I. Project Title: SNP Panels for Chinook Salmon. II. Project Number: PCSRF Objective: RM&E

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1 Alaska Sustainable Salmon Fund Statement of Work Revision #1: Extend end date; revise SOW/budget; reduce match to required amount (eliminate ADF&G over-match) I. Project Title: SNP Panels for Chinook Salmon II. Project Number: PCSRF Objective: RM&E III. Principal Investigator Lisa W. Seeb, Research Professor School of Aquatic and Fishery Sciences (SAFS) University of Washington (UW) 1122 NE Boat Street; Box Seattle, WA Phone: (206) ; William D. Templin, Fisheries Scientist Gene Conservation Laboratory (GCL) ADF&G, Commercial Fisheries Division 333 Raspberry Road Anchorage, Alaska Phone: (907) ; IV. Amended Project Period: 9/1/10 12/31/13 V. Project Description 1. Synopsis This project will screen 288 single nucleotide polymorphisms (SNPs) to improve genetic stock identification in the Arctic-Yukon-Kuskokwim (AYK) region. This large SNP set will enable the use of linked SNPs, an approach that reveals clear population differences not otherwise apparent. Additionally, this large SNP set will enable individual panels that may be enriched with high-resolution outlier SNPs that have discriminating power specific to problem identification within Norton Sound, the Yukon, Kuskokwim, and Nushagak River drainages, as well as in the Bering Sea bycatch. 2. Introduction The fine-scale genetic structure and migration corridors of Chinook salmon spawning along the Bering Sea coast of Western Alaska are poorly understood. Traditional genetic stock identification techniques do not accurately identify individual drainages within the complex. Chinook salmon represent a critically important component of the Western Alaskan ecosystem and serve as an important subsistence and commercial resource for residents throughout the region. The inability to differentiate drainages has resulted in considerable uncertainty in understanding the migration of the Western Alaska Coastal group in the Bering Sea. Migrating salmon, especially those originating from Western Alaska and the Yukon River, are caught as SNP Panels for Chinook Salmon Page 1 of 11 1/30/2013

2 bycatch in the Bering Sea walleye pollock fishery. Interception in other mixed stock fisheries is uncertain. Information on the interception on a scale finer than the large Western Alaska Coastal group will be highly advantageous. Recent advances in molecular technology provide almost unlimited sequence information from any organism, allowing the development of thousands of SNPs as genetic markers for population identification. The availability of large numbers of SNPs allows the identification of selected markers with particularly high levels of differentiation, which can be used for specific applications such as compositional analyses of closely-related stocks and assignment tests. This project relies on and extends work currently funded by AKSSF project High Resolution SNPs for Chinook. That project includes three objectives and is a collaboration between the University of Washington (UW), School of Aquatic and Fishery Sciences (SAFS), and the ADF&G Gene Conservation Laboratory (GCL). Objective 1 uses high-throughput DNA sequencing to discover SNPs for mixed stock analysis, Objective 2 identifies a panel of 96 high-resolution SNPs for Western Alaska, and Objective 3 genotypes 2,400 individuals for the 96 high-resolution SNPs. These individuals will be chosen by ADF&G and will originate from previously archived material and newly sampled collections. This project also relies on and extends research on a project funded by the Chinook Technical Committee (CTC) of the Pacific Salmon Commission (PSC) entitled Moving GSI into the Next Decade: SNP Coordination for Pacific Salmon Treaty Fisheries (CTC SNP Coordination). That project is a collaboration between Washington Department of Fish and Wildlife (WDFW), SAFS, and ADF&G; its first objective is to coordinate and develop standardized SNP panels among North American laboratories. The second objective is to evaluate an initial pool of approximately 288 newly-available SNPs and select 192 for screening in 40 core populations which are heavily weighted towards the larger PSC area. The final objective is to conduct a power analysis to identify high resolution panels for PSC fisheries. This project extends the CTC SNP Coordination project to Western Alaska by genotyping the 2,400 individuals identified in AKSSF project for the 192 SNPs identified in the CTC project. This will allow a full evaluation of the 288 SNPs for Western Alaska populations to provide the most informative panels for Western Alaska systems. NOAA Fisheries, Auke Bay Laboratory, is currently using the existing SNP baseline developed by scientists at ADF&G and the UW to investigate the origin of the pollock bycatch in the Bering Sea. Markers developed in this project will be incorporated into the panel to improve accuracy and precision of the estimates. SNP Panels for Chinook Salmon Page 2 of 11 1/30/2013

