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.D MSc 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 i COMMITMENT ii LIST OF contents .iii LIST OF TABLES v LIST OF FIGURES vi SUMMARY vii CHAPTER INTRODUCTION 1.1 Introduction 1.1.2.Objectives 1.1.3 Requirements CHAPTER LITERATURE REVIEW 2.1 Genetic resources of rice 2.1.1 Genetic resources 2.1.2 Global genetic resources of rice 2.1.3 Genetic resources of rice in Vietnam 2.1.4 Pigmented rice genetic resources 2.2 Pigmented rice 2.2.1 Properties of pigmented rice 2.2.2 Anthocyanin 2.2.3 The studies about genetic diversity of rice 14 Chapter Materials and methods 16 3.1 Materials 16 3.2 METHODS 19 3.2.1 Field layout 19 3.2.2 DNA extraction with potasium acetate (CH3COOK) 20 3.2.3 PCR reaction: 20 3.2.4 Electrophoresis: 21 3.2.5 Data processing 21 iii 3.2.6 Anthocyanin content measurement 22 Chapter Results and discussions 24 4.1 The result of the DNA extraction of 30 accessions 24 4.3 Analysis the genetic relationships between the studied pigmented rice samples 27 4.3 Total anthocyanin content 28 CHAPTER CONCLUSIONS AND SUGGESTIONS 32 5.1 Conclusions 32 5.2 Suggestions 32 REFERENTS 34 APPENDIX 42 iv LIST OF TABLES Table Several anthocyanins in pigmented rice (Deng et al., 2013) 12 Table 35 SSR molecular markers were used in the experiment 16 Table 30 pigmented rice varieties was used in this research 17 Table The compositions of PCR 21 Table the PIC index of SSR markers 24 Table Total anthocyanin content of 30 accessions 28 Table Classification of 30 accessions by anthocyanin content 29 Table Classification of color of rice pericarp 31 v LIST OF FIGURES Figure Black rice, Red rice and other pigmented rice varieties Figure The Basic structur of Anthocyanin (Athanasios Valavanidis et al, Studies in Natural Products Chemistry, 2013) 10 Figure Chemical diagram of color-changing anthocyanin pH reaction Under different pH conditions(Kan, Vargo et al 2017) 11 Figure The field layout 19 Figure The extraction anthocyanin content in rice 22 Figure The test results the quality of DNA on Agarose 1% gel 24 Figure Electrophoresis images of several SSR pairs of primers 26 Figure The dendrogram of the genetic similarity coefficients Jaccard of 30 pigmented rice varieties 28 Figure Total anthocyanin content (mg/100g) of 30 accessions 29 Figure 10 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 0.00 to 0.99 with an average value of 0.27 The rice cultivars were divided into 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 92.3 mg/100g The data obtained in this study would provide important information for the breeding of hight quality rice accessions by molecular markers vii CHAPTER INTRODUCTION 1.1 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 2014) Some studies report that the nutritional quality of white rice is poor ycompared to that of pigmented variants(Mbanjo, Kretzschmar et al 2020) 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 2007) Currently, most pigmented rice varieties are low-yielding, being grown only for local markets (Mbanjo, Jones et al 2019) 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 2017) These compounds also have lots of nutritional value (Mbanjo, Kretzschmar et al 2020) 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 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 1.1.2.