Genetic stock identification in 6a Herring Preliminary Results 6a Herring Meeting, Pelagic AC, Dublin, 9th December 2016

Transcription

1 Genetic stock identification in 6a Herring Preliminary Results 6a Herring Meeting, Pelagic AC, Dublin, 9th December 2016 Edward D. Farrell, Jeanette E.L. Carlsson & Jens Carlsson University College Dublin

2 Stage 1 Recap Development of New Baseline Deliverables 1. Screening of baseline samples and confirmation of temporal stability of genetic structure. WESTHER Fst-values: IRL SCT BLT IRL SCT BLT 0 P-values: IRL SCT BLT IRL SCT BLT Pilot Study Fst-values: IRL SCT BLT IRL SCT BLT 0 P-values: IRL SCT BLT IRL SCT BLT 0 2. A panel of high-graded temporally stable informative genetic markers. 3. A baseline with which to compare mixed survey and fishery samples. 4. Development of automated genotyping application for data analyses.

3 Progress at 9/12 months Sampling Completed DNA extraction Completed Genetic marker development Completed Sample screening with markers - Completed Sequencing - First run completed Sequence analyses Underway Preliminary Results Genotyping app - Manual version only

4 Baseline Samples # Baseline samples Code Year n 1 WESTHER Donegal 6AS WESTHER Cape Wrath 6AN WESTHER western Baltic BLT VIaS 6AS VIaS 6AS VIaN 6AN North Sea NS15a North Sea NS15b Irish Sea IS Celtic Sea CS Total , 4, 5 2, 6 9 7, 8 Small geographic area Discrete spawning grounds Temporally separated spawning Northern area = summer/autumn Southern area = winter High gene flow species Baltic outgroup = + ctrl 10

5 Genetic Markers Revised panel of 100 microsatellite markers DNA derived - putatively neutral 31 novel from shotgun sequencing McPherson et al. (2001) 1 3 O'Connell et al. (1998) 2 2 Olsen et al. (2002) 1 14 Libungan et al. (2012) 3 RNA derived - putatively adaptive 43 Teacher et al. (2012) 4 1 Used in WESTHER, HERGEN: 2 Pacific Herring 3 Used in NSSH & ISSH: 4 Used in Baltic

6 Neutral markers Neutral V Adaptive Markers Affected by evolutionary forces (e.g. gene flow and random genetic drift) Not affected by natural selection Useful if strong genetic structure e.g. boarfish Good for demographics (e.g. connectivity, estimates of gene flow, N e ) Adaptive markers Selected by environmental conditions Diversifying selection may reflect distinctive features of local stocks Good for specific questions (e.g. stock discrimination, assigning catches)

7 Data Analyses Genotyping Application manual version 46 out of 100 markers scored 17 DNA derived & 29 RNA derived Individuals >75% genotyping success retained 881 out of 1008

8 Marker Tests Initial QC Scoring errors, large allele dropout, null alleles Neutral expectations (Hardy Weinberg equilibrium) 38 markers retained 8 markers with flags (revisit)

9 38 marker dataset Confirm structure detected in the pilot study? Pairwise multi-locus F ST POWSIM Expected F ST of = 99% power Baltic is different (+ve ctrl, scale of differentiation) WESTHER 6aS 2003 sample = mixed signals Little or no differentiation across sampling area

10 Revisit 8 flagged markers Can flags be explained? Genotyping errors etc.? Are any of the markers under diversifying selection? 5 markers for further analysis Pairwise multi-locus F ST

11 North South difference (AMOVA) Her502 and Her514

12 Preliminary assignment attempts GeneClass2 - select/exclude populations as origins of individuals 6aS versus 6aN-NS Subsample from each = unknown(s) & unknown (N) Assign unknown groups to baseline sample Successful assignment rates of % BUT needs rigorous testing (Stage 2) Mixed sample assignment not run yet

13 What next? Her502 and Her514 North South difference Potential for assignment Adaptive markers Related to what? Are there more available? Map to genome (1Gb) Identify additional markers

14 Recent advance in Herring Genetics Martinez Barrio et al 2016 Draft Herring genome 400,000 SNPs 70k SNP array 500 loci related to Atlantic v Baltic 71 SNPs related to spring v autumn spawning Relevant to 6a? Mapping Shotgun data to genome Mapping microsats to genome Her514 Heat shock & sodium chloride tolerance genes

15 1. Genome sequencing Screen baseline samples for SNPs Initial cost very high 2. Affymetrix 70k SNP array c. 120 per sample (min 96 samples) 3. Fluidigm or similar up to 96 SNPs c. 20 per sample Option 3 in January Remaining consumable budget

16 Stage 1 Development of New Baseline Progress Deliverables 1. Screening of baseline samples and confirmation of temporal stability of genetic structure. 2. A panel of high-graded temporally stable informative genetic markers. 3. A baseline with which to compare mixed survey and fishery samples. 4. Development of automated genotyping application for data analyses

17 Preparing for next stage Current project (Stage 1) ends 9 th March 2017 Next stage needs to be in place by end of 2016 to ensure continuity in UCD and avoid delays. EU Commission Work Programme Tender documents being developed Earliest start date if successful = Q3/ One year funding for survey sample analysis (Stage 2) Long term funding for development and implementation? SFI Strategic Partnership Programme? Industry-SFI co-funding Industry non cash contribution acknowledged

18 Stage 2 Mixed Stock Analysis of Survey Samples (EU?) SGHERWAY samples Status Location n 2014 Collected UCD Collected UCD Collected UCD 400 Total 2061 Duration: 12 months Deliverables 1. Development and validation of mixed stock analysis model. 2. Screening of SGHERWAY samples and comparison with morphometric analyses for retrospective splitting of survey series. 3. Development of a commercial sampling protocol and determination of required sampling levels.

19 2017 Interim Period Industry Priority? Industry survey samples or NWHAS samples Cost depends on proposed work programme Example cost Description Cost to budget Research fellow (full time 8 months) 46,412 (35,497 basic salary + 10,915 employer costs) Sequencing and Lab consumables 22,000 Subtotal ex overheads 68,412 UCD 5% 3,421 Subtotal ex VAT 71,833 23% 16,521 Total 88,354