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

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.

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.

Project Introduction

A typical metazoan mitochondrial genome

16kbp circular molecule 37 genes coding proteins, rRNAs

and tRNAs compactly arrayed conservatively ordered

Project Introduction

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

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.

Project Introduction

Hexactinellida (glass sponges)

Demospongiae (demosponges)

Calcarea (calcareous sponges)

Project Introduction

Project Introduction

Project Introduction

Choanocyte Choanoflagellate - Collar Cell in Sponges -Protist Ancestor

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

Project Introduction

However,

the Calcarea are thought to be more closely related to higher metazoan organisms than the other two groups.

Scypha ciliata

Strategy

The aim of the project is to obtain the complete mitochondrial genome sequence for a calcareous sponge, Scypha ciliata, 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.

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.

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

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

Results- 16S

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

Results-16S

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.

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.

Future Perspectives

Order other general primers to amplify other regions

XL-PCR Shearing A Clone Library Sequencing Assembling Phylogenetic Analysis

Acknowledgement Many thanks to everyone in the lab

Especially thanks to Mikael

Anna-Sofie

Olga

Guan Na

Johan Viklund

Yin Jun