Molecular Ecology (1995) 4,389-393

SHORT COMMUNICATION

A single-locus minisatellite discriminates chinook salmon (Oncorhynchus tshawytscha) populations D. D. HEATH, N. J. BERNIER' and T. A. MOUSSEAU Department of Biological Sciences, Unhersity of South Carolina, Columbia, SC, USA, 29208 and *Department OfzOOlogy, Uniuersity of British Columbia, 6270 University Bltnf., Vancouver, BC, Canada V6T 2A9

Abstract

A knowledge of genetic structure in natural populations is often necessary for conservation and management purposes, especially in declining Pacific salmon populations. To test for genetic differentiation betweennine populations of chinook salmon, Oncorhynchus fsshawyfscha, from south-western British Columbia, Canada, DNA was extracted from 603 fish and hybridized with a single-locus minisatellite probe. Multivariate statistical analyses of the resulting allele size data permitted successful overall population identification of 52% (individual population range: 2678%; P < 0.0051, indicating a high level of genetic differentiation among the nine populations. Two of the nine populations were further analysed using data from a second minisatellite locus. The discrimination success rate improved from 81.1% (one-locus analyses) to 90.0% (two-locus analyses), indicating the potential for greatly increased resolution gained by the addition of more loci. These results indicate that variation at minisatellite loci can be used for assessing population-level genetic structure, even with artificial gene flow.

Keywords: disaiminan t analysis, DNA fingerprint, population differentiation, principle component analysis

Received 26 August 1994; revision received 5 December 1994; accepted 9 December 1994

Introduction

One of the challenges facing biologists today is the conser- vation of biological and genetic diversity in wild popula- tions. In some systems the identification of genetically di- vergent populations of organisms is complicated by dispersal of the adults. The first step towards effective management of such populations is the identification of genetically distinct groups, or populations, since geneti- cally differentiated populations should be managed sepa- rately (Utter et al. 1993). A number of genetic markers have been employed to

identify population-level genetic structure, two of the most common are allozymes and mitochondria1 DNA se- quence and restriction fragment length polporphism~, or W s (see Avise 1994). Although nuclear DNA ~ e -

quences, m s , and random amplification of polymor-

Comspondence: D. D. Heath Fax +I 803 777 4002 E-mail heath@biol.scarolina.edu.

phic DNA (RAPD) have potential for discriminating be- tween populations, they have only recently come into gen- eral use (Hadrys et al. 1992; Avise 1994). Variable number of tandem repeat (VNTR) loci have been widely applied in ecological and evolutionary studies (see Burke ef al. 1991). High levels of variation in the number of tandem repeats at minisatellite VNTR loci are due to very high mutation rates (Saibner et al. 1994). Such variability might be ex- pected to limit the usefulness of minisatellite loci for iden- hfying population genetic differentiation (Degnan 1993; ! h i h e r et af. 1994). Nevertheless, there'are examples where minisatellite loci have been shown to be useful for identifying population differentiation (Gilbert et al. 1990; Wirgin etal. 1991;Triggs ef al. 1992; Degnan 1993; Scribner et ul. 1994; Taylor et al. 1994). Ethnic and geographical sub- groups in human populations have also been identified using minisatellite data (8alazs el d. 1992).

Chinook salmon ( O r ~ ~ m ~ c h u s tshmtrytscha) OCCUT in North America from California to Alaska. They return to their natal freshwater spawning grounds to reproduce with low levels of straying between streams (Vtter et al.

0 1995 Blackwell Science Ltd

390 D. D . H E A T H e t d .

1993). Although genetic differentiation between popula- tions of Pacific salmon has long been accepted (Utter & Ryman 19931, it has proven difficult to directly demon- strate genetic differences between adjacent populations (Wood et nl. 1989; Utteret al. 1989; Brodziak et a!. 1992). In this study, we analyse allele data from a minisatellite locus for nine geographically close populations of chinook salmon from south-western British Columbia, Canada, to test for genetic differentiation. Data from a second mini- satellite locus were available for two of the nine popula- tions and these data were used to determine if the addition of more loa would significantly improve the resolution of our analyses.

