THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE VIET TRINH PCHARACTERIZATION AND MAP-BASED CLONING OF A NOVEL MUTANT CAUSING ABNORMAL LEAF IN Arabidopsis Thaliana BACHELOR THESIS Study Mode: Full Time Major: Biotechnology Faculty: Biotechnology & Food Technology Batch: 2012-2016 Thai Nguyen, 2016 n THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE VIET TRINH PHENOTYPIC CHARACTERIZATION AND MAP-BASED CLONING OF A NOVEL MUTANT CAUSING ABNORMAL LEAF IN Arabidopsis Thaliana BACHELOR THESIS Study Mode: Full Time Major: Biotechnology Faculty: Biotechnology & Food Technology Batch: 2012-2016 Supervisors: Professor Soon- Ki Park. Doctor Bang Phuong Pham. Thai Nguyen, 2016 n DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Viet Trinh Le Student ID DTN1153150084 Thesis title PHENOTYPIC CHARACTERIZATION AND MAP-BASED CLONING OF A NOVEL MUTANT CAUSING ABNORMAL LEAF IN ARABIDOPSIS THALIANA Supervisor(s) Professor Soon-Ki Park Dr. Bang Phuong Pham Abstract: This study was carried out to identify the mutant gene causing Leaf Rolled Inside (LRI) phenotype of mutant line named as AP-44-1.
Mutant line obviously showed curly leaf for all rosette leaves, less leaf number compared to wild type. To identify mutant gene, F2 mapping population was generated for map-based cloning using SSLP markers. Based on PCR analysis, the LRI gene was predicted to locate between 32160 and 32580 markers that containing 49 candidate in the region of approximately 163kb. At2g32460 gene that was identified by sequencing and compared with previous report (An et al., 2014) is a strong candidate causing abnormal leaf phenotype.
Based on the Arabidopsis database (TAIR; http://www.org), At2g32460 is the gene coding for a member of the R2R3-MYB transcription factor family and was designated MYB101. The expression and genetic complementation of At2g32460 is being carried out to investigate the responsible of gene to abnormal leaf phenotype. Keyword: Arabidopsis, map-based, cloning, curly leaf, leaf-rolled inside Number of pages: 36 Date of 2016/08/29 submission: n ACKNOWLEDGEMENT I would like to sincerely thank my supervisor, Prof. Soon-Ki Park, for all the guidance and support me to develop an understanding of the subject.
I am also thankful to Dr. Sung-Aeong Oh and Dr. Tien- Dung Nguyen for all advices. I also would like to thank MSc.
Thi Hoai Thuong Nguyen, MSc. Hyo-Jin Park for their technical support. I wish to thank to graduate students Rupesh Tayade, Saima Samin and Thu Huong Nguyen, for their help and friendship. It is a pleasure to thank those who made this thesis possible to complete.
I would like to give a very special thanks to my supervisor committee members, Dr. Bang Phuong Pham. Despite the geographical distance, my family was always nearby. I am grateful for their love and believing in me.
This thesis would not have been possible unless their support. Lastly, I would like to thank Faculty of Biotechnology and Food Technology members for their support through my internship. n CONTENT LIST OF FIGURES. i LIST OF TABLES.
ii LIST OF ABBREVIATIONS. MATERIALS AND METHODS. Plant materials and growth condition. Genetic analysis using SSLP markers for positional cloning of AP- 44-1.
Phenotypic characterization of a novel mutant causing abnormal leaf. RESULT AND DISCUSSION. Plant morphological analysis. Morphological phenotypes of AP-44-1 mutants showing defects and sterility.
Comparative analysis of growth and biological activity. Fine mapping of AP-44-1 locus. SUMMARY AND CONCLUSION. 33 n LIST OF FIGURE Figure No.
Title Page Figure 1 Arabidopsis Thaliana model 2 Figure 2 Procedure of map and clone mutation 6 Figure 3 Arabidopsis growth room 8 Figure 4 Generation of F2 mapping population 14 Figure 5 Procedure of a typical map-based cloning experiment 15 Figure 6 Principle of PCR-based mapping using SSLP markers 15 Figure 7 Steps of PCR purification 17 Figure 8 Comparative analysis of the morphological phenotypes of 19 wild-type, and AP-44-1 plants Figure 9 Plants at flower stage 20 Figure 10 Example of linkage analysis with four markers using wild- 22 type plants in the mapping population (AP-44-1) Figure 11 Example of linkage analysis with four SSLP markers to 23 narrow down the region of mutant gene using wild-type plant in the mapping population (AP-44-1) Figure 12 Result of PCR analysis with three sets of primer 32460 24 Figure 13 Schematic representation of SSLP markers positions used 25 in the genetic mapping experiment Figure 14 A schematic of the positional cloning of the AP-44-1 gene 28 (A) and structure of candidate gene i n LIST OF TABLE Table Title Page No. Table 1 Primer sequences used in this study 12 Table 2 Identification of chromosome containing the gene of 27 interest Table 3 Recombinants by PCR analysis of a large mapping 28 population with flanking markers ii n LIST OF ABBREVIATIONS ADW Autoclaved distilled water Col-0 Columbia CTAB Cetyltriethy-ammonium bromide Ler-0 Landsberg erecta DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid IAA Isoamyl Alcohol LRI Leaf rolled inside PCR Polymerase Chain Reaction SSLP Simple Sequnce Length Polymophic TAE Tris-acetate-EDTA dNTPs Deoxynucleotide WB Ethanol 75% CLF Curling leaf CTAB Cetyl trimethylammonium bromide iii n PART I. INTRODUCTION Arabidopsis Thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis has been used as an ideal model for studying the plant biology and genetics.
