Leading the world in aquaculture

1. Leading the world in aquaculture Robert Deerenberg, Victoria Cepeda, Ruth Gonzalez Analysis of Transcriptome Sequencing Differences Between Resistant & Susceptible Strains of Atlantic Salmon to ISAv

2. Introduction • Introduction (ISA, Tackle, Group) • Trial (Challenging Fish) • Molecular Genetics & Analysis • Conclusions

3. Introduction: ISA crisis • Infectious Salmon Anemia or ISA • Disease that affects Atlantic salmon • Has affected fish farming in Canada, Norway, Scotland, Faroe Islands and Chile • Economical Losses can be considerable • From 2007 to 2009 huge impact on the Chilean salmon industry

4. Introduction: How to Tackle? VACCINES ISA GENETICS REGULATIONS PRACTICES

5. Introduction: Group Marine Harvest Chile: Reproduction & Genetics Fish $$ & Resources Breeding Know-How PROJECT USACH Genomics Group $$ & Resources Molecular Genetics Know-How $$$$ CORFO Governmental Fonding

6. Trial: Questions & Objectives • Do differences in ISA resistance exist between families? • Are there differences between strains/lines? • Is there a difference between vaccinated and non-vaccinated fish? • Does a vaccine-genetic interaction exist on strain level? • Gather genetic material of families and strain during the trials, of dead and surviving fish

7. Trial: 1: Challenging LM Families 120 LM 40 families x 3 fish/fam 80 TROJAN ISAv 7B 5 x

8. Trial: 1: Challenging LM Families

9. Trial: 2: Challenging Strains (+/- vac) 40 LM 40 LM + vac 40 HM 40 HM + vac 110 TROJAN ISAv 7B 2 x

10. Trial: 2: Challenging Strains (+/- vac)

11. Molecular Genetics: Objectives • Identify novel genes or transcripts in Atlantic salmon • Annotation of genes • Differential gene expression from salmon infected with ISAv • qPCR pending

12. M.G.: Work Model Phenotypes Work Model HM LM DIFFERENTIAL EXPRESSION (Desired) Gene Expression Profile • Vaccine testing • Immune Stimulators • Development of Genetic Markers for selection

13. M.G.: BioInformatic Pipeline Bioinformatic Pipeline Illumina Reads

14. M.G.: Illumina Libraries

15. M.G.: Step 1 Step I- Pre-processing

16. M.G.: Step 2 Step II- RNA-seq De Novo Assembly • Trinity • Minimum Length contig :300 • Kmer=25

17. M.G.: Transcriptome Assembly Reference Transcriptome Assembly

18. M.G.: Annotation Functional Annotation

19. M.G.: Assignments GO assignments

20. M.G.: Expression Analysis Differential Expression Analysis Factorial design containing eight libraries Table 1: An example factorial design experiment in which samples 1 and 2 are subjected to experimental conditions A and C, samples 3 and 4 are subjected to conditions A and D, samples 5 and 6 are subjected to conditions B and C and samples 7 and 8 are subjected to conditions B and D.

21. M.G.: Expression Analysis Differential Expression Analysis Factorial design containing six libraries

22. M.G.: Expression Analysis Differential Expression Analysis

23. M.G.: Expressed Genes GO Differential Expressed Genes

24. Conclusions • We Confirmed Increased Resistance in One Atlantic Salmon Line • We Showed Lower Mortality in the Vaccinated “Resistant” Line Compared to a “Normal” Vaccinated Line, Suggesting a Genotype-Vaccine Interaction • We found 47 Genes that are Involved In Response to a Vaccine • We found 68 Genes Involved in Resistance of No-Vaccinated Fish • Quantifying the level of Expression of Those Genes with qPCR is still pending, but part of this project