• • • • Who is the audience? 
        • Upper-level high school students, college students in intro biology or a first course in evolutionary biology
      • What do they need to learn? 
        • How to use evolutionary trees and sequence data to you formulate and test hypotheses related to cool phenotypes
      • What do they need to learn how to do? 
        • Generate a hypothesis about the genetic basis of a cool phenotype 
        •  Identify what NCBI resources are relevant to the evolutionary biology research question 
        •  Build a simple phylogenetic tree using sequence data 
        •  Identify sequence orthologs related to the phenotype 
        • Evaluate changes in sequence orthologs that could  
        • Use visualization tools to support hypothesis and share findings with others 
      • What is a relevant and interesting story for the audience? 
        • Evolution of a relatable sensory phenotypes
      • What kind of NCBI data & resources are relevant?
        • Sequence Analysis Tools
          
          • BLAST alignment tool
          • COBALT sequence aligner
          • Multiple Sequence Alignment Viewer (Graphics)
        • Biological Sequence resources
          • Gene
          • Nucleotide
          • Protein
        •  Biological structure resources
          
          • Structure
          • iCn3D (if time)
      • What workflow will help find the data and provide the information to solve the case study? 
        • Workflow: See below
      • How can they assess that they've mastered the content? 
        • Opportunities to check work against answer keys
        • Practice with other phenotypic examples

1. Come up with an evolutionary question and plan to test it
2. Gather data to build phylogenetic tree
3. Use data to build evolutionary tree 
4. Find gene sequences and map them onto phylogenetic tree 
5. Identify specific changes to protein sequences and finish the story!

Step 1: Come up with an evolutionary question

Have you ever noticed…
That you have a bigger sweet tooth than your friends? 
Your dog seems more interested in your candy than your cat? 
The recommended food for hummingbirds is sugar water? 
And wondered...why is this? is there a genetic component to the ability to taste and seek out sugar? 

can you tell which ones are responsible for sweet (carb) perception? 

T1R1: https://www.ncbi.nlm.nih.gov/gene/80835/
T1R2: https://www.ncbi.nlm.nih.gov/gene/80834
T1R3: https://www.ncbi.nlm.nih.gov/gene/83756

Step 2: Gather sequence data for building an evolutionary tree

From Guo et al. 2022

• Go to the page for the ruby-throated hummingbird mitchondrial genome sequence (Accession NC_010094.1) 
• Once you are there, click "Send to" in the top left corner of the page. 
• Leave "Complete Record" selected and Choose Destination set to "Collections". Click "Add to Collections" 
  • 
• Choose "Create new collection" and name it something that makes sense, like "Sweet Mitochondrial Genomes" and Press "Save" 
• From now on, you can add all mitochondrial sequences to that collection by choosing "Append to existing collection" 

• From nearly any NCBI page, simply go to the top right corner of the whole page, click your username -> Dashboard. 
• Under Collections, click on the name of the collection.
• The following two items should now be in your collection: 

• Navigate to the NCBI Nucleotide Database: NCBI Nucleotide database
• In the search bar type "Human mitochondrial genome" and click Search
• A result box like this will pop up - click the main title "Human mitochondrial reference genome" 
• Just like you did above, send this record to your "Sweet Mitochondrial Genomes" collection

• • Ruby-throated hummingbird - Archilobus colubris -  tasting (DONE)
  • Common swift - Apus apus - non-tasting 
  • Little blue penguin - Edyptula minor - non-tasting 
  • African ostrich - Struthio camelus - non-tasting 
  • Grey parrot - Psittacus erithacus -  non-tasting
  • Zebra finch - Taeniopygia guttata - non-tasting 
  • Human - Homo sapiens - tasting (DONE) 
  • House mouse - Mus musculus - tasting 
  • Domestic cat - Felis catus - non-tasting
  • Dog - Canis lupus familiaris - tasting
  • Little brown bat - Myotis lucifugus - tasting
  • Cattle - Bos taurus - tasting
  • Outgroup: Zebrafish (DONE)

Step 3: Infer evolutionary tree and use it to look at patterns

Once all thirteen of the above sequences have been gathered, you have what we need to build a tree of the relationships between the species and use it to refine your hypotheses. We are going to use NCBI's BLAST tool to align the sequences, and then visualize the output as a tree using Tree Viewer. 

A. Send Data from Collection to BLAST 

• From nearly any NCBI page, simply go to the top right corner of the whole page, click your username -> Dashboard. 
• Under Collections, click on the name of the collection.
• Once inside collection – send to -> Analysis tool -> BLAST 

• Click “Align two or more sequences”  
• Copy and paste the list of sequences IDs into the "Enter Subject Sequence" box too 
• Make job title something informative: "MitoGenomes"
• Choose “more dissimilar sequences” to use a version of BLAST that is good for cross-species comparisons 
• Click "BLAST"!

• Choose Tools -> “Edit labels” Select common names 
• The only “scientific name” left should be the hummingbird, which may be missing common name data - we can always re-label the sequences in other software program.  

