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Molecular genetic analysis of Tobamovirus resistance in Capsicum spp. : 고추 토바모바이러스 저항성의 분자생물학적 분석
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 강병철 | - |
| dc.contributor.author | 양희범 | - |
| dc.date.accessioned | 2017-07-13T17:41:38Z | - |
| dc.date.available | 2017-07-13T17:41:38Z | - |
| dc.date.issued | 2014-02 | - |
| dc.identifier.other | 000000018117 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/121050 | - |
| dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부(원예과학전공), 2014. 2. 강병철. | - |
| dc.description.abstract | 토바모바이러스(Tobamovirus)는 고추에서 가장 큰 피해를 주는 바이러스 중 하나이다. 바이러스는 화학적 약제에 의한 방제가 거의 불가능하고 식물 위생만으로는 토바모바이러스 감염을 막는 데 한계가 있다. 토바모바이러스를 방제하는 가장 좋은 방법은 저항성 품종을 재배하는 것으로 방제에 필요한 노력이나 비용을 절감할 수 있다. 토바모바이러스 저항성 유전자에 대한 분자유전학 연구는 토바모바이러스 저항성에 대한 깊은 이해를 가능케하며 저항성 육종에 유용하게 활용될 수 있다.
첫 번째 장에서는 토마토의 I2, 감자의 R3, 고추의 L 사이의 비교유전학적 연구를 통해 I2C-1과 R3/7에 상응하는 BAC 클론을 선발했다. 선발한 BAC을 PCR, 염기서열 분석, 유전자 지도 작성을 통해 분류했고, 그 중 082F03 BAC이 TG36 근처에 위치했다. 082F03 염기서열을 이용하여 추가적인 BAC 클론을 선발했고, 이들로 구성된 224kb의 컨티그(contig)를 작성했다. 컨티그 염기서열로부터 개발한 3개의 분자표지는 L4 유전자와 약 1.2 cM 거리에 위치했다. L3 유전자와 완전하게 공분리하는 분자표지를 L4 분리 집단에서 연관 분석을 수행한 결과 L3와 L4가 같은 유전자좌에 위치하는 대립유전자가 아닌 가깝게 연관된 서로 다른 유전자일 가능성이 있음을 확인했다. 두 번째 장에서는 동정된 L3 유전자의 동질성을 이용한 PCR로 찾은 L4 후보유전자로부터 개발한 분자표지(L4segF&R)의 연관 분석을 통해 L4 후보유전자를 검정했다. L4segF&R은 L4 분리집단에서 완벽하게 공분리하지 않고 약 0.3cM 거리에 위치했다. 육종 재료의 L4segF&R 분자표지 유전형을 분석한 결과 추가적인 재조합체를 발견했다. 재조합체의 정보를 바탕으로 유전적 구조 모델을 고안했고, 토바모바이러스 저항성이 L4 후보 유전자에 의해서 완전히 설명되지 않다는 것을 확인했다. 마지막으로 L4 후보 유전자의 LRR 연기서열로부터 L4RP-3F/L4RP-3R HRM 분자표지를 개발했다. 세 번째 장에서는 L4를 보유한 PI260429의 전체 전사체 염기서열을 이용하여 L과 상동성이 높은 전사체의 de novo assembly를 수행했다. De novo assembly 결과를 검증하기 위하여 2개의 지표(컨티그 수, 최대 컨티그 길이)를 고안했다. k-mer가 59일 때 Velvet 전사체는 2개의 지표에서 모두 가장 높은 점수를 얻어 추후 분석에 사용되었다. 총 96개의 컨티그가 L과 상동성을 보였고, 4개의 토바모바이러스 저항성 계통과 4개의 이병성 계통의 전체 전사체 염기서열을 이용하여 디지털 발현 분석을 수행했다. 8개의 contig가 저항성 계통에서만 특이적으로 발현되었는데, 이들 contig는 4개의 전사체인 L4 후보유전자, PIX-4, CA00g34020_G4_C3, L_homologue 98_99로부터 유래한 것으로 추정된다. 이 결과는 저항성에 중요한 역할을 하는 L4 후보유전자를 제외한, 다른 3개의 전사체 역시 토바모바이러스 저항성에 중요한 역할을 할 가능성이 있음을 시사한다. 본 연구의 이상의 성과는 고추 토바모바이러스 저항성의 분자유전학적인 이해를 돕고, 더 나아가 토바모바이러스 저항성 분자 육종에 기여할 수 있을 것으로 기대한다. | - |
| dc.description.abstract | Tobamovirus is one of the most destructive viruses in Capsicum. Because it is hard to control Tobamovirus by chemical agents or sanitation, development of resistant cultivars would be the best option to manage Tobamovirus. Molecular genetic analysis provides deep understanding of Tobamovirus resistance, which leads usefully application to Tobamovirus resistance breeding.
