With 99.99% probability, you ARE the father - CRITFC · 2012/12/7 · Broodstock sampled 5,151 5,761 5,282 5,931 Genotyped 5,070 5,636 5,198 5,765 “Tagging” Rate of Offspring

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

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 Candidate parents

??

2

3

4

5

Parentage analysis, exclusion approach

Possible outcomes:

1.) All, but one parent-pair, are excluded Assign parentage

2.) More than one parent cannot be excluded

Need more loci or use another approach (i.e., likelihood)

3.) All potential parents are excluded Un-sampled parents

What do we need for parentage analysis?

Powerful set of loci- simulations to evaluate power- empirical test using known parent-offspring

Ass

ign

men

t ra

te t

o

corr

ect

par

ent(

s)

# microsatellite loci

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Marker type # loci needed Variability

Microsatellites Usually few

n ~ 12-15

High

many alleles

SNPs Many

n > 60

Low

bi-allelic

Genetic markers


Sampling of most potential parents- trap fish as they return to spawn- trap below majority of spawninghabitat

- carcass surveys can recoverun-sampled fish

Sampling a parent genetically ‘tags’ ALL of that individual’s offspring

Crossed in a hatchery or mated in the wild

Representative sample of offspring- Sample as adults or juveniles


http://fish101-spring2011.community.uaf.edu/page/5/

http://gorgenewscenter.com/57/

Example project using parentage analysis

Parentage based tagging (SNPs)

What is parentage based tagging (PBT)?

• 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

• Non-lethal sample to recover tag from offspring

Genetic tags (PBT)

Hatchery broodstock

offspring

AdultJuvenile

X

Stock contribution to various fisheries Survival trends Escapement estimatesOrigin of strays and kelts

• Sample all hatchery broodstock:~5,000 steelhead/yr~9,000 Chinook salmon/yr

• Parental sampling complete for 2008 -2011, 2012 ongoing

Snake River basin PBT

• Permanently “tag” ~20 million smolts/yr

• All hatcheries record spawn dates and gender (many provide lengths and spawn cross)

BY2008

or

Sampling PBT-tagged offspring

Age Hatchery Parents

Sawtooth Hatchery

adultsjuveniles

Non-lethal tissue sample

Assignment accuracy of offspring to known broodstock parents

We only need 1 96-SNP panel to assign accurate parentage!

• High number of juveniles assigned (97.3%)• 100% accuracy to stock

Spring/Summer Chinook

Spawn Year

2008 2009 2010 2011

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

Steelhead

Spawn Year

2008 2009 2010 2011

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

Bendbulletin.com

Sampled in 2011 at Sherars Falls, Deschutes River: 446 ad-clipped steelhead 278 ad-intact steelhead

Deschutes River

Snake River basin

*Collaboration with Rod French, ODFW

Example application of PBT:

Origin of hatchery strays?

SherarsFalls

Ad-clipped Steelhead354 / 446 = 79.4% Assigned

Dworshak, n = 27

Oxbow, n = 95

E. Fk. Salmon, n = 2

Sawtooth, n = 66

GrandeRonde, n = 4

Squaw Creek, n = 7

Wallowa, n = 48

Pahsimeroi, n = 105


SherarsFalls

Ad-intact Hatchery Origin Fish32 / 278 = 11.5% Assigned

Sawtooth, n = 26

Wallowa, n = 1

Pahsimeroi, n = 5


Dworshak

Oxbow

Sawtooth

Grande Ronde

Wallowa

PahsimeroiM

B

1,500 adipose-clipped steelhead collected in 2011 Zone 6 fishery

http://wildsteelheadcoalition.org

2008 and 2009 Snake River PBT baselines

Lyons Ferry

Example application of PBT:

Origin and age of harvest fishery?

Zone 6 Fishery

Of the 1,482 steelhead genotyped, 1,117 (75%) assigned to two parents

Zone 6 2011, n=1,117

Dworshak

Pahsimeroi

Oxbow

Sawtooth

Wallowa

Lyons Ferry

46%

19%

14%

10%

7%5%

10% (n=153) estimated mid

or upper Columbia River origin

76%

8%

7%

7%

2% 0%

BY2008 (2-ocean), n=633

*Wallowa unsampled SY2008

BY2009 (1-ocean), n=484

6%

33%

23%

13%

12%

12%

Origin and age of harvest fishery?

