Title Page
Contents
LIST OF ABBREVIATIONS 14
ABSTRACT 17
I. INTRODUCTION 19
1. The genus Narcissus spp. 21
2. The genus Hippeastrum hybridum spp. 26
II. MATERIALS AND METHODS 30
1. Bulb sources of Narcissus and Hippeastrum hybridum 30
2. Extraction of total RNAs from the bulb tissues 33
3. Reverse transcription (RT) 34
4. Polymerase chain reaction (PCR) using universal primers 34
5. Cloning and sequencing of PCR products of Narcissus and Hippeastrum hybridum infected with potyvirus and potexvirus 37
6. Design of primers 40
7. PCR using virus-specific primers 42
8. Cloning and sequencing PCR products of Narcissus and H. hybridum infected with CMV, CyEVA, HiMV, NeLV, NLSYV, NMV and NYSV 42
9. Sequence analysis 44
III. RESULTS 46
1. Detection of potyvirus and potexvirus by RT-PCR using the universal primers 46
2. Application of RT-PCR using virus-specific primers in the bulbs of Narcissus and Hippeastrum hybridum 50
3. Phylogenetic tree of virus isolates from Narcissus and H. hybridum obtained in this study 56
3-1. CMV isolates from Hippeastrum hybridum 57
3-2. CyEVA isolates from Narcissus 61
3-3. HiMV isolates from Hippeastrum hybridum 65
3-4. NeLV isolates from Narcissus and Hippeastrum hybridum 69
3-5. NLSYV isolates from Narcissus 73
3-6. NMV isolates from Narcissus 77
3-7. NYSV isolates from Narcissus 81
IV. DISCUSSION 85
V. REFERENCES 88
ABSTRACT IN KOREAN 98
Table 1. Horticultural classification of Narcissus cultivars 23
Table 2. Narcissus-infecting viruses and symptoms (continued) 24
Table 3. H. hybridum-infecting viruses and symptoms (continued) 28
Table 4. List of universal primers used for RT-PCR to detect potyvirus and potexvirus 36
Table 5. RT-PCR primer pairs specific to the segment of CMV, CyEVA, HiMV, NeLV, NLSYV, NMV and NYSV RNA 41
Table 6. Comparison of nucleotide sequences for potyvirus and potexvirus from Narcissus and H. hybridum with other potyvirus and potexvirus isolates 49
Table 7. Occurrence of different viruses on Narcissus and H. hybridum 55
Table 8. Incidence of mixed virus infection in Narcissus and H. hybridum 55
Table 9. Mixed infection of viruses in Narcissus and H. hybridum 55
Table 10. Distance matrix based on the nucleotide sequences of the coat protein gene isolated from the bulb tissues of H. hybridum in this study with other CMV isolates, other isolates of cucumovirus and an outgroup 58
Table 11. Distance matrix based on the amino acid sequences of the coat protein gene isolated from the bulb tissues of Narcissus in this study with other CyEVA isolates, other isolates of potyvirus and an outgroup 62
Table 12. Distance matrix based on the nucleotide sequences of the coat protein gene isolated from the bulbs of H. hybridum in this study with other HiMV isolates, other isolates of potyvirus and an outgroup 66
Table 13. Distance matrix based on the amino acid sequences of the coat protein gene isolated from the bulb tissues of Narcissus and H. hybridum in this study with other NeLV isolates, other isolates of carlavirus and an outgroup 70
Table 14. Distance matrix based on the amino acid sequences of the coat protein gene isolated from the bulb tissues of Narcissus in this study with other NLSYV isolates, other isolates of potyvirus and an outgroup 74
Table 15. Distance matrix based on the amino acid sequences of the first triple gene block protein gene isolated from the bulb tissues of Narcissus in this study with other NMV isolates, other isolates of potexvirus and an outgroup 78
Table 16. Distance matrix based on the amino acid sequences of the coat protein gene isolated from the bulb tissues of Narcissus in this study with other NYSV isolates, other isolates of potyvirus and an outgroup 82
Figure 1. Ratio of the number of discarded inspections of major bulbous plants imported into Korea and discarded during inspections from 2017 to 2021. 20
Figure 2. The collected bulbs of Narcissus for detection of viruses in this study. All the following cultivars belong to Narcissus. 1-5, 'Tete-a-tete' 1-5; 6-10, 'Tete Boucle'... 31
Figure 3. The collected bulbs of H. hybridum for detection of viruses in this study. Two varieties were used: 1-10, 'Red Lion' 1-10; 11-15, 'Alfresco' 1-5. 32
Figure 4. pGEM-T Easy vector (Promega, Madison, Wisconsin, USA) was used for cloning of cDNA. To confirm of cDNA clones, the clones were cut using EcoRІ. 39
Figure 5. RT-PCR analysis using potyvirus and potexvirus universal primers to identify virus infection in total RNA isolated from the bulbs of Narcissus and H.... 48
Figure 6. RT-PCR analysis using virus-specific primers for detection of CMV, CyEVA, HiMV, NeLV, NLSYV, NMV and NYSV in the bulbs of Narcissus and H.... 53
Figure 7. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the nucleotide sequences of CMV coat protein gene isolated from the bulb... 60
Figure 8. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the amino acid sequences of CyEVA coat protein gene isolated from the bulb... 64
Figure 9. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the nucleotide sequences of HiMV coat protein gene isolated from the bulb... 68
Figure 10. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the amino acid sequences of NeLV coat protein gene isolated from the bulb... 72
Figure 11. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the amino acid sequences of NLSYV coat protein gene isolated from the... 76
Figure 12. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based the amino acid sequences of the first triple gene block protein gene isolated... 80
Figure 13. Phylogenetic tree using the neighbor-joining method with 1,000 bootstraps based on the amino acid sequences of the coat protein gene isolated from the bulb... 84