- Email: [email protected]
No evidence for overdominance at the phosphoglucomutase-2 locus in Pacific oysters (Crassostrea gigas) from New Zealand
Aquaculture 259 (2006) 74 – 80 www.elsevier.com/locate/aqua-online
No evidence for overdominance at the phosphoglucomutase-2 locus in Pacific oysters (Crassostrea gigas) from New Zealand Ann R. Wood ⁎, Jonathan P.A. Gardner Centre for Marine Environmental and Economic Research, School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand Received 28 November 2005; received in revised form 11 May 2006; accepted 15 May 2006
Abstract Overdominance for enzyme specific activity at the phosphoglucomutase-2 (PGM-2) locus has been demonstrated for wild, adult Pacific oysters (Crassostrea gigas) that are formed from heterozygous combinations of the allele at highest frequency and any other allele at this locus. Overdominant genotypes have been shown to have higher catalytic capacities than other genotypes, and consequently it was proposed that this effect might account for genotype-dependent weight differences among Pacific oysters. From an aquaculture perspective, it would be relatively easy for a selective breeding programme to exploit a genotype-dependent effect on production traits (growth rate, wet body weight) if this phenomenon could be demonstrated among hatchery-reared oysters. However, a previous test of this failed to detect either an overdominant effect or a genotype-dependent effect among wild juvenile oysters (9 months of age), among adults of this same batch 12 months later, or among juveniles (5 months of age) from 8 full-sib hatchery-reared families. In the present paper, we extend this work by examining the relationship between PGM-2 genotype and production traits in adult market-size Pacific oysters from five full-sib families specifically selected because they contain putatively overdominant and other genotypic combinations. Within-families, there were significant weight differences among genotypes, but there was no evidence of a consistent PGM-2 genotype-dependent effect, overdominant or otherwise, on wet tissue weight, dry shell weight or total weight (=wet tissue weight plus dry shell weight). Among families, tests of pooled data showed no significant difference between putative overdominant and other genotypes for wet tissue weight, dry shell weight or total weight. We conclude that there is no evidence on which to base a Pacific oyster-breeding programme using PGM-2 as a marker for increased production. © 2006 Elsevier B.V. All rights reserved. Keywords: Pacific oyster; Crassostrea gigas; Overdominance; Phosphoglucomutase; PGM-2; Growth
1. Introduction Positive correlations between allozyme heterozygosity and various fitness-related traits have been observed frequently in many species, and in particular among marine bivalves (e.g. Fujio, 1982; Gaffney and Scott, ⁎ Corresponding author. Tel.: +64 4 463 5578; fax: +64 4 463 5331. E-mail address: [email protected] (A.R. Wood). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.05.015
1984; Mitton and Grant, 1984; Gentili and Beaumont, 1988; Britten, 1996), and various suggestions have been advanced for genetic mechanisms by which this heterozygote advantage (=heterosis) can occur. Debate has centred around two main hypotheses: overdominance, which involves the direct action of selection on allozyme genotypes and superiority of heterozygotes at one or more loci influencing fitness, and associative overdominance, where allozyme loci are not under
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
selection directly but act as markers of selection against deleterious recessive alleles at linked loci which influence fitness (e.g., Koehn et al., 1988; Gaffney et al., 1990; Zouros, 1993; Pogson and Zouros, 1994; Thelen and Allendorf, 2001). Regardless of its mechanistic basis, the utility of the heterosis phenomenon to the aquaculture production of shellfish and other species has long been of interest (e.g., Gosling, 1982; Gaffney and Scott, 1984; Hedgecock et al., 1995, 1996, 1997). In Japanese populations of the Pacific oyster, Crassostrea gigas, Fujio (1982) described a positive correlation between body weight and heterozygosity at five enzyme loci, which translated into a pronounced growth advantage for heterozygotes compared to homozygotes. At the phosphoglucomutase-2 (PGM-2) locus, the mean body weight of heterozygotes across all 20 populations was 21% greater than that of homozygotes. Because multi-locus