Studi Fisiologi dan Molekuler Terkait Karakter Pati Pada Ubi Kayu (Manihot esculenta Crantz.)
Date
2021-08-12Author
Dianasari, MIta
Ardie, Sintho Wahyuning
Khumaida, Nurul
Putranto, Riza Arief
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Show full item recordAbstract
Ubi kayu saat ini memiliki potensi pemanfaatan sangat besar sebagai bahan pangan, pakan, dan industri dengan produk turunan yang beragam. Oleh karena itu perlu didukung dengan penyediaan genotipe ubi kayu yang unggul dari aspek kandungan pati dan produktivitasnya. Ubi kayu umumnya diperbanyak menggunakan stek batang sehingga potensi keragamannya kurang berkembang dengan cepat, peningkatan variasi genetik ubi kayu dapat dilakukan dengan metode persilangan. Pada penelitian ini digunakan materi genetik berupa populasi tanaman ubi kayu yang dihasilkan dari biji penyerbukan terbuka (open pollinated/OP) tanaman generasi M1G4. Penelitian ini mencakup identifikasi karakterisasi panen dan komponen pati pada populasi penyerbukan terbuka untuk memperoleh data profil kualitas umbi, serta percobaan molekuler secara in silico dan identifikasi marka SNP pada gen penting yang terlibat dalam biosintesis pati. Luaran yang diharapkan berupa data karakter hasil dan pati dari populasi OP, sekuens primer gen spesifik untuk analisis situs SNP dan informasi situs SNP pada gen pengendali sintesis pati. Materi yang digunakan pada penelitian ini adalah tanaman dan umbi segar beberapa mutan putatif ubi kayu pada populasi OP, data genom ubi kayu AM560-2 asal Colombia dan KU-50 asal Thailand serta sekuens gen yang diduga sebagai pengendali sintesis pati, dan isolat DNA tanaman ubi kayu genotipe Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah, dan Manggu. Pengamatan terhadap tanaman OP yang berumur 9 bulan setelah tanam (BST), meliputi keragaan tanaman sebelum panen dan umbi saat panen. Keragaman fenotipe karakter kualitatif meliputi warna korteks batang, warna luar batang, warna pucuk, tipe cabang, tipe leher umbi, bentuk umbi, warna korteks umbi, dan rasa umbi. Pengelompokan populasi berdasarkan karakter kualitatif membentuk dendogram dengan tiga kelompok besar. Sebaran 214 genotipe berdasarkan hubungan bobot umbi per tanaman dengan karakter diameter batang, tinggi tanaman, level percabangan, jumlah umbi, dan tebal korteks umbi menunjukkan terdapat 8 genotipe yang potensial dikembangkan ke tahap pengujian lanjutan menggunakan perbanyakan vegetatif. Teknik in silico dapat digunakan untuk akselerasi pemuliaan tanaman ubi kayu yang mudah dan murah untuk mengidentifikasi gen putatif pada jalur sintesis pati ubi kayu menggunakan data sekuens genom pada basis data bioinformatika. Beberapa gen dipilih berdasarkan aktivitasnya yang diketahui dalam proses biosintesis pati meliputi ERF (ethylene response factor), ethylene, hexose, LEA (late embryogenesis abundant), starch synthase, dan sucrose. Berdasarkan hasil analisis komparasi genom diperoleh informasi terdapat 24 sekuens gen putatif yang terdapat pada genom ubi kayu asal Colombia dan Thailand namun terdapat dua sekuens putatif yang hanya ditemukan pada genom Colombia yaitu sekuens putatif gen Lea14-A-like dan SS4-isoform X1. Sebanyak 14 gen putatif yang teridentifikasi, memiliki peran dalam proses biologis dan fungsi molekuler dan lokasinya tersebar pada 12 koromosom yaitu pada kromosom LG 01, LG 02, LG 03, LG 04, LG 05, LG 08, LG 09, LG 11, LG 14, LG 15, LG 16, dan LG 18. Berdasarkan peran masing-masing gen terpilih gen GBSS1 dan Lea yang diduga mengendalikan proses biosintesis pati. Gen putatif ini dapat dijadikan sebagai acuan analisis marka molekuler dan analisis ekspresi gen untuk akselerasi program pemuliaan ubi kayu melalui teknik pengeditan gen spesifik sehingga lebih efisien dari segi waktu dan biaya. Selanjutnya terpilih gen GBSS1 dan Lea yang diduga berkaitan erat terkait kualitas pati yang dihasilkan, kemudian di susun primer untuk diisolasi pada ubi kayu genotipe Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah, dan Manggu. Fragmen parsial gen GBSS1 berhasil diamplifikasi dari genom ubi kayu genotipe Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah dan Manggu. Situs SNP pada gen GBSS1 ketujuh genotipe ubi kayu menunjukkan terdapat 10 situs SNP baik sinonimus dan non sinonimus pada intron 1, ekson 2, intron 12 dam ekson 14, namun hasil dugaan situs SNP ini belum dapat digunakan sebagai marka molekuler dikarenakan kualitas sekuensing yang kurang baik. Amplifikasi gen Lea5 pada tujuh genotipe ubi kayu hanya menghasilkan fragmen parsial yang diperoleh dari lima genotipe ubi kayu yaitu genotipe Jame-jame, Adira-4, Malang-4, Gajah dan Manggu. Fragmen gen Lea5 yang memiliki kemiripan dekat hanya terdapat pada genotipe Adira-4, Jame-jame, Malang-4 dan Manggu. Fragmen gen Lea5 yang diperoleh belum dapat digunakan untuk analisis SNP dikarenakan variasi sekuens antar genotipenya cukup tinggi. Kombinasi teknik pemuliaan tanaman konvensional dan penggunaan marka molekuler diharapkan dapat digunakan untuk proses seleksi guna