Map based gene cloning of arabidopsis thaliana gametophytic mutant

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY HA THI LIEU MAP-BASED GENE CLONING OF ARABIDOPSIS THALIANA GAMETOPHYTIC MUTANT BACHELOR THESIS Study mode : Full time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2013 – 2017 Supervisor : Professor Park Soon Ki Doctor Pham Bang Phuong Thai Nguyen – 2017 ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Ha Thi Lieu Student ID DTN1353150045 Thesis title Map-based gene cloning of Arabidopsis thaliana gametophytic mutant Professor Park Soon Ki Supervisor(s) Doctor Pham Bang Phuong Arabidopsis thaliana has recently became the organism of choice for a wide range of studies in plant sciences Map-based cloning is the method of identifying a mutation gene by looking for linkage to markers that physical location in Arabidopsis genome To find mutation gene, mutant line, named AP -29 – 38 of Landsberg erecta (Ler) was out – crossed with Columbia (Col) wild type plant The F generation was allowed to self – pollination and seeds were collected Abstract Individual F are planted and performed phenotype and genotype analysis DNA genome was extracted from leaves of F population PCR analysis was conducted using 10 Simple Sequence Length Polymorphisms (SSLPs) markers to define the chromosome is the region where mutation located For fine mapping of mutant gene on chromosome 1, known or self-made markers were used to narrow genetic interval down Candidate genes were sequenced to identify the mutant gene Keywords Arabidopsis thaliana, map-based cloning, AP-29-38 Number of pages 28 ACKNOWLEDGEMENT To accomplish this thesis, first and foremost, I would like to express my sincere gratitude to the Professor Park Soon Ki and Doctor Oh Sung Aeong, who gave me helpful advice and guidance during the whole period I am thankful to Saima Akhter for her contributions and helpful discussions during the course I would like to thank the members of Sexual Plant Reproduction Laboratory for their help I would also like to extend my heartfelt thanks to Doctor Pham Bang Phuong, who helped me with useful advice during my internship I am grateful to the members of the faculty of Biotechnology and Food Technology for their support Finally, I am especially gratitude to my family and my friends for their encouragement, generous support and understanding during all of this long campaign Ha Thi Lieu CONTENTS LIST OF FIGURES i LIST OF TABLES ii LIST OF ABBREVIATIONS iii PART I INTRODUCTION PART II MATERIALS AND METHODS 2.1.Materials and equipment 2.1.1.Plant material for map-base cloning and growth conditions 2.1.2.Equipment 2.2.Methods 2.1.1.Create population 2.2.2.DNA extraction 2.2.3.Genetic analysis using SSLP markers 2.2.4.Electrophoresis 11 2.2.5.DNA purification 12 PART III RESULTS AND DISCUSSION 13 3.1.DNA genomic extraction 13 3.2.Genetic analysis using SSLP markers 13 3.2.1.Identify chromosomes containing mutation region 13 3.2.2.Narrow mutation region down on chromosome 14 REFERENCES 18 RESULT PAPERS 18 WEBSITE 19 Appendices Result of PCR-based analysis using SSLP markers in chromosome 20 LIST OF FIGURES Figure Arabidopsis thaliana Figure Process of create F population Figure Procedure of AP – 29 -38 map –based cloning Figure Process of DNA extraction (CTAB method) Figure Location of 10 SSLP markers in each chromosome Figure Location of markers in chromosome 10 Figure Principle of SSLP mapping 11 Figure Process of DNA purification 12 Figure Examples of electrophoresis with wild type plant in mapping line (AP-29-38) for linkage analysis 15 Figure 10 A schematic diagram