The assessment of the fragrant trait in a collection of vietnamese rice landrace varieties

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - GRADUATION THESIS “THE ASSESSMENT OF THE FRAGRANT TRAIT IN A COLLECTION OF VIETNAMESE RICE LANDRACE VARIETIES” Hanoi - 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - GRADUATION THESIS “THE ASSESSMENT OF THE FRAGRANT TRAIT IN A COLLECTION OF VIETNAMESE RICE LANDRACE VARIETIES” Student’s name : Vu Manh An Class : K61CNSHE Faculty : Biotechnology Supervisor : Dr Hoang Thi Giang Dr Nguyen Thi Lam Hai Hanoi - 2021 STATEMENT OF ORIGINAL AUTHORSHIP The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made Hanoi, February 23rd, 2021 Student Vu Manh An i ACKNOWLEDGMENTS This thesis, like any other, would not have been possible without the involvement and support of many people They have all earned my deepest gratitude, even if it has been poorly expressed at times First and foremost, my sincere thank gives my academy supervisor, Dr Hoang Thi Giang, for her enthusiastic support and useful suggestions for my graduation thesis Secondly, I would like to express my thankfulness to Dr Nguyen Thi Lam Hai for guiding me to carry out my tasks in the Faculty of Biotechnology at Vietnam National University of Agriculture Without them, this project would not have been possible Next, I would like to thank to all staff at International joint laboratory on Functional Genomics & Biotechnology for Plants and Associated Microorganisms (LMI RICE-2), Agricultural Genetics Institute who have stood by me for a long period of time for completing my graduation thesis and shared me a number of worthy working experiences They are all friendly and helpful to me, I was lucky to study in a great working environment Last but not least, I want to thank my family and friends for all their help, encouragement and supporting me over the past years Hanoi, February 23rd, 2021 Student Vu Manh An ii CONTENTS STATEMENT OF ORIGINAL AUTHORSHIP .i ACKNOWLEDGMENTS ii CONTENTS iii THE LIST OF TABLES v THE LIST OF FIGURES .vi THE LIST OF ABBREVIATIONS vii ABSTRACT viii PART I INTRODUCTION 1.1 Preface 1.2 Objectives 1.3 Requirements PART II LITERATURE REVIEW 2.1 Rice grain quality .3 2.1.1 Milling quality .3 2.1.2 Appearance quality 2.1.3 Cooking and eating quality 2.1.4 Nutritional quality 2.2 The fragrant trait in rice 2.2.1 Fragrance in rice 2.2.2 Origin and diversity of fragrant rice varieties .8 2.2.3 Genetic basis of fragrance on rice .11 2.3 Molecular breeding of fragrant rice 13 2.3.1 Marker-assisted selection 13 2.3.2 Breeding of the fragrant rice in the world and in Vietnam 14 PART III MATERIALS AND METHODS 16 3.1 Materials 16 3.1.1 Plant materials .16 3.1.2 PCR-based molecular markers 16 3.2 Research contents 17 3.3 Location and time 17 iii 3.4 Research methodology and techniques 17 3.4.1 The sensory assessment method of fragrance .17 3.4.3 Identification of mutation types of Badh2 gene associated with fragrance by using functional markers 18 PART IV RESULTS AND DISCUSSION 20 4.1 Evaluation of fragrance for the collection of Vietnam rice landrace varieties .20 4.2 Identification of mutation types of Badh2 gene associated with fragrance in the collection of Vietnamese rice landrace varieties by using functional markers .24 4.2.1 Total genomic DNA extraction 24 4.2.2 Marker badh2-E7 25 4.2.4 Marker badh2-E2 33 4.2.3 Marker FMbadh2-E4-5 34 4.3 The selection of promising high-quality fragrant local rice varieties for rice breeding program 36 PART V CONCLUSION AND PROPOSALS 39 5.1 Conclusion 39 5.2 Proposals 40 PART V REFERENCES 41 APPENDIX 47 iv THE LIST OF TABLES Table 2.1 Some common fragrant rice cultivars of the world (Pachaurl et al., 2010) Table 3.1 List of the molecular