweet Potatoes and Yams Picked in the Yap Islands

107classified) were tested (Table 2.). Picked leaves were stored - 80 ℃ before DNA extraction.
DNA extraction
Total DNA was isolated from frozen leaf sample by modified SDS method. About 1 g leaf
tissue per sample was ground in liquid nitrogen. To remove polyphenol, sample was vortexedwith 5ml of washing buffer (100mM HEPES, 0.1% Polyvinylpyrrolidone K-30, 4% 2Mercaptoethanol) and then centrifuged at 18,000 x g for 3min. This washing process was repeated at least 4 to 5 times up to take out stickiness. The precipitant was solved with 5ofextraction buffer (15% sucrose, 50 mM Tris-HCl, 50 mM EDTA, 500 mM NaCl, pH 8.0) andcentrifuged at 4,600 x g for 5min. The precipitant was resolved with 2of resuspension buffer℃(20 mM Tris-HCl, 10 mM EDTA, pH 8.0) and incubated at 70 for 15 min within 250of
10 % SDS. 1of 7.5 M ammonium acetate was added and cooled on ice for more than 30 min.
After centrifuge for 15 min. at 18,000 x g, the supernatant was transferred to a new tube.
Equivalent cold isopropanol was added and centrifuged for 15 min at 18,000 x g. DNA pellet
was washed by 70 % ethanol and dried and dissolved in 500
TE buffer. 10 g /RNAase
was added and incubated at 55 ℃ for 10 min. Lastly, DNA solution was centrifuged for 5 min.
at 18,000 x g. The supernatant was transferred a 1.5
tube and stored - 20 ℃ until use.
PCR and electrophoresis
PCR was performed in a 0.2
tube for use on a GeneAmp PCR systems 9700 (PEBiosystems). The reaction consisted of 20 ng of DNA, 10 pmol primer, 2.5
10 x PCR buffer,
200 M of each dNTP and Taq polymerase (TAKARA Taq) in 25
volume. Used primers forwild sweet potato were CMN-A17, A19, A47, D1, D2, D3 (Bex) that were selected for classification of its diploid spices (Sakai, unpublished). In yam, OPA-2, C-15, C19, C-20, E-12, W-7,X-1, X-9 (Operon) were used (Shiwachi et al. , 2000). Those are possible to classify into D.
alata, D.opposita and D. japonica. The thermocycler was set at 94 ℃ for 5 min. and repeated 30sec. at 94 ℃, 1 min. for annealing, and 2 min. at 72℃ . That annealing temperature was degraded
4 ℃ every 3 cycles from 56 ℃ to 44 ℃, and the reaction kept on 35 cycles at 40℃ . This was followed by a final cycle of 72 ℃ for 7 min. The amplified DNA sample with dye was loaded on
1% agarose and 0.5 % synergel (Diversified biotech) in 1 x TAE buffer at 60 V for 70min, andstained by ethidium bromide. The electrophoresed gel was photographed under UV light.
RAPD analysis
All DNA samples were tested and divided into some groups by RAPD band patterns. Oneor two samples were picked up from each group. About 16 PCR samples including control samples were tested again for reconfirmation of relationship between them. Polymorphic bands werescored 1 for present or 0 for absent. Cluster analysis using flock average method (UPGMA) was
done by STATISTICA soft wear.

Results and Discussions
Wild sweet potatoes
Total 27 samples were tested and divided into 4 groups, I. triloba group, I. trifida group,8203 group and group of Yaps own, different from control plants (Table 1. and Fig. 2.). I.
purpurea group wasn’t recognized in Yaps samples (Fig. 3.). Most of samples belong to I.
triloba group. In this group, seeds set and seedling spread every where of roadside (YW 4, YW

Satoru TAURA et al.

108

Table 1. Wild sweet potatoes picked in the Yap islands and used control plants
Lines
YW 1
YW 2
YW 3
YW 4YW 5
YW 6
YW 7
YW 8
YW 9
YW 10
YW 11
YW 12
YW 13
YW 14
YW 15
YW 16
YW 17
YW 18
YW 19
YW 20
YW 21
YW 22
YW 23
YW 24
YW 25
YW 26
YW 27
K 150
K 148
K 121
7926
K 221
7930
8203
K4

