Genetic variability in tomato germplasm (solanum lycopersicum l ) using morphological characteristics and simple sequence repeat (SSR) markers

KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY KUMASI, GHANA SCHOOL OF GRADUATE STUDIES DEPARTMENT OF CROPS AND SOIL SCIENCES GENETIC VARIABILITY IN TOMATO GERMPLASM (Solanum lycopersicum L.) USING MORPHOLOGICAL CHARACTERISTICS AND SIMPLE SEQUENCE REPEAT (SSR) MARKERS JACINTA ADOMA OPOKU AUGUST, 2015 GENETIC VARIABILITY IN TOMATO GERMPLASM (Solanum lycopersicum L.) USING MORPHOLOGICAL CHARACTERISTICS AND SIMPLE SEQUENCE REPEAT (SSR) MARKERS A Thesis submitted to the Department of Crop and Soil Sciences, Faculty of Agriculture, Kwame Nkrumah University of Science and Technology in Partial Fulfilment of the Requirements for the Degree of MASTER OF PHILOSOPHY IN AGRONOMY (PLANT BREEDING) JACINTA ADOMA OPOKU (BSc Hons Agriculture) AUGUST, 2015 DECLARATION I hereby declare that this work is a direct result of my field and laboratory research undertaken and are supported by cited references in relation to other previous and similar work performed and therefore, this thesis has not been presented anywhere for a degree All references to other works have been duly acknowledged Jacinta Adoma Opoku (Student ID: 20288128) …………………… Signature …………… Date Prof Richard Akromah (Supervisor) …………………… Signature …………… Date Dr Charles Kwoseh (Co-Supervisor) …………………… Signature …………… Date …………………… Signature …………… Date Certified by: Dr Enoch Adjei Osekre (Head of Department) i ABSTRACT Tomato is an essential ingredient in the daily diets of majority of Ghanaians and is cultivated in almost all the agro-ecological zones in Ghana, however, not much information is available on the genetic and morphological variability of tomatoes cultivated by farmers The need for varietal identification of tomato cannot be overemphasised It was therefore, necessary to collate information and collect tomato genotypes from the farmers for morphological and molecular evaluation to assess the variations among and within the genotypes A survey was conducted and semi structured questionnaires administered to obtain information about farmers’ knowledge of varietal differences, preferences and production practices and constraints Preferred tomatoes were documented and fruits were obtained from the farmers’ field to extract seeds for the study A pot experiment was conducted to study the genetic variability among and within 12 tomato genotypes obtained from the farmers in three-selected tomato growing areas, namely Afari, Akomadan and Kumawu and their environs in Ashanti Region and planted in a randomised complete block design DNA was extracted from eight (8) plants per genotype without bulking to check the purity of the genotypes using the CTAB method with modification by Takrama, (2000) CRIG adopted by Kirkhouse Trust Mobile Laboratory Data taken included 16 qualitative and 16 quantitative traits and scored using AVRDC descriptor list with additions from the IPRGI descriptor list It was realised that most (65 %) of the farmers saved seeds from previous harvest for the subsequent planting Analysis of variance revealed highly significant difference (P≤ 0.01) among the genotypes for all quantitative traits studied except plant height and number of diseased and pest damaged fruits Genotypic (GCV) and phenotypic coefficient of variation (PCV) were high but narrow for most of the traits studied except for days to ii first flowering, days