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 1.1.3 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 Figure Total anthocyanin content (mg/100g) of 30 accessions Base on the result obtained, we divided 30 local pigmented rice accessions into group in following table : Table Classification of 30 accessions by anthocyanin content Group I TAC Number of (mg/100g) accessions > 50 11 Codes C1, C2, C6, C12, C22, C34, C41, C49, C51, C52, C55 II 20-50 C8, C14, C27, C37, C47, C48, C50, C53 III < 20 11 C3, C4, C5, C9, C10, C11, C15, C35, C36, C42, C54 From table 4.4, there are 36.7% of accessions belonged to the high anthocyanin content with more than 50 mg/100g, 26.7% belonged to the intermediate content ranged from 20 to 50 mg/100g The remaining 36.6% of accessions had low anthocyanin content ( less than 20 mg/100g) 29 Figure 10 color of pericarp of 30 accessions To compare corelation between anthocyanin content and color of rice pericarp, we took pictures of grain and dehulled rice Then, we classified into color groups according to the table below: 30 Table Classification of color of rice pericarp Color Group Codes Black A C1, C2, C6, C12, C14, C22, C27, C37,C51, C55 Black and Black is more than brown brown B1 C8, C10, C15, C34, C47, C48, C49, C50, C52, C53 Brown is more than B2 C4, C5, C9, C11 C C54 black Red phenotypes Red , green D C3 phenotypes Brown, green, red E C35, C36 phenotypes Brown, black, white, F C41, C42 green From table 4.4 and 4.5, we remarked that almost black rice among 30 accessions had high anthocyanin content ( more than 50mg/100g) The group B1 had lower than black rice and belong to intermediate anthocyanin content The low anthocyanin content was in red rice, brown or green rice The highest anthocyanin content was C6 and C22 with concentration 92.3 mg/100g This concentration is higher than concentration of blue corn (70.5 mg/100g) (Herrera-Sotero, Cruz-Hernandez et al 2017) The lowest anthocyanin content belonged to group D that is C3 with content mg/100g Anthocyanins, which are responsible for purple pigmentation, represent the bulk of the flavonoids present in black and purple rice Red and white rice grains have been classified as lacking anthocyanin (Boue, Daigle et al 2016) However, the other study indicated anthocyanin has been able to detect a low level in both red and brown rice accessions (Ghasemzadeh, Karbalaii et al 2018) In our study, we also found the back rice has high anthocyanin concentration and the red and brown rice have low anthocyanin content 31 CHAPTER CONCLUSIONS AND SUGGESTIONS 5.1 Conclusions In this thesis, we analyze the genetic diversity of 30 newly collected local pigmented rice varieties in Vietnam by DNA markers In all 35 SSR markers were used, 23 markers present a total of 47 alleles, 20 markers present polymorphism The average number of alleles per locus was 2.04 alleles/ locus The PIC index of RM5, RM133, RM484, RM514, RM5509 more than 0.5 so these five loci have the polymorphism rate at a high level Genetic similarity coefficients of 30 pigmented rice varieties ranged from 0.45 to 0.97 According to the phylogenetic tree, Cam Pe sticky rice variety collected from Thanh Hoa (C10) has the lowest genetic similarity coefficient, about 0.45, the other 29 varieties of pigmented rice divided into distinct groups: Group I includes 17 rice varieties collected from different regions, Group II consists of 12 varieties of black rice, mainly Khau Cham Chuoi and sticky rice from Dien Bien The two varieties with the highest similarity coefficients were Nep Cam Den in Sapa-Lao Cai and Bach Hop variety collected from China Lastly, the experiment in this thesis shows that anthocyanin content in black rice was higher than brown rice and red rice The accessions C6 and C22 had the highest anthocyanin content 92.3 mg/100g that is higher than the concentration of blue corn (70.5 mg/100g) (Herrera-Sotero et al., 2017) 5.2 Suggestions Vietnam is a country with very diverse rice genetic resources especially pigmented rice gene resources However, there are only a few studies on the genetic diversity of these