It All Started in Ulithi, Micronesia Colonized a long time ago - - PowerPoint PPT Presentation

It All Started in Ulithi, Micronesia

• Colonized a long time ago

by polynesian voyagers

• Isolated: Distance to

nearest island (YAP) >100km

• Took us 24+ hours and 4

plane rides to get to the small atoll. Ulithi Atoll

Australia Japan India G u a m Hawaii

Bio 80S:

Applied Research Technologies: “Genetic Barcoding of Micronesian Fish”

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Introduction

• We work on a research project of

One People One Reef program, which is a collaboration between scientists and local communities in Micronesia for sustainable

• cean management.
• The samples we used were fin

clippings collected by local fisherman in Micronesia.

Class Goals

• Identify species being fished in Ulithi, and match to the names used

locally

• Compare with samples of fish of same species from other countries
• Help identify populations or cryptic species
• Help focus future fisheries research
• Skill and knowledge building for the students

Introduction

• All lab work was done on

Cabrillo College’s campus by students

• We fundraised for a new

PCR machine for Cabrillo

What We Did

• DNA digestion/extraction
• PCR and Electrophoresis Gel
• Amplified DNA sequences, sent for sequencing at

UC Berkeley

• Use computer programs to:

○ Clean genetic sequences ○ ID species (Match genetic sequences to online databases) ○ Evaluate evolutionary relationships

Mitochondrial Gene CO1

• Cytochrome Oxidase Subunit 1
• Sometimes called COX 1 (in

humans called MT-CO1)

• Gene region for DNA “barcoding”
• f almost all animal groups
• This means you can use this gene

to tell species apart

Challenges

Sample:

• Degradation

○ Quality ○ Age ○ Made it hard to amplify the DNA ○ Tested by using gel to see quality of DNA - showed some degradation

• We extracted DNA from

○ Fish ○ Corals ○ Sea cucumbers

Challenges

PCR troubleshooting:

• Nuanced differences in procedure

for different species

• Unknown optimal annealing

temperatures for each species

Challenges

Human Error:

• Cross-contamination

○ Working with several samples at once

• Pipetting incorrect volumes

○ Microscopic amounts

Results

• Extracted DNA of 93 samples

○ Fish, sea cucumbers & corals

• Amplified COI of 49 fish samples
• Found 14 fish species
• 10 were new to our genetic

database!

• At the end of class, successfully

amplified sea cucumber DNA ○ The COI gene of Holothuria atra ○ To be continued next semester!

Results: species added to genetic database (Fall ‘17):

Bothus mancus Flowery flounder Gymnosarda unicolor Dogtooth tuna Kyphosus vagiensis Brassy chub Parupeneus barberinus

Dash-and-dot goatfish

Siganus punctuatus Gold spotted spinefoot Stolephorus andhraensis (?) Anchovy species Siganus stellatus Brown spotted spinefoot

Found in Fall ‘17, but also during Spring class

Thunnus albacares Yellowfin tuna Lethrinus rubrioperculatus Spotcheek Emperor Plectropomous laevis Blacksaddled coral grouper Acanthurus lineatus Lined surgeonfish

Katsuwonus pelamis Skipjack tuna Juveniles! Benthosema fibulatum Lanternfish

Results: Samples collected in 2017

Cheilopogon abei Abe’s flyingfish

Identified species:

Results (previous semester, Spring ‘17)

Acanthurus triostegus Convict surgeonfish Acanthurus lineatus Lined surgeonfish Caranx lugubris Black jack Caranx sexfasciatus Bigeye trevally Crenimugil crenilabis Fringelip mullet Kyphosus cinerascens Blue sea chub Lethrinus rubrioperculatus Spotcheek Emperor

Results (previous semester, Spring ‘17)

Identified species (continued):

Variola albimarginata White edged lyretail Variola louti Yellow edge lyretail Thunnus albacares Yellowfin tuna Plectropomus laevis Blacksaddled coral grouper Myripristis berndti Blotch eye soldierfish Lutjanus kasmira Blue striped snapper Lutjanus gibbus Humpback red snapper Oxycheilinus unifasciatus Ring tail maori wrasse

Results

Phylogenetic tree of a Bacteria???

Stolephorus andhraensis (?)

Results

Potential cryptic species

Parupeneus barberinus

Dash-and-dot goatfish

Graph: fish commonly caught in Ulithi

• We confirmed that the fish

identified, are the ones reported, giving their database more credibility

• Mostly herbivores-this is found

across all research angles (surveys, genetics, fisheries data)

• On top of that, some species we

found appear to be either new species or cryptic

Applications

• Our genetic sampling can

highlight unique genetic differences of the fish in the Micronesian region

• Our results are a starting point,

that can help guide future research

• And this future research can

inform sustainable management

• f fish populations

Experience Gained

• Hands on experience in a genetics

laboratory

• Learn how to use academic software to

analyze data

• Real world research experience

○ Troubleshooting ○ Team building

• DIPLOMA to certify these experiences!!

Thank you!

Cabrillo College Biology Teachers:

• Eva Salas
• Nicole Crane
• John Carothers
• Yves Tan

UCSC Professor Giacomo Bernardi

Questions

DNA extraction: DNEasy Blood and Tissue Kit

• Cell lysis: Digestion. Lysis & Incubate @ 56°C for 6hrs
• Purification: Prepares sample to bind to clay in spin

column.

• Washing: Membrane w/ DNA, but has salts and

proteins also

• Dry spin: Residual ethanol is BAD!
• Elution: Releases DNA into solution from spin

column. We now have extracted DNA!!!

What’s so Special About CO1?

• The mitochondrial CO1 gene is a

homologous gene found in all species.

• The mutation rate for the gene

found in animals is high enough that species can be differentiated.

• Short enough to be easily

sequenced (658 base pairs, depending on the species)

PCR and Gel Electrophoresis

• Using our extracted DNA samples we ran PCR
• Used specific primers to amplify COI gene for DNA barcoding
• PCR protocol with steps for denaturation, replication and annealing
• Run gel electrophoresis to visualize PCR products
• PCR products sent to UC Berkeley for sequencing

Computational Analysis: Geneious

• Sequence data analyzed in

Geneious

• Align Forward and Reverse

sequences

• Performed a multiple sequence

alignment; cleaned and trimmed results to create consensus sequence

Results

Phylogenetic tree of Thunnus albacares and closely related species

Computational Analysis: Genbank and BOLD

• Blast consensus sequences using genomic browsers (Genbank & BOLD)

to determine what species we have by matching COI sequences

• Once species were identified, we downloaded sequences from Genbank

and created neighbor- joining phylogenetic trees

Benefits

• Troubleshoot issues in lab

procedure/results

• Research experience and

expectations

• Team building in lab environment
• Practice with essential lab

techniques