heterozygote deficiencies were also observed, Fujio (1982) suggested that inbreeding may have resulted in an excess of homozygotes and thus of individuals homozygous for deleterious genes. More recently, overdominance of enzyme specific activity in C. gigas was reported at the PGM-2 locus (Pogson, 1991). Heterozygotes for the most common allele and any other allele showed specific activities approximately 20% greater than those observed for all homozygotes and all other heterozygotes. Pogson (1991) suggested that the heterozygosity/growth correlation observed for C. gigas at the PGM-2 locus by Fujio (1982) might be explained by the greater specific activities of heterozygotes for the most common allele. If the specific-activity overdominance observed by Pogson (1991) translates to faster growth or larger market size, it would be relatively easy for a hatcherybased aquaculture programme to breed overdominant genotypes to take advantage of this. To investigate this possibility, Gardner and Lobkov (2005) looked, but found no support, for a relationship between PGM-2 genotype and body weight in juvenile hatcheryproduced New Zealand C. gigas. Gardner and Lobkov (2005) suggested that an overdominant effect might for some reason only be visible in adult oysters (as analysed by Fujio, 1982; Pogson, 1991) and not in juveniles. In the present study, we have investigated further this putative overdominant effect by analysing individual PGM-2 genotypes and body weights for adult marketsize Pacific oysters from five new hatchery-produced full-sib families, specifically selected for testing because they each contain putative overdominant and other
75
PGM-2 genotypes to facilitate within-family and among-family comparisons. 2. Materials and methods 2.1. Hatchery oysters During December 2003, 23 full-sib families were produced at the Cawthron Institute's Glenhaven hatchery, from wild parents collected from Kaipara Harbour in northern New Zealand. Tissue from the parents was stored at − 80 °C. Offspring were reared in the hatchery for the first month, transferred to nursery upwellers for a further 2 months, and then grown in intertidal grow-out bags at Orongo Bay (Bay of Islands, northern New Zealand) from March 2004 until they were harvested in December 2004. Particularly small larvae were lost from each family during routine sieving in the hatchery, but no further culling of oysters took place. Samples of digestive gland were taken from each parent and analysed for PGM-2 genotype as described below. As far as possible, the observed genotypes were matched to those found by Gardner and Lobkov (2005) and Pogson (1989, 1991). Allele frequencies for the parent oysters genotyped in this study were similar to those found in previous studies (Buroker et al., 1975, 1979; Ozaki and Fujio, 1985; Smith et al., 1986; Pogson, 1989, 1991; Gardner and Lobkov, 2005). It therefore seems unlikely that the most common allele observed in the present study (Pgm-23) is not the same as the most common allele observed by Pogson (1989, 1991). Based on parent genotypes, five families (F800, F805, F810, F816 and F818) that would allow within family comparison of putative overdominant and other genotypes were chosen for this study. In the present context, “overdominant” refers to the Pgm-23 allele in combination with any other allele, and “other” refers to all other PGM-2 genotypic combinations including heterozygous combinations not involving the Pgm-23 allele. 2.2. Sample processing Approximately 110 haphazardly selected 12-monthold oysters from each of the five families were couriered on ice to Victoria University of Wellington in December 2004 and stored at −20 °C for 1–2 months prior to analysis. A total of 538 oysters were analysed (mean ± SD of 107.6 ± 2.7 per family). Small batches of oysters were removed from the freezer and held on ice during processing. Shells were opened whilst the oysters were still frozen and a sample of digestive gland was taken for
76
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
