Judul: Phylogenetic Analysis and Design sgRNA of Gn1a Gene Encoding Cytokinin Oxidase/Dehydrogenase in Rice (Oryza sativa L.) Japonica Group
Abstrak: Gn1a adalah gen yang menyandi sitokinin oksidase/dehidrogenase (CKX) pada padi. Adanya regulasi sitokinin menyebabkan tanaman memiliki jumlah malai yang banyak. Jumlah malai menjadi karakter agronomi penting dalam menentukan tingkat produktivitas padi. Genotipe padi dapat dibagi menjadi dua tipe berdasarkan jumlah malai yang dihasilkan yaitu genotipe padi malai banyak dan malai sedikit. Padi dengan genotipe malai sedikit dapat dimanipulasi secara genetik dengan merekayasa gen utama terkait malai, salah satunya gen Gn1a. Oleh karena itu, penelitian ini bertujuan menganalisis filogeni sekuen protein CKX di dalam famili Poaceae serta mendesain guide-RNA CRISPR/Cas9 (sgRNA) dari sekuen gen Gn1a untuk penyuntingan gen pada padi. Penelitian ini dilakukan dengan pendekatan bioinformatika dengan menguji disimilaritas gen dan protein CKX pada berbagai aksesi padi beserta menganalisis potensi sekuen fungsional gen Gn1a untuk dijadikan kandidat sgRNA. Hasil penelitian ini menunjukkan bahwa gen Gnla secara filogeni terbukti menyandi sitokinin oksidase/dehidrogenase, sehingga gen Gnla pada padi grup Japonika dapat dipilih sebagai target posisi mutasi untuk menghilangkan fungsi dari sitokinin oksidase/dehidrogenase. Berdasarkan hasil desain sgRNA pada gen Gnla diperoleh 3 kandidat terbaik untuk dikontruksi ke vektor CRISPR/Cas9 yaitu sgRNA pada peringkat 2, 4, dan 8., Gn1a is a gene encoding cytokinin oxidase/dehydrogenase in rice (CKX). The presence of cytokinin regulation causes plants to have high number in panicles. The number of panicles is an important agronomic character in determining the rice productivity. Rice genotypes can be divided into two types based on the number of panicles produced, i.e.: high and low panicle rice genotypes. Rice with low panicle genotype can be genetically manipulated by engineering the main panicle-related gene, one of which is Gn1a. Therefore, this study aimed to analyze the phylogeny of the CKX protein in the Poaceae family and to design the CRISPR/Cas9 (sgRNA) guide-RNA from the Gn1a gene for gene editing in rice. This research was carried out using a bioinformatics approach by analyzing the dissimilarity of the Gn1a gene and CKX protein sequences in rice and analyzing the potential functional sequence of the Gn1a gene to be a candidate for sgRNA design. The results of the study showed that the Gnla gene is phylogenically proven to encode cytokinin oxidase/ dehydrogenase, so that the Gnla gene in Japonika Group rice can be selected as a mutation target to eliminate cytokinin dehydrogenase function. Based on the results of the sgRNA design on the Gnla gene, we found the 3 best candidates for sgRNA sequence targets to be incorporated into the CRISPR/Cas9 vector, i.e. sgRNA numbers 2, 4, and 8.
