DNA barcoding using the cytochrome c oxidase I (COI) gene will allow species identification within...

1
DNA barcoding using the cytochrome c oxidase I (COI) gene will allow species identification within the genus Agrilus. This will allow the positive identification of not only Agrilus males but females, larvae, and eggs. Availability of good quality specimens for initial DNA extraction seems to be a major limiting factor in studies of this kind. The ability to amplify DNA from archived specimens provides a large sample size to begin barcoding, along with demonstrating the value of collections. Future research will involve expanding the number of species within the database. Currently I have sequence data for 31 of the approximately 70 species of Agrilus native to Indiana and the surrounding Midwest. Once complete, a DNA barcode database of native Agrilus species could be used to identify unknown samples or facilitate ecological studies. ATCCCGCTTTGATATCCGGCTTGAGTCGGTGTGTGCCAACGCGATATGACGGACGTGTGTGCGAGGGTCTCAACTACAGGGATTAGATAGATGATATTTTAGATATTAGAGGAA AAGAGAGGGGAGCGACGAGGCGAGGGCGAGCTGTAGCTACGGGATCATGCATGCGAAGGGATCGAGCTGACCCACACACCCGCGGCGCGGCATATGCATCTCTCTCAGCGAGAG ACATATATATACGATTTTTTATGAGAGTAGCAGGAGGCGAGGCCCCGAGCGCGAGATATATAAAATATAGAGAATAGTATTTTTTAGATATACGCCGCGAGCGCGCGGCGCGCT ATATATATATTCTCGCTACGATGTAGCATCGATGCAGATGCGATTATATATATGATTATTGGCTAGCTATGCGCGGCGATGGAGACATATATATACGATTTTTTATGAGAGTAG CAGGAGGCGAGGCCCCGAGCGCGAG mtDNA Barcoding for Taxonomic Identification within mtDNA Barcoding for Taxonomic Identification within the Genus the Genus Agrilus Agrilus John T. Shukle and Jeffrey D. Holland John T. Shukle and Jeffrey D. Holland Department of Entomology, College of Agriculture, Purdue University, 901 West State Street, West Lafayette, Department of Entomology, College of Agriculture, Purdue University, 901 West State Street, West Lafayette, IN 47907, USA (4/04/07) IN 47907, USA (4/04/07) Introduction Introduction Ecological studies are constantly refining our image of what an ecosystem is and how it works; however, these studies are often complicated and time consuming due to several limiting factors, one of which is the need for species level identifications. Studies involving insects especially rely on fast and accurate identification. Unfortunately, many groups of insects require a high level of expertise to identify to the species level. Insects have a major effect on natural ecosystems, either by driving ecosystem services or as disruptive invasive species. There is a clear need for a faster method of species identification within these important groups of organisms. The main objectives of my research are: 1. Test the hypothesis that a standard DNA sequence can differentiate species within the genus Agrilus, and 2. Develop a searchable DNA barcode database for the genus in the Midwest. 5` 3` LCOF 1490* HCOR 2198* Ag1718F° Ag1859R° mtDNA Genome tRNA Coding Regions Gene Coding Regions Control Region Barcoding Region Forward Primer Reverse Primer 1401 2942 DNA was extracted from three legs of specimens collected on purple sticky traps or from museum specimens, using either a Qiagen DNeasy kit or a modified version of the method of Lis et al. (1983). Some museum specimens were more than forty years old. PCR reactions followed the protocol described by Hebert et al. (2002). Forward and reverse primers from Hebert et al. (2002) were used to Fig. 5. The order of genes on the mitochondrial genome of Tribolium. The enlarged region shows the position of the barcoding region within the COI gene and positions of forward and reverse primers. * Indicates primers from Hebert et al. (2002). ° Indicates degenerate versions of primers from Simon et al. (1994). Concepts of Barcoding Concepts of Barcoding DNA barcoding uses sequences of DNA to identify species based on base pair comparisons. While the concepts behind DNA barcoding should work for most rapidly evolving genes, mitochondrial genes have become the preferred choice because of their maternal inheritance, low recombination potential, and lack of insertions and deletions. Within the mitochondrial genome the cytochrome c oxidase I (COI) gene was chosen as a standard for DNA barcoding because of the presence of robust primer locations. One of the main advantages of DNA barcoding is the ability to identify an individual to species using very small amounts of tissue, for example, a single insect leg or the base of a bird’s feather. While initial studies have proven the effectiveness of barcoding as a tool for species identification, the validity of the technique still needs to be tested at finer resolutions. I chose