allozyme electrophoresis. Dry shell weight and blotted wet soft tissue weight were determined to 0.001 g. The digestive gland sample weight was estimated after preparation of allozyme wicks (see below), by centrifuging the sample for 10 min at 13 000 rpm, removing the supernatant (comprising the added distilled water plus the original water content of the sample, minus the liquid absorbed by the wick), weighing the pelleted tissue left in the microtube and subtracting the weight of the empty microtube. An alternative method, weighing samples whilst they were still frozen, gave similar results (results not shown). 2.3. Allozyme electrophoresis Allozyme electrophoresis methods were essentially the same as those of Gardner and Lobkov (2005), using approximately 100 mg of digestive gland tissue for each individual. Alleles were numbered Pgm-21 to Pgm-25 with Pgm-21 being the most anodal, following Gardner and Lobkov (2005). 2.4. Data analysis PGM-2 genotypes of parents were used to determine the expected genotypes of offspring in each family. The observed genotypic frequencies in the offspring were tested against expected frequencies calculated on assumptions of Mendelian inheritance (ratios of 1:1, 1:2:1 or 1:1:1:1), using a chi-square “goodness-of-fit” test. The software package SPSS (v 12.0, SPSS Inc., Chicago, Illinois, USA) was used for analysis of dry shell weight, blotted wet soft tissue weight (wet tissue weight) and total weight (dry shell weight+ wet tissue weight) estimates. For each family a one-way ANOVA was used to test for differences in the weight variables among genotypes. In all cases, Levene's test for homogeneity of variances among genotype classes gave non-significant results, and in all cases except one, the weight variables were normally distributed (except F810 — wet tissue weight (Kolmogorov-Smirnov test; P = 0.006) and total weight (P = 0.017)). For family F810, because of the nonnormally distributed weight variables, a non-parametric Kruskal-Wallis analysis of ranks was also used. In cases where ANOVA showed significant differences in weight variables among genotypes, Tukey's HSD post-hoc test was used to identify the location of significant differences. For families F805, F810 and F816 (which had more than 2 genotypes) and for pooled data from the five families, oysters were coded into two groups, “putative overdominant genotypes” and “all other genotypes”. t-tests were used to test for weight
differences between these two groups. Where appropriate, the sequential Bonferroni correction for multiple testing was employed (Rice, 1989). To allow for the possibility that our scoring did not match that from previous studies, rather than simply assuming that the allele at highest frequency in this study (our Pgm-23 allele) was the same as that observed by Pogson (1991), each allele in our study was individually tested for an overdominant effect. As appropriate, depending on homogeneity of variances and/or normal distribution of data, we used either a t-test (all alleles except Pgm-25) or the Mann Whitney U-test (only the Pgm-25 allele, for which Levene's test showed significant heterogeneity of variances) to test for classical overdominance. Heterozygotes for each allele in turn were tested against all other genotypes to examine the possible relationship between allelespecific overdominance and mean weight (dry shell, wet body, and total body weight). We tested the data from this study and that of Gardner and Lobkov (2005) for a difference in mean values for each weight variable between putative overdominant and other genotypes within each family using a randomization test procedure (Resampling Stats software, v5.0.2, Resampling Stats Inc., Arlington, Virginia, USA). This test assigns at random the individual weight values (dry shell, wet body or total body) to individual genotype scores. For each test, 100,000 randomizations of the data were performed to produce a distribution of the difference in mean values that should apply under the null hypothesis of no overdominant effect. Given this distribution, the probability of obtaining the observed difference between mean weights was calculated to permit a test of the effect (if any) of putative overdominant versus other genotypes on production characteristics. 3. Results PGM-2 genotypic frequencies observed in offspring were significantly different from those expected assuming Mendelian inheritance in three of the five families (Table 1). Family F800 exhibited a deficiency of the Pgm-23/4 and an excess of the Pgm-23/3 genotype. In F805, an excess of the Pgm-22/2 genotype was observed along with deficiencies of the Pgm-22/3 and Pgm-23/3 genotypes. In F816, genotypes Pgm-22/3 and Pgm-23/3 were approximately in accordance with expectations but there was an excess of the Pgm-22/4 and a deficit of the Pgm-23/4 genotypes. Genotype frequencies of families F810 and F818 were not significantly different from expectations.
