Billington, N. 1996. Genetic markers and stock Identification. Pages 323-330 in R. C. Summerfelt, 

editor. The walleye culture manual. NCRAC Culture Series 101. North Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Genetic Markers and 

Stock Identification 

Neil Billington, Fisheries Research Laboratorx Zoology Department, Southern Illinois University, Carbondale, 

IL 62901-657 1. 

Introduction 

Genetic differences among geographically separate 

stocks of walleye have important implications for 

aquaculture. For example, the natural distribution of 

walleye ranges from the Mackenzie River in northwestem Canada to northeastern Mississippi (Scott and 

Crossman 19731, areas with substantial dfferences in 

climate to which walleye have had to adapt. If aquaculturists wish to raise walleye in outdoor facilities or 

produce fish that will be sold as fry or fingerlings to 

stock lakes, ponds or impoundments (currently the 

major market for walleye aquaculture), broodstock 

should be chosen from an area with a climate similar to 

that where they will be cultured. In indoor facilities, 

temperature and other conditions can be optimized for 

characteristics of the stock. 

The economic viability of walleye aquaculture to foodsize fish lies in the development of strains of that are 

adapted to the environmental conditions and feeding 

regimens found in commercial aquaculture. Development of a selective breeding program for walleye 

should begin with a profile of the genetic variation 

within and among dfferent walleye populations. 

Population genetic data is needed to recognize unique 

strains that may be of value for selective breeding 

programs, to maintain pure strains for aquaculture, and 

for the protection of native populations. The latter are 

natural gene banks which are sources of broodstock for 

future aquacultural manipulation. Within population 

genetic variation provides a basis for positive gains 

possible from selective breeding programs as populations with little or no genetic variation have little or no 

potential. It is important to choose a population with a 

high level of genetic variation when starting a selective 

breeding program. Knowledge of the amount of genetic 

variation, particularly the level of heterozygosity, is thus 

important in choosing a founder stock for initiating a 

selective breeding program. In addition, there is the 

possibility that traits that are to be selected for may be 

linked to a genetic polymorphism, greatly enhancing 

the ability to select individuals for use in a broodstock 

improvement program (Liebowitz et al. 1987). 

Genetic markers and stock identification 

Programs to ensure the effective management and/or 

conservation of genetically dvergent stocks of a fish 

species require the identification of such stocks and 

depend on the availability of genetic markers that can 

be used to monitor the success of stocking programs. 

Protein electrophoresis and mitochondrial DNA 

(mtDNA) analysis have been used to determine genetic 

markers and stock structure in walleye populations. 

These techniques are described here and findings on use 

of protein variation as genetic markers to evaluate 

stocking strategies will be discussed. In the following 

review, alleles and loci are given using the nomenclature recommended by Shaklee et al. (1990), although in 

some cases alleles and loci are described as they were 

in the original papers. 

Protein electrophoresis 

Protein molecules tend to have &fferent net electrical 

charges. When allelic differences occur at a proteincodmg locus, the net charge of the protein often 

changes. Electrophoresis separates mixtures of proteins 

according to charge, allowing alleles to be distinguished. Generally, electrophoresis involves introducing 

a solution of soluble proteins into a starch gel, following whch the proteins are separated by passing a drect 

electrical current through the gel. Factors such as the 

NCRAC Culture Series 101 - The Walleye Culture Manual 323 
chemical composition, ionic strength, and pH of the 

buffer solution can affect the time needed for electrophoresis and the resolution of the protein bands. Most 

proteins studied are enzymes that are histochemically 

stained. Details and interpretation of protein electrophoresis and staining methods for fishes can be found in 

Aebersold et al. (1987), Utter et al. (1987), Buth (1990), 

and Morizot and Schmidt (1 990); information about 

walleye can be found in Uthe et al. (1966), Clayton et 

al. (1 97 l), Ward et al. (1 989), Billington et al. (1 990), 

Todd (1990), and McInerny et al. (1991). Proteins can 

also be separated in the buffer-film above a cellulose 

acetate plate (Hebert and Beaton 1989), and this 

technique has been shown to be as effective as starch 

for many polymorphic systems in walleye (McInerny et 

al. 1991). 