mempercepat perakitan varietas ubi kayu unggul yang baru serta sesuai dengan kriteria konsumen. Cassava currently has enormous potential for use as food, feed, and industry with various derivative products. Therefore, it is necessary to support the provision of superior cassava genotypes in terms of starch content and productivity. Cassava is generally propagated using stem cuttings so that the potential for diversity is not developed quickly, increasing genetic variation of cassava can be done by crossing methods. In this study, genetic material was used in the form of a population of cassava plants produced from open pollinated seeds (OP) of M1G4 generation plants. This study scope was including identification of harvest characterization and starch components in OP populations to obtain tuber quality profile data, as well as in silico molecular experiments and identification of SNP markers on important genes involved in starch biosynthesis. The expected outcomes are yield and starch character data from the OP population, primary sequences of specific genes for SNP site analysis and SNP site information on genes controlling starch synthesis. The materials used in this study were fresh plants and tubers of several putative mutants of cassava in the OP population, genomic data of AM560-2 cassava from Colombia and KU-50 from Thailand as well as gene sequences suspected to control starch synthesis, and DNA isolates of cassava plants from Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah, and Manggu genotypes. Cassava OP population observed on 9 months after planting (MAP) including plant characteristics before harvesting phase and tubers in harvesting phase. Qualitative characters of plant phenotypic diversity observed including stem cortex color, stem exterior color, color of end branches, branching habit, extent of root peduncle, root shape, tuber cortex color, and tuber taste. Grouping based on qualitative characters forms a dendogram with three large groups. Distribution of 214 OP genotypes based on tuber weight per plant and stem diameters, plant height, level of branching, number of tubers, and tuber cortex thickness shows that 8 genotypes are potential to be developed in the next testing stage by vegetative propagation. In silico technique can be used as cassava breeding acceleration for simple and cheaper ways to identifying putatif genes in cassava starch pathway using genome data sequences in bioinformatics database. Several genes are screened based on its known activity in starch biosynthesis including ERF (ethylene response factor), ethylene, hexose, LEA (late embryogenesis abundant), starch synthase, and sucrose. Based on comparative genome analysis, there were 24 putative gene sequences found in Colombia and Thailand cassava genome, but there were two putative sequences only found in Colombian genome, which were Lea14-A-like and SS4-isoform X1 genes. A total of 14 identified putative genes have roles in biological processes and molecular functions and their locations are scattered on 12 chromosomes, on chromosomes LG 01, LG 02, LG 03, LG 04, LG 05, LG 08, LG 09, LG 11, LG 14, LG 15, LG 16, and LG 18. Based on the role of each gene, GBSS1 and Lea genes were selected suspected controlling the starch biosynthesis process. This putative gene can be used as a reference for molecular marker analysis and gene expression analysis to accelerate cassava breeding programs through specific gene editing techniques to get efficiency in terms of time and cost. From in silico result then GBSS1 and Lea genes were selected which were thought to be closely related to the quality of the starch produced, then primers were designed to be isolated in cassava genotypes Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah, and Manggu. The GBSS1 gene partial fragment was successfully amplified from the cassava genome of the Jame-jame, Ratim, UJ-5, Malang-4, Adira-4, Gajah and Manggu genotypes. SNP sites in the GBSS1 gene of the seven cassava genotypes showed that there were 10 SNP sites including synonymous and non-synonymous SNP on 1st intron, 2nd exon, 12th intron, and 14th exon, but this putative SNP sites still cannot being used for molecular marker due to its low sequencing quality. Amplification of the Lea5 gene in seven cassava genotypes only resulted partial fragments obtained from five cassava genotypes, namely the Jame-jame, Adira-4, Malang-4, Gajah, and Manggu genotypes. Lea5 gene fragments that have close similarities are only found in the Adira-4, Jame-jame, Malang-4 and Manggu genotypes. Fragment of Lea5 gene that successfully amplified still cannot being used for SNP site analysis due to its sequence high variance among used genotypes. The combination of conventional plant breeding techniques and the use of molecular markers is expected can accelerate selection process to creating of new high yielding cassava varieties according to consumer criteria.
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- MT - Agriculture [3683]