Linkage Map Construction and Genetic Diversity Analysis based on Transcriptome-derived Markers in Brassica oleracea L.

학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부(작물생명과학전공), 2014. 2. Tae-Jin Yang.Molecular markers have been proven to be an essential and powerful tool for plant geneticists. The advent of molecular markers helps the geneticists in many genetic applications. Transcriptome sequencing, which is DNA sequencing of the mRNA pool of a given tissue, has enabled rapid marker discovery. In present study, two cabbage parental lines, C1184 and C1234, which is susceptible and resistant to black rot disease, respectively, were used for transcriptome sequencing. A total of 92,255 and 127,522 reads were generated, and clustered into 34,688 and 40,947 unigenes, respectively. 27 ESTs were found to have different expression between parental lines, and the most differentially expressed EST were known as Ankyrin repeat domain and RING finger-containing protein XBAT31 that could be considered as candidate gene for resistance to black rot. In addition, 2,405 SSR motifs derived from the unigenes of the black rot resistant parent C1234 were detected. Of these, trinucleotide motifs were the most abundant one (66.15%) observed among all the repeat motifs. Moreover, 1,167 SNPs were also identified between two parental lines. A total of 937 EST-based SSR and 97 EST-based dCAPS markers were designed and used for polymorphism analysis. These newly designed primers, together with the published SSRs and intron-based polymorphism (IBP) markers, were used to construct a genetic linkage map using an F2 segregation population of 97 individuals. The constructed genetic map composed of 265 loci (98 EST-based SSRs, 21 EST-based dCAPS, 91 public SSRs and 55 IBP markers) distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The genetic map constructed in this study could serve as framework for identifying marker-trait associations through quantitative trait loci (QTL) mapping, map-based cloning and physical map alignment. On the other hand, molecular DNA markers were also useful for genetic diversity analysis. Of which, analysis of genetic diversity of crop plants is a key component for crop improvement program. In this study, 91 commercial B. oleracea cultivars belonging to six subspecies, which obtained from seed companies worldwide, have been characterized using 69 SSR markers. A total of 359 different alleles have been observed, with an average number of 5.20 alleles per locus. Polymorphism information content (PIC) values ranged from 0.06 to 0.73, with an average of 0.40. Kohlrabi cultivars showed the highest heterozygosity level among six subspeciesmeanwhile kale cultivars exhibited the lowest one. Based on genetic similarity values, an UPGMA clustering dendrogram and principal coordinate analysis (PCoA) were generated to analyze genetic diversity. All of 91 cultivars were clearly separated into six different clusters with a propensity to cluster into its subspecies. Structure analysis exhibited six genetic groups, of which cabbage cultivars differentiated into two subgroups based on their head shapes, whereas cauliflower and kai-lan cultivars clustered together in the same group. In addition, 18 SSR markers were found to have 27 specific alleles that can be used to differentiate 22 cultivars. Genetic diversity and population structure analysis generated in this study presents new insight into the genetic structure and relationships among 91 B. oleracea cultivars and provides valuable information for future breeding of B. oleracea species.GENERAL ABSTRACT I CONTENTS IV LIST OF TABLES VIII LIST OF FIGURES X LIST OF ABBREVIATIONS XII GENERAL INTRODUCTION 1 LITERATURE REVIEW 5 Development of molecular DNA marker 5 Expressed sequence tags (ESTs)-based SSR markers 6 Frequency and distribution of SSRs in plant ESTs 8 Single nucleotide polymorphism (SNP) markers 9 Analysis of genetic diversity in B. oleracea L. 11 Genetic mapping in Brassica oleracea L. 12 CHAPTER I. Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var. capitata L.) and construction of an EST-based genetic map ABSTRACT 14 INTRODUCTION 16 MATERIALS AND METHODS 19 Plant materials and genomic DNA extraction 19 454 transcriptome sequencing and assembly 19 Functional annotation 20 Marker development 21 Selection of EST sequences containing SSRs and primer design 21 SNP discovery and primer design 21 Molecular marker analysis 22 SSR and IBP analysis 23 dCAPS analysis 23 Linkage analysis and map construction 24 RESULTS 25 Sequence assembly and functional annotation 25 Differentially expressed ESTs 30 NBS-encoding genes in Black rot resistant line 33 Frequency and distribution of SSRs in cabbage ESTs 36 Development of EST-based SSR markers 39 Development of EST-based dCAPS markers 41 Construction of genetic linkage map 43 Segregation distortion of polymorphic markers 64 DISCUSSION 67 Transcriptome sequencing, assembly and gene annotation 67 General features of EST-SSRs in cabbage genome 69 Marker development and polymorphism level of EST-based SSR markers 70 Validation and polymorphism analysis of EST-based dCAPS markers 71 Linkage map construction and its comparison with pseudo chromosome map 71 Segregation distortion phenomenon in cabbage genetic map 74 CHAPTER II. Genetic diversity analysis of 91 commercial Brassica oleracea L. cultivars belonging to six subspecies based on microsatellite markers ABSTRACT 76 INTRODUCTION 78 MATERIALS AND METHODS 82 Plant materials and DNA extraction 82 Analysis of SSR markers 89 Data analysis 99 RESULTS 102 Allelic diversity of SSR markers 102 Genetic diversity and phylogenetic relationships among 91 F1 hybrid cultivars 109 Cabbage (Group I) 112 Broccoli (Group II) 112 Kohlrabi and Kale (Group III and IV) 113 Cauliflower and Kai-lan (Group V and VI) 114 Analysis of population structure 114 Genetic diversity among members in each of the six subspecies 125 Variation in heterozygosity 127 DISCUSSION 128 Transferability and diversity of SSR markers 128 Phylogenetic relationships between varietal groups according to UPGMA and population structure analyses 129 Allele diversity and heterozygosity 131 REFERENCES 134 ABSTRACT IN KOREAN 152 APPENDIX 155 ACKNOWLEDGEMENT 184Docto