Biology 164 Laboratory

Artificial Selection in Brassica, Part I (Based on a lab exercise originally developed by Bruce Fall, Univ. of Minnesota and revised by Tim Christensen, Colby College)

I. Objectives

1. Understand the process of artificial selection. 2. Become familiar with some plant cultivars in the genus Brassica, which are the products of artificial selection. 3. Participate in an on-going artificial selection study involving three related cultivars of Brassica rapa in which you will: a. quantify the variability of a specific trait in the three cultivars, b. measure how the expression of the specific trait has responded to varying numbers of rounds of artificial selection, c. subject one of the cultivars to another round of artificial selection, and d. determine if there is evidence for an upper limit having been reached in the expression of the specific trait as additional rounds of artificial selection were employed.

II. Introduction

A visit to a farm, supermarket, pet store or plant nursery will offer many examples of selective breeding of plants and animals by humans. Over hundreds and even thousands of years, humans have altered various species of plants and animals for our own use by selecting individuals for breeding that possessed certain desirable traits. This selective breeding process was continued for generation after generation. Often the products of such selective breeding are remarkable. Quite diverse domestic dog breeds, from chihuahuas and miniature poodles to Newfoundlands and Irish wolfhounds are all related to a common wild ancestor, the wolf (Canis lupus). Domestic chickens are all derived from the wild Jungle Fowl (Gallus gallus).

The success of plant and animal breeders in dramatically changing the appearance of various lineages of organisms in a relatively short period of time is an obvious yet profound fact. This fact did not escape Charles Darwin as the first chapter of The Origin of Species concerns artificial selection by humans (“Variation under Domestication”). Darwin used many examples of artificial selection by humans to help support the case for his proposed mechanism for the evolution of natural populations – natural selection. For example, Darwin was particularly taken with the number of pigeon varieties such as tumblers, pouters, fantails and many others. All of these pigeon varieties were derived from wild Rock Doves (Columba livia) over the past 5,000 years. One can find similar examples of selective breeding among plants, including those humans have bred for food.

To gain a better understanding of selection and inheritance, biologists have experimented with artificial selection involving a variety of traits in many different species of bacteria, yeast, plants and animals. The results, obtained in a relatively short period of time, are often impressive.

A general finding of these studies is that most variable traits in organisms respond to artificial selection. In other words, it is usually possible to increase or decrease the frequency or average value of a trait in a lineage through careful selective breeding. In our lab study you will have a chance to examine whether or not Colby students have been successful plant breeders using artificial selection to alter the genotype of a plant.

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How artificial selection differs from natural selection

In contrast to natural selection, artificial selection 1) favors traits that for some reason are preferred by humans; 2) has a goal or direction toward which the selection process is directed; and 3) generally is much faster than natural selection because the next generation can be absolutely restricted to offspring of only those parents that meet the desired criteria (natural selection is rarely so absolute). In artificial selection, humans perform the selecting, intentionally restricting breeding to individuals with certain characteristics. In natural selection, the environment accomplishes the selecting – individuals that survive and reproduce better in a given environment, are “naturally selected”. The ‘environment’ includes the myriad of factors such as predators, food supply, weather, etc. that determine which traits make it into the next generation.

Some important plant cultivars that have arisen by artificial selection

A specific plant type or variety that is cultivated for human use is known as a cultivar (cultivated variety). The genus Brassica of the mustard family (Brassicaceae) includes many cultivars that are important in agriculture. A number of nutritious and tasty vegetable cultivars have originated from three species of Brassica (Brassica rapa, B. oleracea and B. juncea). Some cultivars have been bred for root production, others for leaves, flower buds or oil production. You may be surprised to learn that these familiar vegetables, though very different in appearance, are actually descendents of the same ancestral species. Centuries of artificial selection have produced greatly divergent cultivars within these species (Fig. 1).

The following cultivars originate from the three wild species indicated in italics:

Brassica oleracea – kale, cauliflower, broccoli, cabbage, Brussels sprouts, kohlrabi, collards

Brassica juncea – leaf mustard, root mustard, head mustard and lots of other mustard varieties

Brassica rapa – turnip, Chinese cabbage, pak choi, rapid-cycling

turnip Chinese cabbage pak choi rapid cycling Fig. 1: Examples of four cultivars of the species, Brassica rapa.

The rapid-cycling cultivar of Brassica rapa was developed at the University of Wisconsin and has been used extensively in plant research and education. This cultivar was developed for its ability to rapidly flower and mature, produce lots of seeds, have a short stature, and thrive under artificial light. The generation time (from seed to maturation to fertilization to mature seed) is 6-7 weeks, short enough to allow us to study one complete generation this semester. The life cycle of these so-called Wisconsin Fast Plants is shown in Fig. 2.