of the positional cloning 16 i LIST OF TABLES No of tables Title Page Table List of primers and sequences used in this 10 study Table Result of PCR-based analysis using SSLP 14 markers in each chromosome ii LIST OF ABBREVIATIONS ADW Autoclaved distilled water Col Columbia CTAB Cetyltriethy-ammonium bromide DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid IAA Isoamyl Alcolhol Ler Landsberg erecta PCR Polymerase Chain Reaction SSLP Simple Sequence Length Polymorphism TAE Tris-acetate-EDTA dNTPs Deoxynucleotide triphosphates WB Washing buffer iii PART I INTRODUCTION Arabidopsis thaliana is a small flowering, double leaf plant, native to Eurasia Arabidopsis thaliana is considered a weed; it is found by roadsides and in disturbed land [8] Arabidopsis thaliana is an annual (rarely biennial) plant, usually growing to 20–25 cm tall [12] The leaves form a rosette at the base of the plant, with a few leaves also on the flowering stem The basal leaves are green to slightly purplish in color, 1.5–5 cm long and 2–10 mm broad, with an entire to coarsely serrated margin; the stem leaves are smaller and unstalked, usually with an entire margin Leaves are covered with small, unicellular hairs (called trichomes) [7] The flowers are 3mm in diameter, arranged in a corymb; their structure is that of the typical Brassicaceae The fruit is a siliqua 5–20 mm long, containing 20–30 seeds Roots are simple in structure, with a single primary root that grows vertically downward, later producing smaller lateral roots Arabidopsis thaliana can complete its entire life cycle in 6-8 weeks [18] The central stem that produces flowers grows after about three weeks, and the flowers naturally self-pollinate In the lab, Arabidopsis thaliana may be grown in petri plates, pots, or hydroponics, under fluorescent lights or in a green house The Arabidopsis thaliana genome was sequenced in 2000 by the Arabidopsis Genome Initiative (AGI) The genome has five chromosomes and a total size of approximately 135-megabases Physical maps of all chromosomes completed in 1997 The biggest is chromosome and the smallest is chromosome [19] The first mutant in A thaliana was documented in 1873 by Alexander Braun, describing a double flower phenotype (the mutated gene was likely Agamous, cloned and characterized in 1990) [3] However, not until 1943 did Friedrich Laibach (who had published the chromosome number in 1907) propose A thaliana as a model organism In the 1980s, A thaliana started to become widely used in plant research laboratories around the world It was one of several candidates that included maize , petunia, and tobacco [3] The first gene sequences were published in 1986, and TDNA- mediated transformation of Arabidopsis was also first established in 1986 This was followed by the first restriction fragment-length polymorphism map in 1988, T1 DNA insertional cloning, map-based cloning, and the extremely efficient vacuum infiltration method of transformation Each method was developed to solve specific biological problems, and each added to the reasons to use Arabidopsis in laboratory [3] The fowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions The developing flower has four basic organs: sepals, petals, stamens, and carpels (which go on to form pistils) Observations of homeotic mutations led to the formulation of the ABC model of flower development by E Coen and E Meyerowitz [4] According to this model, floral organ identity genes are divided into three classes: class A genes (which affect sepals and petals), class B genes (which affect petals and stamens), and class C genes (which affect stamens