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Unanticipated departures from breeding designs can be detected using microsatellite DNA parentage analyses
Aquaculture 280 (2008) 71–75
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Unanticipated departures from breeding designs can be detected using microsatellite DNA parentage analyses Andrew K. Gray ⁎, John J. Joyce, Alex C. Wertheimer National Marine Fisheries Service, Auke Bay Laboratory, 11305 Glacier Highway, Juneau, Alaska 99801, USA
A R T I C L E
I N F O
Article history: Received 6 September 2006 Received in revised form 24 March 2008 Accepted 11 April 2008 Keywords: Oncorhynchus tshawytscha Chinook Microsatellite Parentage analysis Pedigree
A B S T R A C T Using microsatellite analysis, a partial pedigree was constructed from an experiment designed to evaluate differences between hatchery Chinook salmon (Oncorhynchus tshawytscha) and their wild donor stocks. First generation (F1) progeny were created by crossing both hatchery and wild females with hatchery and wild males to create hybrid and control lines. The pedigree analysis revealed an accidental switch of a half-sib control and hybrid F1 family, which most likely occurred during incubation and led to the misclassification of individuals in those families. The experimental error propagated into the F2 lines. The pedigree also revealed a number of unintended full-sib matings in the F2 crosses. This resulted from the small numbers (5 females, 14 males) of wild fish available for the experiment and differential family survival of the F1 fish. Because researchers used group-specific physical marks to track typed crosses and not family groups, and because physical marks cannot detect inadvertent movement of fish until they are large enough to mark, experimental error occurred that would have influenced the results had a DNA-based analysis not been employed to verify the integrity of the experiment. Published by Elsevier B.V.
1. Introduction Aquaculture researchers often use external physical marks (e.g., fin clipping, spaghetti tags, PIT tags, coded-wire tags) to track groups of experimental fish. External physical marks are relatively easy to apply and are a cost-effective way to mark large numbers of fish, which make them attractive to researchers. However, for all the advantages, external physical marks used to track experimental fish have disadvantages that limit their utility. One disadvantage of external marks is that most fish and other aquatic animals are too small to be marked at hatching and marks can only be applied after individuals are sufficiently large (Herbinger et al., 1999). Consequently, experimental integrity can be compromised by the inability to track individuals, families, or groups from fertilization of gametes without error until the fishes are large enough to be marked. Gilk et al. (2004) demonstrated this problem during a study of outbreeding depression in even-and odd-year pink salmon. Although control and hybrid first generation (F1) progeny were marked with distinct fin marks upon release, when returning F1 adults were genotyped using microsatellite DNA analysis, 58 out of 1266 individuals were identified that did not correspond to any parental combinations used in the original matings. Two individuals marked as hybrids were identified as controls. The investigators were able to use this information to avoid corruption of F1 data and subsequent F2 crosses. In another paper, Hedrick et al. (2000) described the detection ⁎ Corresponding author. Tel.: +1 907 789 6047; fax: +1 907 789 6094. E-mail address: [email protected] (A.K. Gray). 0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2008.04.021
of classification errors within experimental groups of winter-run Chinook salmon (Oncorhynchus tshawytscha) in the Sacramento River, California. Using microsatellite DNA analysis researchers discovered that a number of non-winter-run Chinook salmon had been accidentally used as spawners for the winter-run supplementation program. Lack of pedigree information can potentially confound the effects for which a study was designed. In order to track families with external marks, progeny from family groups must be isolated and reared together until they are large enough to be marked. This approach is both labor and space intensive (Norris et al., 2000), and for these reasons, many researchers choose not to or are unable to collect family information. However, in the absence of pedigree information researchers run the risk of inadvertent matings of related individuals resulting in inbreeding, which in salmonids includes depression of survival and growth traits (Gjerde et al., 1983; Kincaid, 1983). A study by Jackson et al. (2003) underlines this point. Investigators used five microsatellites to assess the parentage of an F1 broodstock of Atlantic halibut (Hippoglossus hippoglossus) derived from wild adults. They observed that full and half-sib groups were disproportionately represented and only 36% of the attempted parental crosses were represented. As Rodgveller et al. (2005) demonstrated in size data from Chinook salmon, without pedigree information researchers also lack variability data within families. In this instance the magnitude of family variation was approximately as large as the variation among individuals, suggesting that in some cases much of the experimental variation may be attributed to family origin, and not to the treatment being tested. In 1996, an experiment to evaluate differences between hatchery Chinook salmon and their wild donor stocks after 20 years of hatchery
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culture was designed and implemented at the Little Port Walter (LPW) marine research station located on southern Baranof Island in Southeast Alaska. At the time DNA-based analysis was not available to track parentage, so coded-wire tags were used to track four experimental categories of fish. In 2002, microsatellite genotyping became available to the researchers and was used to accomplish two tasks: the first was to develop a pedigree and verify to crosstype the returning 2001 and 2002 F1 spawners; the second was to check for inadvertent inbreeding in the F2 lines.
Table 1 Number of F1 adults returning to hatchery, used as broodstock, and sampled for DNA 2001 F1 Adults Categories
Returns
Broodstock
Sampled for DNA
H×H H×W W×H W×W
162 322 103 61
111 85 54 46
62 77 45 40
2002 F1 Adults
2. Materials and methods
Categories
Returns
Broodstock
Sampled for DNA
2.1. Crosses and incubation procedures
H×H H×W W×H W×W
77 94 66 29
46 48 36 17
33 48 36 21
On August 12, 1996, gametes from 20 females and 18 males were collected from LPW Chickamin River stock hatchery returns. At the same time, with the help of the Alaska Department of Fish and Game personel (ADF&G), mature gametes from 5 wild female and 14 wild male Chinook salmon were collected from Barrier Creek, a tributary of the South Fork of the Chickamin River. The gametes were then transferred by helicopter to LPW. Four types of F1 progeny were made by crossing hatchery and wild females with hatchery and wild males in two by two, one by two, two by one, and one by one blocks (Fig. 1). The progeny types were: 1) hatchery
female × hatchery male (H× H), 2) hatchery female× wild male (H× W), 3) wild female × hatchery male (W× H), and 4) wild female × wild male (W × W). Each set of half-sib families was assigned to cells within a single tray and placed in vertical tray incubators. Prior to hatching, families were consolidated by type within a single tray in order to minimize the effects of potential movement between tray cells. At hatching, families of each category were transferred from incubators and reared communally in four separate freshwater vertical raceways (VRs) according to the type of cross. Fish were reared approximately 1 year in the VRs, tagged with category specific coded-wire tags, and released to the estuary at LPW in May of 1998. In 2001 and 2002, returning adult F1 were collected and a portion of these were used as broodstock (Table 1). The type of cross of each fish was confirmed by coded-wire tag detection and code verification before F2 crosses were conducted. Heart tissue samples were collected from F1 fish at spawning and preserved in 100% EtOH (Table 1). DNA analysis of the F1 fish was conducted on the samples after the F2 crosses were made. Six types of F2 progeny (HH × HH, HH × WW, WW × HH, WH × WH, HW × HW, and WW × WW) were made by crossing F1 spawners according to tag code in 1 by 1 and 1 by 2 blocks. DNA analysis has not been performed on returning F2 fish. 2.2. Microsatellite and parentage analysis
Fig. 1. The 1996 experimental design used to create F1 control and hybrid groups. Five hatchery (H) females and males were crossed with five wild (W) females and males creating five 2 by 2 blocks. Nine hatchery females were crossed with nine hatchery and nine wild males creating nine 1 by 2 blocks. Hatchery controls were also created by crossing hatchery females and males in two 2 by 1, and two 1 by 1 blocks.
