KRISTIANTO NUGROHO, KRISTIANTO
Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian, Jl. Tentara Pelajar 3A, Bogor 16111 Indonesia Telp. (0251) 8337975

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METODE EKSTRAKSI DNA TANAMAN TANPA PRESIPITASI ETANOL UNTUK KEGIATAN POLYMERASE CHAIN REACTION (PCR) Nugroho, Kristianto; Terryana, Rerenstradika Tizar; Reflinur, .; Lestari, Puji
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol 6, No 1 (2019): June 2019
Publisher : Badan Pengkajian dan Penerapan Teknologi (BPPT)

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A Simplified Plant DNA Extraction Protocol without Ethanol Precipitation for Polymerase Chain Reaction (PCR) Activities ABSTRACTMolecular-based research in agriculture includes DNA extraction stage involving DNA precipitation using ethanol or isopropanol which tends to take a long time. The purpose of this study was to obtain a plant DNA extraction method for Polymerase Chain Reaction (PCR) activities without going through the ethanol precipitation stage. Five important agricultural commodity crops, namely rice, corn, soybeans, chilies, and shallots were extracted by DNA using the modified Doyle and Doyle method. After the extraction phase using chloroform and isoamil alcohol solvents, the supernatant obtained was not precipitated using ethanol but was directly diluted and used as a template in PCR activities using two pairs of Simple Sequence Repeat (SSR) markers. The results showed that all samples could be well amplified, and amplicon tape visualized in both 1% agarose gel and 6% polyacrylamide gel were clearly visible. This method could save time and material, and reduce the dependence on liquid nitrogen. But this method is still limited to PCR requirements only, and cannot be used for activities that require high quality and quantity of DNA such as Next Generation Sequencing (NGS), digestion, and hybridization.Keywords: DNA extraction, ethanol precipitation, liquid nitrogen, PCR, SSR,  ABSTRAKPenelitian berbasis molekuler pada bidang pertanian mencakup tahapan ekstraksi DNA yang melibatkan presipitasi DNA menggunakan etanol atau isopropanol yang cenderung memakan waktu lama. Tujuan penelitian ini adalah untuk memperoleh metode ekstraksi DNA tanaman untuk kegiatan Polymerase Chain Reaction (PCR) tanpa melalui tahapan presipitasi etanol. Lima tanaman komoditas pertanian penting yaitu padi, jagung, kedelai, cabai, dan bawang merah diekstraksi DNA-nya menggunakan metode Doyle and Doyle yang dimodifikasi. Setelah tahap ekstraksi menggunakan pelarut kloroform dan isoamil alkohol, supernatan yang terbentuk tidak dipresipistasi menggunakan etanol melainkan langsung diencerkan dan digunakan sebagai template dalam kegiatan PCR menggunakan dua pasang marka Simple Sequence Repeat (SSR). Hasil menunjukkan bahwa seluruh sampel dapat teramplifikasi dengan baik serta pita hasil amplikon yang tervisualisasi baik pada gel agarosa 1% maupun gel poliakrilamid 6% terlihat jelas. Metode ini dapat menghemat waktu dan bahan serta mengurangi ketergantungan pemakaian nitrogen cair. Tetapi metode ini masih terbatas hanya untuk kebutuhan PCR saja dan tidak dapat digunakan untuk kegiatan yang membutuhkan DNA dengan kualitas serta kuantitas tinggi seperti Next Generation Sequencing (NGS), digesti, maupun hibridisasi.Kata Kunci: ekstraksi DNA, nitrogen cair, PCR, presipitasi etanol, SSR
Genetic Diversity Analysis and F2 Population Development for Breeding of Long Juvenile Trait in Soybean Tasma, I Made; Yani, N. P. Mega Gena; Purwaningdyah, Rosliana; Satyawan, Dani; Nugroho, Kristianto; Lestari, Puji; Trijatmiko, Kurniawan R.; Mastur, Mastur
Jurnal AgroBiogen Vol 14, No 1 (2018): June
Publisher : Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

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Genetic diversity analysis using molecular markers is an important step for selecting appropriate parents in a soybean breeding program. The aims of this study were to (1) analyze genetic diversity of 29 soybean genotypes assessed with 27 SSR markers for selecting appropriate parents and (2) develop F2 populations to be used for breeding long juvenile (LJ) trait in soybean tobe cultivated in short photoperiod condition. The soybean genotypes used consisted of 11 Indonesian soybean genotypes and 18 genotypes introduced from the USA. F2 populations were developed by crossing Grobogan with three introduced genotypes carrying LJ character. The PIC values of the 27 SSR markers ranged from 0.87 to 0.96. Cluster analysis resulted in three mainclusters at coefficient similarity of 0.76. The five LJ introduced accessions and the nine Indonesian genotypes showed high genetic distances and are useful as parent pairs for developing breeding populations. The F1 progeny phenotypicperformances of the cross far exceeded the performaces of both parents. Three F2 populations were developed by crossing the distantly related soybean genotypes. The F2 populations were verified by using SSR markers and it was found that they segregated in a 1:2:1 ratio confirming the segregation ratio of codominant SSR markers. The F2 populations should be useful for breeding LJ characters to improve soybean productivity in low latitude tropical countries such as Indonesia, which has day length of approximately 12 h all year round.
KARAKTERISASI KERAGAMAN GENETIK 27 GENOTIPE CABAI BERDASARKAN MARKA SSR (SIMPLE SEQUENCE REPEAT) Terryana, Rerenstradika Tizar; Nugroho, Kristianto; Rijzaani, Habib; Lestari, Puji
BERITA BIOLOGI Vol 17, No 2 (2018)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