pigmented rice This leads to difficulties in the conservation of local pigmented rice varieties Results of the research can be used for providing information on genetic resources, selecting good materials 32 for high-quality rice breeding in Vietnam The information of Anthocyanin content also can use in the study about the nutrition value of pigmented rice In the future, we should study the nutritional value of anthocyanin in these 30 local pigmented rice varieties, more specifically is determine the antioxidant content of these pigmented rice varieties to develop them into the product like functional food 33 REFERENTS Ahuja, U., S Ahuja, N Chaudhary and R Thakrar (2007) "Red rices–past, present and future." Asian Agri-History 11(4): 291-304 Alvarez, A., J L Fuentes, V Puldón, P J Gómez, L Mora, M C Duque, G Gallego and J M Tohme (2007) "Genetic diversity analysis of Cuban traditional rice (Oryza sativa L.) varieties based on microsatellite markers." Genetics and Molecular Biology 30(4): 1109-1117 Banjerdpongchai, R., B Wudtiwai and K Sringarm (2013) "Cytotoxic and apoptotic-inducing effects of purple rice extracts and chemotherapeutic drugs on human cancer cell lines." Asian Pacific Journal of Cancer Prevention 14(11): 6541-6548 Boue, S M., K W Daigle, M H Chen, H Cao and M L Heiman (2016) "Antidiabetic Potential of Purple and Red Rice (Oryza sativa L.) Bran Extracts." J Agric Food Chem 64(26): 5345-5353 Bowen-Forbes, C S., Y Zhang and M G Nair (2010) "Anthocyanin content, antioxidant, anti-inflammatory and anticancer properties of blackberry and raspberry fruits." Journal of food composition and analysis 23(6): 554-560 Brush, S B (1991) "A farmer-based approach to conserving crop germplasm." Economic Botany 45(2): 153-165 Cuc, H T K and P C Huynh; (2004) "Determination of anthocyanin content in fruits and vegetables by ph-differential spectrophotometry." Cuevas‐Pérez, F E., E P Guimarães, L BerrÍo and D I González (1992) "Genetic base of irrigated rice in Latin America and the Caribbean, 1971 to 1989." Crop Science 32(4): 1054-1059 Chakuton, K., D Puangpronpitag and M Nakornriab (2012) "Phytochemical content and antioxidant activity of colored and non-colored Thai rice cultivars." Asian Journal of Plant Sciences 11(6): 285-293 34 10 Chalker‐Scott, L (1999) "Environmental significance of anthocyanins in plant stress responses." Photochemistry and photobiology 70(1): 1-9 11 Chang, T (1984) "Conservation of rice genetic resources: luxury or necessity?" Science: 251-256 12 CHENG, Z.-q., F.-y YING, D.-q LI, T.-q YU, F Jian, H.-j YAN, Q.-f ZHONG, D.-y ZHANG, W.-j LI and X.-q HUANG (2012) "Genetic diversity of wild rice species in Yunnan province of China." Rice Science 19(1): 21-28 13 Chiou, S Y., J Y Lai, J A Liao, J M Sung and S D Lin (2018) "In vitro inhibition of lipase, α‐amylase, α‐glucosidase, and angiotensin‐converting enzyme by defatted rice bran extracts of red‐pericarp rice mutant." Cereal Chemistry 95(1): 167-176 14 Dipti, S S., C Bergman, S D Indrasari, T Herath, R Hall, H Lee, F Habibi, P Z Bassinello, E Graterol and J P Ferraz (2012) "The potential of rice to offer solutions for malnutrition and chronic diseases." Rice 5(1): 1-18 15 DoiHongHanh (2017) "Nghiên cứu khai thác CSDL nguồn gen lúa gạo màu nhằm khuyến khích sử dụng." 16 Dowling, N G., S M Greenfield and K Fischer (1998) Sustainability of rice in the global food system, Int Rice Res Inst 17 Fossen, T., L Cabrita and O M Andersen (1998) "Colour and stability of pure anthocyanins influenced by pH including the alkaline region." Food chemistry 63(4): 435-440 18 Fukuoka, S., N Alpatyeva, K Ebana, N Luu and T Nagamine (2003) "Analysis of Vietnamese rice germplasm provides an insight into japonica rice differentiation." Plant breeding 122(6): 497-502 19 Garris, A J., T H Tai, J Coburn, S Kresovich and S McCouch (2005) "Genetic structure and diversity in Oryza sativa L." Genetics 169(3): 16311638 35 20 Gephart, N., G Blate, C McQuistan and C Thompson (2010) "New Blood: Greater Mekong New Species Discoveries 2009." WWF report 21 Ghasemzadeh, A., M T Karbalaii, H Z E Jaafar and A Rahmat (2018) "Phytochemical constituents, antioxidant activity, and antiproliferative properties of black, red, and brown rice bran." Chem Cent J 12(1): 17 22 Glaszmann, J.