During the collection of wild gametes in August 1996, scales from 35 wild Chickamin River (WCR) Chinook salmon spawners (16 females and 19 males) were collected and placed on scale cards by the ADF&G personnel. These samples included scales from the wild spawners used for the LPW experiment, but the log book describing which of the 35 WCR fish contributed gametes to the LPW experiment was incomplete. Consequently DNA was isolated from all 35 WCR fish because each needed to be considered as a possible parent to the returning LPW 2001 and 2002 F1 H × W, W × H, and W × W LPW experimental fish. Tissues for DNA analysis were not sampled from the 1996 LPW hatchery brood stock. Total DNA was isolated from both scale and preserved heart tissue using DNeasy ™ Tissue Kit (QIAGEN, Inc., Valencia, CA) following the procedure for isolating DNA from mammal tissue provided in the kit instructions. Two scales or approximately 25 mg of heart tissue per individual provided sufficient DNA for the analyses. Polymerase chain reaction (PCR) amplification was done in 96-well microtitre plates in a DNA Engine™ (MJ Research, Inc., Reno, NV). Amplification reactions were in 10 μL volumes [10 mM Tris–HCl at pH 8.3, 50 mM KCl, 25 mM MgCl2, 2.5 mM each deoxyribonucleotide triphosphate (dNTP), 0.5 units Taq polymerase, 0.1–0.5 μM each primer, and 50–100 ng DNA template]. In addition to unlabeled forward and reverse primers for each locus, each mixture included a forward primer labeled with an infrared-sensitive dye, IRDye™ (LICOR, Inc., Lincoln, NE).
A.K. Gray et al. / Aquaculture 280 (2008) 71–75 Table 2 Expected and observed contribution of wild female and male gametes in F1 families Expected
Observed⁎
Categories
# of families
Female
Male
Female
Male
H×H H×W W×H W×W
20 14 5 5
0 0 5 5
0 14 0 5
0 0 5 5
0 14 1 4
⁎Recreated using microsatellite analysis.
After evaluating 10 microsatellite loci, 5 were chosen based on their ability to separate each of the 35 wild Chickamin Chinook salmon into unique genotypes. The five loci were: Ots100, Ots104, Ots107 (Nelson and Beacham, 1999), Ogo4 (Olsen et al., 1998), and Omy325 (O'Connell et al., 1997). In general, PCR conditions were as follows: 1 cycle at 95 °C for 3 min; 28 cycles at 95 °C for 30 s, 58 °C for 15 s, 72 °C for 15 s, and 1 cycle at 72 °C for 1 min. After amplification, DNA products were denatured by adding an equal volume of stop buffer (95% formamide, 0.1% Bromophenol Blue) and heating for 3 min at 95 °C. One μl of PCR product was loaded onto polyacrylamide denaturing gels composed of 6.5% KBPlus gel matrix (LI-COR, Inc., Lincoln, NE) in a reaction catalyzed by ammonium persulfate and TEMED (N,N,N',N' tetramethylethylenediamine). Alleles were separated electrophoretically and detected on an automated sequencer (LongReadIR 4200™, LI-COR, Inc., Lincoln, NE) in 1 × TBE running buffer, with running parameters 31.5 W, 1500 V, 35 mA, and 50 °C plate temperature. The size of microsatellite alleles was estimated using Saga™ Generation 2 automated microsatellite software (Version 3.0, LI-COR, Inc., Lincoln, NE). Allele sizes were estimated by comparing allele band patterns with IRD700TM or IRD800TM standard ladders (LI-COR, Inc., Lincoln, NE) and standardized using reference alleles. Parentage of returning W × W F1 spawners was deduced using PROBMAX (version 1.2, Danzmann, 1997) executable program PROBMAX2, which assigns progeny to a mixture of possible contributing parents given that the genotypes are known for both progeny and possible parents. The PROBMAX executable program PROBMAXN was used to test for possible missed assignments due to the presence of nonamplifying (null) alleles. The default tolerance levels and
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disallowing for mis-scoring were used for both executable programs. All known parental genotypes not considered by PROBMAX analyses were systematically examined to confirm PROBMAX assignments. Assignments using PROBMAX are based on known genotypes for the mother, father, and offspring and cannot make assignments when one parental genotype is unknown. Therefore returning F1 H × W and W × H fish were assigned a parent by systematically comparing their genotypes with the 35 WCR parental genotypes. Assignments were only made when one of the 35 possible parents had an allele that matched at each of the loci examined. Although we did not have tissue samples of the hatchery parents used as broodstock in 1996, paternal and maternal hatchery alleles were derived by comparing individual genotypes in each F1 W × H and F1 H × W family array with their known WW maternal and paternal genotypes respectively. Paternal and maternal hatchery alleles were first identified manually and then confirmed using the program GERUD 2.0 (Jones, 2005). GERUD 2.0 performs an exhaustive search of the minimal set of parents necessary to explain a set of offspring or ‘progeny array' known to have a single parent. For progeny arrays with multiple minimum parent solutions, GERUD 2.0 chooses the most likely solution based on Mendelian segregation probabilities. The inferred parental hatchery genotypes reconstructed by GERUD 2.0 were used to assign H × H F1 fish to families using PROBMAX2 with the same tolerance levels as stated above. PROBMAX could only be used to assign individuals to the 2 by 2 blocks (Fig. 1) because the program makes assignments based on both sets of parental genotypes. Since we had inferred genotypes for nine 1996 hatchery females reconstructed from nine H × W 1 by 2 blocks (Fig. 1), the F1 H × H fish not assigned by PROBMAX were analyzed manually and with the program ML Relate (Kalinowski et al., 2006), which uses maximum likelihood estimates to calculate relatedness and relationships between individuals of unknown ancestry, to create fullsib arrays. Each array was then assigned to reconstructed hatchery parent if all individuals in the array had a common allele at each locus with the inferred hatchery parental genotype. The progeny arrays which did not match any recreated hatchery parent were assumed to belong to one of the H × H blocks, from which we have no data to infer hatchery parental genotypes (Fig. 1). The parentage analysis provided information to establish whether inbreeding was imposed as a result of limited parents in the F2 crosses.
