Parentage analysis. Every person receives a unique set of genetic information from their parents - half from Mom and half from Dad

Transcription

1 Parentage analysis Similar techniques as those used in human parentage testing! With 99.99% probability, you ARE the father Every person receives a unique set of genetic information from their parents - half from Mom and half from Dad Different approaches depending on goals and system - Exclusion, likelihood

2 Parentage analysis, exclusion approach 1.) Determine multi-locus genotypes for all potential mothers, fathers, and offspring 2.) A parent must share at least 1 allele at every locus with a potential offspring Offspring?? 3 Candidate parents 4 2 5

3 Parentage analysis, exclusion approach Possible outcomes: 1.) All, but one parent-pair, are excluded 2.) More than one parent cannot be excluded 3.) All potential parents are excluded Assign parentage Need more loci or use another approach (i.e., likelihood) Un-sampled parents

4 Assignment rate to correct parent(s) What do we need for parentage analysis? Genetic markers Marker type # loci needed Variability Microsatellites SNPs Usually few n ~ Many n > 60 High many alleles Low bi-allelic Powerful set of loci - simulations to evaluate power - empirical test using known parent-offspring 100% 80% 60% 40% 20% 0% # microsatellite loci

5 What do we need for parentage analysis? Sampling of most potential parents - trap fish as they return to spawn - trap below majority of spawning habitat - carcass surveys can recover un-sampled fish Sampling a parent genetically tags ALL of that individual s offspring Crossed in a hatchery or mated in the wild

6 What do we need for parentage analysis? Representative sample of offspring - Sample as adults or juveniles

7 Example project using parentage analysis Parentage based tagging (SNPs)

8 What is parentage based tagging (PBT)? Genetic tags (PBT) Hatchery broodstock X Genetic tagging of hatchery broodstock = can identify hatchery of origin and age for ANY offspring produced Passive mark (no handling of juveniles) Nearly 100% tagging rate of hatchery fish Eliminates issues with tag loss, tag detection, handling mortality Juvenile offspring Adult Non-lethal sample to recover tag from offspring Stock contribution to various fisheries Survival trends Escapement estimates Origin of strays and kelts

9 Snake River basin PBT Sample all hatchery broodstock: ~5,000 steelhead/yr ~9,000 Chinook salmon/yr Parental sampling complete for , 2012 ongoing All hatcheries record spawn dates and gender (many provide lengths and spawn cross) Permanently tag ~20 million smolts/yr

10 Sampling PBT-tagged offspring or juveniles adults Non-lethal tissue sample Age Hatchery Parents BY2008 Sawtooth Hatchery

11 Assignment accuracy of offspring to known broodstock parents High number of juveniles assigned (97.3%) 100% accuracy to stock We only need 1 96-SNP panel to assign accurate parentage!

12 Spring/Summer Chinook Spawn Year Broodstock sampled 10,836 8,849 8,290 8,187 Genotyped 10,630 8,493 8,235 In progress Tagging Rate of Offspring 96.2% 92.1% 98.7% Smolts Produced * ~18.96 mil ~15.49 mil ~14.51 mil Smolts Tagged ~18.25 mil ~14.26 mil ~14.32 mil * Assuming 3,500 smolts produced per broodstock pair

13 Steelhead Spawn Year Broodstock sampled 5,151 5,761 5,282 5,931 Genotyped 5,070 5,636 5,198 5,765 Tagging Rate of Offspring 96.9% 95.7% 96.9% 94.5% Smolts Produced * ~9.01 mil ~10.08 mil ~9.24 mil ~10.38 mil Smolts Tagged ~8.74 mil ~9.65 mil ~8.96 mil ~9.81 mil * Assuming 3,500 smolts produced per broodstock pair

14 Example application of PBT: Origin of hatchery strays? Sampled in 2011 at Sherars Falls, Deschutes River: 446 ad-clipped steelhead 278 ad-intact steelhead Deschutes River Snake River basin Bendbulletin.com *Collaboration with Rod French, ODFW

