Genetic diversity of the purple waxy inbred lines revealed by phenotype and ssr markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY UNDERGRADUATE THESIS “GENETIC DIVERSITY OF THE PURPLE WAXY INBRED LINES REVEALED BY PHENOTYPE AND SSR MARKERS” STUDENT : NGUYEN THI THUY LINH CODE : 621691 CLASS : K62KHCTT SUPERVISOR : PROF DR VU VAN LIET DEPARTMENT : PLANT GENETICS AND BREEDING HA NOI- 2022 COMMITMENT I pledge that the contents of this report are honestly conducted by me under the guidance of Prof DR Vu Van Liet I hereby undertake that all references in this document are quoted which are the name of the authors, name of research, time, location, and origin Hanoi, March 2022 Student Nguyen Thi Thuy Linh ACKNOWLEDGMENT During the implement this graduation thesis, I am not the only effort but also received a lot of support in many aspects of leaders, organizations, and individuals First of all, I would like to express my sincere thanks to the dean of the Agronomy Faculty in general, my teachers in the Department of Plant Genetics and Breeding in particular who taught and created most conditions for students in the process of learning and performing this research The project would not have been possible if it lacks support from the mentors of the teachers Therefore, I would like to express my deep gratitude and respect to Prof Dr Vu Van Liet has dedicatedly instructed, cared for, and helped me during the last time Their ideas, inputs, and guidance were most helpful and have assisted me in making the research Moreover, I also would like to thank the dean and all the teachers who have imparted knowledge to me, facilitating and supporting me during the implementation of the topic This is a great opportunity I was able to practice my skills in class At the same time, it was good to help to give me more confidence in myself Finally, I would like to send my sincere thanks to my family, friends, and relatives for encouraging, supporting, and helping me during the thesis Hanoi, March 2022 Student Nguyen Thi Thuy Linh CONTENTS COMMITMENT i ACKNOWLEDGMENT i LIST OF TABLES v LIST OF FIGURES i LIST OF ACRONYMS i PART I INTRODUCTION 1.1 Research gap 1.2 Objective and requirements 1.2.1 Objective 1.2.2 Requirements PART II LITERATURE REVIEWS 2.1 History original of the purple waxy corn 2.1.1 Waxy corn classification 2.1.2 Characteristics of waxy corn 2.1.3 Purple waxy corn 2.1.4 Nutritional value 2.2 Research and production of waxy corn in the world 2.2.1 Genetic studies on traits in waxy corn 11 2.2.2 Inheritance of quality traits in waxy corn 13 2.2.3 Genetics of purple waxy corn 14 2.2.4 Improved quality of waxy corn 16 2.2.5 Achievements in breeding anthocyanin-rich purple waxy corn 19 2.3 Situation of research and production of waxy corn in VIETNAM 19 2.4 SSR markers and it function in plant breeding 23 2.5 Research on waxy corn genetic diversity analysis 25 2.5.1 Research on waxy corn genetic diversity analysis based on phenotypes and molecular markers in the world 25 2.5.2 Research on genetic variation based on phenotype and molecular markers in Vietnam 27 PART III: MATERIALS AND METHODS 30 3.1 Plant materials 30 ii 3.2 Research place and time 30 3.3 Research content 