• Tools -> Download PDF, which can be pasted into a slide or document 
• Tools -> Download Newick file (to import into other tree-visualization software as an extension!)
• Can also take a screenshot in a pinch!

• What is the best bird species for comparison with the hummingbird if we want to learn how their sequences evolved?  
• You could probably already guess this, but what is the best animal for comparison with domestic cat? 

Step 4: Tying things together: Find orthologs and map presence/absence on our tree

Let's move on to look at the actual basis of sweet tasting perception. As a reminder, according to the paper we read at the beginning of this lesson:

"In vertebrates, sweet and savory (“umami”) tastes are sensed by G protein–coupled receptors (GPCRs) termed T1Rs. Most vertebrates have three T1Rs, with the T1R1-T1R3 heterodimer mediating umami taste and the T1R2-T1R3 heterodimer mediating sweet taste. Human T1R2-T1R3 detects carbohydrates and artificial sweeteners (10), and knockout mice lacking T1R2 or T1R3 have defective sweet taste perception.“  

Now, we are going to be tying the genetic basis of sweet perception together with the patterns of trait evolution we mapped above. This will help us test our hypothesis - DOES each animal that can perceive sweetness have copies of both the T1R2 and T1R3 gene family? To to this, we need to 1) Find whether or not each species in our tree has an ortholog (related sequence) for each gene, and 2) Label this presence/absence on our tree.

We are going to take advantage of pre-computed NCBI Orthologs to do this! These have been calculated for many animals and more orthologs are being calculated and added all the time. 

• Human T1R2 Gene Page
• Human T1R3 Gene Page

• Use what you know about animal relationships: Tetrapods -> Mammals -> Placentals -> Bats
• Use the Taxonomic search box to check directly! 
• Either way, we see that bats do have orthologs of this sequence. We can check YES next to our bat species for T1R2. 

Almost all mammals, including dogs, have the ability to detect sugars (think about ice cream – yum!). Why can’t cats (big AND small!) do this too? 
All other birds have lost the ability to taste sugars, due to the loss of one of the T1R genes. How can hummingbirds detect the sugar in the flower nectar that they eat?  

Step 5: Tying things together -  specific genetic changes

Through our hard work, we have found some specific exceptions to the idea that presence/absence of the gene 

A. Looking for specific changes between cat and dog T1R2 and T1R3 genes

T1R2 - Starting on the NCBI Orthologs of the Human T1R2 gene page again.

• See “canis lupus familiaris” sequence in first result table. Select checkbox and click “Add to Cart”.  
• Use Taxonomy search function and do the same for “domestic cat”  
• Go to cart -> Align -> One Sequence per gene  
• You will be taken to COBALT aligner - go ahead and click through 
• View alignment and customize view - share link if you want!- Mine looked roughly like this: What are some differences? 

On your own: Align the cat and dog T1R3 sequences to see if there are also major changes. 


Link to one possible alignment view: https://www.ncbi.nlm.nih.gov/projects/msaviewer/?anchor=-1&coloring=cons&key=vAMq0twBCyAnLz0nDD4DKVBUUlVaV3ZdfkVwb-VhHi9LGVrF9Z3E8mBI6jG_LeY19B2jHOAC8BPqCfoWzDv1OdUJ8w&track_config=protein_default&columns=d:120,b:55,x:17,aln,e:55,o:150 

Of note, these changes match previous research comparing cat and dog taste receptors!



From Xia et al. 2006


B. Do humans have within-species variation?

Thus far, we have focused on major species-level differences in whether or not. Can smaller changes within the human species help account for the differences between how big a sweet tooth each of us have? 

We can begin to answer this question by going back yet again to the T1R2 human gene page, and scrolling until we find Bibliography. The short answer is YES there is meaningful human variation in this gene! 

• Have students think about how they would tell a sibling about cats and dessert preferences
• Pick the two most important figures and create custom versions to include in short presentation
• Your idea here? 

All biology research projects involve finding relevant literature and identifying related biological information. Often high school or new undergraduate students need guidance on how to effectively search for helpful information. For almost 35 years, the NCBI has provided free access to high-quality biological databases for the research community. In addition, we’ve created tools to help organize selected literature and biological data records for quick and easy access.

In this online, interactive workshop we will take you step-by-step through how to:

• Use the web interface to effectively search NCBI databases
• Create and use your NCBI account to store selected records in an online folder to access and share them
• Find information related to a proposed research project topic across linked NCBI databases, from publications and associated genome sequences down to protein structures
• Begin to interpret the information you’ve found in the context of the project

Note:  This workshop was designed for both student researchers, and the educators and mentors who want to help students use these resources.

Specifically, students will accomplish: 

• Learning about a project from a publication
• Finding data about an organism
• Finding available genome data for an orgnansim
• Get details about an interesting gene
• Using BLAST to find orthologs of an interesting gene 
• Explore protein structure and sequence for your gene of interest

• have a good understanding of biology
• are comfortable trying new things and using technology
• are a hard worker, learn quickly, and are eager to support their research efforts

Today's scenario

Your plan