The level of band sharing among unrelated parents was 8.18. Probe 3.15.34 hybridized .... 1 99 I), and other nonfish DNA (Georges et al. 1988; Castelli et al.
A New Mu ocus Probe for DNA Fingerprinting in Chinook Sa Oncorhynchus tshawytscha ,and Comparisons with a e-Locus Probe Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Fisheries and Oceans on 08/14/15 For personal use only.
T.A. Stevens and R.E. Withier Department of Fisheries and Oceans, Biological Sciences Branch, Pacific Biological Station, Nanaimo, BC V9R 5K4, Canada
FMG Integrated Biotechnical Laboratories btd., 244-9080 River Road, Richmond, BC V6X 7x5, Canada
and T. D. Beacham Department sf Fisheries and Oceans, Biological Sciences Branch, Pacific Biological Station, Nanaims, BC V9R 5K6, Canada
Stevens, T.A., R.E. Withler, S.H. Goh, and T.D. Beacham. 7993. A new rnultilocus probe for DNA fingerprinting in chinook salmon (Oncorhynchus tshawytscha), and comparisons with a single-locus probe. Can. J. Fish. Aquat. Sci. 58: 1559-1 567. A rnulti%ocusDNA probe, 82-2, isolated from chinook salmon (Oncorhynchus tshawytscha) and a single-locus Atlantic salmon (Salma saiar) probe, 3.1 5.34, were examined for discriminatory ability among seven parents and 33-37 juveniles from five families of chinook salmon. DNA fingerprint patterns were observed in Hae Ill-digested chinook salmon DNA probed with B2-2. Between 8 and 20 fragments, from 2.20 kilobase pairs (kbp) to 8 9.0 kbp, were detected in each individual. The level of band sharing among unrelated parents was 8.18. Probe 3.15.34 hybridized with a total of nine DNA fragments, from 3.35 to 6.08 kbp, in the chinook salmon parents and progeny. One or two fragments were detected in each individual. Pedigree analysis confirmed that 3.15.34 detected both alleles of a single polymorphic locus whereas 52-2 detected autosornal, unlinked, predominantly heterozygsus DNA fragments that were inherited in a Mendelian fashion at a minimum of 7 0 polymorphic loci. Among juvenile chinook salmon, levels of band sharing detected with probe €32-2 increased with increasing relatedness, dnd clustering based on differences in banding patterns distinguished unrelated progeny, half sibs, and full sibs even in the absence of parental genotypic data. Une sonde d'ABN sur plusieurs locus, la B2-2, isolee A partir du saumoa-equinnat (Oncorhynchus tshawytschaj ainsi qu'une sonde sur un seul locus, la 3.1 5.34, trsuvee chez le saun-8on de IfAtlantique (Sdlrno salar) ont 6t6 soumises A des tests pour juger de leur psuvoir discriminant lorsqu1appliqu6es3 sept parents et 33-37 juv6niles de cinq familles de quinnat. On a observe des signatures d'ABN dans I'ADN de quinnat soumis ji une digestion dans le Hae ill et mis en prksence de la sonde BP-2. Entre 8 et 28 fragments, de 2,20 paires de kilobases (kbp) A 19,O kbp, ont 6tk detectees dans chaque sujet. he niveau de partage des bandes entre sujets non apparent& ktait de 0,18. La sonde 3.1 5.34 s'est hybrid& avec un total de neuf fragments dlADN, de 3,35 A 6,00 kbp, chez des quinnats parents et leur descendance. Un ou deux fragments ant 6te detectks chez chaque sujet. b'analyse de la provenance a confirm6 que la sonde 3.15.34 detectait les deux alleles d'un m@melocus polymorphe unique, alors que la sonde 52-2 detectait des fragments dlADN principalement hktkronygotes, non lies et autosomiques, qui ktaient herites par h6r6dite mendelienne2 un n-~inimurn de 10 locus polyrnorphes. Chez les quinnats juv6niles1 les niveaux de partage des bandes detectees avec la sonde B2-2 augmentaient avec le degr6 de parent6 et le regroupement par grappes a partir de differences dans la repartition des bandes permeetait de reconnaitre les descendants sans parentk, les demi-soeurs et freres et les frPres et soeuss, meme en I'absence de donnees sur le genotype parental. Received August 2 l , 1992 Accepted january 28, 1 993
(JB604)
enetic fingerprints, the individual-specific patterns of DNA fragments that result from examination of extremely polymorphic regions of nuclear DNA, have been produced for a wide variety of organisms (Jeffrey%et al. 1985a, 298%; Hillel et al. 1989; Meng et al. 1990; Reeve et al. 1990), including a number of fish species (Castelli et al. 1990;Taggart and Ferguson 1990b; Bentzen et aI. 199B ;Wirgin "resent address: G.F. Strong Laboratory, Vancouver General Hospital, 2733 Heather Street. Brancouver, BC B75Z 355, Canada. Can. J . Fish, Aquat. Sci., Vo1. 50, 1993
et al. 1991). For the most part, these polymorphisrns arise from the existence of multiple alleles at a number of noncsding minisatellite loci dispersed throughout the genome. At each Escus, each allele carries a different number of repeat copies sf a short DNA '"core9' sequence. A DNA fragment termed a probe, itself containing multiple copies of a core sequence, is used to simultaneously visualize alleles at all of the minisatellite loci that contain that particular core sequence, producing the complex but genetically informative fingerprint pattern. DNA probes developed from different core sequences detect 1559
Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by Fisheries and Oceans on 08/14/15 For personal use only.