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Chili pepper (Capsicum annuum) is one of the high economical horticultural comodity in Indonesia and its genetic diversity contributes to the success of breeding programs. Simple sequence repeat (SSR) markers can be used to analyze genetic diversity among chili pepper genotypes. The aim of this research was to analyze the genetic diversity of twenty-seven genotypes of chili pepper by using 24 SSR markers. The collected data was analyzed using cluster analysis and principle coordinate analysis (PCoA). The result showed that high allele variation (4–17 alleles) was observed among chili pepper genotypes tested, with an average allele number and Polymorphism Information Content (PIC) value was 7.708 and 0.758 (0.598–0.920) respectively. All of SSR markers showed PIC value >0.5 which indicated that these markers were suitable for chili pepper diversity studies with a high differentiation and with the average value of genetic diversity was 0.78. The clustering and principle coordinate analysis showed that twenty-seven genotypes of chili pepper were divided into two groups (coefficient of similarity 0.74 in cluster analysis) indicating a high genetic variability among them. Genetic diversity analysis in this study will be useful as an initial basis of selection for appropriate parents with desired traits to assist the breeding program of chili pepper in Indonesia.
Genetic Diversity Analysis Using Resistance Gene Analog-Based Markers to Support Morphological Characterization of Shallots Herlina, Lina; Reflinur, Reflinur; Nugroho, Kristianto; Terryana, Rerenstradika T.; Sobir, Sobir; Maharijaya, Awang; Wiyono, Suryo
Jurnal AgroBiogen Vol 14, No 2 (2018): December
Publisher : Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

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Abstract

Shallot (Allium cepa var. aggregatum) is one of the most important vegetable crops grown in Indonesia. The limited knowledge available on the genetic diversity and the threat of plant disease have been major problems to maintain high shallot production in Indonesia. Development of molecular markers linked to disease resistance is required for molecular breeding activity in this crop. This study aimed to assess the genetic diversity at conserved domain of resistance gene analog (RGA) in a set of 36 Indonesian shallot genotypes to complement morphological characterization. Twelve morphological and fifteen molecular markers traits were investigated in an attempt to characterize and to discriminate the Indonesian shallots genotypes. Characterization at orphological level indicated that phenotypic variance was highest for total bulb weight (TWB, cv = 99.39%) and the least for the plant height (PH, cv = 28.16%). The correlation analysis between traits showed that TWB and number of bulb (NB), TWB and bulb weight per plant (WB), NB and WB, and WB and PH were positively correlated. Molecular analysis revealed a total of 1,512 alleles with an average of 1.946 alleles per locus. The Polymorphism Information Content (PIC) values ranged from 0.253 to 0.676 and six out of 15 RGA markers were highly informative with PIC values ≥0.50. Based on cluster analysis, the 36 Indonesian shallot genotypes were clearly discriminated into six major groups. These results revealed that the RGA-based markers could support the morphological characterization in evaluating the genetic diversity of shallots. 
KERAGAMAN GENETIK 24 VARIETAS PADI SAWAH DAN PADI GOGO (Oryza sativa L.) INDONESIA BERDASARKAN MARKA SSR Nugroho, Kristianto; Slamet, Slamet; Lestari, Puji
Scripta Biologica Vol 4, No 1 (2017)
Publisher : Fakultas Biologi | Universitas Jenderal Soedirman

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Abstract

The use of molecular marker is an efficient approach to analyze the genetic diversity and it can be used widely in biological studies. The characterization of rice germplasms by using molecular markers technique is more precise because it is not influenced by environmental factors. The purpose of this study was to analyze the genetic diversity of 24 varieties of lowland and upland rice by using 15 SSR markers. The results showed as many as 86 alleles were detected in 24 rice varieties, with the average number of alleles per marker was 5.73 and the range of alleles per locus was 2-10. The average of major allele frequency was 43% with the lowest score was 26% on RM6997 and RM536 markers and the highest score was 65% on RM60 marker. A total of 14 SSR markers were able to discriminate heterozygous alleles within a range between 0.17 (RM105) to 1.00 (RM201, RM263, RM416, RM518 and RM223). The value of gene diversity ranged from 0.48 (RM60) to 0.81 (RM536) with an average of 0.70. The value of PIC (Polymorphic Information Content) ranged from 0.38 (RM105) to 0.78 (RM536) with an average of 0.65. The phylogenetic analysis showed that 24 rice varieties separate into two main clusters in the coefficient of 0.63. The first cluster consists of 12 lowland varieties and the second cluster consists of 12 upland varieties. The genetic diversity data in this study were expected could be a valuable information in the rice plant breeding activities in the future.
Studi Kekerabatan Tetua Persilangan dan Polimorfisme Marka Mikrosatelit pada Bawang Merah (Allium cepa var ascalonium L. (Back)) Nugroho, Kristianto
Scripta Biologica Articles in Press
Publisher : Fakultas Biologi | Universitas Jenderal Soedirman