Materials and Methods

Nine populations of chinook salmon were sampled from south-western British Columbia, Canada (Fig. 1). Fish from populations 2,3,5,6,7,8 and 9 were all sampled as sexually mature adult salmon that had returned to the river to spawn. The Robertson Creek (pop" 4) fish were part of a first generation corneraally reared population (seeHeathetd. 1!?94).TheNiadaCreek(popnl)fishwere sampled from under-yearling juveniles held at a salmonid enhancement hatchery in the interior of British Columbia (see Bernier et al. 1993).

Fish were selected at random and blood and/or liver samples were taken. DNA was extracted (see Devlin et nl. 19911, restriction digested (restriction endonuclease HaeIII), fractionated on 0.6% agarose gels, and transferred to nylon membranes (Hybond N, BRL-Amersham Corp. 1 following Sambmk et al. (1989). Individuals from each population were divided among at least two gels, and

each gel had molecular size marker DNA lanes (I-kb lad- der) on both sides, and in a centre lane. All membranes were hybridized with the chinook salmon singlelocus minisatellite probe OtSLl (Heath et al. 1993). The mem- branes from the Robertson Creek and Nicola Creek popu- lations were also hybridized with the Atlantic salmon (Sulrno salar) singlelocus VNTR probe Ssul (Bentzen & Wright 1993). The probes were radiolabelled by random- priming (Feinberg & Vogelstein 19841, and hybridized as described in Heath et af. (1993). The membranes were then exposed to X-ray film with intensifying screens at -70 "C for 2-4 days. Allele (or band) position was measured as the distance between the loading wells and the centre of each band for all lanes be. fish). These distances were con- verted to molecular sizes by using a log-log-transformed linear relation generated from the molecular size marker (1 kb DNA ladder) for each gel (see Heath et nl. 1994). All single bands were scored as homozygotes (Fig. 2).

The observed range of allele sizes was divided into a set of discrete intervals, or 'bins' (Pascali et nl. 1991). In this analysis, the range of allele sizes was divided into allele bins using a bin width of 5% of the median allele size (il- lustrated in Fig. 2). Note that this definition of allele bin size makes the bins wider at smaller fragment sizes, and narrower at larger fragments sizes (see Fig. 2). The 5% bin width was chosen as slightly wider than the total observed range in band position of the molecular markers and a p proximated the 95% confidence interval (see Heath et al. 1994). The resulting data consisted of an allele matrix of columns corresponding to individual fish (i.e. lanes in Fig. 2), and rows corresponding to the allele bins. The choice of bin width may be critical for analyses based on binning procedures (Pascali et a/ . 1991; Heath et al. 1994). To determine the sensitivity of our analyses to bin width

Fig. 1 A map of south-western British Columbia and Vancouver Island showing the location of the nine chinook salmon populations vmpled. The populations shown are: Nicola Creek (popn 11, Capilano River (popn 21, Big Qualicum River (popn 31, Robertson Creek (popn 41, Puntledge River, summer migation returns (popn 5) and fall migration returns (popn 6), Quinsam River (popn 7), Salmon River (popn 81, and Nimpkish River (popn 9). Quinsam River chinook salmon eggs and juveniles have been introduced into the Salmon River and the Puntledge River fall popu- lations. Big Quallcum River chinook salmon eggs and juveniles have also been introduced into the Capilano River and puntledge River fall populations.

