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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張繼堯(Chi-Yao Chang) | |
dc.contributor.author | Bou-Yun Lin | en |
dc.contributor.author | 林渤昀 | zh_TW |
dc.date.accessioned | 2021-06-15T06:24:16Z | - |
dc.date.available | 2011-07-04 | |
dc.date.copyright | 2010-08-18 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-09 | |
dc.identifier.citation | References :
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(1992) Oligonucleotide DNA fingerprinting detects a multiallelic locus in box elder (Acer negundo). Molecular Ecology 1: 65-7. McConnell, S.K., Franck, J.P.C. and Wright, J.M. (1997) Molecular genetic markers and their applications to the Tilapias. In Mair, G., ed. Reviews in Applied Genetics of Tilapia. Manila: ICLARM (in press). O'Connell, M. and Wright, J.M. (2004) Microsatellite DNA in fishes. Reviews in Fish Biology and Fisheries 7: 331-63. Rivera, M.A.J., Graham, G.C. and Roderick, G.K. (2003) Isolation and characterization of nine microsatellite loci from the Hawaiian Grouper Epinephelus Quernus (Serranidae) for population genetic analyses. Marine Biotechnology 5:126-129. Suci, A., Uthairat, N.N., Worawut, K. (2006) Study of genetic diversity of orange-spotted grouper, Epinephelus coioides, from Thailand and Indonesia using microsatellite markers. Marine Biotechnology 8:17-26. Tautz, D. (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Research 17, 6463-71. Yeh, S.P., Chang, C.A., Chang, C.Y., Liu, C.H. and Cheng, W. (2008) Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides. Fish & Shellfish Immunology 25 (1-2):19-27. Zane, L., Bargelloni, L., Patamello, T. (2002) Strategies for microsatellite isolation: a review. Molecular Ecology 11:1-16. Zhao, L.L., Shao, C.W., Liao, X.L. and Chen, S.L. (2008) Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification. Conservation Genetics 10 (3):627-629. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47881 | - |
dc.description.abstract | 近年來微衛星序列 (microsatellites) 被應用在許多領域的研究而成為相當重要的分子標記工具。微衛星基因座有下列特性:普遍存在於基因體(Genome)中,故能夠很方便的經由聚合酵素連鎖反應(PCR)的方式偵測其多型性。微衛星基因座優於其他分子標記的主要原因為其能夠顯現來自父親及母親的遺傳訊息,這就是所謂的共顯性(Co-dominance)。目前,這種標誌在水產上被應用於親源鑑定及純化品系的工具,以增加養殖魚隻的競爭力造就更大的市場價值。
本實驗目的在於從點帶石斑魚(Epinephelus coioieds)中分離出微衛星基因座並分析其特性。以了解各基因座之總性狀數及性狀出現頻度,以便計算各基因座在種內的鑑別力(Polymorphic Information Content, PIC)。本實驗分離微衛星基因座的策略是依照其本身的一些特性所設計。微衛星基因座之片段長度約為 300 bp 到 500bp;以及含有 2 個鹼基重複數次或以上的重覆序列。首先將基因體核酸從點帶石斑魚中萃取出來,利用特定酵素將長且完整得片段作用成更小的片段,接下來利用特定含有 2 個鹼基重複序列的探針(probe) 去和這些小片段進行雜合反應(hybridization),最後篩選出微衛星基因座。 有 28 個微衛星基因座經過篩選後被分離出來,針對其游離區域(flankingregions)設計引子(primer),在分析樣本前,嘗試找出最佳的黏合溫度(annealingtemperature)以降低非專一性的產物干擾分析結果。經過數據分析結果發現 Ec-21之 PIC 值為 0.85 是這些基因座中最高的,同時代表 Ec-21 為很有潛力的分子標誌,將來可用來分析種內個體之間的差異。而在跨物種的分析中也發現 Ec-19 這個基因座能夠分辨同科魚種和點帶石斑魚之間的差異。此外本實驗五個高度多型性的基因座被篩選出來,未來可發展多基因座增幅(multiloci amplification),如此一來就能同時分析其中的兩個基因座,將能夠更有效率的進行更深入的分析。 | zh_TW |
dc.description.abstract | In recent years, microsatellites have become one of the most popular molecular markers used with applications in many fields. The objective of this study is to isolate the microsatellite loci from orange-spotted grouper, Epinephelus coioides and characterize those microsatellite loci.