Table 3 Familiy origins of F1 progeny returning in 2001 and 2002 2 by 2 blocks
1 by 2 blocks
Tag code
Recreated Families
2001 Individuals
2002 Individuals
Tag code
Recreated Families
2001 Individuals
2002 Individuals
H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×W
H1 × H1 H1 × W1 W1 × H1 W1 × W1 H2 × H2 H2 × W2 W2 × H2 W2 × w2 H3 × H3 H3 × W3 W3 × H3 W3 × W3 H4 × H4 H4 × W4 W4 × H4 W4 × W4 H5 × H5 H5 × W5 W5 × W5⁎ W5 × H5⁎ W1H1⁎
5 8 20 19 7 3 4 0 10 4 7 8 1 1 8 3 4 5 3 10 1
7 2 20 5 4 11 9 4 2 6 0 1 3 3 4 8 2 3 3 3 0
H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W W×H W×H H×W
H6 × H6 H6 × W6 H7 × H7 H7 × W7 H8 × H8 H8 × W8 H9 × H9 H9 × W9 H10 × H10 H10 × W10 H11 × H11 H11 × W11 H12 × W12 H12 × W12 H13 × H13 H13 × W13 H14 × H14 H14 × W14 H10 × W10⁎ Unknown⁎⁎ Unknown⁎⁎
1 3 16 17 2 4 2 4 3 5 5 5 6 10 0 4 0 1 1 2 1
1 2 2 6 2 1 4 1 2 5 1 4 3 1 0 2 0 1 0 0 0
Individuals observed per tag code and recreated families. W = wild parent and H = Hatchery parent. ⁎ Misclassified by Tag code. ⁎⁎ Individuals for which genotype data was incomplete.
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3. Results
Table 4 The number of full-sib and misclassified F2 families created in 2001 and 2002
3.1. Parentage assignment
Families
HH × HH HH × WW WW × HH WH × WH HW × HW WW × WW
2001 2002 Total
59 41 100
31 32 63
34 18 52
54 38 92
78 48 126
23 20 43
Full-sib Families 2001 2002 Total
5 2 7
0 0 0
0 0 0
10 15 25
6 5 11
9 4 13
Misclassified Families 2001 2002 Total
0 0 0
3 10 13
8 1 9
7 4 11
2 0 2
8 6 14
PROBMAX analysis of the 2001 and 2002 W × W F1 control line indicated that four WCR parental pairs could be assigned to the majority of the fish. The W × W F1 fish that were not assigned a parental pair were systematically assessed to determine if any of the 35 WCR fish could have contributed as a parent. All of the unassigned fish had one allele at each locus that could be assigned to a single WCR female, suggesting they were actually W × H fish. Because the same five females were used to create the W × W and W × H F1 lines, the above discrepancy suggested an inadvertent mixing of experimental lines (Table 2). Upon the discovery of a possible mixing of experimental lines based on the W × W F1 parentage assignments, the rest of the 2001 and 2002 F1 experimental fish were examined to determine parentage. Of the 81 W × H F1 individuals assessed, 6 fish (3 from 2001 and 3 from 2002) could be assigned to a single WCR pair. The female from the WCR pair was the same fish that was assigned to the W × H F1 individuals found in the W × W offspring (Table 3). By systematically comparing allele scores of the 75 W × H F1 individuals that were not assigned a parental pair by PROBMAX, 65 could be assigned to a total of 4 WCR females and 1 fish could be assigned to one WCR male. Two W × H F1 fish were not assigned to any of the possible WCR parents (Table 3). PROBMAX analysis of 124 H × W F1 revealed zero fish that could be assigned to any WCR pair; however, 122 individuals were systematically traced back to 14 WCR males (Table 3) which is consistent with the mating design (Table 2). One H × W F1 fish was assigned to a WCR female and 1 H × W F1 fish was not assigned to any of the possible WCR parents (Table 3). A WCR pair or single individual was not assigned to any of the 112 H × H F1 fish that were analyzed systematically or with PROBMAX. Parentage analysis taking into account nulls did not produce any unique assignments across any of the experimental lines. The retrospective analysis using genotypes of the F1 individuals revealed that no more than 5 of 16 possible wild parental females and 14 of 19 possible wild parental males could account for the majority of the F1 individuals coded as W × W, W × H, and H × W, which is consistent with the original experimental design and lends strength to the reconstructed pedigree. Reconstruction of hatchery parental alleles using W × H and H × W F1 progeny arrays and the program GERUD 2.0, followed by analysis of the 112 H × H F1 using PROBMAX, allocated 39 H × H F1 fish among the 2 × 2 experimental blocks (Table 3). Using ML Relate and manual conformation of progeny arrays, 56 H × H F1 fish were assigned among the 1by 2 H × W blocks (Table 3). The remaining 17 H × H F1 fish were separated into 4 full-sib arrays and are assumed to belong to one of the H × H blocks (Fig. 1). 3.2. Inbreeding and misclassification error in F2 lines The pedigree analysis revealed that 8% of HH × HH, 8% of HW × HW, 19% of WH × WH, and 39% of WW × WW F2 families were created by full-sib matings during spawning in 2001 (Table 4). Similarly in 2002, 4% of HH × HH, 10% of HW × HW, 39% of WH × WH, and 20% of WW × WW F2 families were created by full-sib matings (Table 4). The WW × HH and HH × WW F2 families in 2001and 2002 did not contain any full-sib families (Table 4). With the exception of HH × HH F2 types, most F2 cross types created in 2001 and 2002 contained misclassified families. In 2001, 10% of HH × WW, 23% of WW × HH, 3% of HW × HW, 12% of WH × WH, and 35% of WW × WW F2 families were created with a misclassified parent (Table 4). In 2002, 31% of HH × WW, 5% of WW × HH, 0% of HW × HW, 4% of WH × WH, and 30% of WW × WW F2 families were created with a misclassified parent (Table 4). Both the number of F2 inbred and
misclassified families in 2001 and 2002 were exacerbated due to a few F1 families in the W × W and W × H categories dominating as spawners in 2001 and 2002 (Table 3). 4. Discussion There are many opportunities for experimental errors to occur when conducting experiments that involve complex mating designs and large numbers of families. Using external markers to track groups or families has been the standard for many years. However, with the recent advances in molecular techniques researchers now have additional tools to help track groups, families, and even individuals with more precision than with external marks alone. We used microsatellite analysis to verify the category of returning F1 experimental fish and investigate inadvertent inbreeding in the F2 crosses created during a hatchery wild interaction experiment conducted at LPW hatchery starting in 1996. Because of the small numbers of wild females and males used and the simple experimental design, we were able to reconstruct a pedigree for the F1 W × W, W × H, H × W, H × H spawners, even in the absence of DNA samples from the hatchery parents and lack of information for which of 35 wild fish contributed gametes to the LPW experiment. Analysis of the pedigree revealed that two F1 half-sib families were misclassified, which probably occurred as a result of a switch between incubator cells either when eggs were placed in the tray, or when families were moved between incubator trays at egg picking. The switch led to these family groups being misidentified during culture and subsequently marked with the inappropriate codedwire tag. We also observed one H × W and one W × H F1 individuals that were misclassified as W × H and H × W, respectively. This seems consistent with movement of a few individuals within the vertical incubator trays due to leaky seals around some cells, which were observed during hatching. There were also a small number of individual in the H × W and W × H categories which had genotypes that could not be traced to a particular wild parent. This was due to lack of information for one or more loci for these individuals. Such events led to the corruption of some families in every F2 category except the 2001 and 2002 HH ×HH controls and the 2002 HW × HW line. To what degree corruption of lines influenced the experimental results is difficult to quantify. However, one could speculate that the homogenizing of experimental lines would bias results towards type 2 statistical errors and decrease the power of statistical comparisons. Our parentage analysis has been used to validate data from F1 fish produced in 1996 and returning in subsequent years. These data form the basis for the estimation of hatchery impacts on important life history traits such as female maturation timing, fecundity and egg size (Joyce et. al., unpublished data); however, because of the corrupted families statistical power was limited. A loss of genetic diversity due to inbreeding can also influence fitness traits if essential alleles are lost or deleterious alleles are
