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Molecular Techniques and Conservation Biology

By Julie Pederson, Ph.D., and Neal Cosby, Ph.D.
Promega Corporation

There are 4 pages to this Feature:
Overview - Goals - Technology - Photos & References

Editor's Note: This Feature is offered as a supplement to the Applications article, Screening of the Gamµ-7 Microsatellite Locus to Determine the Sex of Captive Whooping Cranes. The goal of the Feature section of eNotes is to place a specific application in the context of science and research affecting all of us.

Composite image composed of (clockwise, from upper left) a whooping crane, STR sequencing gel results, DNA forensics studies and agarose gel analysis of PCR products. The photographs are linked by a cartoon representation of the PCR process, illustrated by a single cycle of amplification and including template DNA, oligonucleotide primers, thermostable DNA polymerase and nucleotides. Image of the whooping crane courtesy of the International Crane Foundation.

Overview

"As for other areas of molecular biotechnology, conservation genetics is an applied science with the important goal of describing explicitly the composite genomes of small endangered populations" (1).
--Stephen J. O'Brien

Analyzing DNA is common enough these days but for the most part the techniques are applied to readily available samples. The case for threatened or endangered animals, captive or in the wild, is much different. Extracting a tissue sample, much less locating or being granted access to such animals, presents real problems before applying molecular techniques. This story focuses on some of the current molecular techniques being applied in conservation biology.

Threatened species are those having an uncertain chance of continued survival, and likely to become an endangered species. Endangered species are those 'threatened' with extinction. These may not be the legally-binding definitions, but each conveys the meaning of the species being challenged in the face of a dwindling habitat ("threatened") versus the species consisting of only a few extant breeding individuals ("endangered"). Species are removed from the lists by one of two main ways: they recover or become extinct. For more information on threatened and endangered animals, see the U.S. Fish and Wildlife Service web site.

Name the threatened or endangered species and molecular techniques are more than likely being applied. For example, scientists in the genetics laboratory at the Brookfield Zoo in Illinois are collaborating on projects using i) PCR amplification, mtDNA sequence and enzymatic restriction to determine gender of monomorphic birds, such as the Humboldt penguin (endangered); ii) determine the species and subspecies of three genera of captive New World primates, owl monkeys (Aotus), spider monkeys (Ateles) and squirrel monkeys (Saimiri) distributed throughout Central and South America; iii) microsatellite loci and DNA fingerprinting methods for paternity analysis in gorillas and dolphins; and in studies on African wild lions, iv) PCR amplification, sequencing of cytochrome b and NADH dehydrogenase 5 genes, microsatellite analysis and DNA fingerprinting; v) multi-locus DNA fingerprint methods were used to determine that offspring born to a captive female Arafuran Filesnake at Brookfield Zoo had no father---apparently females can reproduce by parthenogenesis; vi) mtDNA sequence of both NADH dehydrogenase 5 and 6 subunits and cytochrome b were used to study the mountain brushtail possum in Australia. And this just from the files of a single institution. For an explanation of these and other techniques, see the Technology section of this article.

International Crane Foundation

The International Crane Foundation, or ICF, is one place where the current technology is aiding the 15 species of cranes, seven of which are endangered. The ICF was founded in 1973 by Ron Sauey and George Archibald, and has since concentrated efforts on recovering these populations of birds in five ways: research, education, habitat protection, captive breeding and restocking. The research efforts involve collaborations with scientists and conservationists in Europe, Asia, Australia and Africa, in addition to North America. Ken Jones, currently at the University of Chicago, is just one of many scientists who have traveled to Wisconsin to study cranes and apply current molecular techniques to the study of this species. Jones along with Travis Glenn of the Savannah River Ecology Laboratory in South Carolina, address the use of PCR for confirming sex in breeding pairs of whooping cranes in their article, Screening of the Gamµ-7 Microsatellite Locus to Determine the Sex of Captive Whooping Cranes.

There are 4 pages to this Feature:
Overview - Goals - Technology - Photos & References

Goals

The techniques of population genetics and molecular biology offer methods for developing conservation plans for species that are at risk of extinction (1). The application of population genetics to conservation work takes advantage of the wealth of early basic research performed in the laboratory to the characterization of genomic structure of dwindling species populations. The application of established population genetics to the conservation field is on several levels.

