Ways to measure Genetic variation;
Over the last century and a half, various population or ecologist geneticists and evolutionists have attempted to estimate the genetic variability of species and of their populations. Why should so many be interested in the genetic diversity of populations?
Before we start in with techniques which researchers do use to measure this diversify, let us review some reasons for population variability
Potential causes for differing levels of genetic variability include:
Can you add other factors which may alter genetic variability?
Now to the question, how did or do these evolutionary biologists
actually measure or estimate population variability. There are four
major techniques employed listed in chronological order of initial
development:
Measurement of phenotypic traits of a populationIdentification of chromosomal variation
DNA fingerprinting and Mitochondrial DNA analysis
Measurement of phenotypic traits
The concept that morphological variation has a genetic basis goes back thousands of years. The selection by humans of crops which would produce larger seed or farm animals that were tamer or dogs with pointy ears and longer legs is long withstanding. Thus it is no surprise that a number of evolutionists can find morphological differences either between or within populations that is genetically based.
Perhaps one of the best studied examples of natural phenotypic variation is that of the yarrow plant, Achillea. studied by Clausen, Keck and Heisey in the late '40's. Since the species can be found in the Sierra Nevadas from the lowlands to high in the tundra it is not unexpected that it would vary in size along a transect - plants generally decrease in size along most altitudinal gradients. The questions was, was this phenotypic variation a plastic response or expression of a single genotype due to the lack of resources i.e.. moisture, shorter growing season or due to true genetic differences between subpopulations along the slope.
They resolved the question by transplanting seedlings from high, low and medium elevation sites to gardens located at the coast, mid elevation [1300 meters] and at timberline [ 3400 meters].
lowland pop mid elevation pop timberline pop coastal garden 83.6 cm. 58.2 15.5 mid elevation 79.6 82.4 34.3 timberline 21.2 31.6 23.7
Some populations are obviously more plastic than others - why?
What are the pros and cons of this transplanting technique to
determine heritability of phenotypic traits?
Identification of
chromosomal variation
Chromosomes may vary in size, shape ( one or two armed) , banding patterns with staining, numbers and position ( some cases they may form rings or odd configurations).
A number of critical studies are based on the analysis of chromosomal variants or karyotypes of fruit flies along geographical gradients. Dobzhansky was able to identify 4 major gene arrangements in the 3d chromosome of Drosophila pseudoobscura, He interpreted these arrangements as coadapted gene complexes protected from recombination via inversion.
Others have used Geimsa staining band patterns to reveal patterns which correlated with environmental gradients or with mating restrictions.
Pros: With training, differences in karotypes may be readily studied
Con? DNA: It is unlikely that there is always a molecular connection
with karyotypes differences in wildlife. However in the better studied human
populations, a number of deviations have been related to specific genetic diseases
.
Allozymes identify protein variants of the same gene; they are due to amino acid substitutions in proteins. .
After grinding tissue to release the cytoplasm, wicks are used to absorb the the resulting extract and placed in a slit cut into a starch gel, Acrylamide gels or agarose gels are also used in which case the extract is loaded into a well. A low current is run across the gel resulting in a positive and negative ends. Proteins are separated by charge and size, with the smaller and more highly charged molecules moving more quickly across the gel.
After the run is completed the gel is sliced horizontally to produce 2 or more slices. Each slice is stained with the appropriate substrate, cofactors and energy source under controlled conditions of temperature and pH. Given there is genetic variability reflected by alleles that produce proteins of different charge and size, these protein variants will show up as bands. A heterozygote may show 2 bands, homozygote one and a polyploid many.
Cons
This techniques does underestimate true genetic variability as there may be an amino acid substitution but if the aa is not charged differently than the original no differential migration will appear.- approximately 1/3 of the true genetic variation is thus not expressed by this technique. A an example follows:
Genetic variability in African catfish
Genetic variability in both domestic and wild populations has been assessed using allozymes and microsatellite DNA as markers. Microsatellite markers, which are comprised of non- coding chromatin material, have a greater degree of polymorphism than allozymes and have been found to be more useful in assessing variability in populations. They also have the added advantage of being easy to use in population studies carried out in the field, since preservation in ethanol is a simple process.
