Background Information for "Reconstruction the Evolution of Cauliflower and Broccoli"
The domestication of crop plants over 10,000 years provides rich opportunities to explore evolution through artificial selection. The subject also places genetic engineering in a broader context, as many students associate the term only with recombinant DNA. The laboratory experience is built around the findings reported by Smith and King (200) and Purugganan, Boyles and Suddith (2000) on the evolution of domesticated Brassica oleracea focused on the CAULIFLOWER (CAL) gene.
Single gene versus multigenic appproaches to understanding domestication. Before delving into specifics, it is important to recognize that there are limitations to putting too much weight on one or two genes in understanding evolution. Quantitative trait loci mapping studies reveal the variability in the number and effect of loci, ranging form a couple to many, that affect plant adaptive traits (Ehrenreich and Purugganan 2006). We intentionally chose the CAL example because the molecular evolutionary data is compelling and because considering adaptive traits controlled by many loci adds a layer of complexity that leads to confusion rather than learning progress for beginning college students. We have found that examples involving multiple loci are more effectively integrated into our mid-level genetics, development, and evolution courses. Recognizing the difference in single gene and quantitative trait locus approaches also reflects new conceptualizations in the emerging fields of evolution of development and ecological and evolutionary functional genomics. The laboratory experience, as we have designed it, leaves opportunities for further discussion and exploration should the instructor choose to adapt it to an upper level course. Most pointed is the guided inquiry where we lead students to a final conclusion that both broccoli and cauliflower plants have the same stop mutation in the CAL gene. Clearly broccoli and cauliflower do not look the same and the only reasonable conclusion one can draw is that there are other genetic differences that have been selected for during the ongoing domestication of cauliflower and broccoli.
Origins of the CAL gene. With the above caveats about plant adaptive traits, some background information on the CAL, the gene of interest in this lab, may be helpful. The gene was first identified through mutant analysis in the model plant Arabidopsis, a close relative of the broccoli and cauliflower (Kempin et al. 1995). Arabidopsis plants with two copies of the cal mutation have a wild type phenotype. It is only in the double mutant apetala1 (ap1) cal that the inflorescences (branching portion of the plant containing the flowers) resemble cauliflower rather than the simple, single flower per node architecture of wild type Arabidopsis. [Images of these plants and others can be found in the Description and Teaching Materials section of the laboratory website.] Sequence analysis reveals that CAL and AP1 have 82% maximum identity (you can try this using one of the CAL accession numbers (see student lab) in a BLAST search. The most likely explanation is a duplication event. AP1 is highly conserved among flowering plants, thus CAL is most likely the duplicated gene that diverged. Our students are intrigued by redundant genes that arise through gene duplication and begin diverging. The redundancy or partial redundancy explains why only the double mutant has the extreme cauliflower phenotype (ap1 alone does have a noticeable phenotype, but cal does not). The flexibility that redundant genes provide in evolution is worth a brief discussion with students.
Gene duplication. Understanding how the duplication gave rise to two identical and, over evolutionary time, similar genes, is a helpful way to begin exploring what paralogous genes are. This naturally leads to an exploration of orthologs and the question of whether or not CAL exists in other plants (e.g. has orthologs) and whether the mutant phenotype in Arabidopsis is coincidental or relates to the phenotype of cauliflower plants one can purchase in the supermarket.
Phylogenetic evidence. Lowman and Purugganan (1999) provided phylogenetic evidence that the cal mutation arose within the Brassica family. This work and later research also supports the conclusion that the duplication event producing CAL occurred within the Brassicas. Two points may be of interest to students based on this conclusion. First, while it is possible that other plant families could evolve inflorescences that look like cauliflower, the responsible gene, even if it were an AP1 duplicate would not be orthologous to CAL, because the families diverged before the Brassica CAL gene appeared on the evolutionary scene. The second point is to be clear about the difference between the cal mutation that was studied in Arabidopsis where mutations are made for developmental genetic studies and the cal mutation that naturally arose in the Brassica oleracea and were selected for by early agricultural societies. Both are examples of artificial selection. The Arabidopsis data can be used to understand the evolution of broccoli and cauliflower, but the starting information from Arabidopsis was not causal in the evolution of the subspecies of Brassica oleracae.
Role of AP1. In the student version of the laboratory description, there is a paragraph about the role of AP1 and the double mutant necessary for the cauliflower phenotype. Lowman and Purugganan's (1999) work also supports the role of the double mutant in cauliflower evolution. This paragraph can be eliminated if the instructor chooses to focus exclusively on the cal mutation or can be used as a springboard for discussing content outlined in the preceding paragraph in this background information document.
Supporting on-line images. The student laboratory and the link to Brassica oleracea images under Description and Teaching Materials offer the opportunity to explore the concept of speciation and what constitutes a species. The morphologically obvious difference among the subspecies and the molecular genetic conclusion the students arrive at regarding one aspect of the morphological differences could be used to reinforce what students have already learned about the species concept and agents of evolutionary change if Hardy-Weinberg equilibrium has been covered. That is the approach we have used in the past few years. In past years we have used the laboratory experience as a jumping off point for classroom work on speciation and found that to be effective in terms of student learning as well.
Speciation and Hardy-Weinberg equilibrium. Our students have been involved in a multi-week research project to determine whether or not gall fly genetic differentiation that could lead to speciation is occurring in our local arboretum before they begin the broccoli and cauliflower lab. They have already worked through real world data, using Hardy-Weinberg and Chi square analyses, to determine whether or not the gall flies are undergoing genetic differentiation for host species preference. While we are able to integrate the two learning experiences during student reflection and discussion times, the "Reconstructing the Evolution of Cauliflower and Broccoli" module is not dependent on the previous laboratory experience.
Our goal is for students to integrate their understanding of genetics, evolution, and development over multiple levels of organization - DNA sequence level to whole plant morphology in this case. For those who prefer to narrow the focus to molecular genetics and evolution, the module should also be effective.
Ehrenreich, I.M., M.D. Purugganan (2006) The molecular genetic basis of plant adaptation. American Journal of Botany 93: 953-962. electronic resource
Kempin, S., S. Savidge, M.F. Yanofsky (1995) Molecular basis of the cauliflower phenotype in Arabidopsis. Science 267: 522-525. Lowman, A.C., M.D. Purugganan (1999) Duplication of the Brassica oleracea APETALA1 floral homeotic gene and the evolution of domesticated cauliflower. Journal of Heredity 90: 514-520 electronic resource
Purugganan, M.D., A.L. Boyles, J.I. Suddith (2000) Variation and selection at the CAULIFLOWER floral homeotic gene accompanying the evolution of domesticated Brassica oleracea. Genetics 155: 855-862. electronic resource
Smith, L.B., G.J. King (2000) The distribution of BoCAL-a alleles in Brassica oleracea is consistent with a genetic model for curd development and domestication of the cauliflower. Molecular Breeding 6: 603-613.