Module 6: Exploring macro- and micro-synteny with the OMA Browser

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When comparing two related species, the position of orthologous genes is often conserved. Positional conservation can be at the chromosomal level—e.g. when there are entire chromosomes or chromosomal segments that are orthologous between species; or it can be more local—e.g. neighboring genes in one genome are orthologous to neighboring genes in the other genome. Additionally, synteny between orthologs in extant genomes can be used to infer the ancestral gene order.

In OMA, we have several tools to help visualize and analyze synteny at different levels of resolution. The Synteny Dot Plot can be used to compare chromosomes (global synteny). The Local Synteny Viewer can be used to compare genes in a local gene neighborhood (microsynteny). Finally, the Ancestral Synteny View of Hierarchical Orthologous Groups can be used to deduce the likely order of ancestral genes at any taxonomic level. In this module, we will focus on using these three synteny viewers: how to use them and what types of useful biological information we can obtain from them.

TODO: Where to find documentation For more information on OMA standalone, please see this blog post and the extensive documentation available here.

1. Go to the Synteny Dot Plot by Tools -> Synteny dotplot on the omabrowser or by clicking here. The Synteny Dot Plot displays the pairwise orthologs (or homoeologs) between two chromosomes. Select the cat (genome 1) and dog (genome 2) to compare.

Cat = Felis catus, or FELCA species code; Dog = Canis lupus familiaris, or CANLF species code
2. Which pair of chromosomes between cat and dog have the highest number of orthologs between them, and how many?

Mouse over the cells of the heatmap to see the exact values.

Cat = A2, dog = 20; 820 pairwise orthologs.
Select the chromosomes with the highest number of orthologs and click Launch Dotpot. Note that the synteny dot plot does not necessarily show the chromosome in its entirety. What is the general level of synteny conservation for the segments displayed for these two pairs of chromosomes?

Very high, because nearly all the points are on the diagonal.
Zoom in on the region of the chromosome with an abnormality: ~10.5-10.9 on FELCA A2 and ~47.2 - 47.7 on CANLF 20. It should look like a little nodule along the diagonal. What do you observe in this region in terms of chromosomal events?

There are many duplications in this region.
Select the top “row” of duplicated genes using the Select tool, as indicated below:

TODO: add synteny_select_orthologs.png

How can you interpret the relationship between these genes?

Consult the table which appears below upon selection of the orthologs.

There are many genes in cat which are orthologous to the CANLF07749 gene in dog. In other words, there were lineage-specific duplicates in the cat lineage.
Where are the physical locations of the duplicated cat genes from the previous question? (i.e. the locus coordinates along the chromosome)

10561828, 10465336, 10774323, 10637607, 10802221 on cat chromosome A2
Additionally, we can see that FELCA01637 has several orthologs in dog, thus meaning there have also been lineage-specific duplications in dog. What are the functions of these genes? Use the OMA browser to find the functions of these genes.

Go to the Gene Information and GO Annotations pages for one of the duplicates

These genes are olfactory receptors. The GO terms include: G protein-coupled receptor activity, olfactory receptor activity, and integral component of membrane.
Let’s look at another pair of chromosomes. Go back to the Synteny Dot Plot home page and display cat chromosome E2 versus dog chromosome 1. What kinds of structural rearrangements do you observe?

We can observe two deletions, 1 inversion, and 1 inversion/translocation.

TODO: insert synteny_rearrangements.png
Notice the region of the chromosomes which nonsyntenic genes (see answer to previous question). What can you say about these four pairs of orthologs, in terms of rate of evolution and duplication?

Observe the phylogenetic/evolutionary distance based on color.

The CANLF01158 gene is in one copy and cat has 4 copies (FELCA16265, FELCA16274, FELCA16277, and FELCA16288). Thus there have been several lineage-specific duplications in the cat lineage. These duplications are clustered in the cat genome, and appear to be the result of tandem duplications. The Distance is a measure of amino acid substitution and rate of evolution. Based on the color, we can see that these orthologs have a higher rate of evolution. We can confirm this by selecting the nonsyntenic orthologs and looking at the extract distance in the table.