A.K. Gray et al. / Aquaculture 280 (2008) 71–75
expressed (Nevo et al., 1986; Emlen, 1991). After one generation of full-sib mating (inbreeding coefficient [F] = 0.25), rainbow trout show an increase in the number of crippled fry, and a decrease in feed conversion efficiency, growth rate and fry survival (Kincaid, 1976a). Juveniles and adults had a slower growth rate (Kincaid, 1976b). The reduction of fitness traits by close inbreeding could influence experimental results if not accounted for. Inbreeding was a concern in the F2 families due to the small number of wild females and males used to create the F1 generation. The pedigree analysis determined that this concern was warranted; differential family contribution at spawning in each of the H × H, H × W, W × H, and W × W F1 lines contributed to a number of full-sib families in the 2001 and 2002 HH × HH, HW × HW, WH × WH, and WW × WW F2 lines. The small numbers of wild fish use in this experimental design led to an inadvertent increase in inbreeding. This situation would have been avoided by researchers if they had increased the number of wild spawners. However, in many cases collections of wild gametes for experimental purposes is complicated by their remote locations, small population sizes, or endangered status. It should be emphasized that in experimental designs which unavoidably use small numbers of fish, researchers should incorporate a method to track individual families to avoided unintended inbreeding. Although experimental design and execution bear equal responsibility for the validity of an experiment, execution error can intrude at more points in an experiment and is often more subtle than design error (Hurlbert, 1984). Whereas uncovering execution error in experimental data is not a pleasant experience for any researcher, the importance of detecting such error cannot be overstated. Recent papers such as Hedrick et al. (2000) and Gilk et al. (2004) confirm the fact that experimental execution errors do occur. In our case, both problems of inbreeding and misclassification would have gone unnoticed and could have influenced the results of the experiment if the parental origins were not verified using a DNA-based approach. External marks alone were not sufficient. References Danzmann, R.G., 1997. PROBMAX: a computer program for assigning unknown parentage in pedigree analysis from known genotypic pools of parents and progeny. Journal of Heredity 88, 333.
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Emlen, J.M., 1991. Heterosis and outbreeding depression: a multilocus model and an application to salmon production. Fisheries Research 12, 187–212. Gilk, S.E., Wang, I.A., Hoover, C.L., Smoker, W.W., Taylor, S.G., Gray, A.K., Gharrett, A.J., 2004. Outbreeding depression in hybrids between spatially separated pink salmon, Oncorhyncus gorbuscha, populations: marine survival, homing ability, and variability in family size. Enviromental Biology of Fishes 69, 287–297. Gjerde, B., Gunnes, K., Gjedrem, T., 1983. Effect of inbreeding on survival and growth in rainbow trout. Aquculture 34, 327–332. Herbinger, C.M., O’Reilly, P.T., Doyle, R.W., Wright, J.M., O’Flynn, F., 1999. Early growth performance of Atlantic salmon full-sib families reared in single family tanks versus in mixed family tanks. Aquaculture 173, 105–116. Hedrick, P.W., Hedgecock, D., Hamelberg, S., Croci, S.J., 2000. The impact of supplementation in winter-run chinook salmon on effective population size. Journal of Heredity 91 (2), 112–116. Hurlbert, S.H., 1984. Pseudoreplication and the design of ecological field experiments. Ecological Monographs 54 (2), 187–211. Jackson, T.R., Martin-Robichaud, D.J., Reith, M.E., 2003. Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock. Aquaculture 220, 245–259. Jones, A.G., 2005. Gerud 2.0: a computer program for the reconstruction of parental genotypes from half-sib progeny arrays with known or unknown parents. Molecular Ecology Notes 5, 708–711. Kalinowski, S.T., Wagner, A.P., Taper, M.L., 2006. ML-Relate: a computer program for maximum likelihood estimation of relatedness and relationship. Molecular Ecology Notes 6, 576–579. Kincaid, H.L., 1976a. Effects of inbreeding on rainbow trout populations. Transactions of the American Fisheries Society 105, 273–280. Kincaid, H.L., 1976b. Inbreeding in rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 33, 2420–2426. Kincaid, H.L., 1983. Inbreeding in fish populations used for aquaculture. Aquaculture 33, 215–227. Nelson, R.J., Beacham, T.D., 1999. Isolation and cross species amplification of microsatellite loci useful for study of Pacific salmon. Animal Genetics 30, 228–229. Nevo, E., Noy, R., Lavie, B., Beiles, B., Muchtar, S., 1986. Genetic diversity and resistance to marine pollution. Biolology Journal of the Linnean Society 29, 139–144. Norris, A.T., Bradley, D.G., Cunningham, E.P., 2000. Parentage and relatedness determination in farmed Atlantic salmon (Salmo salar) using microsatellite markers. Aquaculture 182, 73–83. O’Connell, M., Danzmann, R.G., Cornuet, J., Wright, J.M., Ferguson, M.M., 1997. Differentiation of rainbow trout (Oncorhynchus mykiss) populations in Lake Ontario and the evaluation of the stepwise mutation and infinite allele mutation models using microsatellite variability. Canadian Journal of Fisheries and Aquatic Sciences 54, 1391–1398. Olsen, J.B., Bentzen, P., Seeb, J.E., 1998. Characterization of seven microsatellite loci derived from pink salmon. Molecular Ecology 7, 1083–1090. Rodgveller, C.J., Smoker, W.W., Gray, A.K., Joyce, J.E., Gharret, A.J., 2005. Effects of inbreeding and family origin on variation of size of Chinook salmon Oncorhynchus tshawytscha fry. Alaska Fisheries Research Bulletin 11 (2), 73–81.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Unanticipated departures from breeding designs can be detected using microsatellite DNA parentage analyses Andrew K. Gray ⁎, John J. Joyce, Alex C. Wertheimer National Marine Fisheries Service, Auke Bay Laboratory, 11305 Glacier Highway, Juneau, Alaska 99801, USA
A R T I C L E
I N F O
Article history: Received 6 September 2006 Received in revised form 24 March 2008 Accepted 11 April 2008 Keywords: Oncorhynchus tshawytscha Chinook Microsatellite Parentage analysis Pedigree
A B S T R A C T Using microsatellite analysis, a partial pedigree was constructed from an experiment designed to evaluate differences between hatchery Chinook salmon (Oncorhynchus tshawytscha) and their wild donor stocks. First generation (F1) progeny were created by crossing both hatchery and wild females with hatchery and wild males to create hybrid and control lines. The pedigree analysis revealed an accidental switch of a half-sib control and hybrid F1 family, which most likely occurred during incubation and led to the misclassification of individuals in those families. The experimental error propagated into the F2 lines. The pedigree also revealed a number of unintended full-sib matings in the F2 crosses. This resulted from the small numbers (5 females, 14 males) of wild fish available for the experiment and differential family survival of the F1 fish. Because researchers used group-specific physical marks to track typed crosses and not family groups, and because physical marks cannot detect inadvertent movement of fish until they are large enough to mark, experimental error occurred that would have influenced the results had a DNA-based analysis not been employed to verify the integrity of the experiment. Published by Elsevier B.V.
