Genetic and Genomic Study of Shoot Branching in Soybean

학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부(작물생명과학전공), 2018. 8. 이석하.The number of soybean branch directly affected yield components of pod and seed number per plant by generating more pod bearing sites on the branches. The number of branches in soybean is immensely affected by various environmental factors, numerous genetic factors and their interactions. For this reason, a little genetic factors associated with branch number has been identified. The causal genes controlling branch development has not been identified in soybean. QTLs associated with number of branches and promising candidate genes including BRANCHED1 (BRC1) and REVOLUTA (REV) for branch development were identified using bi-parental mapping population and high-resolution genetic map. To validate the major QTL, genetic association within a set of 430 soybean germplasms under three geographic replications was analyzed. Resultantly, a total of six out of 45 markers, which were located in the major QTL for branch number, were significantly associated with branch numbers that measured in at least two geographical replications. The six markers located in exon of BRANCHED1 (BRC1) gene, intron of gene encoding transcription factor TFIIE alpha subunit and intergenic regions. In the meantime, using a set of near-isogenic lines (NILs) derived from a F6 residual heterozygous line (RHL) for the major QTL, expression of two candidate genes between the NILs were compared. The result showed in that the BRC1 gene was significantly down-regulated in NIL that exhibited more branches (p-value RNA-seq analysis for the set of NILs was conducted to provide global view of comprehensive mechanism for branch development in soybean. As a result, a total 376 differentially expressed genes were identified by comparison of the set of NILs. These DEGs were mapped to biological pathways such as abiotic stress signaling, plant-hormonal, secondary metabolism and development. Hormonal pathways, including auxin, cytokinin, gibberellin and abscisic acid, which were associated with branch development, were observed. Through a series of study about soybean branch development, strong candidate gene (BRC1) controlling branch development in soybean was identified. This gene encodes TEOSINTE BRANCHED 1/CYCLODEA/PROLIFERATING CELL FACTORS (TCP) transcription factor 18. The TCP transcription factor (TF) family are involved in developmental growth, such as leaf development, flower symmetry and shoot branching. Based on bioinformatics analysis such as phylogenetic tree, gene structure, motif conservation, synteny and expression pattern analysis, we could construct the duplication network of soybean TCPs. Through the duplication network, it was revealed that soybean TCP family was duplicated by whole genome duplication (WGD) and tandem duplication. Thus, the retention and structural/transcriptional divergence which could be supported by gene balance hypothesis and sub-/neo-functionalization were observed. These results will be helpful for understanding of branch development, breeding high yield soybean by increased branches and evolution of TCP TF genes. Keywords: soybean, branch, QTL, association, BRC1, evolutionGENERAL INTRODUCTION 1 LITERATURE REVIEW 5 Branch of soybean 5 QTL for branching 7 Development of near-isogenic line (NIL) from residual heterozygous line (RHL) 8 Evolution of TCP transcription factors 9 REFERENCES 11 CHAPTER 1. Identification of QTLs for branching in soybean (Glycine max (L.) Merill) 16 ABSTRACT 16 INTRODUCTION 18 MATERIALS AND METHODS 21 Plant materials 21 Phenotype assesment 21 DNA extraction and SNP genotyping 22 Genetic linkage map construction and QTL analysis 22 Comparison of the identified QTLs with the previously reported QTLs 23 Expression patterns of candidate genes 23 RESULTS 25 Phenotypic evaluation for traits related to branching and total pod number 25 Map construction and QTL analysis of branching and total pod number 25 Survey of candidate genes residing in QTL regions and their expression patterns 27 DISCUSSION 30 REFERENCES 35 CHAPTER 2. Genetic variations underlying branching in soybean (Glycine max (L.) Merill) 57 ABSTRACT 57 INTRODUCTION 59 MATERIALS AND METHODS 63 Plant materials and phenotype assessment 63 Genotypes of soybean core collection 64 Association analysis in germplasms 64 Genome-wide association analysis 65 DNA extraction of NILs and germplasms provided by GRIN and resequencing of NILs 66 RNA extraction and qRT-PCR of candidate genes in NILs 67 Miltiple protein sequence alignment of BRC1 gene, G. max paralogues and homologues in A.thaliana and O. sativa 68 RESULTS 69 Number of soybean branches in different geographic regions of Korea 69 Association of genetic variations underlying branching using 430 germplasms 69 Foreground and background selection of NILs 70 Difference of phenotype and candidate gene expression in NILs 71 Validation of genetics association of BRC1 and its upstream sequences 72 Protein sequence alignment of BRC1 gene, G. max paralogues and homologue in A. thaliana and O. sativa 73 DISCUSSION 74 REFERENCES 77 CHAPTER 3. Transcriptome profiling of soybean NILs for a major QTL for branching, qBR6-1 94 ABSTRACT 94 INTRODUCTION 96 MATERIALS AND METHODS 99 Plant materials and growth conditions 99 RNA extraction and RNA-seq analysis of NILs 100 Pathway analysis of DEGs from RNA-seq result 101 RESULTS 102 RNA-seq mapping 102 Expression of genes located in qBR6-1 102 Differentially expressed gene and pathway mapping overview 103 Plant hormone related DEGs 103 DEGs associated with secondary metabolism 105 Development associated DEGs 106 DEGs related to drought stress 107 DISCUSSION 108 REFERENCES 113 CHAPTER 4. Duplication and diversification mechanisms of TCP family of transcription factors in soybean (Glycine max [L.] Merrill) 131 ABSTRACT 131 INTRODUCTION 133 MATERIALS AND METHODS 137 TCP TFs identification in soybean genome 137 Phylogenetic analysis 138 Gene structure and conserved motif identification of G. max TCP TFs 138 Expression analysis of TCP TF genes using soybean RNA-seq Atlas database 139 Chromosomal location and duplication of G. max TCP genes 140 RESULTS 142 Genome-wide identification of the TCP TF gene family in G. max and their chromosomal distribution 142 Phylogenetic analysis of TCP TF gene family 143 Gene structure and conserved motifs 144 Expression analysis of TCP genes 145 Duplication of TCP TF genes in G. max 146 DISCUSSION 153 REFERENCES 161 CONCLUSION 190 ABSTRACT IN KOREAN 193Docto