and carpels) These genes code for transcription factors that combine to cause tissue specification in their respective regions during development Although developed through study of A thaliana flowers, this model is generally applicable to other flowering plants Gametophytic mutants affecting various aspects of pollen development and function in Arabidopsis thaliana have been identified through genetic screens for segregation distortion [11] Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development [1] In flowering plants, development of the haploid male gametophytes (pollen grains) takes place in a specialized structure called the anther Successful pollen development, and thus reproduction, requires high secretory activity in both anther tissues and pollen The life cycle of flowering plants alternates between a haploid organism, the gametophyte, and a diploid organism, the sporophyte Plants have male and female gametophytes, both of which are multicellular The male gametophyte, the pollen grain, is, at the mature stage, a threecell organism consisting of a vegetative cell and two sperm cells Pollen development starts inside the anther, which is a specialized structure of the flower, with the meiotic divisions of the microsporocytes to form a tetrad of haploid spores [10] Although pollen-specific genes have been studied extensively in most cases it remains unclear what roles these genes play We have used a mutational approach to identify genes involved in the control of pollen development [14] The gametophyte is a sexual stage in the life cycle of plants and algae undergoes generational changes A mature sporophyte produces spores by meiosis, a process which reduces the number of chromosomes to half, from 2n to n The haploid spores germinate and grow into a haploid gametophyte At maturity, the gametophyte produces gametes by mitosis, which does not alter the number of chromosomes Two gametes (originating from different organisms of the same species or from the same organism) fuse to produce a zygote, which develops into a diploid sporophyte This cycle, from gametophyte to gametophyte (or equally from sporophyte to sporophyte), is the way in which all land plants and many algae undergo sexual reproduction [15] Mutations that arise during the gametocyte degeneration, which occur in certain genital cells through fertilization, enter the zygote Change the molecular structure of the gene can lead to structural changes of the type of protein that it encodes, eventually could lead to changes in the phenotype Identification of a gametophyte mutant gene is important because this gene might affect on reproduction process In this study, I performed a population consisting of 852 plants in Arabidopsis used map-based cloning method to identify a gene involved gametophytic in Arabidopsis, the mutant line called AP-29-38 Arabidopsis was chosen as the model plant by many advantages: small and easy to grow, a short life cycle (see a complete plant life cycle in 6-8 weeks), known full sequence, easily mutated, not consuming plantings but create many seeds, selfpollination and relationship closeness with edible plants, adapted to live in many different environments [2] It has been introduced and naturalized worldwide Arabidopsis genome possesses almost the smallest of the higher plant with about 125MB contains approximately 26,000 genes distributed on five chromosomes; The genome is 7.5 times smaller than the tomato genome, 19 times corn, and 128 times more wheat [6] Characterized accessions and mutant lines of A thaliana serve as experimental material in laboratory studies