VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UDERGRADUATE THESIS TITTLE: CHARACTERIZATION OF LOCAL PIGMENTED RICE GENETIC RESOURCES IN VIETNAM Student : DO THI MINH NGUYET Faculty : BIOTECHNOLOGY Supervisors : LE DUC THAO, Phd NGUYEN QUOC TRUNG, Msc Class : K61CNSHE Student ID : 610654 Ha Noi - 2021 COMMITMENTS I guarantee that 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, 30th January , 2021 Student Do Thi Minh Nguyet i ACKNOWLEDGEMENTS Firstly, I would like to express my appreciation to the directory of Vietnam National University of Agriculture, board of deans and lectures in faculty of biotechnology that created the best condition for me to perform and complete my thesis Secondly, I’m deeply indebted to my supervisor, MSc Nguyen Quoc Trung Who guided me to conduct experiments, advised me on professional knowledge His support pushed me to sharpen my thinking and patience to accomplish the thesis I would also like to thank guides from Crop Research and Development Institute who helped me building the field layout, shared useful experience in rice cultivation and the way to measure agronomic characteristics I gratefully acknowledge my colleagues in laboratory for their wonderful assistance Thank to them I can complete my research and they made me fun and optimistic Last but not least, I would like to thank my parents for encouragement in study as well as in life I have never finished my dissertation without their love and their support Hanoi, 30th January , 2021 Student Do Thi Minh Nguyet ii CONTENTS COMMITMENTS i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii Summary viii CHAPTER INTRODUCTION 1.1 Introduction 1.2 Objectives 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 12 2.2.3 The studies about genetic diversity of rice 17 CHAPTER MATERIALS AND METHODS 20 3.1 Materials 20 3.2 Field layout 21 3.2.1 Experimental layout 21 3.2.2 Cultivation techniques 22 3.3 Methods 23 3.3.1 Agronomical characterization 23 iii 3.3.2 Identification of four genes: kala1, kala3, kala4 and rc 24 3.3.3 Data analysis 26 CHAPTER RESULTS AND DISCUSSIONS 27 4.1 Agronomical characteristics and yield 27 4.1.1 Agronomical characteristics 27 4.1.2 Grain yield, yield components and yield actual 30 4.2 Identification of four genes: kala1, kala3, kala4 and rc 32 4.2.1 The result of genomic DNA extraction 32 4.2.2 Identification of four genes: kala1, kala3, kala4 and rc 33 CHAPTER CONCLUSIONS AND SUGGESTIONS 39 5.1 Conclusions 39 5.2 Suggestions 39 REFERENCES 40 APPENDIX 44 iv LIST OF TABLES Table The number of pigmented rice accessions collected Table 2 Anthocyanin content of some kind of food 15 Table List of 30 colored rice accessions 20 Table Four new SSR markers for genes controlling pigment of pericarp 26 Table 3 The compositions of PCR 26 Table Morphological characteristics of the 30 accessions 27 Table Grain yield, yield components and yield actual of the 30 local pigmented rice accessions 31 Table Classification of color of rice pericarp 36 Table 4 Identification and characterization of pigment pericarp of 30 accessions 37 v LIST OF FIGURES Figure Phylogenetic tree of the genus Oryza (Wing et al., 2005) Figure 2 White, red, brown and black rice Figure Metabolic pathways for the synthesis of proanthocyanidins and anthocyanins (ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; DFR, dihydroflavonol‐4‐reductase; F3H, flavanone‐3‐hydroxylase; GT, anthocyanidin glucosyltransferase; LAR, leucoanthocyanidin reductase) (Xie and Dixon, 2005) 10 Figure Chemical structures of several anthocyanins in pigmented rice (Deng et al., 2013) 13 Figure 5.Predominant structural forms of anthocyanins present at different pH levels (Wrolstad et al., 2001) 14 Figure Field layout 22 Figure Days to heading (more than 50% emergence of all panicles) 28 Figure Plant height of 30 accessions 