1. Introduction Aquaculture researchers often use external physical marks (e.g., fin clipping, spaghetti tags, PIT tags, coded-wire tags) to track groups of experimental fish. External physical marks are relatively easy to apply and are a cost-effective way to mark large numbers of fish, which make them attractive to researchers. However, for all the advantages, external physical marks used to track experimental fish have disadvantages that limit their utility. One disadvantage of external marks is that most fish and other aquatic animals are too small to be marked at hatching and marks can only be applied after individuals are sufficiently large (Herbinger et al., 1999). Consequently, experimental integrity can be compromised by the inability to track individuals, families, or groups from fertilization of gametes without error until the fishes are large enough to be marked. Gilk et al. (2004) demonstrated this problem during a study of outbreeding depression in even-and odd-year pink salmon. Although control and hybrid first generation (F1) progeny were marked with distinct fin marks upon release, when returning F1 adults were genotyped using microsatellite DNA analysis, 58 out of 1266 individuals were identified that did not correspond to any parental combinations used in the original matings. Two individuals marked as hybrids were identified as controls. The investigators were able to use this information to avoid corruption of F1 data and subsequent F2 crosses. In another paper, Hedrick et al. (2000) described the detection ⁎ Corresponding author. Tel.: +1 907 789 6047; fax: +1 907 789 6094. E-mail address: [email protected] (A.K. Gray). 0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2008.04.021
of classification errors within experimental groups of winter-run Chinook salmon (Oncorhynchus tshawytscha) in the Sacramento River, California. Using microsatellite DNA analysis researchers discovered that a number of non-winter-run Chinook salmon had been accidentally used as spawners for the winter-run supplementation program. Lack of pedigree information can potentially confound the effects for which a study was designed. In order to track families with external marks, progeny from family groups must be isolated and reared together until they are large enough to be marked. This approach is both labor and space intensive (Norris et al., 2000), and for these reasons, many researchers choose not to or are unable to collect family information. However, in the absence of pedigree information researchers run the risk of inadvertent matings of related individuals resulting in inbreeding, which in salmonids includes depression of survival and growth traits (Gjerde et al., 1983; Kincaid, 1983). A study by Jackson et al. (2003) underlines this point. Investigators used five microsatellites to assess the parentage of an F1 broodstock of Atlantic halibut (Hippoglossus hippoglossus) derived from wild adults. They observed that full and half-sib groups were disproportionately represented and only 36% of the attempted parental crosses were represented. As Rodgveller et al. (2005) demonstrated in size data from Chinook salmon, without pedigree information researchers also lack variability data within families. In this instance the magnitude of family variation was approximately as large as the variation among individuals, suggesting that in some cases much of the experimental variation may be attributed to family origin, and not to the treatment being tested. In 1996, an experiment to evaluate differences between hatchery Chinook salmon and their wild donor stocks after 20 years of hatchery
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culture was designed and implemented at the Little Port Walter (LPW) marine research station located on southern Baranof Island in Southeast Alaska. At the time DNA-based analysis was not available to track parentage, so coded-wire tags were used to track four experimental categories of fish. In 2002, microsatellite genotyping became available to the researchers and was used to accomplish two tasks: the first was to develop a pedigree and verify to crosstype the returning 2001 and 2002 F1 spawners; the second was to check for inadvertent inbreeding in the F2 lines.
Table 1 Number of F1 adults returning to hatchery, used as broodstock, and sampled for DNA 2001 F1 Adults Categories
Returns
Broodstock
Sampled for DNA
H×H H×W W×H W×W
162 322 103 61
111 85 54 46
62 77 45 40
2002 F1 Adults
2. Materials and methods
Categories
Returns
Broodstock
Sampled for DNA
2.1. Crosses and incubation procedures
H×H H×W W×H W×W
77 94 66 29
46 48 36 17
33 48 36 21
On August 12, 1996, gametes from 20 females and 18 males were collected from LPW Chickamin River stock hatchery returns. At the same time, with the help of the Alaska Department of Fish and Game personel (ADF&G), mature gametes from 5 wild female and 14 wild male Chinook salmon were collected from Barrier Creek, a tributary of the South Fork of the Chickamin River. The gametes were then transferred by helicopter to LPW. Four types of F1 progeny were made by crossing hatchery and wild females with hatchery and wild males in two by two, one by two, two by one, and one by one blocks (Fig. 1). The progeny types were: 1) hatchery
female × hatchery male (H× H), 2) hatchery female× wild male (H× W), 3) wild female × hatchery male (W× H), and 4) wild female × wild male (W × W). Each set of half-sib families was assigned to cells within a single tray and placed in vertical tray incubators. Prior to hatching, families were consolidated by type within a single tray in order to minimize the effects of potential movement between tray cells. At hatching, families of each category were transferred from incubators and reared communally in four separate freshwater vertical raceways (VRs) according to the type of cross. Fish were reared approximately 1 year in the VRs, tagged with category specific coded-wire tags, and released to the estuary at LPW in May of 1998. In 2001 and 2002, returning adult F1 were collected and a portion of these were used as broodstock (Table 1). The type of cross of each fish was confirmed by coded-wire tag detection and code verification before F2 crosses were conducted. Heart tissue samples were collected from F1 fish at spawning and preserved in 100% EtOH (Table 1). DNA analysis of the F1 fish was conducted on the samples after the F2 crosses were made. Six types of F2 progeny (HH × HH, HH × WW, WW × HH, WH × WH, HW × HW, and WW × WW) were made by crossing F1 spawners according to tag code in 1 by 1 and 1 by 2 blocks. DNA analysis has not been performed on returning F2 fish. 2.2. Microsatellite and parentage analysis
Fig. 1. The 1996 experimental design used to create F1 control and hybrid groups. Five hatchery (H) females and males were crossed with five wild (W) females and males creating five 2 by 2 blocks. Nine hatchery females were crossed with nine hatchery and nine wild males creating nine 1 by 2 blocks. Hatchery controls were also created by crossing hatchery females and males in two 2 by 1, and two 1 by 1 blocks.
