Putranto, Riza Arief
Center for Forest Biotechnology and Tree Improvement (CFBTI)

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Purification, characterization, and bioassay of putative protease inhibitors from Hevea brasiliensis latex PUTRANTO, Riza Arief; SISWANTO, .; MULYATNI, Agustin Sri; BUDIANI, Asmini; TISTAMA, Radite
E-Journal Menara Perkebunan Vol 84, No 2 (2016): Desember 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1452.514 KB) | DOI: 10.22302/iribb.jur.mp.v84i2.220

Abstract

Lateks yang menyerupai cairan susu putih diperoleh dari penyadapan kulit batang tanaman karet (Hevea brasiliensis). Lateks merupakan sitoplasma dari jaringan pembuluh bernama latisifer yang didalamnya terkandung berbagai macam komponen, termasuk protein-protein penting. Berbagai jenis enzim yang memiliki fungsi terkait pertahanan tanaman dari serangan patogen dan pelukaan telah berhasil dideteksi di dalam lateks, di antaranya protease inhibitor (PI). Protease inhibitor memiliki aktivitas senyawa antifungi sehingga berpotensi untuk  dimanfaatkan sebagai biofungisida. Pada penelitian ini, protease  inhibitor putatif yang berasal dari serum B (lutoid) lateks tanaman karet telah berhasil diisolasi menggunakan teknik Ion Exchange Chroma-tography. Dari total 70 fraksi protein yang diekstrak dari kolom, hanya 26 fraksi yang menunjukkan kadar protein yang terukur. Kandungan protease inhibitor putatif yang di-peroleh berkisar antara 0,0067 hingga 0,022 mL/g serum B dari hasil 3 fraksi terpilih. Aktivitas penghambatan terhadap empat enzim protease (subtilisin A, tripsin, α-kimotripsin, dan papain) menunjukkan karakteristik protease inhibitor putatif tersebut sebagai serine dan/atau cysteine inhibitor protease dengan persentase hambatan di atas 15% terhadap protease target. Hasil SDS-PAGE memperlihatkan pemisahan protein dominan yang diperkirakan merupakan protease inhibitor putatif dengan berat molekul sebesar 21,5 kDa. Uji bioassay aktivitas antifungi secara in vitro dari protease inhibitor memperlihatkan penghambatan pertumbuhan miselium dari fungi Ganoderma boninense, Sclerotium sp., dan Rigidosporus lignosus. [Kata kunci : protease inhibitor, Hevea brasiliensis, lateks, serum B, ion exchange chromatography]AbstractLatex, a milky white liquid, is the main product from rubber tree (Hevea brasiliensis). Latex is the cytoplasm of complex cellular networks named laticifers in which it contains many different components, including important proteins. Various types of enzymes carrying functions associated with plant defense against pathogen and wounding have been detected in latex in which one of these enzymes is protease inhibitor (PI). Plant protease inhibitor has tremendous potential as an antifungal agent which can be developed as biofungicide. In this work, protease inhibitors from B-serum (lutoid) of rubber tree latex were isolated and purified using Ion Exchange Chromatography (IEC) technique. Of the total 70 fractions of proteins extracted from the columns, only 26 fractions showed measurable levels of protein. The concentration of obtained putative protease inhibitors (three fractions of IEC) ranged from 0.007 to 0.022 mL/g B-serum. Inhibitory activity against four protease enzymes (subtilisin A, trypsin, α-chymotrypsin, and papain) showed the characteristics of Hevea putative protease inhibitors from B-serum as serine and/or cysteine protease inhibitors with more than 15% inhibitory activity of target protease. Based on SDS-PAGE visualization, the molecular weight of dominant protein considered as Hevea putative protease inhibitors was 21.5 kDa. In vitro bioassay test of antifungal activity for Hevea putative protease inhibitors showed reduced mycelium growth of Ganoderma boninense, Sclerotium sp., and Rigidosporus lignosus.[Keywords: protease inhibitor, Hevea brasiliensis, latex, B-serum, ion exchange chromatography]
Evaluation of eleven reference genes for Reverse Transcriptase Quantitative PCR of rubber tree under water deficit Evaluasi sebelas gen referensi untuk Reverse Transcriptase Quantitative PCR pada tanaman karet tercekam kekeringan PUTRANTO, Riza Arief; LECLERCQ, Julie; MONTORO, Pascal
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (280.989 KB) | DOI: 10.22302/iribb.jur.mp.v83i2.5