The earliest work on protein variation in walleye was 

conducted by Uthe et al. (1966), who studed muscle 

myogen proteins in two Canadian populations. They 

found three phenotypes at the fastest migrating protein 

zone (locus), with six fish from Great Slave Lake being 

homozygous for the A pattern, while all three phenotypes (A 10 indviduals; B 7 homozygotes; and AB 14 

heterozygotes) were found in 3 1 fish from Lake St. 

Clair. In addition, other genetically interpretable 

polymorphisms were found in some minor protein 

zones. In an expanded survey that included Lake 

Winnipeg (N = 61), Lake Superior (N = 31), Lake 

Huron (N = 32), Lake St. Clair (N = 51) and Baptiste 

Lake in eastern Ontario (N = 38).Uthe and Ryder 

(1970) found the same three main phenotypes. Although there were differences in phenotypic frequencies 

among lakes, observed frequencies within lakes 

generally agreed with Hardy-Weinberg expectations. 

Moreover, they suggested that the geographcal 

distribution of allelic frequencies among the lakes was 

consistent with the hypothesis of post-Pleistocene 

glacial recolonization from two separate refugia, with 

the *A allele being associated with a Mississippian 

refugium and the *B allele being associated with an 

Atlantic refugium. Ward et al. (1989) also examined 

genetic variation in muscle proteins, identifying four 

loci, two of which were polymorphic: PROT-4" (the 

most anodal locus corresponded to the fast migrating 

locus of Uthe et al. [1966]), and PROT-2* (which 

corresponded to their minor zone polymorphisms). The 

PROT-4*100 allele was equivalent to the *A allele and 

the PROT-4 *I 60 allele was equivalent to the *B allele 

of Uthe et al. (1966). Ward et al. (1989) observed 

geographic distributions of allelic frequencies similar to 

those observed by Uthe et al. (1966). 

The next protein system that was found to be polymorphic in walleye was malate dehydrogenase (MDH) 

(Enzyme Commission number, E.C. 1.1.1.37). Clayton 

et al. (1971) found six MDH phenotypes at the second 

cytosolic (supernatant) MDH locus (sMDH-B *) in 

walleye populations from central Canada, the inheritance of which were confirmed in a breeding experiment. They confirmed the presence of three codominant 

alleles at this locus, which they labeled CI, C2 and C3, 

and which correspond to the "70, *loo, and "140 

alleles at the MDH-3" of Ward et al. (1989). Clayton et 

al. (1971) found large differences in the frequencies of 

each allele among four populations: CI ranged from 

zero to 0.556, C2 ranged from 0.143 to 0.641, and C3 

ranged from 0.302 to 0.535. They suggested that this 

variation could be used as a genetic marker in studying 

the effects of fry stockmgs. Clayton et al. (1974) 

extended their previous work by surveying 19 walleye 

populations from central and western Canada for 

genetic variation at the sMDH-B* locus. They found the 

same three alleles, and observed that their allelic 

frequencies varied considerably (Cl , 0.000-0.622; C2 , 

0.007-0.702; and C3 ,0.174-0.650). Of particular 

interest was the fact that the frequency of the CI allele 

exceeded 0.500 in only three populations (Lake 

Waskesiu, Crean Lake, and Montreal Lake) that were at 

the headwaters of the Montreal River, in the Prince 

Albert National Park, Saskatchewan. The 99% confidence intervals for the frequencies of the C1 alleles 

from these populations did not overlap with those of 

any of the other populations, except for the next lake 

down stream, Egg Lake, which also had a relatively 

high C1 allele frequency (0.375). Thus, Clayton et al. 

(1974) considered the three headwater lakes to have a 

genetically different walleye stock compared to the 

other lakes surveyed, with a steep cline in Cf allele 

frequencies occurring along the Montreal River. 

