## 2 Materials and Methods
### Molecular Methods  
DNA was isolated from fin-clips using the DNEasy kit (Qiagen, Germantown, MD, USA). The _Svi17_ and _Svi33_ microsatellite loci [10], at which markers for upper New River loci were established by [PERSON] et al. [9], were screened. Each PCR reaction contained 4.5 \(\upmu\)L of 5X Promega reaction buffer, 2.25 \(\upmu\)L of 25 mM MgCl\({}_{2}\), 2.5 mM dNTPs (Lucigen, Middleton, WI, USA), 0.18 U/\(\upmu\)LoGTaQ DNA polymerase (Promega, Madison, WI, USA), 5 mM forward primer and 5 mM reverse primer, 2 \(\upmu\)L of sample DNA (25-200 ng/\(\upmu\)L), and DI water to 17 mL. PCR reactions were performed in a T-100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The thermocycling protocol consisted of 35 cycles of the following: denaturation for 30 s at 94 \({}^{\circ}\)C, annealing at a primer-specific temperature (Table 1) for 30 s, and elongation for 30 s at 72 \({}^{\circ}\)C; and a final elongation step for 10 min at 72 \({}^{\circ}\)C. Amplification products were separated on a 2.5% TBE agarose gel for 2 h. Amplicon sizes were estimated by visualizing bands using ethidium bromide and UV illumination, comparing band mobilities relative to those of walleye of known genotype (determined using an Applied Biosystems (Waltham, MA, USA) ABI 3730 xl DNA sequencer, Institute of Biotechnology, Cornell University) and a 20 base-pair molecular weight size ladder (Takara Bio USA, Mountain View, CA, USA). Genotyping results were reported to the Virginia Department of Wildlife Resources (VDWR) within 24 h of capture. Because individuals bearing four native alleles proved rare, individuals bearing at least three of the marker alleles associated with the native upper New River stocks at the two diagnostic loci (_Svi17_-99 bp and _Svi_ 33-78 bp, [9]) were selected and their gametes crossed by VDWR hatchery personnel. Eggs from each female stripped were fertilized using malt from 3 to 4 males. The young were reared at the Front Royal, King and Queen, and Buller hatcheries, and fry or fingerlings subsequently were stocked in the upper New River. Adults bearing at least three native marker alleles were returned to the New River. To provide quality assurance for hatchery operations after the progeny of selected wall-eye broodsk had been produced and reared, VDWR collected 30 individuals from ponds \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Year**} & **Females** & **Eggs** & \multirow{2}{*}{**Fry Stocked**} & **Fingerlings** & \multirow{2}{*}{**Locations Stocked**} & **No. Stocked** \\  & **Spawned** & & & & **Stocked** & **per km** \\ \hline
2000 & 9 & 0.5 million & - & 10,000 & Allisonia to Buck Dam & 283 \\
2001 & 20 & 2.2 million & 500,000 & 51,840 & Allisonia to Buck Dam & 1463 \\
2002 & 9 & 0.9 million & - & 156,200 & Allisonia to Fries Dam & 3031 \\
2003 & 20 & 1.7 million & 100,000 & 90,080 & Allisonia to Fries Dam & 1748 \\
2004 & 17 & 2.0 million & - & 106,000 & Allisonia to Fields Dam & 889 \\
2005 & 23 & 2.5 million & - & 20,000 & Allisonia to Buck Dam & 564 \\
2006 & 11 & 1.0 million & - & 143,000 & Allisonia to Fields Dam & 1200 \\
2007 & 11 & 0.66 million & - & 67,140 & Allisonia to Buck Dam & 1895 \\
2008 & 13 & 0.87 million & - & 33,250 & Allisonia to Buck Dam & 938 \\
2009 & 17 & - & - & 143,000 & Allisonia to Fields Dam & 1200 \\
2010 & 14 & - & - & 40,612 & Allisonia & N/A \\
2011 & 14 & - & - & 151,912 & Allisonia to Fries Dam & 2948 \\
2014 & 18 & - & - & 26,354 & Allisonia & N/A \\
2015 & 17 & - & - & 150,100 & Allisonia to Fries Dam & 2913 \\
2016 & 24 & - & - & 150,000 & Allisonia to Fries Dam & 2911 \\
2017 & 36 & - & - & 105,000 & Allisonia to Fries Dam & 2038 \\
2018 & 30 & - & - & 10,000 & Allisonia to Buck Dam & 283 \\
2019 & 23 & - & - & 51,840 & Allisonia to Buck Dam & 1463 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Stocking of walleye into the upper New River during the study period. Stocking was not practiced in 2012–2013 to assess natural reproduction. at each of the hatcheries in 2017.