Abstract

AbstrakReverse Transcriptase Quantitative PCR (RT-qPCR) merupakan teknik yang sangat ampuh untuk mendeteksi jumlah mRNA yang rendah dalam sel tanaman. Pengukuran akumulasi transkrip tersebut relatif terhadap kontrol ekspresi seperti gen-gen housekeeping. Keandalan teknik RT-qPCR ber-gantung pada pemilihan kontrol internal yang disebut pula gen referensi. Hal tersebut menjadi alasan kenapa validasi gen referensi disarankan untuk setiap set sampel cDNAs yang akan diguna-kan pada eksperimen RT-qPCR baru. Penelitian ini bertujuan untuk menganalisis stabilitas sebelas gen-gen housekeeping terpilih pada tiga organ Hevea brasiliensis (daun, kulit batang dan akar) tercekam kekeringan moderat selama 15 hari. RNA total diisolasi dari 18 sampel yang terdiri dari tanaman kontrol dan tercekam kekeringan pada hari ke-0 (D0), ke-5 (D5) dan ke-15 (D15). Kualitas cDNA yang disintesis divalidasi dengan amplifikasi PCR menggunakan primer HbActin. Kesebelas pasangan primer penyandi gen-gen housekeeping pada Hevea (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S dan HbUBI) divalidasi dengan amplifikasi PCR. Nilai Crossing-point (Cp) yang diukur dengan metode derivatif kedua pasca analisis RT-qPCR mengungkapkan nilai rerata Cp yang lebih tinggi secara signifikan untuk kesebelas gen housekeeping pada titik sampling D5 dibanding D0 dan D15. Studi ini menyarankan bahwa metode perhitungan koefisien keragaman (CV) sederhana dapat digunakan untuk menentu-kan peringkat gen referensi pada tanaman karet berdasarkan ekspresinya yang stabil. Lima gen housekeeping (HbRH2b, HbRH8, HbUBC4, HbαTUB dan HbActin) dapat digunakan sebagai gen referensi untuk analisis RT-qPCR pada Hevea brasiliensis yang tercekam kekeringan moderat. Gen HbRH2b memiliki ekspresi paling stabil dibanding yang lain.AbstractReverse Transcriptase Quantitative PCR (RT-qPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected house-keeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others.
Identifikasi famili gen putatif penyandi protease inhibitor dengan pendekatan in silico komparatif pada genom Hevea brasiliensis Muell. Arg (Identification of putative gene family encoding protease inhibitors by in silico comparative analysis in Hevea brasiliensis Muell. Arg genome) MARTIANSYAH, Irfan; PUTRANTO, Riza Arief; KHUMAIDA, Nurul
E-Journal Menara Perkebunan Vol 85, No 2 (2017): Oktober 2017
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (807.039 KB) | DOI: 10.22302/iribb.jur.mp.v85i2.257