30 3.4 Methods 30 3.4.1 Experimental design 30 3.4.2 Applied technical measures 32 3.4.3 Methods of monitoring experiments 33 3.3.4 DNA extraction and SSR analyses 37 3.3.5 Data analysis 40 PART IV: RESEARCH RESULTS AND DISCUSSION 41 4.1 Growth and development stages of purple corn lines in Autumn-Winter season 2021 in Gia Lam, Hanoi 41 4.1.1 The stage from sowing seeds to spreading pollen 43 4.1.2 The stage from sowing seeds to spraying beard 43 4.1.3 The difference between tossing pollen spraying beard 44 4.1.4 The period from sowing to harvesting fresh corn 44 4.1.5 The period from sowing to physiological maturity 45 4.2 Some agronomic characteristics of purple corn lines in Autumn-Winter season 2021 in Gia Lam, Hanoi 45 4.2.1 Plant height (PH) 47 4.2.2 Ear height (EH) 48 4.2.3 Ratio of ear height/plant height 49 4.2.4 Tassel length (TL) 50 4.3 Pests and diseases of purple waxy inbred lines in Winter season 2021 at Hanoi 52 Rhizoctonia solani Kuhn 52 4.3.1 Chilo partellus 54 4.3.2 Rhizoctonia solani Kuhn 55 4.3.3 Pour the roots 55 4.4 Yield and yield components of purple waxy corn inbred line in Winter season 2021 at Hanoi 55 4.4.1 Ear length (EL) 59 4.4.2 Ear diameter (ED) 59 iii 4.4.3 Number of kernel rows per ear (RE) 59 4.4.4 Number of kernels per row (KR) 60 4.4.5 Weight 1000 seeds (g) (P1000) 60 4.5.6 Potential yield (TY) 60 4.5.7 Grain yield (GY) 61 4.5 Quality parameters of of purple waxy corn inbred line in Winter season 2021 at Hanoi 62 4.6 Correlation between agronomic traits of purple waxy corn lines in Spring 2021 crop in Gia Lam, Hanoi 64 4.7 Selection of elite purple waxy corn inbred lines based in phenotypes 65 4.8 Genetic diversity of purple corn lines based on SSR marker 68 PART V: CONCLUSIONS AND RECOMMENDATIONS 73 5.1 Conclusions 73 5.2 Recommendations 73 REFERENCES 72 APENDIX EXPERIMENTAL IMAGES 75 APENDIX STATISTICAL ANALYSIS 82 iv LIST OF TABLES Table 3.1 Plant materials used in this study 31 Table 3.2 List of 14 directives used for surveying 39 Table 4.1 Growth duration of purple waxy corn inbred line in Winter season at Hanoi, 2021 41 Table 4.2 Morphological traits of purple waxy corn inbred lines 46 Table 4.3 Pest and disease tolerance of purple waxy corn inbred line in Winter season 2021 at Hanoi 52 Table 4.4 Yield and yield components of purple waxy corn inbred line in Winter season 2021 at Hanoi 55 Table 4.5 Quality of purple waxy corn inbred line in Winter season 2021 at Hanoi 62 Table 4.6 MGIDI index 65 Table 4.7 Genetic similarity matrix 71 v LIST OF FIGURES Figure 2.1 Dried waxy corn containing different types of seeds Figure 2.2 Carbohydrate compositions (mg g-1 fresh weight) of six waxy corn 10 Figure 2.3 Schematic illustration of the WX gene 13 Figure 2.4 Kernels color of waxy corn genotypes at full maturity 16 Figure 2.5 Diversity in colored corn 18 Figure 4.1 Correlation heatmap of phenotypic traits measurement on purple waxy corn inbred lines 65 Figure 4.2 Selection of elite purple waxy corn based on phenotypic traits with 25% selection intensity4.8 Genetic diversity of purple corn lines based on phenotypes 67 Figure 4.3 Cluster dendrogram presented genetic diversity of purple waxy corn based on phenotypic traits 68 Figure 