Mitsuru HAYASHI, Hironobu SHIWACHI and Michio ONJO RAPD RAPD RAPD. (D.japonica)

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1 Kagoshima University Research Center for the Pacific Islands, Occasional Papers No.34, , 2001 Part 2, Section 2, Report 2. The Progress Report of the 1999 Survey of the Research Project Social Homeostasis of Small Islands in an Islandzone 145 RAPD 3 (D.alata) 3RAPD5 RAPD RAPD (D.japonica) RAPD Mitsuru HAYASHI, Hironobu SHIWACHI and Michio ONJO Abstract Water yams (D. alata) could be classified on the characteristicsof leaves, stemsand aerial tubers. It was difficult to distinguish some strains of chinese yam (D. opposita) from jinenjo (D. japonica) because of similarity of morphological characteristics. However, jinenjo was distinguished from chinese yams by using RAPD method. This suggests that the random amplified polymorphic DNA (RAPD) method is useful for identification of species of yams. The dendrogram based on the RAPD method corresponded with cluster analysis based on the morphological characteristics among the three species of yams. On the other hand, water yams were classified into three groups by cluster analysis based on the morphological characteristics, but were classified into five groups by the RAPD method. In RAPD analysis, morphological characteristics of strains may differ from each other. Therefore to classify yam strains using the RAPD method,it is necessary to use RAPD marker related to the specific characteristics. It was able to use that the appearance of anthocyanin on the leaf axil or petiol made indicator for clasification in the strains of water yam and jinenjo roughly. However,circumstantial classification of the strains should be combined with another indicator. Based on both analaysis of morphological characteristics and the RAPD method in water yam,

2 146 the strains from Indonesia were found to be similar to Japannese, Micronesian and Oceanian strains. This result suggests that the variation of water yam in Indonesia is quite large. The strains of water yam in Japan have, characteristics similar to the strains of Indonesia not those of Micronesia and Oceania. This suggests that Japanese strains were originated from SouthEast Asia. Key words: Classification, Morphological characters, RAPD method, Strains, Yams MARTIN MARTIN (IITA) (D. cayenensis/d. rotundata) HAMON1990RAPD (Random Amplified Polymorphic DNA) RFLP (Restriction Fragment Length Polymorphisms) AFLP (Amplified Fragment Length Polymorphism) (MIGNOUNA1998TERAUCHI1992THOTTAPPILLY RAPD (1) g TPN 42890cm50cm 1m 100kg/10aN:P205:K20=10:10:10 20kg/10a 2kg/10a

3 (Dioscorea spp.) RAPD 147 Table 1. Code number, their recent geographic sources and classification based on the morphological criteria of the tested yam Species Code No. Cultivar/strain name Geographic source Classification based on morphological criteria D. alata A 1 Unknown Indonesia A 2 Unknown Indonesia A 3 Unknown Indonesia A 4 Unknown Indonesia III A 5 Unknown Indonesia III A 6 Unknown Indonesia IV A 8 Unknown Indonesia III A 9 Soloyam Indonesia A11 Unknown Nauru III A12 Unknown Papua New Guinea III A16 Unknown Papua New Guinea III A40 Unknown Papua New Guinea IV A41 Unknown Papua New Guinea III A42 Unknown Papua New Guinea IV A43 Unknown Papua New Guinea IV A44 Unknown Papua New Guinea IV A45 Unknown Papua New Guinea III A47 Unknown Papua New Guinea III A48 Unknown Papua New Guinea III A61 Listeye Papua New Guinea III A62 Sulka Papua New Guinea III A63 Nami warumbo Papua New Guinea III A17 Unknown Palau Is. IV A19 Unknown Palau Is. III A20 Unknown Palau Is. III A22 Unknown Palau Is. III A28 Unknown Palau Is. IV A29 Unknown Palau Is. IV A30 Unknown Palau Is. IV A31 Unknown Palau Is. IV A32 Unknown Palau Is. IV A33 Unknown Palau Is. III A36 Unknown Pohnpei Is. III A64 Unknown Pohnpei Is. III A65 Unknown Pohnpei Is. III A34 Unknown Okinawa, Japan III A35 Unknown Okinawa, Japan IV A49 Unknown Kagoshima(Amamioshima), Japan III A54 Unknown Kagoshima(Tokunoshima), Japan IV A55 Tsukuneimo Kagoshima, Japan A56 Arata Kagoshima, Japan A57 Osato no. 1 Kagoshima, Japan A58 Osato no. 2 Kagoshima, Japan A59 Tsukuneimo Kagoshima, Japan A66 Unknown Kagoshima, Japan A67 Unknown Kagoshima, Japan A68 Unknown Kagoshima, Japan A60 Unknown Chiba, Japan D. opposita O 1 Shinsyuu Nagano, Japan II O 2 Tokuri Aomori, Japan II O 3 Himejin Fukui, Japan II O 4 Ichyou(Ichyou strain) Kanto Area, Japan II O 5 Ichyou(Baci strain) Kanto Area, Japan II O 6 Ise Mie, Japan II O 7 Kagamaru Fukui, Japan II O 8 Yamanoimo Hyogo, Japan II D. japonica J 1 Jinenjo Kagoshima, Japan I J 2 Jinenjo Kagoshima, Japan II

