Lestari, Arvi Wahyu and Dr. Ir. Damanhuri, MS. and Dr. Ir. Muhamad Yunus, M.Si. (2020) Seleksi Foreground Dan Background Tanaman Populasi BC5F1 Turunan Esensial Ciherang Tahan Wereng Batang Cokelat (Nilaparvata lugens Stal.) Menggunakan Marka SSR. Sarjana thesis, Universitas Brawijaya.
Abstract
Wereng batang cokelat (Nilaparvata lugens Stal.) ialah salah satu Organisme Pengganggu Tanaman (OPT) yang merupakan hama utama tanaman padi. Selain merusak tanaman padi secara langsung, wereng batang cokelat juga berperan sebagai vektor virus penyakit kerdil hampa (Rice Ragged Stunt Virus) dan penyakit kerdil rumput (Rice Grassy Stunt Virus), pada kepadatan populasi rendah dapat menurunkan produksi padi secara signifikan. Salah satu upaya pengendalian wereng batang cokelat yaitu penggunaan varietas tahan. Pengembangan varietas tahan dapat dilakukan dengan metode silang balik (backcross). Namun, metode silang balik secara konvensional membutuhkan waktu lama, kesulitan dalam seleksi gen target, dan adanya peristiwa linkage (pautan gen) saat persilangan. Pengembangan varietas tahan dengan bantuan marka molekuler mampu menyeleksi gen target tanpa dipengaruhi oleh lingkungan dan dapat digunakan untuk membedakan varietas maupun individu dalam varietas, sehingga kegiatan seleksi tanaman menjadi lebih efektif. Ciherang dipilih sebagai tetua penerima yang diperbaiki ketahanannya terhadap wereng batang cokelat karena sampai saat ini merupakan varietas yang pertanamannya paling luas dibandingkan varietas unggul lainnya. Tujuan dari penelitian ini ialah untuk mendapatkan individu pada populasi BC5F1 yang memiliki lokus gen ketahanan Bph3 dengan persentase latar belakang genetik Ciherang yang besar berdasarkan marka Simple Sequence Repeat (SSR). Penelitian dilaksanakan pada bulan Januari – Maret 2020 di Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian, Bogor. Bahan yang digunakan dalam penelitian ialah sampel daun populasi BC5F1 sebanyak 120 progeni hasil silang balik antara Ciherang x NIL1 (Bph3 dan Bph6), CTAB (cetyl trimethyl ammonium bromide), natrium metabisulfit 0,38%, NaOAc (natrium asetat) 3 M, chloroform : isoamyl alcohol (24 : 1), etanol absolut 96%, etanol 70%, bufer TE 1X (Tris HCl pH 8,0; EDTA pH 8.0), MyTaq HS Red Mix, nano pure water, DNA Ladder 100 bp, 142 primer RM untuk SSR-PCR, akrilamid-bisakrilamid 8%, APS (Ammonium persulfate) 10%, TEMED (N’-tetramethylethane-1,2-diamine), bufer TBE 1X (Tris-borate EDTA pH 8.0), tube 1,5 ml, tube 2 ml, dan kertas tisu KimWipes. Alat yang digunakan ialah mesin Centrifuge 5427 R – Eppendorf, microwave, waterbath 65C, vortex, NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, 2009), Mini Plate Spinner, mesin Polymerase Chain Reaction (PCR) T100 Thermocycler, Polyacrylamide Gel Electrophoresis (PAGE), Agarose Gel Electrophoresis (AGE), Gel Doc EZ System dan Chemidoc EQ UV Transilluminator (Bio-Rad, 2011). Metode penelitian menggunakan analisis molekuler dengan mengisolasi DNA, uji kuantitatif dan kualitas DNA, PCR-SSR, elektroforesis, dan visualisasi hasil elektroforesis. Variabel pengamatan berupa pita DNA hasil elektroforesis gel poliakrilamid yang divisualisasi menggunakan software Graphical GenoTypes (GGT 2.0). Data yang diperoleh dari seleksi foreground dianalis menggunakan uji χ2 dan G2 (goodness of fit) pada taraf 1% sedangkan, data seleksi background dianalisis menggunakan statistik deskriptif berupa perhitungan distribusi frekuensi dan analisis statistik inferensial menggunakan uji t satu sampel pada taraf 1%. Hasil penelitian seleksi foreground menunjukkan terdapat 33 individu BC5F1 memiliki lokus gen ketahanan Bph3 yang berasal dari tetua Rathu Heenati berdasarkan uji molekuler menggunakan marka yang terpaut erat dan marka pengapit lokus gen Bph3 (RM586, RM588, dan RM8072) sedangkan, seleksi background didapatkan tujuh individu dari tanaman yang terseleksi memiliki persentase pemulihan genom tetua penerima diatas 98,4% yaitu individu P2/8.21 (98,5%), P2/8.51 (99%), P2/8.17 (99,3%), P2/8.54 (99,3%), P2/8.12 (99,4%), P2/8.38 (99,4%), dan P2/8.90 (99,8%) berdasarkan 60 pasang marka SSR polimorfik pada lokus non-target. Oleh karena itu, individu nomor P2/8.12, P2/8.17, P2/8.21, P2/8.38, P2/8.51, P2/8.54, dan P2/8.90 dapat dijadikan bahan genetik untuk seleksi selanjutnya.
