VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- UNDERGRADUATION THESIS TITLE: STRUCTURAL ANALYSIS OF THE RC GENE IN THE LOCAL PURPLE STICKY “YEN BAI” RICE VARIETY HANOI, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- UNDERGRADUATION THESIS TITLE: STRUCTURAL ANALYSIS OF THE RC GENE IN THE LOCAL PURPLE STICKY “YEN BAI” RICE VARIETY Student : Bui Thi Thanh Hien Class : K62CNSHE Faculty : Biotechnology Supervisor : Nguyen Quoc Trung, MSc. Le Duc Thao, PhD. HANOI, 2022 COMMITMENT This thesis is composed of my original works, and contains no material previously published or written by another person. Hanoi, 20th March, 2022 Student Bui Thi Thanh Hien i ACKNOWLEGEMENTS During the process of studying, researching and completing the thesis, I have received the help of many individual First and foremost, I would like to express my gratitude to my supervisor MSc.
Nguyen Quoc Trung, Department of Molecular Biology, Vietnam National University of Agriculture for their patience, motivation, and immense knowledge. Their enthusiasm guidance helped me all the time of researching and writing of this thesis. My sincere thanks also go to teachers in the Faculty of Biotechnology, Vietnam National University of Agriculture, whose encouragement, guidance, and inspiration supported me during my studying time at higher education. Besides that, I am thankful to my dear classmates, class K62CNSHE, for all the time we had in the last four years.
Last but not least, I am grateful to my family and my friends for supporting me spiritually throughout writing this thesis and my life in general I sincerely thank you! Hanoi, 20th March , 2022 Student Bui Thi Thanh Hien ii CONTENT COMMITMENT. iii LIST OF TABLES. v LIST OF FIGURES. vi LIST OF ABBREVIATIONS.
Evolution and domestication of ricecultivar. Purple sticky rice. Overview of anthocyanin. Roles and characteristics of the Rc gene.
Diversity of rice genetic resources in Vietnam. Studies on phylogeny of Vietnamese local varieties. MATERIAL AND METHODS. Sequencing and identification of gene model.
Sequence polymorphism analysis. Amplification and sequencing of Rc gene. Sequence Rc fragments. Structural analysis of Rc gene.
Intron-exon structures :. 14-base deletion detection:. Sequence polymorphism of Rc gene. Sub cellular localization and 3Dmodel.
CONCLUSION AND SUGGESTION. 43 iv LIST OF TABLES Table 2. Component of the PCR reation. List of 18 primer pairs for amplifying DNA fragments of Rc gene.
Rc gene sequences of 8 accessions from the NCBI. Physical and chemical parameters of protein of NepCam YB with 8 reference varieties. 38 v LIST OF FIGURES Figure1.The evolution of rice (Purugganan 2010). Awns of wild and cultivated rice.
28 samples of rice varieties after husking. Basic anthocyanin structure (Khoo, Azlan et al. Different R radicals of anthociadin (Chaves-Silva, Dos Santos et al. Metabolic pathways for the synthesis of pro‐anthocyanidins and anthocyanins (Chaves-Silva, Dos Santos et al.
Genetic diversity of rice color. Rc allele phenotypes (Sweeney, Thomson et al. Population structure and location of the Indica and Japonica subpopulations within Vietnam. (Higgins, Santos et al.
Population structure and location of the Indica and Japonica subpopulations within Vietnam. (Higgins, Santos et al. Rice grain of NepCam YB. Electrophoresis photo of Rc gene fragments amplified by PCR (DNA ladder was KAPPA universal ladder 100 bp).
Part of the gene sequencing results expressed through Chromas software. Full sequence of NepCam YB. Exon intron structure of Nepcam YB. Collinear analysis of the fifth intron's partial sequence of Rc.
33 Gene of NepCam YB. The SNPs in RC gene of NepCam YB compared with 8 representative rice accessions. G-C content, A-T content chart of each variety. Phylogenetic tree of Nepcam YB with 8 rice varieties.Model structure Rc of each variety.