Abstract

AbstractProtease inhibitors (PIs) are small proteins that form complexes with proteases and inhibits their proteolytic activity. Its potential application as an antimicrobial agent has been studied. Most of PIs' molecule size is around 8-22 kDa depending on their protein families.To date, on the basis of sequence homologies of inhibitor domains, PIs have been classified into 48 families in all organisms. In plant, more than 13 families of PIs have been identified but they were not widely identified in the rubber tree (Hevea brasiliensis Muell.Arg). In the present study, 40 putative HbPI genes, designated as HbPI01 to HbPI36, were identified from whole-genome sequence of rubber tree clone Reyan 7-33-97 using 7453 scaffolds available online in NCBI with the accession code: LVXX01000000. Multiple sequence alignment using MUSCLE algorithm discovered seven conserved motifs (Motifs I-VII) among HbPIs. Phylogenetic analysis of 50 and 36 PI amino acid residues of 32 scaffolds containing putative PI genes from Arabidopsis thaliana and H. brasiliensis showed three clusters (families): LTP-I, SERPIN and LTP-II. LTP-I has 23 putative HbPI genes (HbPI05 to HbPI27) and 12 AtPI genes. SERPIN, a family member of serine protease inhibitor group, has 11 putative HbPI genes (HbPI01 to HbPI04 and HbPI28 to HbPI34) and 22 AtPI genes. LTP-II has 2 putative HbPI genes (HbPI35 to HbPI36) and 16 AtPI genes. In conclusion, this work provides valuable information for further functional characterization of HbPI genes in H. brasiliensis.[Key words: protease inhibitor, genome-wide, scaffold, in silico, Hevea brasiliensis]. AbstrakProtease inhibitor (PI) merupakan protein yang membentuk kompleks dengan protease dan menghambat aktivitas proteolitik dari enzim tersebut. Potensi penggunaan protease inhibitor sebagai agensia antimikroba telah diketahui. Kebanyakan PI memiliki ukuran molekul sekitar 8-22 kDa bergantung pada familinya. Saat ini, PI dapat diklasifikasikan menjadi 48 famili di seluruh organisme berdasarkan kemiripan sekuen dari domain inhibitornya. Pada tanaman, lebih dari 13 famili PI telah diketahui tetapi pada tanaman karet (Hevea brasiliensis Muell.Arg) belum diidentifikasi. Pada penelitian ini, sebanyak 40 gen putatif penyandi PI (HbPI01 hingga HbPI36) telah berhasil diidentifikasi dari 7453 scaffold genom utuh tanaman karet klon Reyan 7-33-97 yang tersedia secara daring dengan kode aksesi LVXX01000000. Penjajaran sekuen menggunakan algoritma MUSCLE memper-lihatkan tujuh konservasi motif (Motif I-VIII) pada famili gen putatif HbPIs. Analisis pohon filogenetik dari tanaman Arabidopsis thaliana dan H. brasiliensis sebanyak 50 dan 36 sekuen residu asam amino dari 32 scaffold yang mengandung gen putatif PI menunjukkan adanya tiga klaster besar, yaitu famili LTP-I, SERPIN dan LTP-II. LTP-I terdiri dari 23 gen putatif HbPI (HbPI05 hingga HbPI27) dan 12 gen AtPI. SERPIN yang merupakan anggota kelas protease inhibitor serin terdiri dari 11 gen putatif HbPI (HbPI01hingga HbPI04 dan HbPI28 hingga HbPI34) dan 22 gen AtPI. LTP-II terdiri dari 2 gen putatif HbPI (HbPI35 hingga HbPI36) dan 16 gen AtPIs. Penelitian ini menghasilkan informasi penting untuk melakukan karakterisasi fungsional lebih mendalam pada gen HbPI tanaman karet ke depannya.[Kata kunci: protease inhibitor, genome-wide,scaffold, in silico, Hevea brasiliensis].
Ekspresi dan kloning gen penyandi ADP-Glucose Phyrophosphorylase dari tanaman sagu (Metroxylon sagu Rottb.) Expression and cloning of gene encoding ADP-Glucose Phyrophosphorylase from sago palm (Metroxylon sagu Rottb.) BUDIANI1, Asmini; PUTRANTO, Riza Arief; MINARSIH, Hayati; RIYADI, Imron; SUMARYONO, .; ABBAS, Barahima
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (413.009 KB) | DOI: 10.22302/iribb.jur.mp.v83i2.4

