According to my proposal Academic year 2021-2022 School of life sciences and medicine Major: Biotechnology Class: Biotchnology 18 (1) Name: Rostane Darnie Nyangone Bidza Tutor: Andres Student ID:2018532860006 Date of Entrance : 2018/9 Source topic :widespread 1. Introduction 1.1 Research Background and Significance Ranunculus japonicus belongs to the order Ranunculeae and the family Ranunculaceae. This plant is used as a medicinal herb in many countries, especially in China, Korea and Japan as a treatment for diseases like jaundice, malaria, severe headaches and migraines, analgesic issues like stomach aches, inflammations, ulcers, etc. (Dai, Jia, & Zhao, 2015; Rui, Chen, Tan, Zhong, & Feng, 2010; Yun et al., 2021). Several studies have revealed through a total glycosides of Ranunculus japonicus that it contains anti-inflammatory, anti-malarial, analgesic, and anti-angiotensin effects (R. Wang & Tan, 2008; R. Wang, Tan, & Luo, 2009; Yun et al., 2021; ZHANG, LIU, TAN, ZHAO, & LIU, 2013; Zhicheng, Shoujia, Yuan, Qing, & Yunzhi, 2011). This plant is recorded as a part of the Standard of traditional Chinese medicine and is used for relieving many ailments. However, there is a lack of a complete chloroplast genome characterization of this plant in past studies. The past research reveals that chloroplasts have a different and a circular genome as compared to other parts of angiosperm plants (B. Li & Zheng, 2018; Liang et al., 2020; B. Zhu et al., 2020). This circular genome can play a vital role in the process of photosynthesis as well as the overall physiology and development in most of the plants. In comparison to the nuclear genome of plants, the chloroplast (cp) genome is considered to be more conserved i.e., the gene size and content in the chloroplast, the cp genome structure, and linear order of the genes in the cp genome is more easily understandable and clear (Ruhfel, Gitzendanner, Soltis, Soltis, & Burleigh, 2014; Wicke, Schneeweiss, Depamphilis, Müller, & Quandt, 2011). This is why it is ideal to conduct a characterization of the cp genome of plants as it allows to understand the genomic evolution and phylogenetic relationships that are present in complex angiosperm families (Huang, Shi, Liu, Mao, & Gao, 2014; Walker, Zanis, & Emery, 2014). The current study will be valuable as Ranunculaceae family is known for its medicinal properties and indication of evolution history of Ranunculus japonicus may indicate medicinal nature of the plant beyond the characteristics that have already been explored. 1.2 Research Aims and Objectives Overall, the aim of this study is to enhance understanding of the of Ranunculus japonicus plant cp genome as it can allow to create an insight into the evolutionary history of the plant. Moreover, the objectives of the study also include a comparison of the plant cp genome with the cp genomes of other species in the same family so that medicinal properties can be indicated for further research in the future. 2. Content 2.1 Research advance of chloroplast genome As already discussed in previous section cp genome is more conserved and as a circular construction that enables researchers to better uncover genetic information due to gene density and very minimal cases of gene loss (Costa, Lin, Macaya, Fernández-García, & Verbruggen, 2016). Much research has been conducted in the area of phylogenetic analysis in the past two decades and most researchers agree that chloroplast DNA (cp DNA) and nuclear DNA (n DNA) are two tools that have an ability to uncover the secrets of plant evolution. However, in comparison to n-DNA, cp-DNA has received more praise due to its lack of genetic recombination, which is common in n-DNA and the high rate of conservation of genes and gene sequence (Baev et al., 2021; Q. Li et al., 2018; Yang et al., 2020) making it easier to conduct phylogenetic analysis (Assefa et al., 2015; Assefa et al., 2011; Espelund, Bekele, Holst‐Jensen, Jakobsen, & Nordal, 2000). Many studies in the past have been conducted to uncover the phylogenetic and evolutionary history of plants in recent years (Hong et al., 2017; W. Li, Zhang, Guo, Liu, & Wang, 2019; J. Park, Xi, & Kim, 2020; Wei et al., 2020), however, there is only one study in the family of Ranunculaceae i.e., complete chloroplast genome of Ranunculus Cantoniensis (T. Li et al., 2019). 