to 50 percent flowering, 100 percent flowering and plant height which recorded lower phenotypic and genotypic coefficients of variation (PCV and GCV), indicating the little environmental influence and hence, highly heritable Genotypes with higher fruit yield per plant (PF-AF and PM-AF) were from Afari and its environs The correlation analysis indicated that number of fruits per plant was highly significant and positive with fruit yield per plant Similar observation was made with plant height and days to 50 % flowering The SSR markers were highly informative as generated by the PowerMaker V3.25 software NTSyS PC software 2.2 grouped the 96 DNA samples of the 12 genotypes into two major groups The samples were reduced to 48 for clear dendrogram Samples from DW-KU were all put into one group while the rest were in the other group The outcome of this study should be useful to manage the germplasm conservation and future tomato genetic improvement However, all the genotypes may be cultivated over time at different locations on field to ascertain their stability and purity iii DEDICATION To the glory of God, I dedicate this thesis to my uncle, Mr Akwasi Adjei Adjekum iv ACKNOWLEDGEMENT I am forever grateful to the Almighty God for His protection throughout my studies I wish to express my sincere gratitude to my supervisors: Prof Richard Akromah (Provost, College of Agriculture and Natural Resources, KNUST) and Dr Charles Kwoseh (Head of Crop and Soil Science Department, KNUST) for their adept supervision and support that enabled me to complete this research successfully I thank Dr Daniel Nyadanu,(Crop and Soil Sciences Department, KNUST), Dr Amoah (CRI, Fumesua), Dr Francis Appiah (Head of Horticulture Department, KNUST), Dr B K Banful (Horticulture Department, KNUST), Mr Prince Pobi Owusu and all the lecturers and Staff of Department of Crop and Soil Sciences for their incessant support, affection and encouragement I acknowledge the following individuals for their immense technical support; Mr Ben Armooh (CRIG, Tafo), Mr Fuseini, Jamal, Ruchia (Biotechnology Lab), Mr Koranteng, Mr Osei, Mr Malik, Bismark, Pieterson, Eunice and Jemima (Pathology Lab), I also thank David Gaikpa, Kirpal, Ambrose, Ulzen, Gifty Marian, Naomi and Nana Yaw for their selfless support in data taken and analysis My utmost gratitude goes to my sponsors, AGRA (Alliance for Green Revolution in Africa) for the financial support throughout the Masters programme May God replenish everything you all lost while supporting me To all my family and loved ones especially my mum, Georgina Cylacia Adjei, my late father, Mr Collins Opoku, Mad Faustina Akoto, my sibblings, all my aunties, uncle and cousins, I say God bless you profusely v TABLE OF CONTENT DECLARATION i ABSTRACT ii DEDICATION iv ACKNOWLEDGEMENT v TABLE OF CONTENT vi LIST OF FIGURES ix LIST OF TABLES x LIST OF PLATES xi LIST OF APPENDICES xii CHAPTER ONE 1.0 INTRODUCTION CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Introduction 2.2 Tomato production Tomato production in the world Tomato Production in Ghana 2.3 Types or groups of tomato 2.4 Genotypes of tomatoes grown in Ghana 2.5 Genetic divergence 2.6 The tomato quality 2.7 Characterization Morphological characteristics of Tomato 10 Molecular characterization 11 Marker Assisted Selection (MAS) 13 Molecular marker techniques 14 Non - PCR based technique 14 2.8 PCR-based techniques 16 Limitation of PCR 17 Optimal PCR-primers 17 Amplified Fragment Length