Site and species
Looraag
Tamargil
Tamargil
Maa
Maa
Machalead
Kanif
Runuw
Kanif
Kanif
Kanif
Madeqdeq
Madeqdeq
Looraag
Looraag
Fanif
Fanif
Fanif
Fanif2)Agri. Exp. Sta.
Agri. Exp. Sta
Maa
ColoniaAgri. Exp. Sta
Wacholab
Runuw
Maqweach
Ipomoea purpurea
Ipomoea purpureaIpomoea triloba
Ipomoea triloba
Ipomoea trilfida
Ipomoea trilfida
unknown
unknown

Band patterns
Yap own1)
Yap own
Yap own
I. triloba
I. triloba
I. triloba
Yap own
I. triloba
I. trilfida
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. triloba
I. trilfida
I. triloba
8203
I. triloba
I. triloba
I. purpurea
I. purpurea
I. triloba
I. triloba
I. trilfida
I. trilfida
820382031): band pattern different from controls
2): Agricultural experiment station

Fig. 2. DNA polymorphisms of wild sweet potatoes detected by amplification of total DNAusing CMN-A17 primer.

Random Amplified Polymorphic DNA (RAPD) Analysis of Wild Sweet Potatoes and Yams Picked in the Yap Islands

109

6 and YW 20). These plants were similar to I. triloba in shape of leaf and flower. This kind ofI. triloba was called “Giliy” in the Yap islands and used for medicine or food formerly. Otherplants in the I. triloba group showed lignification in part of stem (YW 11, YW 12 and YW 14).
Those plants intertwined with fernbrake that grow in barren and damp clayey soil, and bornflowers but didn’t set seeds. Two of 27 samples were found to belong to I. trifida (YW 2 andYW 9). I. trifida is origin of sweet potato, because only I. trifida is able to cross to I. batatas.
Yap islands locate the end of Kamote route on the sweet potato distribution. It is interesting thatI. trifida groups exist in the Yap islands far from the origin.

Fig. 3. Cluster diagram for wild sweet potatoes picked in the Yap islands using RAPD maker.

Yams
29 samples were tested and showed 9 different band patterns (Table 2. and Fig. 4). Theband pattern 1 (YY5) was the largest group and was found in all the islands. The group belongs
to D. alata and D. opposita that are typical cultivated species in tropical zone. Second largest
group (YY2 and band pattern 4) showed different band patterns from used as control plants’. The
YY2 group is the farthest one on genetic distance among the tested materials (Fig. 5). Two wildtypes picked at roadside were located in same group, D. domitorium and D. japonica cluster. InFig. 4. DNA polymorphisms of yams detected by amplification of total DNA using OPA-2 primer.

Satoru TAURA et al.

110

Table 2. Yams (Dioscorea spp.) picked in theYap islands and used control plants
Lines and species

Site

YY 2
YY 3
YY 4
YY 5
YY 6
YY 7 (wild)
YY 8
YY 10
YY 11
YY 12
YY 13
YY 14
YY 15
YY 16
YY 17
YY 18
YY 19
YY 20
YY 21
YY 22
YY 23
YY 24
YY 25
YY 26
YY 27
YY 28YY 29
YY 30
YY 31 (wild)
D. domitorium
D. bulbiferea
D. bulbiferea (Airyam)
D. alata (arata)
D. alata (Soloyam)
D. opposita (Shinsyu zairai)
D. opposita (Yamanoimo)
D. Japonica (Jinen-jo)
Unknown (Nepal wild)

Doomchuy
Doomchuy
Doomchuy
Doomchuy
Doomchuy
Doomchuy
Doomchuy
Doomchuy
Thol
Tamargil
Fitilaw
Amun
Amun
Tamargil
Tamargil
Tamargil
Tamargil
Maa
Qatliw
Balabata
Balabata
Okaw
Runuw
Kanif
Anoth
Anoth
Anoth
Anoth
Madeqdeq

Band patterns and clusters
43411244454531972118176916172Ⅰ
Ⅲ
ⅡⅤ

ⅡⅢⅤⅢ

ⅣⅤⅣ
ⅡⅡⅤⅣFig. 5. Cluster diagram for yams picked in the Yap islands using RAPD maker.

Random Amplified Polymorphic DNA (RAPD) Analysis of Wild Sweet Potatoes and Yams Picked in the Yap Islands

111

result, 29 samples were divided into 5 clusters on 3.8 Euclidian distance.
In this survey, variation of yams in the Yap islands were recognized high and many different kinds of yams were cultivated in one site.

References
COURSEY, D. G. 1967. “Yams.” Longmans, (London) 230 pp.
SHIWACHI, H. et al. 2000. Jpn. J. Trop. Agr., 44:229-237.
Y EN, D. E. 1974. Bernice P. Bishop Museum Bull., 236:1-389.