15 Origin of hatchery strays? Ad-clipped Steelhead 354 / 446 = 79.4% Assigned Grande Ronde, n = 4 Dworshak, n = 27 Pahsimeroi, n = 105 Sherars Falls Wallowa, n = 48 Oxbow, n = 95 Sawtooth, n = 66 Squaw Creek, n = 7 E. Fk. Salmon, n = 2

16 Origin of hatchery strays? Ad-intact Hatchery Origin Fish 32 / 278 = 11.5% Assigned Pahsimeroi, n = 5 Wallowa, n = 1 Sherars Falls Sawtooth, n = 26

17 Example application of PBT: Origin and age of harvest fishery? 1,500 adipose-clipped steelhead collected in 2011 Zone 6 fishery 2008 and 2009 Snake River PBT baselines B Zone 6 Fishery Lyons Ferry M Grande Ronde Wallowa Dworshak Pahsimeroi Oxbow Sawtooth

18 Origin and age of harvest fishery? Of the 1,482 steelhead genotyped, 1,117 (75%) assigned to two parents BY2008 (2-ocean), n=633 2% 0% Zone , n=1,117 BY2009 (1-ocean), n=484 8% 7% 7% 76% 10% 14% 7% 5% 19% 46% 12% 13% 12% 23% 6% 33% *Wallowa unsampled SY2008 Dworshak Pahsimeroi Sawtooth Oxbow Lyons Ferry Wallowa 10% (n=153) estimated mid or upper Columbia River origin

19 Goal: Extend PBT sampling to ALL Chinook salmon and Steelhead hatcheries above Bonneville Dam beginning 2012 Large collaborative effort between the tribes, ODFW, WDFW, IDFG, USFWS, NOAA Required as fish are spawned: Fin tissue Spawn date Gender REQUIRED information OPTIONAL information Sample ID Spawn Date Gender Cross 1 Cross 2 Length PAH /10/2012 M 600 PAH /10/2012 M 730 PAH /10/2012 F PAH PAH PAH /10/2012 F PAH PAH /14/2012 F PAH PAH PAH /14/2012 F PAH PAH /14/2012 M 590 PAH /20/2012 F PAH PAH /20/2012 M 620

20 Goal 2012-ongoing: Chinook salmon, PBT hatcheries Winthrop Methow Leavenworth Cle Elum Wells Eastbank Priest Rapids Lyons Ferry Marion Drain Prosser Willard Klickitat Carson Lookingglass Bonneville Spring Cr. Umatilla Little White Parkdale Catherine Cr. Warm Springs Round Butte Nez Perce Dworshak Lostine Imnaha Rapid R. McCall, JC Sawtooth Powell Pahsimeroi CRITFC/IDFG, SY 2008-present WDFW USFWS ODFW Tribes or co-manager/tribes

21 Goal 2012-ongoing: Steelhead, PBT hatcheries Winthrop Wells Eastbank Ringold Springs Lyons Ferry Dworshak Skamania Parkdale Umatilla Wallowa Little Sheep Oxbow Round Butte Sawtooth Pahsimeroi CRITFC/IDFG, SY 2008-present WDFW USFWS ODFW Tribes or co-manager/tribes

22 Parentage Example

23 Finding Nemo s Parents: A Parentage Example

24 Parentage example 5 candidate parents sampled 1 5 juveniles sampled Match at least 1 allele at every locus? If not, exclude parent Genotype 11 samples for 5 SNPs

25 Approach: Compare each offspring to the pool of candidate parents - focus on 1 locus at a time - Does the candidate parent share at least one allele in common with offspring? - If not, exclude candidate Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Offspring A C A T T A T C T A A Candidate parents C A C C T T T T A G A A T T A A T T A G C A C C A T T T G G C C C C T T C T G G C C C T A T C C A A

26 Parentage assignment of Offspring B Offspring B C A C C T T T T A G Candidate parents 1 Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 C A C C T T T T A G Locus 6 A A C C A A T T A A T T A G C A C C A T T T G G C C C C T T C T G G C C C T A T C C A A A C A A Need more SNP loci for higher resolving power!

