Brief Description of Anticipated Work:
The objective of the proposed effort for the Base option is to:
Objective 1:
Conduct the new Genotyping in Thousands by Sequencing (GT-Seq, Campbell et al.

2015) analysis on up to 1,000 lower Missouri River samples annually during the 4-year study

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period (FY 2022-2025), which will contribute to the development of new genetic markers.

The objectives of the proposed effort for the 4 option years include using the previously established genetic markers on up to 15,000 samples annually (FY 2023-2026) to:
Objective 1:
Screen Missouri River pallid sturgeon broodstock to ensure genetic purity for the pallid sturgeon propagation and population augmentation project.

Objective 2:
Efficiently identify pallid from shovelnose and hybrid sturgeon based on genetic samples provided.

Objective 3:
Research and develop a spawning relatedness matrix to help hatcheries avoid crosses between related individuals utilizing results from Objective 1 and genotypes of males from the cryopreserved repository.

Objective 4:
Use genetic identification to assess natural reproduction of pallid sturgeon in the LMOR.

The work associated with the Base option for this project includes:
1) Use the GT-Seq genotype analysis (Campbell et al.

2015) and ddRAD sequences to develop new and more powerful markers for the identification of pallid sturgeon, shovelnose sturgeon and hybrids.

Once the markers are developed, use a variety of analytical approaches, such as Discriminate Analysis using Principle Components (DAPC, Jombart et al.

2010), STRUCTURE (Pritchard et al.

2000), and NewHybrids (Anderson and Thompson 2002) to provide better resolution of the species status of each individual and identify more reliable criteria for identifying pure pallid sturgeon.

The successful applicant will have the genetic baselines to reliably verify purity of pallid sturgeon based on genetic analysis and have access to the equipment necessary to conduct the new GT-Seq genotype analysis.

The work associated with the 4 option years for this project includes:
1) Use the previously established genetic techniques, including Single Nucleotide Polymorphism (SNP) and microsatellite markers, to identify juvenile pallid sturgeon from shovelnose sturgeon and hybrids in samples collected from the Central Lowlands and Interior Highlands Management Units.

Pallid sturgeon monitoring crews sample small sturgeon from the Missouri and Mississippi Rivers; some of these sturgeon could be wild pallid sturgeon and would represent natural reproduction.

These results will provide the analysis and assessment to determine if small Acipenseriformes species sampled by these crews represent recruitment of wild pallid sturgeon.

2) Analyze the genotypes of pallid sturgeon broodstock against established baselines of wild pallid and shovelnose sturgeon from the same river regions from which the broodstock were collected using three analytical tools:
STRUCTURE (Pritchard et al., 2000), WhichRun (Banks, Eichert, 2000), and NewHybrids (Anderson, Thompson, 2002).

Identify each fish as pallid sturgeon, shovelnose sturgeon, or hybrid (pallid x shovelnose) based on the STRUCTURE-generated Q-value, WhichRun LOD, and NewHybrids scores.

This will be done to ensure that only pure pallid sturgeon, based on the available genetic baselines, are utilized for artificial spawning and propagation efforts.

Therefore, the progeny produced and stocked will follow genetic recommendations outlined by the Pallid Sturgeon Recovery Team and supported by the U. S. Fish and Wildlife Service relative to the collection and stocking of locally collected specimen for population augmentation.

Monitoring and detecting successful reproduction of pallid sturgeon in the Missouri River is a critical factor for assessing the Corps’ management actions under the Biological Opinion and to identify critical threats to the population.