markers used in this study 17 Table 3.2 The criteria for assessing fragrance (IRRI, 2013) 18 Table 4.1 Fragrance score of the Vietnamese rice landraces collection 21 Table 4.2 The statistic result of the fragrant test by the sensory evaluation 24 Table 4.3 PCR results for checking mutations in Badh2 gene .26 Table 4.4 List of promising varieties for rice breeding program 38 v THE LIST OF FIGURES Figure 2.1 Images of rice kernels milled with the three indicated laboratory mills showing the effect of each machine on the shape and size of milled rice kernels (Bautista & Siebenmorgen, 2002) Figure 2.2 Genetic diversity of appearance quality Figure 2.3 Major fragrant rice growing regions in Asia .9 Figure 2.4 Structure of fragrance gene and the three functional sites in rice .13 Figure 3.1 Field experiment to obtain new grain source 16 Figure 4.1 Preparation of samples for sensory evaluation of fragrance 20 Figure 4.2 Electrophoresis of extracted total genomic DNAs 25 Figure 4.3 PCR banding patterns for functional marker badh2-E7 26 Figure 4.4 PCR banding patterns for functional marker badh2-E2 34 Figure 4.5 PCR banding patterns for functional marker FMbadh2-E4-5 35 vi THE LIST OF ABBREVIATIONS Abbreviations Meaning 2AP 2-acetyl-1-pyrroline AAS Atomic absorption spectroscopy BADH2 Betaine aldehyde dehydrogenases BRR Brown rice recovery EAP External Antisense Primer ESP External Sense Primer HRR Head rice recovery ICP-MS Inductively coupled plasma-mass spectrometry ICP-OES Inductively coupled plasma-optical emission spectrometry IFAP Internal Fragrant Antisense Primer INSP Internal Non-fragrant Sense Primer InDels Insertion / Deletions LIBS Laser-induced breakdown spectroscopy MAS The molecular-associated markers MRR Milled rice recovery RNAi RNA interference XRF X-ray fluorescence spectrometry vii ABSTRACT Fragrance is the immensely important criterion in the assessment of rice quality, is known to be controlled by loss-function of the Badh2 gene In this study, a collection of 182 Vietnamese rice landrace varieties was screened for fragrance by sensory test and genotyped for three major mutation types of Badh2 gene using functional markers A total of 18 fragrant varieties were identified, and 13 of them were confirmed by the presence of 8-bp deletion mutation in exon and/or 803-bp deletion mutation between exons and Two detected mutation types were present in some non-fragrant rice varieties, and absent in some fragrant rice varieties No any rice varieties were determined to possess 7-bp deletion mutation in exon The results suggest that these 13 local rice landrace varieties could be potentially provided as genetic resources in breeding for fragrant rice varieties or could be reintroduced in production viii Table 4.4.List of promising varieties for rice breeding program No ID Name of varieties Location Rice group G108 Tám ấp bẹ Ninh Binh Regular rice Slightly fragrant 8-bp deletion in exon G132 Padai tlig jug Khanh Hoa Glutinous rice Slightly fragrant 8-bp deletion in exon G154 Nếp thơm Ha Tay Glutinous rice Slightly fragrant 8-bp deletion in exon and 803-bp deletion between exons and G161 Bn An Giang Regular rice Slightly fragrant 8-bp deletion in exon G177 Chăm hơm Hoa Binh Regular rice Fragrant 8-bp deletion in exon G178 Kháu phủ Hoa Binh Glutinous rice Fragrant 8-bp deletion in exon 7 G200 Chà fu nu Lai Chau Glutinous rice Slightly fragrant 8-bp deletion in exon G214 Blè blậu Lao Cai Glutinous rice Slightly fragrant 8-bp deletion in exon G24 Tám xoan hải hậu Nam Dinh Regular rice Slightly fragrant 8-bp deletion in exon 10 G25 Nếp vàng ong lạc sơn HB Hoa Binh Glutinous rice Fragrant 803-bp deletion between exons and 11 G50 Lúa nếp tháng dạng Quang Nam Glutinous rice Slightly fragrant 