Kagoshima University Research Center for the Pacific Islands Occasional Papers No.39, 77-81, 2003
Section 2, Report 8. The Progress Report of the 2000 and 2001 Survey of the Research Project“Social Homeostasis of Small Islands in an Island-zone”

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RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSISOF YAMS COLLECTED IN THE YAP ISLANDS
AND THE ULITHI ISLANDS.
TAURA Satoru 1) , PARK Byoung-Jae2) , ICHITANI Katuyuki2), ONJO Michio 2), andKAWABE Kotaro1)

AbstractYam (Dioscorea spp.) leaf samples were collected from cultivated crop in the Yap islands (Yapproper) and the Ulithi islands (Mogmog, Asor, Fassarai and Falalop). Most of the materials have been
identified based on morphological characters as D. alata L., but some other species were found, aswell. For genetic analysis, total DNA was obtained by SDS method and polymerase chain reaction(PCR) was done to amplify the segment of DNA generated in the presence of RAPD primers. Within
the 36 samples, due to RAPD markers band polymorphism, 14 band patterns were detected. These results suggest that the presence of large genetic diversity in D. alata from the state of Yap and that
RAPD markers might work well in detecting it.
Keywords: classification, RAPD, yam, Yap islands, genetic resources

Introduction
Tubers (taro, yam, sweet potato etc.) were staple foods in the state of Yap. The weights oftubers transferred to cereals gradually. Still, they are important crops, especially taro and yam.
Taro is cultivated at the hollow called the taro patch located near the farmer’s house. From planting time to harvest takes 4 to 5 years. Yams are cultivated in the forest where small trees are cut
down at planting sites. Yam vines are twined about a bamboo pole stood against a big tree. Yamand taro were transmitted to the state of Yap along with mankind transmission from Asia. So that
we accept the crops have a long history in this area.
Yams as crop exist from temperate to tropical zones in Asia, Africa and Oceania. About 10spices have been used for starch (COURSEY 1967). Classifications of the cultivated species havebeen done by identifying morphological characters. Recently, classification using DNA markers
has been tried. The existence of D. alata, D. opposita and D. japonica is also confirmed byRandom Amplified Polymorphic DNAs (RAPDs) analyses (S HIWACHI et al. 2000). Yam breeding programs need a rapid and easy method to detect genetic variations of geographically different materials. The RAPD analysis by the polymerase chain reaction (PCR) was successfully usedon many crops. So RAPD analyses of yams collected in the state of Yap were done.

1)
2)

Research Center for Life Science Resources, Kagoshima University. Kagoshima 890-0065, Japan.
Faculty of Agriculture, Kagoshima University. Kagoshima 890-0065, Japan.

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TAURA Satoru, PARK Byoung-Jae, I CHITANI Katuyuki, ONJ O Michio, and KAWABE Kotaro

Materials and Methods
Leaf samples
Yam leaf samples were collected from cultivated ones by farmers. Most samples are identified D. alata by morphological characters. A total of 36 samples were collected including 3 different species. Table 1 shows collecting sites and control plants used for testing (D. bulbifera as
Airyam, D. alata as Arata, and D. japonica as Jinen-jo). Collected leaf samples were stored atTable 1. Collected Yams (Dioscorea spp.) in theYap islands and the Ulithi islands
Lines and species
YYA 2
D. alata
YYA 3D. alata
YYA 5
D. alata
YYA 6
D. alata
YYA 7
D. alata
YYA 8D. alata
YYA 9
D. alata
YYA 10D. alata
YYA 11
D. alata
YYA 12D. alata
YYA 13D. alata
YYA 14D. alata
YYA 15D. alata
YYA 16
D. alata
YYA 17
D. alata
YYA 18D. alata
YYA 19D. alata
YYA 20D. alata
YYA 21
D. alata
YYA 22D. alata
YYA 23D. alata
YYA 24D. alata
YYA 25D. alata
YYA 26
D. alata
YYA 27
D. alata
YYA 28D. alata
YYA 29D. alata
YYA 30D. alata
YYA 31
D. alata
YYA 32D. alata
YYA 33D. alata
YYA 34D. liebretsiana
YYA 35D. liebretsiana
YYA 36
D. esculenta
YYA 37
D. bulbiferea
YYA 38
D. esculenta
AiryamD. bulbiferea
Arata
D. alata
Jinen-jo
D. Japonica
N.D = Not detected

Site（Island, District, Village）
Yap
Tomil
Thol
Yap
TomilThol
Yap
Tomil
Thol
MogmogMogmogMogmogAsor
Asor
Fassarai
Fassarai
Fassarai
MogmogMogmogMogmogYap
Gagil
Leabinau
Yap
Gagil
Leabinau
Yap
Rull
Ngof
Yap
Rull
Dachngar
Yap
Maap
Waned
Yap
Maap
WanedYap
Maap
Waned
Yap
Maap
Waned
Yap
Maap
Waned
Yap
Maap
Waned
Yap
TomilThol
Yap
TomilThol
Yap
TomilThol
Yap
TomilThol
Yap
TomilThol
Yap
Maap
Waned
MogmogFalalop
MogmogMogmogMogmogMogmogBand pattern
71112
N.D
213N.D
132N.DN.D
416781075876N.D
1010476N.D
99121412RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS OF YAMS COLLECTED IN THE YAP ISLANDS AND THE ULITHI ISLANDS.