-C (1987) "Isozymes and classification of Asian rice varieties." Theoretical and Applied genetics 74(1): 21-30 23 Glaszmann, J.-C., L Grivet, B Courtois, J.-L Noyer, C Luce, M Jacquot, L Albar, A Ghesquière and G Second (2003) "Asian rice." Genetic Diversity of Cultivated Tropical Plants: 77 24 Gunaratne, A., K Wu, D Li, A Bentota, H Corke and Y.-Z Cai (2013) "Antioxidant activity and nutritional quality of traditional red-grained rice varieties containing proanthocyanidins." Food chemistry 138(2-3): 11531161 25 Hargrove, T R and V L Cabanilla (1979) "The impact of semidwarf varieties on Asian rice-breeding programs." Bioscience 29(12): 731-735 26 Harukaze, A., M Murata and S Homma (1999) "Analyses of free and bound phenolics in rice." Food Science and Technology Research 5(1): 7479 27 Hemamalini, T and V R G Dev (2018) "Comprehensive review on electrospinning of starch polymer for biomedical applications." International journal of biological macromolecules 106: 712-718 28 Herrera-Sotero, M Y., C D Cruz-Hernandez, C Trujillo-Carretero, M Rodriguez-Dorantes, H S Garcia-Galindo, J L Chavez-Servia, R M Oliart-Ros and R I Guzman-Geronimo (2017) "Antioxidant and antiproliferative activity of blue corn and tortilla from native maize." Chem Cent J 11(1): 110 36 29 Hossain, M and K Fischer (1995) "Rice research for food security and sustainable agricultural development in Asia: achievements and future challenges." GeoJournal 35(3): 286-298 30 Hudson, E A., P A Dinh, T Kokubun, M S Simmonds and A Gescher (2000) "Characterization of potentially chemopreventive phenols in extracts of brown rice that inhibit the growth of human breast and colon cancer cells." Cancer Epidemiology and Prevention Biomarkers 9(11): 1163-1170 31 Kan, V., E Vargo, N Machover, H Ishii, S Pan, W Chen and Y Kakehi (2017) Organic primitives: Synthesis and design of pH-reactive materials using molecular I/O for sensing, actuation, and interaction Proceedings of the 2017 CHI conference on human factors in computing systems 32 Kong, J.-M., L.-S Chia, N.-K Goh, T.-F Chia and R Brouillard (2003) "Analysis and biological activities of anthocyanins." Phytochemistry 64(5): 923-933 33 Lam, D T., B C Buu, N T Lang, K Toriyama, I Nakamura and R Ishikawa (2019) "Genetic diversity among perennial wild rice Oryza rufipogon Griff., in the Mekong Delta." Ecology and evolution 9(5): 29642977 34 Lang, N T and B C Buu (2007) "Rice breeding and inheritance of herbicide resistance in clearfield rice (Oryza sativa L.)." Omonrice 15: 3645 35 Li, D., Y Zhang, Y Liu, R Sun and M Xia (2015) "Purified anthocyanin supplementation reduces dyslipidemia, enhances antioxidant capacity, and prevents insulin resistance in diabetic patients." The Journal of nutrition 145(4): 742-748 36 Mazza, G (2018) Anthocyanins in fruits, vegetables, and grains, CRC press 37 37 Mbanjo, E G N., H Jones, X G I Caguiat, S Carandang, J C Ignacio, M C Ferrer, L A Boyd and T Kretzschmar (2019) "Exploring the genetic diversity within traditional Philippine pigmented Rice." Rice 12(1): 1-18 38 Mbanjo, E G N., T Kretzschmar, H Jones, N Ereful, C Blanchard, L A Boyd and N Sreenivasulu (2020) "The Genetic Basis and Nutritional Benefits of Pigmented Rice Grain." Frontiers in Genetics 11 39 Muthayya, S., J D Sugimoto, S Montgomery and G F Maberly (2014) "An overview of global rice production, supply, trade, and consumption." Annals of the new york Academy of Sciences 1324(1): 7-14 40 NgoThiHongTuoi.2014 "Analysis of Genetic Diversity in Black Rice by SSR Markers." 