to test this technique within the genus Agrilus. Biology of the Biology of the Agrilus Agrilus Agrilus larvae are borers that feed on the living tissue of their host. Most species attack the cambial tissue of woody trees or shrubs, although a few species do feed on herbaceous plants, generally attacking root or stem tissue. In native habitats Agrilus target stressed or dying host plants, providing an ecosystem service by eliminating diseased individuals of a population. However, when introduced into ecosystems where host plants lack co-evolutionary resistance, or where natural predators and parasites are absent, they can become severe pests. The recent infestation of Emerald Ash Borer, Agrilus planipennis, is an excellent example of this. A B Taxonomic Relationships Taxonomic Relationships vittaticollis ruficollis bilineatus ferrisi planipennis cladrastis1 celti lacustris1 cyanescens cephalicus1 fuscipennis H_memnonius 60 61 58 100 60 60 41 37 67 Fig. 4. Phylogram showing the taxonomic relationships of 11 species of Agrilus based on full (708 bp) and micro (369 bp) mtDNA barcodes. For distance/neighbor-joining analysis 1000 bootstrap replicates were performed. The values at nodes refer to the percentage of replications supporting each node. The click beetle H. memnonius was used as an outgroup. Hebert, Paul D. N, et al. 2002. “Biological identifications through DNA barcodes.” Proc. R. Soc. Lond. Online. Lis, J. T., et al. 1983. “New heat shock puffs and β-galactosidase activity from transformation of Drosophila with an hsp70-lacZ hybrid gene.” Cell 35:403-410. Simon, N., et al. 1994. “Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87:651-70. % Sequence Variation % Sequence Variation % Occurrence % Occurrence H. memnonius A. fuscipennis A. cephalicus A. cyanescens A. lacustris A. celti A. cladrastis A. planipennis A. ferrisi A. bilineatus A. ruficollis A. vittaticollis Fig. 2. Galleries in oak caused by A. bilineatus. High levels of infestation girdle the host tree. Fig. 1. Mitochondria contain large numbers of replicates of their circular genomes. This provides an excellent amount of starting template for PCR reactions. Mitochondrial Mitochondrial DNA strands DNA strands Conclusions Conclusions Summary Summary Methods Methods The genus Agrilus is the largest genus within the family Buprestidae, order Coleoptera, and contains nearly 3,000 described species. amplify the full 708 bp barcoding region. For amplification from degraded DNA, degenerate versions of primers from Simon et al. (1994) were used to recover 369 bp and 480 bp overlapping fragments internal to the barcoding region (Fig. 3). DNA was direct sequenced bi-directionally through either MWG Biotech or the low throughput genomics facility at Purdue. Pairwise sequence comparisons were done in PAUP. The program Clustal X was used for multiple sequence alignments and to generate a neighbor-joined tree. Overlapping fragments were merged using the Merger tool in EMBOSS. Fig. 3. The success of PCR amplification depended on the quality of the template DNA: 1. Fresh collected A. planipennis; 2. Dried specimen of A. bilineatus. Three sets of primers were used to amplify either (A) the whole 708 bp barcoding region, or two overlapping internal fragments of (B) 480 bp and (C) 369 bp respectively. Sequence Variation Sequence Variation 1 Fresh 2 Dried A C B A B C 1 Kb 1 Kb COI Acknowledgements Acknowledgements References References While DNA barcoding is primarily a tool for species identification, barcode-based trees can provide a preliminary phylogenetic placement for species. The number of taxa (species) supported by a tree based on barcode sequence data is related to the number of determining characters within the sequence. Therefore, the longer the sequence the more taxa can be successfully placed. Fig. 6. Larva of A. bilineatus showing the characteristic morphology for the genus Within Species Between Species Recent studies have shown the efficacy of mtDNA barcoding to differentiate morphologically similar species. I used a 708 bp fragment internal in the cytochrome c oxidase I (COI) gene to evaluate the effectiveness of this technique within the genus Agrilus. Results indicate that mtDNA barcoding using the COI gene will provide a viable method to distinguish between species in the genus. This work was supported through undergraduate research scholarships from the Purdue College of Agriculture. Additional financial support came from the Department of Entomology. Specimens and identification material were generously provided by Arwin Provonsha. 708 bp Barcoding Region 1.0% Divergence SCALE