79-80 °C before DNA extraction.
DNA extractionTotal DNA was isolated from frozen leaf sample by modified SDS method, about 1 g leaf
tissue per sample ground in liquid nitrogen. To remove polyphenol, sample was vortexed with5 ml of washing buffer (100 mM HEPES, 0.1% Polyvinylpyrrolidone K-30, 4% 2Mercaptoethanol) and then centrifuged at 18,000 × g for 3 min. This washing process was repeated at least 4 to 5 times up to take out stickiness. The precipitant was solved with 5 m
extraction buffer (15% sucrose, 50 mM Tris-HCl, 50 mM EDTA, 500 mM NaCl, pH 8.0) andcentrifuged at 4,600 × g for 5 min. The precipitant was resolved with 2 m of resuspension
buffer (20 mM Tris-HCl, 10 mM EDTA, pH 8.0) and incubated at 70 °C for 15 min within 250
μ of 10% SDS. 1 m of 7.5 M ammonium acetate was added and cooled on ice for more than
30 min. After centrifuge for 15 min. at 18,000 × g, the supernatant was transferred to a new cube.
Equivalent cold isopropanol was added and centrifuged for 15 min. at 18,000 × g. DNA pellet
was washed in 70% ethanol and dried and dissolved in 500 μ TE buffer. 10 μg /μ RNAase
was added and incubated at 55 °C for 10 min. Lastly, DNA solution was centrifuged for 5 min.
at 18,000 × g. The supernatant was transferred a 1.5 μ cube and stored - 20 °C until use.
PCR and electrophoresis
PCR was performed in a 0.2 m tube for use on a GeneAmp PCR systems 9700 (PEBiosystems). The reaction consisted of 20 ng of DNA, 10 pmol primer, 2.5μ 10 × PCR buffer,
200 μM of each dNTP and Taq polymerase (TAKARA Taq) in 25 μ volume. Primers used foryam were OPA-2, C-15, C-19, C-20, E-12, W-2, W-7, X-1, X-9 and Y-9 Operon(S HIWACHI et al. 2000). It was possible to classify these into D. alata, D.opposita and D. japonica. The thermocycler was set at 94 °C for 5 min. and repeated 30 sec. at 94 °C, 1 min. forannealing, and 2 min. at 72 °C. That annealing temperature was degraded 4 °C every 3 cycles
from 56 °C to 44 °C, and the reaction kept on 35 cycles at 40 °C. This was followed by a final
cycle of 72 °C for 7 min. The amplified DNA sample with dye was loaded on 1% agarose and0.5% synergel (Diversified biotech) in 1 × TAE buffer at 60 V for 70 min., and stained byethidium 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. One
sample was picked from each group. About 16 PCR samples including 3 controls were tested
again for reconfirmation of relationship between them.

Results and discussions
Total 36 samples were tested. Most of them did not show any amplified DNA bands
because of the weakness of the SDS methods. And then, phenol treatment was applied to all samples. Some samples (YYA7, 10, 14, 15, 27 and 33) gave no bands, so they could not be used inclassification.
Table 1 and Fig. 2 showed 14 different band patterns that were detected by OPA-2 in thistest. In this survey, the D. alata species was mainly collected. D. alata characterized by about1200 bp fragment was found in Arata which is accepted as model population for this D. alataspecies. Other examples differ in length or position. These can be accepted as signs of genetic80TAURA Satoru, PARK Byoung-Jae, I CHITANI Katuyuki, ONJ O Michio, and KAWABE Kotaro

Fig. 1. Collecting sites of yams in Yap, Asor and Mogmog islands.

Fig. 2. DNA polymorphisms of yams detected by amplification of total DNA using OPA- 2 primer

diversity. Those samples with no the band of 1200 bp (YYA 9, 36 and 37) were considered asother species. According to morphological characters, YYA 36 was identified as D. esculenta
and YYA37 as D. bulbiferea. The sample YYA37 morphologically relates to D. bulbiferea
which shows a different band pattern, so this may mean genetic variation can be found also inD. bulbiferea.
Genetic variation of yams in the Yap islands and Ulithi islands can be easily detected byRAPD analysis. The OPA-2 primer seems to be fitted for detecting genetic diversity in the D.
alata species.

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS OF YAMS COLLECTED IN THE YAP ISLANDS AND THE ULITHI ISLANDS.

References
COURSEY, D. G. 1967. Yams. 230 pp. London: Longmans.
SHIWACHI, H. et al. 2000. Jpn. J. Trop. Agr., 44: 229–237.

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