VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- GRADUATION THESIS STUDY ON SEQUENCE VARIATION IN Rc GENE OF VIETNAMESE LOCAL PIGMENTED RICE HANOI, 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------oOo------- GRADUATION THESIS STUDY ON SEQUENCE VARIATION IN Rc GENE OF VIETNAMESE LOCAL PIGMENTED RICE Student : Nguyen Thi Yen Chi Class : K61CNSHE Faculty : Biotechnology Supervisor : Nguyen Quoc Trung, MSc. Chu Duc Ha, PhD. HANOI, 2021 COMMITMENT This thesis is composed of my original works, and contains no material previously published or written by another person. Hanoi, 30th January, 2021 Student Nguyen Thi Yen Chi 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 Biology, Vietnam National University of Agriculture, and Dr. Chu Duc Ha, Agricultural Genetics Institude, for their patience, motivation, and immense knowledge. Their enthusiasm guidance helped me all the time of researching and writing of this thesis. Besides my supervisors, I would like to thank guides and people working in Crop Research and Development Institute for sharing their experiences, time, and commitment during my work to prepare my materials as rice samples.
I also would like to offer all other friends, who also do thesis in Center of International Plant Research Vietnam and Japan for their kindly help, care and lift me up all the trouble in the time doing experiments. 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 K61CNSHE, 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, 30th January, 2021 Student Nguyen Thi Yen Chi ii TABLE OF CONTENTS COMMITMENT.
ii TABLE OF CONTENTS.iii LIST OF TABLES. v LIST OF FIGURES. vi LIST OF ABBREVIATIONS. Evolution and domestication of rice cultivar.
Overview of theRc gene in rice. Studies on genes controlling anthocyanin synthesis. Studies on Rc gene. MATERIAL AND METHODS.
RESULTS AND DISCUSSION. Results of cloning and amplification of Rc gene fragments. Design the primers for sequencing. Optimization of PCR for cloning fragments.
Results of sequencing Rc gene fragments. Alignments of Rc gene. Rc gene in Nep cam dang 2. Rc gene in Nep cam (Tuyen Quang).
Rc gene in Nep cam den. Rc gen in Nep cam (Hoa Binh). Identification of 14 nucleotide deltion in seventh exon. Sequence variations in Rc gene (SNP).
CONCLUSIONS AND SUGGESTIONS. 42 iv LIST OF TABLES Table 1. Example of rice genes involved in flavonoid biosynthesis (Furukawa et al. Information of four local pigmented rice varieties used.
18 in this study. Component of the PCR reation. The Annealing temperature of each primer. The optimal annealing temperature in PCR for each reasponsible primers.
Total 17 fragments were sequenced. Collinear analysis of the seventh exon's partial sequence of Rc gene for 4 local pigmented rice varieties. The number of SNP identified by comparising the Nep cam dang 2 variety with 2 varieties ( O. sativa indica and O.
39 v LIST OF FIGURES Figure1. Basic anthocyanin structure. Two-dimensional structure of flavylium ion. Metabolic pathways for the synthesis of pro‐anthocyanidins and anthocyanins.
Rc allele phenotypes (Sweeney, et al. The primers were designed locating overlap along Rc gene sequence. PCR product electrophoresis before and after determining the optimal annealing temperature. Some fragments with single bands.
The product electrophoresis is Rc_8 fragment. Phylogenetic tree of 'Nep cam dang 2' variety. 40 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 LIST OF APPENDIX Appendix table 1. Sequences of 17 fragments in Nep cam dang 2.
Sequences of 17 fragments in Nep cam (Tuyen Quang). Sequences of 17 fragments in Nep cam den. Sequences of 17 fragments in Nep cam (Hoa Binh). 55 viii ABSTRACT The aim of study to detect the genetic variations in Rc gene of Vietnamese local pigmented rice.
In this study, we selected 4 rice cultivars, then, sequenced the full lengths of their Rc genes (approximately 6.4 kb) by a set of 17 pairs of primers. In addition, we collected 6 different Rc genes in the Oryza genus from the literature and from NCBI database. Phylogenetic analyses showed that the Rc genes of Nep cam dang 2 from Yen Bai were rc genotypes found in South Asia and Southeast Asia. Introduction Rice (Oryza sativa) has been considering as one of the most important cereal crops that widely cultivated in many regions.
Briefly, rice is a staple food of more than 100 countries as the main carbohydrate source for more than half of the global population. Depending on the type of rice, it may contain a high concentration of fibers, proteins, vitamins B, irons, and manganese. Up till now, a number of rice varieties have been cultivated in the world. Based on the color of the grain, white rice varieties have been reportedly the most popular cultivars due to their storable ability and cooking qualities.