During the collection of wild gametes in August 1996, scales from 35 wild Chickamin River (WCR) Chinook salmon spawners (16 females and 19 males) were collected and placed on scale cards by the ADF&G personnel. These samples included scales from the wild spawners used for the LPW experiment, but the log book describing which of the 35 WCR fish contributed gametes to the LPW experiment was incomplete. Consequently DNA was isolated from all 35 WCR fish because each needed to be considered as a possible parent to the returning LPW 2001 and 2002 F1 H × W, W × H, and W × W LPW experimental fish. Tissues for DNA analysis were not sampled from the 1996 LPW hatchery brood stock. Total DNA was isolated from both scale and preserved heart tissue using DNeasy ™ Tissue Kit (QIAGEN, Inc., Valencia, CA) following the procedure for isolating DNA from mammal tissue provided in the kit instructions. Two scales or approximately 25 mg of heart tissue per individual provided sufficient DNA for the analyses. Polymerase chain reaction (PCR) amplification was done in 96-well microtitre plates in a DNA Engine™ (MJ Research, Inc., Reno, NV). Amplification reactions were in 10 μL volumes [10 mM Tris–HCl at pH 8.3, 50 mM KCl, 25 mM MgCl2, 2.5 mM each deoxyribonucleotide triphosphate (dNTP), 0.5 units Taq polymerase, 0.1–0.5 μM each primer, and 50–100 ng DNA template]. In addition to unlabeled forward and reverse primers for each locus, each mixture included a forward primer labeled with an infrared-sensitive dye, IRDye™ (LICOR, Inc., Lincoln, NE).
A.K. Gray et al. / Aquaculture 280 (2008) 71–75 Table 2 Expected and observed contribution of wild female and male gametes in F1 families Expected
Observed⁎
Categories
# of families
Female
Male
Female
Male
H×H H×W W×H W×W
20 14 5 5
0 0 5 5
0 14 0 5
0 0 5 5
0 14 1 4
⁎Recreated using microsatellite analysis.
After evaluating 10 microsatellite loci, 5 were chosen based on their ability to separate each of the 35 wild Chickamin Chinook salmon into unique genotypes. The five loci were: Ots100, Ots104, Ots107 (Nelson and Beacham, 1999), Ogo4 (Olsen et al., 1998), and Omy325 (O'Connell et al., 1997). In general, PCR conditions were as follows: 1 cycle at 95 °C for 3 min; 28 cycles at 95 °C for 30 s, 58 °C for 15 s, 72 °C for 15 s, and 1 cycle at 72 °C for 1 min. After amplification, DNA products were denatured by adding an equal volume of stop buffer (95% formamide, 0.1% Bromophenol Blue) and heating for 3 min at 95 °C. One μl of PCR product was loaded onto polyacrylamide denaturing gels composed of 6.5% KBPlus gel matrix (LI-COR, Inc., Lincoln, NE) in a reaction catalyzed by ammonium persulfate and TEMED (N,N,N',N' tetramethylethylenediamine). Alleles were separated electrophoretically and detected on an automated sequencer (LongReadIR 4200™, LI-COR, Inc., Lincoln, NE) in 1 × TBE running buffer, with running parameters 31.5 W, 1500 V, 35 mA, and 50 °C plate temperature. The size of microsatellite alleles was estimated using Saga™ Generation 2 automated microsatellite software (Version 3.0, LI-COR, Inc., Lincoln, NE). Allele sizes were estimated by comparing allele band patterns with IRD700TM or IRD800TM standard ladders (LI-COR, Inc., Lincoln, NE) and standardized using reference alleles. Parentage of returning W × W F1 spawners was deduced using PROBMAX (version 1.2, Danzmann, 1997) executable program PROBMAX2, which assigns progeny to a mixture of possible contributing parents given that the genotypes are known for both progeny and possible parents. The PROBMAX executable program PROBMAXN was used to test for possible missed assignments due to the presence of nonamplifying (null) alleles. The default tolerance levels and
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disallowing for mis-scoring were used for both executable programs. All known parental genotypes not considered by PROBMAX analyses were systematically examined to confirm PROBMAX assignments. Assignments using PROBMAX are based on known genotypes for the mother, father, and offspring and cannot make assignments when one parental genotype is unknown. Therefore returning F1 H × W and W × H fish were assigned a parent by systematically comparing their genotypes with the 35 WCR parental genotypes. Assignments were only made when one of the 35 possible parents had an allele that matched at each of the loci examined. Although we did not have tissue samples of the hatchery parents used as broodstock in 1996, paternal and maternal hatchery alleles were derived by comparing individual genotypes in each F1 W × H and F1 H × W family array with their known WW maternal and paternal genotypes respectively. Paternal and maternal hatchery alleles were first identified manually and then confirmed using the program GERUD 2.0 (Jones, 2005). GERUD 2.0 performs an exhaustive search of the minimal set of parents necessary to explain a set of offspring or ‘progeny array' known to have a single parent. For progeny arrays with multiple minimum parent solutions, GERUD 2.0 chooses the most likely solution based on Mendelian segregation probabilities. The inferred parental hatchery genotypes reconstructed by GERUD 2.0 were used to assign H × H F1 fish to families using PROBMAX2 with the same tolerance levels as stated above. PROBMAX could only be used to assign individuals to the 2 by 2 blocks (Fig. 1) because the program makes assignments based on both sets of parental genotypes. Since we had inferred genotypes for nine 1996 hatchery females reconstructed from nine H × W 1 by 2 blocks (Fig. 1), the F1 H × H fish not assigned by PROBMAX were analyzed manually and with the program ML Relate (Kalinowski et al., 2006), which uses maximum likelihood estimates to calculate relatedness and relationships between individuals of unknown ancestry, to create fullsib arrays. Each array was then assigned to reconstructed hatchery parent if all individuals in the array had a common allele at each locus with the inferred hatchery parental genotype. The progeny arrays which did not match any recreated hatchery parent were assumed to belong to one of the H × H blocks, from which we have no data to infer hatchery parental genotypes (Fig. 1). The parentage analysis provided information to establish whether inbreeding was imposed as a result of limited parents in the F2 crosses.