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Figure 2. Life cycle of rapid-cycling cultivars of Brassica rapa.

Variable traits: continuous or discreet? There are a number of fairly obvious variable traits that one can observe in a large population of Fast Plants. A brief list might include: number of flowers, color of hypocotyl, number of leaves, type of petiole, size of first true leaf, and plant height. In contrast, other traits usually don’t vary at all: number of petals per flower (four), petal color (yellow), number of cotyledons (two), and leaf color (green). Refer to Fig. 2 if you are unfamiliar with any of these terms.

Artificial Selection in Brassica, Part I Page 3 Your lab instructor will give each student a container with five plants. Treat these plants gently; they are young and tender, and easily damaged! Examine the small sample of plants that you and your lab partners have at your table and note the variability you can see among the plants. Remember, the plants are the same age so differences you see (such as height or leaf number) are not due to differences in age.

Note how some traits vary continuously over a range of possibilities, while others fall into discreet (either/or) categories. For the traits below indicate whether they vary continuously or discreetly.

Number of flowers:

Color of hypocotyl:

Number of leaves:

Type of petiole:

Size of first true leaf:

Plant height:

One variable that you may have overlooked is “hairiness” of leaves, petioles and stems. Look at some plants more closely using a hand lens, and see if you can observe these hairs, also known as trichomes.

Does the hairiness trait vary continuously or discreetly?

Trichomes have been demonstrated to have specific functions, which differ from species to species. In the space below, hypothesize what these functions might be.

What do you speculate are some functions of trichomes?

III. Experimental Materials and Methods

A. The Three Cultivars Involved in the Study You and the other members of your lab section will participate in an artificial selection study that began two years ago and involves subjecting a base population of a rapid-cycling cultivar of Brassica rapa to a number of rounds of artificial selection. You will complete the first portion of the study today but will not complete the second portion of the study for several weeks, when you will be able to observe the expression of traits in plants that you have subjected to a further round of artificial selection.

The three different cultivars used in this year’s study include a) the base population (BP), b) a population that has undergone one round of artificial selection (AS1), and b) a population that has undergone two rounds of artificial selection (AS2). Note that these cultivars are related to one another, representing three generations of plants that were successively selected for the expression of an extreme form of a specific trait. Each round of artificial selection involved producing offspring from only the top 10% of individuals within each population that expressed the most extreme form of the trait.

We established populations of the three cultivars by planting seeds approximately 17 days prior to the lab. All plants were grown identically, at 23°C, using commercial potting soil, a constant feeding regime, and continual light of 200µE. brightness. As you begin this study, these plants will have begun flowering (see Figure 2 above).

Artificial Selection in Brassica, Part I Page 4 B. Experimental Design and Purpose of Study

The study is divided into two parts. In the first part of the study you will a) determine how the three cultivars differ from one another with respect to the specific variable trait in question, b) graphically plot the rate at which the expression of the trait has changed in response to successive rounds of artificial selection, and c) predict the outcome of employing an additional round of artificial selection on the AS2 cultivar. In the second part of the study you will a) determine how the AS2 cultivar responded to a further round of artificial selection, b) decide whether or not your prediction on the response to a further round of artificial selection was accurate, and c) conclude whether or not there is evidence that the expression of the trait is approaching some upper limit.

C. Screening of Cultivars For Numbers of Petiole Trichomes

Counting “hairs” As you may have guessed, the variable trait we will investigate in this study is “hairiness”. In order to compare plants to one another we need a way to quantify hairiness. For this measurement we could count all the trichomes on all parts of the plant. This task would be quite time-consuming. Since we know that the hairiness of one part of a plant is strongly correlated with hairiness on other parts, a plant’s hairiness in general can be quantified by assessing hairiness of a specific structure. The structure we will use in this study is the petiole where the trichomes are large, conspicuous and easily counted. The petiole is relatively small with an easily defined starting and ending point, and quantifying hairiness by counting the trichomes on this structure will make data gathering more manageable.

To be consistent, we will use the petiole of the first (lowermost) true leaf, and we will define the limits of the petiole as follows: from its junction with the main stem (usually marked by a small bulge or ridge, often differently colored to its junction with the lowermost leaf vein. See Figure 3 below.

Figure 3. Young Brassica rapa showing first true leaf, petiole and trichomes.

