Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors

The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the SiFMBP, SiDof4, SiBADH2, SiGBSS1, and SiIPK1 genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an Agrobacterium-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T0 generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the SiALS and SiACC genes of foxtail millet. By utilizing CBE to target the SiALS gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.

Pengaruh Metode Isolasi Terhadap Komposisi Proksimat, Sifat Pasting dan Morfologi Pati Sekoi (Setaria italica (L)P. Bauv) Varietas Lokal Bengkulu

Beberapa penelitian mengenai pati sekoi telah dilakukan, namun belum ada metode yang direkomendasikan untuk isolasi pati sekoi. Dalam penelitian ini, dilakukan isolasi pati sekoi varietas lokal Bengkulu, menggunakan basa NaOH dengan 1x, 2x dan 3x siklus, menggunakan hexan-NaOH, dan dibandingkan dengan isolasi menggunakan aquadest. Tujuan penelitian adalah untuk menentukan metode isolasi pati sekoi yang menghasilkan pati dengan kemurnian tinggi, mempelajari sifat fisikokimia dan morfologinya. Penelitian ini dilakukan menggunakan rancangan acak lengkap dengan metode isolasi sebagi faktornya. Hail penelitian menunjukkan bahwa metode isolasi pati sekoi dengan NaOH 0,3% 3 siklus, menghasilkan pati sekoi dengan kadar pati dan amilosa tertinggi, yakni 83,15%, dan 19,87%. Kadar air, kadar abu, kadar protein, kadar lemak dan kadar serat kasar pati sekoi tersebut berturut-turut adalah 8,48%, 0,47%, 4,60%, 0,09% dan 0,22%. Suhu gelatinisasi, peak time, viskositas, dan nilai break down viscosity berturut-turut adalah 78 oC, 7,6 menit, 4228 cp, dan 2738 cp. Pati ini memiliki kecerahan (L*) paling tinggi dan bentuk granula pati polygone.

Genome-Wide Analysis of the MADS-Box Gene Family in Foxtail Millet (Setaria Italica L.) and Expression Analyses to Reveal Their Role During Drought Stress

MADS-box gene family is a key regulatory factor family, which controls vegetative growth, reproductive development and can be used to mediate abiotic stresses in many plants. However, Knowledge of this gene family is still limited in Setaria italica. In the present study, a total of 70 SitMADS genes were identified and renamed on the basis of the chromosomal distribution of the SitMADS genes. According to gene structure, conserved motif and phylogenetic feature, the 70 SitMADSs were classified into type-Ⅰ (Mα, Mβ, Mγ) and type-Ⅱ (MIKCC and MIKC*). All of the SitMADS genes were randomly distributed on nine chromosomes, and five tandem duplicated genes and 12 pairs of duplicated gene segments were detected in the SitMADS genes family. Synteny analysis provided a high homology between SitMADS genes and OsMADS genes. A cis-element analysis inferred that SitMADS genes, except for SitMADS23, possessed at least one drought stress response and ABA(Abscisic Acid)-induced response cis-element. Real-time quantitative PCR analysis was used to detect the expression patterns of SitMADS genes in various tissues and demonstrated that the genes responded drought stress and ABA treatments. SitMADS23, SitMADS42, SitMADS51, SitMADS52, SitMADS58 and SitMADS64 were highly expressed in PEG(Polyethylene glycol) and drought stress, which suggested its important role in drought stress response. SitMADS51, SitMADS63 and SitMADS64 seemed to be responsive to ABA hormone signaling, suggesting that they were involved in the ABA signaling pathways. This paper provided a deep insight into the evolutionary characteristics of SitMADS genes. The results provide comprehensive information for further analyses of the molecular functions of the MADS-box gene family in Setaria italica.

Keragaman genetik koleksi plasmanutfah jewawut (Setaria italica (L.) P. Beauv.) berkerabat dekat menggunakan markah SSR

Keragaman genetik sangat penting dalam pengembangan tanaman, oleh karena itu informasi genetik sangat dibutuhkan dalam kegiatan pemuliaan untuk seleksi. Teknologi molekuler dengan menggunakan marka SSR saat ini banyak digunakan untuk penelitian diversitas genetik karena keakuratan informasi yang tinggi dan sangat polimorfik bahkan untuk spesies atau galur yang berkerabat dekat. Penelitian ini dilakukan untuk mengidentifikasi keragaman genetik dari 14 koleksi plasmanutfah jewawut berkerabat dekat dengan menggunakan 27 marka SSR yang menyebar pada semua kromosom jewawut. Hasil penelitian menunjukkan bahwa koleksi plasmanutfah jewawut berkerabat dekat mengelompok pada klaster yang sama, kecuali pada koleksi aksesi Wete yang menyebar pada kelompok yang berbeda pada kisaran nilai koefisien kemiripan genetik 0.28-0.85 dengan nilai matriks korelasi (r) sebesar 0.954. Koleksi Jewawut yang memiliki koefisien kemiripan genetik yang tinggi (0.85) ditunjukkan pada koleksi ICE-276-11A dengan ICE-276-12A dan yang memiliki koefisien kemiripan terkecil (0.61) ditujukkan pada koleksi 2007-ICE-1335 dan 2007-ICE-1335B. Koleksi jewawut yang berasal dari NTT (WeteGha dan WeteWolowea) dan Introduksi (2007-ICE-1335B dan 2007-ICE-1335) memiliki tingkat variasi genetik yang tinggi yang ditunjukkan oleh penyebaran aksesi pada klaster yang berbeda dengan nilai jarak genetik rata-rata yang cukup tinggi terhadap aksesi koleksi. Adanya keragaman yang tinggi memberikan peluang untuk pemuliaan tanaman jewawut dalam menghasilkan variasi yang lebih banyak dari persilangan aksesi-aksesi tersebut.

​Development and Nutritional Evaluation of Foxtail Millet (Setaria italica) based Laddu

Backround: Foxtail millet (Setaria italica) is known for its health benefits as it is nutritionally superior to conventional food grains. Laddu are ball-shaped sweets which are popular in India and are often served at festive or religious occasions. Hence, an attempt was made to develop foxtail millet based value added laddu. Methods: Besan laddu was taken as control. Optimized foxtail millet laddu were nutritionally evaluated for proximate composition, sugars and minerals. Paired ‘t’ test was used to compare the physical parameters and nutrient composition of besan and foxtail millet laddu. Result: The developed foxtail millet laddu had the 11.61, 17.52, 2.64, 1.11 and 66.85% per 100g of protein, fat, crude fibre, ash and carbohydrate respectively. There was no significant difference in sugar composition between the control and the optimised laddu. However, it was observed that the calcium, iron, zinc, copper and manganese of foxtail millet laddu were high as compared to control laddu.