Ward and Clayton (1975) used variation at the sMDHB* locus as a genetic marker to study the effects of 

walleye fry introductions to West Blue Lake, an isolated 

lake in Manitoba. As the CI allele was not present in 

West Blue Lake, walleye fry that had been produced by 

ClCl x CICI and ClCf x CIC3 matings were stocked 

in the lake to evaluate the contribution of the stocked 

fry to the 1971 and 1972 year-class (Ward and Clayton 

1975). Despite high fry mortalities in 1971, stocked fry 

324 NCRAC Culture Series 101- The Walleye Culture Manual 
contributed 43% to the 1971 year-class, while in 1972 

stocked fry appeared to contribute approximately 100% 

to the 1972 year-class. Schweigert et al. (1977) continued to monitor the contribution of these genetically 

marked fish to the total catch from 1973 through 1975. 

They found that the 1971 stocking augmented the total 

catch between September, 1974 and August, 1975 by 

only 1.3%, while the 1972 stochng contributed 37.8% 

of the total catch over the same period. Genetically 

marked (and also fin-clipped) fingerlings had also been 

stocked in 1972, and these contributed only 7% to the 

1974-1 975 total catch, suggesting that fry stockings had 

made a more successful contribution to the fishery than 

had stocking fingerlings. Similarly, Mathias et al. 

(1992) used walleye fry genetically marked by the 

sMDH-B *70 allele to determine that stocked walleye 

contributed about 2.9% to the 1985 year class in 

Dauphin Lake, Manitoba. 

Murphy et al. (1983) also used variation at the sMDHB* locus as a genetic marker to evaluate stochng 

success of walleye in Claytor Lake, Virginia. The lake 

was originally stocked with walleye from Pennsylvania 

between 1939-1946 and a self sustaining walleye 

population became established. In the early 1970's, the 

walleye population declined and the lake was stocked in 

1974, 1975, 1977, and 1979 with fish from Nebraska 

and Kansas reservoirs that contained walleyes that may 

have originated in Minnesota. Despite the fact that the 

stocked fish did not possess unique alleles or phenotypes. Murphy et al. (1983) found that the frequency of 

the sMDH-B "-2 allele differed considerably between 

stocked and unstocked cohorts, which allowed them to 

estimate the success of supplemental stocking by 

quantifying changes in cohort allele frequency. 

Electrophoretic analysis of MDH and isocitrate dehydrogenase (IDHP) (E.C. 1.1.1.42) was used by 

Paragamian (1988) to study three Iowa walleye stocks 

(Mississippi River, Cedar River, and Spirit Lake) to 

assess the genetic impact of fry stocking in the Cedar 

River. He found that while phenotypic frequencies of 

IDHP-1 *I and *2 alleles did not differ significantly 

among the three populations, phenotypic frequencies of 

the MDH-B *2 and *3 alleles were significantly different 

in Spirit Lake than in the two river populations, which 

were similar. Paragamian (I 988) used these genetic 

markers to determine that fry stockings of Spirit Lake 

walleyes (and their progeny) into Cedar River only 

contributed about 19% to the Cedar River population. 

Therefore, he recommended that the stocking program 

needed reevaluation. 

The two alleles at the IDHP-I* locus have been used as 

genetic markers in two studies on the comparative 

stochng success of three size groups of walleye. 

Jennings and Philipp (1 992) produced three groups of 

walleye each with one of the three possible genotypes 

(IDHP-I * 80/80, *80/100 and * I00/100) to compare 

the success of stocking walleye as fry, as 50-mm 

fingerlings, or as 100-mm fingerlings in five northern 

Illinois impoundments. They quantified the relative 

success of each stoclung strategy by sampling surviving 

walleyes by electrofishmg and by assigning indwiduals 

to each of the test groups by electrophoretic analysis of 

tissue from fin clips. They found that survival was 

highly variable among test groups, lakes, and years. 

Koppelman et al. (1992), used a similar approach to 

monitor the stochng success of walleye fry, 38-mm 

fingerlings, and 102-mm fingerlings in two Missouri 

impoundments. They found that small fingerlings gave 

the best survival rates. 

While other significantly polymorphc loci (~0.95 

frequency of the most common allele) have been found 

in later walleye electrophoretic studies (Ward et al. 