3 SNPs for Chinook salmon are independently being discovered and shared by the Columbia River Intertribal Fish Commission and the NOAA Fisheries Southwest Fisheries Science Center. WDFW has numerous collaborations with the UW for SNP discovery. 3. AKSSF Objective AYK: 2B-1 VI. Objectives 1. Project Objectives Objective 1: Genotype 2,400 individuals from Western Alaska for the 192 additional SNPs chosen by the CTC SNP Coordination project. Objective 2: Merge the genotypes from the 2,400 individuals for 192 SNPs with the genotypes derived from the 96 next-generation SNPs derived in AKSSF project Objective 3: Evaluate the results from 288 SNPs to develop panels of 96 highresolution SNPs optimized for specific drainages and also for the Bering Sea bycatch for compositional analysis and individual assignment. 2. Methods SNP Genotyping Genomic DNA will be extracted using a DNeasy 96 Tissue Kit by QIAGEN. DNA will be extracted at the SAFS and ADF&G laboratories with replicate tissues and/or DNA stored at each facility and coordinated among collaborating projects. Genotyping of the markers will be performed using the BioMark Dynamic Array described above. A set of 2,400 individuals will be chosen from available samples after collaboration with the ADF&G AYK regional staff. This set will include the Western Alaska subset of the samples used in the CTC SNP Coordination project which have been identified as: Andreafsky River (Lower Yukon), Henshaw Creek (Middle Yukon), Mayo River (Upper Yukon), Kogrukluk River (Lower Kuskokwim), Stuyahok River (Bristol Bay), and Steelhead Creek (North Alaska Peninsula). Genotypes will be entered into the Progeny database (Progeny Software, South Bend, Indiana) installed at SAFS and the Oracle database, LOKI, installed at ADF&G. This project will conduct quality control measures to ensure that all assays and procedures are transferred to ADF&G from SAFS. These will include: 1) staff exchanges, 2) replicate analysis of 96 samples in the SAFS and ADF&G laboratories to verify data standardization, and 3) reanalysis of 8% of each collection for all markers to ensure that genotypes are reproducible and to identify laboratory errors and measure rates of inconsistencies during repeated analyses. Final genotypes for all individuals for all markers and appropriate metadata will be maintained at each facility. Use of the Progeny and LOKI database will ensure that all data are correctly matched across individuals and across the various projects. SNP Evaluation The SNP results will be evaluated based on both laboratory and statistical properties. Laboratory evaluation will include properties such as scatterplot spread and success SNP Panels for Chinook Salmon Page 3 of 11 1/30/2013

4 rate of assays. Descriptive population genetics statistics will include allele frequency distributions, departure from Hardy-Weinberg expectation, and genotypic linkage disequilibrium using GenepopV4. Participants will then conduct several tests to detect outlier loci (those exhibiting high values of F ST ). The methods rely on the property that loci influenced by directional selection will show a larger genetic differentiation than neutral loci while loci subject to balancing selection will show a lower genetic differentiation. This project will also evaluate pairs of linked SNPs for improved resolution. The two linked loci can be combined using statistical methods into a single locus by phasing the alleles into haplotypes for additional fine-scale resolution. These types of analysis can also provide valuable information when looking across major evolutionary lineages on a range-wide basis. The power and resolution for genetic stock identification will be evaluated in two ways. First, participants will evaluate discriminatory power for genetic mixture and individual assignment analyses using variations on the multilocus cross-validation method. Known individuals removed from the baseline will be used to construct proof tests. This will allow evaluation of both compositional and individual assignment. The laboratory and statistical results will be used to design final panels of 96 SNPs composed of neutral and selective markers to assess population structure. It is anticipated that multiple panels of SNPs each optimized for a particular region or mixed stock fishery including the Bering Sea bycatch will be developed. Prior to commencement of project activity, PI will obtain ADF&G Fish Collection, Fish Resource, Fish Transport, and/or other required permits, as appropriate. VII. Benefits The results of this project will significantly advance the understanding of geographic and temporal diversity of AYK region Chinook salmon and the development of a database for use in the Bering Sea bycatch. These data will be integral in ensuring the success of research aimed at high-seas migration and interception and improving and maintaining effective, biologically sound salmon management systems for Chinook stocks that support subsistence fisheries. VIII. Products, Milestones, and Timelines Revision #1 moves the project end date from 12/31/12 to 12/31/13 and modifies the timeline accordingly. October July 2011: Obtain DNA sequence information for 192 SNPs from colleague labs in California, Idaho, and Washington. June-November 2012: Screen 2,400 individuals (from AKSSF project 44515) for 192 additional SNPs. December 2012: Conduct quality control reruns for 8% of fish for all SNPs. February-August 30, 2013: Analyze data and write project report. December 31, 2013: Provide final report to AKSSF. SNP information from this study will be made available to the consortium of state, federal, and other agencies that are cooperating on Pacific Rim baseline dataset SNP Panels for Chinook Salmon Page 4 of 11 1/30/2013