Various strategies for the identification of plant genes have been used Analyzing DNA using molecular markers was very fast and accurate strategy and map-based cloning was used to identify the mutated gene in this study Map-based cloning (or positional cloning) is the process of identifying the genetic Table Result of PCR-based analysis using 10 SSLP markers in each chromosome No Samples Phenotype nga63 nga280 nga168 nga6 nga162 17200 nga1107 CTR1 PHYC M1324 Chro# Chro# Chro# Chro# Chro# Chro# Chro# Chro# Chro# Chro# At1g09910 At1g55840 At2g39010 At3g62220 At3g13950 At4g17200 At4g38770 At5g03740 At5g35840 At5g40750 Sample-1 Wild type Sample-2 Wild type Sample-3 Wild type Sample-4 Wild type Sample-5 Wild type Sample-6 Wild type Sample-7 Wild type Sample-8 Wild type 10 Sample-9 Wild type Sample-10 Wild type 11 Sample-11 Wild type L C C/L C/L C C/L L C C/L C/L L C/L 14 Sample-14 Wild type C/L 15 Sample-15 Wild type C/L 16 Sample-16 Wild type C/L 17 Sample-17 Wild type C 18 Sample-18 Wild type C/L 19 Sample-19 Wild type C 20 Sample-20 Wild type C/L Recombinant gene/ Total gene 11/20 12 Sample-12 Wild type 13 Sample-13 Wild type C C C C C C C C C C C C C C C C C C C C 0/20 C C/L C C/L C C/L C L L C/L C/L C/L C/L C/L C/L C/L L L C C/L C/L L C/L C/L L L C/L C/L C C/L C/L L C L C/L C C L C/L C/L L C/L L C/L C/L C C L L C/L C/L C/L C C C C/L C/L C C L C C C/L C/L C L C/L C/L C/L C L L C/L L L C C/L C C/L C/L C/L C/L C/L L C/L L C/L C/L C/L 10/20 C/L C/L C/L C/L C/L C/L C/L C/L L 10/20 C C C/L C/L C C C/L L C/L 10/20 C C/L C/L L C C/L C/L C/L C/L 10/20 C C/L L C C L L C/L L 8/20 C/L C L C/L C C/L C/L C/L C 7/20 C C/L C C L C/L C/L C C/L 9/20 L C/L L L C C/L C/L L C/L 8/20 3.2.2 Narrow mutation region down on chromosome Once a chromosome closely linked to mutation of interest is defined, fine mapping can be effectively carried out by searching the recombinants in the vicinity of the mutation using two markers flanking mutation on both sides [13] As a beginning of fine mapping process, two flanking markers, nga111 and nga280, successively found mutation point in chromosome We attempted to narrow the genetic distance down by checked the recombinants from nga111 and nga280 using 56130 and 59620 markers The recombinant samples from these two markers are sent for sequencing to continuos narrow down the mutant region 14 A B C D Figure Examples of electrophoresis with wild type plant in mapping line (AP-2938) for linkage analysis A & B Marker: nga111 (chromosome 1) with 26 samples C & D Marker: nga280 (chromosome 1) with 26 samples (Control samples L: Ler-0; C: Col-0; Ht: Heterozygous) 15 Figure 10 show the schematic diagram of the position cloning of mutant line AP – 29 -38 In this figure, the mutant region was narrowed down to about 400kb using two markers 56630 and 58360 Figure 10 A schematic diagram of the positional cloning 16 PART IV CONCLUSION Map-based cloning (position cloning) is a unique approach that identifies underlying genetic cause of a mutant phenotype by looking for the linkage to markers whose physical location in the genome is known For map-based gene cloning, mutant plant is out-crossed with opposite ecotype F seeds were collected from self- pollinated plants F progeny is used for mapping population DNA genomic was extracted from individual F leaf To indentify the location of the mutant gene, we test 20 wildtype samples with 10 markers cover Arabidopsis chromosome In each chromosome would be have to set of SSLP markers Result suggested that the mutant gene was located in chromosome Finally, the region containing mutant gene was narrowed down to find candidates