Assessment of some black rice varieties for drought tolerance

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATION THESIS TITLE: ASSESSMENT OF SOME BLACK RICE VARIETIES FOR DROUGHT TOLERANCE HANOI, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY � � � UNDERGRADUATION THESIS TITLE: ASSESSMENT OF SOME BLACK RICE VARIETIES FOR DROUGHT TOLERANCE Student name : Nguyen Thi Khanh Huyen Student code : 620499 Class : K62CNSHE Faculty : Biotechnology Supervisor : Phan Huu Ton, Prof Ph.D HANOI, 2022 COMMITMENT This thesis is composed of my original works, and contains no material previously published or written by another person Hanoi, 20th March, 2022 Student Nguyen Thi Khanh Huyen i ACKNOWLEDGEMNETS First of all, with the deepest and most sincere affection, allow me to express my gratitude to all the individuals and organizations that have facilitated and supported me throughout the process of studying and researching this thesis In order to successfully complete the undergraduation thesis, in addition to my own efforts, I also received a lot of attention and support from teachers, friends as well as family members Second, I would like to express my deep gratitude to Prof Ph.D Phan Huu Ton, who enthusiastically guided and taught me valuable professional knowledge and lessons during the process of completing my undergraduation thesis Besides, I would also like to thank the masters and engineers who are working and working at the Center for Conservation and Development of Crop Genetic Resources, for helping and creating the most favorable conditions for me in the process internship at the center Third, I would like to express my deepest gratitude to the teachers of the Faculty of Biotechnology - Vietnam National University of Agriculture With the understanding and dedication of the teachers, they have imparted to me a lot of valuable knowledge during my years of studying at the school so that I can equip myself with the necessary knowledge to be able to successfully complete the undergraduation thesis Finally, I would like to thank my parents and loved ones for their love and support so that I can successfully complete this undergraduation thesis I would like to convey to them my deep gratitude Hanoi, 20th March, 2022 Student Nguyen Thi Khanh Huyen ii CONTENTS COMMITMENT i ACKNOWLEDGEMNETS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES AND GRAPHS vi CHAPTER INTRODUCTION 1.1 Introduction 1.2 Aim 1.3 Requirements CHAPTER LITERATURE REVIEW 2.1 Origins of rice 2.2 Origin and some characteristics of black rice 2.3 Overview of drought tolerant rice 2.3.1 Definition of drought 2.3.2 Drought stress in plants 2.3.3 Physiological and biochemical basis of drought tolerance in rice 2.4 Research methods to assess genetic diversity 12 2.4.1 Morphological marker 13 2.4.2 Biochemical marker 13 2.4.3 DNA molecular marker 14 2.5 Selection of drought-resistant rice varieties 16 2.5.1 Selecting and creating drought-tolerant rice varieties in the world 16 2.5.2 Selecting and creating drought-tolerant rice varieties in Vietnam 17 CHAPTER MATERIALS AND METHODS 19 3.1 Materials 19 3.2 Research location and time 19 3.3 Research content 20 3.4 Research methods 20 iii 3.4.1 Evaluation method of drought tolerance at the germination stage by artificial seed treatment 20 3.4.2 Methods of assessing drought tolerance degree (DTD) (Bibha Rani et al., 2016) 21 3.5 Methods to assess genetic diversity 24 3.5.1 Total DNA extraction 24 3.5.2 PCR reaction 25 CHAPTER RESULTS AND DISCUSSION 28 4.1 Evaluation results of drought tolerance at sprout stage by artificial seed treatment 28 4.1.1 Results of evaluation of drought tolerance at sprout stage by KClO3 solution 28 4.1.2 Results of evaluation of drought tolerance at germination stage by PEG 6000 solution 29 4.2 Results of evaluation of drought tolerance degree (DTD) 33 4.2.1 Response of varieties of black rice to drought stress treatments in drought – tolerance trials 33 4.2.2 DTD values after severe drought treatment 34 4.3 Results of genetic diversity analysis of studied black rice varieties 36 4.3.1 Results of total DNA extraction 36 4.3.2 Results of electrophoresis of PCR products of studied black rice varieties 37 4.3.3 Results of polymorphism analysis of drought-tolerant black rice varieties by molecular marker SSR 40 4.3.4 Results of polymorphism analysis and genetic relationships of studied rice varieties 42 CHAPTER CONCLUSION AND SUGGESTION 44 5.1 Conclusion 44 5.2 Suggestion 45 REFERENCES 46 iv LIST OF TABLES Table 3.1 Samples of black rice varieties as research materials 19 Table 3.2 SSR markers used in the experiment 26 Table 4.1 Results of evaluation of drought tolerance of varieties of rice with 3% KClO3 solution 28 Table 4.2 Germination rate of varieties of black rice by measurements after seed treatment through PEG 6000 30 Table 4.3 Results of evaluation of germination rate (GR) and relative germination rate (RGR) of varieties of black rice 32 Table 4.4 The DTD values of cultivars