0 1995 Blackwell Science Ltd, Molecular Ecology, 4,391-395

POPULATION D I S C R I M I N A T I O N WITH M I N I S A T E L L I T E S 391

a b c d e f g

- x+ m 9.0 - - -- Y

.I, 5.0 - w

4.0 -

Fig. 2 An autoradiogam of a membrane hybridized with the OtSLl hypervariable singIeloclrs probe, showing bands (or alleles) for seven fish from the Robertson Creek population (mo- lecular sizes on the left). The right-hand magin shows the allele bins used in our analysis (5% of the median allele size). note that the allele bins were fixed, and that the width of the bins incmsed with smaller allele molecular size. Lane g shows how the bands for each fish were assigned to discrete allele bins (all other bins in the column were assigned zeros). This produced an allele matrix consisting of columns correpponding to individual fish (or lanes), and rows corresponding to allele sizes.

choice, a series of bin widths were generated, larger and smaller than the selected 5% width. The resulting allele matrices were analyxed as described below to determine the effect of altering allele bin size.

Our analysis consisted of three sequential steps: (1) principle component analysis was performed on the allele matrix to reduce the number of variables (i.e. diminate noninfonna tive alleles) and to ensure independence; (2) the prinaple components (PO) were tested for significant population effects using a one-way ANOVA and all nonsig- nificant PCs (P > 0.05) were dropped; and (3) a discrimi- nant function analysis was performed on the remaining PCs to generate a quantitative estimate of the population differentiation. All analyses were performed using the SAS statistical package (SAS Institute Inc.). The Robertson Creek and Nimla Creek populations were analysed using just the OtSLl data, then using data from the combined OtSLl and Ssal loci.

100

A ao

L I

0 5 10 15 20 25 30 35

BIN WIDTH (96 MEDIAN ALLELE SIZE) 3 2

-t z E 2 100

B

#8 #3 97 1)6 #2 #4 #5 # I #9 POPULA TION

Fig. 3 (A) A sensitivity analysis showing the mean dixrimination success (as a percentage) for nine chinook salmon populations using a range of bin widths (as a percentage of median allele size). The 5% bin width was chosen as the most appropriate based on estimated measurement error, and is marked by an arrow. (B) Individual disaimination success (as a percentage) for each of the nine chinook salmon populations based on data from one mini- satellite locus. The populations are numbered as in Fig. 1. The dashed line represents the expected success based on random as- signment. All disaimina tion values were significantly higher than expected by chance (P < 0.005).

Results and discussion

Our analysis of the allele frequency distributions for the nine populations gave an overall population discrimina- tion success of 52% at the 5% (fixed) bin width (Fig. 3a). Furthermore, the sensitivity analysis indicated that our results were relatively insensitive to bin size, since changes in the bin width did not lead to krge changes in the discrimination power of our analysis (Fig. 3a). This level of discrimination is remarkable, since it was based on only one marker at one locus. Although there was consid- erable variation in the discrimination success between populations (Fig. 3b), all population discrimination levels were signrficantly greater than that expected by chance (XI

test; P < 0.005). The variation in discrimination success between individual populations probably reflects artifi- aa l gene flow due to transplantation of juvenile fish. The four best discriminated populations (i.e. popns 4,5,1 and

Q 1995 Blackwell Science Ltd, Motrmlar €wlw, 4,391-395

392 D. D. HEATHetal.

9) were not only the most widely geographically sepa- rated (Fig. l), but also had experienced little or no artificial gene flow. The least discriminated populations (i.e. 8,3,7 and 6) had all been involved in transfers. The analysis of the Robertson Creek and Nicola Creek populations using the OtSLZ locus alone gave a disaimination success of 81.196, this increased to 90% when two loci (OtSLI and Ssul) were included in the analysis. It is thus likely that high levels of discrimination between Pacific salmon populations would be possible using multiple VNTR loa.