Of our interest, some local pigmented rice varieties, including black/purple rice, brown rice, and red rice have been characterized to have high nutritional value, especially black rice such as a 1/2 cup serving of prepared black rice, made from about 1/4 cup of uncooked rice, contains 160 calories, 1 1/2 grams of fat, 34 grams of carbohydrates, 2 grams of fiber, 5 grams of protein, and 4 percent of iron, carotene, 8 kinds of amino acids and trace elements (iron, zinc) necessary for the body (Tuoi, et al. Specifically, the color of the grain is thought that that the result of the accumulation of flavonoids, carotenoids, and betalains. In while, flavonoid compounds such as anthocyanins, flavonols, and proanthocyanidins are major secondary metabolites in plants and are red, purple (black), and brown in color. However, little information on pericarp in these rice varieties has been reported.
Rc has been well-characterized as one of the most important regulatory genes in the pathway of proanthocyanidin accumulation in the rice pericarp(Li, et al. Briefly, Rc locates on rice chromosome 7, contains eight exons, and encodes a basic helix-loop-helix (bHLH) regulatory protein (Furukawa, Maekawa et al. 2007, Sweeney, Thomson, et al. The major Rc domestication allele which has been occurred in more than 97% of non- 1 pigmented rice cultivars, is characterized by a 14-bp fragment deletion in the seventh exon (Li, Qiang et al.
This mutation generates a truncated, non- fuctional gene product and the non-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 (Sweeney, Thomson, et al. Furthermore, “(Brooks, Yan, et al. 2008) identified the spontaneous mutant red pericarp rice cultivar Wells in the USA, which exhibits one G-base deletion located at the 20- bp site upstream of the absent 14-bp fragment in the seventh exon.
This new mutation restores the reading frame and allows the re-accumulation of proanthocyanidins in the pigmented rice. (Lee, Lupotto, et al. 2009) confirmed that the red pericarp rice variety Perla Rosso in Italy is a spontaneous mutant with one G-base deletion at the 44-bp site upstream of the absent 14-bp fragment in the seventh exon. This new Rc allele is designated as Rcr”(Li, Qiang et al.
The red pericarp rice cultivated in most African appears a transversion mutiation (i., A to T) occurs in the seventh exon transforms the pericarp color of African cultivated rice from red to white (Gross, Steffen et al. “The allele of Rc is known as rc-g1. So far, six alleles of the Rc gene have been reported: Rc (wild type); rc and Rc-s, which are domestication alleles found in Asian cultivated rice; Rc-g and Rcr, revertants of rc; and rc-g1, which is found only in African cultivars” (Li, Qiang et al. 2014) The aim of this study was to show the domestication progress of rice in Vietnam by studying the sequence variation of Rc gene found in local pigmented rice.
Objectives The objectives of this study are to detect sequence variation in Rc gene of local pigmented rice for rice domestication progress in Vietnam 2 1. Requirements - Design primers for clone fragments of Rc gene in rice - Optimization of PCR conditions for cloning Rc gene fragments - Sequencing gene fragments and assemply full sequence of the gene - Sequence analyzation of Rc gene in 4 varieties 3 CHAPTER 2. Evolution and domestication of rice cultivar 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 Gramineae 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. nivara (also considered to be an ecotype of 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. barthii (formally called O.
sativa was domesticated from O. rufipogon According to Cao et al. 2006, 4000 BC, rice grains sieved from the oldest known paddy fields in the lower Yangzi River Valley (Cao et al., 2006), 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. Communities that foraged wild, shattering rice seeds would likely gather them before maturity since most of the mature grains 4 quickly fall to the ground. Immature rice grains have a smaller width than fully mature seeds because rice grains reach their full length early in seed development, and subsequent grain filling increases the width of the seeds. A survey of diverse modern rices has shown that mature modern cultivated grains rarely have a width, 2 mm, although some mature wild grains do (Fuller et al.
Therefore, if width of the assemblage of ancient grains from a site falls below 2 mm it is unlikely that they represent mature domesticated grains. What can be documented is that seeds with measurements similar to mature, modern O. sativa do not appear until 4500 BC at Chengtoushan in the Middle Yangzte and approx. 4000 BC in the Lower Yangzte area (Fuller et al.
These seeds are certainly domesticated. Before this time the genetic changes conditioning a lack of shattering and/or the mutations leading to thicker grains had not been selected. While these mutations are genetically independent, they result in the same grain width phenotype. Rice moved north to the Yellow River basin in Central China beginning in 3000 – 2000 BC (Crawford, 2005).