4.4 Gel electrometric of 14 SSR markers on 14 elite purple waxy corn inbred lines with check inbred line NT111 70 Figure 4.5 UPGMA dendrogram based on the SSR markers of 14 elite purple waxy corn inbred lines with check variety NT111 72 LIST OF ACRONYMS Abbreviate a1 ANOVA Description Anthocyanin biosynthetic gene, anthocyanins-1 Analysis of Variance CV Coefficient of Variation LAI Leaf Area Index LSD Least Significant Different MAS Marker Assist Selection MGIDI Multitrait Genotype-Ideotype Distance Index QCVN Vietnamese Standards RCBD Randomized Complete Block Design Se Sugary enhanced Su1 Sugary Sh2 Shrunken UDP-Glucose Uridine diphosphate glucose VCU Value of Cultivation and Use ABSTRACT Limited labor, cost, and time regarding hybrid formation and yield trials encourages corn breeders to identify potential F1 hybrids without crossing all possible combinations by line screening based SSR-genetic distance Therefore, this study aimed to determine combining ability and heterosis of parental lines included 55 purple waxy corn inbred lines, denoted from D01 to D55, with check is purple waxy corn inbred line NT111 , to save costs only 14 promising lines + control line, NT111, were selected to analyze genetic diversity based on SSR molecular markers.(parental line of hybrid purple waxy corn VNUA141) developed by the Institute of Crop Research and Development, Vietnam National University of Agriculture Genetic diversity analysis based on 20 phenotypic traits showed that purple corn lines were divided into main groups that exhibited relatively high levels of genetic diversity Based on SSR markers , with the coefficient 0.8, those purple waxy corn inbred lines were divided into groups There were was a poor correlation between phenotypic diversity and genetic divserity of those studied purple waxy corn inbred lines ii 4.8 Genetic diversity of purple corn lines based on phenotypes Figure 4.3 Cluster dendrogram presented genetic diversity of purple waxy corn based on phenotypic traits Genetic diversity analysis based on 20 agronomic traits showed that purple corn lines divided into main groups exhibited relatively high levels of genetic diversity 4.9 Genetic diversity of purple corn lines based on SSR markers Total 14 elite purple waxy corn inbred line with check inbred line NT111 were used for SSR marker analysis Total 14 SSR markers were used to analysis the genetic diversity 68 69 Figure 4.4 Gel electrometric of 14 SSR markers on 14 elite purple waxy corn inbred lines with check inbred line NT111 70 Table 4.7 Genetic similarity matrix D03 (H1) D03 (H1) D30 (H2) D28 (H3) D39 (H4) D10 (H5) D08 (H6) D04 (H7) D50 (H8) D26 (H9) D38 (H10) D48 (H11) D05 (H12) D09 (H13) D17 (H14) NT111 (H15) D30 (H2) D28 (H3) D39 (H4) D10 (H5) D08 (H6) D04 (H7) D50 (H8) D26 (H9) D38 (H10) D48 (H11) D05 (H12) D09 (H13) D17 (H14) NT111 (H15) 1.00 0.82 1.00 0.82 0.95 1.00 0.71 0.71 0.72 1.00 0.77 0.75 0.77 0.82 1.00 0.81 0.92 0.95 0.73 0.78 1.00 0.76 0.78 0.82 0.79 0.84 0.82 1.00 0.72 0.71 0.75 0.68 0.77 0.77 0.81 1.00 0.77 0.79 0.83 0.79 0.80 0.84 0.88 0.86 1.00 0.80 0.93 0.94 0.71 0.79 0.91 0.82 0.75 0.82 1.00 0.77 0.90 0.90 0.73 0.79 0.90 0.83 0.76 0.84 0.94 1.00 0.78 0.95 0.95 0.72 0.78 0.92 