27 Parentage assignment of Offspring C Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Offspring C A A T C A T T T A A Candidate parents C A C C T T T T A G A A T T A A T T A G C A C C A T T T G G C C C C T T C T G G C C C T A T C C A A

28 Parentage assignment of Offspring D Offspring D C C C T T T C C A G Candidate parents C A C C T T T T A G A A T T A A T T A G C A C C A T T T G G C C C C T T C T G G C C C T A T C C A A

29 Parentage assignment of Offspring E Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Offspring E A A T C T T C C A G Candidate parents C A C C T T T T A G A A T T A A T T A G C A C C A T T T G G C C C C T T C T G G C C C T A T C C A A Unsampled parent, or parent poor quality DNA (not included in evaluation)

30 Parentage Exercise

31 Fishery Chinook salmon used for lab exercise Lower Granite Dam Bonneville Dam Adult fish Basin-wide Stock Composition of Lower Mainstem Harvest (CH)

32 True composition of test mixture file based on CWT data. Genetic samples for lab exercise

33 True composition of test mixture file based on CWT data. Genetic samples for lab exercise PBT baseline Snake River Basin Spring Chinook 2008-present

34 Genetic samples for lab exercise True composition of test mixture file based on CWT data. Reporting Group Hatchery Source N W_Cascade_sp Cowlitz R. 3 Lewis R. 2 5 Willamette_sp Clackamas R. early 2 McKenzie Hatchery 3 Santiam North Fork 3 Willamette R. Mid. Fork 2 10 Klickitat_sp Klickitat Hatchery 5 5 Deschutes_R_sp Deschutes R. 3 Warm Springs R. 2 5 Yakima_sp Yakima R. Upper 4 4 Upper_Columbia_R_sp Chiwawa R. 3 Leavenworth Hatchery 3 Ltl. White Salmon NFH 3 Umatilla R RapidR_Clearwater_sp Clearwater Hatchery 9 Dworshak Natl. Hatchery 5 Kooskia Natl. Hatchery 7 Lookingglass Hatchery 5 Nez Perce Tribal H. 6 Rapid River Hatchery 6 38 SF_Salmon_sp McCall Hatchery 5 5 Upper_Salmon_sp Sawtooth Hatchery 5 5 Grand Total 88

35 Genetic samples for lab exercise 8 mystery samples!! Different species Degraded sample Out of basin

36 Parent and offspring samples SY , parent sample ~18,000 Chinook sample from fishery: 88 samples (48 with CWT in Snake, collected ) B Lookingglass Tucannon Lostine Grand Ronde Catherine Cr Imnaha Dworshak SF Clearwater Rapid R NPTH SF Salmon Powell Johnson Cr Pahsimeroi Sawtooth

37 Parentage assignment expectations 48 with CWT in Snake River 43 BY 2008 or BY Clearwater Dworshak Rapid R NPTH BY and 4 yr. old BY yr. old Lookingglass SF Salmon 5 Sawtooth 5

38 Compile input file for parentage analysis program (SNPPIT) Parent dataset: 2008 and 2009 PBT parental baseline, n~18,000 Offspring dataset: 88 samples that you genotyped (48 CWT Snake) SNPPIT approach: exclusion of trios based on Mendelian incompatibility:

39 SNPPIT - parentage analysis program Approach: exclusion of trios based on Mendelian incompatibility 1.) Exclude individual candidate parents offspring AA?? 10k candidate parents AA AC CC CC AC 2.) Compare non-excluded candidates as parent pairs & exclude offspring AA locus 2 TT AA AC locus 2 locus 2 TT X AC GG GT X AC GG TRIO! AC GT X AA TT

40 SNNPIT output of results Key parameters for accepting parentage assignment: FDR, p value, LOD, most likely relationship, # mismatching loci

41 Summary of parentage results 40 of the expected 43, assigned to parents 3 offspring that should have assigned did not have parents in the sample 2 Lookingglass (likely unsampled parents, missed some broodstock) 1 Sawtooth (matched 1 parent, cross record-1 parent missing data) No false assignments (i.e., 45 samples that should not have assigned based on CWT origin or collection yr expectedly did not assign to parents in our baseline) 39 PBT assignments concordant with CWT 1 CWT = Sawtooth, but PBT = Dworshak FDR and p value < , likelihood supports trio, zero mismatches