8-bp deletion in exon 12 G61 Nếp rằn Quang Binh Glutinous rice Slightly fragrant 803-bp deletion between exons and 13 G86 Tan ngần Yen Bai Glutinous rice Slightly fragrant 803-bp deletion between exons and 38 Level of fragrance Mutation types of Badh2 gene PART V CONCLUSION AND PROPOSALS 5.1 Conclusion - Among 181 local Vietnamese rice varieties evaluated by sensory method, were categorized as fragrant, 14 as slightly fragrant, the rest of 163 varieties as nonfragrant - Two mutation types of Badh2 gene responsible for fragrance in rice, 8-bp deletion in exon and 803-bp deletion between exons and 5, were determined in 13 fragrant varieties, including G108, G132, G154, G161, G177, G178, G200, G214, G24, G25, G50, G61 and G86 No any rice varieties were observed to possess 7-bp deletion mutation in exon Two detected mutation types were present in some nonfragrant rice varieties, and absent in some fragrant rice varieties, it is thus interesting to explore the genetic basis of fragrance in Vietnamese rice landrace varieties - Determined 13 promising fragrant varieties which have 8-bp deletion mutation in exon and/or 803-bp deletion mutation between exons and 5, including G108, G132, G154, G161, G177, G178, G200, G214, G24, G25, G50, G61 and G86 These selected varieties could be potentially introduced as genetic resources for fragrant rice breeding programs or could be reintroduced in production 39 5.2 Proposals - A requirement for LMI RICE-2 team is continuing to examine the badh2-E2 allele for all the left varieties in the study rice collection by the functional marker badh2-E2 From the final data, the whole of the collection should be assessed genetic diversity and then, additionally choose promising varieties for breeding program - The determination and discovery of other mutations related to this fragrant trait in the studied rice collection also should be conducted to provide with information source for next studies - The promising fragrant rice varieties should be used in breeding program or developed for production 40 PART V REFERENCES Vietnamese references Duong Xuan Tu, Nguyen Van Khoi, Le Thi Thanh, Nguyen Thi Huong, Nguyen The Duong, Tran Thi Dieu and Phan Huu Ton (2014) Molecular Marker for using to detect 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gié Tám tròn hải dương Rằn trắng Nếp rẫy Nàng thiệt Koi lòi Khảo sang L26 Tám cao vĩnh phúc Bảy thánh Cá rơ Nếp đen Nếp nương Khâu đắm địi Khâu đăm Lc 93-2 Lúa da bò Padai long khánh Padai tlig jug A 330 Padai calòc Phước long Nàng quất 47 Location Hai Phong Nam Dinh Tuyen Quang Tay Nguyen Thua Thien Hue Thua Thien Hue Thua Thien Hue Ha Giang Ninh Binh Ninh Binh Ninh Binh Quang Binh Hai Duong Binh Thuan Binh Thuan Vung Tau na Quang Tri na Vinh Phuc Ca Mau Tay Ninh Quang Ninh Quang Ninh Nghe An Nghe An Khanh Hoa Khanh Hoa Khanh Hoa Khanh Hoa Khanh Hoa Khanh Hoa Khanh Hoa Ben Tre Group I I J J I J I I J J m I I I I I I J I I I I J I J J I J J I I J I I No 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 ID G139 G14 G140 G141 G142 G143 G144 G145 G146 G147 G150 G152 G153 G154 G155 G156 G157 G158 G16 G160 G161 G162 G163 G165 G166 G167 G168 G169 G17 G170 G171 G172 G173 G177 G178 G179 Name of varieties Lúa nàng đen Tám nhỡ bắc ninh Lúa bẩy đảnh Lúa nàng niếu chùm Lúa trà Nếp trời cho Lúa mùa địa phương Bà rịa Nàng loan hạt tròn Lúa loan hạt dài Nếp địa phương Lốc sớm Tẻ nương Nếp thơm Khẩu pe lạnh Lúa k Sợ crioong Va tai ana acu Nếp vằn ruộng hịa bình Jasmine Bn Neang Cà choch chấp Giống 90 ngày Chín tèo Thần nơng mùa Om 1490 Jasmine 95 Nếp gà gáy hải dương Om 504 japan Nếp thái Vnd 95-20 Tám thơm trung quốc Chăm hơm Kháu phủ Blao pu lau 48 Location Ben Tre Bac Ninh Ben Tre Ben Tre Ben Tre Ben Tre Ben Tre Ben Tre Ben Tre Ben Tre