Genetic studies first attempt to characterize the amount of genetic diversity of a species on a molecular level and determine an estimate of heterozygosity (a measure of genetic variation within a population). Further studies may look at the pedigrees of existing populations, which allows assessment of mating success and correlation of certain genetic profiles with genetic relatedness and mating success. Armed with the data from genetics and other disciplines (epidemiology, reproductive biology, etc.), plans may be developed for setting up captive breeding matches or plans to introduce new diversity into the population, if possible. The genetic data also can be used to verify or correct taxonomic classification and to reconstruct natural histories for a population.

A number of the molecular techniques of population genetics are described in the following section, Technology (right arrowhead below).

There are 4 pages to this Feature:
Overview - Goals - Technology - Photos & References

Technology

There are many methods used for conservation work to identify individuals in key populations, including allozyme analysis, two-dimensional polyacrylamide gel electrophoresis (PAGE), sequence analysis, restriction fragment length polymorphism (RFLP) analysis, PCR-RFLP and microsatellite analysis.

Allozyme analysis, the earliest population genetic measure using a molecular technique involves protein electrophoresis to distinguish allelic forms of an enzyme. The first application of this work to population genetics was by Lewontin and Hubby in 1966 to survey gene-controlled protein variants in natural populations of Drosophila pseudoobscura and provide measures of heterozygosity for those populations (2). This form of analysis is still in use today.

Two-dimensional PAGE combines SDS-PAGE in the first dimension with isoelectric focusing in the second dimension to reveal heterogeneity of charge in proteins and can be used to show genetic polymorphisms in a population.

Sequence analysis can be applied to studies of mitochondrial DNA (mtDNA) diversity. For example, the D-loop of mitochondria is a segment of 500-600 bases in mammalian mitochondria where an opening of the duplex DNA forms and replication initiates. The D-loop is displaced by a short stretch of RNA base-paired to the complementary strand of mitochondrial DNA. Sequence analysis of the D-loop (control region) has revealed that this region is polymorphic in many species. When used with phylogenetic analyses, information about this sequence can be used to trace maternal lineage of an individual.

The mtDNA also exhibits restriction fragment length polymorphisms (RFLP). To determine polymorphisms in restriction fragment length of mtDNA, the mtDNA is first amplified by PCR and then analyzed for RFLPs using a number of restriction enzymes. This combination of PCR and RFLP (PCR-RFLP) has been used to detect genetic polymorphisms in cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes, for example.

RFLP analysis has been used for some time in the analysis of polymorphisms of mammalian major histocompatibility complex (MHC) genes and has revealed a large degree of polymorphism, making this region of DNA targeted in assessments of genetic diversity in many animals. Exploiting the diversity in the MHC RFLP and other genetic locations, researchers have made "DNA fingerprints" for different populations. In the DNA fingerprinting technique, DNA from different individuals is digested with restriction enzymes and resolved on an agarose gel. Then, using labeled probes to specific sequences in the polymorphic region, Southern blot analysis is performed to show the bands of interest. In general, the pattern displayed on the gel is specific for a particular individual and can be used to establish family relationships or degree of relatedness between individuals.

Other forms of analyses use PCR-based techniques to reveal polymorphic loci. Microsatellite, or short tandem repeat (STR), loci consist of short repetitive sequence elements 3 to 7 base pairs in length (3-6). These repeats are well distributed throughout the genomes of many species and are a rich source of highly polymorphic markers that may be detected using PCR (7-10). Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and can be distinguished from one another using radioactive, silver stain or fluorescence detection following electrophoretic separation.

The application of genetic techniques to the analysis of population structure and management allows a rigorous analysis of endangered species. Improvements in DNA analysis techniques, as well as theoretical and statistical work adapted from basic science, provide the conservation biologist with a powerful set of tools for potentially saving populations at risk.