Previous reports using minisatellite variation to test for population-level genetic structure have often been based on organisms exhibiting extremely low levels of genetic variation. Such studies have been reported in striped bass from rivers draining into the Gulf of Mexico and the At- lantic Ocean (Wirgin et al. 1991), blue duck populations in Nova Scotia Crriggs et uf. 19921, island populations of silvereyes (Degnan 1993), and Channel Island fox (Gilbert ef ul. 1990). In these cases, the level of genetic variation observed using ahzymes was low. Saibner et al. (1994) analysed allozyme, minisatellite, and microsatellite data for three populations of Bufo bufo and found that the three types of markers gave concordant estimates of population genetic divergence and diversity. Saibner et af. (1994) con- cluded that VNTR markers were potentially very useful for studying population-level genetic structure. Taylor el ul. (1994) analysed minisatellite data for 42 populations of chum salmon, Oncurhynchw kta, from the north Pacific. They found significant regional groupings; however, they were not as successful at identifying finesale population genetic structure. These results, and ours, indicate that VIVl'R loci can be surressfully used to idenhfy population- level genetic differentiation for ecological and manage- ment studies, even in populations that exhibit normal lev- els of genetic variation.

The origin of Pacific salmon has been successfully de- termined using cambinations of biological and genetic markers (Fournier ef d. 1984; Wood ef al. 1989; see Shaklee & Phelps 1990; Utter ct el. 1993). Such analyses generally employ many aHozyme loci (> 201, andlor morphological, meristic, and life history data. Our study was not designed to develop a practical method of population identification for fisheries r n a ~ g e r ~ , but rather to test for genetic differ- entiation between populations for mnserva tion purposes. It is clear, nevertheless, that minisatellite data is poten- tially useful far population identification, and may pro- vide even greater resolution than traditional markers.

Although singblocus minisatellite data are easier to analyse than multilocus data (Degnan 1993; Scribner et al. 19941, they still require allele binning procedures. Re- cently, polymerase chain reaction (PCR) techniques have been developed that assay variation at microsatellite VNTR 106 (see Wright & Bentzen 1994). These techniques allow the exact measurement of allele size, eliminating the

need for allele binning. Thus, the development of PCR primers for microsatellite loci may be a practical approach for fisheries managers interested in the identification of specific Pacific salmon populations.

It is clear that the use of minisatellite and microsatellite allelic variation to identify popula tion-level genetic differ- entiation has many potential applications for the conser- vation and management of wild populations of plants and animals. Our specific application to Pacific salmon is of pressing concern, given the uncertain future of many salmon stocks.

Acknowledgements We would like to thank Dr R H. Devlin for his advice and sugges- tions, and Dr P. Bentzen for the use of the Ssal single-lonts probe. We would also like to acknowledge the staff and volunteersof the Salmonid Enhancement Program hatcheries. This research was supported in part by Natural Science and Engineering Research Council (Canada) operating grants and Canadian Bacterial Dis- eases Network funding to Dr G. K. Iwama. DDH was supported by a Nahual Science and Engineering Research Council (Canada) postdoctoral fellowship.

References Avise JC (1994) Molecular Markers, Natural History and Ewlution.

Chapman and Hall, Inc., New York Balazs I, Neuweiler J, Gunn P, Kidd J, Kidd KK, Kuhl J, Mingjun L

(1992) Human population genetic studies using hypervariable loci. I. Analysis of Assamese, Australian, Cam- bodian, Caucasian, Chinese and Melanesian populations. Ge-

Benken P, Wright JM (1993) Nucleotide sequence and evolution- ary conservation of minisatellite variable number tandem re- peat cloned from Atlantic salmon. Salmo salar. Genome, 36, 271-277.

Bemier NJ, Heath DD, Randall DJ, Iwama GK (1993) Repeat sexual maturation of precocious male chinook salmon (Onmrhynchw tshazuytscha) transferred to sea water. Cnnadkn

Brodziak J, Bentley B, Bartley D, Gall GAE, Gomulkiewia R, Mange1 M (1992) Tests of genetic stock identification using coded wire tagged fish. G d k n Jouml of Fisheries and Aquntic Sciences, 49,1507-1517.

Burke T, Hanotte 0, Bruford MW, Cairns E (1991) Multilocus and single locus minisatellite analysis in population biological studies. In: D N A Fingmprhting: Apprmches and Applications (eds Burke T, Dolf G, Jeffreys AJ, Wolff R), pp. 154-168. Birkhauser Verlag, Basel.