Ben Tre Bac Giang Thanh Hoa Ha Tay Son La na Sekong Ninh Thuan Hoa Binh An Giang An Giang An Giang An Giang Kien Giang Kien Giang Kien Giang Kien Giang Kien Giang Hai Duong Kien Giang Kien Giang Kien Giang Kien Giang Hoa Binh Hoa Binh Hoa Binh Group I I I I I I I J I I I J I J I I J J J I I I I I I I I I I I I I I J J J No 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 ID G18 G180 G181 G182 G183 G186 G187 G189 G19 G190 G191 G192 G193 G194 G195 G196 G2 G20 G200 G201 G202 G203 G204 G205 G206 G207 G208 G209 G21 G210 G211 G212 G214 G216 G217 G219 Name of varieties Nếp quýt hải dương Cà đung hạt Blau plan pieng Khẩu mổ Khẩu pe lạnh Khẩu nỏ Khẩu đường phưởng Khẩu năm rinh Ỏn Plề phmả chua Khẩu tan Khẩu bao thai Blề pê xá Blề blậu lia Blề bdề Blề có pịn Tà lào cai Tẻ lề hịa bình Chà fu nu Chà xư phu lu Nống to Plàu cà bành Plê đờ Blề blẩu Blê blẩu Khẩu lứa Khẩu boong lăm Blề Gié trắng hịa bình Khẩu lếch Plầu ngồng plặc Plầu bulặt Blè blậu Tồm bèo bua Blè blậu soa Khẩu la lạnh 49 Location Hai Duong na Son La Son La Son La Son La Son La Dien Bien na Dien Bien Dien Bien Dien Bien Dien Bien Dien Bien Dien Bien Dien Bien Lao Cai Hoa Binh Lai Chau Lai Chau Lai Chau Dien Bien Dien Bien Son La Son La Son La Son La Lai Chau Hoa Binh Lao Cai Lao Cai Lao Cai Lao Cai Lao Cai Lao Cai Son La Group I I I I I I J I I I J I J J J m I I J I J J J I J m I I I J I J J J J I No 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 ID G22 G220 G221 G222 G223 G24 G25 G26 G299 G3 G30 G300 G31 G32 G35 G36 G37 G38 G39 G4 G40 G41 G42 G43 G45 G46 G47 G48 G49 G5 G50 G51 G52 G53 G54 G56 Name of varieties Trứng trắng tuyên quang Plề la Khẩu mắc có Plề mà mủ Blè blậu tan Tám xoan hải hậu Nếp vàng ong lạc sơn HB Khẩu cai noi Blao sinh sái An tu đỏ vỏ Tiêu chệt Nàng quớt biển Nàng chi Nàng đùm Sanaki Nàng tây Nếp cẩm Nếp nương Nếp cẩm Nhông đỏ hải dương Nếp đo Lúa đỏ Lúa hịn cơi Thành tua Nếp cúc Nếp bà lão Nếp ông lão Lúa ngoi DT10 Nhông trắng hải phòng Lúa nếp tháng dạng Ba trăng hướng Ba trăng Lúa can đỏ Lúa lốc đỏ Lúa mặn 50 Location na na na na na Nam Dinh Hoa Binh Tay Bac Hoa Binh na Can Tho Bac Lieu Can Tho Can Tho Can Tho Can Tho Ha Giang Ha Giang Ha Giang Hai Duong Kien Giang Kien Giang Kien Giang Kien Giang Ninh Binh Nam Dinh Nam Dinh Nam Dinh na Hai Phong Quang Nam Quang Nam Quang Nam na Quang Nam Quang Nam Group I J J J J m J J J I I I I I I I I J I I I I I I J J J J I I J I I I I I No 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 ID G57 G58 G59 G6 G61 G62 G63 G64 G65 G67 G68 G69 G7 G70 G72 G73 G74 G77 G78 G79 G8 G80 G81 G83 G84 G85 G86 G87 G88 G89 G9 G90 G91 G92 G93 G94 Name of varieties Nếp ghim hương Nếp hương lăng Nếp mậm Sớm giai hưng yên Nếp rằn Quảng trắng Chiêm đỏ Ven đỏ Nước mặn dạng Lúa trì đỏ dạng Nếp tháng Cốc dạng Tẻ trắng hịa bình Cốc dạng Lúa cang dạng Lúa cang dạng Nếp quạ có râu dạng Cang kiến dạng Cang kiến dạng Lúa đá dạng Chọn từ 502 học viện Ba ktong Ba đỏ dạng Nếp vàng Ba chơ k'tê Chành trụi Tan ngần Khẩu pan pua Ble mạ mùa Khẩu bò Lốc trắng sớm plei cầu Blào clía Blào cố kén Blào cô cẩm Pờ lề pờ lẩu xá Lúa đỏ 51 Location Quang Nam Quang Nam Quang Nam na Quang Binh Quang Tri Quang Tri Quang Tri Quang Tri Binh Dinh Binh Dinh Binh Dinh Hoa Binh Binh Dinh Binh Dinh Binh Dinh Binh Dinh Binh Dinh Binh Dinh Binh Dinh na Quang Ngai Quang Ngai Quang Ngai Binh Dinh Thanh Hoa Yen Bai na na na na Hoa Binh Hoa Binh Hoa Binh Nghe An Thua Thien Hue Group I I I I J I I I I I m I I I I I I I I I I J m J J J J J J J I J J J I I No 179 180 181 182 183 184 ID G95 G96 G98 G99 BT7 KD18 Name of varieties Lúa chăm Chiêm rong Ngoi tía Lúa chăm biển Bắc Thơm Khang Dân 18 I: indica; J: japonica; M: admix 52 Location Nam Dinh Nam Dinh Nam Dinh Ninh Binh Commercial Commercial Group I I J I Control Control