1989; Todd 1990; McInerny et al. 1991), the MDH, 

IDHP, and muscle myogen or PROT protein systems are 

particularly useful for electrophoretic studies, as they 

have been shown to remain stable in storage for up to 

10 months in a regular kitchen freezer and for more 

than three years at ultra-low (-70°C) temperatures 

(Murphy et al. 1990). The stability of these protein 

systems enables researchers to conduct electrophoretic 

analysis months after the samples have been collected. 

In most of these studes, protein variation was used as a 

genetic marker to evaluate stocking strategies. Protein 

electrophoresis has also been used to discriminate 

among walleye populations. Wingo (1982) found that a 

walleye population in the Tombigbee River in northeastern Mississippi exhbited &fferent blood serum 

protein bands than walleyes from Iowa, New York, and 

Pennsylvania. Walleye from the Tombigbee River were 

also unusual in that they were monomorphic at four 

protein loci that are usually highly polymorphic in 

walleye (Murphy 1990), providing evidence that they 

might represent a unique southern stock of walleye. 

Waltner (1 988) found significant differences in allele 

frequencies at two loci (sMDH-B* and PROT-4") that 

NCRAC Culture Series 101 - The Walleye Culture Manual 325 
allowed discrimination between four walleye populations from South Dakota and a Mississippi River 

population. She also found highly significant allele 

frequency differences at PROT-4 * that allowed a glacial 

lake stock in South Dakota to be discriminated from 

three upper Missouri River tributary populations. Ward 

et al. (1989) found that five loci (ADH*, IDHP-f *, 

sMDH-B *, PROT-2 *, and PROT-4 *) were significantly 

polymorphc among 15 walleye populations from 

across the Great Lakes and Manitoba. Four of these loci 

(IDHP-I *, sMDH-B*, PROT-2*, and PROT-4*) showed 

statistically significant allelic frequency differentiation 

among populations. Ten loci were found to be polymorphc in walleyes sampled from 11 locations in Minnesota, with four loci (ADH*, IDHP-1 *, sMDH-B *, and 

PRO7'-4") exhbiting statistically significant dfferences 

in allelic frequencies among populations (McInerny et 

al. 1991). Walleye from Lake Pepin were genetically 

different from all of the other populations, probably 

reflecting isolation during the Pleistocene, as Lake 

Pepin walleye likely originated from the Mississippian 

refugium whereas the other populations likely originated from a Missourian refugium. Todd (1 990) found 

that the same four loci were sufficiently polymorphic to 

differentiate walleye populations from western Lake 

Erie (6 spawning sites) and Lake St. Clair (2 spawning 

sites). Allelic frequencies differentiating the two 

walleye stocks were stable between year-classes, sexes, 

and sample years, demonstrating that fry hatched in a 

particular area returned there to spawn. Stock-specific 

homing behavior in Lake St. Clair and western Lake 

Erie walleye populations was confirmed by additional 

genetic and physical tagging studes (Todd and Haas 

1993). 

Mitochondrial DNA analysis 

Due to its maternal mode of inheritance, rapid rate of 

evolution (compared to single copy nuclear DNA), and 

enhanced susceptibility to genetic bottlenecks (four 

times that of nuclear DNA), mtDNA has often proved 

to be a useful genetic marker for stock discrimination in 

fish populations (Billington and Hebert 1991). Typically, in studies of mtDNA variation, mtDNA is 

extracted and purified from fresh tissue and digested 

with restriction endonucleases (enzymes that cut DNA 

at specific 4-, 5-, or 6-base pair sequences). The 

resulting fragments are separated according to molecular weight by gel electrophoresis and then visualized 

with either ethidium bromide staining or by autoradiography (exposing the gel to an X-ray film) after labeling 

the ends of the DNA fragments with radioactive 

nucleotides (Ferris and Berg 1987; Billington and 

Hebert 1988). However, other protocols are available 

that allow semi-pure mtDNA to be rapidly obtained 

(Chapman and Brown 1990). If pure mtDNA is not 

available for analysis on certain specimens, total DNA 

(mitochondnal and nuclear) can be extracted from 

frozen or ethanol preserved tissue and then dgested 

with restriction endonucleases. The DNA fragments are 

separated by electrophoresis, following which the DNA 

is denatured (to make it single stranded), and transferred and bound to a nylon membrane. Pure mtDNA is 

radolabeled, denatured, and used as a hybridization 

probe to the membrane, where it will only bind to the 

mtDNA fragments. These fragments are then visualized 

by autoradiography (Ferris and Berg 1987; Chapman 

and Brown 1990; Billington and Hebert 1990). Modifications of this technique allow the determination of 

mtDNA fragment patterns without the need to sacrifice 

fish (Billington and Hebert 1990). 

