INTRODUCTION TO DNA BARCODING This is the introduction to DNA barcoding for the Let’s Talk Science Fish Market Survey Youth action project INTRODUCTION TO DNA BARCODING Let’s Talk Science Fish Market Survey Action Project

What is the elementary unit for a physicist? A metre The distance traveled by light in absolute vacuum in 1/299,792,458 of a second To get us started, let’s think about the elementary units in science. One of the elementary units for a physicist is a metre. This has a very specific definition.

What is the elementary unit for a chemist? A mole One mole contains Avogadro's number (approximately 6.022×1023) entities. An elementary unit for chemistry might be a mole. Again there is only one specific definition of what a mole is.

What is the elementary unit for a biologist? A species There are 23 – 36 definitions for what a species is What about biology? Can you think about what the elementary unit for a biologist might be? On click: Although there are many answers, the one that is most important for this presentation is a species. Think about how you would define a species (you may want to pause here and have students discuss with an elbow partner) On click: There are many valid definitions of what a species is. All of these are used by different kinds of biologists or in studying different kinds of organisms. In short, there is a lot of debate about what a species really is.

Ed Wiley (1978), modified from George Simpson (1961) “... are groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups” Ernst Mayr (1942) “... is a single lineage of ancestor-descendent populations which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fate” Ed Wiley (1978), modified from George Simpson (1961) “... is the most inclusive population of individuals having the potential for phenotypic cohesion through intrinsic cohesion mechanisms” Alan Templeton (1989) “... is the smallest diagnosable cluster of individual organisms within which there is a parental pattern of ancestry and descent. ” Joel Cracraft (1983) “… are segments of population-level lineages.” Kevin De Queiroz (1998) “... is what a good taxonomist says it is.” Anonymous In the end, a species is what a good taxonomist says it is!

This is a taxonomist in part of a typical museum collection This is a taxonomist in part of a typical museum collection. They generally do two jobs, one is to describe new species and the other is to sort through hundreds or even thousands of specimens and identify what species they are.

Who tells us what a species is? How? Taxonomists How? Taxonomic Keys Identification by morphological traits They do this using taxonomic keys which focus on identification by morphological traits or what the specimen looks like. Sometimes this can be simple. You can probably tell the difference between many species yourself. But it can get very complicated as well.

Worker ant head measurements EL EL Worker ant head measurements HL HL This image shows some of the measurements required to identify ant species. This is one example of where an expert is required to make an identification. For some species or groups there are only a handful of people in the entire world who are able to make these identifications. HW HW

What about unknown species? Traditional methods have identified about 1.7 million species Estimates of the total number of species are between 10 million-100 million Using traditional methods of identification over the past 250 years, scientists have identified about 1.7 million species on the planet. Estimates of the total biodiversity on Earth are between 10 and 100 million species. So we have a long way to go. Understanding biodiversity is important if we want to protect it.

DNA Barcode: A short standardized sequence enabling species discrimination in a large block of life DNA barcoding is one method that can speed up the process of identifying species. A DNA barcode is a short standardized sequence enabling species discrimination in a large block of life.

The Barcode Region For animals, the DNA barcode is a 650 base pair region of the cytochrome C oxidase subunit 1 or CO1 gene. This gene is found in the mitochondria and is involved in the electron transport chain. Since there are many mitochondria in each cell, there are many more copies of mitochondrial DNA than nuclear DNA.

DNA-based identification system The DNA barcode analogy can be explained by comparing a DNA barcode sequence, at the right, with a traditional UPC barcode, at the left. UPC barcodes use a combination of numbers to identify the products we purchase by comparing the combination to a database. A DNA barcode does a similar thing for species. The barcode region of CO1 can be used to identify the species from which it originated by searching a database of DNA barcode sequences for a match. This database will be reviewed later. The barcode region can differentiate between species because the DNA sequence - the unique arrangement of nucleotides – is usually highly variable between species but not very variable within the same species.

Why DNA-based identification? Known species: >150,000 >250,000 >300,000 > 30,000 flies, mosquitoes flowering plants beetles crabs, lobsters So, what are the advantages of DNA-based identification? First, it can be much quicker than traditional methods, and since there are so many species out there, this is very important. …because there are many, many species!

