VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNERGRADUATE THESIS ANALYSIS OF GENETIC DIVERSITY OF LOCAL PIGMENTED RICE IN VIETNAM Student : NGUYEN VU KIEU OANH Supervivor : LE DUC THAO, Ph. NGUYEN QUOC CHUNG ID : 614076 Class : K61CNSHE Hanoi - 2021 ACKNOWLEDGEMENTS Firstly, I would like to express my appreciation to the directory of the Vietnam National University of Agriculture, the board of deans, and lectures in the faculty of biotechnology that created the best condition for me to perform and complete my thesis. Secondly, I’m deeply indebted to my supervisors, Dr. Le Duc Thao and MSc.
Nguyen Quoc Trung, who guided me to conduct experiments and advised me on professional knowledge. Their support gave me knowledge and patience to accomplish the thesis. I would also like to thank guides from Crop Research and Development Institute who shared useful experiences in building field layout and rice cultivation for me. I gratefully acknowledge my colleagues in the laboratory for their wonderful assistance.
Thank to them, I can complete my research with high excitement. Last but not least, I would like to thank my parents for their support in the study as well as in life. I have never finished my dissertation without their love and their support. I sincerely thank all of you! Hanoi ……………… Student Nguyen Vu Kieu Oanh i COMMITMENT I guarantee that this thesis is my original works and all the data and results in this thesis are truthful and have never been used in any report yet.
I also assure that the information cited in the thesis is indicated the origin and all the help is thankful. Hanoi ……………… Student Nguyen Vu Kieu Oanh ii LIST OF CONTENTS Acknowledgements. ii LIST OF contents.iii LIST OF TABLES. v LIST OF FIGURES.
Genetic resources of rice. Global genetic resources of rice. Genetic resources of rice in Vietnam. Pigmented rice genetic resources.
Properties of pigmented rice. The studies about genetic diversity of rice. Materials and methods. DNA extraction with potasium acetate (CH3COOK).
Anthocyanin content measurement. Results and discussions. The result of the DNA extraction of 30 accessions. Analysis the genetic relationships between the studied pigmented rice samples.
Total anthocyanin content. CONCLUSIONS AND SUGGESTIONS. 42 iv LIST OF TABLES Table 1. Several anthocyanins in pigmented rice (Deng et al.
35 SSR molecular markers were used in the experiment. 30 pigmented rice varieties was used in this research. The compositions of PCR. the PIC index of 3 SSR markers.
Total anthocyanin content of 30 accessions. Classification of 30 accessions by anthocyanin content. Classification of color of rice pericarp. 31 v LIST OF FIGURES Figure 1.
Black rice, Red rice and other pigmented rice varieties. The Basic structur of Anthocyanin (Athanasios Valavanidis et al, Studies in Natural Products Chemistry, 2013). Chemical diagram of color-changing anthocyanin pH reaction Under different pH conditions(Kan, Vargo et al. The field layout.
The extraction anthocyanin content in rice. The test results the quality of DNA on Agarose 1% gel. Electrophoresis images of several SSR pairs of primers. The dendrogram of the genetic similarity coefficients Jaccard of 30 pigmented rice varieties.
Total anthocyanin content (mg/100g) of 30 accessions. color of pericarp of 30 accessions. 30 vi SUMMARY The experiment aimed to analyze the genetic diversity of 30 local pigmented rice accessions based on the presence and polymorphism level of SSR molecular markers. The experiment used 35 SSR molecular markers with a total of 47 alleles, 20 markers present polymorphism, an average of 2.04 alleles per locus.
Polymorphic Information Content (PIC) ranged from 0.99 with an average value of 0. The rice cultivars were divided into 2 main groups. In addition, experiments also determined the anthocyanin content of varieties. Two varieties with the highest anthocyanin content were C6 and C22 with concentration 92.
The data obtained in this study would provide important information for the breeding of hight quality rice accessions by molecular markers. Introduction Rice (Oryza sativa) makes a major contribution to the calorific intake of populations in countries where rice is the main food source. However, white rice is a poor source of vitamins and minerals, Therefore, the diet is too dependent on it is a risk of the lack of some nutritional factors (Dipti, Bergman et al. 2012) (Muthayya, Sugimoto et al.
Some studies report that the nutritional quality of white rice is poor ycompared to that of pigmented variants(Mbanjo, Kretzschmar et al. Today, the increasing consumer interest in health-promoting food products is creating a significant market for rice with higher nutritional value, creating health benefits for large numbers of people. However, the high market demand of humans for white rice leading to the depletion of pigmented varieties (Ahuja, Ahuja et al. Currently, most pigmented rice varieties are low-yielding, being grown only for local markets (Mbanjo, Jones et al.
The pigmented (black, purple, red-orange, or brown) rice have the compounds responsible for these color variations like flavonoids, anthocyanin, and proanthocyanidin in their pericarps. Black and purple pericarps are the results of the accumulation of anthocyanin, while red pericarps are due to proanthocyanidins (Gunaratne, Wu et al. 2013, Samyor, Das et al. These compounds also have lots of nutritional value (Mbanjo, Kretzschmar et al.
Vietnam is a country with very diverse rice genetic resources especially pigmented rice gene resources. Most pigmented rice varieties are specialty rice varieties that have been grown for a long time and are used for many different purposes in the lives of Vietnamese people. The products made from it are present at vast of majority festivals and it creates civilization bearing national cultural identity. Pigmented rice is grown in many localities, different ecological regions, and is diverse in style and color.