In the first chapter, comparative genetic relationship between I2 of tomato, R3 of potato, and L of pepper were investigated and bacterial artificial chromosome (BAC) clones corresponding to I2C-1 and R3/7 were isolated. Selected BAC clones were sorted by PCR screening, sequencing, and genetic mapping. The BAC clone 082F03 of one sorted group was anchored near TG36. Additional BAC clones were isolated using 082F03 sequence and 224kb contig was constructed. Three markers developed based on contig sequence were closely linked to the L4 gene (about 1.2cM). The linkage analysis of that marker co-segregated with the L3 gene was performed in L4-segregating populations. The result suggests the possibility that L3 and L4 may be different genes that are closely linked to each other instead of different alleles in same locus. In the second chapter, the L4 gene candidate isolated by homology based PCR using the L3 gene was validated by linkage analysis of L4segF&R marker developed based on LRR sequence of L4 gene candidate. L4segF&R was closely linked to the L4 gene (0.3cM), however it did not co-segregate perfectly in L4-segregating population. Linkage analysis of L4segF&R of breeding materials provided by three seeds companies was performed to confirm the mapping result and several recombinants were also found. A number of genetic architecture models were postulated based on recombinants data. They demonstrated that Tobamovirus resistance is not fully explained by L4 gene candidate alone. Finally allele specific HRM marker, L4RP-3F/L4RP-3R, was developed based on LRR sequence of the L4 gene candidate sequence. In the third chapter, de novo assembly of L-homologous transcripts using transcriptome sequence of C. chacoense PI260429 containing L4 was performed. Two indexes including the number of contigs and maximum contig length, were designed to validate de novo assembly products. Transcripts assembled by Velvet with single k-mer = 59 showed the highest score in both indices, and these were chosen for next analysis. A total of 96 L-homologous contigs were isolated and digital expression analysis was performed between four Tobamovirus resistant accessions and four susceptible accessions. Eight contigs were expressed only in resistance accessions. These are derived from four transcripts, the L4 candidate, PIX-4, CA00g34020_CNL_G4_C3 and L_homologue 97_98. This indicates the other three transcripts except the L4 candidate which is known to be responsible for Tobamovirus resistance may also play an important role for Tobamovirus resistance. The results of this research are expected to provide molecular genetic understanding of Tobamovirus resistance in Capsicum spp. It is expected that this study will consequently contribute to developing molecular breeding system for Tobamovirus resistance. | - |
| dc.description.tableofcontents | CONTENTS
ABSTRACT i CONTENTS iii LIST OF TABLES viii LIST OF FIGURES x LIST OF ABBREVIATIONS xiii GENERAL INTRODUCTION 1 LITERATURE REVIEW 5 CHAPTER I Development of SNP Markers Linked to the L Locus in Capsicum spp. by a Comparative Genetic Analysis ABSTRACT 28 INTRODUCTION 30 MATERIALS AND METHODS Plant and virus materials 35 Virus strains and resistance screening 35 BAC clone analysis and sequencing 36 BAC sequence annotation 38 Development of markers linked to the L locus based on BAC sequences 39 Linkage analysis of molecular markers 40 RESULTS Identification of BAC clones cross-hybridized with tomato I2C-1 gene and potato R3/R7 44 Mapping of the candidate BAC clones 45 Construction of a partial contig 50 Development of SNP markers linked to the L alleles 50 Linkage analysis and allelic relationship of L-linked markers 56 Gene annotation from contig sequence and the genomic relationship of RGA 60 DISCUSSION 67 REFERENCES 73 CHAPTER II Development and Validation of the L Allele Specific Markers in Capsicum spp. ABSTRACT 80 INTRODUCTION 82 MATERIALS AND METHODS Plant materials 86 Virus strains and resistance screening 86 Genomic DNA extraction 87 DNA sequence analysis 87 SNP marker development and analysis 88 Linkage analysis of molecular markers linked to the L4 gene 91 RESULTS Development of markers based on the L4 candidate gene 92 Linkage analysis of the L4 candidate 92 Analysis of breeding lines using markers flanking the L4 gene 96 Genetic structures around the L4 locus in different breeding lines 104 Development of L allele specific HRM marker 107 DISCUSSION 119 REFERENCES 126 CHAPTER III De novo Assembly and Digital Expression Profiling of the L Homologs in Capsicum