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Genetic Diversity of Falcataria Moluccana and Its Relationship to the Resistance of Gall Rust Disease

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Genetic Diversity of Falcataria Moluccana and Its Relationship to the Resistance of Gall Rust Disease BIODIVERSITAS ISSN: 1412-033X Volume 19, Number 1, January 2018 E-ISSN: 2085-4722 Pages: 12-17 DOI: 10.13057/biodiv/d190102 Genetic diversity of Falcataria moluccana and its relationship to the resistance of gall rust disease NEO ENDRA LELANA1,2,♥, SURYO WIYONO2, GIYANTO2, ISKANDAR Z. SIREGAR3,♥♥ 1Forest Protection Division, Centre for Forest Research and Development, Ministry of Environment and Forestry, Indonesia. Jl. Gunung Batu No. 5 Bogor 16610, West Java, Indonesia. Tel.: +62-251-8633234, Fax.: +62-251-8638111, ♥email: [email protected] 2Department of Plant Protection, Faculty of Agriculture, Institut Pertanian Bogor. Jl. Kamper, Kampus IPB Dramaga Bogor 16680, West Java, Indonesia. 3Department of Silviculture, Faculty of Forestry, Institut Pertanian Bogor. Jl. Lingkar Akademik Kampus IPB Dramaga Bogor 16680, West Java, Indonesia, ♥♥email: [email protected] Manuscript received: 21 August 2017. Revision accepted: 7 November 2017. Abstract. Lelana Ne, Wiyono S, Giyanto, Siregar IZ. 2018. Genetic diversity of Falcataria moluccana and its relationship to the resistance of gall rust disease. Biodiversitas 19: 12-17. The use of cultivars that are resistant to a particular disease is one strategy that could mitigate the incidence of gall rust disease on Falcataria moluccana. Previous studies on the genetic diversity of F. moluccana did not attempt to link that genetic diversity to gall rust disease resistance. This research was carried out using RAPD analysis to determine the preliminary information on the association between different markers and the resistance to gall rust disease. The analysis evaluated a total of 20 pairs of healthy and infected F. moluccana trees that were classified based on their disease severity level. The RAPD primers used in this study were as follows: OPA-05, OPA-08, OPA-10, OPA-13, OPA-18, OPB-07, OPD-13, OPF-02, and OPG-05. The results showed that each RAPD primer produced a varying number of polymorphic bands, ranging from 3 to 12 bands, with a total of 80 polymorphic bands. Despite the number of loci analyzed, however, no specific polymorphic bands were found that could distinguish between healthy and diseased trees. This was supported by principal component analysis, which showed that healthy and diseased populations were not distributed separately. The structure analysis also showed that the healthy and diseased populations were not different. Keywords: Diseased, healthy, pairing, polymorphic band, RAPD INTRODUCTION (Rahayu et al. 2010). In 2015, Doungsa-ard et al. (2015) proposed a new name for this fungus as Uromycladium Falcataria moluccana (Miq.) Barneby & J. W. Grimes, falcatarium. This disease has become a serious threat to F. also known as sengon in Indonesia, is native to Indonesia, moluccana plantations in Indonesia. The disease not only Papua New Guinea, and the Solomon Islands (Soerianegara causes significant economic losses, but also makes farmers and Lemmens 1993). Since the 1870s, Falcataria worried. In Indonesia, gall rust disease was first reported moluccana has spread throughout Southeast Asia from on Seram Island, Moluccas (Maluku). In Java, the disease Burma to the Philippines (NAS 1979). Currently, F. was first detected in Eastern Java in 2004. Since then, the moluccana is the most prevalent tree found in small-scale disease has spread throughout Java (Anggraeni 2008). community (private) forests and its population is In addition to environmental factors and pathogenic increasing. Based on the results of the agricultural census virulence, the resistance or susceptibility of plants to of 2013, the population of F. moluccana increased almost pathogens is also important in influencing the epidemic rate five times since 2003 (BPS 2013). Its rapid growth rate and of the disease (Burdon and Thrall 2008). Susceptible plants stable market availability is the reason why this species is will support the spread of the disease, while plants that preferred by farmers. F. moluccana is widely grown as a show high resistance to the pathogens will inhibit the shade tree for reforestation, afforestation, and wood spread of the disease. Therefore, the use of resistant production (Soerianegara and Lemmens 1993). The wood genotypes is often proposed as the first line of defense for of F. moluccana can be used for various purposes, such as controlling diseases. veneer, plywood, light construction, lightweight packaging Identifying molecular markers associated with host material, toys, firewood, and pulp (Soerianegara and resistance to disease is typically the first step applied in the Lemmens 1993; Krisnawati et al. 2011). Recently, the use breeding strategy for resistant varieties for plant disease of this wood for bare-core industry has been preferred control. Some markers, such as random amplified because of demands for exports (BPS 2014). polymorphic DNA (RAPD), restriction fragment length Various pests and diseases are known to be associated polymorphism (RFLP), amplified fragment length