Abstract

AbstractSago palm (Metroxylon sagu Rottb.) is a potential food and energy resources becouse it is the highest starch producing plant.  Breeding of sago palm should be directed to produce elite genotype with superior characters such as high starch content, wider pith diameter, without spine and high starch quality. However, research on sago palm in Indonesia so far is limited espescially in the field of cultivation and breeding, and attempt to produce such elite would take long time. Availability of molecular marker for starch content would be beneficial to shorten the length period of breeding. ADP-Glucose Phyrophosphorylase is one of the important enzymes in starch biosynthesis. Therefore its gene is an interesting subject in order to develope molecular marker of high starch content.  This research was aimed to study the expression of gene encoding AGP in the sago palm with high starch content versus low starch content, and to clone the full cds of the gene. RNA was isolated from leaf and pith of both palms. Exspression analysis and amplify-cation of full cds were conducted by Reverse Transcryptase-Polymerase Chain Reaction (RT-PCR) using specific primers. The results showed that sago palm with higher starch content expressed AGP higher than that of sago palm with lower  starch content. Expression of AGP in the full developing leaf was higher than in the young leaf, and there was no expression detected in the pith. The full cds of AGP was successfully amplified and cloned. Even though the DNA sequence showed high homology with DNA sequence of the same gene that has been deposited in GenBank, there were differences in severall nucleotide including that in the active domain of the enzyme.AbstrakTanaman sagu merupakan sumber pangan dan energi yang sangat potensial untuk dikembangkan karena merupakan tanaman penghasil karbihidrat tertinggi. Pemuliaan tanaman sagu mestinya diarah-kan untuk menghasilkan bibit sagu yang selain memiliki rendemen pati tinggi, juga memiliki diameter empulur besar, tidak berduri dan memiliki cita rasa pati yang enak. Namun, sampai saat ini riset mengenai sagu di Indonesia masih sangat terbatas, sehingga pemuliaan sagu untuk menghasilkan bibit unggul demikian akan memerlukan waktu lama. Ketersediaan penanda rendemen pati akan sangat membantu mempercepat pemuliaan tanaman tersebut. ADP-Glucose Pyrophosphorylase adalah salah satu enzim yang berperan penting dalam biosintesis pati, sehingga gene penyandinya merupakan subjek yang menarik dalam pengembangan marka kandungan pati tinggi.  Sebagai bagian dari upaya untuk mendapat-kan penanda rendemen pati tinggi pada tanaman sagu, penelitian ini bertujuan untuk mempelajari ekspresi gen penyandi AGP. RNA diisolasi dari daun tanaman sagu rendemen pati rendah dan tanaman sagu rendemen pati tinggi. Perbedaan tingkat ekspresi gen penyandi AGP dari tanaman sagu rendemen pati tinggi vs rendemen pati rendah, dianalisis dengan teknik Reverse-Transcryptase PCR menggunakan primer spesifik. Hasil penelitian menunjukkan bahwa tanaman sagu rendemen pati tinggi mengekspresikan AGP lebih tinggi dibandingkan dengan tanaman sagu rendemen pati rendah. Ekspresi gen tersebut pada daun tua (full developing leaf) lebih tinggi di-bandingkan dengan pada daun muda, dan pada empulur tidak dideteksi ekspresi gen tersebut. Daerah penyandi lengkap AGP subunit kecil telah diklon. Meskipun memiliki homologi yang tinggi dengan sekuen DNA gen yang sama yang telah dideposit pada  GenBank,  namun terdapat perbedaan beberapa nukleotida termasuk pada daerah domain aktif dari enzim tersebut. 
Respons molekuler Hevea brasiliensis ethylene response factors (HbERFs) sebagai marka ekspresi gen terhadap stimulasi ethephon pada klon-klon tanaman karet Moleculer response of Hevea brasiliensis ethylene response factors (HbERFs) as expression marker genes in response to ethephon stimulation in rubber tree clones PUTRANTO, Riza Arief; KUSWANHADI, .; MONTORO, Pascal
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (376.771 KB) | DOI: 10.22302/iribb.jur.mp.v82i2.22