5 developments for parental based tagging, migration, and harvest assessment studies. These agencies include Idaho Fish and Game, WDFW, NOAA Fisheries (Northwest, Southwest, and Auke Bay centers), US Fish and Wildlife (Abernathy), Columbia River Intertribal Fish Commission, Oregon State University, and Department of Fisheries and Oceans Canada. PI will submit PCSRF performance metrics, semiannual, and project completion reports according to the AKSSF schedule, as well as copies of any other report/product/ deliverable produced with this funding. All invoices will be submitted for payment within 30 days of the project end date in accordance with the current AKSSF invoicing P&P. Any report or product distributed as a result of this funding will include the following language: This [report/video/website] was prepared by [recipient/author name] under award NA09NMF from the National Oceanic and Atmospheric Administration, U.S. Department of Commerce, administered by the Alaska Department of Fish and Game. The statements, findings, conclusions, and recommendations are those of the author(s) and do not necessarily reflect the views of the National Oceanic and Atmospheric Administration, the U.S. Department of Commerce, or the Alaska Department of Fish and Game. IX. Partners NOAA Fisheries Auke Bay Laboratory, Columbia River Intertribal Fish Commission, NOAA Fisheries Southwest Fisheries Science Center, WDFW X. Project Budget Original Budget FY11 FY12 Total 100 Personnel $61,404 $53,988 $115, Travel $5,300 $5,300 $10, Contractual $9,978 $9,978 $19, Supplies $9,000 $9,000 $18, Equipment $0 $0 $0 Subtotal $85,682 $78,266 $163,948 MTDC $75,704 $68,288 $143, Indirect 26% $19,683 $17,755 $37,438 Total $105,365 $96,021 $201,386 ADF&G 3% $6,042 Total $207,428 SNP Panels for Chinook Salmon Page 5 of 11 1/30/2013

6 Revision #1 Budget FY11 FY12 FY13 FY14 Total 100 Personnel $0 $6,780 $62,964 $37,778 $107, Travel $0 $1,743 $3,502 $2,000 $7, Contractual $0 $5,049 $36,112 $4,944 $46, Supplies $0 $0 $0 $0 $0 500 Equipment $0 $0 $0 $0 $0 Subtotal $0 $13,572 $102,578 $44,722 $160,872 MTDC $0 $8,523 $102,578 $44,722 $155, Indirect 26% $0 $2,216 $26,670 $11,628 $40,514 Total $0 $15,788 $129,248 $56,350 $201,386 ADF&G 3% $0 $474 $3,877 $1,691 $6,042 Total $0 $16,261 $133,126 $58,041 $207,428 Original Budget Narrative: Line 100: Personnel ($115,392) J. Seeb and L. Seeb are in charge of design, writing, and implementation. The graduate student is responsible for laboratory work and statistics and will write a Master s thesis based upon this project. Personnel FY11 FY12 Monthly Months Fraction Amount Months Fraction Amount Total Salary L Seeb $10, $18, $24,000 $42,000 J. Seeb $10, $18, $6,000 $24,000 Grad student $1, $14, $14,976 $29,952 Total Salary $50,976 $44,976 $95,952 Benefits L. Seeb 23.6% $4,248 $5,664 $9,912 J. Seeb 23.6% $4,248 $1,416 $5,664 Grad student 12.9% $1,932 $1,932 $3,864 Total benefits $10,428 $9,012 $19,440 Total $61,404 $53,988 $115,392 Line 200: Travel ($10,600) Each year the graduate student will travel to Anchorage to work with the ADF&G laboratory and/or Dillingham to perform lab work at a UW field station for 30 days. UW camp fees are $150/day. This figure also covers meals and lodging for work at the ADF&G laboratory in Anchorage. Travel FY11 FY12 Total Seattle/Dillingham/Anchorage airfare $800 $800 $1,600 Camp fees: 30 $4,500 $4,500 $9,000 SNP Panels for Chinook Salmon Page 6 of 11 1/30/2013

