An update on CSIRO research into AGD in Tasmania

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1 An update on CSIRO research into AGD in Tasmania Mathew T Cook, Richard Taylor, Ben Maynard, Paula Lima, Peter Kube, Klara Verbyla and Nick Elliott FOOD FUTURES FLAGSHIP

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4 Environment water sampling, traditionally labour intensive and replication difficult 4

5 Semi-automatic sampling in situ 5

6 Results Semi-automated water sampling 40 RNA analysis DNA Analysis Average Ct Concentration (cells/l) 6

7 2 nd Generation sampler Computer controlled set and forget Capable of taking and archiving 24 samples Pumps water across filter and then floods with appropriate buffer Will be deployed 2 nd ½ of

8 Non-destructive sampling of gills - swabs 8

9 RNA vs DNA results GS 4 GS 2 GS 0 GS 3 GS 1 34 Average Cycle Threshold Value per GS Score Ct Average Lineær (Ct Average)

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11 Development of Amoebae Bioassay Assay based on the observation that P. perurans actively takes up Neutral red dye Apply treatments to observe uptake Requires appropriate controls (+ve and ve) 11

12 Indicative results % viability of N. pemaquidensis after treatment with candidate compound

13 Transcriptomic analysis Compare genetic information of amoeba P. Perurans (infective) and closely related species P. Pemaquidensis (non-infective) to identify differences. N. perurans N. pemaquidensis Compare the two datasets to identify: Seq 01 : ACGGTAGGCTAGACTAGATATTAACG Seq 02 : CCTGAGTACCTGGACTAGATAC Seq 03 : GATGCGGTTACGTACGATCCATGGA Seq 04 : CATTTATTATATATACGCGCGCGA Seq 05 : TTTCGATAGGGGATATATTAACGCCG Seq 06 : GTAGGTAGGTGGAGGCCCGCAGACGC Seq 07? : GATAGACTCGCGCCGATATATAG Seq 08 : ATATATTTCCTAGATCGAGAGATAC Seq 09 : GATAGGTTAATTAATTTCCTATAT Seq 01 : ACGGTAGGCTAGACTAGATATTAACG Seq 10 : TGGATTGGATAGCGCGATAGATC Seq 02 : CCTGAGTACCTGGACTAGATAC Seq 11 : AAAAGTCGATAAGGCTAGAGCTAG Seq 03 : GATGCGGTTACGTACGATCCATGGA Seq 12 : GGATATAGATATATCTAGATATC Seq 04 : CATTTATTATATATACGCGCGCGA Seq 13 : CGATATAGCCCTCTAGAGATACTTT Seq 05 : TTTCGATAGGGGATATATTAACGCCG Seq 14 : GATACCCGCGATATATCAT Seq 06 : GTAGGTAGGTGGAGGCCCGCAGACGC Seq 15 : TAGATCCCCGAGATAGAGACT Seq 07 : GATAGACTCGCGCCGATATATAG Seq 16 : CACCATAGAAGACTGATCGAGATAG Seq 08 : ATATATTTCCTAGATCGAGAGATAC Seq 09 : GATAGGTTAATTAATTTCCTATAT Seq 10 : TGGATTGGATAGCGCGATAGATC Seq 11 : AAAAGTCGATAAGGCTAGAGCTAG Seq 12 : GGATATAGATATATCTAGATATC Seq 13 : CGATATAGCCCTCTAGAGATACTTT Seq 14 : GATACCCGCGATATATCAT Seq 15 : TAGATCCCCGAGATAGAGACT Seq 16 : CACCATAGAAGACTGATCGAGATAG 1. Transcripts (i.e., functionality) present in one dataset that are missing in the other dataset? 2. Of those transcripts that are similar, how similar are they? 13 Amoeba Transcriptome Analysis

