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PART A: TRANSCRIPTOME
Materials
We collected total 12 RNA samples from the three species:
Methods
We made cDNA libraries from the RNA, and sent them for Illumina short-read sequencing
From Illumina sequencing, we assembled 303,238 – 646,586 transcripts with an average contig size of 561 – 650 bp.
We created separate datasets for each species and annotated the genes and looked at differential Expression
Results
Many genes were differentially expressed (i.e. more highly expressed in a type of tissue compared to other)
An example heatmap:
Each row/line represents a single gene
In this example we compared skin vs blood vs spleen within Rana tagoi
Yellow = gene is highly expressed in this particular tissue
Purple= gene is not highly expressed in this particular tissue
As you can see, there are thousands of genes that are diffentially expressed, so we did GO analyses and focussed on immune-related genes that might have expression differences.
What is GO analyses?
Gene ontology (GO) analysis looks at a whole group of upregulated genes to find 'GO terms' that are over-represented.
On the left axis there is a list of the GO terms under three parent categories (Biological processes, Cellular component and Molecular function)
You can see that the 'immune system process' GO term has higher expression in the spleen (blue bar) in all three species
We then focused on just looked at these immune-related genes.
Focus on immune related GO-terms:
- Spleen (blue) contained the most enriched immune-related GO terms, 9 terms common in all three species, including inflammatory response and T cell differentiation.
- In skin (green), only one enriched term common in all three species- regulation of inflammatory response.
- MHC class II related GO term (black square ) was enriched in various samples (no pattern).
We then studied MHC class II (MHC-II) in several individuals
PART B: MHC class II
Using PCR, we amplified ~ 750 bp of MHC class II beta 1 and beta 2 domain.
From the phylogenetic tree, we can see that MHC-II sequences did not group into species-specific clusters. Also, the beta-2 domain of Rata-01 had high similarity to sequences from the American bullfrog (Lica). This supports something called 'trans-species polymorphism'
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