Degnan SM (1993) Genetic variability and population differentia- tion inferred from DNA fingerprinting in silvereyes (aves: zosteropidae). Ewlution, 47, 1105-1117.

Devlin RH, McNeil BK, Groves DD, Donaldson EM (1991) Isola- tion of a Y-chromosomal DNA probe capable of determining genetic sex in chinook salmon (Onwrhynchus tshawytscha). Canadian Journal of Fishnies and Aquatic Sciences, 48. 1-161 2.

Feinberg AP, Vogelstein B (1984) Addendum. Analytical Biochem- istry, W7,266-267.

Fournier DA, Beacham TD, Riddell BE, Busadc CA (1984) Esti-

n~ficS, Wl, 191-198.

l o u d of Zoology, n, 683-686.

(D 1995 Blackwell Science Ltd, Molecular Ecology, 4,391-395

P O P U L A T I O N DISC R I M I N AT1 0 N W I T H MI N IS A T E L LIT E S 393

mating stock composition in mixed stock fisheries using morphometrlc, meristic, and electrophoretic characteristics. Canadian Journal of Fishcria and Aquatic Sciences, 41,400-408.

Gilbert DA, Lehman N, CYBrian SJ, Wayne RK (1990) Genetic h- gerprinting refleck population differentiation in the CaliEor- nia Channel Island fox Nuhm, 344,764-767.

Hadrys H, Balick M, Schierwak B (1992) Applications of ran- dom amplified polymorphic DNA (RAPD) in molecular ecol- ogy. Molecular Ewlogy, 1,M.

Heath DD, Iwama GK, Devh RH (1993) FCR primed with VNTR core sequences yields species speafic patterns and hypervariable probes. Nuclrie Acids Research, 21,5782-5785.

Heath DD, Iwama GK, Devh RH (1994) DNA fingerprinting used to test for family effects on precodous sexual maturation in two populations of Onwrhynchus tshmuytsdur (chinook salmon). Hcredify, 73,616-624.

PasCali VL, d'Aloja E, Dobosz M, P S ~ ~ ~ O M M (1991) Estimat- ing allele frequencies of hypervariable DNA systems. Formsic Science International, 51,273-280.

Sambrook J, Frikch EF, Maniatis T (1989) MoIeculat Ckming: a Lahatory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

Scribner KT, AmtzenJW, BurkeT (1994) Comparative analysis of inha- and interpopulation genetic diversity in Bufo bufo, using allozyme, single-locus microsatellite, minisatellite, and multi- locus minisatellite data. Molccllhr Biology and Eoolution, 11,

Shaklee JB, Phelps SR (1990) Operation of a large-scale, multi- agency program for genetic stodc identification. M a n Fkhcrics Society SvmpoJium, 7,817-830.

Taylor EB, Beacham TD, Kaeriyama M (1994) Population struc- ture and identification of north Pacific Ocean churn salmon OnwrhynJrlls krta) revealed by an analysis of minisatellite DNA variation. Canadian Journal of Fisheries and Aquatic Sci- ences, 51,1430-1442

737-748.

Triggs SJ, Williams MJ, Marshall SJ, Chambers GK (1992) Genetic struchue of blue duck (Hjmunolnirnus mnlncorhunchos) popula- tions revealed by DNA fingerprinting. Auk, 109,8&89.

Utter F, M i e r G, StaN G,Teel D (1989) Genetic ppula tion smc- ture of chinook salmon Onmhynchus tshawytscha, in the Pa- cific Northwest. Fishery Bulletin US, 87,239-264.

Utter F, Ryman N (1993) Genetic markers and mixed stock fisher-

Utter FM, Seeb JE, Seeb LW (1993) Complimentary uses of eco- logical and biochemical genetic data in identifying and con- serving salmon populations. Fishm'es Research, l8,59-76.

