Download Biology 164 Laboratory Inbreeding Depression and the Evolutionary

Biology 164 Laboratory
Inbreeding Depression and the Evolutionary Advantage of Outbreeding
in Brassica rapa, Part I
I. Introduction
Inbreeding depression can be defined as the reduction in fitness of offspring derived from mating
between relatives (inbreeding) compared to offspring resulting from mating among unrelated individuals
(out-crossing). The harmful effects of close inbreeding were well known long before any formal
scientific investigation of the phenomenon. In humans, about 42% of offspring from sister/brother
marriages die before they reach reproductive age. Hence, most human cultures have strong traditions
with respect to incest. Plant and animal breeders have also known for centuries of the superior vigor and
yield associated with out-breeding compared to inbreeding. The importance of inbreeding depression
was first established in scientific terms by none other than Charles Darwin in his 1876 book, The Effects
of Cross and Self Fertilization. Darwin described the results of extensive experiments involving 57
species of plants; these experiments showed that inbreeding depression is widespread and a significant
evolutionary force.
The most likely cause for this reduction in fitness upon inbreeding involves the expression of deleterious
recessive alleles. Recessive alleles are expressed in homozygotes but remain unexpressed when they
occur with a dominant allele in a heterozygote. Deleterious alleles originate when the underlying DNA
sequence of a functional allele is altered by mutation to code for a gene product which is either harmful
or simply does not work. Mutation is a universal feature of DNA, and all species have individuals with
deleterious recessive alleles. At any given locus, however, deleterious alleles are usually so rare that
offspring produced through matings among unrelated individuals are almost never homozygous for the
harmful gene. With inbreeding, the odds of producing an offspring homozygous for a deleterious allele
are much higher. Since rare deleterious mutations are transmitted along family lines, brothers and
sisters are much more likely to carry the same deleterious allele than any two unrelated individuals.
Very close inbreeding is possible in plants. More than 70% of flowering plant species are
hermaphroditic (i.e., they possess both female and male sex organs) so it is possible for an individual to
mate with itself. Such a process is termed self-fertilization.
Let’s consider a population of out-crossing hermaphrodites in which a self-fertilizing mutant arises.
That individual can be mother and father to its offspring (contributing two copies of each gene to its
offspring) as well as contributing sperm to other individuals (contributing one copy of a gene to those
out-crossed offspring). Such a mutant should have a great advantage, transmitting three copies of its
genes to the next generation while other out-crossing individuals can only transmit two copies of their
genes to the next generation.
Why then is self-incompatibility so common? Many evolutionary biologists believe that inbreeding
depression can provide an explanation. These scientists would argue that even though a selfing mutant
does send three copies of its genes into next generation, the offspring resulting from self-fertilization are
less fit than out-crossed offspring.
Although some plants do self-fertilize, other plants species have a number of ways to prevent selffertilization. Male and female sex organs may be separated within a flower or may function at different
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times during the life of the flower. In other species (like Brassica rapa which we will study), selfincompatibility results in a plant recognizing and rejecting its own pollen.
Measuring Inbreeding Depression
Plants can be excellent study organisms for measuring inbreeding depression. It is relatively easy to
regulate who mates with whom, and thereby produce both selfed and out-crossed offspring through
experimental hand-pollinations. In addition, the relative fitness of selfed and out-crossed offspring can
be compared by growing them in a common environment.
The term fitness has been used a couple of times in this handout already. Before proceeding, we should
define this important concept in evolutionary biology. (The concept will be covered in lecture as well.)
We can define fitness as the relative ability of an individual to leave descendants and hence transmit its
genes to future generations. Fitness has two components. The first, often called fecundity, is the
production of viable offspring, which, in turn, go on to produce their own offspring.
The other component is survival. Throughout an organism’s life, the probability of dying may differ
from one life-history stage to the next. Biologists measuring the survival of plants often break the plant
life cycle into four life-history stages: seed; seed to seedling (germination); seedling to adult; and
reproductive period as an adult. Figure 1 below depicts sources of mortality for plants in different lifehistory stages.
Figure 1. Stage-specific mortality sources for a plant.
Quantifying survival for different life-history stages is difficult for long-lived organisms but can be
easily done in organisms with short generation times. Brassica rapa Fast Plants completes its life cycle
in 30 days so you will be able to estimate survival for all life-history stages.
We need to realize that the environment in which organisms occur can influence the expression of
inbreeding depression. For instead inbred and out-bred progeny may show differences in fitness in
harsh environments but those differences may not be observed in more moderate, benign environments.
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II. Experimental Procedure
For our investigation of inbreeding depression, we will use the rapid cycling Brassica rapa, that remain
from your study of artificial selection. The short generation time of the Fast Plants make them ideal
subjects for a study of inbreeding depression.
Today you will produce matings through both self- and cross-pollination. Each pair of students will
work with six flowering plants. On three of these, flowers will be self-pollinated. On the remaining
three, flowers will be outcrossed. Performing controlled pollinations, whether selfed or outcrossed,
merely requires removing pollen from the anthers of the male parent and applying that pollen to the
stigma of the female parent. As you will recall, B. rapa has a physiological self-incompatibility
mechanism which we will have to circumvent.
We will overcome the self-incompatibility with the use of salt. You will treat the stigma surface of a
flower with salt just before pollination. The incompatibility reaction occurs on the surface of the stigma.
Altering the stigmatic environment by the application of a weak salt solution abolishes the
incompatibility reaction and permits self-fertilization.
Performing Pollinations
1)
With colored tape and a waterproof marker, label the sides of the container for each of your
six chosen plants. Write your name and lab section on each label as well as an O (for outcrossed) for three of the labels and S (for selfed) for the remaining three labels.
2)
Find the most recently opened flower and locate the first three buds up the stem (the oldest
buds). You will pollinate these three buds today.
3)
Using forceps and a dissecting needle, gently open the bud to expose the stigma as illustrated
in Figure 2. Do not remove the sepals and petals; simply tease them apart to expose the
stigma. Work ever so gently and carefully in this procedure in order to prevent
damaging the flowers!! One team member can be used to steady flower and hold sepals
and petals apart, while the other team member uses hand lens to locate stigma and
apply treatment.
Figure 2. Bud-pollination technique.
4)
Note that the anthers are not yet mature and should NOT be releasing pollen now.
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5)
Apply a drop of salt solution to each stigma using a Q-Tip. Wait 15 minutes for the salt to
break down the incompatibility system and then dry the stigma off with a fresh, dry Q-Tip to
remove any remaining salt solution.
6)
Using a bee-stick, collect pollen from mature anthers of recently opened flowers on one of
your other plants (for out-crossing) or the same plant (for selfing) and rub the pollen-covered
surface of the bee-stick lightly across the exposed stigmas of the three buds (as shown in
Figure 2), making sure some pollen adheres to the stigma surfaces.
7)
Gently mark the base of each pollinated bud with a small loop of thread to help identify it
later.
8)
Remove all old flowers (i.e., those below the buds you have pollinated).
9)
Return your plant container to the appropriate table in the Arey greenhouse.
10)
Come back two days later to retrieve your plants from the greenhouse and pollinate another
three buds and mark them with loops of thread as before. A table will be set up outside the
greenhouse with equipment and supplies so that you can perform the pollination procedure
there. Return your plants to the greenhouse table when you are finished working.
11)
During next week’s lab session, remove all un-pollinated buds plus open flowers that you
have not pollinated. Cut off the apical meristem and remove all other inflorescences and
axillary buds. Do not remove any leaves. With the flowers, un-pollinated buds and other
meristems removed, the plant can devote more resources toward maturing the buds you
pollinated.
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