39 vi LIST OF ABBREVIATIONS bHLH basic helix-loop-helix BC Before Century AD Anno Domini QTL Quantitative trait locus FNP Functional nucleotide polymorphism ABP Anthocyanins biosynthesis pathway DFR Dihydroflavonol-4-reductase PCR Polymerase chain reaction vii ABSTRACT Rc is one of the most important regulatory genes for proanthocyanidin accumulation in the rice pericarp. A study on mutation and sequence polymorphism of the Rc gene will provide evidence for understanding anthocyanin synthesis and evolution of rice cultivars. The aim of this study was to a Rc hive a full-length genomic DNA sequence and analyze polymorphism in the Rc gene of Nepcam YB variety, a local pigmented rice in Yen Bai province. By using 18 primer pairs, full- length sequence along with down-stream and up- stream segment of the Rc gene was successfully assembled and aligned by BioEdit software with 7500 bp.
We found that the Rc gene from NepCam YB variety contains 8 introns and 9 exons. Sequence polymorphism analysis has revealed 14 bp deletions in the fifth introns and 54 SNPs was detected by comparing with 8 representative rice cultivars. Identification of physicochemical parameters of Rc protein showed hydrophilic and unstable properties. The Rc protein was determined to be located in the mitochondria.
Phylogeny analysis revealed close relationship of Nepcam YB with japonica Jefferson cultivar. In this study, full-length sequence of the Rc gene in Nepcam YB variety was successfully sequenced and is preliminary data for further study on domestication of Vietnamese localrice. Keywords: Proanthocyanidin, pigmented rice, gene model, SNPs, phylogenetic tree, Rc gene. viii INTRODUCTION Introduction Rice (Oryza sativa) has been considering as one of the most important cereal crops that is widely cultivated in Asia and has a long domestication history.
Rice is a staple food of more than 100 countries as the main carbohydrate source for more than half of the global population. A large number of rice varieties, including wild and cultivated rice plants, have been obtained in the world, of which they were varied on grain properties, like shape, amylose content and pericarp color. Among them, pericarp color, caused by the high accumulation of non- Chl pigments (like anthocyanins, flavanols and pro- anthocyanidins), was noted as a rare characteristic of rice plants. According to, the color of rice pericarp was varied from red (from weedy rice), purple (purple rice), brown (brown rice) and white (cultivated rice).
Therefore, it would be very interesting to get insight into the purple sticky rice. In Vietnam, purple sticky rice is a specialty rice variety that has been grown for a long time, in many localities, in many different ecological regions, and is very diverse in phenotypes. Products made from purple sticky rice are used for many different purposes in people's lives; purple sticky rice is present in many festivals. That creates bearing Vietnamese cultural identity.
However, studies on anthocyanins at molecular level in Vietnamese purple sticky rice are still limited, making it difficult to exploit and use native genetic resoues Rc. Rc has been well-characterized as one of the most important regulatory genes in the pathway of proanthocyanidin accumulation in the rice pericarp. Briefly, Rc locates on rice chromosome 7, contains eight exons, and encodes a basic helix-loop-helix (bHLH) regulatory protein. The major Rc domestication allele which has been occurred in more than 97% of non-pigmented rice cultivars, is characterized by a 14-bp fragment deletion in the seventh exon.
This mutation generates a truncated, non-functional gene product and the non- 1 pigmented („white‟) pericarp of the domesticated rice. An independently evolved domestication allele, Rc-s is found in other white pericarp rice genotypes with the frequency of less than 3% and exhibits a base transversion (i., C to A) in the seventh exon instead of the absence of a 14-bp fragment. The aim of this study was to analyze the sequence polymorphism in Rc gene of Nepcam pigmented YB rice, a local cultivar in Yen Bai province, Vietnam. Objectives The objectives of this study were to analyze the sequence polymorphism and structural properites of Rc gene to reveal phylogeny of Nepcam pigmented YB rice, a local cultivar in Yen Bai province, Vietnam.