Transcript of DNA barcoding using the cytochrome c oxidase I (COI) gene will allow species identification within...

Page 1: DNA barcoding using the cytochrome c oxidase I (COI) gene will allow species identification within the genus Agrilus. This will allow the positive identification.

DNA barcoding using the cytochrome c oxidase I (COI) gene will allow species identification within the genus Agrilus. This will allow the positive identification of not only Agrilus males but females, larvae, and eggs.

Availability of good quality specimens for initial DNA extraction seems to be a major limiting factor in studies of this kind. The ability to amplify DNA from archived specimens provides a large sample size to begin barcoding, along with demonstrating the value of collections.

Future research will involve expanding the number of species within the database. Currently I have sequence data for 31 of the approximately 70 species of Agrilus native to Indiana and the surrounding Midwest. Once complete, a DNA barcode database of native Agrilus species could be used to identify unknown samples or facilitate ecological studies.

ATCCCGCTTTGATATCCGGCTTGAGTCGGTGTGTGCCAACGCGATATGACGGACGTGTGTGCGAGGGTCTCAACTACAGGGATTAGATAGATGATATTTTAGATATTAGAGGAAAAGAGAGGGGAGCGACGAGGCGAGGGCGAGCTGTAGCTACGGGATCATGCATGCGAAGGGATCGAGCTGACCCACACACCCGCGGCGCGGCATATGCATCTCTCTCAGCGAGAGACATATATATACGATTTTTTATGAGAGTAGCAGGAGGCGAGGCCCCGAGCGCGAGATATATAAAATATAGAGAATAGTATTTTTTAGATATACGCCGCGAGCGCGCGGCGCGCTATATATATATTCTCGCTACGATGTAGCATCGATGCAGATGCGATTATATATATGATTATTGGCTAGCTATGCGCGGCGATGGAGACATATATATACGATTTTTTATGAGAGTAGCAGGAGGCGAGGCCCCGAGCGCGAG

mtDNA Barcoding for Taxonomic Identification within the Genus mtDNA Barcoding for Taxonomic Identification within the Genus AgrilusAgrilusJohn T. Shukle and Jeffrey D. HollandJohn T. Shukle and Jeffrey D. Holland

Department of Entomology, College of Agriculture, Purdue University, 901 West State Street, West Lafayette, IN 47907, USA (4/04/07)Department of Entomology, College of Agriculture, Purdue University, 901 West State Street, West Lafayette, IN 47907, USA (4/04/07)

IntroductionIntroduction Ecological studies are constantly refining our image of what an ecosystem is and how it works; however, these studies are often complicated and time consuming due to several limiting factors, one of which is the need for species level identifications. Studies involving insects especially rely on fast and accurate identification. Unfortunately, many groups of insects require a high level of expertise to identify to the species level. Insects have a major effect on natural ecosystems, either by driving ecosystem services or as disruptive invasive species. There is a clear need for a faster method of species identification within these important groups of organisms. The main objectives of my research are: 1. Test the hypothesis that a standard DNA sequence can differentiate species within the genus Agrilus, and 2. Develop a searchable DNA barcode database for the genus in the Midwest.

5` 3`

LCOF 1490*

HCOR 2198*

Ag1718F°

Ag1859R°

mtDNA GenometRNA Coding Regions

Gene Coding Regions

Control Region

Barcoding Region

Forward Primer

Reverse Primer

1401 2942 DNA was extracted from three legs of specimens collected on purple sticky traps or from museum specimens, using either a Qiagen DNeasy kit or a modified version of the method of Lis et al. (1983). Some museum specimens were more than forty years old. PCR reactions followed the protocol described by Hebert et al. (2002). Forward and reverse primers from Hebert et al. (2002) were used to

Fig. 5. The order of genes on the mitochondrial genome of Tribolium. The enlarged region shows the position of the barcoding region within the COI gene and positions of forward and reverse primers. * Indicates primers from Hebert et al. (2002). ° Indicates degenerate versions of primers from Simon et al. (1994).

Concepts of BarcodingConcepts of Barcoding DNA barcoding uses sequences of DNA to identify species based on base pair comparisons. While the concepts behind DNA barcoding should work for most rapidly evolving genes, mitochondrial genes have become the preferred choice because of theirmaternal inheritance, low recombination potential, and lack of insertions and deletions. Within the mitochondrial genome the cytochrome c oxidase I (COI) gene was chosen as a standard for DNA barcoding because of the presence of robust primer locations. One of the main advantages of DNA barcoding is the ability to identify an individual to species using very small amounts of tissue, for example, a single insect leg or the base of a bird’s feather. While initial studies have proven the effectiveness of barcoding as a tool for species identification, the validity of the technique still needs to be tested at finer resolutions. I chose to test this technique within the genus Agrilus.