Polymorphism (AFLP) 18 vi 2.9 Simple Sequence Repeats (SSR) markers 19 2.10 Evaluation of molecular markers 19 CHAPTER THREE 21 3.0 MATERIALS AND METHODS 21 3.1 Introduction 21 Survey of tomato fields in selected major growing area and collection of tomato genotypes 21 Tomato genotypes obtained from farmers’ fields in the selected major growing areas 22 Extraction of tomato seeds from fruits collected from farmers during the survey 22 Field experiment: Evaluation of morphological characteristics of the tomato genotype 23 Soil used for the pot experiment 23 Nursery and transplanting of the tomato seedlings 24 Agronomic practices 24 3.2 Data collected 24 Field layout and experimental design 25 3.3 Morphological data analysis 25 Quantitative characters measured 27 Qualitative character studies 29 3.4 Laboratory experiment: Evaluation of 12 tomato genotypes from major tomato growing areas in the Ashanti Region of Ghana using SSR markers 29 Genomic DNA extraction and purification 31 DNA quality testing and quantification 32 Test run for SSR primers 33 Molecular markers and polymerase chain reactions 34 Agarose gel electrophoresis (AGE) 34 Gel scoring of DNA fragment 35 Statistical Data analysis 35 vii CHAPTER FOUR 37 4.0 RESULTS 37 4.1 Information on Farmers and the Study Areas 37 General socio-economic characteristics of the areas of study 37 Socio-demographic information on farmers and farming activities 37 4.2 Agro-morphological traits of the selected tomato genotypes 41 Variability in qualitative traits 41 Variability in quantitative traits 43 Cluster analysis of the morphological data 51 4.3 Correlation among yield components 53 4.4 Summary Statistics about the SSR markers used 55 Analysis of Molecular Diversity of the tomato genotypes 56 CHAPTER FIVE 61 5.0 DISCUSSIONS 61 5.1 Introduction 61 5.2 Survey of tomato fields in selected major growing area and collection of tomato genotypes 61 5.3 Agro-morphological traits of the tomato genotypes 62 5.4 Phenotypic and genotypic coefficient of variation and heritability estimates 63 5.5 Morphological relationship among genotypes 64 5.6 Correlation of yield and yield component 65 5.7 Evaluation of SSR markers in tomato genotypes using dendrogram 66 5.8 Genetic diversity within and among the genotypes 66 CHAPTER SIX 68 6.0 CONCLUSIONS AND RECOMMENDATION 68 6.1 Conclusion 68 6.2 Recommendation 70 REFERENCES 71 viii Valpuesta, V and Botella, M (2004) Biosynthesis of L-ascorbic acid in plants: new pathways for an old antioxidant Trends Plant 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Theoretical and Applied Genetics, 100: 368–375 86 LIST OF APPENDICES APPENDIX I: QUESTIONNAIRES FOR TOMATO FARMERS AT AGOGO, AKOMADAN AND KUMAWU SECTION A: Demographical Information Please circle the one that answers the question below Name of town / village: …………………… District: ….……….……………… Name of farmer: ……… ……………………………………………………… Age of farmer: …… ……….…………………………….……………………… Sex: ………………………………………… Marital status: ………… a married Educational background…… Number of children: ……………………………………………………………… Number of children in school: …………………………………………………… a JSS b single a male b female c divorced b SSS c Form four widowed d Tertiary SECTION B: Occupational background Are you a tomato farmer? a Yes b No 10 Did you cultivate tomato this year? a Yes b No 11 If yes, what are the names of tomato genotypes you cultivated ……………….…… 12 What other tomato type(s) have you cultivated before? 