spp. ABSTRACT 132 INTRODUCTION 134 MATERIALS AND METHODS Plant materials 137 Total RNA extraction and transcriptome sequencing 137 De novo assembly of transcriptome 140 Annotation and in silico mapping 140 Digital expression analysis 141 RESULTS Optimization of de novo assembly of the L homologs 142 Annotation of L homologs 149 In silico mapping of L homologs 156 Digital expression of L homologs 162 DISCUSSION 167 REFERENCES 175 ABSTRACT IN KOREAN 180 LIST OF TABLES CHAPTER I Table 1. Primer information lists in this research 41 Table 2. L4-linked SNP markers developed based on BAC sequences 54 Table 3. Number of recombinants in SP, CP and MS L-segregating populations 58 Table 4. Summary of gene predictions on the BAC contig sequence 63 CHAPTER II Table 1. Primers used in this study 89 Table 2. Analysis of co-segregation of three L-linked markers with Tobamovirus resistance 94 Table 3. L4-linked marker genotypes of recombinants 95 Table 4. Marker analysis of De Ruiter progeny groups 99 Table 5. Summary of progeny tests of Monsanto F2 breeding populations and Nongwoo breeding lines 100 Table 6. Progeny tests of each Monsanto F2 breeding populations 101 Table 7. Genotype analysis of L4RP-3F / L4RP-3R and L4RP-3F / 3end marker sets for Enza Zaden breeding lines 110 CHAPTER III Table 1. Transcriptome sequences of plant materials 139 Table 2. The number of transcripts, N50 and maximum transcripts length of de novo assembly of PI260429 for each k-mer 144 Table 3. The number of transcripts, N50 and maximum transcripts length of Oases products 146 Table 4. Identities, domain prediction and matching region in the L4 functional homolog sequence of L-homologous de novo assembly products of PI260429 151 Table 5. In silico mapping of L homologous transcripts of PI260429 158 Table 6. Digital expression level of L-homologous transcripts in eight Capsicum accessions 164 LIST OF FIGURES CHAPTER I Figure 1. Comparative map of lower arm of chromosome 11 47 Figure 2. Fingerprinting result of 89 BAC clones 48 Figure 3. Partial contig of 13 BAC clones 49 Figure 4. Analysis of molecular markers developed in this study 55 Figure 5. Genetic maps of four molecular markers around the L4 locus 59 Figure 6. Multiple alignment of the R3a, I2, and three deduced RIGAs 65 Figure 7. Phylogenetic analysis of RIGAs, R3a, and I2 with other resistance genes in Solanaceae by neighbor-joining algorithm 66 CHAPTER II Figure 1. Sequence comparison of L3 and four L candidates and development of markers based on the L4 gene candidate sequence 90 Figure 2. Putative genetic structures of the region around L4 in different breeding lines deduced form marker genotypes 106 Figure 3. PCR amplification of L4RP-3F&3end primer set in accessions containing each L alleles 111 Figure 4. SNP marker profiles developed based on the 3 LRR-encoding domain of the L4 candidate 112 Figure 5. Melting curve patterns of homozygous genotypes of six L alleles and heterozygous genotypes of each pair of these alleles in HRM analysis of L4RP-3F / L4RP-3R primer set 113 Figure 6. Melting curve patterns of homozygous genotypes of six L alleles and heterozygous genotypes of each pair of these alleles in HRM analysis of L4RP-3F / 3end primer set 116 Figure 7. Haplotypes models representing introgression of the L4 gene from of C. chacoense into C. annuum. 125 CHAPTER III Figure 1. Maximum contig length and coverage rate of the L4 functional homologs of de novo assembly of PI260429 for each k-mer from 35 to 99 147 Figure 2. Comparison of coverage rates and maximum contig length of Oases products (k-mer = 47 - 67) and Velvet product (k-mer = 59) 148 Figure 3. Short reads mapping of PI260429 to the L4 functional homolog sequence 155 Figure 4. Phylogenetic tree of L alleles 174 | - |
| dc.format | application/pdf | - |
| dc.format.extent | 2849808 bytes | - |
| dc.format.medium | application/pdf | - |
| dc.language.iso | en | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | 고추 | - |
| dc.subject | 토바모바이러스 | - |
| dc.subject | 병 저항성 | - |
| dc.subject | 분자표지 | - |
| dc.subject | de novo assembly | - |
| dc.subject.ddc | 635 | - |
| dc.title | Molecular genetic analysis of Tobamovirus resistance in Capsicum spp. | - |
| dc.title.alternative | 고추 토바모바이러스 저항성의 분자생물학적 분석 | - |
| dc.type | Thesis | - |
| dc.contributor.AlternativeAuthor | Yang Hee-Bum | - |
| dc.description.degree | Doctor | - |
| dc.citation.pages | xiv, 182 | - |
| dc.contributor.affiliation | 농업생명과학대학 식물생산과학부(원예과학전공) | - |
| dc.date.awarded | 2014-02 | - |
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