Biology of the Biology of the AgrilusAgrilus

Agrilus larvae are borers that feed on the living tissue of their host. Most species attack the cambial tissue of woody trees or shrubs, although a few species do feed on herbaceous plants, generally attacking root or stem tissue. In native habitats Agrilus target stressed or dying host plants, providing an ecosystem service by eliminating diseased individuals of a population. However, when introduced into ecosystems where host plants lack co-evolutionary resistance, or where natural predators and parasites are absent, they can become severe pests. The recent infestation of Emerald Ash Borer, Agrilus planipennis, is an excellent example of this.

A B

Taxonomic RelationshipsTaxonomic Relationships

vittaticollis

ruficollis

bilineatus

ferrisi

planipennis

cladrastis1

celti

lacustris1

cyanescens

cephalicus1

fuscipennis

H_memnonius0.01

60

61

58

100

60

60

41

37

67

Fig. 4. Phylogram showing the taxonomic relationships of 11 species of Agrilus based on full (708 bp) and micro (369 bp) mtDNA barcodes. For distance/neighbor-joining analysis 1000 bootstrap replicates were performed. The values at nodes refer to the percentage of replications supporting each node. The click beetle H. memnonius was used as an outgroup.

Hebert, Paul D. N, et al. 2002. “Biological identifications through DNA barcodes.” Proc. R. Soc. Lond. Online.

Lis, J. T., et al. 1983. “New heat shock puffs and β-galactosidase activity from transformation of Drosophila with an hsp70-lacZ hybrid gene.” Cell 35:403-410.

Simon, N., et al. 1994. “Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87:651-70.

% Sequence Variation % Sequence Variation

% O

cc

urr

en

ce

% O

cc

urr

en

ce

H. memnonius

A. fuscipennis

A. cephalicus

A. cyanescens

A. lacustris

A. celti

A. cladrastis

A. planipennis

A. ferrisi

A. bilineatus

A. ruficollis

A. vittaticollis

Fig. 2. Galleries in oak caused by A. bilineatus. High levels of infestation girdle the host tree.

Fig. 1. Mitochondria contain large numbers of replicates of their circular genomes. This provides an excellent amount of starting template for PCR reactions.

MitochondrialMitochondrial

DNA strandsDNA strands

ConclusionsConclusions

SummarySummary

MethodsMethods

The genus Agrilus is the largest genus within the family Buprestidae, order Coleoptera, and contains nearly 3,000 described species.

amplify the full 708 bp barcoding region. For amplification from degraded DNA, degenerate versions of primers from Simon et al. (1994) were used to recover 369 bp and 480 bp overlapping fragments internal to the barcoding region (Fig. 3). DNA was direct sequenced bi-directionally through either MWG Biotech or the low throughput genomics facility at Purdue. Pairwise sequence comparisons were done in PAUP. The program Clustal X was used for multiple sequence alignments and to generate a neighbor-joined tree. Overlapping fragments were merged using the Merger tool in EMBOSS.

Fig. 3. The success of PCR amplification depended on the quality of the template DNA: 1. Fresh collected A. planipennis; 2. Dried specimen of A. bilineatus. Three sets of primers were used to amplify either (A) the whole 708 bp barcoding region, or two overlapping internal fragments of (B) 480 bp and (C) 369 bp respectively.

Sequence VariationSequence Variation

1 Fresh 2 Dried

A

CB

A

BC

1 Kb1 Kb

COI

AcknowledgementsAcknowledgements

ReferencesReferences

While DNA barcoding is primarily a tool for species identification, barcode-based trees can provide a preliminary phylogenetic placement for species. The number of taxa (species) supported by a tree based on barcode sequence data is related to the number of determining characters within the sequence. Therefore, the longer the sequence the more taxa can be successfully placed. Fig. 6. Larva of A. bilineatus showing the

characteristic morphology for the genus

Within Species Between Species

Recent studies have shown the efficacy of mtDNA barcoding to differentiate morphologically similar species. I used a 708 bp fragment internal in the cytochrome c oxidase I (COI) gene to evaluate the effectiveness of this technique within the genus Agrilus. Results indicate that mtDNA barcoding using the COI gene will provide a viable method to distinguish between species in the genus.

This work was supported through undergraduate research scholarships from the Purdue College of Agriculture. Additional financial support came from the Department of Entomology. Specimens and identification material were generously provided by Arwin Provonsha.

708 bp Barcoding Region

1.0%Divergence

SCALE