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Laboratory heritability (I): The evolution of quantitative traits by artificial selection Objectives

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Heritability (I): The evolution of quantitative

traits by artificial selection

1) Become familiar with the concept of quantitative inheritance and its connection to Mendelian genetics.
2) Understand the relationship between selection, heritability, and response to selection.
3) Learn how to set up and implement an artificial selection experiment.
Terms of interest: selection differential (S), response to selection (R), heritability, variation, genetic variation,
Changes in allele frequencies within a population over time = evolution.

The characteristics (phenotype) of an organism, its size, shape, chemical properties, behavior, etc. result from its developmental stage, its environment and its genetic makeup (genotype). The phenotypic traits that you have studied in previous courses, such as eye color in Drosophila melanogaster or seed color in peas, likely had two or three possible states (e.g., red versus white eyes) and followed Mendelian inheritance patterns (see textbook). Such traits were determined by two alleles at one or two loci. However, the genetic component of the vast majority of phenotypic traits results from the actions and interactions of alleles at numerous loci scattered across the chromosomes. That is most traits are polygeneic. Variation in these traits tends to be quantitative and continuous rather than discrete as in Mendelian traits. Even traits that seem to follow simple Mendelian patterns often reveal quantitative underpinnings. Think about your own body. We know a single gene determines “blue” versus “brown” eye color, but what does “blue” mean? Some blue eyes are darker some lighter, some greener, others grayer. These differences are the product of small effects from many genes, as well as the environment.
Quantitative trait variation

Because the variation in quantitative traits most often results from many genes of relatively small effect whose exact number is unknown, description and analysis of variation and selection on such traits is based on statistical measures and relations. Statistical variance quantifies variation around the average value of the trait. Such phenotypic variance (VP) can be divided into a genetic component, the genetic variance (VG) an environmental component the environmental variance (VE) and a genotype by environment interaction (VGE), thus;

VP = VG + VE + VGE.

Offspring will tend to resemble their parents both because of common environment and because they share a common genetic background for the trait. However, selection operates only on the genetic variance, thus a trait can only evolve if variation has a genetic component. Thus assessing the genetic contribution to traits, VG is critical to understanding how traits might evolve. The genotype by environment interaction term accounts for the fact that differences in the environment do not have a uniform effect among all genotypes in the population. For example, one genotype might grow better at 20C but another better at 30C. The environment does not have a uniform effect among genotypes.

Natural selection and artificial selection

Natural selection stands as one corner stone of evolutionary biology and explanation for the diversity of life on earth. Some have called natural selection a deceptively simple concept, or instilled it with purposeful intent. However, natural selection is in essence a mathematical process. It is simply differential survival and reproduction. Natural selection does not lead to differential survival and reproduction, nor is it brought about by differential survival and reproduction, natural selection is differential survival and reproduction. In this lab exercise with artificial selection you will become familiar with the processes surrounding evolution and natural selection. Fundamentally, artificial selection and natural selection are the same. Artificial selection differs from natural selection primarily in that the reproductive success of the organism hinges on a single character or small set of characters chosen by the human investigator rather than by the organism’s overall survival and reproduction. Natural selection also lacks the purposeful “directedness” of artificial selection. Throughout the lab you should consider what part of the selective process you are implementing or observing. For example, you as the investigator become the agent of selection (i.e., you determine the strength of selection and the trait that determines fitness) for the plants rather than some aspect of the physical and biological environment.

Heritability and Selection

The rate of evolution by artificial or natural selection depends on the level of genetic variation in the trait and the strength of the selection applied to the population. The process is best illustrated with an example. Figure 1 shows the frequency distribution of a given trait, let’s say beak size, in a population of finches. The distribution of individuals is bell-shaped (although this need not be the case) and XP indicates the mean value of the trait for the whole population. Suppose that in the population, birds that have larger beaks are able to crack open a wider variety of seeds and therefore store more fat and survive cold winters more often than birds that have smaller beaks. During one winter, only finches with beaks of larger than a certain size survive, so that the average beak size among the survivors is XS (Fig 1a). The differential survival resulting from larger beak size is directional selection. If beak size is heritable, that is if the variance in size of a bird’s beak is at least partially determined by genes,

  1. Selection differential

b) Selection response

c) realized heritability
h2 = R/S
Fig. 1 Illustration of selection differential and response for a population with normally distributed trait values

then the directional selection will produce a shift in average beak size in the next generation. The difference between the average beak size among survivors and that in the original population (which includes the survivors) is know as the selection differential (S: Fig. 1a).