7 $150/day Total $5,300 $5,300 $10,600 Line 300: Contractual ($19,956) FY11 & FY 12 Graduate student tuition: $9,978/year (exempt from indirect cost recovery) Line 400: Supplies ($18,000) FY11 & FY12 BioMark chips for genotyping: $9,000/year Line 600: Indirect ($37,438) UW s indirect cost recovery rate for this project is 26% of Modified Total Direct Costs (MTDC). FY11: $19,683 FY12: $17,755 Revision #1 Budget Narrative: Line 100: Personnel ($107,522) J. Seeb and L. Seeb are in charge of design, writing, and implementation. The graduate student position is responsible for laboratory work and statistics and will write a Master s thesis based upon this project. Revision #1 reduces personnel budget and incorporates detail for the extended project period. Personnel FY11 FY12 FY13 FY14 Monthl y M o Fracti on Amo unt M o Frac tion Amoun t M o Frac tion Amoun t M o Fra ctio n Amount Total Salary L Seeb $9, $ $ $29, $19,800 $49,500 J. Seeb $9, $ $ $19, $9,900 $29,700 Grad student $1, $0 3 1 $5, $0 0 1 $0 $5,840 Total Salary $0 $5,840 $49,500 $29,700 $85,040 Benefits L. Seeb 27.20% $0 $0 $8,078 $5,385 $13,463 J. Seeb 27.20% $0 $0 $5,386 $2,693 $8,079 Grad student 16.10% $0 $940 $0 $0 $940 Total benefits $0 $940 $13,464 $8,078 $22,482 Total $0 $6,780 $62,964 $37,778 $107,522 SNP Panels for Chinook Salmon Page 7 of 11 1/30/2013

8 Line 200: Travel ($7,245) Project activity requires graduate student and PI travel from Washington to Anchorage to work with the ADF&G laboratory and/or Dillingham to perform lab work at a UW field station. Budget also includes meals and lodging for work at the ADF&G laboratory in Anchorage and camp fees at the UW field station. Revision #1 reduces travel budget and incorporates detail for the extended project period. Travel FY11 FY12 FY13 FY14 Total Seattle/Dillingham/Anchorage airfare $0 $1,168 $1,800 $1,200 $4,168 Meals and Lodging, UW Camp fees $0 $575 $1,702 $800 $3,077 Total $0 $1,743 $3,502 $2,000 $7,245 Line 300: Contractual ($46,105) (Increased with Revision #1) FY12 Graduate student tuition: $5,049 (exempt from indirect cost recovery) UW Cost Center charges for all supplies and analyses including BioMark chips, plastic consumables, TaqMan assays reagents and primers, and Qiagen extraction kits for 2,400 samples for 192 SNP Assays: $41,056. FY13: $36,112 FY14: $4,944 Line 400: Supplies (Reduced to $0 with Revision #1) With Revision #1, because supplies are now procured via contractual billing to the UW Cost Center, these costs were moved to line 300. Line 600: Indirect ($40,514) UW s indirect cost recovery rate for this project is 26% of Modified Total Direct Costs (MTDC) consisting of all salaries and wages, fringe benefits, materials and supplies, services, travel, and subgrants up to the first $25,000. Equipment, capital expenditures, scholarships, fellowships, the portion of each subgrant and subcontract in excess of $25,000, and other costs, are excluded from MTDC. FY11: $0 FY12: $2,216 FY13: $26,670 FY14: $11,628 Budget adjustments between line items are allowed for amounts equal to or less than 10% of the total award without prior AKSSF approval; any amount above 10% requires prior AKSSF approval. All costs in lines must represent actual cash expenditures. All invoices shall be adequately documented with both documentation of expense and proof of payment for both direct and match expenses. Examples of expense documentation include copies of invoices, pay stubs, and receipts. Examples of proof of payment documentation include copies of checks, check numbers, and credit card SNP Panels for Chinook Salmon Page 8 of 11 1/30/2013

