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05 SS14-070 Pieter the Molecular Lay-Akhir2nd Makara J. Sci. 19/1 (2015), 27-36 doi: 10.7454/mss.v19i1.4479 The Molecular Diversity-based ISSR of Durio tanjungpurensis Originating from West Kalimantan, Indonesia Pieter Agusthinus Riupassa 1,2 , Tatik Chikmawati 1*, Miftahudin 1, and Suharsono 1 1. Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Bogor 16680, Indonesia 2. Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Pattimura, Ambon 97233, Indonesia *E-mail: [email protected] Abstract The Durian Tengkurak ( Durio tanjungpurensis Navia) is one of the endangered exotic species in the Malvaceae family. The species is important for conservation of the germplasm and is considered a potential genetic resource for the development of durian in the future. The objective of this research project was to assess the molecular diversity of D. tanjungpurensis in West Kalimantan, based on Inter Simple Sequence Repeat (ISSR) markers. We applied ten ISSR primers to reveal the genetic diversity of 60 individuals from six natural endemic D. tanjungpurensis populations. The genetic diversity parameters were estimated based on binary data about PCR products (present or absent bands). The results showed that the mean number of observed alleles, the mean number of effective alleles, the genetic diversity, the Shannon’s Information Index score, the number of polymorphic loci, and the percentage of polymorphic loci were 1.53, 1.29, 0.17, 0.26, 77.83, and 52.59, respectively. An analysis of molecular variance (AMOVA) showed that the genetic diversity within a population (65%) was higher than that found between the populations (35%). UPGMA clustering and principal coordinate analysis, based on the DICE similarity matrix, were used to classify the populations into three groups: 1) Hutan Rejunak and Tembaga, 2) Bukit Merindang, and 3) Hutan Rawak, Bukit Sagu 1, and Bukit Sagu 2. Further analysis of the population structure using STRUCTURE software was used to classify all the individuals into two major categories, thus uniting Groups 2 and 3 as one major category. In conclusion, a high level of genetic diversity in the Durian Tengkurak was revealed utilizing the ISSR markers employed in the study. Abstrak Diversitas Molekuler Berbasis ISSR pada Durio tanjungpurensis Asal Kalimantan Barat, Indonesia . Durian Tengkurak ( Durio tanjungpurensis Navia) adalah salah satu spesies langka yang eksotis dari suku Malvaceae. Durian tersebut bernilai penting untuk konservasi plasma nutfah dan berpotensi sebagai sumber daya genetik untuk pengembangan durian di masa depan. Tujuan penelitian adalah mengetahui keragaman molekuler D. tanjungpurensis asal Kalimantan Barat berdasarkan penanda Inter Simple Sequence Repeat (ISSR). Sepuluh primer ISSR digunakan untuk mengetahui keragaman genetik 60 individu Durian Tengkurak dari enam populasi endemik alami D. tanjungpurensis . Parameter keragaman genetik didasarkan pada data biner pita DNA produk PCR, yaitu ada atau tidak- ada pita. Hasil penelitian menunjukkan bahwa rata-rata jumlah alel, rata-rata jumlah efektif alel, diversitas genetik, indeks informasi Shannon, jumlah polimorfik lokus, dan persentase polimorfik lokus berturut-turut adalah 1,53, 1,29, 0,17, 0,26, 77,83 dan 52,59. Analisis ragam molekuler (AMOVA) menunjukkan keragaman genetik yang lebih tinggi di dalam populasi (65%) dibandingkan antar populasi (35%). Analisis gugus menggunakan metode UPGMA berdasarkan matriks keserupaan Dice dan analisis koordinat utama digunakan untuk mengelompokkan semua individu populasi ke dalam tiga kelompok, yaitu grup 1 (Hutan Rejunak dan Tembaga), grup 2 (Bukit Merindang), dan grup 3 (Hutan Rawak, Bukit Sagu 1 dan Bukit Sagu 2). Analisis lebih lanjut terhadap struktur populasi menggunakan program STRUCTURE menyatukan grup 2 dan 3 ke dalam satu grup utama. Penelitian ini berhasil mengungkap keragaman genetik Durian Tengkurak menggunakan penanda ISSR. Keywords: Durian Kura, Durian Tengkurak, endemic species, genetic diversity, ISSR marker Introduction countries in Southeast Asia, including Thailand and the Philippines. It is a popular fruit because of its unique The durian ( Durio spp.) is a fruit with a high economic taste and its wide availability. It varies in terms of its value, which is grown in Indonesia and several other size and smell, the color of its skin, and the color of its 27 March 2015 | Vol. 19 | No. 1 28 Riupassa, et al. flesh (seed arillus), which depend on the species and and high levels of polymorphism [6]. In certain cultivar. This plant was first found in the forests of circumstances, they can act as co-dominant markers that Malaya (or Malaysia) by Murray, and was given the title are able to identify individuals with heterozygote alleles the King of Fruit by Wallace [1]. Morphologically, the [7-8]. Within the PCR process, oligonucleotide primers durian is a prickly fruit that grows on a tree branch and can be determined randomly based on di-, tri-, or tetra- is classified as part of the Malvaceae Family. nucleotide repeats, which, at the end of the 3' or 5', could be added with 1-3 bases. ISSR-marker