87 13 Which of the above-mention tomato genotypes did you prefer? ………………………………………………………………….…… Why? ……………………………………………………………………………… 14 For how long have you been cultivating the tomatoes? …………………………………………………………………………………………… 15 How many acre(s) of land did you cultivate this year? ……… ………………… 16 Where did you obtain the seeds? a From the seed shop 17 b Farmer saved seed c Others If you saved your own seeds, for how long did you save the seeds before planting this year 18 How long did it take the tomato to flower? 19 How many boxes (52kg/box) of tomatoes did you harvest? ….………………… 20 How long can you store the tomatoes when there is no ready market? … ……… 21 Do you want a different type of tomato if possible? If yes, describe the characteristics of the a Yes tomato b No you would like …………………………………………………………………………………….……… ………………………………………………………………………………….………… ……………………………………………………………… ………………………… 88 APPENDIX II: Analysis of Physical and chemical properties of soil S/NO I II Porperties Physical Properties Sand (%) Silt (%) Clay (%) Texture Chemical Properties Soil pH (1:1) Organic Carbon (%) Total Nitrogen Available P Available K Value 69.16 15.64 15.20 Sandy Loam 6.90 1.97 0.14 23.2 66.7 89 APPENDIX III: Morphological Characteristics observed in the experiment based on IPGRI tomato descriptor (Darwin et al., 2003) Leaf type, (LT) 1- dwarf 2-potato leaf type 5-Pimpinellifolium -Hirsutum descriptor) Leaf arrangement, (LA) Plant growth habit (PGH) 4-indeterminate x-mixture 3-standard 4-Peruvianum 7-other (specify in 3-semi erect 5-horizontal 7-drooping x-mixture 1-dwarf 2-determinate 3-semi determinate Plant height, (PH) Stem diameter/ girth (SD/G) Flower cluster (FC) Flowering date, (FD) Anthocyanin colouration of hypocotyls (ACH) 0-absent 1-present x- mixture Stem pubescence, (SP) 0-absent 10 Style type (ST) 1-inserted 7-highly exserted x-mixture 11 3-weak 5-medium 7-strong 3-same level as stamen 5-slightly exserted Exterior colour of immature fruit, (ECIF) 3-light 5-medium 7-dark 9- very dark x-mixture 12 Blossom end shape, (BES) 1-indented 13 Exterior colour of mature fruit, (ECMF) Red 5-Pink x-Mixture 2-flat 1-Green 14 Easiness of fruit to detach from the pedicel (EFD) difficult 90 3-pointed x-mixture 2-Yellow 3-Orange 4- 3-easy 5-intermediate 7- 15 Predominant fruit shape, (PFS) 1-flattened 2-slightly flattened 3-round 4-high round 5-heartshaped 6-lengthened cylindrical 7-pearshaped 8-plumshaped 16 Fruit size at maturity (FS) 1-very small ( 10 cm) 17 Fruit number (FN) a b Diseased fruits (DF) Cracked fruits (CF) 18 Transverse section (TS) 19 Fruit weight (g) (FSSWg) 20 Fruit length (mm) (FL) 21 Fruit width (mm) (FW) 1-round 2-angular 22 Fruit shoulder shape (FSS) 1- Flat depressed 7- strongly depressed 3-slightly 23 Shape of pistil scar (SPS) 2-stellate 24 Brix (oBx / TSS) 25 Seed colour (SC) 1- Light 2- dark Yellow 1- dot 91 3-lrregular x-mixture depressed 3-Linear 5-moderately 4- Irregular 3-Grey 4-Brown 5- Dark Brown APPENDIX IV: Survey Results Farmers who had cultivated tomato Afari Akomadan Frequency Percent Frequency percent Male 11 55.0 17 85.0 Female 45.0 15.0 Total 20 100 20 100 Number of Years of cultivating tomatoes Afari Frequency Percent less years 5.0 5-10 years 40.0 10-20 years 45.0 Above 20 years 5.0 Total 19 95 Kumawu Frequency percent 19 95.0 5.0 20 100 Akomadan Frequency percent 5.0 45.0 45.0 0.0 19 95 Kumawu Frequency percent 20.0 15 75.0 5.0 0.0 20 100 Size of land cultivated 1acre and below acres acres acres above acres Total Afari Frequency