We measure strength of selection as the selection differential (S). The selection differential is simply the difference between the mean of the trait in the selected group of parents (XS) and that in the entire base population (XP, Fig. 1a).

Selection response (R) is the change in the average value of the trait in the offspring generation compared to that of the entire parental generation (not only the selected parents, Fig. 1b). A response to selection thus provides evidence for a genetic basis to the trait. Such a response also represents an evolutionary change. In lab, you will be imposing selection on Brassica rapa plants, and attempting to produce an evolutionary response!

The relation between S and R can be used to provide an estimate of the genetic component of the trait of interest. Selection response can be expressed as a proportion of the selection differential, to yield h2 the realized heritability (Fig. 1c).

h2 = R/S (1)

Thus, if the realized heritability of a trait in a population = 1, then all the variation of that trait in the population is due to genetic factors. If h2 = 0, then, R had to be zero and therefore, there was no response to the selection. If the next generation shows no response to a selection force, then that trait does not have a genetic component. That is, all the variation comes from the environment or factors other than genetic make-up.

Figure 2 The life cycle of rapid B. rapa Wisconsin Fast Plants ™

Week 1 Selection With Rapid Brassica rapa

In lab today, you will begin an exercise that will cover parts of 2 labs over the next 8 weeks. You will work as plant breeders in an attempt to artificially select for a particular variable trait in rapid-cycling Brassica rapa. The plants you will be working with are the same species as cultivated turnip, pak choi, and Chinese cabbage, each a product of selective breeding for specific phenotypic traits. Brassica rapa forms part of a complex of related species that comprise many of the most important vegetable crops world-wide, for example,

B. rapa: pak choi, Chinese cabbage, turnip, saichin
B. oleracea: kale, cauliflower, broccoli, head cabbage, Brussel sprouts, kohlrabi, collard greens
B. juncea: brown mustard, mustard greens B. napus: canola
Earlier in the semester, the course staff established a number of separate populations of about 100 plants, all from the same basic “wild type” B. rapa seed stock (Rbr 1-33). Within each lineage, called a selected line, we planted all seeds on the same date, 14 days prior to your lab.

As discussed in the Introduction, genetic variation is crucial for any evolutionary response to selection. The first task in lab today is to identify trait variation. Spend the next 5-10 minutes examining the plants before you. 1) Look for and list different traits that vary among the individuals 2) Determine how you might go about quantifying these traits (measure, weigh, count etc.). Could you make a standardized measure that would allow you to calculate variation in the population? 3) Rank each different trait that you recorded based on how variable it is and how easy it is to score.

Attention: Treat these young plants carefully, they are tender, and you can damage their leaves or stems.

List set of traits on which you might select

Trait Variability Scoreable

­ 1. _____________________________________ ________ ________


2. _____________________________________ ________ ________


3. _____________________________________ ________ ________


4. _____________________________________ ________ ________


Trichomes – plant hairs

In this population, individual plants are covered with different numbers of hairs. Look particularly at the leaf petiole and margin of the first true leaves (Fig. 3). Some plants are very hairy, many are slightly hairy and others hairless (bald, if you like). Hairiness is a particularly good trait to use for an investigation of selection. The number of hairs is a fairly straightforward trait to count and varies among individual plants in the population. Because all plants are the same age and all grown in identical conditions, it is unlikely that such variability arises from age or environmental differences. The variability very likely has a genetic component, making it a good candidate for a selection experiment. The other thing that makes these simple hairs, called trichomes, more interesting is that, as mundane as they look, they serve a role in defense against certain herbivores in some species.

1st True Leaves

Fig. 3 12-day old B. rapa with close-up view of leaves

In this lab, you will attempt to increase the average hairiness of plants in the population, through artificial selection. Prior studies show that hairiness comprises a genetic component and it can respond to selection (Ågren and Schemske 1992). We will use the artificial selection experiment to explore variation in quantitative traits, selection, heritability and response to selection–all fundamental concepts in biology.