analysis Borneo Island is considered to be the epicenter of durian has been performed on many plant species, with various diversity. There are 21 species of durian tree found on objectives, such as the collection and conservation of the island [2], one of which is the Durian Tengkurak the African citrus [6], the conservation of japonica tea in (Durio tanjungpurensis Navia), which is endemic to China and Japan [9], the characterization of the sweet West Kalimantan. potato Brazilian germplasm [5], the sex determination of green potato plants from India [10], determination of The Durian Tengkurak is relatively unknown, compared the homogeneity of in-vitro clones in grapes [11], and with the common durian, Durio zibethinus Murray. the study of the evolution and speciation of the Interestingly, bunches of Durian Tengkurak fruit are Asteraceae [12]. ISSR markers have also been used to found at the base of the tree’s trunk, whereas common assess the genetic diversity of D. zibethinus cultivars in durian fruit is found hanging from a tree branch, as Thailand [13], but they have not been utilized previously stated. Phenotypically, the fruit of Durian previously to reveal the genetic diversity of the Durian Tengkurak is slightly different from that of the common Tengkurak of West Kalimantan. Durian Kura (D. testudinarum Becc.), being smaller (5-8 cm in diameter), with a brown rind, and with the white The objective of this research project was to analyze the aril of the seeds being less than 1 mm thick [3]. genetic diversity of the Durian Tengkurak of West Kalimantan using ISSR markers. It is hoped that in The high rate of deforestation on Borneo Island and the future, the genetic diversity profile for the Durian conversion of much land to monocultural practices have Tengkurak could be used for the purpose of conserving caused the indigenous durian to become endangered. Durian Tengkurak germplasms. Therefore, conservation efforts in relation to the indigenous durian germplasm, including the Durian Materials and Methods Tengkurak, must be encouraged. One of the efforts being made by researchers is in the identification of the Plant materials. A total of 60 samples of Durian diversity within the durian germplasm. The development Tengkurak leaves from West Kalimantan, spread over of genetic tools for plants is needed in order for the six locations (Table 1 and Figure 1) were examined in genetic profile of the durian to be revealed. One current this research. Durian Tengkurak trees grow naturally in set of applications available for such a procedure are the indigenous forest and hill communities on the slope ISSR molecular markers. lands. ISSR markers are multilocus markers that are based on DNA isolation. DNA was isolated using the CTAB the amplification of DNA fragments, which are flanked method, with some modifications [14]. Fresh leaves by types of repetitive nucleotide regions (repeat were weighed into sample amounts of 0.2 g and then cut sequences) with inverted orientations and are scattered up and crushed to a powder with liquid nitrogen using a throughout genome chromosomes. ISSR markers are mortar, before being transferred into 2 mL eppendorf dominant markers and have several advantages, when tubes. 1 mL of CTAB lysis buffer was added, followed compared with other dominant markers, such as RAPD by 2 µL of 2-mercaptoethanol (Sigma Chemical Co., markers [4]. In studies, ISSRs have revealed high USA). The sample tubes were incubated in a water bath discrimination ratios, high genetic variability levels [5], Table 1. Locations and Numbers of Samples of Durian Tengkurak Leaves, West Kalimantan Locations Coordinates Altitude (m asl) Code Samples Hutan Rejunak, Rawak District, Sekadau Regency 0o09’16.0”S – 110 o51’13.5”E 125 HRJ (H) 11 Hutan Rawak, Rawak District, Sekadau Regency 0o09’15.8”S – 110 o51’13.9”E 120 HRW (R) 11 Tembaga, Nanga Mahap District, Sekadau Regency 0o30’37.4”S – 110 o40’48.9”E 93 TBG (T) 10 Bukit Merindang, Nanga Taman District, Sekadau Regency 0o18’30.5”S – 110 o55’49.3”E 105 MER (E) 11 Bukit Sagu 1, Nanga Silat Hilir District, Kapuas Hulu Regency 0o16’09.2”N – 111 o56’11.8”E 59 BS1 (B) 8 Bukit Sagu 2, Nanga Silat Hilir District, Kapuas Hulu Regency 0o16’09.9”N – 111 o56’12.1”E 74 BS2 (S) 9 ∑ samples 60 Makara J. Sci. March 2015 | Vol. 19 | No. 1 The Molecular Diversity-based ISSR of D. tanjungpurensis 29 Figure 1. Distribution Map of Durian Tengkurak Populations in the West Kalimantan Province of Borneo, Indonesia (Red Circles) Table 1. List of ISSR Primers, Primer Lengths, and Sequences, as well as the PCR annealing Temperatures used in the Study ISSR primer Primer sequence (5’–3’) Primer length Annealing temperature ( oC) References ISSR1 (AGG) 5 15 49.5 [13] ISSR3 (AGA) 4AGT 15 42.5 “ ISSR4 (GAG) 5AC 17 50.0 “ ISSR5 (GAG) 5AT 17 51.6 “ ISSR9 (GGGGT) 3 15 53.0 “ PKBT2 (AC) 8TT 18 52.0 [15] PKBT3 (AG) 8T 17 47.5 “ PKBT7 (GA)9A 19 50.7 “ PKBT8 (GA) 9C 19 52.8 “ PKBT12 (GT) 9T 19 54.0 “ (temperature: 65 °C) for 60 min, with the tubes being purified from RNA contamination using RNAse A inverted every 15 min.
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