Wirgin 11, Grunwald C, Carte SJ (1991) Use of DNA fingerprint- ing in the identification and management of a striped bass population in the southeastern United States. Trnnsactions of the M a n Fisheries Socicty, 120,273-282.

Wood CC, Rutherford DT, Md(inneU S (1989) Identification of sockeye salmon (Oncmhynchus nerh) stocks in mixed-stodc fishen- in British Columbia and Southeast Alaska using bio- logical markers. Canadian Journal of Fisheries and Aquatic Sci- mcs, %, 2108.2120.

Wright Jh4, Benken P (1994) Microsatellites: genetic markers for the future. Reviews in Fish Biology and Fisheries, 4,384-388.

ies. FiJur ia , l8,11-21.

~~ ~ ~

Thh paper represents a collaboration of effort from a salmon molecular genetics laboratory (Robert Devh, Department of Fish- eries and Oceans, Canada), a salmonid physiology/aquaculhm laboratory (GeorgeIwama,Universityof BritishCo1umbia)and an evolutionary genetics laboratory (Timothy Mousseau). Daniel Heath is usingmolecular techniques to study patterns of variation in genetic diversity in natural populations. Nicholas Bernier is studying saknonid physiology at the University of Ottawa (Canada). Timothy Mousseau is using hypervariable marken to address questions concerning sexual selection in insects.

0 1995 Bladcwell Sdence Ltd, Molmlar Ecology, 4,391-395

Molecular Ecology (1995) 4,394

Forthcoming papers 1

2

3

4

5

6

7

8

9

10

11

12

13

14

Genetic differentiation in Fomitopsis pinicolu (=warts: Fr.) Karst studied by means of arbitrary primed - PCR N. Hagbe% J. Stenlid and J.-0. Karlsson Marked mitochondrial DNA differences between Mediterranean and Atlantic populations of the swordfish, Xiphias gladius G. KototrLas, A. Magoulas , N. Tsimenides and E. Zouros Mitochondria1 DNA variation among populations of Oeduru reticulnta (Gekkonidae) in remnant vegetation: implications for metapopulation structure and population decline s. S v r e Miaosatellite variation in grey seals (hizlichoerus g'ypKS) shows evidence of genetic differentiation between two British breeding colonies P. J. Allen, W. Amos, P. P. Pomeroy and S. D. Twiss, The effects on genetic variability following a recent colonization event: the Australian Sheep Blowfly, Lucilia cuprina (Weidemann 1830) (Diptera: Calliphoridae) amves in New Zealand. D. M. Gleeson PCR-based identification of wheat genomes R Sallares, R G. Allaby and T. A. Brown Patterns of covert infection by invertebrate pathogens: iridescent viruses of blackflies. Trevor Williams Multiple paternity in the red-eyed treefrog, Agnlychnis cnllidryas (Cope) C. A. d'Urgeix and 8. J. Turner RAF'D assessment of California phylloxera diversity G. L Fong, M. A. Walker, J. Granett, G. Fong, M. A. Walker PCR Amplification of intergenic spacers in the ribosomal DNA of Drosophila rnelnnogaster reveals high levels of turnover in length and copy-number of spacers in geographically separated populations T. Bowen and G. A. Dover

SHORT COMMUNICATION Resolving genetic relationships with microsatellite markers: a parentage testing system for the swallow Hirundo rilstica C R Primmer, A. P. Msller and H. Ellegren Amplification of hypervariable simple sequence repeats (microsatellites) from excremental DNA of wild living Bonobos (Pan pmriscus) U. Gerloff, C Schlottercr, K. Rassmann, 1. Rambold, G. Hohmann, B. F ~ t h , D. Tautz

TECHNICAL NOTE The molecular basis and evolutionary history of a microsatellite null allele in bears D. Paetkru and C Strobe&

BOOK REVIEW Mdmlar Erology of Rhizosphere Microorganisms. Biotechnofogy nnd the Release of CMOS