Prac 6 Genetic Diversity

Ecosystems &Biodiversity Measuring Genetic Diversity 1

Practical 6 Measuring Genetic Diversity

Reading

Burgman, M.A. and Lindenmayer, D.B. (1998). Conservation Biology for the

Australian Environment. Surrey Beatty & Sons, Sydney. Chapter 6 Loss of genetic

diversity, populations and species.

Eldridge M. (1998). Trouble in Paradise? Nature Australia 26, 24-31.

Van Dyke F. ( 2003) Conservation Biology. McGraw-Hill. Chapter 6 Conservation of genetic diversity.

Introduction

If you look at any group of of individuals from the same species – humans, cats, dogs –

you will quickly see that they are no all identical. Chromosomes are made up of a

collection of genes, and the external expression of genes (the individual’s phenotype)

can vary considerably. This is because genes can have alternative forms called alleles,

and the observed differences in appearance are caused by variations in which alleles are

present in the genetic make-up an individuals. The “gene pool” of a species is the whole

collection of all the possible alleles of all the genes in the species.

Conservation ecologists cannot maintain all the genetic diversity in species, but they do

try to maintain the genetic diversity found in individuals in local populations of plants

and animals. For example, if the population of an orchid species in one valley has

evolved adaptations that are different from the population of the same orchid species in

the next valley, it is desirable to keep both populations to maintain the full evolutionary

potential of the species. But, if both populations are genetically identical then

conserving just one population will capture all the “gene pool” of the species. However,

there are also problems which must be dealt with if only one population is conserved!


Why does it matter ? Well, when collecting seed, or tissue samples, or animals for

reintroduction programs, or animals for a zoo, how representative are these samples of

the genetic variation of the whole species? What are the implications of using only a

small proportion of the available genetic material? These “founder” populations may

lead to “bottlenecks”, genetic “drift”, loss of “fitness”, and may limit the ability of a

population to adapt to changes in its environment, and therefor to persist.

Of course, to make the decisions about how much variation we need to conserve for the

best chance of long term species survival, we must first be able to “measure” the

genetic diversity within populations and secondly, determine how many populations we

need to conserve. This practical is based on Gibbs et al. (1998). Problem-solving in

Conservation Biology and Wildlife Management. Blackwell Science.

The task

Work slowly through the calculations for this task. You DO NOT need to remember how to carry out these calculations - the task aims to demonstrate one of the genetic tools available to conservationists to help make objective decisions about the genetic base of the populations they must manage.

You are working for a conservation agency and are faced with a crisis decision: a piece

of land with six different wetlands hosting the only known populations of two rare

orchids is about to be developed as an industrial site.

Your organization has only have enough funds to

purchase and protect four of these wetlands. Which of

the six populations should you protect ? The wetlands

are of two types. Three are marshes with the world’s

only known populations of Pterostylis isozymus. The

other three are swamps where the only populations of

the closely related but distinct species Pterostylis

polyzymous lives. You send some leaf samples off to a colleague for genetic analysis,

and receive back the data below, which are in the form of protein electrophoresis gels

for one allozyme locus that is polymorphic in both these species. The locus has two


alleles, “Fast” and “Slow” (because they move at different rates through the gel), that

appear on the gel with the faster allele below the slower allele.

Divide into groups of two or three, and carry out the genetic assessment together, by

following the four steps and filling in the worksheets as you go.

Step 1 To measure how genetic variation is spread within and between populations

you first need to determine allele frequencies in each population. The particular

allozyme locus examined has two alternate forms. The identity of the two alleles in

each individual is reflected directly by the banding patterns within each lane on the gel.

For example, the first individual in the first lane of the first gel is heterozygous, that is,

the two alleles it has are different and are indicated (+) by both a Fast and a Slow

moving band on the gel. In contrast, the individual in the second lane is homozygous, as

indicated by having a single band representing two Slow alleles.

Determine the allele frequencies in each population for the Fast-moving allele

(p) and the Slow-moving allele (q) by counting the number of alleles for

individuals in each population (remember that a homozygote has two alleles the

same so you have to count the + twice, and the total number of alleles for the 15

individuals is 2 x 15 = 30). The, divide that by the total number of alleles

present in the population (always equal to two times the number of individuals).

Read the page explaining gel electrophoresis and

look at some of the web sites – they have good

explanations


Pterostylis isozymus, Population 1 (individual 1 to 15 from left to right)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 23

Fast 7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 12

Fast 18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 4

Fast 26

Pterostylis polyzymus, Population 1 (individual 1 to 15 from left to right)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 12

Fast 18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 14

Fast 16


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 total

Slow 8

Fast 22


Step 2. Next, you need a measure of genetic difference between populations. A

commonly used measure is Wright’s fixation index, or Fst, which ranges from 0,

indicationg no difference between populations, upwards, indicating increasing

difference. To determine Fst, you need to calculate the expected heterozygosity for each

species (Hs). Do this by multiplying 2pq for each population and then averaging these

values over all three populations within each species.