Counting hairs

In order to begin the selection you first need to quantify hairiness of the population. Counting all of the hairs on each plant would take a great deal of time; however, there are several regions of the plant on which hairs could be reliably and repeatably scored. Two in particular are the petiole of the first true leaf and the margin of the first true leaf (Fig. 3). Both have large, conspicuous trichomes. Both regions are relatively small and have defined starting and ending points. Both regions can serve as indices for overall hairiness. We will use the number of hairs on the petiole as an index for hairiness, but we could just as easily use the margin of the leaf.

Count the number of trichomes on the petiole for each plant in your sample. To count, illuminate the leaf from above with the light from your microscope, if this helps. The trichomes are most conspicuous if illuminated against a black background such as your lab bench. Most trichomes, if they are present, are concentrated on its underside. Be certain that you are not counting looking at one of the cotyledons, which never have trichomes. For each plant count a second time to verify accuracy then record your data in Data Box 1. Also record the number on the side of the pot. Again, handle the plants carefully, bent stems can be fatal.

Data Box 1

Enter your data: the number of trichomes on the petiole of the first true leaf.

____ ____ ____ ____ ____ ____

____ ____ ____ ____ ____ ____

Record the number of individuals in each category in the combined class data table supplied by your instructor. Copy the whole data set for the class into your own table. Next, plot a frequency histogram of these data in the space provided. Mark the population average (mean) on the graph.

Selecting parents of the next generation.

The next task is to select the plants that will be the parents for the new generation (the selected parents). You are applying directional selection to increase hairiness. A straightforward and standard way to apply directional selection, truncation selection, is to breed only a predetermined percentage of individuals possessing the most extreme values for the trait. You will be selecting approximately the 10% hairiest plants from your section to be parents for the next generation. Identify the 10 or so (you will need to make the calculation on the class data) hairiest plants from the population (the whole lab section). Mark these with RED TAPE on the pot. You may find that there are several plants that possess equal numbers of trichomes. From these plants randomly select the number needed to make 10% of the total population. (NOTE, when choosing these plants avoid any that look particularly weak or damaged, but do not choose plants that look particularly, tall, large, green, etc. Carefully place a bamboo stake in the soil near the base and attach the plant to the stake with the tygon-loop. Record the trichome numbers for each of these plants. Place these plants back in the wick tub at the front of the room.

On your histogram, use cross hatching to mark the categories of the selected parents and label their average trait value. Then record the selected parents’ values and calculate the difference between the average number of trichomes on petiole or leaf margin for these selected parents and for the population as a whole (Data Box 2). What does this difference measure?

Data Box 2

Selected parent values selected parent’s

____ ____ ____ average _________

____ ____ ____ population

average _________ ____ ____ ____

____ ____ difference ________

Finally, discard all of the unselected plants in the trash bins provided. These plants will not contribute to the next generation, their fitness is zero. PLACE THE EMPTY GROWING CANISTERS IN THE COLLECTION BINS PROVIDED.

Predictions for response to selection.

Based on your reading of the lab manual what do you think the distribution of hairs will look like in the next generation. The next generation is grown from the seeds produced by random (panmictic) mating among the selected parents. Below are three examples. These are not necessarily right answers, but are intended to generate thinking about how the distribution of a trait might respond to directional selection. Your predicted histogram should include the range of values (lowest and highest number of trichomes), as well as the frequency of trichomes in each category. This exercise seems very simple at first, but accuracy will depend on your consideration of many factors.

Pollination of selected parents
The plants that you scored are now in full flower. Today you need to pollinate these plants so that they can complete sexual reproduction. Brassica rapa plants rely completely on several types of insects to transfer pollen from the anthers of one plant to receptive stigmas on a different plants. Although all flowers have both male and female sexual parts, pollen is incapable of fertilizing seeds on the same plant. Such self-incompatability ensures outcrossing.