Variability in mtDNA fragment patterns is interpreted 

as genetic variation at the nucleotide level. Thus, a 

nucleotide substitution in the molecule that causes the 

loss or gain of a nucleotide sequence recognized by a 

specific restriction enzyme will cause a change in the 

observed fragment pattern. Composite restriction 

fragment patterns are determined for a suite of restriction endonucleases that reveal polymorphisms (more 

than one fragment pattern) and each composite is 

designated as a mtDNA haplotype. Detailed accounts of 

mtDNA extraction and analytical protocols can be 

found elsewhere (Ferris and Berg 1987; Billington and 

Hebert 1988, 1990, 1991; Chapman and Brown 1990). 

An initial study of mtDNA variation in central and 

eastern Great Lakes walleye populations revealed nine 

mtDNA haplotypes, which fell into two main groupings 

(Billington and Hebert 1988). These two groups of 

haplotypes were thought to represent fish that had 

recolonized the Great Lakes from two separate Pleistocene glacial refugia, an Atlantic refugium in the east 

(group A) and the Mississippian refugium (Group B). 

Ward et al. (1989) expanded their survey westwards to 

include walleye populations from Lake Superior and 

northern Manitoba. They found an additional mtDNA 

haplotype that was present in populations that would 

have been recolonized with fish from a Missourian 

refugium (Group C fish). An extensive survey of 847 

walleyes from 68 populations from across the whole 

326 NCRAC Culture Series 101- The Walleye Culture Manual 
range of the species revealed 34 rntDNA haplotypes, of 

which 21 were only observed in single fish (Billington 

et al. 1992). Three major walleye haplotypes were 

identified which had distinct geographic distributions 

that reflected their origins from the three separate 

glacial refugia hypothesized by Ward et al. (1989). The 

other ten haplotypes were locally hstributed and could 

be used for stock identification and to detect past 

transfers of fish. Analysis of walleyes collected from 

the Tombigbee River system in northeastern Mississippi 

revealed that they had a unique divergent mtDNA 

haplotype (Billington et al. 1992; Billington and 

Strange 1995), confirming the suggestion by Wingo 

(1982) and Murphy (1990) that these fish are a genetically unique stock. 

The relative resolution of protein electrophoresis and 

mtDNA analysis in walleye stock identification was 

examined by Ward et al. (1989), who showed that, 

consistent with theoretical expectations (Birky et al. 

1983), mtDNA data were more effective for stock 

discrimination than were allozyme data; 37% of the 

total variation among walleye mtDNA haplotype 

frequencies could be attributed to inter-population 

variation, while only 10% of allelic frequency differentiation was linked to inter-populational variation. Ward 

et al. (1 989) also found that mtDNA data provided 

evidence that the Great Lakes were recolonized by 

walleye from two glacial refugia, whereas the protein 

data suggested only a single origin for these populations. This result is a likely consequence of the different 

modes of inheritance of mtDNA and nuclear DNA 

markers (Billington and Hebert 1991). 

Other uses of genetic markers 

Detection of waleye-sauger hybrids 

Walleye and sauger hybridize naturally, but rarely. 

However, saugeye (walleye female x sauger male 

hybrids) are stocked in a number of states, usually into 

impoundments as they often perform well in these 

environments (Humphreys et al. 1984; Leeds 1988). 

There is also an interest in using saugeye in aquaculture 

(see chapter on hybrid walleye). Despite the fact that 

saugeye can have certain aquacultural advantages over 

walleye, the reciprocal hybrid (sauger female x walleye 

male) generally does not perform as well (Malison et al. 

1990). Hybrids are able to interbreed (backcross) with 

both walleye and sauger (Hearn 1986), which can result 

in the introgression of walleye or sauger genomes 

(introgression is where the genome of one species 

becomes diluted with that of another species). Introgressive hybridization will likely occur if hybrids 

escape into waters containing walleye and/or sauger. 