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Bawang merah merupakan salah satu tanaman hortikultura yang memiliki nilai ekonomi tinggi sebagai bumbu masakan. Ketersediaan bawang merah seringkali bersifat fluktuatif akibat pengaruh cuaca serta serangan hama dan penyakit yang berakibat terjadinya lonjakan harga di pasaran. Busuk pangkal umbi atau moler yang disebabkan oleh cendawan Fusarium oxysporum f. sp. cepae merupakan salah satu penyakit penting pada bawang merah yang secara signifikan dapat menyebabkan kehilangan hasil. Oleh karena itu, perakitan varietas unggul baru (VUB) bawang merah yang memiliki karakter ketahanan terhadap penyakit tersebut perlu terus diupayakan. Penelitian ini bertujuan untuk menganalisis kekerabatan empat genotipe bawang merah menggunakan 40 pasang marka mikrosatelit dan menganalisis polimorfisme dari marka-marka tersebut. Penelitian ini dilaksanakan pada bulan Maret hingga Agustus 2018 di Laboratorium Biologi Molekuler BB Biogen, Bogor. Hasil amplifikasi diskor sebagai data biner dan dianalisis menggunakan perangkat lunak NTSYS dan PowerMarker. Hasil penelitian menunjukkan bahwa keempat varietas bawang merah mengelompok menjadi dua klaster utama pada koefisien kemiripan genetik 0,58. Berdasarkan hasil estimasi nilai jarak genetik, sebanyak lima kombinasi tetua persilangan, yaitu antara varietas Mentes dan Sembrani (jarak genetik sebesar 45%), Mentes dan Tiron (jarak genetik sebesar 46%), Sembrani dan Kuning (jarak genetik sebesar 41%), Sembrani dan Tiron (jarak genetik sebesar 29%), serta Kuning dan Tiron (jarak genetik sebesar 37%) direkomendasikan sebagai kombinasi persilangan yang ideal untuk perakitan VUB tahan terhadap penyakit busuk umbi Fusarium. Di antara 40 marka yang digunakan, sebanyak 25 marka bersifat polimorfik dan akan menjadi bagian set marka molekuler yang akan digunakan dalam pemetaan gen terkait karakter ketahanan Fusarium ke depannya. Kata kunci: bawang merah (Allium cepa var ascalonium L. (Back)), mikrosatelit, marka molekuler, kekerabatan 
METODE EKSTRAKSI DNA CABAI (Capsicum annuum L.) MENGGUNAKAN MODIFIKASI BUFFER CTAB (CETHYL TRIMETHYL AMMONIUM BROMIDE) TANPA NITROGEN CAIR Nugroho, Kristianto; Terryana, Rerenstradika T; Lestari, Puji
Scripta Biologica Vol 4, No 2 (2017)
Publisher : Fakultas Biologi | Universitas Jenderal Soedirman

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Chili pepper is an agricultural commodity having high economic value. The production and supply of chili pepper frequently did not match the increased demand; it caused the market price fluctuated. It is important to create new varieties of chili pepper with high production trait to overcome the scarcity. Therefore the plant breeding activities for chili pepper should be done intensively in both conventional and molecular-based to obtain varieties of chili pepper with expected qualities. In molecular breeding, DNA extraction is the crucial steps of the process. If extracted DNA has an excellent quality and quantity,  the next processes normally could be completed with the high-quality result. To date, most methods of DNA extraction used liquid nitrogen to destroy the tough carbohydrates of plant tissue. Liquid nitrogen is nitrogen gas in a fluid state which quite difficult to be distributed to the remote laboratory wit no available storage facility. This study aimed to obtain a modified DNA extraction method, in particular for chili pepper, which capable to produce DNA with high quality and quantity without using liquid nitrogen. The sample used consisted of eight F2 plants including their hybrid-parental of the Kencana and the 0207. This research applied modified Doyle and Doyle method for extraction. Modification of extraction buffer is done through the addition of the 1% (w/v) PVP (Polyvinylpyrrolidone) and 0.2% (v/v) β-mercaptoethanol. The results showed that the DNA extracted using this method has good quality and quantity, capable of being amplified by using SSR (Simple Sequence Repeat) primer and could be digested by restriction enzyme EcoRI. Besides, this method can reduce dependence on the use of liquid nitrogen, in particular for remote laboratories with no available storage facility.