Abstract

Abstract Real-Time quantitative RT-PCR technique is a sensitive method for measuring the accumulation of gene transcripts. This widely used technique in a variety of plant species; including rubber tree (Hevea brasiliensis) must meet basic criteria in order to produce accurate gene expression markers. Gene expression markers associated to the response of ethephon stimulation such as the Hevea brasiliensis Ethylene Response Factors (HbERFs) family has been characterized in a single rubber clone. It is known that the effect of genotype on rubber tree clones can give different expression of the same gene. This difference can be converted into a profile that characterizes clones to a certain trait. This study aimed to identify gene expression profile in response to ethephon stimulation using six HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1) in three rubber tree clones having contrasted latex metabolism (PB 260, SP 217, and RRIM 600). Total RNA was isolated from 18 samples and used for cDNA synthesis. The quality of cDNAs was examined by PCR using HbActin primer. HbRH2b was selected among the 11 housekeeping genes to be used as an internal control in gene expression analysis. Gene expression analysis resulted to an induction and inhibition of  HbERFs by ethephon stimulation which are specific to a particular clone. Expression profile of three Hevea clones showed distinct characteristics. The high latex metabolism clone PB 260 was characterized by the upregulated expression of  HbRAP2.3 and HbERF12. The low latex metabolism clone SP 217 was characterized by the upregulated expression of HbRAP2.3 and HbRRTF1. Meanwhile, the profile of intermediate latex metabolism clone RRIM 600 was shown by downregulated expression of HbORA47 and up-regulated expression of HbABR1. This study shows that HbERFs gene family is an important expression marker because it can inform physiological conditions of rubber clones associated in response to ethephon. AbstrakTeknik Real-Time quantitative RT-PCR merupakan metode sensitif untuk mengukur akumulasi transkrip dari gen. Teknik yang telah banyak digunakan pada berbagai spesies tanaman, termasuk tanaman karet (Hevea brasiliensis) ini harus memenuhi kriteria dasar agar meng-hasilkan marka ekspresi gen yang akurat. Beberapa marka ekspresi gen terkait respons terhadap stimulasi ethephon seperti famili gen Hevea brasiliensis Ethylene Response Factors (HbERFs) telah dikarakterisasi pada satu klon tanaman karet. Sebagaimana diketahui, efek genotip pada klon tanaman karet dapat memberikan ekspresi yang berbeda dari gen yang sama. Perbedaan ekspresi tersebut dapat dikonversi menjadi sebuah profil yang menjadi karakteristik klon karet terhadap perlakuan tertentu. Penelitian ini bertujuan untuk mengidentifikasi profil ekspresi gen HbERFs pada tiga klon tanaman karet (PB 260, SP 217, dan RRIM 600) yang memiliki metabolisme lateks yang berbeda terhadap respons stimulasi ethephon dengan menggunakan enam gen HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1). RNA total diisolasi dari 18 sampel dan digunakan untuk sintesis cDNA. Kualitas cDNA diperiksa dengan PCR menggunakan primer HbActin. Gen HbRH2b terseleksi diantara 11 gen housekeeping digunakan sebagai kontrol internal pada analisis ekspresi gen. Hasil dari analisis ekspresi gen menunjukkan bahwa stimulasi ethephon memiliki efek induksi dan inhibisi gen yang spesifik untuk klon tertentu. Profil ekspresi dari tiga klon tanaman karet yang diuji memperlihatkan perbedaan karakteristik. Klon metabolisme tinggi PB 260 ditunjukkan dengan ekspresi positif dari gen HbRAP2.3 dan HbERF12. Klon meta-bolisme rendah SP 217 ditunjukkan oleh ekspresi positif gen HbRAP2.3 dan HbRRTF1. Sedangkan klon metabo-lisme intermedier RRIM 600 memiliki profil ekspresi negatif dari HbORA47 dan ekspresi positif dari HbABR1. Penelitian ini memperlihatkan bahwa famili gen HbERFs merupakan marka ekspresi yang penting karena dapat menginformasikan kondisi fisiologis klon tanaman karet terkait respons terhadap ethephon.
Molecular markers and their application for DNA fingerprinting and genetic diversity studies in Coffea species Marka molekuler dan penerapannya untuk studi sidik jari DNA dan keragaman genetik pada spesies Coffea PRIYONO, .; PUTRANTO, Riza Arief
E-Journal Menara Perkebunan Vol 82, No 1: Juni 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (324.678 KB) | DOI: 10.22302/iribb.jur.mp.v82i1.30

