Development of Pre-breeding Materials in Capsicum spp. and Map-based Cloning of a Male Sterile Gene, ms1035 in Tomato

학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부(원예과학전공), 2014. 2. 강병철.Tomato and pepper belong to the Solanaceae family and economically important vegetable crops worldwide. The main objectives of tomato and pepper breeding are developing high yield, pest resistant, and quality cultivars. Narrowed genetic diversity in commercial lines due to under-utilization of germlasm is a serious problem in tomato and pepper breeding. Therefore, discovering of allelic diversity and characterization of useful traits are essential for breeding. In the first chapter, species identification markers for Capsicum spp. were developed. Identification of Capsicum species has always been controversial because of ambiguous morphological behaviors. Moreover, most previous studies for species identification using molecular markers also had disadvantages that were low reproducibility and unsuitability for handling large quantities of samples. Here, SNP markers derivd from conserved ortholog set II (COS II) and the Waxy gene were developed as candidate maker set for species identification. Furthermore, phylogenetic analysis supported that C. annuum, C. baccatum, and C. pubescens clades were clearly separated. Closely related species C. chinense and C. frutescens were divided into subclades, indicating that the two species are apparently different. C. chacoense, the most controversial species, was belonged to C. baccatum clade. These results provide the information of genetic relationship of Capsicum species and reliable species classification method. In the second chapter, allele mining was performed to find useful allelic variation of a virus resistance gene in natural germplasm pool and ethyl methanesulfonate (EMS) mutagenized population to enhance the genetic diversity of Capsicum species. A fast and accurate method was developed to mine the useful alleles in significant number of germplasm using high resolution melting (HRM) analysis. Of 248 pepper germplasm, 13 newly obtained allelic variations of eIF4E were identified and one accession of them, C03946 (pvrHRM113) from C. baccatum was shown to carry strong resistance to TEV-HAT virus. Furthremore, five more allelic variations from EMS-mutageneized M1 population were also identified. Therefore, this result provides the strength of genetic diversity in Capsicum spp. and a useful tool for mining novel alleles. In the third chapter, a male sterile gene of tomato was characterized. Male sterility is the one of the most important characteristics for plant breeding. Here, the map-based cloning of the ms1035 mutant of tomato was performed and a responsible gene encoding a bHLH transcription factor was discovered. With morphological defects and molecular evidence, Ms1035 played a pivotal role for pollen development after meiotic stage. ms1035 was firstly isolated gene in tomato discovered by map-based cloning among the pollen development related genes at early stage. This provides that the insight of molecular evidence for tomato pollen development and utilization of male sterility line to improve tomato breeding.Contents Abstract i Contents iv List of tables ix List of figures xi List of abbreviations xiii General introduction 1 Literature review 6 Chapter I Identification of Capsicum Species Using SNP Markers Based on High-Resolution Melting Analysis Abstract 61 Introduction 63 Materials and methods Plant materials 67 Morphological trait observation 67 Genomic DNA extraction 67 Marker screening 70 HRM analysis conditions 70 DNA sequence analysis 71 Dendrogram tree and genetic distance analysis 71 Results Species identification based on morphology 72 Development of species identification markers using nuclear DNA sequences 74 Phylogenetic analysis of Capsicum species 77 Discussion 83 References 88 Chapter II A Survey of Natural and EMS-induced Variations of eIF4E Using High-Resolution Melting Analysis in Capsicum Species Abstract 94 Introduction 96 Materials and methods Plant materials 99 Chemical mutagenesis 99 DNA extraction 101 HRM analysis 101 Sequencing and sequence alignment of eIF4E fragment 102 Virus inoculation and disease resistant evaluation 103 Results Screening of pvrHRM variation in Capsicum germplasm using HRM 104 Nucleotide and amino acid changes in natural variants 105 Virus resistance test on new allelic variants 108 Generation of an EMS mutant population 111 Application of HRM to mutation screening 116 Discussion 119 References 124 Chapter III Tomato Male Sterile Gene, ms1035 Regulates Tapetum Degeneration and Meiosis Abstract 131 Introduction 133 Materials and methods Plant materials 137 Phenotypic observation and genetic analysis of ms1035 137 Scanning electron microscopic (SEM) analysis 138 Light microscopic analysis and transmission electron microscopic (TEM) observation 138 DAPI staining analysis for meiotic process 139 DNA extraction 140 Alignment of BAC clone and marker development using high-resolution melting (HRM) analysis 140 Fine mapping of ms1035 141 Total RNA isolation and RT-PCR 142 5 RACE analysis for transcription start site survey 142 Phylogenetic analysis 145 Complementation of the dyt1 mutant 145 Localization of expressed Ms1035 by in situ hybridization 146 RNA transcriptome analysis by RNAseq 147 Results ms1035 shows complete sterility with no ability to produce viable pollen 149 Pollen development of ms1035 is arrested at tetrad stages 149 Defe cts in degeneration of tapetal cells result in failure of pollen development in ms1035 154 Meiosis of ms1035 is arrested at anaphase I 158 ms1035 encodes a bHLH transcription factor 160 Localization of the Ms1035 gene by RNA in situ hybridization 166 Complementation of male sterility with a full length Ms1035 gene 169 Comparison of global gene expression between male sterilie and fertile plants by RNAseq analysis 173 Discussion 186 References 193 Abstract in Korean 203Docto