From Bugs to Barcodes: Using
Molecular Tools to Study
Biodiversity
Mandy Butler, Heather Henter, Stephanie Mel
University 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 BARCODE
specific 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 activity
2. Compare your sequence data to published sequences
3. ID specimen
4. Analyze data
HOW?
!!
!!
Publish
Student collaboration
ecology courses
Also, independent study students
molecular biology
courses
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 once
OK: dry
Worst: 70% ethanol
Be sure to label each specimen with date, locality, collector
Collaborations
San Diego – Biodiversity hotspot
San Diego – BOL?
Education –BOL?
Today’s insect collecting
Each group:
insect net
aspirator & aspirator vials
2 mL vials with 70% ethanol
blank labels
white paper plate
fine paint brush
sharp pencil
kill jar
History of Barcoding
http://www.dnabarcoding101.org/
Barcoding protocol
Insect collection
DNA extraction
Amplification of COI gene by PCR
Direct sequencing of cleaned up PCR
product
Analysis of sequence
DNA EXTRACTION
• Remove leg if large insect
• Grind up entire insect if very small
• Follow extraction protocol
An Internal ID System for All Animals
The Mitochondrial Genome
Small ribosomal
DRNA
Loop
DNA
Cytochrome b
ND1
ND6
Typical Animal Cell
mtDNA
COI
ND5
Hstrand
ND4
L-strand
ND4L
ND3
Mitochondrion
ND2
COIII
COII
ATPase subunit 8
ATPase subunit 6
Slide from David E. Schinde
PCR - Polymerase Chain Reaction
5’
3’
CO1 DNA
3’
5’
30+ cycles of amplification
PCR product: Billions of copies of CO1 DNA
sequence from your organism of interest
Run PCR product on a gel
Check to see if correct size PCR product
Follow clean-up protocol
Send samples out for sequencing…
Compare CO1 sequences from different organisms
Org 1 - CTGCTGACATCGATGCTGATCGGAGTATCATAA
Org 2 - CTGCTGACATCGATGCTGATCGGACTATCATAA
Org 3 - CTGCTGACATTGATGCTGATCGGACTATCATAA
Org 4 - CTGCTGACATTGATGCTGATCGGACTATCATAA
Org 5 - CTGCTGACATCGATGCTGATCGGACTATCATAA
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)