0.80 0.75 0.83 0.97 0.95 1.00 0.79 0.93 0.94 0.73 0.77 0.95 0.81 0.75 0.84 0.93 0.92 0.95 1.00 0.81 0.95 0.95 0.72 0.77 0.95 0.82 0.76 0.84 0.94 0.90 0.95 0.98 1.00 0.80 0.95 0.92 0.70 0.76 0.92 0.79 0.75 0.82 0.95 0.90 0.94 0.96 0.98 71 1.00 From Figure 4.5, it can be seen that with the coefficient 0.8, those purple waxy corn inbred lines were divided into groups Figure 4.5 UPGMA dendrogram based on the SSR markers of 14 elite purple waxy corn inbred lines with check variety NT111 Contrasting dendogram pattern and insignificantly poor correlation between SSR and phenotypic based-genetic distance indicated that there was a lack of agreement between SSR and phenotypic markers 72 PART V: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions The purple waxy corn lines tested for the Autumn-Winter crop 2021 in Gia Lam, Hanoi have a growth period of 93-107 days Liệt kê dòng ngắn ngày đối chứng NT111 mức có ý nghĩa thống kê: Grain yield of purple waxy corn inbred lines ranged from … to … Liệt kê dòng cao đối chứng NT111 mức có ý nghĩa thống kê: The representative sweetness index oBrix of sweet corn lines ranged from 10.3 to 16.0% Liệt kê dòng cao đối chứng NT111 mức có ý nghĩa thống kê: The pericarp thickness of purple waxy corn inbred lines ranged from 45.1 to 97.3μm Liệt kê dòng thấp đối chứng NT111 mức có ý nghĩa thống kê: Multivariable selection based on MGIDI index with 25% selection intensity have selected 14 elite purple waxy corn inbred lines lines viz., D03, D30, D28, D39, D10, D08, D04, D50, D26, D38, D48, D05, D09, and D17 Genetic diversity analysis based on 20 phenotypic traits showed that purple corn lines were divided into main groups exhibited relatively high levels of genetic diversity Based on SSR markers, with the coefficient 0.8, those purple waxy corn inbred lines were divided into groups There were poor correlation between phenotypic diversity and genetic divserity of those studied purple waxy corn inbred lines 5.2 Recommendations Continue to evaluate the promising lines and estimate combining abilities of those purple waxy corn inbred lines in the next season 73 REFERENCES English referemces Angevin F., Klein E K., Choimet C., Gauffreteau A., Lavigne C., Messéan A & Meynard J M (2008) Modelling impacts of cropping systems and climate on maize cross-pollination in agricultural landscapes: The MAPOD model European Journal of 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Thị Thanh Hà, Nguyễn Thị Hồng Ngát, Nguyễn Văn Hà, Dương Thị Loan, Vũ Thị Bích Hạnh, Vũ Văn Liết (2013) Chọn lọc vật liệu có tính trạng vỏ hạt mỏng phục vụ tạo giống ngơ nếp ăn tươi chất lượng cao Tạp chí Khoa học Phát triển 11(2): 135-144 Phạm Quang Tuân, Nguyễn Thế Hùng, Nguyễn Việt Long, Vũ Văn Liết, Nguyễn Trung Đức, Nguyễn Thị Nguyệt Anh (2018) Cải thiện độ dịng ngơ nếp phương pháp lai trở lại Tạp chí Khoa học Nơng nghiệp Việt Nam 16(3): 197-206 74 APENDIX EXPERIMENTAL IMAGES Figure Purple waxy corn inbred lines at flowering time 75 Figure Variation in tassel of purple waxy corn inbred lines 76 Figure Variation in purple corn cob inbred lines 77 APENDIX STATISTICAL ANALYSIS MGIDI components Tab 1 10 11 12 13 14 15 16 17 18 19 20 PC Eigenvalues Variance (%) Cum variance (%) PC1 4.533768 22.66884 22.66884 PC2 2.320178 11.60089 34.26973 PC3 1.985962 9.92981 44.19954 PC4 1.953281 9.766407 53.96595 PC5 1.54142 7.7071 61.67305 PC6 1.292087 6.460436 68.13348 PC7 1.219297 6.096487 74.22997 PC8 1.031321 5.156604 79.38657 PC9 0.908924 4.544618 83.93119 PC10 0.808799 4.043994 87.97518 PC11 0.616589 3.082945 91.05813 PC12 0.48479 2.423948 93.48208 PC13 0.394986 1.974929 95.45701 PC14 0.30053 1.502648 96.95965 PC15 0.276164 1.38082 98.34047 PC16 0.149617 0.748085 99.08856 PC17 0.134152 0.670761 99.75932 PC18 0.040435 0.202175 99.9615 PC19 0.00572 0.0286 99.9901 PC20 0.001981 0.009905 100 Tab 2 10 11 12 13 14 15 16 17 18 19 20 VAR GD EL KR PY GY MY LN LAI TL LW P1000 PH EH BRIX TPB LL ED RE EP PER Factor FA1 FA1 FA1 FA1 FA1 FA1 FA2 FA2 FA3 FA3 FA3 FA4 FA4 FA4 FA5 FA5 FA6 FA6 FA7 FA8 Xo 99.73214 12.825 23.83214 4.271429 2.448214 4.523214 8.767857 2.625 21.79286 8.575 179.375 148.8089 59.17857 12.36429 7.971429 69.65357 4.121429 14.36607 1.042857 65.45179 Xs 98.28571 13 24.7 4.928571 2.792857 4.814286 3.014286 20.8 8.985714 190.1786 143.8 58.75714 12.49286 7.764286 74.95714 4.271429 14.82143 1.071429 64.92143 82 SD -1.44643 0.175 0.867857 0.657143 0.344643 0.291071 0.232143 0.389286 -0.99286 0.410714 10.80357 -5.00893 -0.42143 0.128571 -0.20714 5.303571 0.15 0.455357 0.028571 -0.53036 SDperc -1.45031 1.364522 3.641541 15.38462 14.07732 6.435057 2.647658 14.82993 -4.55588 4.789671 6.022897 -3.36601 -0.71213 1.039861 -2.59857 7.614213 3.639515 3.169671 2.739726 -0.8103 sense goal decrease 100 increase 100 increase 100 increase 100 increase 100 increase 100 increase 100 increase 100 increase increase 100 increase 100 decrease 100 decrease 100 increase 100 increase increase 100 increase 100 increase 100 increase 100 decrease 100 Tab VAR GD PH EH PER TL TPB LL LW LN 10 LAI 11 EP 12 EL 13 ED 14 RE 15 KR 16 P1000 17 PY 18 GY 19 MY 20 BRIX FA1 FA2 FA3 FA4 FA5 FA6 FA7 FA8 Communality Uniquenesses -0.50868 -0.03229 -0.19791 0.179163 -0.50177 0.023519 -0.29917 -0.07151 0.678006 0.321994 0.136705 0.079289 -0.06584 0.886739 -0.09305 -0.10043 0.001848 0.144422 0.85522 0.14478 0.257216 0.145208 0.011199 0.80757 0.023796 0.161206 -0.12123 -0.02733 0.781536 0.218464 -0.10051 -0.0139 0.038378 0.096184 0.109999 0.129598 -0.03015 0.901099 0.862804 0.137196 0.093311 -0.24328 0.589211 0.035604 -0.0147 0.022322 -0.30271 -0.31425 0.607431 0.392569 0.068589 -0.10793 -0.13651 -0.27067 -0.53649 0.23753 -0.06024 -0.12518 0.471789 0.528211 0.077812 0.209551 0.139977 0.127648 -0.82034 -0.04343 0.169001 0.022575 0.789772 0.210228 0.02223 0.262401 0.811043 -0.06947 -0.06083 -0.12935 0.006781 0.275507 0.828349 0.171651 -0.06421 0.918702 -0.12474 0.104145 0.103101 0.101752 -0.08033 -0.03732 0.903371 0.096629 -0.00515 0.876885 0.335657 0.103877 -0.28773 -0.03235 -1.2E-05 0.084768 0.983429 0.016571 -0.04276 -0.08188 -0.06754 -0.05041 -0.05635 0.082238 0.901986 -0.04341 0.841037 0.158963 -0.57362 0.320885 -0.08692 -0.31633 0.3712 0.238084 0.052558 0.074331 0.742389 0.257611 -0.34751 0.108716 -0.05806 -0.1537 0.147633 -0.81434 0.054598 -0.07434 0.853024 0.146976 -0.1139 -0.20197 0.15384 0.04658 -0.01092 -0.8568 -0.1579 -0.05562 0.841852 0.158148 -0.67422 0.069611 0.27987 -0.12269 