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80 Table 1 Parent and offspring PGM-2 genotypes and results of χ2 test for observed vs. expected genotype frequencies in 5 hatchery-produced Crassostrea gigas families Family
PGM-2 genotype Dam
Sire
Offspring
800
3/4
3/3
805
2/3
2/3
810
3/4
4/5
816
3/4
2/3
818
3/3
1/3
3/3 3/4 2/2 2/3 3/3 3/4 3/5 4/4 4/5 2/3 2/4 3/3 3/4 1/3 3/3
Nexp
Nobs
χ2 test of genotype frequencies
54 54 27.5 55 27.5 24.5 24.5 24.5 24.5 25.8 25.8 25.8 25.8 54 54
78 30 63 32 15 26 19 28 25 26 41 29 7 52 56
χ2 = 21.333, P < 0.001 χ2 = 61.127, P < 0.001 χ2 = 1.837, P = 0.607
χ2 = 23.097, P < 0.001 χ2 = 0.148, P = 0.700
Nexp = number of oysters expected for each genotype assuming Mendelian inheritance. Nobs = number of oysters observed for each genotype. P values shown in bold typeface are statistically significant.
Three of the five families (F800, F805 and F810) exhibited mean values of dry shell weight, wet tissue weight and total body weight for putative overdom-
77
inant genotypes (those with allele Pgm-23) which were slightly greater than those for other genotypes, however, families F816 and F818 showed the opposite trend (Table 2). After within-family correction for multiple testing (Rice, 1989), only the test for wet tissue weight in F810 showed a statistically significant difference among genotypes. For F810 wet tissue weight, Tukey's HSD post-hoc test showed that the putative overdominant genotype Pgm-23/4 had significantly greater wet tissue weight than the other genotypes Pgm-24/4 and Pgm-24/5, but Pgm-23/5 was not significantly different from either Pgm-23/4, Pgm24/4 or Pgm-24/5. Within-family t-test comparisons of difference in mean weight between putative overdominant and other genotypes were significant in only two instances. For family F810 the mean wet tissue weight for putative overdominant genotypes was greater than the mean weight for other genotypes (Pgm-23/4 + Pgm-23/5 > Pgm24/4 + Pgm-24/5, P = 0.002), whereas for family F816 the mean total weight for other genotypes was greater than that for putatively overdominant genotypes (Pgm22/4 + Pgm-23/3 > Pgm-22/3 + Pgm-23/4 , P = 0.008). The same test on pooled data from all five families showed no significant difference between putative overdominant and other genotypes for mean dry shell weight (t = − 0.849, P = 0.397), mean wet tissue weight (t = − 0.704, P = 0.482) or mean total weight (t = − 1.068, P = 0.286).
Table 2 Mean estimates of dry shell weight, wet tissue weight and total weight for each genotype in 5 hatchery-produced Crassostrea gigas families Family
800 805
810
816
818
PGM-2 genotype
Class1
3/4 3/3 2/3 2/2 3/3 3/4 3/5 4/4 4/5 2/3 3/4 2/4 3/3 1/3 3/3
O N O N N O O N N O O N N O N
N2
30 78 32 (27) 63 (49) 15 (14) 26 (25) 19 28 (25) 25 (22) 26 7 (6) 41 (35) 29 (28) 52 56
Dry shell weight
Wet tissue weight
Total weight
Mean (g)
P value
Mean (g)
P value
Mean (g)
P value
26.648 24.725 22.528 22.229 21.886 23.308 21.546 21.786 21.129 20.730 20.059 25.206 22.326 23.173 24.617
PA = 0.126
12.592 11.425 8.577 8.459 8.529 9.173a 8.330a,b 7.698b 7.411b 7.381 7.686 9.137 8.614 8.480 9.232
PA = 0.092
39.240 36.151 31.085 31.116 30.606 32.410 29.876 29.459 28.423 28.110 27.264 34.692 30.913 31.653 33.849
PA = 0.091
PA = 0.923
PA = 0.477, PKW = 0.601
PA = 0.021
PA = 0.261
PA = 0.977 PA = 0.007, PKW = 0.007
PA = 0.109
PA = 0.193
PA = 0.972
PA = 0.199, PKW = 0.241
PA = 0.019
PA = 0.223
PA = P value from one-way ANOVA, PKW = P value from Kruskal-Wallis analysis of ranks. P values shown in bold typeface are statistically significant after within-family correction for multiple testing (Rice, 1989). a and b indicate the two homogeneous subsets identified by ANOVA post-hoc tests (Tukey HSD) for family 810 wet tissue weight. 1 Genotype class: O = putative overdominant genotypes, N = other genotypes. 2 Sample size. Numbers in parentheses indicate sample size for dry shell weight and total tissue weight where this differs from the sample size for wet tissue weight due to shells of several individuals being fused together.