2.2 Biological Characteristics and Research Status of Ranunculus japonicus Ranunculus japonicus was first recorded in Zhou Hou Beji Fang over 1800 years ago as a medicinal herb (Z.-Y. Wang et al., 2021). It can be used as a whole i.e., all parts of the plant possess properties that make it valuable to the traditional Chinese medicine. However, it is characterized by a pungent flavor, warm in nature, and mostly toxic if consumed orally, so it is recommended in most cases for external application instead of oral administration (Sharif et al., 2020; Z.-Y. Wang et al., 2021; Yun et al., 2021). According to traditional Chinese medicine, the application of fresh Ranunculus japonicus can reduce effects of jaundice, relieve asthma, pain in general, and prevent malaria (Z.-Y. Wang et al., 2021). Moreover, it is found to be possessing anti-inflammatory, cardiovascular-modulatory, anti-cancer and anti-bacterial properties in accordance to modern pharmacological studies (Bo, 2011; Dai et al., 2015; Gao, Liu, Yang, & Tan, 2014; Hakan & Karagöz, 2018; Sharif et al., 2020; Sultana, 2011; R. Wang & Tan, 2008; Z.-Y. Wang et al., 2021; Yun et al., 2021; F.-Y. ZHU, 2013). While it is evident that there is ample research in the biological and pharmacological actions of this plant, there is a lack of a complete chloroplast genome characterization. 3. Method 3.1 Materials The researcher will collect the leaves of Ranunculus japonicus from Chun’an, Zhejiang, China (GPS: E118°46’57’’, N29°50’45’’). First of all, leaves will be prepared by drying them using silica and then DNA Plantzol Reagent will be used by the researcher to extract the DNA from the silica-dried leaves. The researcher will conduct analysis and generate the total genomic sequences using the Illumina HiSeq 2500 platform as it is an instrument of choice for major genome centers and research institutions throughout the world (Gorbachev et al., 2019; Mak et al., 2017). MAFFT software will be used for assembling the chloroplast genome for the plant. Moreover, BLAST, GeSeq and GENEIOUS prime software will be used for ensuring proper aligning and annotation of the genome material as the use of these programs is the norm in the latest similar researches (Candassamy et al., 2020; Jin et al., 2019; Maurya et al., 2020). 3.2 Technical Route The overall process of the study is as followed. • The first stage is the collection of sample material • After sample collection, the researcher will make morphological observations of the plant. • Next, the researcher will prepare the plant material for DNA extraction. Extraction will be carried out using DNA Plantzol Reagents which is to be used for ensuring primer designing and as a filtrate. • High-throughput sequencing techniques will be used for Cp DNA amplification • Next, the researcher will use techniques of trimming data to ensure the accurate de novo assembly of the genome. • In the next step, MAFFT will be used to encode the alignment and annotation of the sequence of cp DNA of Ranunculus japonicus. It will allow accurate analysis of the genome organization as well as the gene order and gene content. This data will then be used to draw a circular sequence of chloroplast genome of the studied plant. • Phylogenetic analysis will be carried out and phylogenetic tree will be drawn to analyze the evolution relationship of Ranunculus japonicus. • Comparison of the cp genomes pf Ranunculus japonicus will be carried from other plants of same genus using genes that are conserved in IR, LSC and SSC regions and the difference between the coding regions and noncoding regions. 3.3 Bioinformatic analysis The researcher will draw the chloroplast genome structure of Ranunculus japonicus using the online open website OGDRAW after collection of all the data of chloroplast genes (Greiner, Lehwark, & Bock, 2019; Lohse, Drechsel, Kahlau, & Bock, 2013). In the past, such analysis were expensive but with the development of software for sequencing technology, it has become less costly and time taking to conduct phylogenetic analysis and create phylogenetic trees. The drawing of phylogenetic tree for the Ranunculus japonicus allows information gathering about divergences and similarities between species, population, and within the populations (Mello, Tao, Barba‐Montoya, & Kumar, 2021; Mello, Tao, & Kumar, 2019). In this study, the researcher will conduct an inter-species analysis. The researcher plans to search, filtrate and download the genome data of the species that fall in family Ranunculus from NCBI as references to accurately compare and draw the phylogenetic tree. Each branch of the tree will be representative of a different kind of species. The researcher will draw the phylogenetic tree using the Maximum Likelihood (ML) method (Liu, Anderson, Pearl, & Edwards, 2019; Yoon & Kim, 2020) which relies on statistical models of DNA mutation occurrences among the species and allows permitting of different rates of flexibility of evolution, 80% will be set in this study which may be relaxed to 70% in special cases. 