gene responsible for mutation phenotype The region was narrowed down to more than 400Kb In Arabidopsis a genetic distance of 1% recombination corresponds, on average, to a physical distance of about 250Kb However, the ratio between genetic and physical distance is by no means constant and it varies with respect to position on the chromosome as well as with respect to different mapping populations Mapping resolution is mainly determined by the size of a mapping population [13] The ultimate goal of fine mapping is to narrow down the region containing the gene of interest to 40 kb or less (approximately 0.16 cM genetic distance in Arabidopsis) There would ideally be several recombination events in this interval to define the position of the mutation that is being mapped So we are working to reduce the genetic distance on the chromosome to find the position of the mutation 17 REFERENCES RESULT PAPERS Antonia Procissi, Solveig de Laissardie`re, Madina Fe´rault, Daniel Vezon, Georges Pelletier, & Sandrine Bonhomme (2001) Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana Genetics 158, p 1773–1783 David W Meinke, J Michael Cherry, Caroline Dean, Steven D Rounsley, & Maarten Koornneef (1998) Arabidopsis thaliana: a model plant for genenome analysis Science 23 , pp 662-682 Elliot M Meyerowitz (2001) Prehistory and History of Arabidopsis Research Plant Physiology, pp 15–19 Enrico S Coen, & Elliot M Meyerowitz (1991) The war of the whorls: genetic interactions controlling flower development pp 31-37 Jander G, Norris SR, Rounsley SD, Bush DF, Levin IM, & Last RL (2002) Arabidopsis Map-based Cloning in the Post-Genome Era Plant Physiol, pp 440–450 Kirankumar S Mysore, Robert P Tuori, & Gregory B Martin (2001) Arabidopsis genome sequence as a tool for functional genomics in tomato Genome Biology, reviews1003.1–1003.4 Maarten Koornneef, & Ben Scheres (2011) Arabidopsis thaliana as an experimental organism Encyclopedia of Life Sciences, pp 662–682 Matthias H., & Hoffmann (2002) Biogeography of Arabidopsis thaliana (L.) Heynh (Brassicaceae) Journal of Biogeography, pp 125–134 Mi Kwon, Hyun Kyung Lee, & Sunghwa Choe (2005) Novel Simple Sequence Length Polymorphic (SSLP) Markers for Positional Cloning in Arabidopsis thaliana The Genetics Society of Korea, pp 1-8 10 Mia Kyed Jakobsen, Lisbeth R Poulsen, Alexander Schulz, Pierrette Fleurat-Lessard, Annette Møller, Søren Husted, et al (2005) Pollen development and fertilization in 18 Arabidopsis is dependent on the male gametogenesis impaired anthers gene encoding a Type V P-type ATPase Genes Development, pp 2757–2769 11 S Bonhomme, C Horlow, D Vezon, S de Laissardière, A Guyon, M Férault, et al (1998) T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone Molecular and General Genetics MGG, pp 444–452 12 Ute Krämer (2015) Planting molecular functions in an ecological context with Arabidopsis thaliana elife sciences, pp 4222-4240 13 Wolfgang Lukowitz, C Stewart Gillmor, & Wolf-Rüdiger Scheible (2000) Positional Cloning in Arabidopsis Why It Feels Good to Have a Genome Initiative Working for You Plant Physiology, pp.123.3.795 14 Y.C Chen, & S McCormick (1996) Sidecar pollen, an Arabidopsis thaliana male gametophytic mutant with aberrant cell divisions during pollen development Development, pp 3243-3253 WEBSITE 15 http://biotechcrunch.blogspot.kr/2011/11/9-factors-affecting-dna-extractionfrom.html 16 https://en.wikipedia.org/wiki/Alternation_of_generations 17 https://www.arabidopsis.org/portals/education/aboutarabidopsis.jsp 18 https://www.arabidopsis.org/portals/genAnnotation/gene_structural_annotation/agico mplete.jsp 19 