1. How to set up a reverse genetics experiment with an Arabidopsis thaliana mutant Mining Phenotypes 1

2. The Arabidopsis Information Portal is funded by a grant from the National Science Foundation (#DBI-1262414) and co-funded by a grant from the Biotechnology and Biological Sciences Research Council (BB/L027151/1). These lessons were developed during the summer of 2015 as education outreach for the www.Araport.org portal in conjunction with the J. Craig Venter Institute, Rockville, MD, 20850, USA. We appreciate the lesson review and edits by David Lally at the Partnership for Research and Education with Plants.www.Araport.org Contact information General information: araport@jcvi.orgaraport@jcvi.org Jason Miller, Grant Co-Principal Investigator, JCVI jmiller@jcvi.org This lesson was prepared by Andrea Cobb, Ph.D. (adcobb@fcps.edu)adcobb@fcps.edu with the help of Margot Goldberg (mgoldberg1@pghboe.net)mgoldberg1@pghboe.net 2

3. Phenotype A detectable trait Examples of detection: – Visual (number of flower petals per flower) – Immunological (human blood type) – Biochemical (antibiotic resistant bacteria) 3

4. Genotype DNA sequence of a gene Gene variants Homozygous or heterozygous 4

5. Phenotype  Genotype Traditional “forward” genetics known phenotype  work out genotype https://blogs.brandeis.edu/flyonthewall/translational-findings-how-frui t-fly-research-has-already-contributed-to-human-health  find and sequence gene http:// cubocube.com/dashboard.php?a=1181&b=1260&c=103 http://www.sciencedirect.com/science/article/ pii/S0005273699000644 5

6. “Reverse” genetics Scientists alter the genotype (mutate) Detect resulting change in phenotype Learn something about the gene’s function Genotype   Phenotype https://www.youtube.com/watch?v=5gyl_ODu ZdY This links to a short video with Dr. Gillaspy describing her reverse genetics research. 6

7. Aren’t all Arabidopsis thaliana phenotypes known? >13% of all Arabidopsis genes coding for proteins have a completely unknown function domains of unknown function >30% of the Arabidopsis proteome is poorly characterized 7

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9. Variations in phenotype might result from: Environmental interactions Epigenetic regulation Copy number variants Multiple gene interactions Pleiotropy-one gene has multiple effects Mutations—knockouts, knock-downs and adding genes (transgenic) 9

10. Loss of function mutants-”knockouts” Point mutations –chemicals /EMS Insertions/deletions (indels) T-insertions CRISPR-Cas9 Must be back-crossed to reduce heterozygosity but sometimes that results in a lethal combination https://www.youtube.com/watch?v=QEbVpj7Ebw Uhttps://www.youtube.com/watch?v=QEbVpj7Ebw U shows how plant scientists can change Arabidopsis genes 10

11. Mutants with reduced gene expression-knock down Works well when homozygous knockouts prove lethal RNAi mediated From Wikipedia 11

12. Transgenic plants How adding a gene may affect a plant’s ability to respond to the environment https://www.youtube.com/watch?v=EW- 3hgE6XpA Links to a PREP video 12

13. How do scientists screen phenotypes? Visually-abnormal morphology, growth rate, color, flowering, fertility, etc. Biochemically—alterations in basic cell processes (replication, protein synthesis, etc.) Microscopically (use fluorescent tags to see overexpression) and image analysis http://www.illuminatedcell.com/autophagosomes.html 13

14. Phenotype screening, continued Developmental Metabolic or stress- dependent changes Chemical genetic screens http://abrcoutreach.osu.edu/educational-kits http://web.stanford.edu/group/gcep/cgi-bin/gcep- research/all/lignin-management-optimizing-yield-and-composition- in-lignin-modified-plants/ http://journal.frontiersin.org/article/10.3389/fpls.2012.00004/full 14

15. Phenotype screening, continued Co-expression data Protein-protein interactions From the following article: Proteomics: Protein complexes take the bait Anuj Kumar and Michael Snyder Nature 415, 123-124(10 January 2002) doi:10.1038/415123a http://www.learner.org/courses/biology/textb ook/proteo/index.htmlhttp://www.learner.org/courses/biology/textb ook/proteo/index.html provides helpful information on functional proteomics. 15

16. Challenges to determining phenotype-genotype relationships Mutants usually have more than one induced mutation—which mutation is crucial to effect? Genetic redundancy in gene families-not all copies in the gene will be mutated, so normal phenotype may persist. High throughput screening methods needed. http://www.ncbi.nlm.nih.gov/pubmed/235171 22http://www.ncbi.nlm.nih.gov/pubmed/235171 22 * 16