14 Raw Read Support 14 BLAST Amoeba Transcriptome Analysis ACGTTA TTAACG TACGTA TACAGT CATAGT AGTCAT GTACAT CATGTA ACGTTA TTAACG TACGTA TACAGT CATAGT AGTCAT GTACAT CATGTA TTAACG GGCACG AACGTA TACAGT CATAGT AGTCAT GTACAT CATGTA ACGTTA TTAACG TACGTA TACAGT CATAGT AGTCAT GTACAT CATGTA ACCGCA TATACG CCGGTA TACAGT CATAGT AGTCAT GTACAT CATGTA ACGTTA TTAACG TACGTA TACAGT CATAGT AGTCAT GTACAT CATGTA ACGGTAGGCTAGACTAGATATTAACG CCTGAGTACCTGGACTAGATAC GATGCGGTTACGTACGATCCATGGA CATTTATTATATATACGCGCGCGA TTTCGATAGGGGATATATTAACGCCG GTAGGTAGGTGGAGGCCCGCAGACGC GATAGACTCGCGCCGATATATAG ATATATTTCCTAGATCGAGAGATAC CCTGAGTACCTGGACTAGATAC GTAGGTAGGTGGAGGCCCGCAGACGC ACGGTAGGCTAGACTAGATATTAACG GTAGGTAGGTGGAGGCCCGCAGACGC GTAGGTACGTGGAGGCCCGCAGACGC GTAGGTACG CATGTA TGTA AGTCAT AGTCAT ACGTTA GCCCGCAGACG CATGTA ATGTA AGTCAT CATGTA TCAACG AGTCAT ACGTTA TACGTA

15 We can turn off genes in P. perurans GSP for Target cdna gdna Average Ct time Target 18S Untreated hr hr hr week NTC

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17 Hybrids can we unlock the key to resistance SxS SxT TxS TxT 17 AGD functional feed and hybrid trials Dr Ben Maynard

18 18 AGD functional feed and hybrid trials Dr Ben Maynard

19 Hybrid vigour 63% hybrid vigour observed at AGD4 Best parent performance 19 AGD functional feed and hybrid trials Dr Ben Maynard

20 Breeding for AGD Resistance Broodstock never go to sea 20

21 Gill Score is a consistent heritable trait All infections have significant genetic variation (heritability range is h2 = to ) Two distinct traits, one is measured at first infection and the other at all subsequent infections First infection has lower genetic variation (h2 = 0.14) Subsequent infections have stronger genetic expression, especially when measured during summer Gill score 3rd infection Gill score 4rd infection AGD1 versus AGD Gill score 1st infection AGD3 versus AGD Gill score 3rd infection 21

22 Selection is reducing the number of bathes MONTH Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan 2005 YC 2006 YC Founders 2007 YC 2008 YC 2009 YC 1 st generation selection 2010 YC * 2011 YC 2 nd generation selection Water temp o 11.3 o 11.6 o 12.5 o 14 o 15.1 o 16.5 o 17.2 o 16.1 o 14.9 o 13.6 o 12.2 o 11.5 o 11.3 o 11.6 o 12.5 o 14 o 15.1 o 16.5 o Marine grow-out period AGD infections (1 to 6) and measurement events Low score, prophylactic treatment 22

23 Selection is working A deliberate Bad family AVERAGE FAMILY VALUES 2012 YC (SBP COHORT) SBP BAD AGD families family Best AGD family EBV AGDac (Bath interval) * 25% -12% 49% Mean AGD1 Gill score Mean AGD2 Gill score NB Following GS2, Best AGD Family 5% GS1 23

24 Within vs between family variation Genetic change after progeny testing 60% 50% 40% 30% 20% 10% 0% -10% AGD predictions before and after progeny testing 2009YC y = x R² = % -20% -10% 0% 10% 20% 30% 40% 50% Predicted genetic change before progeny testing Highlights the power of whole genome selection (WGS) to increase genetic gains as the broodstock remain in freshwater (biosecurity) 24

25 Can SNP explain some of the AGD resistance observed? SNP # LG %Variance 1 Ssa Ssa Ssa

26 Summary Activities are focused on HOST, PATHOGEN and ENVIRONMENT Focus is on delivering solutions/information to assist in alleviating the issue Partnership with the end users (industry) is crucial AGD R&D needs to be dynamic to adjust to changing priorities International collaboration is key no need to reinvent the wheel 26

27 The CSIRO AGD Team Amoeba Biology Mat Cook Richard Taylor Natasha Botwright Paula Lima AGD Resilience Richard Taylor Peter Kube Selective Breeding Nick Elliott Peter Kube Richard Taylor Mat Cook Sonja Dominik John Henshall Scott Cooper Amoeba Detection Mat Cook Richard Taylor Sharon Appleyard Paula Lima Dietary Intervention Brett Glencross Mat Cook Ben Maynard Richard Taylor 27

28 Funding bodies and Industry and Academic collaborators 28

29 Thank you CSIRO Food Futures Flagship Mathew Cook Stream Leader t e mathew.cook@csiro.au w