Analysis (using 7 microsattelite and 25 allozyme markers) of natural populations from 11 localities within the native range indicated a generally high genetic variability (mean expected heterozygosity He up to 78%), particularly when comparing populations from difference catchments. Variability in African catfish is generally higher than in other riverine catfish species such as Eutropius niloticus, Schilbe mystus, Chrysichthys nigrodigitatus and C. anguillaris. There are however some significant departures from this finding, particularly in South Africa where the population from the van der Kloof Dam (Orange River) has a much lower degree of variability. Domesticated populations have generally been found to have a low genetic variability in comparison with wild populations.
Galbusera et al 1996; Galbusera 1997; Teugels et al 1992; Volckaert & Agnese 1996
Additional problems:
....there is the possibility that a particular isozyme may not be expressed in the organism at that time, thus underestimating heterozygosity. There may be developmental or environmental interactions.
....Also there must be some real attempt to determine the genetic basis of these banding patterns which is not typically done- this requires crossing individuals of known genotypes which is pretty difficult to do with wild animals populations. Other wise bands expressed due to protein-molecular bondings may be interpreted as allozymes.
Pros:
Rather than just measuring one gene, you can estimate variability of an individual using as many 20-30 genes products [ or more if you can afford it]. Majority of these proteins are common to all organisms so you can compare across species.
Since proteins are direct products of genes, we are closer to the true gene level
Since you know the enzyme and its role in the organism you can more directly understand the relationship between frequency differences and the given environment. This is not always true but can be if you are willing to further investigate physiological associations between enzyme activity and a given environmental parameter.
Although not cheap, it can be accomplished at a reasonable cost.
Results from thousands of analyses: How would you interpret the following results?
biotic grouping % of polymorphic loci heterozygosity plants .26 .07 invertebrates .40 .10 insects .33 .07 fruit flies .43 .14 amphibia .27 .08 reptiles .22 .05 birds .15 .05 mammals .15 .04
Data obtained may be used to also estimate divergence between populations, species.
Minisatellites or hypervariable regions that are made up of short segments continuing multiple copies of a short sequence that can be analyzed readily. The variability of individual minisatellite loci is the result of mutations that cause the gain or loss of repeat units- these units are thought to be noncoding regions.
A DNA fingerprint is constructed by first extracting a DNA sample from body tissue or fluid. The sample is then segmented using restriction enzymes. The segments are marked with probes and exposed on X-ray film, where they form a pattern of black bars&emdash;the DNA fingerprint. If the DNA fingerprints produced from two different samples match, the samples probably came from the same organism.
The proposal copied below is an actual example of how DNA analysis is being used to genetically analyze wild populations: Please read through!
Title: Genetic Diversity Monitoring in Plants and Wildlife
Agency: U.S. Environmental Protection Agency // Laboratory: Environmental Monitoring Systems Laboratory //Proposal ID: #246
Problem Statement:
Goal: The goal of this project is to monitor the genetic diversity of feral populations in ecologically sensitive areas using DNA fingerprinting technologies. Loss of diversity resulting from habitat destruction and pollution is a major concern in wildlife populations. The genetic diversity or total gene ensemble of a population reflects its intrinsic robustness. Loss of genetic diversity leaves a species less able to adapt to new environmental stressors; therefore, loss of population genetic diversity can foreshadow species loss, with resultant loss of biological diversity within an ecosystem.
Background: Undisturbed natural populations tend to maintain a high degree of biological diversity or polymorphism, but any environmental stress that eliminates a large fraction of individuals from the breeding population can eliminate (by pure chance) important genetic variants. This phenomenon, known as a genetic "bottleneck", leads to a reduction of heterozygosity in succeeding generations. The overall effect is populations with greater vulnerability to future stresses. Therefore, quantitative measures of genetic diversity can be useful as indicators of past environmental insult as well as criteria for targeting potentially sensitive, i.e., genetically homogeneous populations.
Measurement of population genetic diversity directly supports the SERDP Conservation thrust area as an assessment tool to identify vulnerable populations and subpopulations of many species of animals and plants, and to monitor their responses to ongoing conservation and protection efforts. This project will have both basic research and applied research components. Enhancement of fingerprinting technologies and statistical evaluations will continue, especially as new species are examined. Once the analytical strategy for a species or genus is established it will be applied to a myriad of situations confronting member populations. This proposal is for continuation and enhancement of the efforts to develop fingerprinting technologies as genetic diversity measures currently underway in this laboratory with SERDP support.
Project Description:
Technical Objectives: The technique of DNA fingerprinting is being widely used to determine the identity and relatedness of individuals (particularly humans), and is also attracting attention as a tool for assessment of genetic variations within and between populations. Genetic distinctions between individuals will be demonstrated by analyzing unique differences in DNA, even in species that are otherwise genetically uncharacterized. The summation of DNA fingerprint differences of many individuals will provide a measure of genetic diversity in the population from which those individuals are derived.