Table 3 Familiy origins of F1 progeny returning in 2001 and 2002 2 by 2 blocks
1 by 2 blocks
Tag code
Recreated Families
2001 Individuals
2002 Individuals
Tag code
Recreated Families
2001 Individuals
2002 Individuals
H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×H H×W W×H W×W H×W
H1 × H1 H1 × W1 W1 × H1 W1 × W1 H2 × H2 H2 × W2 W2 × H2 W2 × w2 H3 × H3 H3 × W3 W3 × H3 W3 × W3 H4 × H4 H4 × W4 W4 × H4 W4 × W4 H5 × H5 H5 × W5 W5 × W5⁎ W5 × H5⁎ W1H1⁎
5 8 20 19 7 3 4 0 10 4 7 8 1 1 8 3 4 5 3 10 1
7 2 20 5 4 11 9 4 2 6 0 1 3 3 4 8 2 3 3 3 0
H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W H×H H×W W×H W×H H×W
H6 × H6 H6 × W6 H7 × H7 H7 × W7 H8 × H8 H8 × W8 H9 × H9 H9 × W9 H10 × H10 H10 × W10 H11 × H11 H11 × W11 H12 × W12 H12 × W12 H13 × H13 H13 × W13 H14 × H14 H14 × W14 H10 × W10⁎ Unknown⁎⁎ Unknown⁎⁎
1 3 16 17 2 4 2 4 3 5 5 5 6 10 0 4 0 1 1 2 1
1 2 2 6 2 1 4 1 2 5 1 4 3 1 0 2 0 1 0 0 0
Individuals observed per tag code and recreated families. W = wild parent and H = Hatchery parent. ⁎ Misclassified by Tag code. ⁎⁎ Individuals for which genotype data was incomplete.
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3. Results
Table 4 The number of full-sib and misclassified F2 families created in 2001 and 2002
3.1. Parentage assignment
Families
HH × HH HH × WW WW × HH WH × WH HW × HW WW × WW
2001 2002 Total
59 41 100
31 32 63
34 18 52
54 38 92
78 48 126
23 20 43
Full-sib Families 2001 2002 Total
5 2 7
0 0 0
0 0 0
10 15 25
6 5 11
9 4 13
Misclassified Families 2001 2002 Total
0 0 0
3 10 13
8 1 9
7 4 11
2 0 2
8 6 14
PROBMAX analysis of the 2001 and 2002 W × W F1 control line indicated that four WCR parental pairs could be assigned to the majority of the fish. The W × W F1 fish that were not assigned a parental pair were systematically assessed to determine if any of the 35 WCR fish could have contributed as a parent. All of the unassigned fish had one allele at each locus that could be assigned to a single WCR female, suggesting they were actually W × H fish. Because the same five females were used to create the W × W and W × H F1 lines, the above discrepancy suggested an inadvertent mixing of experimental lines (Table 2). Upon the discovery of a possible mixing of experimental lines based on the W × W F1 parentage assignments, the rest of the 2001 and 2002 F1 experimental fish were examined to determine parentage. Of the 81 W × H F1 individuals assessed, 6 fish (3 from 2001 and 3 from 2002) could be assigned to a single WCR pair. The female from the WCR pair was the same fish that was assigned to the W × H F1 individuals found in the W × W offspring (Table 3). By systematically comparing allele scores of the 75 W × H F1 individuals that were not assigned a parental pair by PROBMAX, 65 could be assigned to a total of 4 WCR females and 1 fish could be assigned to one WCR male. Two W × H F1 fish were not assigned to any of the possible WCR parents (Table 3). PROBMAX analysis of 124 H × W F1 revealed zero fish that could be assigned to any WCR pair; however, 122 individuals were systematically traced back to 14 WCR males (Table 3) which is consistent with the mating design (Table 2). One H × W F1 fish was assigned to a WCR female and 1 H × W F1 fish was not assigned to any of the possible WCR parents (Table 3). A WCR pair or single individual was not assigned to any of the 112 H × H F1 fish that were analyzed systematically or with PROBMAX. Parentage analysis taking into account nulls did not produce any unique assignments across any of the experimental lines. The retrospective analysis using genotypes of the F1 individuals revealed that no more than 5 of 16 possible wild parental females and 14 of 19 possible wild parental males could account for the majority of the F1 individuals coded as W × W, W × H, and H × W, which is consistent with the original experimental design and lends strength to the reconstructed pedigree. Reconstruction of hatchery parental alleles using W × H and H × W F1 progeny arrays and the program GERUD 2.0, followed by analysis of the 112 H × H F1 using PROBMAX, allocated 39 H × H F1 fish among the 2 × 2 experimental blocks (Table 3). Using ML Relate and manual conformation of progeny arrays, 56 H × H F1 fish were assigned among the 1by 2 H × W blocks (Table 3). The remaining 17 H × H F1 fish were separated into 4 full-sib arrays and are assumed to belong to one of the H × H blocks (Fig. 1). 3.2. Inbreeding and misclassification error in F2 lines The pedigree analysis revealed that 8% of HH × HH, 8% of HW × HW, 19% of WH × WH, and 39% of WW × WW F2 families were created by full-sib matings during spawning in 2001 (Table 4). Similarly in 2002, 4% of HH × HH, 10% of HW × HW, 39% of WH × WH, and 20% of WW × WW F2 families were created by full-sib matings (Table 4). The WW × HH and HH × WW F2 families in 2001and 2002 did not contain any full-sib families (Table 4). With the exception of HH × HH F2 types, most F2 cross types created in 2001 and 2002 contained misclassified families. In 2001, 10% of HH × WW, 23% of WW × HH, 3% of HW × HW, 12% of WH × WH, and 35% of WW × WW F2 families were created with a misclassified parent (Table 4). In 2002, 31% of HH × WW, 5% of WW × HH, 0% of HW × HW, 4% of WH × WH, and 30% of WW × WW F2 families were created with a misclassified parent (Table 4). Both the number of F2 inbred and
misclassified families in 2001 and 2002 were exacerbated due to a few F1 families in the W × W and W × H categories dominating as spawners in 2001 and 2002 (Table 3). 4. Discussion There are many opportunities for experimental errors to occur when conducting experiments that involve complex mating designs and large numbers of families. Using external markers to track groups or families has been the standard for many years. However, with the recent advances in molecular techniques researchers now have additional tools to help track groups, families, and even individuals with more precision than with external marks alone. We used microsatellite analysis to verify the category of returning F1 experimental fish and investigate inadvertent inbreeding in the F2 crosses created during a hatchery wild interaction experiment conducted at LPW hatchery starting in 1996. Because of the small numbers of wild females and males used and the simple experimental design, we were able to reconstruct a pedigree for the F1 W × W, W × H, H × W, H × H spawners, even in the absence of DNA samples from the hatchery parents and lack of information for which of 35 wild fish contributed gametes to the LPW experiment. Analysis of the pedigree revealed that two F1 half-sib families were misclassified, which probably occurred as a result of a switch between incubator cells either when eggs were placed in the tray, or when families were moved between incubator trays at egg picking. The switch led to these family groups being misidentified during culture and subsequently marked with the inappropriate codedwire tag. We also observed one H × W and one W × H F1 individuals that were misclassified as W × H and H × W, respectively. This seems