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AbstrakStrategi klasik yang meliputi perbandingan anatomi, fisiologi dan sitogenetika telah banyak diterapkan untuk mengidentifikasi karakter tertentu serta untuk menentukan keragaman dan hubungan antar dan intra spesies. Namun, saat ini penanda molekuler telah melengkapi strategi sebelumnya dengan sangat cepat. Berbagai jenis penanda molekuler digunakan untuk menilai tingkat polimorfisme DNA. Penanda molekuler ini diklasifikasikan sebagai penanda berbasis hibridisasi dan berbasis Polymerase Chain Reaction (PCR). Dalam beberapa tahun terakhir, sistem penanda DNA yang berbeda seperti Restriction Fragment Length Polymorphisms (RFLPs), Random Amplied Polymorphic DNAs (RAPDs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) yang juga disebut Mikrosatelit, Single Nucleotide Polymorphims (SNPs) dan lain-lain telah dikembangkan dan diterapkan pada berbagai spesies tanaman. Penanda molekuler ini dapat digunakan untuk sidik jari DNA dan studi keragaman genetik. Sidik jari berdasarkan DNA telah banyak digunakan dalam ilmu forensik, juga memiliki berbagai aplikasi dalam pemuliaan tanaman. Tulisan ini memberikan overview tentang berbagai penanda molekuler dan aplikasinya untuk sidik jari dan kajian keragaman genetik tanaman berdasarkan DNA pada berbagai spesies tanaman, dan secara khusus pada Coffea sp.AbstractConventional strategies including comparative anatomy, physiology and cytogenetics were applied to identify the certain character as well as to determine inter- and intra-species diversity and relationships. However, more recently molecular markers have very rapidly complemented the previous strategies. Various types of molecular markers are used to assess DNA polymorphism. They are classified as hybridization-based markers and polymerase chain reaction (PCR) based markers. In recent years, different DNA marker systems such as Restriction Fragment Length Polymorphisms (RFLPs), Random Amplied Polymorphic DNAs (RAPDs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) which also called as microsatellites, Single Nucleotide  Polymorphims  (SNPs)  and  others  have been developed and applied to a range of plant species. These molecular markers can be used for DNA fingerprinting and genetic diversity study. DNA fingerprinting has been widely used in forensic science, but is has also a variety of application in plant breeding. This paper provides an overview about various molecular markers and their application for DNA plant fingerprinting and genetic diversity, especially in Coffea sp.
IDENTIFIKASI TIGA ISOLAT CENDAWAN PENGHASIL GAHARU DARI NUSA TENGGARA BARAT DENGAN MENGGUNAKAN PRIMER ITS DAN TEF 1-α Nugraheni, Y.M.M Anita; Anggadhania, Lutfi; Putranto, Riza Arief
Jurnal Pemuliaan Tanaman Hutan Vol 9, No 2 (2015): Jurnal pemuliaan Tanaman Hutan
Publisher : Center for Forest Biotechnology and Tree Improvement (CFBTI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (518.459 KB) | DOI: 10.20886/jpth.2015.9.2.77-90

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Several isolates of agarwood-forming fungus in Gyrinops versteegii have been isolated from the result of exploration in Lombok and Sumbawa Islands. This study aimed to identify the three fungus associated with the agarwood formation in G. versteegii originated from Lombok Tengah, Alas, and Lombok Barat, Nusa Tenggara Barat. The three fungus cultured in liquid medium PDB (Potato Dextrose Broth) and incubated for 1 month in shaken culture. The mycelium of each fungus was harvested for DNA isolation. Amplification is done by Polymerase Chain Reaction (PCR) using the Primers ITS and TEF with obtained amplicon having base length ranging of 300-600 bs. BLAST analysis showed that the three fungus have similarity with Fusarium solani. These results were confirmed by phylogenetic tree where all fungus has genetic relationship with F. solani.
Kloning dan karakterisasi daerah promoter gen penyandi ADP glucose pyrophosphorylase dari Metroxylon sagu rendemen pati-tinggi dan -rendah [Cloning and characterization of promoter region of ADP glucose pyrophosphorylase-encoding gene from Metroxylon sagu with high- and low-starch content] BUDIANI, Asmini; PUTRANTO, Riza Arief; MINARSIH, Hayati; RIYADI, Imron; SUMARYONO, .; ABBAS, Barahima
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (52.299 KB) | DOI: 10.22302/iribb.jur.mp.v84i1.200