Technical Approach: In this laboratory, we are currently adapting two different, but complementary, fingerprinting techniques for use in several species of fish and terrestrial animals. The first method relies on the presence of short repetitive DNA sequences interspersed throughout the genomes of most organisms. These DNA sequences, called VNTRs (variable number of tandem repeats), exhibit high variability within a population. Bands visualized on a Southern blot of target genomic DNA, using radiolabeled probes specific to the repeat sequence, are the genetic identity of an individual. Comparison of the banding patterns among individuals from a population yields a measure of genetic variation within that population. Comparisons across populations yield measures of relative genetic variation and also of the degree genetic relatedness of the populations. This method is being applied to a test sample of DNAs purified from more than seventy individual brown bullhead catfish representing three populations from both environmentally impacted and clean areas.
The second fingerprint method is based on thermal cycle polymerase chain reaction (PCR). In this procedure, DNA marker bands are biochemically multiplied by a cyclical enzymatic reaction with target DNA. This amplification process occurs when synthetic 10-base DNA molecules, used in the reaction, match exactly with regions in the DNA of interest. This is termed DAF, or DNA Amplification Fingerprinting. DAF reactions, for each of 400 commercially available synthetic 10-base molecules, yield several distinct bands or amplification products - depending on the species surveyed. These products ( 5,000 base pairs) from PCR reactions are visually analyzed by gel electrophoresis in agarose or polyacrylamide. Genomic DNA from two individuals within a species often produce disparate amplification banding patterns. A particular DNA band which is generated from the genome of one individual, but absent in a second individual, represents a polymorphism which can serve as a genetic marker. These markers, presumed to be allelic, are inherited in a Mendelian fashion. By statistically analyzing the segregation of these marker among the progeny of a sexual cross, or individual members of a population, genetic maps and indices of heterozygosity of virtually any species can be assembled.
Also, we have developed an ecologically based method of tissue/DNA acquisition for direct use in DNA Amplification Fingerprint reactions. This simple and rapid technique, which is non-intrusive to terrestrial and aquatic animals, obviates need for radionuclides and isolation, purification, and quantitation of genomic DNA for thermal cycle amplification reactions. This method combines the powerful tools of genetic analysis with an ecologically favorable means of sample acquisition. The strategy is particularly useful when collecting field specimens for population or forensic analysis, or species verification on field specimens and endangered species.
Using the raw fingerprint data from both of these methods, several mathematical treatments for assessing DNA fingerprint diversity are being examined and compared in order to determine the best statistically valid approach. This part of the effort is being done in conjunction with Dr. Vicki Hertzberg with a Cooperative Agreement funded by SERDP.
Since methods of DNA fingerprinting are under continuous and rapid advancement within the scientific community, we are requesting continuing SERDP support for development of the most efficacious system for each new species to which population genetic diversity measures are applied. Most particularly we will be expanding our efforts to examine plant population genetics. We intend to use an expanding battery of VNTR probes, such as PCR generated synthetic tandem repeat (STR) probes. We intend to adapt the new non-isotopic probe labeling techniques for use with our fingerprinting methods, which would avoid the use of radioisotopes, providing the advantages of standardization due to a long probe shelf-life and portability of methods to laboratories not equipped for radioisotopes usage.
We also will require continuous statistical support in order to tabulate and analyze data generated as each new population and species is examined but to continue to develop and refine the statistical methods required. Each new species will present not only a unique set of banding patterns to be analyzed, but these analyses will need to take into account characteristic higher-order population dynamics, most notably differences in breeding strategies.
We expect ever-increasing liaison with field ecologists who are experienced with and possess detailed knowledge of each relevant population. When appropriate, these interactions will be formalized as cooperative agreements. This will provide detailed expertise and assistance in field sample collection.
These fingerprint measures of population genetic diversity will directly support the conservation efforts of DoD/DOE by providing assessment tools for monitoring, protecting and rehabilitating natural ecosystems.
Expected Payoff:
This method will provide a rapid, non-invasive, and cost effective monitoring method in the form of an assessment of population genetic robustness for virtually any species, animal or plant, aquatic or terrestrial. It is anticipated that it can be modified into a commercially available, field usable tool and marketed via a CRADA.