29 Figure The yield of 30 pigmented rice accessions 32 Figure 4 Genomic DNA extracted of 30 accessions 32 Figure The product of primer RM7405 33 Figure The product of primer RM3400 33 Figure 7.The product of primer RM7210 34 Figure 8.The product of qPC7 34 Figure Color of pericarp of 30 accessions 35 vi LIST OF ABBREVIATIONS Abbreviation Meaning bHLH Basic-helix-loop-helix CBD Convention on Biological Diversity SSRs Simple sequence repeats IRGSP International Rice Genome Sequencing Project IRRI International Rice Research Institute SNP Single nucleotide polymorphism ISSR Inter-simple sequence repeat RFLP Restriction fragment length polymorphism QTL quantitative trait loci vii Summary Recently, ‘colored rices’ ternminology is used to refer to three types of rice: Purple Rice, Black Rice, Red Rice that are the seed of the glass species Oryza sativa L Black and purple pericarps are the result of accumulation of anthocyanin, while red pericarps are due to proanthocyanidins (Gunaratne et al., 2013; Samyor et al., 2017) These natural products are well known for their beneficial effects on health: anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme function (Panche et al., 2016) In this study, we focused characterization of agronomical characters, genes controlling pigment in pericarp: kala1, kala3, kala4 and Rc genes of 30 accessions that were collected from different locations in Vietnam According to the methods of IRRI (2013), the results showed that the pigmented rice accessions varied in heading time (62 to 101 days), plant height (85.6 to 153.6 cm) The grain yield of 30 accessions reached from 1.3 to 5.9 t/ha whereas the yield actual got from 0.7 to 2.5 t/ha Four genes controlling pigment in pericarp kala1, kala3, kala4 and Rc were identified by SSR markers (Gu et al., 2011; McCouch et al., 2002) This study contributes to the efficient exploitability and cultivation of pigmented rice resources Key words: pigmented rice, kala, Rc, yield viii CHAPTER INTRODUCTION 1.1 Introduction Rice (Oryza sativa L.) is one of the most important food crops worldwide, being used as the main food source by more than half of the global population (Mahender et al., 2016) In the developing world, rice provides 27% of dietary energy and 20% of dietary protein For Vietnamese people, ordinary, white rice is main dish and present in all three meal of day White rice has been domesticated and preferred since ancient times, so very geneticists find ways to select white rice varieties Recently, ‘colored rices’ ternminology is used to refer to three types of rice: Purple Rice, Black Rice, Red Rice that are the seed of the glass species Oryza sativa L Black and purple pericarps are the result of accumulation of anthocyanin, while red pericarps are due to proanthocyanidins (Gunaratne et al., 2013; Samyor et al., 2017) Anthocyanins and proanthocyanidins belonging to plant flavonoids, contribute to the intense color of many fruits, vegetables, and pigmented cereals These natural products are well known for their beneficial effects on health: anti-oxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme function (Panche et al., 2016) The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp (Furukawa et al., 2007) Interaction of Rc (on chromosome 7), which encodes a basic helix-loop-helix (bHLH) transcription factor, and Rd (chromosome 1) encoding a form of dihydroflavonol 4-reductase, an enzyme which enhances the accumulation of proanthocyanidin (Mbanjo et al., 2020) The purple pericarp color in rice was controlled by two dominant complementary genes, Pb and Pp (Rahman et al., 2013) A whole genome survey of the introgression line using DNA markers suggested that three Figure 7.The product of primer RM7210 The primer RM7210 is a marker on chromosome and produces predicted product with size 158 bp In