41 Onwueme, I and T Sinha (1991) Field crop production in tropical Africa: principles and practice, CTA 42 Petroni, K., M Trinei, M Fornari, V Calvenzani, A Marinelli, L Micheli, R Pilu, A Matros, H.-P Mock and C Tonelli (2017) "Dietary cyanidin 3glucoside from purple corn ameliorates doxorubicin-induced cardiotoxicity in mice." Nutrition, metabolism and cardiovascular diseases 27(5): 462-469 43 Plucknett, D L and N J Smith (2014) Gene banks and the world's food, Princeton University Press 44 Portères, R (1956) "Taxonomie Agrobotanique des Riz cultivés O sativa L et O glaberrima Steudel." Journal d'agriculture traditionnelle et de botanique appliquée 3(7): 341-384 45 Quỳnh, Đ T., N T Hảo, V T T Hiền and T V Quang "ĐÁNH GIÁ ĐA DẠNG DI TRUYỀN NGUỒN GEN LÚA NẾP ĐỊA PHƯƠNG DỰA TRÊN KIỂU HÌNH VÀ CHỈ THỊ PHÂN TỬ." 46 Ramarathnam, N., T Osawa, M Namiki and S Kawakishi (1989) "Chemical studies on novel rice hull antioxidants Identification of isovitexin, a C-glycosyl flavonoid." Journal of Agricultural and Food Chemistry 37(2): 316-319 38 47 Rogers, E., S Rice, R Nicolosi, D Carpenter, C McClelland and L Romanczyk Jr (1993) "Identification and quantitation of γ‐oryzanol components and simultaneous assessment of tocols in rice bran oil." Journal of the American Oil Chemists' Society 70(3): 301-307 48 Roy, S C and B Sharma (2014) "Assessment of genetic diversity in rice [Oryza sativa L.] germplasm based on agro-morphology traits and zinc-iron content for crop improvement." Physiology and Molecular Biology of Plants 20(2): 209-224 49 Ryu., S N., S Z Park and C T Ho (1998) " High performance liquid chromatographic determination of anthocyanin pigments in some varieties of black rice." J Food Drug Anal 6(4): 729-736 50 Sampath, S (1951) "Interrelationships between species in genus Oryza." Ind J Genet Plant Breed 11: 14-17 51 Sampath, S (1958) "Wild rices of Orissa and their relationship to the cultivated varieties." Rice News Teller 6(3): 17-20 52 Samyor, D., A B Das and S C Deka (2017) "Pigmented rice a potential source of bioactive compounds: a review." International journal of food science & technology 52(5): 1073-1081 53 Second, G (1982) "Origin of the genic diversity of cultivated rice (Oryza spp.): study of the polymorphism scored at 40 isozyme locil)." Jpn J Genet 57: 25257 54 Smeriglio, A., D Barreca, E Bellocco and D Trombetta (2016) "Chemistry, pharmacology and health benefits of anthocyanins." Phytotherapy Research 30(8): 1265-1286 55 Steyn, W J., S Wand, D Holcroft and G Jacobs (2002) "Anthocyanins in vegetative tissues: a proposed unified function in photoprotection." New Phytologist 155(3): 349-361 56 Strack, D and V Wray (1986) "The anthocyanins." The flavonoids: advances in research since: 1-22 39 57 Tantipaiboonwonga, P., K Pinthaa, W Chaiwangyena, T Chewonarinb, K Pangjitc, O Chumphukama, N Kangwand and M Suttajita (2017) "Antihyperglycaemic and anti-hyperlipidaemic effects of black and red rice in streptozotocin-induced diabetic rats." ScienceAsia 43: 281-288 58 Tsai, P.