0.276911 0.167807 0.358248 0.031625 0.786982 0.213018 -0.56928 -0.01146 -0.64968 -0.07343 -0.17682 -0.00513 -0.15147 0.166399 0.833605 0.166395 -0.80714 -0.0727 -0.25874 -0.13717 -0.04303 -0.28528 0.296676 0.110479 0.925975 0.074025 -0.89424 0.040079 -0.16867 -0.06811 -0.04786 -0.28857 0.03738 0.0922 0.929826 0.070174 -0.83043 0.011775 0.034562 -0.1096 0.075893 -0.19426 -0.18148 -0.1074 0.790921 0.209079 0.231015 0.12254 -0.14166 -0.45776 -0.15125 -0.31279 -0.04536 0.3863 0.569997 0.430003 83 Tab TPB LL LN LAI P1000 PY GY MY GD GD 1.000 0.042 -0.043 0.036 -0.023 -0.245 0.166 -0.052 -0.072 0.081 -0.090 0.020 -0.138 0.091 -0.464 -0.344 -0.412 -0.332 0.136 -0.027 PH 0.042 1.000 0.736 -0.012 0.102 -0.195 0.047 -0.140 -0.157 0.088 0.347 0.097 -0.049 0.180 0.049 0.150 0.109 0.202 0.150 0.137 EH -0.043 0.736 1.000 -0.049 0.084 -0.133 0.025 -0.157 -0.152 0.200 0.256 0.304 0.167 0.189 0.233 0.415 0.299 0.248 0.182 -0.019 TL PH EH TL LW EP EL ED RE KR BRIX PER 0.036 -0.012 -0.049 1.000 -0.055 -0.002 0.205 -0.176 -0.028 -0.165 -0.224 -0.034 0.085 -0.059 -0.334 -0.283 -0.267 -0.022 -0.065 0.191 TPB -0.023 0.102 0.084 -0.055 1.000 0.221 -0.120 -0.095 -0.039 -0.036 -0.134 -0.148 -0.120 -0.091 0.104 -0.034 -0.044 -0.065 0.033 0.120 LL -0.245 -0.195 -0.133 -0.002 0.221 1.000 0.114 0.005 0.450 0.061 -0.230 -0.072 -0.039 -0.133 -0.055 -0.081 -0.006 -0.171 0.137 0.084 LW 0.166 0.047 0.025 0.205 -0.120 0.114 1.000 0.070 0.560 -0.067 -0.055 0.046 0.154 0.142 -0.413 -0.152 -0.093 0.021 0.042 -0.180 LN -0.052 -0.140 -0.157 -0.176 -0.095 0.005 0.070 1.000 0.763 -0.074 0.291 0.027 -0.227 0.051 0.097 -0.022 0.043 0.041 -0.042 -0.069 LAI -0.072 -0.157 -0.152 -0.028 -0.039 0.450 0.560 0.763 1.000 -0.056 0.083 0.029 -0.092 0.031 -0.143 -0.117 -0.007 -0.016 0.053 -0.075 EP 0.081 0.088 0.200 -0.165 -0.036 0.061 -0.067 -0.074 -0.056 1.000 0.043 -0.029 -0.208 0.194 -0.037 0.320 0.017 -0.113 -0.061 0.008 EL -0.090 0.347 0.256 -0.224 -0.134 -0.230 -0.055 0.291 0.083 0.043 1.000 0.180 -0.202 0.587 0.268 0.407 0.469 0.401 0.011 -0.146 ED 0.020 0.097 0.304 -0.034 -0.148 -0.072 0.046 0.027 0.029 -0.029 0.180 1.000 0.646 0.143 0.204 0.492 0.564 0.432 0.144 0.087 RE -0.138 -0.049 0.167 0.085 -0.120 -0.039 0.154 -0.227 -0.092 -0.208 -0.202 0.646 1.000 -0.098 -0.087 0.274 0.262 0.235 0.058 0.088 KR 0.091 0.180 0.189 -0.059 -0.091 -0.133 0.142 0.051 0.031 0.194 0.587 0.143 -0.098 1.000 0.067 0.550 0.561 0.477 -0.087 -0.069 P1000 -0.464 0.049 0.233 -0.334 0.104 -0.055 -0.413 0.097 -0.143 -0.037 0.268 0.204 -0.087 0.067 1.000 0.687 0.614 0.434 0.073 -0.136 PY -0.344 0.150 0.415 -0.283 -0.034 -0.081 -0.152 -0.022 -0.117 0.320 0.407 0.492 0.274 0.550 0.687 1.000 0.841 0.626 0.053 -0.086 GY -0.412 0.109 0.299 -0.267 -0.044 -0.006 -0.093 0.043 -0.007 0.017 0.469 0.564 0.262 0.561 0.614 0.841 1.000 0.804 0.017 -0.100 MY -0.332 0.202 0.248 -0.022 -0.065 -0.171 0.021 0.041 -0.016 -0.113 0.401 0.432 0.235 0.477 0.434 0.626 0.804 1.000 -0.050 0.055 BRIX 0.136 0.150 0.182 -0.065 0.033 0.137 0.042 -0.042 0.053 -0.061 0.011 0.144 0.058 -0.087 0.073 0.053 0.017 -0.050 1.000 -0.072 PER -0.027 0.137 -0.019 0.191 0.120 0.084 -0.180 -0.069 -0.075 0.008 -0.146 0.087 0.088 -0.069 -0.136 -0.086 -0.100 0.055 -0.072 1.000 84