There are 4 pages to this Feature:
Overview - Goals - Technology - Photos & References

Photos and References

International Conservation Center, just ahead! Although the International Crane Foundation (ICF) sports a roadside billboard on Wisconsin Highway 12 just a few miles from its headquarters in Baraboo, the foundation is anything but a roadside operation. The ICF breeds and raises all fifteen species of cranes and collaborates with scientists around the world on crane ecology, behavior and genetics studies. For more information on the ICF, visit their web site at: www.savingcranes.org.
Photograph by Neal Cosby.

Gated entrance to the International Crane Foundation (Baraboo, WI). The gate features an exquisite rendering of cranes in wrought iron.
Photograph by Neal Cosby.

Claire M. Mirande and Kate Fitzwilliams of the International Crane Foundation (Baraboo, WI). Mirande is Conservation Services Director, and Fitzwilliams is the Director of Public Relations and Outreach.
Photograph by Neal Cosby.

The Amoco Whooping Crane Exhibit at the International Crane Foundation affords visitors the chance to view cranes much as they might appear in the wild.
Photograph by Neal Cosby.

A whooping crane photographed on site at the International Crane Foundation Baraboo, WI.
Image courtesy of the International Crane Foundation.

References

1. O'Brien, S.J. (1994) A role for molecular genetics in biological conservation. Proc. Natl. Acad. Sci. USA 91, 5748.
2. Lewontin, R.C. and Hubby, J.L. (1966) A molecular approach to the study of genic heterozygosity in natural populations. II. Amount of variation and degree of heterozygosity in natural populations of Drosophila pseudoobscura. Genetics 54, 594
3. Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: polymorphic loci, detection systems, and population genetics. In: The Second International Symposium on Human Identification 1991, Promega Corporation, 31.
4. Edwards, A. et al. (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49, 746.
5. Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241.
6. Warne, D. et al. (1991) Tetranucleotide repeat polymorphism at the human beta-actin related pseudogene 2 (ACTBP2) detected using the polymerase chain reaction. Nucl. Acids Res. 19, 6980.
7. Ausubel, F.M. et al. (1993) Unit 15: the polymerase chain reaction. In: Current Protocols in Molecular Biology, Vol. 2, Greene Publishing Associates, Inc., and John Wiley and Sons, NY.
8. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Chapter 14: in vitro amplification of DNA by the polymerase chain reaction. In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
9. PCR Technology: Principles and Applications for DNA Amplification(1989), ed., Erlich, H.A., Stockton Press, New York.
10. PCR Protocols: A Guide to Methods and Applications (1990) eds., Innis, M.A. et al. Academic Press, San Diego.

Additional Reading:

Ding, B., Zhang, Y.-P. and Ryder, O.A. (1998) Extraction, PCR amplification and sequencing of mitochondrial DNA from scent mark and feces in the giant panda. Zoo Biol. 17, 499.

Eizirik, E. et al. (1998) Phylogeographic patterns and evolution of the mitochondrial DNA control region in two neotropical cats (Mammalia, Felidae). J. Mol. Evol. 47, 613.

Palumbi, S.R. and Cipriano, F. (1998) Species identification using genetic tools: The value of nuclear and mitochondrial gene sequences in whale conservation. J. Heredity 89, 459.

Waits, L.P. et al. (1998) Mitochondrial DNA phylogeography of the North American brown bear and implications for conservation. Conservation Biology 12, 408.

Paxinos, E. et al. (1997) A noninvasive method for distinguishing among canid species: Amplification and enzyme restriction of DNA from dung. Mol. Ecol. 6, 483.

Butler, M.A., Templeton, A.R. and Read, B. (1994) DNA fingerprinting in Speke's gazelle: A test for genetic distinctness, and the correlation between relatedness and similarity. Mol. Ecol. 3, 355.

Ali, S. et al. (1998) Analysis of the evolutionary conserved repeat motifs in the genome of the highly endangered central Indian swamp deer Cervus duvauceli branderi. Gene 223, 361.

Wasser, S.K. et al. (1997) Techniques for application of faecal DNA methods to field studies of Ursids. Mol. Ecol. 6, 1091.

Encalada, S.E. et al. (1996) Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment. Mol. Ecol. 5, 473.

Altmann, J. et al. (1996) Behavior predicts genetic structure in a wild primate group. Proc. Natl. Acad. Sci. USA 93, 5797.

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