10384 200126008 UDC Isolation of microsatellite markers from Porphyra haitanensis and their application in genetic diversity of Porphyra 2004 10 10 2004 10 13 2004 10

...1...3...5 1...5 2...6 2.1...6 2.2...8 2.3...8 2.4...9 3...10 3.1...10 3.2...11 3.3 PCR...12 3.4...12 4...12 4.1...12 4.2...13 4.3......13

4.4...14 5...14 6...15...17 1...17 1.1...17 1.2...17 1.3...17 2...18 3...19 3.1...19 3.2...19 3.3...19 3.4...19 3.5...19 3.6...19 4...21 4.1...21 4.2...25

4.3...28 4.4...28...31 1...31 1.1 DNA...31 1.2 puc18...31 1.3...32 2...34 2.1...34 2.2...35 2.3...36 2.4...38 3...40 4...41 4.1 PCR...41 4.2...43 4.3...45 4.4...47...48 1...48

2...50 3...51 3.1 PCR PAGE...51 3.2...53 3.3...55...56...57...66...67

Content Content Abstract in Chinese...1 Abstract in English...3 Intruction...5 1 The genetic markers...5 2 Microsatellite (simple sequence repeats,ssrs)...6 2.1 Discovery of SSRs...6 2.2 Definition and characterization of SSRs...8 2.3 Length polymorphism of SSRs...8 2.4 Advantage of SSRs technique... 9 3 Procedure of SSRs analysis...10 3.1 Isolation of SSRs DNA...10 3.2 Design of primers for SSRs PCR...11 3.3 Amplification of SSRs...12 3.4 Detection of amplification results...12 4 Applications of SSRs markers...13 4.1 Diagnose the genetic diseases...13 4.2 Construction of genetic map...13 4.3 Identification of germplasm...14

Content 4.4 Gene location and molecular markers assistant breeding14 5 The application of Porphyra molecular markers in genetics and breeding...14 6 The goal of this research...15 Materials and methods...17 1 Materials...17 1.1 Porphyra samples...17 1.2 Bactirial strain...17 1.3 Plasmids...17 2 Experiment instruments...18 3 Reagments...19 3.1 Common reagments...19 3.2 Restriction endonucleases and others enzymes...19 3.3 DNA markers...19 3.4 Kits...19 3.5 Cultural medium...19 3.6 Solutions...19 4 Methods...21 4.1 Construction a small fragment library of Porphyra haitanensis...21

Content 4.2 Identification of positive clones containing SSRs...26 4.3 Design and synthesis the primers...28 4.4 Analysis the PCR products...28 Results and analysis...31 1 The results of small fragment library and screening of positive clones...31 1.1 DNA extraction and digested...31 1.2 PUC18 DNA extraction and digested...31 1.3 Isolation and characteration of positive clones...32 2 Screening and analysis of positive clones containing SSRs..34 2.1 Probe and detection of labeling efficiency...34 2.2 In situ hybridization...35 2.3 Identification of positive clones...36 2.4 Sequencing and analysis of positive hybridization blots.38 3 Design and synthesis of primers...40 4 Analysis the PCR products...41 4.1 Electrophoresis...41 4.2 Information matrix and the number of DNA bands...43 4.3 Similarity of coefficient matrix and dendrogram of the 9

Content samples...43 4.4 Specific DNA bands...47 Discussion...48 1 Analysis of small-sized library and screening of the positive clones...48 2 Analysis of screening the positive clones containing SSRs...50 3 Analysis of SSRs amplification...51 3.1 PAGE electrophoresis analysis...51 3.2 Genetic diversity of Pophyra haitanensis...53 3.3 Application of SSRs in genetic diversity...55 Conclusion and prospect...56 References...57 The papers published and the subjects attended during the period of study for master degree...66 Acknowledgements...67 Appendix

1500 215 4 2 ATN4 GAN5 GeneBank 3 AJ512812AJ512806 AJ512804 9 LYGRDCSGL1 DXYBBDJJSNH LSD PCR 5 31 DNA AJ512812 AJ512804 1 48 31 26 83.9% Between groups linkage Nei NHJJS LSD 96.8 58.1 96.8% 9 NH JJS LSD 4 LYG RD CS GL1 5 9 6 1

LYG ATN4-1140 ATN4-175 CS AJ512812-1214AJ512812-168AJ512806-404 AJ512806-313 2

Abstract Porphyra haitanensis is seaweeds of high nutiitive value, and it has a long history of culture in Fujian province. In this thesis, we first constructed a small-size genomic library of P.haitanensis and got 1500 positive recombinants.among which we obtained 4 microsatellite sequences from 215 positive clones.choosing 2 of the microsatellite loci in P.haitanensis and 3 microsatellite loci of Gracilariopsis(AJ512812AJ512806 and AJ512804) from GenBank as primers.9 DNA samples(lygrdcsgl1dxybbdjjsnh LSD)of Porphyra were amplified,and a totle 31 DNA bands were detected. AJ512812 amplified 12 DNA bands were the most while AJ512804 only amplified 1 DNA bands, which was the least of them. Among 31 DNA bands, 83.9% were polymorphic. The data was analyzed by means of Neis similarity coefficient, and a phenogram was constructed by using between groups linkage method. The result showed that the similarity coefficient of the 7 P. haitanensis from 58.% to 96.8%.The highest similarity coefficient is 96.8% which among 3 wide P.haitanensis.The results indicated a high genetic varitions of P.haitanensis,and make a genetic foundation for excellent breed select. According to the dendrogram we can find that the 9 Porphyra were divided into two teams. The 7 P.haitanensis samples had closely relationship in the same team and the 2 P.yezoensis had closely relationship. However Neis genetic distance between CS and GL1 was larger than that between P.haitanensis and P.yezoensis.It indicated that different geographical localities where the sample were 4

collected tend to make the genetic diversity more obviously, sometimes it maybe the distance between two species. In this thesis, we obtain 6 specific bands,atn4-1140 and ATN4-168 of P.yezoensis LYGAJ512812-1214AJ512812-360AJ512806-404 and AJ512806-313 of excellent strain CS of P.haitanensis. We repeated the experiment for several times and these specific bands appeared stable. Forther study on the linkage between specific bands and excellent trait will be hopefully for molecular gentic breeding of P.haitanensis. Keyword: Porphyra haitanensismicrosatellitegenetic marker 4

1 [1][2] Genetic makers [3][4] 1 2 3 4 5 Morphological markers [5][6][7] 20 60 [3] Cytological markers [8] G C N 20 80 DNA DNA PCR DNA DNA Molecular genetic markers [2] [8] DNA 5

Restriction fragement length pymorphism RFLP [9] Single strand conformation pymorphismsscp [10] DNAMinisatellite DNA [11] DNA Microsatellite DNA [12] DNARandom amplified polymorphic DNARAPD [13] [14] Amplified fragment length polymorphismaflp [15] DNAMitochondrial DNAmtDNA [16] Expressed sequence tagsests [17] 1 2 2.1 20 70 1982 Hamada [12] TGn Tautz Renz [18] DNA 1985 Jeffreys [19] DNA Minisatellites Variable number of tandem rapeatsvntrs [20] [21 ] 6

Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.