Note that the two lowermost, squarish, two-lobed and rather thick leaves are actually cotyledons (seed-leaves) and not true leaves. The first true leaf is just above the cotyledons. You now need to count the total number of trichomes on the petiole of the first true leaf of each plant in your sample. Your data will then be combined with data from the other students in this laboratory section.

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Use a hand lens and desk lamp. The trichomes are most conspicuous if strongly illuminated against a dark background, such as the black surface of the lab table. If present, the trichomes will generally be concentrated on the lower side of the petiole, but some can occur on the top or sides as well. Count a second time for verification. Use lab tape and indelible marker to record on each cell of your planter the number of trichomes for each plant.

Enter your raw data in the data box below, and determine the median number of trichomes per petiole for your sample. The median is the middle value in a distribution, above and below which are found an equal number of values. (Please note that these median values will not actually be used as part of the class results, but are only determined so that you have a clear notion of what a median value is and how it is calculated.)

Enter your data into the computer spreadsheet for the class.

To avoid confusion, we will collect data for the BP cultivar first. Once that data has been recorded, we will repeat the process for the other two cultivars. Note also that each student will measure five plants each for the BP and AS1 cultivars, but ten plants for the AS2 cultivar.

Data Box for Recording Petiole Trichome Numbers for Three Brassica Cultivars

Enter the number of trichomes on the petiole of the first true leaf, per plant: Median:

BP cultivar: ______

AS1 cultivar: ______

AS2 cultivar: ______

D. Selecting Parents for the Next Round of Artificial Selection

Once the trichome data has been collected for all the cultivars, we will discard plants of the BP and AS1 cultivars, and we will use only the plants of the AS2 cultivar to select parents for the next (third) round of artificial selection. As in the earlier stages of the study, the top 10% hairiest AS2 cultivar plants will be selected to be parents of the next generation (which will become the AS3 cultivar). As a class, we need to identify the 14-18 AS2 plants that had the highest petiole trichome counts by referring to the label you created for each plant’s container.

Record the trichome values of the selected AS2 individuals (parents-to-be) in the data box below.

Data Box for AS2 Cultivar Selected Parents (number of trichomes on petiole of first true leaf)

AS2 cultivar selected parents median value

Artificial Selection in Brassica, Part I Page 6 E. Pollination of Selected AS2 Cultivar Parental Plants

Brassica rapa plants need assistance in sexual reproduction because in nature they are primarily dependent on certain insects for transferring sperm-bearing pollen from the male part of the flowers of one plant to the female part of the flowers of another plant. In these plants, the most conspicuous floral structures are the yellow petals. These petals enclose the male sexual structures (stamens, terminating in the pollen-producing anthers) and female structures (pistil, with the pollen-receiving stigma, style and egg-producing ovary), as shown in Figure 4 below.

Figure 4. Cross-section of flower of Brassica rapa.

Although each flower has both male and female parts, sperm (pollen) from one plant usually do not fertilize the eggs for the same plant. This self-incompatibility ensures that out-crossing (mating between different individuals) normally occurs.

The insect pollinators do not transfer pollen out of courtesy. Rather, the insects are lured to the flowers by the reward of nectar and edible pollen. The insects inadvertently pick up the sticky pollen on various body parts and then carry it with them to the next flower. Honeybees are common pollinators of Brassica in the field, and we will use honeybees (dead ones) to help us pollinate the plants.

Figure 5. Pollen on a bee.

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“Bee-sticks” have been prepared for you from the thoraxes of dead honeybees (collected from beekeepers after the bees died naturally – worker bees are short-lived). Glued to the end of a toothpick, these bee thoraxes make quite efficient pollinating devices; pollen grains cling to the many fine hairs on the bee’s body and are easily transferred to the stigmas of other flowers.

Performing pollination (a.k.a. group sex) Each student should obtain one of the selected ‘parental’ plants. Your objective is to transfer pollen from each plant to every other plant at your table. This process can be done in the following way. Using a single bee-stick, each student will lightly rub and twirl the bee end for several seconds on the anthers and stigma of each open flower of her/his plant. Each then passes the bee-stick, now loaded with yellow pollen, to the neighboring student to the right, who will repeat the process. After each bee stick has made a complete round of the table (all open flower on all plants have been “visited” by all bee-sticks), the pollination process is completed. When finished discard the used bee-sticks; do not return the bee-sticks to their original container where other students might confuse them with fresh, unused ones.