without drought treatment and after severe drought treatment 35 Table 4.5 Results of electrophoresis of PCR products in studied black rice varieties 39 Table 4.6 The number of alleles appearing and the PIC coefficients of the primer pairs 41 Table 4.7 Genetic relationship between studied black rice varieties 43 v LIST OF FIGURES AND GRAPHS Figure 2.1 Schematic representation of the evolutionary pathways of Asian and African cultivated rice Figure 2.2 Annotation mechanisms of growth/yield decline in plants under drought stress conditions Figure 3.1 A schematic diagram of parts of leaves used to calculate DTD values F1, S1, and T1 represent the lengths of the green parts of the first, second, and third leaves, respectively, whereas F2, S2, and T2 denote the entire lengths of the first, second, and third leaves, respectively The dried parts of leaves resulting from drought stress are shown in brown 23 Figure 4.1 Soil moisture of varieties of black rice from sowing to severedrought treatment Drought treatment is applied from the cessation of irrigation until re-irrigation Blue, yellow, and orange arrowheads indicate mild, moderate and severe drought-stress stages, respectively 33 Figure 4.2 Response of varieties of black rice to different water conditions (a) Control pots containing the varieties without drought stress treatment (b) The drought-tolerance response of the varieties at the severe-drought treatment stage 34 Figure 4.3 Total DNA of black rice varieties studied 36 Figure 4.4 Electrophoresis image of PCR products of studied rice varieties (left: primer pair RM212 and right: primer pair RM215) 37 Figure 4.5 Electrophoresis image of PCR products of studied rice varieties (left: primer pair RM218 and right: primer pair RM5964) 38 Figure 4.6 Electrophoresis image of PCR products of studied rice varieties with primer pair RM546 38 Figure 4.7 Tree diagram showing the genetic relationship between studied rice varieties with drought tolerance 42 vi LIST OF ABBREVIATIONS PEG: Polyethylene glycogen DTD: Drought tolerance degree SSR: Simple sequence repeat ABA: Abscisic acid ROS: Reactive oxygen species Pas: Polyamines RAPD: Random Amplified Plymorphic DNA AFLP: Amplified Fragment Length Polymorphism RFLD: Restriction Fragment Length Polymorphism CTAB: Cetyltrimethylammonium bromide: TE: Tris-EDTA vii CHAPTER INTRODUCTION 1.1 Introduction Rice (Oryza sativa L.) is one of the main food crops, providing food for more than 65% of the world's population Currently, there are more than 100 rice-producing countries in the world, and Asia is the main rice-producing region, accounting for 90% of both output and area, and is home to the oldest agriculture associated with rice cultivation water To be able to produce and create high-yielding, good-quality rice varieties that are resistant to all climates, it is also necessary to mention the great merits of breeders One of the quality rice varieties is black rice Black rice has the scientific name of Oryza sativa L Glutinosa Tanaka Black rice is also known as tonic blood It is a type of rice with high nutritional content such as: protein content in black rice is 6.8% higher, fat is 20% higher than other rice In particular, in black rice containing large amounts of anthocyanin with antioxidant effects, preventing the hazardous effects of free radicals, which are beneficial to the user's health Currently, due to changes in the global climate, many rice-growing areas suffer from severe drought Drought is the factor that has the greatest impact on world food security, which can reduce overall crop yields by up to 70%, while rice is ranked among the least drought tolerant Therefore, the selection of black rice varieties with good quality and high drought tolerance as starting materials for the selection of drought-resistant rice varieties becomes an urgent issue There have been many studies on drought tolerance and selection of drought tolerant rice varieties for difficult cultivation areas in Vietnam In 2011, Tran Nguyen Thap used an experiment to evaluate the artificial drought tolerance of rice plants in the room and recommended the use of 2% - 3% KClO3 or 0.8-1% sucrose for seed treatment Researches on drought-tolerant rice in the past time have focused on evaluating the morphological, biochemical and genetic have poor drought tolerance and rice varieties KhauCang and KhauLech have the best drought tolerance among varieties of black rice After evaluating the artificial drought tolerance (evaluation of drought tolerance at germination stage by KClO3 and PEG 6000 solution) and assessing the drought tolerance degree of studied black rice varieties, the drought tolerance of these rice varieties is quite high In which, the variety with the best drought tolerance is Khau Cang And KhauDam has the worst drought tolerance among studied black rice varieties 4.3 Results of genetic diversity analysis of studied black rice varieties 4.3.1 Results of total DNA extraction In this study, we chose the