9 receipts. Match documentation for expenses equal to or greater than 33% of the reimbursable expenses must be submitted with each invoice. All invoices shall be submitted for payment within 30 days of the project end date in accordance with the current AKSSF invoicing P&P. Invoices submitted after that deadline will not be reimbursed. Expenses incurred after the project end date will not be reimbursed. XI. Match Budget Revision #1 eliminates match in excess of the required amount (overmatch). Original Match Summary (33%) FY11 FY12 Total 100 Personnel $35,706 $26,688 $62, Travel $0 $0 $0 300 Contractual $0 $0 $0 400 Supplies $0 $0 $0 500 Equipment $0 $0 $0 Subtotal $35,706 $26,688 $62, Indirect 26% $3,214 $2,892 $6,106 Total $38,920 $29,580 $68,500* *Match budget table shows more match than is required for this project. Total required match: $66,458 (unchanged with Revision #1) UW match: $29,590 ADF&G match: $36,868 Revision #1 Match Summary (33%) FY11 FY12 FY13 FY14 Total 100 Personnel $31,107 $23,309 $5,683 $0 $60, Travel $0 $0 $0 $0 $0 300 Contractual $0 $0 $0 $0 $0 400 Supplies $0 $0 $253 $0 $ Equipment $0 $0 $0 $0 $0 Subtotal $31,107 $23,309 $5,936 $0 $60, Indirect 26% $1,720 $2,909 $1,477 $0 $6,106 Total $32,827 $26,218 $7,413 $0 $66,458 UW Match Budget Original UW Match FY11 FY12 Total 100 Personnel $12,360 $11,124 $23, Travel $0 $0 $0 300 Contractual $0 $0 $0 400 Supplies $0 $0 $0 500 Equipment $0 $0 $0 SNP Panels for Chinook Salmon Page 9 of 11 1/30/2013

10 Subtotal $12,360 $11,124 $23, % $3,214 $2,892 $6,106 Total $15,574 $14,016 $29,590 Revision #1 UW Match FY11 FY12 FY13 FY14 Total 100 Personnel $6,613 $11,188 $5,683 $0 $23, Travel $0 $0 $0 $0 $0 300 Contractual $0 $0 $0 $0 $0 400 Supplies $0 $0 $0 $0 $0 500 Equipment $0 $0 $0 $0 $0 Subtotal $6,613 $11,188 $5,683 $0 $23, % $1,720 $2,909 $1,477 $0 $6,106 Total $8,333 $14,097 $7,160 $0 $29,590 Original UW Match Budget Narrative: Line 100: Personnel J. Seeb and L. Seeb are in charge of design, writing, and implementation. Line 600: Indirect UW s indirect cost recovery rate for this project is 26%. Revision #1 UW Match Budget Narrative: Line 100: Personnel J. Seeb and L. Seeb are in charge of design, writing, and implementation. Line 600: Indirect UW s indirect cost recovery rate for this project is 26% of Modified Total Direct Costs (MTDC) consisting of all salaries and wages, fringe benefits, materials and supplies, services, travel, and subgrants up to the first $25,000. Equipment, capital expenditures, scholarships, fellowships, the portion of each subgrant and subcontract in excess of $25,000, and other costs, are excluded from MTDC. ADF&G Match Budget Original ADF&G Match FY11 FY12 Total 100 Personnel $23,346 $15,564 $38, Travel $0 $0 $0 300 Contractual $0 $0 $0 400 Supplies $0 $0 $0 500 Equipment $0 $0 $0 Total $23,346 $15,564 $38,910* *Match budget table shows more match than is required for this project. SNP Panels for Chinook Salmon Page 10 of 11 1/30/2013

11 Revision #1 ADF&G Match FY11 FY12 FY13 FY14 Total 100 Personnel $24,494 $12,121 $0 $0 $36, Travel $0 $0 $0 $0 $0 300 Contractual $0 $0 $0 $0 $0 400 Supplies $0 $0 $253 $0 $ Equipment $0 $0 $0 $0 $0 Total $24,494 $12,121 $253 $0 $36,868 Over-match eliminated with Revision #1. Original ADF&G Match Budget Narrative: Bill Templin has assigned two staff members to support this collaboration. Sara Gilk and Nick Decovich will provide help with sample selection, locus selection, and data assimilation into the ADF&G data sets. They will both share credits in reporting at project end. FY11 FY12 Monthly* Months Fraction Amount Months Fraction Amount Total S. Gilk $7, $7, $7,782 $15,564 N. Decovich $7, $15, $7,782 $23,346 Total $23,346 $15,564 $38,910 * Salary and benefits Revision #1 ADF&G Match Budget Narrative: Line 100: Personnel Salaries and benefits for Sara Gilk and Nick Decovich at the rates listed above. FY11: $24,494 FY12: $12,121. Line 400: Supplies FY 13: $253 SNP Panels for Chinook Salmon Page 11 of 11 1/30/2013