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Cluster Analysis of Dioscorea Alata Accessions of Purwodadi Botanic Gardens (Indonesia) Collection Based on Morphological Characteristic and SSR Markers

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Cluster Analysis of Dioscorea Alata Accessions of Purwodadi Botanic Gardens (Indonesia) Collection Based on Morphological Characteristic and SSR Markers CELL BIOLOGY & DEVEL OPMENT Volume 3, Number 1, June 2019 E-ISSN: 2580-4499 Pages: 6-12 DOI: 10.13057/cellbioldev/t030102 Cluster analysis of Dioscorea alata accessions of Purwodadi Botanic Gardens (Indonesia) collection based on morphological characteristic and SSR markers ♥ SHOFIYATUL MAS’UDAH , FAUZIAH, LIA HAPSARI Purwodadi Botanic Gardens, Indonesian Institute of Sciences. Jl. Raya Surabaya-Malang Km. 65, Purwodadi, Pasuruan 67163, East Java, Indonesia. email: [email protected] Manuscript received: 21 February 2019. Revision accepted: 11 May 2019. Abstract. Mas’udah S, Fauziah, Hapsari L. 2019. Cluster analysis of Dioscorea alata accessions of Purwodadi Botanic Gardens (Indonesia) collection based on morphological characteristic and SSR markers. Cell Biol Dev 3: 6-12. Morphological characteristics of D. alata accessions show a high diversity, especially the tubers which have varying form, size, weight, color, and flesh. Classification and naming of D. alata accessions generally using different local names in each region in which causing problems. This study aimed to classify 20 accession numbers of D. alata collection of Purwodadi Botanic Garden collected from East Java based on tuber morphological characteristics and simple sequence repeat (SSR) molecular markers. Based on the morphological characteristic, the accessions showed high variation. From seventeen characters observed, two characters were similar and 15 others were diverse. The main morphological characters that contribute to the cluster were tuber skin color, tuber shape and the color of the inner skin of the tuber. Clustering result based on DNA amplification showed different group compared to the morphological characters cluster although some accessions with close morphological characters were in the same molecular group. The accession origin could not be used as the group marker. Marker E11 and A7 have the highest polymorphisms in this experiment. As conservation strategies, accessions with high- level similarity could be represented by one accession for maintenance efficiency. Conserving species until varieties level that has different characters could be enriched germplasm for breeding development. Keywords: Classification, diversity, local varieties, microsatellite, phenotype, yam INTRODUCTION functional food and supported the food diversification (Hapsari 2014). Some studies of tuber processing were Dioscorea spp. is tuber plant with climbing stem, it found and its tuber could be used for bakery, cookies, belong to Dioscoreaceae. Dioscorea family consists of flakes, muffin, noddle, vermicelli (Hapsari 2014); flour more than 600 species spread in the Tropical and Sub- (Afidin et al. 2014); chips (Putri et al. 2017); fries Tropical regions, but less than 200 species have been (Munawaroh et al. 2018), etc. identified (Ayensu and Coursey 1972), and only about 50 - Dioscorea alata in Indonesia showed high 60 species have been cultivated as food and medicine morphological diversity, especially in tuber shape, tuber (Coursey 1976; Onwueme 1996). Indonesia has many types size, tuber skin , and flesh color. Accessions clustering and of Dioscorea, there is D. warbugiana, D. alata, D. naming of D. alata used different local name depends on keduensis, D. nummularia, D. esculenta, D. pentaphylla, D. the origin where the plant/tuber found which caused sansibarensis, D. hispida, D. bulbifera, etc. (Onwueme confusion on identification (Onwueme and Ganga 1996; 1996; Herison et al. 2010; Trustinah 2013; Fauziah and Herison et al. 2010; Purnomo et al. 2017; Kinasih et al. Mas’udah 2015; Solikin 2017). 2017). Characterization and clustering on D. alata in Dioscorea alata was one of the most important species, Indonesia is needed as base information to make valid much cultivated and utilized by citizen especially in name of accession and arrange development program. Southeast Asia, including Indonesia (Onwueme and Ganga Characterization and clustering could be held by 1996). D. alata used as substitute of rice/sago as staple morphological characters and genetical marker. food in the dry season (Sulistyono and Marpaung 2004). D. Morphological characterization was used in many studies alata locally known as uwi (Javanese), huwi (Sundanese), to observe diversity of plant germplasm that used as base lame (Celebes), obi (Madura) and lutu (Maluku Island) information. Furthermore, some molecular marker was which in other country known as greater yam, water yam, developed to confirm the diversity of plant which identity or ten-months yam (Onwueme dan Ganga 1996), Tuber of by morphological