-J., J McIntosh, P Pearce, B Camden and B R Jordan (2002) "Anthocyanin and antioxidant capacity in Roselle (Hibiscus sabdariffa L.) extract." Food research international 35(4): 351-356 59 Nguyễn, P T M Q and M Thủy "BIỆN PHÁP TIỀN XỬ LÝ VÀ TỐI ƯU HÓA Q TRÌNH TRÍCH LY ANTHOCYANIN TỪ VỎ KHOAI LANG TÍM." 60 Virk, P., H Newbury, M Jackson and B Ford-Lloyd (1995) "The identification of duplicate accessions within a rice germplasm collection using RAPD analysis." Theoretical and Applied Genetics 90(7-8): 10491055 61 Wang, C., X Zheng, Q Xu, X Yuan, L Huang, H Zhou, X Wei and S Ge (2014) "Genetic diversity and classification of Oryza sativa with emphasis on Chinese rice germplasm." Heredity 112(5): 489-496 62 Wang, W., R Mauleon, Z Hu, D Chebotarov, S Tai, Z Wu, M Li, T Zheng, R R Fuentes and F Zhang (2018) "Genomic variation in 3,010 diverse accessions of Asian cultivated rice." Nature 557(7703): 43-49 63 Wrolstad, R E., T E Acree, E A Decker, M H Penner, D S Reid, S J Schwartz, C F Shoemaker, D Smith and P Sporns (2001) "Characterization and Measurement of 64 Anthocyanins by UV-Visible Spectroscopy." Current Protocols in Food Analytical Chemistry 65 Yawadio, R and N Morita (2007) "Color enhancing effect of carboxylic acids on anthocyanins." Food Chemistry 105(1): 421-427 40 66 Yawadio, R., S Tanimori and N Morita (2007) "Identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities." Food Chemistry 101(4): 1616-1625 67 Article Use of Terms - Convention on Biological Diversity 68 Chandramouli B., Latha M M., Narendra K., Mallikarjuna K (2018) Phytochemical and antimicrobial investigations of methanolic extract & ethyl acetate extract of rice husk (Oryza sativa) mentioned in an ancient palm leaf manuscript (Talapatra) World J Pharm Res 41 APPENDIX Appendix the test results of DNA extracted of 30 pigmented rice varieties Appendix Anthocyanin extraction process 42 Appendix table Total anthocyanin content of accessions TAC (mg/100g pH = 510 nm pH = 4.5 700 nm 510 nm Absorbance TAC (g) %TAC sample) 700 nm C1 0.1461 0.0555 0.0918 0.0636 0.0624 0.000104201 0.059886 59.88566 C2 0.1423 0.0288 0.0375 0.0098 0.0858 0.000143276 0.082343 82.34278 C3 0.0347 0.0318 0.0345 0.0231 -0.0085 -1.41941E-05 -0.00816 -8.1575 C4 0.0595 0.042 0.0284 0.0184 0.0075 1.25242E-05 0.007198 7.197795 C5 0.0354 0.0241 0.0161 0.0057 0.0009 1.5029E-06 0.000864 0.863735 C6 0.1742 0.0633 0.0888 0.0741 0.0962 0.000160643 0.092324 92.32372 C8 0.086 0.0437 0.0515 0.0405 0.0313 5.22675E-05 0.030039 30.0388 C9 0.0433 0.0319 0.021 0.0103 0.0007 1.16892E-06 0.000672 0.671794 C10 0.0634 0.0413 0.0301 0.0162 0.0082 1.36931E-05 0.00787 7.869589 C11 0.0684 0.0559 0.0674 0.0662 0.0113 1.88697E-05 0.010845 10.84468 C12 0.1509 0.0582 0.1027 0.0688 0.0588 9.81894E-05 0.056431 56.43071 C14 0.0859 0.0362 0.0869 0.069 0.0318 5.31025E-05 0.030519 30.51865 C15 0.0491 0.0382 0.0713 0.0629 0.0025 4.17472E-06 0.002399 2.399265 C22 0.1868 0.0682 0.0594 0.037 0.0962 0.000160643 0.092324 92.32372 C27 0.0898 0.06 0.0811 0.0727 0.0214 3.57356E-05 0.020538 20.53771 C34 0.0835 0.0039 0.0783 0.061 0.0623 0.000104034 0.05979 59.78969 C35 0.0165 0.0067 0.0298 0.022 0.002 3.33978E-06 0.001919 1.919412 C36 0.0373 0.0227 0.0341 0.0209 0.0014 2.33784E-06 0.001344 1.343588 C37 0.1128 0.053 0.0682 0.0589 0.0505 8.43294E-05 0.048465 48.46515 C41 0.1153 0.0238 0.042 0.021 0.0705 0.000117727 0.067659 67.65927 C42 0.0875 0.0596 0.0734 0.0616 0.0161 2.68852E-05 0.015451 15.45127 C47 0.0831 0.0184 0.0384 0.0202 0.0465 7.76498E-05 0.044626 44.62633 C48 0.0504 0.0025 0.0343 0.0151 0.0287 4.79258E-05 0.027544 27.54356 C49 0.1304 0.0434 0.0569 0.0361 0.0662 0.000110547 0.063533 63.53254 C50 0.0507 0.0021 0.034 0.0137 0.0283 4.72578E-05 0.02716 27.15968 C51 0.1434 0.0516 0.0945 0.0722 0.0695 0.000116057 0.0667 66.69957 C52 0.1235 0.0491 0.057 0.0748 0.0922 0.000153964 0.088485 88.4849 C53 0.2054 0.1213 0.1308 0.093 0.0463 7.73158E-05 0.044434 44.43439 C54 0.0435 0.0301 0.0163 0.0098 0.0069 1.15222E-05 0.006622 6.621972 C55 0.1936 0.1048 0.0241 0.0067 0.0714 0.00011923 0.068523 68.52301 43