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ADP-glucose pyrophosphorylase (AGPase) is one of the key enzymes in the starch biosynthesis. In many plants, the activity of this enzyme was reported to affect the yield and composition of the produced starch. This research is a part of an effort to develop molecular markers for early selection of high starch-yielding of sago palm. The purpose of the research was to isolate promoters of AGP gene and to analyze the differences in their DNA sequences between sago palm with high starch content (MsHS) and low starch content (MsLS). DNA was isolated and purified from the leaves of the two sago palm. The promoter region of AGP was amplified by Genome Walking technique. The specific primers were designed by Primer3 program based on the information of DNA sequence of AGP genes of sago palm from previous studies. Selected DNA fragments resulted from Genome Walking were isolated from the gel, cloned into E. coli, and analyzed its DNA sequence. DNA sequence analysis showed that one DNA fragment from MsHS  (± 1500 bp) and one DNA fragment from MsLS (> 2000 bp) were confirmed as a 5’ upstream of the AGP gene.  Further in silico analysis using MEME program identified various DNA motifs of cis-acting elements, which confirmed that those DNA fragment were promoter region of the gene. Preliminary analysis showed the differences in DNA sequences and motives of cis-acting elements in the promoter region of the two samples which might influence or indirectly associated with the character of the starch yield in sago palm.
The Hevea brasiliensis AP2/ERF superfamily: from ethylene signalling to latex harvesting and physiological disease response PUTRANTO, Riza Arief; MONTORO, Pascal
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (777.018 KB) | DOI: 10.22302/iribb.jur.mp.v84i1.201

Abstract

Ethylene is a hormone known for its involvement in the process of latex harvesting in Hevea brasiliensis. It facilitates latex flow by activation of endogenous metabolism in the anastomosed latex cells called laticifers. In regard to its ambivalent role, ethylene is both favourable to the latex production and unfavourable, to a certain level, to the apparition of a physiological disease termed as tapping panel dryness (TPD). Comprehensive researches have been carried out to reveal the molecular actors in ethylene biosynthesis and signalling pathways in Hevea brasiliensis. One of the most important superfamily implicated as the last transcription factor known in plant ethylene signalling is the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF). Currently, 114 unique sequences related to the Hevea AP2/ERF gene superfamily have been identified and characterized. Specific characterizations under the condition of harvesting stress and the occurrence of TPD have identified 36 gene expression markers (GEMs). Eighteen of these GEMs were predicted as ortholog with 19 Arabidopsis AP2/ERF genes. The characterization was mainly focused on transcriptional regulation, whilst potential post-transcriptional and post-translational regulations of HbAP2/ERF genes were formerly predicted. Three HbERF groups (HbERF-VII, HbERF-VIII and HbERF-IX) were hypothesized to have an important role in Hevea tolerance during latex production as they highly accumulated in laticifers and in response to multiple abiotic stresses. Further functional analysis of several key genes is suggested in order to fully understand the regulation of HbAP2/ERFs. Finally, the molecular markers for future Hevea breeding could be possibly developed from this superfamily.
Evaluasi 18 primer SSR untuk pengembangan sidikjari DNA tanaman karet (Hevea brasiliensis Muell. Arg.) Evaluation of 18 SSR primers to develop DNA fingerprint of rubber tree (Hevea brasiliensis Muell. Arg.) BUDIANI, Asmini; WOELAN, Sekar; MINARSIH, Hayati; NUHAIMI-HARIS, .; PUTRANTO, Riza Arief
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (343.997 KB) | DOI: 10.22302/iribb.jur.mp.v82i2.23