different sets of minisatellite loci (Jeffreys et al. 1986; Georges et al. 1988; Frank et al. 19911, so that a variety of probes can be used to survey genetic variation at many loci. In addition, simplified fingerprints have been produced from probes that detect polymorphism at only one, or a few, hypervariable loci. The genetic variation detected by fingerprinting probes tends to show short-term stability in somatic and gem-line cells, to be distributed throughout the genome and to be inherited in a codominant Mendelian fashion (Jeffreys and Morton 1987; Castelli et al. 1998; Taggat and Ferguson 1990b). Thus, the anaBysis of DNA fingerprints has widespread applications in paternity testing, linkage and pedigree analysis, breeding programs, and the analysis of genetic structure within populations. The ability to detect highly variable regions of the genome in fish species will have substantial impact on aquaculture and fisheries management (Halleman and Beckman 1988; Bentzen et al. 1991). To date, multilocus fingerprinting has been conducted on fish with Jeffreys' human DNA probes (Castelli et al. 1996%;Taggart and Ferguson 1990b; Bentzen et al. 1991; Harris et al. 19911, bacteriophage M13 (Fields et al. 1989; Castelli et al. 1990; Wirgin et al. 199l), BKm minisatellite sequences (Lloyd et al. 1989), the Drosophila Per gene (Castelli et al. 1990; Wirgin et al. 1991;Turner et a%.199 I), and other nonfish DNA (Georges et al. 1988; Castelli et al. 1990; Carter et al. 1991; Rico et al. 1991). DNA probes and polymerase chain reaction techniques for identifying hypervariable single loci have also been developed for fish (Wirgin and Maceda 1992), including salmonids (Taggart and Ferguson 1990a; Bentzen et al. 1991 ;Phillips and Pleyte 1991). The ability of these probes to identify relatedness among individuals in wild or cultured salmonid populations has not yet been tested. In this study, we report on the multilocus genetic fingerprinting capabilities of a DNA probe developed from a chinook salmon (Oncorhynchus fshawytscha) genomic DNA library and examine its capability to detect relatedness within a chinook salmon population. Genetic similarities, based on band sharing between DNA fragment patterns, were compared between parents and offspring, as well as among maternal half sibs, paternal half sibs, and unrelated individuals. Relationships among the chinook salmon were also analyzed using the simpler DNA banding patterns resulting from use of a single locus probe, 3.15.34. isolated by Taggxt and Ferguson (1990a).
capillary tubes. Between 55 and 150 pE of blood was suspended in 3 mE of 2 x sucrose-triton Bysis buffer (0.32 M sucrose; 10 mM Tiis-HCl, pH 8 .O; 5 mM MgCB,; 2.0% Triton X-100). SDS was added to a final concentration of 1.0%, and the cell Bysates were digested overnight at 37°C in the presence of 200 pg Proteinase UmL. Proteins were precipitated by adding NaCl to a find concentration of 1-5 M (Miller et al . 1988). The mixture was shaken vigorously for 95 s, proteins pelleted by centrifugation at 1200 x ^.J? for 25 min, and the clear supernatant transferred to a new tube. DNA was precipitated by the addition of two volumes of absolute ethanol and pelleted by spinning at 12 000 x g for 30 min. The DNA pellet was rinsed in 75% ethanol, dried in a vacuum desiccator, and resuspended in TE buffer (10 rnM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8 .0). DNA Extraction from Tissue Parental DNA was isolated from frozen liver tissue by digesting approximately 100 mg of chopped tissue in 5 mL of Proteinase K lysis buffer (10 mM Tris-HC1, pH 8.0; 10 mM EDTA, pH 8.8; 1% SDS; 2W pg Proteinase WmL) at 37'C overnight. The lysate was extracted twice with phenol - chloroform - isoamyl alcohol (58:49: 11, once with chloroform - isoamy1 alcohol (24: 11, and precipitated with 2.5 volumes of ethanol. The DNA was plleted by spinning at 12 000 x g for 45 min, washed with '75% ethanol, dried under vacuum, and resuspended in TE buffer. Probe Isolation and Labelling We examined three probes for their fingerprinting ability in chinook salmon. The first was the multilocus B2-2 probe, a 1.8 kilobase pair (kbp) fragment isolated from a chinook salmon genomic DNA library in the plasmid vector pUCl8. Sizeselected DNA fragments isolated from colonies containing nonrepetitive chinook salmon sequences were labelled and used to probe membranes containing restricted chinook salmon DNA. Highly polymorphic fragment hybridization patterns resulted from the use of B2-2 on Hae 11%-digestedDNA. The second probe used was the single-locus Atlantic salmon (Sakrno salar) probe 3.15.34, approximately 4 kbp, isolated by Taggart and Ferguson (1998a). Bacteriophage M 13 (Vassxt et al. 1987) was also used as a probe. The probes were labelled using the random primer method of Feinberg and Vogelstein (1983). B2-2 and M 13 were labelled with [ c x ~ ~ P I - ~ Aand T P ,3.15.34 was nonradioactively labelled by incopration of the nucleotide analogue digoxigenin- l l -dUW (Bronstein et al. 1990).
Materials and Methods DNA Samples Gametes were taken from five male and five female chinook salmon collected in October 1989 from the Big Qualicum River on the east coast of Vancouver IsBand, British Columbia. From a factorial mating design, five families derived from four male (A, C, D, E) and t h e e female (1, 3, 4) parents were chosen for analysis. The families (A- 1, D- I , E- 1, E-4, C-3) were selected to include maternal half sibs, paternal half sibs, and unrelated individuals. Genomic DNA was isolated from frozen liver tissue of the parents and from the blmd of six to eight juveniles per family. DNA Extraction from Blood Genomic DNA of the progeny was isolated from blood of juvenile fish