4 148 Table 2. Factor loading and contribution of the first (Z1), second (Z2) and third (Z3) principal component for 23 characters Factor loading Character Plant vigor Branching Number of vines Diameter of stem Stem wings Stem color Cross section of stem Phyllotaxy Leaf shape Leaf color Length of leaf Breadth of leaf Length/breadth ratio Base depth of leaf Shoulder breadth of leaf Petiole length Pigmentation of petiole(reddish color) Pigmentation of leaf axil Aerial tuber formation Aerial tuber shape Aerial tuber size Skin color of aerial tuber Flowering Component Contribution(%) () (2) UPGMA 2. RAPD (A2A5A6A8 A9A12A16A22A31A40A44A47A57A65)J1 J2 O1 17 DNA (SDS) TE(1M TrisHCl0.5M EDTA)20 DNA 20ng/ml10mer (Operon) DNA () PCR(Polymerase chain reaction) PCR PCR1 1 J2 16

5 (Dioscorea spp.) RAPD 149 2J2O1PCR 3 J2O1PCR (2) 1 Fig. 1. Dendrogram of yam strains obtained from the cluster analysis based on the determinations of morphological characters. The code number of each strains show in Table 1. The extreme left indicates to are the grouping based on the dendrogram.

6 J11 8J (1) ( 1 ) (1 ) 1 9 ( 8 1 ) (2) 4 3 Fig. 2. Plots of individual strains for the first and second principal component axes. The symbols of,,, and are the grouping I to V based on the dendrogram.

(Dioscorea spp.) RAPD 151 ( ) J2( ) 2 2. RAPD 1 17A47 A57 J1 O1 4200 PCR RAPD Fig. 3. RAPD profiles generated by random primer OPA2 in 16 strains of yams. M: size marker. Table 3.

7 (Dioscorea spp.) RAPD 151 ( ) J2( ) 2 2. RAPD 1 17A47 A57 J1 O PCR RAPD Fig. 3. RAPD profiles generated by random primer OPA2 in 16 strains of yams. M: size marker. Table 3. Comparison of PCR products among three species by RAPD analysis RAPD marker OPA2a OPA2b OPC15 OPC19a OPC19b OPC20a OPC20b OPE12 OPW7 OPX1 OPX9 : without marker. D. japonica J1 Species and code No. D. opposita O1 D. alata A47

8 152 Fig. 4. Dendrogram of yam strains obtained from the cluster anlysis based on the RAPD data. The number after the code number indicates the grouping based on the morphological characters. Letters on the code number at the extremely left indicates the grouping based on the dendrogram. bp OPA2OPC15OPC19OPC20OPE12 OPW2OPW7OPX1OPX9OPY910 OPA2 PCR PCR 3 bp RAPD OPA RAPDJ1O1A47PCR3 OPA2OPC19OPC202 OPC15OPE12OPW7OPX1OPX J1 O13.0 a b c d e f g 7 a J1 bo1 c d e f g c A47A22A6d A31e A8A12f A16A40A44g A2A5A6A9A57 J1 O1 3RAPD5 A5A6A16A31 2J2O1PCR OPC15OPC19OPC20 J2O1DNAPCR PCR 5 PCR

9 (Dioscorea spp.) RAPD 153 Fig. 5. RAPD profiles generated by random primer OPC15,19 and 20 in 2 kind of yams. J2 :D. japonica, O1 : D. opposita M: size marker. RAPD RAPD RAPD 3RAPD 5 RAPD RAPD MARTIN