Abstract

Abstract Breeding program of rubber tree to produce elite clones is hampered by the length of selection cycles. On the other hand, attempts to increase production by extensification of the plantation area is also facing a problem from the availability of the rootstock, causing the occurence of fake clones without any information of their origin. Therefore, the availability of molecular markers to be used as DNA fingerprint of rubber tree clones is needed. This will help the breeder to shorten the length of selection program and to identify the purity of the clone. This research was aimed to evaluate 18 SSR primer pairs that had been published to identify 17 rubber clones. Pure genomic DNAs were isolated from 17 clones, followed by experiment to optimize annealing tempe-rature for each primer to obtain the best amplification product. Initially, the PCR product was run in both the agarose and polyacrylamide gels. However, the analysis of all PCR products were then conducted on SDS polyacrylamide gel, since this gel can separate DNA fragments with only a few bases differences. The results showed that 14 clones have been identified specifically using 11 primers. Four out of 18 primer pairs used could identify 12 rubber tree clones, which are PR 107, PR 261, SP 217, PB 330, PB 340, IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 and RRIM 712. Each clone can be distinguished from each other using only one primer pair. Identification of the other tree clones (PB 5/51, PB 260, and RRIC 110) has to be conducted by combining the several PCR products using different primer pairs. Although these results showed that SSR markers had high potential to be used as DNA fingerprint on rubber tree clones, the set of the primer pairs should be tested among other clones, as well as other SSR primers should be tested to identify the clones which could not be identified using the 18 primer pairs in this experiment.Abstrak Pemuliaan tanaman karet untuk menghasilkan klon-klon unggul baru menghadapi masalah lamanya siklus seleksi. Di sisi lain, upaya peningkatan produksi melalui pembukaan lahan baru, juga terkendala oleh ketersediaan bibit, yang memicu beredarnya bibit palsu, yang umumnya tidak jelas asal usulnya. Oleh karena itu, diperlukan ketersediaan marka yang dapat digunakan sebagai sidikjari DNA bagi klon-klon tanaman karet yang ada, sehingga dapat membantu mempercepat proses seleksi dan mengetahui kemurnian bibit. Penelitian ini bertujuan untuk mengevaluasi 18 primer SSR yang telah dipublikasikan untuk mengidentifikasi 17 klon karet. DNA yang murni diisolasi dari 17 klon, kemudian dilakukan optimasi suhu annealing untuk setiap jenis primer agar diperoleh hasil amplifikasi terbaik.  Pada awal percobaan hasil PCR dicek pada gel agarosa dan gel poliakrilamida, namun analisis untuk seluruh hasil PCR dilakukan pada gel SDS poliakrilamid, karena gel ini secara nyata dapat memisahkan fragmen DNA yang hanya berbeda beberapa basa. Hasil percobaan menunjukkan bahwa 14 klon dapat diidentifikasi secara spesifik menggunakan 11 primer. Empat dari 18 pasang primer yang diuji dapat mengidentifikasi 12 klon yang dianalisis, yaitu PR 107, PR 261, SP 217, PB 330, PB 340,. IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 dan RRIM 712. Masing-maing klon tersebut dapat dibedakan dari klon lainnya hanya dengan menggunakan satu jenis primer. Sedangkan identifikasi tiga klon lainnya (PB 5/51, PB 260, dan RRIC110) harus dilakukan dengan menggabungkan hasil PCR menggunakan beberapa primer. Meskipun hasil percobaan ini menunjukkan bahwa marka SSR sangat berpotensi untuk digunakan sebagai sidikjari DNA klon-klon karet, namun primer yang sama perlu diuji untuk klon-klon lainnya. Demikian pula primer lain perlu diuji untuk mengidentifikasi klon-klon yang belum teridentifikasi menggunakan 18 primer dalam penelitian ini.