From Bugs to Barcodes: Using Molecular Tools to Study Biodiversity

From Bugs to Barcodes: Using Molecular Tools to Study

Biodiversity

Mandy Butler, Heather Henter, Stephanie MelUniversity of California, San Diego

Biodiversity

Biodiversity knowledge gap

How can we conserve what we don’t know we have?

How can we address the biodiversity knowledge gap?

BARCODING

DNA BARCODEspecific region in an organism's DNA as a genetic marker to identify species

How?compare DNA sequence with sequences in a public reference library (GenBank or BOLD)

HOW?

Why is barcoding useful?

Morphological identification difficult for non-specialist

Anyone that can do PCR and pay for sequencing can generate barcode data

HOW?

1. Keep a record of class activity2. Compare your sequence data to published sequences3. ID specimen4. Analyze data

HOW?

Publish

!!

!!

Student collaborationecology courses molecular biology

courses

Also, independent study students

Invertebrate Animals

Scale=5 mm

Scripps Coastal Reserve

Hypothesis testing vs. Discovery science

Do flower-inhabiting thrips (Thysanoptera) specialize on different species of plant host?

vs.

What thrips species are at our reserve?

DNA Preservation

Best: 95% ethanol & -20 C freezer, change ethanol onceOK: dryWorst: 70% ethanol

Be sure to label each specimen with date, locality, collector

Collaborations

San Diego – Biodiversity hotspotSan Diego – BOL?

Education –BOL?

Today’s insect collecting

Each group:insect netaspirator & aspirator vials2 mL vials with 70% ethanolblank labelswhite paper platefine paint brushsharp pencilkill jar

History of Barcoding

http://www.dnabarcoding101.org/

Insect collection

Amplification of COI gene by PCR

DNA extraction

Direct sequencing of cleaned up PCR product

Analysis of sequence

Barcoding protocol

DNA EXTRACTION

• Remove leg if large insect• Grind up entire insect if very small• Follow extraction protocol

An Internal ID System for All Animals

Typical Animal Cell

Mitochondrion

DNA

mtDNA

D-Loop

ND5

H-strand

ND4

ND4LND3 COIII

L-strand

ND6

ND2

ND1

COII

Small ribosomal RNA

ATPase subunit 8ATPase subunit 6

Cytochrome b

COICOI

The Mitochondrial Genome

Slide from David E. Schindel

PCR - Polymerase Chain Reaction

CO1 DNA

30+ cycles of amplification

PCR product: Billions of copies of CO1 DNAsequence from your organism of interest

5’ 3’

3’ 5’

Run PCR product on a gel

Check to see if correct size PCR productFollow clean-up protocol

Send samples out for sequencing…

Org 1 - CTGCTGACATCGATGCTGATCGGAGTATCATAAOrg 2 - CTGCTGACATCGATGCTGATCGGACTATCATAAOrg 3 - CTGCTGACATTGATGCTGATCGGACTATCATAAOrg 4 - CTGCTGACATTGATGCTGATCGGACTATCATAAOrg 5 - CTGCTGACATCGATGCTGATCGGACTATCATAA

Compare CO1 sequences from different organisms

Discover a new species!

Ask exciting scientific questions!

Brooklyn vs. Bronx Bedbugs

What lives in the subway?

Expose a local restaurant!

Publish!

Why is a mitochondrial gene used for barcoding?

Review of Mitochondrial DNA – Circular DNA, 17000 bp– Hundreds of copies per cell– Inherited from mother only – so haploid– No recombination– Contains 37 genes – no introns

http://www.geneticorigins.org/mito/intro.html

The CO1 gene is the standard gene for barcoding in animals

• CO1 is a protein coding gene of about 1500 bp

• The COI region that is used for barcoding includes the first half of the gene and is approximately 650 basepairs long

• PCR is used to amplify this region; the PCR product is then sent for sequencing

1. Mitochondrial genes are haploid

• In doing barcoding, we want to make the sequencing part as easy as possible – We want to be able to isolate DNA, amplify it and

sequence it without having to clone the DNA

• Thus it is important to use a haploid gene as our identifying gene. – If you used a diploid gene, you would get product with

possible 2 different sequences.

2. Mitochondrial genes are present in high copy numbers in cells

3. Mitochondrial genes exhibit more differences in sequence among species than nuclear genes, and less

difference within species

• Sequence differences among closely related animal species average 5- to 10-fold higher in mitochondrial than nuclear genes.

• Intraspecific variation in mitochondrial DNA is low in most animal species.

• Thus small intraspecific and large interspecific differences make distinguishing genetic boundaries between species easier, enabling more precise identification.

Universal primers• “Universal primers” are primers that are designed to areas in

genes that are highly conserved among different species. – Thus universal primers are not species specific

• This means that the primers are not totally complementary to the sequence in your insect sample but they are similar enough to hybridize at a low annealing temperature sample– The sequence in the CO1 gene in between where the

primers hybridize is less well conserved and thus can be used to differentiate members of different species.

PCR conditions

• Since we do the annealing step at 42oC, it is possible that non-specific PCR products are made

• We run a gel to see if the PCR worked, and if there is a single PCR product of the expected size, we clean up the PCR product and send for sequencing. – Sequencing is done using the forward and reverse primes

in separate runs

• We will then analyze the sequences and use Blast to try determine the genus and species of your samples

Bioinformatics

• First we determine if the sequence for the insect is in Genbank by doing a Blast

• We will consider any hit with an identity of 97% or better the same species.

• If the match is less than 97%, it means the barcode sequence for theinsect is not in Genbank

Intraspecies genetic diversity

• Once we have a set of sequences from the same organisms (i.e., same genus and species) we will also look at genetic diversity within that organism (SNPs)