Juliandari, Ria Reinnata (2018) Analisis Variasi Genetik berdasarkan Simple Sequence Repeat (SSR) dan Profil Genetik Gen Pun1 serta Kandungan Total Capsaicinoid pada Cabai Rawit (Capsicum frutescens L.) Hasil Induksi Mutasi dengan Ethyl Methane Sulfonate (EMS). Magister thesis, Universitas Brawijaya.
Abstract
Induksi mutasi menjadi tahapan penting dalam menghasilkan variasi genetik untuk tujuan pemuliaan tanaman dan program persilangan tanaman cabai rawit. Induksi mutasi dengan Ethyl Methane Sulfonate (EMS) menjadi metode mutasi sederhana dan cepat untuk menghasilkan variasi genetik. Variasi genetik pada tingkat genom cabai rawit dianalisis berdasarkan marka Simple Sequence Repeat (SSR), variasi gen spesifik dianalisis berdasarkan Single Nucleotide Polymorphism (SNP) pada gen Pun1, serta kandungan senyawa pedas yang dihasilkan. Penelitian ini bertujuan untuk menganalisis variasi genetik menggunakan marka SSR meliputi variasi alel dan pengelompokan genetik, variasi profil genetik sekuen ekson satu gen Pun1, serta kandungan total capsaicinoid pada cabai rawit hasil induksi mutasi dengan EMS. Adanya penelitian ini diharapkan dapat menjadi landasan di bidang pemuliaan dan program persilangan tanaman untuk menghasilkan tanaman cabai rawit dengan variasi baru. Biji cabai rawit (C. frutescens L.) genotip 2 dan 11 berasal dari Malang, Jawa Timur, dan cabai rawit genotip 7 berasal dari Lombok, NTB diperlakukan dengan EMS konsentrasi 0%, 0,01%, 0,02%, dan 0,04% selama 6 jam dalam suhu ruang. Biji dari tanaman kontrol dan mutan ditanam pada media tanam berisi campuran tanah, pupuk organik, dan sekam pada polybag. Daun dari tanaman mutan dan kontrol digunakan untuk isolasi DNA genom dengan sedikit modifikasi metode CTAB. Amplifikasi berdasarkan teknik Polymerase Chain Reaction (PCR) dengan 3 pasang primer SSR CA 19, CA 27, CA 62 dan sepasang primer gen Pun1. Hasil amplifikasi dengan primer SSR dianalisis menggunakan gel agarosa 2,5% dan gel poliakrilamida 8% menghasilkan alel. Variasi alel ditentukan berdasarkan jumlah dan ukuran alel (bp). Alel diskoring untuk menghasilkan format data biner dan digunakan dalam menentukan nilai similaritas genetik untuk merekonstruksi dendrogram. Dendrogram diperoleh dengan menggunakan metode UPGMA berdasarkan koefisien Jaccard dengan program PAST 2.17. Hasil amplifikasi dengan primer gen Pun1 dianalisis dengan menggunakan gel agarosa 1% dan disekuensing. Sekuen gen Pun1 dianalisis dengan menggunakan program bioinformatik yaitu FinchTV, Sequencer 4.1.4, BLAST, Bioedit, Clustalx, dan Expasy. Buah digunakan sebagai bahan analisis kandungan total capsaicinoid pada cabai rawit dengan menggunakan uji spektrofotometri UV/Vis pada panjang gelombang 280 nm. Total capsaicinoid dan nilai kepedasan dianalisis menggunakan analisis varian dua arah (ANOVA) dengan program SPSS. Amplifikasi menggunakan primer CA 19, CA 27, dan CA 62 menghasilkan alel yang bervariasi antara mutan dengan kontrol serta antar mutan. Variasi alel mengindikasikan adanya variasi genetik pada tingkat genom. Adanya perubahan genom menyebabkan terpisahnya mutan dari kelompok kontrol seperti pada cabai rawit genotip 2 perlakuan EMS 0,04% (G2K3), cabai rawit genotip 7 perlakuan EMS 0,02% dan 0,04% (G7K2 dan G7K3), serta cabai rawit genotip 11 perlakuan EMS 0,01%, 0,02%, dan 0,04% (G11K1, G11K2, dan G11K3). Cabai rawit genotip 11 bersifat peka terhadap perlakuan EMS menghasilkan perubahan genom yang besar ditunjukkan dengan nilai similaritas genetik yang rendah dibandingkan dengan kontrol. Sedangkan, cabai rawit