with F. moluccana trees. Of these, the most recent major polymorphism (AFLP), microsatellites, single nucleotide disease attacking the F. moluccana plantation is gall rust polymorphism (SNP), and next-generation sequencing disease. The gall rust pathogen was first identified as the (NGS), have been widely used in studies related to plant fungus Uromycladium tepperianum (Sacc.) McAlpine resistance (Zhang et al. 2013; Asad et al. 2015; Klosterman LELANA et al. – Genetic diversity of Falcataria moluccana 13 et al. 2016; Quesada-Ocampo et al. 2016; Vaghefi et al. Bogor, Cianjur, Sukabumi, and Majalengka (West Java 2016). Province); Batang, Purbalingga, Banjarnegara, Salatiga, Despite the rapid development of genetic markers, and Boyolali (Central Java Province); and Lumajang (East information concerning the resistance of a variety or the Java Province) (Figure 1). The number of samples provenance of F. moluccana to gall rust disease is still collected from each district and their geographical limited. Therefore, studies that identify molecular markers locations are presented in Table 1. related to the resistance of F. moluccana to gall rust disease are indispensable. As a first step, RAPD is suitable for this study. RAPD is a molecular marker based on the Table 1. List of Falcataria moluccana leaves samples that were amplification of random segment of DNA with single collected from various locations in Java, Indonesia primers of arbitrary nucleotide sequences (William et al. 1990). The major advantages of this technique are that Healthy Infected there is no requirement for a DNA sequence, it is relatively Location Latitude Longitude plant plant code code quick and easy to perform, and it is efficient to screen DNA polymorphisms at a very large number of loci (William et Bogor -6.66196 106.79884 H01 D01 al. 1990; Kumari and Thakur 2014). Unfortunately, this Bogor -6.66721 106.80596 H02 D02 technique has some limitations, such as low reproducibility Bogor -6.66721 106.80596 H03 D03 and it only produces dominant markers. Several studies Bogor -6.53107 106.42696 H04 D04 using RAPD to identify molecular markers linked to plant Cianjur -7.10665 107.09946 H05 D05 resistance genes have formed the foundation of plant Cianjur -6.99676 107.12364 H06 D06 breeding programs. Mumtaz et al. (2009) and Dhillon and Cianjur -6.93218 107.12387 H07 D07 Dhaliwal (2011) used RAPD markers to identify the Majalengka -7.06058 108.39017 H08 D08 Majalengka -6.99158 108.30591 H09 D09 presence of resistance genes against rust in wheat. Sukabumi -7.02644 106.80064 H10 D10 Meanwhile, Salah et al. (2016) used RAPD markers to Purbalingga -7.22007 109.33750 H11 D11 identify genes related to maize resistance against stalk rot Purbalingga -7.27352 109.34550 H12 D12 disease caused by Fusarium moniliforme. Thus, this study Boyolali -7.55987 110.52350 H13 D13 aimed to identify molecular markers related to the Banjarnegara -7.30036 109.80983 H14 D14 resistance of F. moluccana against gall rust disease using Batang -7.05156 109.78780 H15 D15 RAPD markers. Batang -7.30530 109.78454 H16 D16 Batang -7.08680 109.76557 H17 D17 Salatiga -7.36890 110.45323 H18 D18 Lumajang -8.12772 113.14735 H19 D19 MATERIALS AND METHODS Boyolali -7.56382 110.53833 H20 D20 Boyolali -7.56382 110.53833 H21 D21 Study area The study area covered 10 districts distributed among three provinces in Java, Indonesia. The districts were Figure 1. Location of sample collection covered 10 districts distributed among three provinces in Java, Indonesia. The black square ( ) indicated location of sample collection 14 BIODIVERSITAS 19 (1): 12-17, January 2018 Procedures RAPD amplification Sample collection The RAPD reaction was performed using nine primers Forty-two F. moluccana were sampled from various (Operon Technology) (Table 2). The reaction was locations on a F. moluccana plantation (Table 1). To performed using Dream Taq Green PCR Master Mix analyze F. moluccana resistance, sampling was carried out (Thermo Scientific). The reaction mixture consisted of 200 using a pairing method (Figure 2). Samples were taken μM of each dNTP, 1× GC Buffer, 0.5 μM of each primer, from healthy and diseased trees that were located side-by- 0.02 unit μl-1 of Dream Taq DNA Polymerase, and 100 ng side. Healthy trees were categorized as trees with no of the DNA template. The reaction conditions were as disease or low severity of disease (severity score: 0 or 1). follows: pre denaturation at 94°C for 5 min; 45 cycles of Meanwhile, infected trees were categorized as trees with denaturation at 94°C for 1 min, annealing at 34°C for 1 min, high disease severity (severity score: 4 or 5). The criteria and polymerization at 72°C for 1 min; and a final for the disease severity score were as follows: 0: There was polymerization at 72°C for 5 min. The resulting PCR no disease recorded; 1: There were up to 20% gall on the product was then visualized using electrophoresis of a 1% leaves, twigs, or branches; 2: There were 21-50% of galls (w v-1) agarose gel stained with SYBR®SAFE (Invitrogen) on the leaves, twigs, or branches; 3: There were more than in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) at 50% of galls on the leaves, twigs, or branches, and one or 100 V for 40 min.
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