Percent 45.0 30.0 5.0 20.0 0.0 20 100 Akomadan Frequency percent 40.0 30.0 20.0 5.0 5.0 20 100 Kumawu Frequency Percent 16 0.0 20.0 0.0 0.0 0.0 20 20 Weight of Harvest 1-10 11-20 21-40 above 40 Total Afari Frequency Percent 40.0 10.0 30.0 20.0 20 100 Akomadan Frequency percent 25.0 20.0 30.0 25.0 20 100 Frequency 13 20 Kumawu percent 65.0 30.0 5.0 0.0 100 Afari Frequency Percent Akomadan Frequency percent Kumawu Frequency percent Shelf life less than week 1-3 weeks 3-4 weeks Total 16 20 20.0 80.0 0.0 100 13 20 30.0 65.0 5.0 100 92 17 20 85.0 10.0 5.0 100 Length of Period for saving seed 1- months months-12 months 13 months-24 months Above 24 months Total Afari Akomadan Kumawu Frequency Percent Frequency percent Frequency percent 5.0 5.0 5.0 15 75.0 30.0 30.0 15.0 20.0 35.0 0.0 15.0 20.0 19 95 14 70 18 90 Days to flower weeks and below weeks weeks above weeks Total Afari Akomadan Kumawu Frequency Percent Frequency percent Frequency percent 10.0 18 90.0 45.0 10.0 5.0 10.0 13 65.0 5.0 35.0 15.0 0.0 10.0 20 100 20 100 20 100 93 APPENDIX V: Qualitative Traits Data in percentage Plant Growth Habit, Stem Pubescence, Style Type, Leaf Type and Arrangement, External Colour of Immature Fruits, Anthocyanin Colouration of Hypocotyl, External Colour of Matured and Seed Colour Percentage Trait Description Class Number of plants (%) Dwarf 0 Determinate 0 PGH Indeterminate 0 Semi determinate 179 100 Mixture X 0 Absent 0 Weak 29 16.2 SP Medium 71 39.66 Strong 79 44.13 Inserted 0 Same level as stamen 179 100 ST Slightly exserted 0 Highly exserted 0 Mixture X 0 Dwarf 0 Potato leaf shape 60 33.52 Standard 104 58.1 LT Peruvianum 0 Pimpinellifolium 15 8.38 Hirsutum 0 Semi erect 109 60.89 Horizontal 70 39.11 LA Drooping 0 Mixture 0 Light green 103 57.54 Medium green 76 42.46 ECIF Dark green 0 Very Dark green 0 Mixture green X 0 Absent 0.56 ACH Present 0 Mixture X 178 99.44 Green 0 Yellow 0 Orange 0 ECMF Red 179 100 Pink 11 0 Mixture X 0 Light Yellow 89 49.72 Dark Yellow 30 16.76 SC Grey 30 16.76 Brown 30 16.76 Dark Brown 0 94 Predominant Fruit Shape, Fruit Shoulder Shape, Easiness to detach, Blossom End Shape, Shape of Pistil Scar, Transverse Section, Skin Colour of Ripened Fruit PFS FSS EFD BES SPS TS SCRF Flattened Slightly Flattened Round High Round Heart-Shaped Lengthened cylindrical Pear-shaped Plum-shaped Flat Slightly depressed Moderately depressed Strongly depressed Easy Intermediate Difficult Indented Flat Pointed Mixture Dot Stellate Linear Irregular Round Angular Irregular Mixture Green Yellow Orange Pink Light red Others (specify) 7 X X 95 70 49 15 25 20 16 36 94 33 62 56 61 114 31 29 129 29 16 41 54 84 0 0 179 39.11 27.37 8.38 13.97 11.17 8.94 20.11 52.51 18.44 34.64 31.28 34.08 63.69 17.32 16.2 2.79 72.07 16.2 2.79 8.94 22.91 30.17 46.93 0 0 100 APPENDIX VI: ANOVA for 16 quantitative traits Trait: Plant height Source of variation Replication Genotype Residual Total d.f 11 22 35 s.s 745.1 4504.2 4217.4 9466.7 m.s 372.6 409.5 191.7 v.r 1.94 2.14 F pr d.f 11 22 35 s.s 3.383 218.102 49.553 271.038 m.s 1.691 19.827 2.252 v.r 0.75 8.8 F pr d.f 11 22 35 s.s 53.247 237.32 124.593 415.16 m.s 26.623 21.575 5.663 v.r 4.7 3.81 F pr d.f 11 22 35 s.s 63.722 302.556 134.944 501.222 m.s 31.861 27.505 6.134 v.r 5.19 4.48 F pr s.s 71.056 372.556 208.944 652.556 m.s 35.528 33.869 9.497 v.r 3.74 3.57 F pr s.s 0.7756 62.0489 10.1311 72.9556 m.s 0.3878 5.6408 0.4605 v.r 0.84 12.25 F pr 0.062 Trait: Stem girth Source of variation Replication Genotype Residual Total