genotip 2 bersifat tahan terhadap perlakuan induksi mutasi dengan EMS dibandingkan cabai rawit genotip 7 dan 11. Induksi mutasi dengan EMS 0,04% menyebabkan perubahan yang besar pada DNA genom ketiga genotip cabai rawit (G2, G7, dan G11). Variasi pada gen spesifik dianalisis berdasarkan SNP pada sekuen ekson satu gen Pun1 sepanjang 738 bp. Variasi genetik ditunjukkan oleh adanya SNP di urutan 369 bp pada cabai rawit genotip 7 perlakuan EMS 0,01% (G7K1) dan cabai rawit genotip 11 perlakuan EMS 0,01% dan 0,04% (G11K1 dan G11K3). SNP pada sekuen ekson satu gen Pun1 diidentifikasi sebagai mutasi titik berupa subtitusi yang menyebabkan perubahan basa pirimidin (sitosin/ C) menjadi basa pirimidin (timin/ T) (mutasi transisi). Adanya SNP tersebut tidak menyebabkan perubahan pada profil asam amino yang dihasilkan (mutasi diam) sehingga diduga tidak mengganggu fungsi kerja gen Pun1. Induksi mutasi dengan EMS 0,01%, 0,02%, dan 0,04% pada cabai rawit genotip 2, genotip 7 dan genotip 11 menurunkan kandungan total capsaicinoid dan nilai kepedasan kecuali dengan perlakuan EMS 0,01% pada cabai rawit genotip 2 (G2K1). Variasi pada gen Pun1 (SNP) menghasilkan nilai kesamaan genetik (ident) antara mutan dan kontrol masingmasing genotip sebesar 99%. Dengan demikian, perubahan kandungan total capsaicinoid dan nilai kepedasan pada cabai rawit genotip 2, genotip 7, dan genotip 11 diduga disebabkan oleh adanya peranan gen-gen lain dalam jalur biosintesis capsaicinoid. Level kepedasan cabai rawit genotip 2, genotip 7, dan genotip 11 menunjukkan kategori pedas tinggi hingga sangat pedas. Dengan demikian, secara genetis induksi mutasi dengan EMS tidak menyebabkan perubahan fungsi gen Pun1.
English Abstract
The important step for crop improvement and selective breeding program in Capsicum frutescens L. can be conducted through genetic variation using induction mutation. Ethyl Methane Sulfonate (EMS) induced mutation is one of the simple and quick method to produce genetic variation. Simple Sequence Repeat (SSR) or microsatellite marker is used for detecting genetic variation in the genome level. Variation in specific gene analyzed using Single Nucleotide Polymorphism (SNP) based on profil of sequence in the first exon of Pun1 gene and total capsaicinoid content. The objective of this study was analyzed of genetic variation based on SSR marker (consist of allele variation and genetic clustering), profil sequence of Pun1 gene, and total capsaicinoid content on chili that was induced mutation by EMS. The results of this study are expected to support crop improvement and selective breeding program to produce new variation of chili. Seeds from local chili (C. frutescens L.) genotype 2 and genotype 11 from Malang, East Java and chili genotype 7 from local Lombok, NTB were subjected to mutation induced by EMS with concentration of 0% (control), 0.01%, 0.02%, and 0.04% in 6 hours at room temperature. Seeds from control and mutant plants were planted in polybag consists of a mixed of soil and orgnaic fertilizer. Young and fresh leaves of mutant and control were used for genomic DNA isolation by slightly modified CTAB method. Amplification was conducted using Polymerase Chain Reaction (PCR) technique with 3 SSR markers CA 19, CA 27, CA 62 and a pair of primer Pun1 gene. The SSR amplification results were visualized using 2,5% agarosa gel and 8% polyacrylamide gel produced of allele. Variation of allele was determine based on variation of the number and size of alleles (bp). Alleles were scored to produce binary data and