Fertilization will result in the development of the ovules (each containing an embryo) into mature seeds, which will be contained in the fruit or seedpod (the elongated ovary). The length of time from fertilization to mature seed is about 3-4 weeks, sometimes a little longer. In subsequent weeks we will prune away the apical meristem plus additional flowers and axillary buds that may form in order to concentrate plant resources into the developing fruits and to hasten the seed-maturation process. Next week, you should easily be able to see the elongating fruits. In two weeks, they will have become even longer (2-5 cm) and the individual seeds inside (perhaps 5-20 per pod) will have become visible. In three weeks, the maturing process will be nearly complete. You will harvest and plant these seeds (the AS3 generation) in five weeks.

Continuing pollination of new flowers as they develop during the week

To insure that enough viable seed will be produced from the selected ‘parental’ plants, the cross- pollination procedure will need to be continued for one week. Your lab instructor will devise a lab pollination schedule for which you will sign up to come in once during the week to pollinate the plants for your lab section. Your lab section’s plants will be grown in the Arey greenhouse.

Please note: always use a fresh bee stick for performing pollinations to insure that unwanted pollen is not transferred during the pollination.

Artificial Selection in Brassica, Part I Page 8 IV. Experimental Results

Combined class data When all students have finished quantifying the trichome number of individual plants and entering data into the class spreadsheet, your lab instructor will summarize the class results by generating a frequency table and a descriptive statistics table for the class data. Your instructor will make the summaries available to you so that you can record the information into the tables below.

Table 1: Number of individuals found in different categories of the number of petiole trichomes found on the first true leaf of Brassica cultivars subjected to different levels of artificial selection. Rapid-cycling Brassica Cultivars # of petiole trichomes BP AS1 AS2 0-4 5-9 10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 ≥75

Table 2: Summary descriptive statistics for the number of petiole trichomes found on the first true leaf of Brassica cultivars subjected to different levels of artificial selection. Rapid-cycling Brassica Cultivars Statistic BP AS1 AS2 Sample size (N) Minimum Maximum Median

Artificial Selection in Brassica, Part I Page 9 V. Paper Assignment

A scientific paper on the artificial selection study will be due in lab during the week of March 2, 2008. The paper should be written in standard scientific format and include all of the sections outlined in A Guide to Writing Scientific Papers. For the Materials and Methods you can simply cite the lab handout unless you deviated from the instructions.

It is important to keep in mind that this study is being conducted in two parts: In the first part of the study you will a) summarize how populations of the three cultivars differ from one another with respect to the specific variable trait in question, b) graphically plot the rate at which the expression of the trait has changed in response to successive rounds of artificial selection, and c) predict the outcome of employing an additional round of artificial selection on the AS2 cultivar. In the second part of the study you will a) determine how the AS2 cultivar responded to a further round of artificial selection, b) decide whether or not your prediction on the response to a further round of artificial selection was accurate, and c) conclude whether or not there is evidence that the expression of the trait is approaching some upper limit. Even though the study is being conducted in two parts, your Introduction should address both components of the study. After the second part of the study is completed, you will append your paper with the additional findings.

You will need to cite at least three papers from the primary scientific literature1 on artificial selection. It is not necessary to find only papers on artificial selection in Brassica; appropriate articles covering artificial selection in other organisms are acceptable. Your paper may cite pertinent references from other reliable sources not part of the primary scientific literature, but they cannot be counted toward the three-paper minimum mentioned above.

Your paper must include appropriately labeled graphical summaries of the results. These graphical summaries should permit the reader to observe a) how the populations of the three cultivars differ from one another, and b) how varying rounds of artificial selection influenced the median hairiness value.

In addition to the above graphical summaries, you must include a summary of the appropriate comparative statistical test that compares the three cultivars to one another. Graphical and statistical summaries must conform to the guidelines outlined in the Style Guide for Papers and Lab Reports.

Here are some questions to consider as you prepare your paper:

1) How did the frequency distributions of the hairiness phenotypes differ among the populations of the three cultivars?

2) How did the median hairiness values differ among the three cultivars?

3) Is there statistical evidence to support the claim that the three cultivars represent three genetically distinct populations?

4) How did the median hairiness values differ between the AS2 cultivar general population and the AS2 cultivar individuals that were selected to be parents of the next generation? What bearing did these differences have on the predictions you made for the hairiness of the next generation?

5) Is there evidence that evolution occurred during any stage of this multi-year study?

6) How did your results compare to findings reported in the scientific literature?

1 Primary scientific literature includes articles in which scientists first publish their original research findings in authoritative peer-reviewed scientific journals. Artificial Selection in Brassica, Part I Page 10