CTAB method of Farhad Masoomi-Aladizgeh et al., 2016 (improved by Dinh Truong Son) to extract genomic DNA from leaves of studied rice varieties The basic principle of this method is the use of cetyltrimethylamonium-bromide (CTAB), which is able to dissolve substances released from the cell membrane after their membrane is broken, DNA is much more soluble than other substances Therefore, CTAB plays a key role in nucleic acid extraction Figure 4.3 Total DNA of black rice varieties studied After obtaining the DNA, check the quality of the DNA by electrophoresis on 1% agarose gel at 100V for 30 minutes Electrophoresis results (Figure 4.3) show that the bands are neat and sharp, the DNA is not broken, and the purity is 36 quite high, ensuring the implementation of PCR reactions and subsequent experiments 4.3.2 Results of electrophoresis of PCR products of studied black rice varieties Based on the results of methods: assessment of drought tolerance at germination stage through seed treatment with KClO3, polyethylene glycogen (PEG) and drought tolerance assessment method (DTD), showed that the variety KhauCang has the best drought tolerance and the variety KhauDam has the worst drought tolerance In addition, varieties T68 and varieties KhauLech are quite drought tolerance From these results, compared with the results of electrophoresis of PCR products, we show that, in the variety KhauDam at primer RM215, the results are bands with sizes of 150bp, 510bp and no band of 345 bp As for the KhauCang variety, it gives bands, of which there are bands of 150 bp and 345 bp similar to the other varieties At primer RM212, the varieties only appeared single band at 136 bp and no polymorphic band appeared (Fig 4.4) Figure 4.4 Electrophoresis image of PCR products of studied rice varieties (left: primer pair RM212 and right: primer pair RM215) At primer RM218, only KhauDam showed band (123bp) As for KhauCang, a 153 bp band appeared similar to the varieties TeTim, TeCam, BlauCam 37 and KhauLech At primer RM5964, in variety KhauDam 3, there was only band of 118 bp As for the KhauCang variety, bands appear, 118 bp and 295 bp And in the varieties T68, Tetim, Khaulech also appear these bands (Fig 4.5) Figure 4.5 Electrophoresis image of PCR products of studied rice varieties (left: primer pair RM218 and right: primer pair RM5964) At primer RM5461, the variety KhauDam had bands of 110 bp, 450 bp, 590 bp and no 310 bp ribbon appeared Meanwhile, KhauCang variety has bands of 110 bp and 310 bp similar to other varieties (Fig 4.6) Figure 4.6 Electrophoresis image of PCR products of studied rice varieties with primer pair RM546 38 Table 4.5 Results of electrophoresis of PCR products in studied black rice varieties SSR Name of sample Number PCR product size markers varieties of bands (bp) RM212 RM215 RM218 Do All varieties T68 TeTim TeCam 150bp, 345bp BlauCam 150 bp, 345 bp 136 bp 418 bp 150 bp, 345 bp, 510 bp 150bp, 345bp, KhauLech KhauDam 150bp, 510 bp T68 200 bp 150bp, 418bp, 510bp Only varieties T68 and 153 bp KhauDam have different bands from the rest KhauDam T68 TeTim 123bp 118 bp, 200 bp, 295 bp, 400bp 118 bp, 295 bp, 400 bp TeCam, BlauCam, not appear 345bp band 345bp, KhauLech RM5964 Only KhauDam does 418bp TeCam, KhauCang, appear 150 bp, 345 bp, BlauCam, not polymorphic bands KhauCang TeTim, Note TeCam, BlauCam and KhauDam only have 118 bp KhauCang 118 bp, 295 bp KhauLech 118 bp, 295 bp, KhauDam 39 band 118bp 400 bp, 495 bp, 1000 bp, 1100bp T68 TeTim 110 bp, 310bp, 450bp, 700 bp 110 bp, 310bp, 450bp, 590 bp 110 bp, 310 bp, RM5461 TeCam 450 bp, 590 bp, Only KhauDam does 700 bp not appear 310bp band BlauCam, KhauCang, 110 bp, 310 bp KhauLech KhauDam 3 110 bp, 450 bp, 590 bp From Table 4.5, it shows that KhauDam has poor drought tolerance and has different bands compared to other varieties The KhauCang variety, which has good drought tolerance, gives the same bands as the rest of the varieties And varieties TeTim, KhauLech have the most similarities with varieties KhauCang Through Table 4.5, it can be seen that primer RM5964 gives more polymorphism than other primers and we can use this indicator to find varieties with good and poor drought tolerance 4.3.3 Results of polymorphism analysis of drought-tolerant black rice varieties by molecular marker SSR In this study, we used a total of SSR markers to investigate some traits related to drought tolerance of rice for studied black rice varieties and obtained good results Following are the results of genetic polymorphism analysis of varieties of black rice with SSR primers on 2.0% agarose gel Analyzing pairs of SSR primers with rice varieties studied, a total of 19 different alleles were obtained, the number of alleles/locus ranged from to 6, with an average of 3.8 alleles/locus In which, pair of primers obtained allele 40 (RM212), pair of primers obtained alleles (RM218), pair of primers obtained alleles (RM215), pair of primers obtained alleles (RM5461) and pair of primers obtained alleles (RM5964) (Table 4.6) Table 4.6 The number of alleles appearing and the PIC coefficients of the primer pairs PCR Numbers SSR marker Chromosomes product size (bp) Number of alleles PIC RM212 136 0.93 RM215 150 - 510 0.85 RM218 123 - 200 0.45 RM5964 118 - 1100 0.73 RM5461 110 - 700 0.76 Total 19 3.72 Average 3.8 0.74 The Polymorphic Information Content (PIC) coefficient is considered a measure of the genetic diversity of alleles at each SSR locus (Smith et al., 1997) The PIC value can be understood as the genetic diversity of the gene With pairs of SSR primers used in the study, pairs of primers gave polymorphic results and pair of primers showed monomorphic results - only allele (RM212) was obtained The total number of alleles obtained on SSR primer pairs is 19 alleles, with an average of 3.8 alleles/primer pair Research results shown in Table 4.6 show that: studied black rice varieties are quite diverse in allele composition at the studied gene loci 41 4.3.4 Results of polymorphism analysis and genetic relationships of studied rice varieties The data obtained from the 5-primer SSR electrophoresis of the group of rice varieties studied were statistically and analyzed using NTSYSpc 2.1 software, thereby establishing a table of genetic similarity coefficients (Table 4.7) and build a phylogeny diagram (Fig 4.7) Figure 4.7 Tree diagram showing the genetic relationship between studied rice varieties with drought tolerance The results obtained in Table 4.7 and Fig 4.7 showed that the genetic similarity coefficients of the studied black rice varieties ranged from 0.42 to 0.89 The rice varieties No and No have the highest similarity coefficient of 0.89 42 Table 4.7 Genetic relationship between studied black rice varieties Number of Sample Sample Sample Sample Sample Sample Sample symbols Sample 1.00 Sample 0.63 1.00 Sample 0.63 0.79 1.00 Sample 0.58 0.74 0.84 1.00 Sample 0.42 0.68 0.68 0.63 1.00 Sample 0.68 0.74 0.74 0.89 0.53 1.00 Sample 0.58 0.74 0.53 0.68 0.42 0.79 1.00 At the genetic similarity level of about 42%, rice varieties were classified into groups of genetic differences: - Group includes variety, which is the variety sample (KhauDam3) - Group includes variety, which is the variety sample (KhauLech) - Group includes varieties: sample (TeTim), sample (TeCam), sample (BlauCam), and sample (KhauCang) The coefficient of genetic similarity among varieties in the group is 0.79 (between variety and variety 3), 0.74 (between varieties sample and sample and sample 6), respectively, 0.84 (between varieties sample and 4) and 0.89 (between varieties sample and 6) In which, variety sample and have the highest genetic similarity coefficient of 0.89 - Group includes variety, which is the variety sample (T68) 43 CHAPTER CONCLUSION AND SUGGESTION 5.1 Conclusion From the results of artificial drought tolerance assessment and drought tolerance degree assessment of studied black rice varieties, it was evaluated that the drought tolerance of the rice varieties was quite high Among them, the best drought-tolerant variety was found, Khau Cang And KhauDam has the worst drought tolerance among studied black rice varieties From the results of electrophoresis of SSR markers in studied black rice varieties, we see that KhauDam has poor drought tolerance and has a different genotype from the rest The KhauCang variety has good drought tolerance and has the same genotype as the other varieties This suggests that varieties that have different bands or not have the same bands as other varieties are likely to be less drought tolerant Through the evaluation results of drought tolerance from methods: artificial drought tolerance assessment and drought tolerance degree assessment and electrophoresis results of PCR products show that KhauDam is the worst drought tolerant variety and KhauCang is the best drought tolerant variety among varieties of black rice studied As for the varieties T68, KhauLech are quite drought tolerant varieties Through analysis of SSR markers for studied black rice varieties, a total of 19 alleles were obtained, with an average of 3.8 alleles/locus The PIC coefficient ranges from 0.45 - 0.93 (average is 0.74) From that, the genetic similarity coefficient of studied black rice varieties ranged from 0.42 to 0.89 In which, rice varieties BlauCam and KhauCang have the highest similarity coefficient of 0.89 44 5.2 Suggestion Continue to study and evaluate the genetic diversity of the drought tolerant rice group at the agro-biological level combined with the evaluation by SSR marker to identify markers associated with drought tolerance to serve for the breeding of drought tolerant rice varieties Continue to study to identify specific alleles, rare alleles to accurately identify elite genetic resources for breeding research and orientate the collection and conservation of diverse genetic resources of drought-tolerant rice at molecular level Plant rice varieties under field conditions to 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