As significant genetic differences exist between walleye 

and sauger for both allozymes and mtDNA (Billington 

et al. 1990), it is possible to use genetic markers to 

detect both hybridization and introgression. Billington 

et al. (1990) found that there were fixed differences at 

four protein loci between walleye and sauger. A fixed 

difference is where one species is fixed (100% frequency) for one allele at a locus and the other species is 

fixed for a different allele at that locus. An F1 hybrid (a 

first generation cross between the two species) will be 

heterozygous at each of these diagnostic loci (i.e., a 

copy of each allele will be found). If two F1 hybrids 

cross with each other, or if an F1 hybrid backcrosses to 

either parent species, some of the hagnostic loci will be 

homozygous and others will be heterozygous. Therefore, these diagnostic loci serve as genetic markers that 

allow F1 walleye-sauger hybrids and walleye that 

contain introgressed sauger alleles to be identified. The 

maternal mode of inheritance of mtDNA allows the 

female parent of an F1 hybrid to be identified. Thus, if 

the female parent of a walleye-sauger hybrid was a 

sauger, the resulting offspring would also have sauger 

mtDNA, as would any offspring of such hybrid females. 

Thus, mtDNA analysis can determine introgression of 

sauger mtDNA into walleye (Billington et al. 1988). 

Introgressed indviduals containing a mixture of 

walleye and sauger genomes may not be the most 

suitable broodstock for developing walleye strains for 

use in aquaculture. Therefore, walleye broodstock for 

use in aquaculture should be pure and not possess 

sauger genes. A careful genetic analysis of potential 

broodstock should be undertaken to check for introgression of sauger alleles before such fish are used in a 

selective breeding program for aquaculture. I conducted 

a survey of allozymes of two walleye populations that 

were canhdates for aquaculture. This survey revealed 

that fish from one population possessed introgressed 

sauger alleles and was thus deemed unsuitable for 

broodstock development. 

Broodstock licensing 

The development, through selective breedmg, of 

broodstock for aquaculture is a lengthy process, often 

taking many generations and may require considerable 

NCRAC Culture Series 101 - The Walleye Culture Manual 327 
financial investment. It is possible to incorporate a Billington, N., and R. M. Strange. 1995. Mitochondrial 

genetic marker into such broodstock as a method of 

identification or as a mark of propriety. A simple 

DNA analysis confirms the existence of a genetically 

divergent walleye population in northeastern Missismethod of achieving this would be to use females with sippi. Transactions of the American Fisheries Society 

a rare and distinctive type of mtDNA in the breeding (1 241770-776). 

scheme. As such mtDNA markers are selectively 

neutral, there will be no effect on the selective breedmg 

process, yet all of the fish produced would possess this 

distinctive mtDNA. This marker could easily be 

screened by a nonlethal sampling technique (Billington 

Billington, N., P. D. N. Hebert, and R. D. Ward. 1988. 

Evidence of introgressive hybridization in the genus 

Stizostedion: interspecific transfer of mitochondrial 

DNA between sauger and walleye. Canadian Journal 

of Fisheries and Aauatic Sciences 45:2035-2041. 

and Hebert 1990), allowing a particular selected strain 

to be distinguished from other strains. Markers based 

on Drotein variation could also be used (Utter and Seeb 

Billington, N., P. D. N. Hebert, and R. D. Ward. 1990. 

Allozyme and mitochondrial DNA variation among I \ 

1990) in a similar manner to those used in the walleye 

stocking studies described earlier. In addition, genetic 

markers could also provide a method for monitoring the 

three species of Stizostedion (Percidae): phylogenetic and zoogeographical implications. Canadian 

Journal of Fisheries and Aquatic Sciences 47:1093- - 1102. frequency of escapees from fish culture operations in 

wild stocks (Billington and Hebert 199 1). Billington, N., R. J. Barrette, and I? D. N. Hebert. 1992. 

Acknowledgments 

I am grateful to funding from the North Central Region 

Aquaculture Center that has supported my research on 

the role of walleye genetics in aquaculture. 

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