The approach to achieve the target is based on the characteristics of the microsatellite loci. First, the genomic DNA extracted from the orange-spotted grouper had to be cut into smaller fragments by RsaI. Then, 6 sets of specific di-nucleotide probe (CG15, GA15, AC15, AT15, TG15 and CT15) were used to select the microsatellite-enriched fragments by the selective hybridization with two different hybridization buffers. Select totally 470 colonies from the microsatellite-enrich library. There were 201 positive colonies confirmed by the digestion check. Among all these positive colonies, 28 microsatellite loci have been successfully isolated. Before the samples were analyzed, finding the optimized annealing temperature was essential. Total 54 samples E. coioides were collected from Cha-Dong, then the allelic distribution and the allele frequency of those microsatellite loci were analyzed. The number of the allele range from 0 to 8 alleles was found. Locus Ec-21 has the most PIC value of 0.85 and consider as the highest polymorphic locus. In the result from the analysis of cross-species amplification, locus Ec-19 can distinguish different species of Serranidae. Base on the distribution of separating alleles, the approach of mutiloci PCR amplification can be used in the further research. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:24:16Z (GMT). No. of bitstreams: 1 ntu-99-R97b45010-1.pdf: 1923291 bytes, checksum: 25b242db9cf476cb8ca45cc9db9f96b4 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Abstract (In Chinese)............................Ⅰ
Abstract (In English).............................Ⅱ Table of contents............................Ⅲ Figure list ................................Ⅵ Table list................................Ⅶ Chapter 1 Introduction...........................1 1.1 E. coioides and the current status in Taiwan................1 1.2 Introduction of Microsatellite DNA...................2 1.3 Mechanisms of polymorphic allele formation.............4 1.4 Applications of microsatellite in aquaculture.................5 1.5 Strategy for microsatellites isolation..................5 1.6 Purpose of this study.........................7 Chapter 2 Materials and Method.......................9 Reagents...............................9 Procedures...............................14 2.1 Sample collection........................14 2.2 Microsatellite-enrich library development................14 2.2.1 Preparation of genomic DNA.................14 2.2.2 Fragmentation of the genomic DNA...............15 2.2.3 Modification of the digested fragments....................16 (1) Preparation of phosphorylated adaptor..............16 (2) Adaptor ligation......................16 2.2.4 PCR amplification of adapter-ligated fragments.........17 2.2.5 Selective hybridization for microsatellite-enrich fragments......17 2.2.6 PCR amplification of microsatellite-enriched fragments.....18 2.2.7 TA-cloning of microsatellite-enriched fragments...........19 (1) Ligation of the microsatellite-enriched fragments with TA-vector.............19 (2) Transformation and plating.................19 2.2.8 Preparation of Plasmid DNA..................19 2.2.9 Checking of the restriction enzyme digestion.............20 2.3 Sequencing and the microsatellite repeat motif searching..........20 2.4 Microsatellite loci the primer sets designing.............21 2.5 PCR amplification of the microsatellite loci.............21 2.6 Alleles detection via vertical electrophoresis of denaturing-polyacryamide gel...................................21 2.6.1 Preparation of the 8% denaturing-polyacryamide gel.......21 2.6.2 Electrophoresis of the denaturing-polyacryamide gel.......22 2.6.3 Silver staining of the denaturing-polyacryamide gel...............22 2.7 Statistic analyze...........................23 2.7.1 Check the optimized annealing temperature of microsatellite loci.............23 2.7.2 Analysis of the Polymorphism Information Content (PIC).....24 2.7.3 Cross-species amplification of the microsatellite loci...................24 Chapter 3 Result...........................25 3.1 Establishments of microsatellite-enrich library..............25 3.2 Analysis of the microsatellite loci...................26 3.3 Designing the primer of the microsatellite loci.................26 3.4 Finding the optimized annealing temperature of microsatellite loci.......27 3.5 Analysis of the allele sizes and PIC of microsatellite loci..........27 3.6 Cross-species amplification with microsatellite loci............28 Chapter 4 Discussion and Conclusion....................29 4.1 Isolation of the microsatellite loci with enrichment method.........29 4.2 Analysis of the microsatellite loci...................29 4.3 Analysis of the allele sizes and PIC of microsatellite loci..........30 4.4 Cross-species amplification of the microsatellite loci.............31 4.5 Conclusion............................32 References................................33 Figures...................................37 Tables.................................48 | |
dc.language.iso | en | |
dc.title | 點帶石斑魚微衛星基因座之選殖與特性分析 | zh_TW |
dc.title | Isolation and characterization of microsatellite loci from
orange-spotted grouper, Epinephelus coioides | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳榮祥,陳哲俊 | |
dc.subject.keyword | 點帶石斑魚,微衛星基因座,跨物種分析,多基因座增幅, | zh_TW |
dc.subject.keyword | Epinephelus coioides,Serranidae,microsatellite DNA,polymorphic information content (PIC), | en |
dc.relation.page | 61 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
顯示於系所單位: | 漁業科學研究所 |
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