consistent with movement of a few individuals within the vertical incubator trays due to leaky seals around some cells, which were observed during hatching. There were also a small number of individual in the H × W and W × H categories which had genotypes that could not be traced to a particular wild parent. This was due to lack of information for one or more loci for these individuals. Such events led to the corruption of some families in every F2 category except the 2001 and 2002 HH ×HH controls and the 2002 HW × HW line. To what degree corruption of lines influenced the experimental results is difficult to quantify. However, one could speculate that the homogenizing of experimental lines would bias results towards type 2 statistical errors and decrease the power of statistical comparisons. Our parentage analysis has been used to validate data from F1 fish produced in 1996 and returning in subsequent years. These data form the basis for the estimation of hatchery impacts on important life history traits such as female maturation timing, fecundity and egg size (Joyce et. al., unpublished data); however, because of the corrupted families statistical power was limited. A loss of genetic diversity due to inbreeding can also influence fitness traits if essential alleles are lost or deleterious alleles are
A.K. Gray et al. / Aquaculture 280 (2008) 71–75
expressed (Nevo et al., 1986; Emlen, 1991). After one generation of full-sib mating (inbreeding coefficient [F] = 0.25), rainbow trout show an increase in the number of crippled fry, and a decrease in feed conversion efficiency, growth rate and fry survival (Kincaid, 1976a). Juveniles and adults had a slower growth rate (Kincaid, 1976b). The reduction of fitness traits by close inbreeding could influence experimental results if not accounted for. Inbreeding was a concern in the F2 families due to the small number of wild females and males used to create the F1 generation. The pedigree analysis determined that this concern was warranted; differential family contribution at spawning in each of the H × H, H × W, W × H, and W × W F1 lines contributed to a number of full-sib families in the 2001 and 2002 HH × HH, HW × HW, WH × WH, and WW × WW F2 lines. The small numbers of wild fish use in this experimental design led to an inadvertent increase in inbreeding. This situation would have been avoided by researchers if they had increased the number of wild spawners. However, in many cases collections of wild gametes for experimental purposes is complicated by their remote locations, small population sizes, or endangered status. It should be emphasized that in experimental designs which unavoidably use small numbers of fish, researchers should incorporate a method to track individual families to avoided unintended inbreeding. Although experimental design and execution bear equal responsibility for the validity of an experiment, execution error can intrude at more points in an experiment and is often more subtle than design error (Hurlbert, 1984). Whereas uncovering execution error in experimental data is not a pleasant experience for any researcher, the importance of detecting such error cannot be overstated. Recent papers such as Hedrick et al. (2000) and Gilk et al. (2004) confirm the fact that experimental execution errors do occur. In our case, both problems of inbreeding and misclassification would have gone unnoticed and could have influenced the results of the experiment if the parental origins were not verified using a DNA-based approach. External marks alone were not sufficient. References Danzmann, R.G., 1997. PROBMAX: a computer program for assigning unknown parentage in pedigree analysis from known genotypic pools of parents and progeny. Journal of Heredity 88, 333.
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Emlen, J.M., 1991. Heterosis and outbreeding depression: a multilocus model and an application to salmon production. Fisheries Research 12, 187–212. Gilk, S.E., Wang, I.A., Hoover, C.L., Smoker, W.W., Taylor, S.G., Gray, A.K., Gharrett, A.J., 2004. Outbreeding depression in hybrids between spatially separated pink salmon, Oncorhyncus gorbuscha, populations: marine survival, homing ability, and variability in family size. Enviromental Biology of Fishes 69, 287–297. Gjerde, B., Gunnes, K., Gjedrem, T., 1983. Effect of inbreeding on survival and growth in rainbow trout. Aquculture 34, 327–332. Herbinger, C.M., O’Reilly, P.T., Doyle, R.W., Wright, J.M., O’Flynn, F., 1999. Early growth performance of Atlantic salmon full-sib families reared in single family tanks versus in mixed family tanks. Aquaculture 173, 105–116. Hedrick, P.W., Hedgecock, D., Hamelberg, S., Croci, S.J., 2000. The impact of supplementation in winter-run chinook salmon on effective population size. Journal of Heredity 91 (2), 112–116. Hurlbert, S.H., 1984. Pseudoreplication and the design of ecological field experiments. Ecological Monographs 54 (2), 187–211. Jackson, T.R., Martin-Robichaud, D.J., Reith, M.E., 2003. Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock. Aquaculture 220, 245–259. Jones, A.G., 2005. Gerud 2.0: a computer program for the reconstruction of parental genotypes from half-sib progeny arrays with known or unknown parents. Molecular Ecology Notes 5, 708–711. Kalinowski, S.T., Wagner, A.P., Taper, M.L., 2006. ML-Relate: a computer program for maximum likelihood estimation of relatedness and relationship. Molecular Ecology Notes 6, 576–579. Kincaid, H.L., 1976a. Effects of inbreeding on rainbow trout populations. Transactions of the American Fisheries Society 105, 273–280. Kincaid, H.L., 1976b. Inbreeding in rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 33, 2420–2426. Kincaid, H.L., 1983. Inbreeding in fish populations used for aquaculture. Aquaculture 33, 215–227. Nelson, R.J., Beacham, T.D., 1999. Isolation and cross species amplification of microsatellite loci useful for study of Pacific salmon. Animal Genetics 30, 228–229. Nevo, E., Noy, R., Lavie, B., Beiles, B., Muchtar, S., 1986. Genetic diversity and resistance to marine pollution. Biolology Journal of the Linnean Society 29, 139–144. Norris, A.T., Bradley, D.G., Cunningham, E.P., 2000. Parentage and relatedness determination in farmed Atlantic salmon (Salmo salar) using microsatellite markers. Aquaculture 182, 73–83. O’Connell, M., Danzmann, R.G., Cornuet, J., Wright, J.M., Ferguson, M.M., 1997. Differentiation of rainbow trout (Oncorhynchus mykiss) populations in Lake Ontario and the evaluation of the stepwise mutation and infinite allele mutation models using microsatellite variability. Canadian Journal of Fisheries and Aquatic Sciences 54, 1391–1398. Olsen, J.B., Bentzen, P., Seeb, J.E., 1998. Characterization of seven microsatellite loci derived from pink salmon. Molecular Ecology 7, 1083–1090. Rodgveller, C.J., Smoker, W.W., Gray, A.K., Joyce, J.E., Gharret, A.J., 2005. Effects of inbreeding and family origin on variation of size of Chinook salmon Oncorhynchus tshawytscha fry. Alaska Fisheries Research Bulletin 11 (2), 73–81.