characters (Azrai 2005; Zulfahmi 2013) D. alata has high content of starch and protein, vitamin C Purwodadi Botanic Garden, Indonesian Institute of and antioxidant but low on sugar. The tuber was also a Science is an ex-situ plant conservation institute that source of minerals that so D. alata was prospective to be collects D. alata from some region in Indonesia especially functional food and supported the diver antioxidant and Java (Lestarini et al. 2012) some Dioscorea collected from tuber was source of mineral which prospective to be Malang (Fauziah 2013), Nganjuk (Trimanto and Hapsari MAS’UDAH et al. – Cluster analysis of Dioscorea alata accessions 7 2015) and Pasuruan (Fauziah and Mas'udah 2015). Some of Amplification processing used Techne PCR Thermocycler. local variety was collected are Uwi Kelopo, Uwi Putih, Amplification cycle consisted of 25 cycle denaturation for Uwi Jaran, Uwi Sego, Uwi Perti, Uwi Bangkulit, Uwi 30 seconds in 95 °C, annealing depends on the primer Ireng, Uwi Alas, Uwi Klelet, Uwi Randu, Uwi Senggrani, (Table 3) for 1 minute and extension for 30 seconds in 72 Uwi Bangkong, Uwi Ndoro, Uwi Dusono, etc. Study of its °C. Final extension was performed using 72 °C for 8 utility and potential was needed to add D. alata tuber value minutes. Result of the amplification confirmed by and support the food security program. The aim of this electrophoresis in 4% agarose gel with 1 μg/ml ethidium study was to cluster accessions of D. alata collected in bromide in TBE buffer and visualized in UV Purwodadi Botanic Garden based on morphological transilluminator (Bio Rad Universal Hood II Gel Doc characteristic and molecular marker used simple sequence System). repeats (SSR) or known as microsatellite. SSR marker was very useful genetic marker as if it is dominant, high Data analysis polymorphism, and could detect variation between Morphological data were analyzed by similarity population in different region, individual variation in coefficient based on Euclidean coefficient and the result population and microsatellite was also cheap in experiment was used to construct dendrograms by unweighted pair (Powell et al. 1996: Siqueira et al. 2012; Tamiru et al. group method with arithmetic mean (UPGMA) (Sokal and 2015; Vieira et al. 2016). Result of this study could be used Michener 1958) using PAST 3.21 program (Hammer et al. as base information to choose valid name on D. alata 2001). Principal component analysis (PCA) of accessions as consideration in conservation strategy and morphological characters was conducted to identify development program. characters with the highest contribution. Molecular data were analyzed by DNA band scoring which showed up in agarose gel. An SSR band in agarose MATERIALS AND METHODS gel represented an allele from each accession. Each band of markers presented shows allele of each accession. The Area of study allele was scored as 1 when it present and 0 when it absent. This research used 20 accessions of Dioscorea alata L. Polymorphism percentage was counted by polymorphism collection of Purwodadi Botanic Garden from Nganjuk, allele in each primer. Cluster analysis used UPGMA Malang, and Pasuruan. The research was divided to two methods and similarity coefficient with Euclidean by experiments. The first experiment was morphological Paleontological Statistics (PAST) 3.21 (Hammer et al. characters observation and the second experiment was 2001). molecular analysis of the accessions. Morphological characters were observed on 2015 and 2016. Molecular Table 1. Twenty accessions of Dioscorea alata observed analysis conducted in Biotechnology Laboratorium, Faculty of Agriculture, University of Brawijaya, Malang. Accession code Local name Origin DA 23 Uwi Kelopo Pasuruan Molecular analysis used fresh leaves from February-March DA 24 Uwi Putih Pasuruan 2015 planting and extract with Purelink® Plant Total DNA DA 27 Uwi Ulo Pasuruan Purification Kit (Invitrogen 2012). DA28 Uwi Perti Pasuruan DA29 Uwi Ulo Pasuruan Methods DA30 Uwi Perti Pasuruan Morphological observation DA31 Uwi Bangkulit Pasuruan Observation on vegetative morphological characters DA32 Uwi Kelopo Pasuruan held on March-April 2015 and 2016. Tuber morphological DA36 Uwi Bangkulit Pasuruan characters were observed after harvest which harvesting DA43 Uwi Bangkulit Nganjuk DA44 Uwi Klelet Nganjuk period was on September until October 2015 and 2016. DA46 Uwi Dusono Nganjuk Morphological characterization used Descriptors for Yams DA48 Uwi Putih Nganjuk (Dioscorea spp) by IPGRI/IITA (1997) with modification. DA57 Uwi Ketan Putih Malang Result of morphologic characterization showed in Table 2 DA58 Uwi Biru Malang DA59 Uwi Lajer Malang Molecular analysis DA63 Uwi Budeng Malang DNA extraction was taken from fresh leaf using DA64 Uwi Ulo Malang Purelink® Plant Total DNA Purification Kit (Invitrogen DA66 Uwi Legi Malang DA67 Uwi Sego Malang 2012). DNA amplification was done using nine SSR marker (Tostain 2006)
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