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Mitochondrial Genome Sequencing of a Calcareous Sponge, Scypha ciliata

Mitochondrial Genome Sequencing of a Calcareous Sponge, Scypha ciliata. Wang Yijing 2005-12-17. Introduction. Department of Molecular Evolution, EGS, EBC October 31st - November 23rd Supervisor: Mikael Thollesson. Laboratory Introduction.

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Mitochondrial Genome Sequencing of a Calcareous Sponge, Scypha ciliata

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  1. Mitochondrial Genome Sequencing of a Calcareous Sponge, Scypha ciliata Wang Yijing 2005-12-17

  2. Introduction • Department of Molecular Evolution, EGS, EBC • October 31st - November 23rd • Supervisor: Mikael Thollesson

  3. Laboratory Introduction • Alpha-proteobacteria: the evolution of symbiotic and parasitic relationships, evolution of mitochondrion and modeling of evolution process • Hyperthermophilic archaeas: the evolutionary, regulatory and mechanistic aspects of cell cycles • Micro arrays, flow cytometry, MegaBace capillary sequencer etc.

  4. Laboratory Introduction • Work hours: 9:00 a.m.- 6:00 p.m. • The whole day at the bench • Breaks: • coffee breaks at 10 am and 3 pm • cakes on Friday afternoons! • Seminars: 4:00-5:00 every Wednesday afternoon.

  5. Project Introduction A typical metazoan mitochondrial genome • 16kbp circular molecule • 37 genes • coding proteins, rRNAs and tRNAs • compactly arrayed • conservatively ordered

  6. A metazoan mtDNA 16kbp circular molecule 37 genes coding proteins, rRNAs and tRNAs compactly arrayed conservatively ordered A unicellular mtDNA about 80 kbp encodes 1.5 times more genes uses a minimally derived genetic code encodes bacterial-like tRNAs and rRNAs. Project Introduction

  7. Project Introduction Why Sponges? • the most primitive metazoans evolved from choanoflagellate-like protist ancestors • at the bottom root of the metazoan phylogenetic tree based on both morphological studies and molecular sequences Thus the mtDNA comparisons between sponges, unicellular ancestor and other animals will provide the insights into the mtDNA evolution and phylogenetic reconstruction.

  8. Project Introduction • Hexactinellida (glass sponges) • Demospongiae (demosponges) • Calcarea (calcareous sponges)

  9. Project Introduction

  10. Project Introduction

  11. Project Introduction Choanocyte Choanoflagellate - Collar Cell in Sponges -Protist Ancestor

  12. Project Introduction mtDNA of Demosponges • resembles most features of metazoan mtDNA • Yet contains some extra genes encoding bacterial-like rRNAs and tRNAs and use a minimally derived genetic code

  13. Project Introduction However, the Calcarea are thought to be more closely related to higher metazoan organisms than the other two groups.

  14. Scypha ciliata

  15. Strategy • The aim of the project is to obtain the complete mitochondrial genome sequence for a calcareous sponge, Scyphaciliata, by using XL-PCR, nebulization and cloning, and sequencing of the clone library. • However, we need to use general primers to get some sequences that we could use to design specific primers for XL-PCR since there are no sequences for the mtDNA of this species published ever.

  16. Methods • Total DNA extracted using CTAB extraction • CO I gene amplified using general primers HCO, LCO at 47℃ • 16S rRNA gene amplified using 16S-ARL, 16S-BRH at 50℃ • Sequencing using MegaBace capillary sequencer • The results were checked against the GenBank database using blastn.

  17. Difficulties • DNA degrading in the specimen? • Little information on the mtDNA sequence • How fit the primers are to the template? Touch-down PCR • Contamination • Nuclear homolog to mitochondrial 16S • Sequence primers

  18. Luckily I got the mitochondrial 16S sequence on the last day!

  19. Results- 16S

  20. Results-16S • The 16S rRNA gene sequence alignment by Clusal X • Scypha ciliata • Calcareous sponges: Grantia, Leucilla,Clathrina • Demosponges: Microciona, Cliona, Tethya, Halichondria and Geodia • Choanoflagellate: Monosiga • Cnidaria: Urticina, Palythoa and Zoanthus • A phylogenetic tree based on these alignments by PAUP

  21. Results-16S

  22. Results-16S • However, 16S rRNA gene of mtDNA is relatively conserved in its sequence during the evolution, thus it is not very suitable for phylogenetic analysis. • But we can use the sequence to design specific primers for XL-PCR.

  23. Results- CO I • We got the amplification products from PCR. • The sequencing of CO I Gene failed. • Reasons: • The products of PCR were not pure enough? • The primers were not suitable to the sequence reactions. • So we propose to clone the PCR products of COI gene before sequencing it.

  24. Future Perspectives • Order other general primers to amplify other regions • XL-PCR • Shearing • A Clone Library • Sequencing • Assembling • Phylogenetic Analysis

  25. Acknowledgement Many thanks to everyone in the lab Especially thanks to Mikael Anna-Sofie Olga Guan Na Johan Viklund Yin Jun

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