figure 4.7, there are bands from well to well 16 including a band with size about 250 bp and a band with size about 158 bp, except well 12,13,14 had blurred band with size about 300 bp At 17, 28, 30 positions, the band wasn’t present The positions from well 19 to well 22 have many nonspecific bands The positions 18, 20, 24, 25, 26, 29 appeared bands with size 250 bp The positions 18, 20, 24, 27 had 300 bp fragments In position 19, 21, 22 , there were bands with size about 280 bp Figure 8.The product of qPC7 Primer qPC7 is a marker for detection of Rc gene on chromosome The product size is produced primer qPC7 is 431 bp The figure 4.8 shows the accessions form C1 to C37 have no band The bands present are product of C41, C42, C47, C48, C49, C50, C51, C52, C53, C54, C55 The product size of the accessions C41, C42, C47, C48, C49, C50, C51, C52, C53, C54 is approximately from 431 bp to 500 bp of length size The bands weren’t in a straight line 34 Figure Color of pericarp of 30 accessions 35 We classified into color groups according to the table below: Table Classification of color of rice pericarp Color Group Codes A C1, C2, C6, C12, C14, C22, Black C27, C37,C51, C55 Black and Black is more than brown B1 brown C8, C10, C15, C34, C47, C48, C49, C50, C52, C53 Brown is more than black B2 C4, C5, C9, C11 C C54 Red phenotypes Red , green D C3 phenotypes Brown, green, red E C35, C36 phenotypes Brown, F C41, C42 black, white, green To better understand the genes involved in pigmented pericarp expression, we raised a table reviewing pigmented pericarp group and genes kala1, kala3, kala4 and Rc of 30 accessions 36 Table 4 Identification and characterization of pigment pericarp of 30 accessions Codes Kala1 Kala3 Kala4 Rc Pigmented pericarp group C1 + + + - A C2 + + + - A C3 + + + - D C4 + + + - B2 C5 + + + - B2 C6 + + + - A C8 + + + - B1 C9 + + + - B2 C10 + + + - B1 C11 + + + - B2 C12 + + + - A C14 + + + - A C15 + + + - B1 C22 + + + - A C27 + + + - A C34 + + + - B1 C35 + + - - E C36 + + + - E C37 + + + - A C41 + + + + F C42 + + + + F C47 + + + + B1 C48 + + - + B1 C49 + + + + B1 C50 + + + + B1 C51 + + - + A C52 + + + + B1 C53 + + - + B1 C54 + + + + C C55 + + - - A Note: ‘+’ means carrying gene, ‘-’ means no carrying gene 37 The table 4.4 shows that most accessions contained gen kala1, kala3, kala4, as for Rc gene, there were only 10 accessions got band so we need to check the accessions with no band again Maeda et al., 2014 indicated that carriers of the three loci Kala1 (chromosome 1), Kala3 (chromosome 3), and Kala4 (chromosome 4) express a black pericarp trait As this demonstration, the accessions in group A carried gen kala1, kala3, kala4 except C51 and C55 lacking Kala4 Since, we also need to look over again All seven pigmented pericarp groups had genes kala1, kala3, kala4 From the results in table 4.6, Rc gene presented in the whole of group C (red rice) and group B (brown rice) even group A (black rice) However, we have to redefine Rc gene on the accessions didn’t have band 38 CHAPTER CONCLUSIONS AND SUGGESTIONS 5.1 Conclusions In this study, these agronomic characters of 30 accessions were different Most accessions belonged to the tall and intermediate plant group Time of heading ranged from 62 to 101 days but C22, C34, C35 and C36 were undefined because of root poisoning The grain yield of 30 accessions reached from 1.3 to 5.9 t/ha whereas the yield actual got from 0.7 to 2.5 t/ha The yield low was caused by the weather as well as environmental conditions The genes kala1, kala3, kala4 and Rc were also identified that influence on pigmented pericarp Almost all 30 accessions contained genes kala1, kala3, kala4 For Rc gene, we need to reassess the accessions with no band to get accurate results In present study, 10 accessions carried Rc gene were red rice, brown rice and black rice 