And many species have a variety of life forms Life cycle of Monarch butterfly Also, the DNA of an individual stays constant for its entire life even though its outer appearance may change substantially, like this Monarch butterfly. This is important as many taxonomic keys can only be used for adult specimens, which means an egg or larva form cannot be identified. The DNA barcode of a male and female of the same species will also be the same. This is actually very important as a male and female of the same species can look very different, for example, think about cardinals (males are red, females are brown) and sometimes taxonomic keys can only be used for the male of a species.

Biodiversity Institute of Ontario & This is the Biodiversity Institute of Ontario and Canadian Centre for DNA Barcoding. This centre is located in Guelph, Ontario and is the world’s largest facility for the production and analysis of DNA barcodes. This is where all of the Fish Market survey samples were analyzed. Biodiversity Institute of Ontario & Canadian Centre for DNA Barcoding World’s largest facility for the production and analysis of DNA barcodes

The Process of DNA Barcoding Photograph Collection Data Tissue Sample Specimen Publicly-Accessible DNA Barcode, Data and Analysis The process of DNA barcoding is relatively straightforward. It starts with a specimen. Click 1: That specimen is photographed a small tissue sample is taken. All collection data from the specimen, such as where and when it was collected is also recorded. Click 2: The sample is subjected to standard DNA barcoding protocols, which will be reviewed in the next few slides. Click 3: Finally, the DNA barcode, photograph and collection data are uploaded to the Barcode of Life Data System or BOLD – a publically accessible database of DNA barcode sequences. The information from this action project was entered into a BOLD project.

A small portion of the specimen is removed for DNA extraction. This is incubated overnight in a chemical cocktail that breaks down cellular structure and proteins. The standard lab protocol for specimens starts with the sub-sampling step. In this step, a small piece of the specimen is removed for DNA extraction and placed in a high throughput 96 well plate. This can be a small piece of muscle, or an insect leg, for example.

DNA extractions are carried out using a robotic system DNA extractions are carried out using a robotic system. The full plate of 96 samples can be extracted in approximately 15 minutes. Extractions are conducted using a dedicated Biomek FX robotic system. 94 specimens can be extracted in approximately 15 minutes.

The gene region of choice is amplified using the polymerase chain reaction (PCR). This is carried out using specific enzymes, primers and temperatures created in these thermocyclers. The next step is to amplify the barcode region using PCR. A specific combination of enzymes and primers are used with optimized thermal settings programmed into thermocyclers, like those pictured here.

Once PCR is complete, it is checked for success by running an agarose gel electrophoresis. The gel contains ethidium bromide, a compound which binds to DNA and causes it to fluoresce, or glow, when exposed to UV light. The success of a PCR reaction is determined by loading PCR product onto an agarose gel containing ethidium bromide. This compound binds to DNA and causes it to fluoresce under UV light.

High-success plates proceed to sequencing. A successful reaction will show a band, whereas a failure will show no band. Plates with high success proceed to sequencing. A successful reaction will display this band – a failure will lack a band. High-success plates proceed to sequencing.

The DNA barcode is sequenced using two ABI 3730xl DNA sequencers. Another robotic system carries out the DNA sequencing reaction. A technician then reviews the output and does any necessary editing. The DNA barcode is sequenced using two ABI 3730xl DNA sequencers.

www.barcodinglife.org The final step is uploading the DNA barcode sequence and all supplementary information for each specimen to BOLD. The database can be used to identify unknown samples.

Current Status and Goals International Barcode of Life (iBOL) Goal: Barcode all life on Earth! Currently 5.9 million DNA barcode sequences for ~545,000 species Goal for 2020 is 1 000 000 species To continue to identify biodiversity and allow for some of the applications mentioned in this presentation, as well as many others, the DNA barcode library must continue to be built and maintained. The International Barcode of Life project is the largest biodiversity genomics initiative ever undertaken. This project plans to DNA barcode all life on Earth, adding digital genetics information to our already existing knowledge about species. In celebration of the one millionth DNA barcode, the CN Tower in Toronto was lit up with the DNA barcode of a beaver. Currently there are 5.9 million DNA barcode sequences for approximately 545, 000 species in BOLD. The goal for 2020 is one million different species Citizen science projects, such as this action project, and other high school barcoding projects, in Canada and around the world, can help achieve this goal.