The Assessment Of Genetic Variability And Taxonomic Affi .

Indonesian JournalSusandariniet al. of Biotechnology, December, 2014Vol. 19, No. 2, I.J.pp.153-158Biotech.The assessment of genetic variability and taxonomic affinity of localpummelo accessions from Yogyakarta, Indonesia based on RAPDRatna Susandarini1*, Rina Arifati1, Abdul Razaq Chasani1, and Siti Subandiyah2Faculty of Biology, Universitas Gadjah Mada, IndonesiaFaculty of Agriculture, Universitas Gadjah Mada, Indonesia12AbstractPummelo (Citrus maxima) is one of three biotypes considered as true species within the genus Citrus.A major issue of pummelo taxonomy in Indonesia is the high number of cultivars showing variability inphenotypic characters but of unknown genetic diversity. In this study, the assessment of genetic variabilityand taxonomic affinity of local accessions of C. maxima from Yogyakarta was examined based on RAPDfingerprinting. The availability of universal primers and technical simplicity makes RAPD as a molecular toolof choice for the assessment of genetic variability at various taxonomic levels. In this study, 13 accessions of C.maxima collected from Yogyakarta were observed for their genetic variability. An additional three registeredcultivars were included for comparative purpose. Two decamer primers used for the amplification of DNAproduced 222 bands with 174 of them were polymorphic. The data was subjected to cluster analysis to observethe grouping of accessions and taxonomic affinity. Results indicated high genetic variability among accessions.The dendrogram constructed using UPGMA method based on simple matching coefficient showed twomain clusters were which was in line to morphological characters. The grouping of accessions showed cleardifferentiation between accessions bearing white and those with reddish fruit flesh, and thus demonstratestaxonomic value of this study in recognizing important agronomic character for this tropical fruit crop.Keywords: Citrus maxima, taxonomic affinity, genetic variability, RAPD.IntroductionCitrus spp. is a group of tropicalfruits with high potential as an importantagricultural commodity in many countries.The importance of Citrus spp. could beobserved from its management efforts in someAsian countries under the program of GlobalEnvironment Facilities (GEF). This programwas developed for the documentation andconservation of exotic tropical fruits, includingmango (Mangifera indica), citrus (Citrusspp.), mangosteen (Garcinia mangostana),and rambutan (Nephelium lappaceum) (GEF,2006).Pummelo (Citrus maxima (Burm.) Merr.)is one species of citrus originated fromtropical areas of Asia, and known as havinghigh phenotypic variability (Niyomdham,1992). Pummelo grows well on various soiltypes at lowland areas to the altitude of 400m above sea level, and could be harvestedtwice to four times a year. One of the seriousproblems of pummelo in Indonesia is thedecline of its genetic variability. Arora (2000)suggested that there was an indication on thedecline of pummelo variability in Indonesiaand Malaysia as a result of genetic erosion.A possible cause of this decline is lack ofconcern in preserving pummelo which isconsidered as underutilized citrus fruits dueto its relatively low economic value comparedto sweet orange and mandarins.A common problem threateningdiversity of tropical fruit species and theiruncultivated close relatives exist in naturalhabitat as landraces also caused by habitatdestruction as a result of unsustainable*Corresponding author:Ratna SusandariniFaculty of Biology, Universitas Gadjah Mada,Indonesia, ratna-susandarini@ugm.ac.id153