10 154 Table 4. The relation between anthocyanin coloration and classification based on morphological criteria, RAPD of the tested yam Classification Anthocyanin coloration based on morphological criteria Tuber Classification Code No. based on RAPD of strain Leaf & petiole I a J 1 II b O 1 " O 2 " O 3 " O 4 " O 5 " O 6 " O 7 " O 8 " J 2 III A 4 " g A 5 " e A 8 " A11 " e A12 " f A16 " A41 " A45 " c A47 " A48 " A61 " A62 " A63 " A19 " A20 " c A22 " A33 " A36 " A64 " c A65 " A34 " A49 IV g A 6 " f A40 " A42 " A43 " f A44 " A17 " A28 " A29 " A30 " d A31 " A32 " A35 " A54 A 1 " g A 2 " A 3 " g A 9 " A55 " A56 " g A57 " A58 " A59 " A66 " A67 " A68 " A60 Anthocyanin coloration, ; pigmentation, ; nopigmentation.

11 (Dioscorea spp.) RAPD RAPD J2 J1 RAPD c ef 3g 5 1 RAPD RAPD c 1 d e 2 f12 g 1 3 A57 RAPD RAPD (Dioscorea spp.) : (Dioscorea spp.) : HAMON, P. and B. TOURE 1990 Characterization of traditional yam varieties belonging to the Dioscorea caynensisrotundata complex by there isozimic patterns. Euphytica 46: 101

12 HAMON, P. and B. TOURE 1990 The classification of the cultivated yams (Dioscorea caynensisrotundata complex) of West Africa. Euphytica 47: (Dioscorea spp.). 1 (Dioscorea alata L.). 34: (Dioscorea spp.) 2. (Dioscorea alata L.). 35: HAYASHI, M., Y. SAKATA, S. TOMINAGA, S. TAURA and M. NAKAMURA 1991 Introduction of tropical plants. Kagoshima Univ. Res. Center S. Pac. Occasional Papers No.21: ,. 15: ). ( ), MARTIN, A. M. and A. M. RHODES 1973 Collelation among greater yams (Dioscorea alata L.) cultivars. Trop. Agric. 50: MARTIN, A. M Tropical Yams and their potential. Part 3. Dioscorea alata. Agriculture Handbook No.495: U.S.D.A. pp MARTIN, A. M. and A. M. RHODES 1977 Interspecific classification of Dioscorea alata. Trop. Agric. 54: MIGNOUNA H. D., N. T. H. ELLIS, M. R. KNOX, R. ASIEDU and N. Ng. QUAT 1998 Analysis of genetic diversity in Guinea yams (Dioscorea spp) using AFLP fingerprinting. Trop. Agric. 75: (Dioscorea spp.). 3 (Dioscorea alata). 43: TERAUCHI, R., V. A. CHIKALEKE, G. THOTTAPPILLY and S. K. HAHN 1992 Origin and phylogeny of Guinea yams as revealed by RFLP analysis of chloroplast DNA and nuclear ribosomal DNA. Theor. Appl. Genet. 83: RHODES, A. M. and A. M. MARTIN 1972 Multivariate studies of variations in yams (Dioscorea alata L.). J. Amer. Soc. Hort. Sci. 97: ( ) p (Dioscorea alata L.) (Dioscorea alata L.) (Dioscorea opposita THUNB.) (Dioscorea japonica THUNB.). 43: (Dioscorea alata L.) (Dioscorea opposita THUNB.) (Dioscorea japonica THUNB.). 44: :

13 (Dioscorea spp.) RAPD 157 : THOTTAPPILLY, G., H. D. MIGNOUNA, A. ONASANYA, M. ABANG, O. OYELAKIN and N. K. SINGH 1999 Identification and differentiation of isolates of colletotrichum gloeosporioides from yam by random amplified polymorphic DNA markers. Afric. Crop Sci. J. 7: (Dioscorea alata L.)

Recurring Somaclonal Variation as a Factor of Intra-Specific Diversity Observed in Dioscorea alata L.

Trop. Agr. Develop. 56(2):71-79,2012 Recurring Somaclonal Variation as a Factor of Intra-Specific Diversity Observed in Dioscorea alata L. P. K. BABIL 1, *,a, Daisuke YASUHARA 2, Keisuke SAKAGUCHI 3, Tsukasa