However, in Vietnam, there are only a few studies on the genetic diversity of local pigmented rice. 1 This leads to difficulties in the conservation of the diversity of this pigmented rice gene resource. Therefore, the study of the genetic diversity of pigmented rice is not only meaningful in providing information on genetic resources, selecting materials for high-quality rice breeding in Vietnam but also has significance in the conservation of local pigmented rice varieties. Plant varieties in general and rice varieties, in particular, have different genetic structures, which is a good source of materials to guide the breeding and selection of new rice varieties.
The evaluation of genetic diversity can be based on phenotype or genotype (using molecular markers). Using molecular markers is a powerful tool in assessing genetic variation, explaining genetic relationships within and between species. The advantage of this type of evaluation is fast, accurate, for high and stable polymorphism. Assessing genetic diversity by molecular markers provides more information and accuracy than morphological methods.
The biggest advantage of this method is to detect DNA level differences and save time as studies can be conducted early when rice is still in the seedling stage.Objectives In this study, we analyze the genetic diversity of 30 newly collected local pigmented rice varieties in Vietnam by DNA markers. Results of the research will be used for conservation, providing information on genetic resources, and exploiting high-yield and good quality rice varieties. Requirements - Extraction of DNA from leaf samples of 30 pigmented rice varieties, PCR with SSR markers, and electrophoresis. - Analysis of genetic diversity by constructing phylogenetic tree( use program NTSYS 2-1) and using Polymorphism Information Content (PIC).
- Determination of the anthocyanin content. Genetic resources of rice. Genetic resources "Genetic resources‟ is defined as the genetic material of actual or potential value. Genetic material means any material containing functional units of heredity( gene) of plant, animal, microbial or other origin and passes it from one generation to the next that passes it from one generation to the next.
Genetic resources are an important source of information for taxonomy, describing, and naming species. Developing the understanding of genetic resources helps the conservation of threatened species and it also helps develop the methods that help safeguard global biodiversity. Global genetic resources of rice According to the History of rice cultivation, rice was first domesticated in the Pearl River valley region of China occurred 8,200–13,500 years ago. Nowadays, there are two species of rice that are widely cultivated in the world, which are Asian rice ( Oryza sativa ) and African rice ( O.
They belong to a group of 25 grass species of the genus Oryza, family Poaceae (Onwueme and Sinha 1991). Asian rice had its origin in South and Southeast Asia and is now cultivated worldwide, whereas African rice was domesticated much later in parts of West Africa (Dowling, Greenfield et al. For thousands of years, rice was selective breeding by farmers to respond to their widely diverse needs. In the process of dispersal since domestication, rice has formed a large range of genetic diversity, reflected in the number of varieties existing today.
sativa is estimated to have more than 140,000 varieties, including primitive varieties and improved varieties (Virk, Newbury et al. 1995) and wad cultivated widely from China, India, Indonesia, Pakistan, Bangladesh, to Vietnam, Thailand, Myanmar, Philippines, and Japan. 3 An overview of the genetic structure of the Oryza genus has been provided by studies using various means: morphological, cytogenetics, interspecific hybridization, and biochemical and molecular markers. Most studies agree that O.
sativa was domesticated from the common wild rice O. rufipogon (Sampath 1951, Sampath 1958) or the annual common wild rice O. glaberrima originated from O. rufipogon still extant in South and Southeast Asia.
sativa varieties into six groups (Glaszmann 1987). The two main groups, I and VI are corresponding to two major subspecies, the sticky, short-grained japonica( sometimes called sinica) , and the nonsticky, long-grained indica. The indica sub-species has been divided into two sub-populations, indica, and aus, while japonica divides into tropical japonica, temperate japonica, and aromatic sub-populations (Garris, Tai et al. In addition to the widely accepted five major rice sub-populations, a new population, Rayada was reported by Wang (Wang, Zheng et al.
Indica is mainly cultivated in tropical and subtropical environments with lower latitudes and altitudes, whereas japonica rice is grown mainly in more temperate environments with higher latitudes and altitudes. Population structure and genetic diversity analyses revealed a higher level of diversity within indica than japonica, and identified additional sub-populations in both sub-species (Wang, Mauleon et al. This classification is well consistent with other biological criteria, so it has been accepted by plant breeders. There some recent studies have shown that indica and japonica varieties are not purebred.
They are mosaics of both indica and japonica alleles (Glaszmann, Grivet et al. 2003) and can be explained by the above hybridization process between indica and japonica varieties. Above the cultivated rice species and their direct ancestors, there more than a dozen other wild species in the genus Oryza that are distributed throughout the tropics of Asia, Africa, and South and Central America. The 4 wild species in Oryza consist of different genomes and have different biosystematic relationships with 2 main cultivated rice.
The adoption of modern rice varieties, the application of modern inputs such as fertilizers and pesticides, and the development of irrigation have increased the food supply and a decrease in rice prices (Hossain and Fischer 1995). But these modern technologies have also contributed to the loss of genetic diversity of rice. This loss, is known as genetic erosion, is defined as the loss of genes from a gene pool attributed to the elimination of populations caused by factors such as the adoption of high-yielding varieties, land clearing, urbanization, and cultural change(Brush 1991, Plucknett and Smith 2014). In Asia, a lot of populations of wild rice species become extinct because their natural habitat is endangered by the extension of cultivation areas and urban pressures.
The study of Chang (1984) estimated that more than 100,000 rice cultivars existed in Asia earlier in the 20th century (Chang 1984).