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)