Local Synteny Viewer

Let’s focus on the gene CANLF07749 in a dog and inspect its local synteny and the gene order conservation of its orthologous genes in other species. To go to the gene page you can either follow the instructions from question 4 in the “Synteny dot plot” section and click on CANFL07749 gene ID in the table below the dotplot. You can also select Explore->Local synteny on top of the OMA webpage and find the gene by inputting the ID into the search box. Your query gene will be in the center of the first row.

To understand the local synteny viewer, check out the documentation: Local Synteny Viewer

What are the first two most closely related species in OMA to the dog?

The first two listed species are the most closely related and to find out the full latin name of a species click on the species OMA identifier.

Vulpes vulpes (Red fox) and Mustela putorius furo (Ferret).
In the red fox (Vulpes vulpes) how many orthologs do we find to the gene CANLF07749 and how would you describe the relationship cardinality of these orthologs?

The query genes and the flanking genes are assigned a different colour to indicate orthologous counterparts in the other species. The definition of relationship cardinality can be found in the glossary under “relation type”.

Only one, VULVU33903. There is a 1:1 orthologous relationship between them.
How much of local synteny around the CANLF07749 genes is conserved between the red fox and the dog? How many orthologous genes from this genomic fragment are co-localized in the two genomes? What evolutionary events can you infer happened in this area?

Two orthologous genes are co-localized between the dog and the red fox. However, between the ortholog of CANFL007748 and the ortholog of CANFL007749 there was an insertion in the red fox genome.
How many ferret genes are orthologous to CANFL07749?

Pay attention to the colours, ferret genes orthologous to CANFL007749 are striped coloured because they are orthologous to the two genes of the same coulour in the query genome. Also, pay attention to the numbers on the genes, they are the unique OMA identifiers. A gene may be displayed more than once in the local synteny viewer if they are the result of tandem duplications.

We find 11 genes orthologous to CANFL00749 and these genes are also orthologous to CANFL007748.
What can we infer about the genomic location of the ferret genes orthologous to CANLF07749?

OMA identifiers are assigned based on their order on the chromosomes. To find out where these genes are in the genome click on the genes and go to Gene Information tab.

MUSPF00909, MUSPF00908 and MUSPF00907 are located next to each other in the genome and so are MUSPF00899 and MUSPF00898. MUSPF10051 and MUSPF10048 are also located relatively close to one another, with two genes in between. These genes are most likely a result of tandem duplications.
What can we infer about the genome assembly of the ferret?

We can also observe that many of the MUSPF rows do not display 10 genes; this means that there is not enough information to display the contiguous chromosome. This is the result of the ferret genome being fragmented into many contigs or scaffolds. For example, MUSPF00233 and MUSPF00234 are the sole genes displayed in their row. This is because they are on a scaffold together, but with no other genes annotated to that scaffold.
Look at the phylogenetic relationship between the 3 species:

TODO: insert synteny_tree.png

Seeing the relationship between species, what would you infer about the evolutionary history of the olfactory genes CANFL007748 and CANFL007749 and their orthologs in the red fox and the ferret?

Consider the type of orthology between the genes (the colours of the boxes), the location of the genes and the number of genes in each of these 3 genomes.

A duplication after speciation of the lineage leading to the ancestor of Canis lupus and Vulpes vulpes led to formation of two genes: one giving rise to the orthologs CANFL007749 and VULVU33903 and the other to CANFL007748 and VULVU33905. Speciation was the process that separated these orthologs in the dog and the ferret. Along the branch leading to Mustela putorius furo there were many duplications of the gene orthologous to CANFL007749 and CANFL007748, some of which were segmental (tandem duplications) as the genes remained in the same genomic location and some of them were non-homologous duplications moving the orthologs to a different location.