English Abstract
Brown planthopper (Nilaparvata lugens Stal.) is one of the major insect pests in rice plant. In addition, brown planthoppers are also known as vectors of viruses diseases such as Rice Ragged Stunt Virus (RRSV) and Rice Grassy Stunt Virus (RGSV), at low densities can cause significant yield loss in rice production. Various strategies that could effectively control brown planthopper is using a host plant resistance method. In such cases, the backcross method is used for the development of a new resistant variety. However, the conventional backcross method requires a long time, difficult in a selection of target genes, and linkage drag during the crossing. The development of resistant varieties with molecular markers can select target genes without being influenced by the environment and can be used to differentiate varieties and individuals in varieties, so that plant selection activities become more effective. Ciherang has been used widely as a recurrent parent because it was the most widely planted variety compared to other superior varieties. This research aimed to obtain individuals in BC5F1 population with Bph3 locus and have a maximum percentage of Ciherang genetic background based on SSR markers. The research was conducted in January – March 2020 at the Indonesian Centre for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor. The materials used in this research is leaf sample of plant population BC5F1 as much 120 progenies backcross from Ciherang x NIL1 (Bph3 and Bph6), CTAB (cetyl trimethyl ammonium bromide), 0.38% sodium bisulfite, 3 M NaOAc (sodium acetate), chloroform : isoamyl alcohol (24 : 1), ethanol absolute, 70% ethanol, 1X TE buffer (Tris HCl pH 8.0, EDTA pH 8.0), MyTaq HS Red Mix, nano pure water, 100 bp DNA ladder, 142 RM primers for SSR-PCR, 8% acrylamide-bisacrylamide, 10% APS (Ammonium Persulfate), TEMED (N’-tetramethylene-1.2-diamine), 1X TBE buffer (Tris-borate EDTA pH 8.0), Eppendorf tube 1.5 ml, Eppendorf tube 2 ml, and KimWipes tissue. The tools used in this research is Centrifuge 5427 R – Eppendorf, microwave, water bath 65C, vortex, NanoDrop 2000 Spectrophotometry (Thermo Fisher Scientific, 2009), Mini Plate Spinner, Polymerase Chain Reaction (PCR) T100 Thermocycler, PAGE (Polyacrylamide Gel Electrophoresis), AGE (Agarose Gel Electrophoresis), Gel Doc EZ System and Chemidoc EQ UV Transilluminator (Bio-Rad, 2011). The research method used molecular analysis by DNA extractions, quantitative and quality analysis of DNA, PCR-SSR, electrophoresis, and visualization. Observation variables were DNA bands from polyacrylamide gel electrophoresis. Genotype visualization of DNA using Graphical GenoTypes (GGT 2.0). Data obtained from foreground selection were analysis using χ2 test and G2 test (goodness of fit) at a significance level of 1% whereas, background selection were analyzed using descriptive statistics which is frequency distribution and inferential statistics used a one-sample t-test at a significance level of 1%. The result of this research showed in foreground selection thirty-three individuals of BC5F1 population have Bph3 locus derived from donor parent Rathu Heenati based on molecular analysis using closely linked and flanking markers to the Bph3 locus (RM586, RM588, and RM8072) while, in background selection out of thirty-three individuals, seven individuals from selected plants had a recurrent parent genome recovery above 98.4%, individuals with number P2/8.21 (98.5%), P2/8.51 (99%), P2/8.17 (99.3%), P2/8.54 (99.3%), P2/8.12 (99.4%), P2/8.38 (99.4%), and P2/8.90 (99.8%) based on 60 pairs of polymorphic SSR markers at non-target loci. Therefore, individuals P2/8.12, P2/8.17, P2/8.21, P2/8.38, P2/8.51, P2/8.54, and P2/8.90 can be used as genetic material for further selection.
Other obstract
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| Item Type: | Thesis (Sarjana) |
|---|---|
| Identification Number: | 0520040029 |
| Subjects: | 600 Technology (Applied sciences) > 633 Field and plantation crops > 633.1 Cereals > 633.18 Rice > 633.182 3 Rice (Development of new varieties) > 633.182 33 Rice (Agricultural genetics) |
| Divisions: | Fakultas Pertanian > Budidaya Pertanian |
| Depositing User: | Nur Cholis |
| Date Deposited: | 03 Feb 2021 06:31 |
| Last Modified: | 03 Oct 2022 01:43 |
| URI: | http://repository.ub.ac.id/id/eprint/182367 |
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