As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production (Boyes et al. There is ample reason to believe that Arabidopsis will serve as a resource base for breeders of crop plant and as a model plant that furthers the knowledge of plant scientists (Hayashi and Nishimura, 2006). Classified in a member of the mustard and cabbage plants, Arabidopsis has several advantages that make it an excellent experimental model (Hartwell et al. Not only the smallest genome makes Arabidopsis useful for genetic mapping and sequencing, also it could be easily grown in the laboratory.
In addition, its small size and rapid life cycle, approximately 6-8 weeks, are also advantageous for research. Finally, mutants are easily induced by treating the seeds with various chemical mutagens. The surviving seeds are the germinated and mutant progeny are recovered for analysis (Hopkins et al. Over 750 natural accessions of Arabidopsis thaliana have been collected from around the world and are available from the two major seed stock centers, ABRC (Arabidopsis Biological Resource Center) and NASC (Nottingham Arabidopsis Stock Centre).
These accessions are quite variable in terms of form and development (e. leaf shape, hairiness) and physiology (e. flowering time, disease resistance). Researchers around the world are using these differences in natural accessions to uncover the complex genetic interactions such as those underlying plant responses to environment and evolution of morphological traits.
While many collections of natural accessions may not meet a strict definition of an ecotype, they are commonly referred to as ecotypes in the scientific literature. Arabidopsis Thaliana model (TAIR) Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues (An et al. Optimum leaf shape and size are very important for photosynthesis process that directly effect on seeds of yield and quality also. Leaf physiological functions are supported by several specialized cell types, such as paired guard cells in the epidermis for gas exchange, mesophyll cells for photosynthesis, and vascular cells for internal fluid and nutrient transport.
As a fundamental component of the plant body, the continuous vascular network provides not only mechanical strength but also the key role of transport: the vascular tissue xylem transports water and minerals, and phloem translocates dissolved photoassimilates efficiently. Leaf morphogenesis corresponds closely with genetic controls and environmental factors and often used to distinguish different plant species (Tsukaya, 2005). Over the past two decades, the isolation of leaf morphological mutants of Arabidopsis thaliana has been commonly used to further genetic studies of leaf development (Scarpella et al. 2 n The morphology of multicellular organisms is attributable to mechanisms that regulate the shapes, sizes, and numbers of the constituent cells.
In higher animals, the body plan is basically established at the stage of gastrulation. By contrast, in plants, the body plan is not strictly determined and, throughout the life cycle of the plant, new organs are added to the body via meristems located at the apices of the roots and shoots. The fundamental unit of each vegetative shoot system can be considered to consist of a leaf, an internode, and a lateral bud (Kim et al. Our current goal is the identification of the various genes that control the development of the leaf, a fundamental component of the shoot.
Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype. The major strength of this approach is the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes (Jander et al. Genetic mapping of a mutation-defined gene is the first step toward isolating and cloning the corresponding normal gene and ultimately identifying its encoded protein. Various techniques are used to produce a genetic map of a chromosome, which indicates the positions of genes relative to one another along the length of the chromosome.
In a physical map, the number of nucleotides between known genes is indicated (Lodish et al. Mapping a novel mutation to a well-defined chromosomal region is an essential step in the genetic analysis of this mutant, and is also (unless the mutant is tagged) a prerequisite for molecular cloning of the corresponding gene. Determining the map position of a gene (as identified by its mutant phenotype) consists in testing linkage with a number of previously mapped markers. Once linkage with a specific marker is detected, a refined mapping can be achieved by analysing linkage relations to more markers in that region (Giraudat et al.
By comparing a genetic map and corresponding physical map, the actual physical position of any gene can be determined. Whereas reverse genetics strategies seek to identify and select mutations in a known sequence, forward genetics requires the cloning of sequences underlying a particular mutant phenotype. 3 n Map-based cloning is tedious, hampering the quick identification of candidate genes. With the unprecedented progress in the sequencing of whole genomes, and perhaps even more with the development of saturating marker technologies, map-based cloning can now be performed so efficiently that, at least for some plant model systems, it has become feasible to identify some candidate genes within a few months (Janny et al.
Historically, mapping in Arabidopsis primarily utilised morphological markers such as mutants with an easily scorable phenotype and a defined map position. Typically, the mutant of interest is crossed to another mutant used as phenotypic marker, the resulting F1 double heterozygote is allowed to self, and the segregation of the two phenotypes is analysed in the F2 population. The mutation used as marker should of course not interfere with the phenotype of the mutant to be mapped. The genetic distance is the number of meiotic recombination events that occur between the two loci in 100 chromosomes.
To facilitate mapping, tester lines that are recessive for several morphological markers have been constructed and can be ordered from NASC (http://nasc. In other word, to map a novel mutation that was generated in ecotype A, this mutant is crossed with a wild-type plant of a polymorphic ecotype B, and the F1 progeny is allowed to self. The resulting F2 population can then be used to analyse the linkage between the mutation of interest and any DNA marker that distinguishes ecotypes A and B. As compared to morphological markers, an additional advantage of molecular markers is that in most cases homozygous and heterozygous individuals can be readily distinguished (Giraudat et al.
• Map-based cloning in Arabidopsis. In the course of map-based cloning, mutant genes are identified through linkage to a sufficiently small region of the genetic map and subsequent DNA sequencing.