4. Critical issue Several important factors and issues need to be kept in mind while conducting this research. These are discussed below • First of all, the basic characteristic and annotation of the needs to be done carefully. While the researcher annotates the Ranunculus japonicus chloroplast genome, it must be kept in mind that the Annotate and Predict function from NCBI to make sure that the nearest species can be recognized for annotation. A similarity level of 80% will be aimed and selected and after this, all the chloroplast genomes from the NCBI database that match the requirements will be downloaded in the same folder. The similarity for some species, that are known to be linked to the plant, will be adjusted to 75%. • Next it must be made sure that alignment of the preliminary content is proper and the file needs to be checked with MAFFT; a program that is used for creating multiple sequence alignments of DNA material (Katoh & Standley, 2013; Rozewicki, Li, Amada, Standley, & Katoh, 2019). It will indicate if the preliminary annotation has any missing annotation genes. • The phylogenetic analysis of adjacent genus needs to be made carefully as well. The genes of Ranunculus japonicus need to be aligned carefully to do so. The files containing genome data of the species need to be named clearly and carefully after downloading them from NCBI. The researcher will use the IQtree software to draw the phylogenetic tree due to its high accuracy rating (Nguyen, Schmidt, Von Haeseler, & Minh, 2015; von Haeseler, Schmidt, Bui, & Nguyen, 2014). 5. Expected accomplishments As a consequence of this study, the researcher aims to gain several accomplishments. First of all, it is expected that new and traditional studies, books and journals will be read and reviewed by the researcher regarding Ranunculus japonicus to learn about its basic morphologic features as described in past literature, the Ranunculus japonicus (Do et al., 2019; Khalil et al., 2020; H. Park, Yoon, Kim, & Kim, 2015) 6. Structure and Schedule The overall research schedule for the proposed thesis is as presented in the table below. Time Content 2021.10-2021.12 • Choose a topic for the thesis • Review available literature on the topic • After reading literature, design an appropriate experiment path of proposed study 2022.1-2021.2 • Consult with supervisor and improve design for the experimental method; • Ensure that the material and instruments required for experimental procedure are prepared. 2022.3-2022.4 • Start the experimental process • Ensure that all the results of experiment are recorded accurately. 2022.4-2022.7 • Summarize the results and interpret them • Complete the writeup for the graduate thesis. 2022.8-2022.9 • Make any suggested or necessary Revision in the graduate thesis and prepare for graduation thesis defenses. The overall chapter structure of the graduate thesis will be as presented in the table below. Chapter Content Chapter 1: Introduction In the first chapter, the researcher will present a detailed background of the topic. The novelty of the research, the objectives and questions driving the research and the significance of the study will be discussed. Chapter 2: Review of Literature The major research in related topic areas will be summarized in this chapter Chapter 3: Methods and Materials The researcher the process of material extraction, preparation, analysis and other necessary details. Chapter 4: Findings and Results The researcher will present the results of genome characterization in this chapter Chapter 5: Discussion and Conclusion In the final chapter, the researcher will present a summary of the findings and will discuss the implications of these findings and the limitations and future research directions for the future studies.