5.2 Suggestions Vietnam is one of the Asian countries with very diverse rice genetic resources However, there are few researches on local pigmented rice resources, so finding information concerned has been difficult The Vietnamese scientists need to more study and explore pigmented rice resources The researches on nutrition in pigmented rice are necessary to develop functional food In this study, we have to redefine Rc gene in accesstions no band We can also use more markers to identify genes influencing pigment of pericarp rice Using more marker on loci helps to learn more the interaction of alleles at loci to understand expression of pigment pericarp 39 REFERENCES Chang, T.-T., A, E., and Bardenas (1965) The morphology and varietal characteristics of the rice plant Technical Bulletin Cheng, A 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Phytochemistry 66, 2127-44 26 Xu, W., Dubos, C., and Lepiniec, L (2015) Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes Trends Plant Sci 20, 176-85 43 APPENDIX Appendix The heading time of accessions Appendix Harvest the accessions 44 Appendix Genomic DNA extracted of 30 accessions Appendix Multi-beads shocker Appendix Running gel electrophoresis 45 Appendix table Yield components and grain yield of accessions Code Number Number of of grains Percentage grain yield panicles number per of filled per hill panicle/m² panicle grains (%) P1000 (g) (t/ha) C1 6.2 155 148.17 79.23 21.67 3.943125 C2 7.4 185 131.47 66.61 22.67 3.672733 C3 6.8 170 157.44 86.22 20 4.615322 C4 10.4 260 130.67 74.66 21 5.326679 C5 200 133 64.68 20.67 3.556249 C6 7.2 180 115.67 43.19 20.33 1.828158 C8 6.2 155 135.57 68.39 23.67 3.401623 C9 7.4 185 173.25 68.62 19.33 4.251357 C10 5.2 130 118 59.8 22.67 2.079592 C11 8.4 210 149 54.52 27.67 4.720311 C12 5.6 140 146.33 75.31 27 4.165602 C14 7.2 180 92.27 44.77 27 2.007631 C15 9.6 240 133.78 60.47 30.33 5.888637 C22 4.6 115 132 51.98 29 2.288264 C27 175 111.33 66.01 26.33 3.386186 C34 125 111.44 33.5 27 1.259969 C35 4.4 110 101.67 46.88 33.67 1.765291 C36 4.2 105 116.17 35.55 35 1.517717 C37 8.4 210 90.33 56.24 24.67 2.631878 C41 5.8 145 144.87 50.78 33.33 3.555285 C42 7.8 195 148.53 71.62 25.33 5.254342 C47 5.6 140 195.83 42.03 28.67 3.303652 C48 6.2 155 185.7 41.13 31.33 3.70905 C49 7.4 185 218.27 45.34 30.67 5.615146 C50 125 168.2 62.69 30.33 3.997668 C51 4.8 120 192.8 60.5 28.67 4.01302 C52 3.2 80 162.58 44.34 29.67 1.71108 C53 3.25 81.25 160.33 49.78 29.33 1.901976 C54 5.8 145 115.47 75.77 19.67 2.495392 C55 150 138.67 68.02 29.33 4.149755 46 Appendix table yield actual of accessions yield Code plant weight actual number (kg) (t/ha) C1 75 0.45 1.5 C2 112 0.4 0.892857 C3 75 0.76 2.533333 C4 34 0.505 3.713235 C5 64 0.575 2.246094 C6 25 0.03 0.3 C8 45 0.41 2.277778 C9 68 0.59 2.169118 C10 73 0.65 2.226027 C11 56 0.46 2.053571 C12 25 0.38 3.8 C14 45 0.27 1.5 C15 11 0.11 2.5 C22 25 0.07 0.7 C27 42 0.51 3.035714 C34 25 0.07 0.7 C35 25 0.04 0.4 C36 25 0.13 1.3 C37 25 0.12 1.2 C41 51 0.125 0.612745 C42 77 0.24 0.779221 C47 61 0.41 1.680328 C48 30 0.43 3.583333 C49 40 0.42 2.625 C50 88 0.775 2.201705 C51 46 0.425 2.309783 C52 53 0.245 1.15566 C53 50 0.38 1.9 C54 52 0.44 2.115385 C55 34 0.395 2.904412 47 Appendix table The time of heading code date of sowing date of heading time of heading (days) C1 2-Jul 18-Sep 76 C2 2-Jul 18-Sep 76 C3 2-Jul 8-Sep 66 C4 2-Jul 10-Sep 68 C5 2-Jul 7-Sep 65 C6 2-Jul 7-Oct 95 C8 2-Jul 4-Sep 62 C9 2-Jul 12-Sep 70 C10 2-Jul 5-Sep 63 C11 2-Jul 30-Aug 58 C12 2-Jul 19-Sep 77 C14 23-Jun 30-Sep 97 C15 23-Jun 29-Sep 96 2-Jul 10-Sep 68 C37 2-Jul 2-Oct 90 C41 2-Jul 18-Sep 76 C42 2-Jul 18-Sep 76 C47 23-Jun 15-Sep 82 C48 23-Jun 12-Sep 79 C49 23-Jun 15-Sep 82 C50 23-Jun 2-Sep 69 C51 2-Jul 12-Sep 70 C52 2-Jul 16-Sep 74 C53 2-Jul 16-Sep 74 C54 2-Jul 7-Sep 65 C55 2-Jul 5-Sep 63 C22 C27 C34 C35 C36 48