Requirements Optimization of PCR conditions for cloning 18 Rc gene fragments. Sequencing18 gene fragments and assembly full sequence of the Rc gene. Sequence and structure analysis of Rc gene in NepCam YB. Construction of phylogenetic tree of NepCam YB.
rice based on full length sequence of Rc gene. Evolution and domestication of ricecultivar Rice has been known as the world‟s largest food crop, providing the caloric needs of millions of people daily. Rice belongs to genus Oryza of family OryzaGramineae that consists of 21 wild-types of the domesticated rice varieties (Vaughan et al.sativa genus is divided into four species complexes, namely O. sativa complex contains two domesticated species, including O.glaberrima, are the only cultivated species grown worldwide, and in some parts of West and Central Africa, then, six wild-types, including O.glumaepatulaare fit into O.sativa is distributed globally with a high concentration in Asia, while O.glaberrima is grown in West Africa.rufipogon can be found throughout Asia and Oceania.
Oryza barthii and O.longistaminata are African species, O.barthii endemic in West Africa and O.longistaminata is found throughout Africa. Oryza meridionalis is native to Australia and O.glumaepatula is endemic in Central and South America. Based on these distributions, it is easy to locate the ancestral pools from which modern rice was extracted. The African cultivars were domesticated from O.sativa was domesticated from O.
Based on the previously presented evidence of independent domestications for indica and japonica, the researchers made some crosses between the two subspecies, the offspring should segregate for wild alleles at several loci and wild characteristics should re-appear among sub-specific populations. Most notably for traits like dormancy and shattering, intra-specific crosses between parents with low dormancy and shattering give rise to progeny that has higher levels of dormancy and shattering than either parent (Lin et al., 1998; Miura et al., 2002; Longbiao et al., 2004; Konishi et al. However, 3 levels of dormancy and shattering in these crosses are not as high as wild accessions, suggesting either that indica and japonica share some domestication alleles or that independent mutations within the same domestication loci occurred in each subspecies which fail to compliment when crossed. Another confirmation that different domestication genes were under selection in different subpopulations comes from QTL studies.
Populations derived from crosses between a single wild accession and diverse cultivars often identity different QTLs for domestication traits (Xiao et al., 1998; Moncada et al., 2001; Septiningsih et al., 2003; Thomson et al., 2003; McCouch et al., 2006; Xie et al.The evolution of rice (Purugganan 2010) According to Cao et al. 2006, 4000 BC, rice grains sieved from the oldest known paddy fields in the lower Yangzi River Valley, giving clear-cut evidence for rice cultivation at this point in time. Genetic changes causing the shift from wild to domesticated rice are harder to pinpoint. Mutations leading to a reduction in the degree of grain shattering are a prerequisite for domestication.
4 Wild grasses are mean little things; for example, the seeds of many wild grass species have awns, large, barbed spikes that can fend off seed-eating animals, assist in seed dispersal, and help plant the seeds. In Triticum spp, changes in humidity cause the awns to flex, which can help bury the seeds. After thousands of years of artificial selection, our domesticated cereal crops have shorter or nonexistent awns to facilitate grain harvesting, handling, and storage. Rice Oryza sativa domestication also involved alterations in many traits, including growth habit, seed shattering, panicle architecture, grain size, and hull color (Sang and Ge,2013).
Awns of wild and cultivated rice. (A) shows the panicles of wild rice ¼ (left) and domesticated rice (right), bar¼ 10cm; (B) shows the seeds of wild rice (top) and a domesticated rice (bottom), bar¼ 1cm; (C) shows the surface of a wild rice awn (left) and a domesticated rice awn (right) under a scanning electron microscope, bar 200mm. (Hua et al.2015) The wild progenitors of cultivated rice were O.rufipogon, two most closely related wild species with the current distribution from southeastern Asia to India. nivara was often regarded as an annual ecotype of O.