https://www.sciencelearn.org.nz/resources/2036-dna-extraction 19 Appendices Result of PCR-based analysis using SSLP markers in chromosome No Samples Phenotype 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 SA-52-01 SA-52-02 SA-52-03 SA-52-04 SA-52-05 SA-52-06 SA-52-07 SA-52-08 SA-52-09 SA-52-10 SA-52-11 SA-52-12 SA-52-13 SA-52-14 SA-52-17 SA-52-18 SA-52-19 SA-52-20 SA-52-21 SA-52-23 SA-52-24 SA-52-25 SA-52-26 SA-52-27 SA-52-29 SA-52-30 SA-52-31 SA-52-32 SA-52-33 SA-52-34 SA-52-35 SA-52-36 SA-52-38 SA-52-39 SA-52-40 SA-52-41 SA-52-42 SA-52-43 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type nga280 Chro# At1g55840 C C/L C C C C C C C/L C C C C/L C C C C C C C C C/L C/L C C C C C/L C C/L C C/L C C C C C/L 59620 Chro# At1g59620 C/L C C C C/L C C C C C/L C C C C C C/L C/L C C/L C/L C C C/L nga111 Chro# At1g72650 C/L C C/L C C C/L C/L C C/L C/L C/L C/L C/L C/L C/L C C/L C/L C/L C C/L C/L C/L C/L C C C/L C L C C/L C C/L C C C/L C/L C/L 20 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 71 72 73 74 75 76 77 78 79 80 SA-52-44 SA-52-45 SA-52-47 SA-52-48 SA-52-49 SA-53-03 SA-53-04 SA-53-06 SA-53-08 SA-53-09 SA-53-10 SA-53-11 SA-53-12 SA-53-14 SA-53-15 SA-53-16 SA-53-17 SA-53-18 SA-53-19 SA-53-20 SA-53-24 SA-53-25 SA-53-26 SA-53-28 SA-53-30 SA-53-31 SA-53-33 SA-53-34 SA-53-36 SA-53-38 SA-53-39 SA-53-41 SA-53-43 SA-53-44 SA-53-46 SA-53-47 SA-53-49 SA-54-03 SA-54-06 SA-54-07 SA-54-12 SA-54-14 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C/L C C C C C C C C C C C C C C C C C C C C C C C C C C C C L C C C C C C C C C C C C C/L C C C C C C C C C C C C L C C C C/L C/L C/L C C C C/L C C C C C/L C C C/L C/L C C C/L C/L C L L C/L C/L C C C C/L C/L C C/L C C C C C C L C L C 21 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 SA-54-16 SA-54-17 SA-54-19 SA-54-21 SA-54-23 SA-54-24 SA-54-25 SA-54-26 SA-54-27 SA-54-28 SA-54-30 SA-54-31 SA-54-34 SA-54-36 SA-54-37 SA-54-38 SA-54-40 SA-54-42 SA-54-45 SA-54-47 SA-54-50 SA-55-01 SA-55-02 SA-55-03 SA-55-04 SA-55-05 SA-55-06 SA-55-07 SA-55-08 SA-55-09 SA-55-10 SA-55-11 SA-55-12 SA-55-13 SA-55-14 SA-55-15 SA-55-16 SA-55-17 SA-55-19 SA-55-20 SA-55-21 SA-55-23 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C C C C C C C L C C C C C C C C C C/L C C C C C C C C C C C C C C C C C C C C C C C C C C C C L C C C C C/L C C C C C C C C C C C C C/L C/L C C C/L C/L C/L C/L C/L C C C C C/L C/L C C/L C C C C/L C C C C C/L C C/L C/L C/L C C/L C/L C C/L C C/L C L C/L L 22 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 SA-55-26 SA-55-27 SA-55-29 SA-55-30 SA-55-34 SA-55-36 SA-55-37 SA-55-38 SA-55-39 SA-55-40 SA-55-41 SA-55-42 SA-55-44 SA-55-48 SA-55-49 SA-61-03 SA-61-04 SA-61-07 SA-61-08 SA-61-11 SA-61-12 SA-61-15 SA-61-17 SA-61-18 SA-61-19 SA-61-22 SA-61-23 SA-61-24 SA-61-25 SA-61-27 SA-61-32 SA-61-33 SA-61-35 SA-61-36 SA-61-37 SA-61-38 SA-61-39 SA-61-41 SA-61-43 SA-61-44 SA-61-45 SA-61-46 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Mutant Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C/L C C C C C C/L C C/L C C C C/L C C C/L C C C/L C/L C C/L C C C L C C C C C C C/L C C C C C C C/L L C C/L C/L 23 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 SA-61-47 SA-61-48 SA-61-49 SA-61-50 SA-62-02 SA-62-07 SA-62-09 SA-62-11 SA-62-12 SA-62-14 SA-62-22 SA-62-23 SA-62-25 SA-62-26 SA-62-27 SA-62-29 SA-62-30 SA-62-31 SA-62-33 SA-62-34 SA-62-37 SA-62-38 SA-62-40 SA-62-41 SA-62-46 SA-62-49 SA-62-50 SA-69-01 SA-69-02 SA-69-03 SA-69-04 SA-69-05 SA-69-06 SA-69-07 SA-69-08 SA-69-09 SA-69-10 SA-69-11 SA-69-13 SA-69-14 SA-69-15 SA-69-16 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type not