Keyword: CRISPR/Cas9, Gn1a, Japonika, malai, sgRNA

Judul: Transformasi Kalus Padi dengan Konstruksi CRISPR-Cas9 untuk Menyunting Gen Terkait Hasil Produksi
Abstrak: High yield improvement of rice (Oryza sativa L.) is the main goal in rice breeding and could be achieved by gene editing through CRISPR-Cas9 technique as a revolution technique in genome editing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas9) has shown a precise and efficient genome editing tool by inducing random mutation in the cleavage region to knock out genes. The objective of this research is to transform sgRNA-CRISPR-Cas9 constructs to Jao Hom Nin (JHN) rice calli to knock out DL, Gn1a, Roc5, and Tad1 genes, which are the yield-related genes. The sgRNA-CRISPR-Cas9 constructs in pRGEB32 plasmid, were transformed into rice callus through Agrobacterium tumefaciens EHA105. Afterwards, the callus genome was verified using PCR technique to identify the sgRNA insert in the rice genome. Also, transformant calli was tested by T7 Endonuclease I (T7EI) assay to detect the mismatches existence shown by digested bands. Mismatches occur when the DNA repairs itself after Cas9 successfully cuts the target gene’s region and causes insertion and deletion (indels) mutation. The result showed that each callus was successfully transformed by the transformant Agrobacterium tumefaciens with sgRNA-CRISPR-Cas9 construction vector and the sgRNA was successfully inserted to the rice genome. When they are tested using PCR, we obtained amplified edited bands of Tad1, DL, and Roc5 genes, except for Gn1a gene target. T7EI assay had been done successfully, but no mismatched fragments were found., Peningkatan hasil produksi tanaman padi (Oryza sativa L.) merupakan tujuan utama dari pemuliaan tanaman padi dan dapat dicapai dengan peyuntingan gen melalui teknik CRISPR/Cas9 sebagai sebuah teknik revolusi dalam penyuntingan genom. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)berasosiasi dengan gen Cas9 telah menjadi sebuah alat penyuntingan genom yang akurat dan efisien dengan menginduksi mutasi acak pada wilayah pemotongan untuk mengubah gen target. Tujuan dari penelitian ini adalah melakukan transformasi kalus padi varietas Jao Hom Nin (JHN) dengan konstruksi sgRNACRISPR-Cas9 untuk mengubah gen Tad1, DL, Roc5, dan Gn1a yang merupakan gen-gen terkait hasil produksi pada tanaman padi. Konstruksi sgRNA-CRISPRCas9 dalam plasmid pRGEB32 diintroduksikan ke dalam kalus padi melalui Agrobacterium tumefaciens EHA105. Selanjutnya, genom kalus tersebut diverifikasi dengan teknik PCR untuk mendeteksi keberhasilan sisipan sgRNA pada genom padi. Selain itu keberadaan mutasi pada gen target diuji dengan uji T7 Endonuclease I (T7EI) untuk mendeteksi keberadaan mismatches yang ditujukkan oleh pita-pita DNA terpotong. Mismatches terjadi ketika DNA memperbaiki dirinya setelah Cas9 berhasil memotong wilayah gen target dan menyebabkan mutasi insersi-delesi. Hasil penelitian menunjukkan bahwa setiap kalus berhasil ditransformasi oleh Agrobacterium tumefaciens dengan pRGEB32 yang membawa konstruksi sgRNA-CRISPR-Cas9 yang ditunjukkan oleh amplikon daerah target gen Tad1, DL, dan Roc5, kecuali Gn1a. Ketika gen target diverifikasi dengan uji T7EI diperoleh amplikon yang sama dengan gen target yang mengindikasikan tidak ditemukan adanya mismatches pada target gen Tad1, DL, dan Roc5.
Keyword: DL, gene editing, Gn1a, Roc5, Tad1

Judul: Hamiltonian formulation for linear waves motion in two layers fluid
Abstrak: The surface wave could be considered as a wave that separates two fluids, namely water and air. Based on this assumption, it is introduced the interfacial wave, a wave between two layers of fluid with different density. The formulation of interfacial waves motion begins with deriving the base equation of irrotational ideal fluid. Furthermore, according to irrotational fluid assumption, the base equation can be stated in velocity potential. In this derivation, the fluids domain is assumed to be restricted by rigid lid boundary conditions, both at the upper and lower limit. Therefore, the interfacial waves motion can be explained in a hamiltonian formulation. In the hamiltonian formulation, total energy is defined as the sum of kinetic and potential energy. The hamiltonian system is obtained from reduction of kinetic energy by using the Dirichlet Neumann Operator. The resulted kinetic energy equation is nonlinear. Therefore, this form is linearized by first part of the Taylor expansion. This linearization gives a dispersion relation of linear wave. Based on this dispersion relation, the phase speed of the linear wave depends on the density ratio of the two layers fluid.
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