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

Sample ID Spawn Date Gender Cross 1 Cross 2 LengthPAH12-0001 4/10/2012 M 600PAH12-0002 4/10/2012 M 730

PAH12-0003 4/10/2012 F PAH12-0001 PAH12-0007 640PAH12-0004 4/10/2012 F PAH12-0009 640

PAH12-0005 4/14/2012 F PAH12-0002 PAH12-0007 620PAH12-0006 4/14/2012 F PAH12-0001 600

PAH12-0007 4/14/2012 M 590PAH12-0008 4/20/2012 F PAH12-0007 620PAH12-0009 4/20/2012 M 620

REQUIRED information OPTIONAL information

Winthrop

Wells

EastbankLeavenworth

Cle Elum

Priest Rapids

ProsserMarion Drain

Klickitat

Spring Cr.

Willard

Little WhiteParkdale

CarsonBonneville

Umatilla

Lyons Ferry

Lookingglass

Catherine Cr.

Lostine

Imnaha

Nez PerceDworshak Powell

Rapid R.

McCall, JC

SawtoothPahsimeroi

Warm SpringsRound Butte

Methow

Goal 2012-ongoing: Chinook salmon, PBT hatcheries

WDFW

USFWS

ODFW

Tribes or co-manager/tribes

CRITFC/IDFG, SY 2008-present

Winthrop

Wells

Eastbank

Ringold Springs

Umatilla

Lyons Ferry

Round Butte

Parkdale

Skamania

Wallowa Little Sheep

Oxbow

Sawtooth

Pahsimeroi

WDFW

USFWS

ODFW

Tribes or co-manager/tribes

CRITFC/IDFG, SY 2008-present

Dworshak

Goal 2012-ongoing: Steelhead, PBT hatcheries

Parentage Example

Finding Nemo’s Parents: A Parentage Example

Match at least 1 allele at every locus? If not, exclude parent

Parentage example

1

2

3

5

4

5 candidate parents sampled

5 juveniles sampled

Genotype 11 samples for 5 SNPs

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

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

1

2

3

4

5

Offspring B C A C C T T T T 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


1

2

3

4

5

Parentage assignment of Offspring B

Locus 6

C C

A A

A C

A A

Need more SNP loci for higher resolving power !

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


1

2

3

4

5

Parentage assignment of Offspring C

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

1

2

3

4

5

Parentage assignment of Offspring D

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


1

2

3

4

5

Parentage assignment of Offspring E

Unsampled parent, or parent poor quality DNA (not included in evaluation)

Parentage Exercise

Fishery

BonnevilleDam

LowerGranite

Dam

Adult fish

Basin-wide Stock Composition of Lower Mainstem Harvest (CH)

Chinook salmon used for lab exercise

Genetic samples for lab exercise

True composition of test

mixture file based on CWT

data.




data.

PBT baseline

Snake River Basin

Spring Chinook

2008-present




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. 2

11

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


8 mystery samples!!Different

species

Degraded

sample

Out of basin

Parent and offspring samples

B

Pahsimeroi

Sawtooth

Johnson Cr

DworshakSF Clearwater

Tucannon

Lookingglass

Catherine Cr

Lostine

SF Salmon

Grand Ronde

SY 2008-2009, parent sample ~18,000

Rapid R NPTH

Powell

Imnaha

Chinook sample from fishery:88 samples (48 with CWT in Snake, collected 2010-2012)

Parentage assignment expectations

08 09 10 12 13

34

5

11 14

3 4 5

BY 2008 - 3 and 4 yr. oldBY 2009 - 3 yr. old

Sawtooth

Clearwater

Lookingglass

SF Salmon

Rapid R

65

5

NPTH

65

5

48 with CWT in Snake River 43 BY 2008 or 2009 5 BY 2007

Dworshak

11

Compile input file for parentage analysis program (SNPPIT)

Parent dataset: 2008 and 2009 PBT parental baseline, n~18,000Offspring dataset: 88 samples that you genotyped (48 CWT Snake)

SNPPIT approach: exclusion of trios based on Mendelian incompatibility:

2.) Compare non-excluded candidates as parent pairs & exclude

offspring X

X

X

AA

AC

AC

AA

AC

AA

AC

SNPPIT - parentage analysis programApproach: exclusion of trios based on Mendelian incompatibility

offspring10k candidate parents

??

1.) Exclude individual candidate parents

AACC

AAAC

CC

AC

GTTT

TT GG

GG

TTGT

locus 2 locus 2

locus 2

TRIO!

SNNPIT output of results

Key parameters for accepting parentage assignment:FDR, p value, LOD, most likely relationship, # mismatching loci

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)

40 of the expected 43, assigned to parents

Summary of parentage results

39 PBT assignments concordant with CWT 1 CWT = Sawtooth, but PBT = Dworshak

FDR and p value < 0.0001, likelihood supports trio, zero mismatches

Documents

With 99.99% probability, you ARE the father - CRITFC · 2012/12/7 · Broodstock sampled 5,151 5,761 5,282 5,931 Genotyped 5,070 5,636 5,198 5,765 “Tagging” Rate of Offspring