17. How does a scientist begin a stress or chemical screening experiment to explore phenotype? https://www.youtube.com/watch?v=foHiKrlY9Qc https://www.youtube.com/watch?v=foHiKrlY9Qc is a PREP video which shows how! Obtain the mutant and its genotype information Mutant and wild type Arabidopsis are available at ABRC.org and some commercial providers. 17

18. Researching treatment ideas may increase the likelihood that you will generate meaningful data. 18

19. A great place to begin desgining your reverse genetics experiment is at www.araport.orgwww.araport.org 19

20. From the Tools pull-down menu, select 50 years of Arabidopsis Research 20

21. Enter the name of the gene into the search box. Scroll to see the related publications If you select one with a great number of citations, you can see the citation network. You can mouse over the network lines to see the citation Add the publications of interest to Google Scholar App is written for Mac but will work for the most part on Windows 50 years of Arabidopsis Research 21

22. Access other detailed information about Arabidopsis genes in ThaleMine 22

23. Mine ThaleMine Enter the gene name into the search box Select the Gene category and you will see the gene information page 23

24. Use the information and links on the gene page to decide: What might I measure about the plant? During what part of the plant’s life cycle should I take measurements? How often should I take measurements? What might the gene product do? How might I measure what the gene product is doing? What treatments might make sense? What part of the plant should I use? 24

25. Gene ontology is a systematic way of recording descriptive info about what a gene does, its involvement in processes, and where it is expressed. Record the GO terms assigned to the gene: Biological processes Molecular function Cellular component These may help you think about what to measure and how to measure 25

26. Gene expression is: -The process of making a gene product (protein or RNA) -The most basic mechanism whereby genotype gives rise to phenotype https://www.youtube.com/watch?v=OEWOZS_JTgk Links to a short overview of gene expression 26

27. A closer look at gene expression What is gene expression? How do scientists measure gene expression? 27

28. Genes can be: Turned on (expressed) Turned off (not expressed) Turned up (increased expression) Turned down (decreased expression) 28

29. Proteins are the machinery of the cells. Proteins do many different jobs for the cell. Genes store information about making those different proteins. When genes are “turned on” (or expressed), the information in the gene’s DNA is used to make RNA. The RNA is then processed and is used to make proteins. It is important to make just the right amount of protein at just the right time and place! Figure 7-5 Molecular Biology of the Cell (© Garland Science 2008 29

30. http://www.ukhairdressers.com/romez.jpg http://newborns.stanford.edu/PhotoGallery/Teeth3.html https://en.wikipedia.org/wiki/Andr%C3%A9_the_Giant Too much gene expression (growth hormone) Wrong timing of gene expression (natal teeth) Wrong location for gene expression (trichosis) Changes in gene expression often change phenotype. 30

31. www.scq.ubc.ca/wp-content/cDNAarray.gif Microarray experiments How do scientists measure gene expression? 31

32. https://www.systembio.com/lncrna- research/disease-long-non-coding-rna/how-it- works Quantitative Reverse- Transcriptase PCR How do scientists measure gene expression? 32

33. How do scientists measure gene expression? http://www.nature.com/ni/journal/v13/n9/fig_tab/ni.2407_F1.html NextGen RNA- sequencing 33

34. Examine Expression First look at the Tissue specific gene expression. This will guide you about what part of the plant to sample or observe.. Look at the legend to see maximum expression. White indicates that data is unavailable or was not tested for that tissue. http://www.hhmi.org/biointeractive/how- analyze-dna-microarray-datahttp://www.hhmi.org/biointeractive/how- analyze-dna-microarray-data links to a lesson on microarrays used to generate this data. 34

35. Examine the Developmental map This will help you think about when to collect data during the plant’s life cycle. 35

36. http://www.prepproject.org/ Matches the developmental stage to the number of days after planting. 36 On which days after planting Arabidopsis seeds might you need to collect data?

37. Look at the other Expression pull- down menus They may give you ideas about how the environment influences gene expression. You might want to select or modify one of the treatments for your stressor (or not). 37

38. Proposal Ideas? Work in groups of 4-6 to review and add to your notes. Brainstorm from your Araport notes and literature searches: What are possible functions for the gene product?. What phenotypic changes might be expected? How might we change the environment to elicit those phenotypic changes? How and when might we measure those phenotypic changes? 38