gem Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C C C C C C C C C C/L C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C/L C C C C C C C C C C C/L C L C C/L C C C C/L C C C C C/L C C C C/L C C C C L C C C C/L C C C/L C C/L C/L C C C C/L C C C/L C 24 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 SA-69-17 SA-69-18 SA-69-19 SA-69-20 SA-69-22 SA-69-24 SA-69-25 SA-69-26 SA-69-27 SA-69-28 SA-69-31 SA-69-32 SA-69-34 SA-69-35 SA-69-36 SA-69-38 SA-69-40 SA-69-41 SA-69-42 SA-69-43 SA-69-44 SA-69-45 SA-69-46 SA-69-47 SA-69-48 SA-69-49 SA-69-50 SA-70-01 SA-70-02 SA-70-03 SA-70-04 SA-70-05 SA-70-06 SA-70-07 SA-70-10 SA-70-11 SA-70-12 SA-70-13 SA-70-14 SA-70-15 SA-70-16 SA-70-17 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C/L C/L C C C C/L C C/L C C C/L C/L C C/L C C/L C/L C C C/L C C/L C/L C C C C/L C C C C C C/L C C/L C C/L C C C/L C C 25 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 SA-70-18 SA-70-19 SA-70-20 SA-70-21 SA-70-22 SA-70-23 SA-70-24 SA-70-25 SA-70-27 SA-70-28 SA-70-29 SA-70-30 SA-70-31 SA-70-32 SA-70-33 SA-70-34 SA-70-35 SA-70-36 SA-70-37 SA-70-38 SA-70-40 SA-70-41 SA-70-42 SA-70-43 SA-70-46 SA-70-47 SA-70-48 SA-70-49 SA-70-50 SA-73-01 SA-73-02 SA-73-03 SA-73-04 SA-73-05 SA-73-07 SA-73-08 SA-73-09 SA-73-10 SA-73-11 SA-73-12 SA-73-13 SA-73-14 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C/L L C/L C C C/L C/L C C C/L C/L C/L C C C C C C/L C/L C/L C/L C C C/L C/L C C/L C C C C C C C C C C C/L C C/L C C 26 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 SA-73-15 SA-73-16 SA-73-18 SA-73-19 SA-73-21 SA-73-22 SA-73-23 SA-73-24 SA-73-25 SA-73-26 SA-73-27 SA-73-28 SA-73-30 SA-73-31 SA-73-32 SA-73-33 SA-73-34 SA-73-36 SA-73-37 SA-73-38 SA-73-39 SA-73-40 SA-73-41 SA-73-42 SA-73-43 SA-73-44 SA-73-45 SA-73-46 SA-73-47 SA-73-48 SA-73-49 SA-73-50 SA-74-01 SA-74-02 SA-74-03 SA-74-04 SA-74-05 SA-74-08 SA-74-09 SA-74-10 SA-74-11 SA-74-12 Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Mutant Wild type Wild type Wild type Wild type C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C/L C C C C C C C/L C C C C C C C C C C C C C C/L C C C C C/L C/L C/L C/L C C C C C C/L C/L C C C/L C/L C/L C C C/L C C/L L C/L C/L C C C/L C C C/L C/L C C C C/L C 27 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 Total SA-74-13 Wild type SA-74-14 Wild type SA-74-16 Wild type SA-74-19 Wild type SA-74-20 Wild type SA-74-21 Wild type SA-74-23 Wild type SA-74-24 Wild type SA-74-25 Wild type SA-74-28 Wild type SA-74-29 Wild type SA-74-30 Wild type SA-74-31 Wild type SA-74-32 Wild type SA-74-33 Wild type SA-74-34 Wild type SA-74-35 Wild type SA-74-36 Wild type SA-74-37 Wild type SA-74-38 Wild type SA-74-39 Wild type SA-74-40 Wild type SA-74-42 Wild type SA-74-43 Wild type SA-74-44 Wild type SA-74-45 Wild type SA-74-47 Wild type SA-74-48 Wild type SA-74-49 Wild type Recombinant/ Chromosome C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 13/360 C C C C C C/L C C C C 14/149 C/L C/L C C C C C C C C C C C C/L C/L C/L C C/L C C C C/L C/L C C/L C C C/L C 141/361 28 ... strategy and map- based cloning was used to identify the mutated gene in this study Map- based cloning (or positional cloning) is the process of identifying the genetic basic of a mutant phenotype... University of Agriculture and Forestry Major Biotechnology Student name Ha Thi Lieu Student ID DTN1353150045 Thesis title Map- based gene cloning of Arabidopsis thaliana gametophytic mutant Professor... mutant gene on chromosome 1, known or self-made markers were used to narrow genetic interval down Candidate genes were sequenced to identify the mutant gene Keywords Arabidopsis thaliana, map- based