Susandarini et al.I.J. Biotech.development (Trinh et al., 2003; GEF, 2006).Inventory study on pummelo geneticvariability is thus worth particular attentionto prevent this potential fruit crop fromvanishing before its biological potential isfully discovered.DNA fingerprinting was defined asany multilocus approach of visualizingDNA polymorphisms, either generatedfrom hybridization or PCR-based techniques(Weising et al., 1995). Random amplifiedpolymorphic DNA (RAPD) developedby Williams et al. (1990) is a PCR-basedfingerprinting technique which uses singleprimer of arbitrary nucleotide sequenceto amplify random DNA segments. Theapplication of RAPD in the assessment ofgenetic variability studies has been reportedat various taxonomic levels, ranging fromfamilial (Prasad, 2014), generic (El-Mouei etal., 2011), to species and cultivar levels (Baiget al., 2009).Previous research on genetic variabilityof pummelo in Indonesia is still limited to asmall number of cultivars from productioncenter areas such as Magetan, East Java asreported by Karsinah et al. (2002), Purwantoet al. (2005), Rakhman et al. (2005), andAgisismanto & Supriyanto (2007). Thesestudies were conducted only on widelycultivated pummelos, and to this point therewas no record on the studies on pummelolandraces occurred as home yard plants.The objective of this study is, therefore,assessing genetic variability of pummeloaccessions occurred as home yard plantsfrom Yogyakarta which is assumed to hostsome local landraces.RAPD reaction was performed in 25 μL tubecontaining 20 μL volume of reaction mixturewith 0.2 μM of primer and 15 ng of genomicDNA. The amplification reaction werecarried out using a BioradTM thermal cyclerwith the following cycling program: 5 mindenaturation step at 95oC; 45 cycles of 30 s at94oC, 45 s at 36oC, 1 min at 72oC; and 5 min at72oC for the final elongation step. Six RAPDprimers were screened for their suitabilityin amplifying C.maxima DNA. These sixprimers were OPA-02, OPE-04, OPK-14,OPN-14, OPN-16, and OPW-19. The criteriafor determining primer suitability were thenumber of amplified DNA fragments, theirreproducibilty, and their quality, in termsof producing clear and unumbiguous DNAbanding patterns. Two decamer primerswere finally chosen based on these criteria:OPA-02 (‘TGCCGAGCTG’) and OPN-16(‘AAGCCGACCTG’). PCR products arevisualized on 2% agarose in 1x TBE buffer,using Good View (Microzone, UK) as nucleicacid stain. The gel was run on electrophoresisapparatus on 110V for 45 min. Gel picturewas observed under UV illuminator anddocumented using digital camera. Thebanding pattern of DNA fragment wascoded numerically to produce binary datarepresenting absence (0) or presence (1) of aband at particular molecular weight. The datawas then subjected to cluster analysis usingMVSP 3.1 (Kovach, 2007).Results and DiscussionTwo primers used in this study weresuccessfully amplified DNA samples from13 accessions of pummelo from Yogyakarta,and additional three registered pummelocultivars (“Pamelo Magetan”, “PangkajeneMerah”, and “Pangkajene Putih”). A totalof 222 scorable bands were produced, with174 being polymorphic (Figure 1). Thereproducibility of RAPD was assured fromidentical results of banding patterns fromtwo consecutive amplification reactions.Taxonomic affinity of pummeloaccessions was examined based on result ofMaterials and MethodsLeaves samples were collected fromhealthy pummelo trees from Gunungkidul,Yogyakarta. Fresh leaves were washed toremove dirt followed by surface sterilizationusing 70% ethanol. DNA extraction from0.2 g of fresh leaf sample was done usingDNAMITE Plant Kit Extraction (Microzone,UK) according to manufacturer’s protocol.154

Susandarini et al.MI.J. Biotech.12345678910 11 12 13141516AM12345678910111213141516BFigure 1. RAPD profile of 13 accessions pummelo from Yogyakarta and three cultivars using primer OPA-02 (A)and OPN-16 (B).M12345678: molecular weight marker (100-bp DNA ladder): TJS-01, unnamed, pink flesh: TJS-02, unnamed, pink flesh: TJS-03, tradionally named as “Bali”, white flesh: TJS-04, unnamed, white flesh: TJS-05, tradionally named as “Bali”, pink flesh: TJS-06, tradionally named as “Gulung”, pink flesh: TJS-07, tradionally named as “Gulung”, pink flesh: TJS-08, tradionally named as “Gulung”, pink flesh910111213141516cluster analysis, represented as