13. Principal Investigator:
Dr. M. Kate Smith USEPA//Ecological Monitoring Research Division//Environmental Monitoring Systems Laboratory//26 West Martin Luther King Drive, Cincinnati, OH 45268//TEL: (513) 569-7577
DNA Fingerprinting
Dateline: 07/06/98
Deoxyribonucleic acid (DNA) is a linear, polymeric molecule composed of four repeating units, or bases: adenine (A), cytosine (C), guanine (G), and thymine (T). A gene is a specific sequence of these bases that directs the cellular synthesis of a particular protein. There are an estimated 100,000 genes in human DNA. However, this represents only 3% to 10% of a human's entire DNA complement; the remaining 90% or more of the DNA does not code for protein, but controls gene expression, orders the three-dimensional structure of the DNA, or has yet undiscovered properties.
This non-gene DNA harbors areas where the base sequences vary between individuals. Most of the time, these variances are without apparent ill effect. Therefore, I (and 10,000 other people) may have an A at one DNA site, whereas you (and 10,000 other people) may have a G in the same location. By contrast, such differences manifesting themselves in genes can have powerful consequences, and are the underpinnings of inherited diseases.
There are three types of DNA variability that are commonly used in DNA fingerprinting: restriction fragment length polymorphism, variable number of tandem repeats, and short tandem repeats (STRs). STR analysis is a popular method of DNA fingerprinting, and will be the technique discussed in this article.
STRs, also known as microsatellite repeats, consist of repeated sequences of two to seven bases. For example, the [GT]4 repeat is GTGTGTGT and [CAG]6 is CAGCAGCAGCAGCAGCAG.
The human genome contains hundreds of thousands of these STRs evenly distributed on all chromosomes. Consequently, there are thousands of each kind of repeat; that is, thousands of [GT]n, [CAG]n, [CTG]n, [GATA]n, etc. As a result, unequivocal DNA fingerprinting depends upon the unique DNA flanking sequences on each side of a repeat. These unique flanking sequences allow the analyst to zero in on a defined area of a hundred or so bases in a human genome that contains three billion bases.
Let's look at the STR designated D7S820 (GenBank number G08616) located on human chromosome 7. D7S820 contains a [GATA]n repeat, where n can range from 6 to 14. The next paragraph shows the DNA sequence of a D7S820 STR with twelve GATA repeats. (DNA is an unbroken, continuous polymer; the example displayed here shows gaps for the sake of readability. The [GATA]12 region is capitalized, and the unique flanking regions are underlined.)
aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa ctaacGATAG ATAGATAGAT AGATAGATAG ATAGATAGAT AGATAGATAG ATAgacagat tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc cctctgagtt tttgatacct cagattttaa ggcc
It is these unique, flanking sequences that are used to amplify the region between them. The amplification procedure, known as the polymerase chain reaction (PCR), enzymatically synthesizes thousands of copies of the intervening DNA region. This amplification enables forensic laboratories to generate enough DNA for analysis from hair roots and blood samples. Only one nanogram of DNA, a billionth of a gram, is required for a successful PCR. In a separate reaction, the PCR products have their DNA sequence determined. This sequence reveals the number of repetitive units in the sample.
A person inherits an equal amount of nuclear DNA from each
parent. Therefore, among all the other DNA passed down from one's father and
mother, one inherits one maternal copy of D7S820, and one paternal copy of D7S820.
The chromosome diagram below tracks STR variability in D7S820 from grandparents
to parents to children. For ease in following the example, each copy of D7S820
has a different number of [GATA] repeat units. For example, the maternal
grandfather has one chromosome 7 with [GATA]7 in its D7S820 STR and
another chromosome 7 with [GATA]8 in its D7S820 STR. His daughter inherited
his [GATA]7 STR and his wife's [GATA]9 STR. The relationship
of child to parent can be followed using this one marker.
In real life situations, more than one STR is analyzed. A hair found at the scene of a crime with [GATA]9 and [GATA]14 in its D7S820 STR does not automatically single out child D as a suspect. There are thousands of unrelated people with this one STR pattern. However, a match in three STRs (D7S820, D13S317, and D16S539, for example) gives more than a 2000 to 1 probability that the DNAs are from the same person. Using nine STRs gives more than a one billion to 1 probability. This degree of certainty in matching a DNA sample to an individual is behind the US military's use of DNA typing for its members. This may eliminate interment of remains as "unknowns."
The same DNA fingerprinting techniques are also being investigated for use in establishing animal pedigrees, as well as matching illegally harvested timber in the