Allele frequencies for Pterostylis isozymus

Fast allele p Slow allele q = 2 x p x q

population 1 7/30 = 0.23 23/30 = 0.77 2 x 0.23 x 0.77 =

0.35

population 2

population 3

average

=

1.06/3

(Hs) = = 1.06/3 =

Allele frequencies for Pterostylis polyzymus

Fast allele p Slow allele q = 2 x p x q

population 1

population 2

population 3

average

=

(Hs) =


Step 3. Now, calculate the expected heterozygosity if all three populations were part

of the same, extended breeding population (Ht). Do this by averaging p and q over all

three populations within each species, and then multiplying 2 x the average p x the

average q This would be the expected frequency of heterozygotes in the population

if it acted as one large breeding pool with no genetic differences at the local population

level.

Expected heterozygosity (Ht) for Pterostylis isozymus

Fast allele p Slow allele q

population 1 7/30 = 0.23 23/30 = 0.77

population 2 0.60

population 3 0.87

average allele

frequency

1.70/3

=0.57

(Ht).= 2 x the average p x the average q = 2 x 0.57 x average q =

Expected heterozygosity (Ht) for Pterostylis polyzymus

Fast allele p Slow allele q

population 1

population 2

population 3

average allele

frequency

(Ht).= 2 x the average p x the average q =


Step 4. OK, now you need to calculate the amount of local, within-population

variation! Deviations of the frequency of heterozygotes in separate populations (Hs)

from what you would expect to find if they were all part of the same larger population

(Ht) provide an index of the amount of genetic variation that is found only in local

populations. Thus, Fst = (Ht - Hs) / Ht, where values of Fst < 0.01 indicate little

divergence between populations, and values Fst > 0.1 indicate great divergence between

populations (that is, the populations are genetically different from each other). Values

in-between indicate some genetic divergence

Follow the examples provided, calculate the fixation index for each species, and

then compare the indices between the two species.

Summary

Pterostylis isozymus

Fst = (Ht - Hs) / Ht = 0.49 – 0.35 / 0.49 = 0.29

Fst > 0.1 and indicates great divergence between populations of P.isozymus

Pterostylis polyzymus

Fst = (Ht - Hs) / Ht =

Fst _______ and indicates no/some/great divergence between populations of

P..polyzymous

Populations of Pterostylis ____________ are more divergent than populations of

Pterostylis_____________. That is, the populations of Pterostylis___________,

are genetically most similar to each other.


Your Report

In your report you should outline the original problem, and discuss the following questions in

an “essay style” report (about 600 words). Include the final summary section of Fst, and use

your calculations to support your discussion. Make sure you correctly reference your

discussion.

Are the populations of each species different from each other ? Does one species

have more “between population” diversity than the other? Which one ?

How will you allocate your scarce funds for wetland acquisition ? Justify your

decision in terms of preserving the maximum amount of genetic diversity that

characterizes these two species.

What considerations other than genetic ones might influence your choice ?

Recall that our goal in this exercise is to capture as much of the genetic diversity

that characterizes these species as is possible, given a limited budget. Note that both

alleles at the locus surveyed are already found in each population of both species.

Why does it matter that more than a single population of each species be protected ?

In the reading by Eldridge(1998) “Trouble in Paradise”, how did the scientists

maximize the genetic diversity in the re-introduced population? Why didn’t they

choose to use animals from just one of the islands ?

Due Date

Check the Assessment Details File for the date. This report is worth 10% of your total

mark.


gel electrophoresis

http://web.utk.edu/~khughes/GEL/index.htmhttp://www.bergen.org/AAST/Projects/Gel/index.html

http://www.life.uiuc.edu/molbio/geldigest/electro.html

Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. Because these differences are determined by genes, the differences in molecules indicates a difference in genetic make-up.

Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. The gel is a jelly-like substance, usually agrose, a substance derived from seaweed. The molecules have an electrical charge, and electrodes at either end of the gel provide the driving force.

The gel acts like a sieve. Large molecules have difficulty getting through the holes in the gel, but small molecules move through easily. Because of this, large molecules will move more slowly than the smaller, faster molecules. As the separation process continues, the separation between the larger and the smaller fragments increases.

Each vertical “lane” belongs to a single individual, and by comparing the positions of the molecules across lanes it is possible to determine if they have the same combinations of molecules – that is, if they are genetically similar or genetically different.

Each lane is an individual

Large DNA or protein molecules will be Slow

Small DNA or proetin molecules will be Fast

http://www.life.uiuc.edu/molbio/geldigest/electro.html

http://www.bergen.org/AAST/Projects/Gel/index.html

http://web.utk.edu/~khughes/GEL/index.htm

Prac 6 Genetic Diversity

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