dendrogram(Figure 2). The dendrogram was constructedbased on Simple Matching Coefficient valuesas a measure of similarity among accessions.The similarity values are indicating thedegree of genetic similarity. The overalltopology of dendrogram is thus representingthe level of genetic variability of pummelo.Results of RAPD fingerprinting showeda high degree of genetic variability amongsamples, indicated with 78.4% polymorphismin the amplification products. This resultwas of comparable extent in defining thedegree of genetic variability as reported bySheidai et al. (2010) in the analysis of geneticdiversity of brown olive (Olea cuspidata). The: TJS-09, tradionally named as “Gulung”, pink flesh: TJS-10, tradionally named as “Bali”, white flesh: TJS-11, named as “Adas Nambangan”, pink flesh: TJS-12, unnamed, pink flesh: TJS-13, unnamed, pink flesh: “Pamelo Magetan”: “Pangkajene Merah”: “Pangkajene Putih”polymorphism of RAPD fingerprinting, asrepresented by the presence or absence ofamplification products, functions as geneticmarkers (Williams et al. 1990; Newbury& Ford-Lloyd, 1993), and provide highlyinformative characters for the evaluation ofgenetic similarity (Weising et al., 1995).The grouping pattern of accessionsin the dendrogram showed the existenceof two main cluster or groups. The firstcluster (group I) consisted of four accessionstraditionally named as “Jeruk Gulung” withpink flesh. This group of accessions is lessprefered pummelo due to its sweet-sourtaste, and even with a reasonable degreeof bitterness. The second cluster (group II)155

Susandarini et al.I.J. Biotech.UPGMA 1.0Simple Matching CoefficientFigure 2. Dendrogram showing genetic variability and taxonomic affinity of pummelo accessionsindicated the existence of infraspecificvariability within pummelo, and showedthe grouping of accessions into recognizabletypes. This finding was in line with the studyof characterization of Citrus cultivars andclones using RAPD as reported by Hvarlevaet al. (2008).The application of RAPD fingerprintingin infraspecific taxa has been concernedwith two major objectives, either for theestimation of genetic variability or cultivaridentification. The combination of thesetwo objectives has been reported in variousfruit crops, such as in Cucumis sativus andBenincasa hispida by Saengprajak & Saensouk(2012) and sugar apple (Annona squamosa) byGuimarães et al. (2013). The ability of RAPDin providing evidence on the occurrenceof infraspecific genetic variability withincultivated plants was reported previously byMahasi et al. (2009) in their study on geneticpolymorphisms in Carthamus tinctorius.consisted of 9 local accessions and 3 namedcultivars of pummelo. This cluster wasclearly divided into three sub-clusters (A,B. and C), with the first sub-cluster beingseparated at substantial degree from theother two. The three accessions within subcluster A could be regarded as a group of“Adas Nambangan”, a typical pummelowith palatable taste of sweet-sour but lack ofbitterness in their fruit taste. The sub-clusterB comprised of accessions with white fleshand sweet fruit taste, with “PangkajenePutih” cultivar was among the members ofthe group. This sub-cluster was then couldbe referred to the commonly known as“Bali Putih” pummelo type. Meanwhile, thesub-cluster C with “Pamelo Magetan” and“Pangkajene Merah” cultvars were nestedtogether with two accessions of pummelowith pink flesh and sweet fruit taste couldbe denoted as those commonly known as“Bali Merah”.The conformity of clustering resultbased on RAPD analysis to morphologicaland agronomical characters observed in thisstudy was in agreement to the finding ofCorazza-Nunes et al. (2002) in the assessmentof genetic variability in Citrus paradisi andCitrus maxima. Results of this study alsoConclusionPummelo (Citrus maxima) accessionsfrom Yogyakarta have a high degree ofgenetic variability as indicated by thehigh percentage of RAPD fingerprintingpolymorphisms. The application of RAPD156