used determine the genetic similarity for construction of dendrogram. The dendrogram was developed using UPGMA method based on the Jaccard’s coefficient in PAST 2.17. The amplification results of Pun1 gene were visualized using 1% agarosa gel and sequenced. Pun1 gene sequences were analyzed using bioinformatic programs such as FinchTv, Sequencher 4.1.4, BLAST, Bioedit, ClustalX, and Expasy. Chili fruit used for analysis total capsaicinoid using UV/Vis spectrophotometer at 280 nm wavelength. Total capsaicinoid and pungency level was analyzed using two-way of variance analysis (ANOVA) with SPSS program. Amplification using CA 19, CA 27, and CA 62 produce variation of alelles in mutant compared to control and other mutant. Variation of alleles indicate genetic variation in the genome level. Genome changes make them separated from control such as chili genotype 2 induced by EMS 0.04% (G2K3), chili genotype 7 induced by EMS 0.02% and 0.04% (G7K2 and G7K3), and chili genotype 11 induced by 0.01%, 0.02%, and 0.04% (G11K1, G11K2, and G11K3). Chili genotype 11 was showed sensitive response to EMS treatments produced the biggest genetic changes with the lowest genetic similarity value compared to control. Moreover, chili genotype 2 was showed resistance response compared to chili genotype 7 and 11. EMS induction with concentration of 0,04% give the biggest effect to the DNA genome in 3 genotypes of chili (G2, G7, and G11). Genetic variation in the specific gene was analyzed based on the first exon sequence of Pun1 gene with 738 bp length. Genetic variation show with the existence of SNP in position 369 bp length in mutant genotype 7 treated with EMS concentration 0.01 % (G7K1) and mutant genotype 11 treated with EMS concentration 0,01% and 0,04% (G11K1 and G11K3). The SNP identified as point mutation especially subtitution are predicted to change pyrimidine (cytosine/ C) to pyrimidine (thymine/ T) (transition mutation). The existence of SNP produces similarity of amino acid product (silent mutation), so there is no interfere with the function of Pun1 gene. EMS induced mutation with concentration of 0.01%, 0.02%, and 0.04% produced the lower of total capsaicinoid content and pungency value in chili genotype 2, genotype 7 and genotype 11 compared to control, except in chili genotype 2 induced by EMS with concentration 0.01% (G2K1). Based on variation in Pun1 gene (SNP) produced similarity genetic (ident) between mutant and control from each genotype of 99%. Thus, the change of total capsaicinoid and pungency value in chili genotype 2, genotype 7, and genotype 11 predicted due to the influence of another gene in capsaicinoid biosynthesis pathway. The pungency level of chili genotype 2, genotype 7, and genotype 11 are highly pungent and very highly pungent. Thus, the EMS induced mutation didn’t cause alteration in Pun1 gene.
Other obstract
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| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/576.549/JUL/p/2018/041804508 |
| Uncontrolled Keywords: | MUTATION (BIOLOGY), PLANT MUTATION BREEDING, CAPSAICINOIDS, ETHYL METHANESULFONATE |
| Subjects: | 500 Natural sciences and mathematics > 576 Genetics and evolution > 576.5 Genetics > 576.54 Variation > 576.549 Mutation |
| Divisions: | S2/S3 > Magister Biologi, Fakultas MIPA |
| Depositing User: | Budi Wahyono Wahyono |
| Date Deposited: | 24 Jan 2020 02:20 |
| Last Modified: | 19 Oct 2021 05:34 |
| URI: | http://repository.ub.ac.id/id/eprint/178322 |
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