The honey-bee commonly pollinates B. rapa in natural and agricultural populations. Honey bees accumulate pollen from the anthers on the hairs that cover their bodies. The thorax in particular is covered with dense hairs that efficiently collect pollen. As bees move to different flowers to collect nectar and pollen on which they feed, they deposit some of the pollen onto the stigmas effecting pollination and fertilization if the recipient is compatible. You will use bee sticks to pollinate your plants. The basic idea for hand-pollinating these parental plants is to replicate the movement of a bee among flowers on different plants.

In the tubs at the front of the room are the plants that you selected to be parents of the next generation, along with a set of plants from two other sections. You will be responsible for pollinating your own plants and those of the other sections. IT IS CRITICAL THAT YOU POLLINATE ALL FLOWERS IN ORDER TO INSURE A GOOD SEED SET. Begin pollinating by taking the bee stick from the block at the end of the tub and carefully twirling the thorax brush across the anthers and stigma of several flowers on a plant. Now move to another plant and repeat the process. Continue this process for several minutes moving between plants every 3-4 flowers. It is okay to go back and pollinate more flowers on a plant that you did earlier, just keep moving around. When you have finished, mark you initials on a sheet in front of the tub. If each of you takes about 5 minutes to pollinate plants during the lab, you will successfully mate your plants and should produce a large crop of seeds for your experiment. These seeds are the basis of the second generation!! Each lab will repeat this process with the plants.

Pollination must continue over the next week to ensure a large seed set to obtain enough seeds for the remainder of this lab. Please make arrangements with lab partners (in groups of four) to come in over the next week and continue pollinating. Every student will come in at least once during the week and pollinate all the flowers for all the labs (remember there will not be many after the selection of the bottom 90% have been culled).

Week 8 Scoring trichomes in the second generation:

Response to selection?

In lab 1, you scored the number of trichomes on the petiole of a population of B. rapa plants. You then selected a subset of the 10 hairiest plants to be parents for the next generation. By doing so you became the agent for selection, (i.e., you imposed a selection differential). You then facilitated random mating within the group of selected parents by cross-pollinating these plants with the bee-stick. In order to assess the heritability and genetic component to trichome number you need to assess the response to the selection. To assess response to selection you need to measure the trait’s values for individuals in the offspring generation (generation 2). Recall from the Introduction to this lab that response to selection equals the difference in the average value of the trait in the population in generation 1 versus generation 2 (Fig. 2).

One person from each pair of students should go to the front of the room to harvest 5 plants from the tub. Count the number of trichomes on the petiole for the 4-5 plants just as you did in lab 1. Refer back to the instructions from lab 1 for details of the methods. You cannot record data on the pot, instead just record number of trichomes in Data Box 3.

Data Box 3

____ ____ ____ ____ ____ ____

____ ____ ____ ____ ____ ____

One member should then go to the front of the room and record your group’s data on the Frequency table on the board. While your instructor is tabulating the data for the class, go back to lab 1 and copy the data from generation 1 histogram into the space provided below. Be sure to include the data for the selected parents on this histogram. Transfer the predicted generation 2 histogram from lab 1. Now, copy the whole data set for generation 2 from the front of the room into your own table. Finally, plot the frequency histogram of these data in the space provided. Mark the population average (mean) on the graph.

WEEK #1: LABORATORY ASSIGNMENT (Due 2 days after your lab).
Turn in the answers to the post-lab questions. From the laboratory website, download the Microsoft Word document for the Post-lab questions. Type out your answers to the questions and upload the Word.doc file to your “Assignment Blog”.
WEEK #9: LABORATORY ASSIGNMENT (Due 2 days after your lab).
Turn in the answers to the post-lab questions. From the laboratory website, download the Microsoft Word document for the Post-lab questions. Type out your answers to the questions and upload the Word.doc file to your “Assignment Blog”.

Ågren and Schemske, D. W. 1992. Artificial selection on trichome number in Brassica rapa. Theor. Appl. Genet. 83:673-678
Fall, B. Fifield, S and Decker, M. Evolution by artifical selection; a nine-week classroom investigation using rapid-cycling Brassica.
Falconer, D. S. Introduction to Quantitative Genetics
Futuyma, D. J. 1999. Evolutionary Biology 3rd Ed. Sinauer NY
Williams, P. H. and Lauffer, D. WFPID” Hairy’s Inheritence. Wisconsin Fast Plants Program. University of Wisconsin, Madison.

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