78
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
To allow for the possibility that our scoring did not match that from previous studies, each allele in our study was individually tested for an overdominant effect. t-tests and Mann-Whitney U-tests on the complete data set showed no significant differences with the exception of the test for Pgm-25, where the putative overdominant genotypes weighed significantly less than the other genotypes (Mann-Whitney U-test, P = 0.024 for dry shell weight, P = 0.004 for wet tissue weight, P = 0.008 for total weight). Randomization tests of the difference in mean weight estimates (dry shell weight, wet body weight or total body weight) between putative overdominant and other genotypes within families were significant after correction for multiple testing for family F810 for wet tissue weight (Pgm-23/4 + Pgm-23/5 > Pgm-24/4 + Pgm-24/5, Pobserved = 0.0007, Pcritical = 0.0167) and for family F816 for wet tissue weight (Pgm-22/4 + Pgm23/3 > Pgm-22/3 + Pgm-23/4, Pobserved = 0.0036, Pcritical = 0.0167), dry shell weight (Pgm-22/4 + Pgm-23/3 > Pgm22/3 + Pgm-23/4, Pobserved = 0.0014, Pcritical = 0.0167) and total weight (Pgm-22/4 + Pgm-23/3 > Pgm-22/3 + Pgm-23/4, Pobserved = 0.0010, Pcritical = 0.0167). Randomization test results for families F800, F805 and F818 (present study) and for families F2, F15 and F20 (these three families contained putatively overdominant genotypes) from Gardner and Lobkov (2005) were not significant, indicating the absence of the putative overdominant effect. 4. Discussion Of the five C. gigas families analysed, three showed significant deviation from expected Mendelian segregation ratios. If overdominance for production traits is occurring at the PGM-2 locus, we would expect to see an excess of putative overdominant genotypes (heterozygotes with the most common allele, Pgm-23), associated with this fitness advantage. However, the observed deviations from Mendelian ratios were instead characterised by a deficit of these putative overdominant genotypes, and no consistent excess or deficit of any particular genotype was observed. Many previous studies of bivalves have reported deviations from Mendelian segregation ratios for both allozyme and DNA markers (e.g. Gaffney and Scott, 1984; Hu and Foltz, 1996; McGoldrick and Hedgecock, 1997; McGoldrick et al., 2000; Launey and Hedgecock, 2001). Segregation distortion increases with age (Gaffney and Scott, 1984; Launey and Hedgecock, 2001) is particularly pronounced in inbred families (McGoldrick and Hedgecock, 1997; Bierne et al., 1998), and can be
explained by selection against deleterious recessive mutations at loci closely linked to the markers (Launey and Hedgecock, 2001). In our study, the parent oysters were collected from the wild Kaipara Harbour population, the major site of spat collection for the New Zealand oyster industry (National Institute of Water and Atmospheric Research website: http://www.niwascience.co. nz/ncfa/aquaspecies/pacific). If there is a history of inbreeding it relates to the natural environment and has not been produced as a consequence of generations of hatchery inbreeding. We have no genetic data for the Kaipara population, but note that Smith et al. (1986) reported no evidence of reduced genetic variation among New Zealand oyster populations. A further possibility is that the loss of small larvae during routine hatchery sieving might, by chance, account for the observed deviations from expected Mendelian segregation ratios. The presence of a null allele at the PGM-2 locus could generate deficiencies of heterozygotes and lower the specific activity of the apparent homozygotes in which it was present relative to all fully expressed heterozygotes. Pogson (1991) observed no difference between homozygotes and those heterozygotes without the most common allele and concluded that it was unlikely that a null allele could explain his results. In our study the presence of a null allele (i.e., a “false” homozygote) was only possible in two families. Family 818 did not show distortion of Mendelian segregation ratios, but family 800 showed a significant deviation from expected ratios in the form of an excess of Pgm23/3 homozygotes and/or a deficiency of Pgm-23/4 heterozygotes. This pattern could result if the family 800 sire was heterozygote for a null allele (Pgm-23/null , observed as Pgm-23/3). However, the presence of a null allele would result in unexpected offspring genotypes (Pgm-24/null in this case, observed as Pgm-24/4), which we did not encounter. We therefore discount the likelihood that our observed genotypic frequencies are distorted by the presence of a null allele. We observed no consistent PGM-2 genotype-dependent effect on wet tissue weight, dry shell weight or total weight in the five families studied. We therefore conclude that there is no evidence for a PGM-2 genotype-dependent effect on growth in the New Zealand C. gigas studied and that this is not a useful marker for a hatchery-based breeding programme. In contrast to this situation, Pogson (1991) demonstrated overdominance for phosphoglucomutase specific activity in Pacific oysters introduced to Canada. Possible explanations for the absence of an overdominant effect on growth among the New Zealand oyster families
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
include differences in alleles or allele frequencies between Canadian and New Zealand C. gigas, variation in expression of overdominance in different environmental conditions, or a lack of translation of overdominance for enzyme specific activity into overdominance for growth. These three explanations are discussed below. 1. Given the similarity in allele frequencies across studies from Japan, USA, Canada and New Zealand (Buroker et al., 1975, 1979; Ozaki and Fujio, 1985; Smith et al., 1986; Pogson, 1989; Gardner and Lobkov, 2005; the present study) it seems unlikely that our Pgm23 allele does not match the overdominant Pgm-2100 allele described by Pogson (1989, 1991). However, we have taken a conservative approach by testing all five individual alleles for the overdominant effect. Overall, we found no evidence of an overdominant effect for any allele. 2. It is possible that environmental conditions in our study were not suited to expression of overdominance. However, the genotypic effect observed by Pogson (1991) was consistent in the sense that the specific activity of both groups (overdominant genotypes and all other genotypes) responded in the same way to changes in season and location. 3. Perhaps the simplest explanation is that the overdominance for enzyme specific activity observed by Pogson (1991) does not translate into overdominance for growth. Phosphoglucomutase functions in glycogen metabolism, catalysing the interconversion of glucose1-phosphate and glucose-6-phosphate (Pogson, 1989). Increased specific activity of PGM-2 is expected to confer on overdominant genotypes a higher catalytic capacity than other genotypes but, to the best of our knowledge, the physiological effect of this on glycogen metabolism has not been investigated. There are examples where variation in specific activity between genotypes at enzyme loci has been associated with differences in physiological performance (e.g. van Delden, 1982; Burton and Feldman, 1983) but this is not always the case (David, 1998). In conclusion, our findings confirm those of the previous investigation (Gardner and Lobkov, 2005) and indicate that the hatchery production of Pacific oysters is unlikely to benefit from selective breeding of particular PGM-2 genotypes because of the absence of overdominance for this locus among tested individuals. It would however be particularly interesting to determine why this phenomenon is absent among New Zealand Pacific oysters. Research looking at PGM-2 enzyme kinetics
79
and perhaps also involving a targeted proteomics-type approach might be particularly informative in this case. Acknowledgements We thank Achim Janke, Henry Kaspar and Rodney Roberts of the Cawthron Institute, Nelson, New Zealand, for providing the oysters. We are grateful to Lesley Milicich for technical assistance. This research was supported by a New Zealand Public Good Science Fund (PGSF) award, as part of The Cawthron Institute's PGSF funded shellfish aquaculture programme (contract CAWX0303). References Bierne, N., Launey, S., Naciri-Graven, Y., Bonhomme, F., 1998. Early effect of inbreeding as revealed by microsatellite analyses on Ostrea edulis larvae. Genetics 148, 1893–1906. Britten, H.B., 1996. Meta-analyses of the association between multilocus heterozygosity and fitness. Evolution 50, 2158–2164. Buroker, N.E., Hershberger, W.K., Chew, K.K., 1975. Genetic variation in the Pacific oyster, Crassostrea gigas. J. Fish. Res. Board Can. 32, 2471–2477. Buroker, N.E., Hershberger, W.K., Chew, K.K., 1979. Population genetics of the Family Ostreidae: I. Intraspecific studies of Crassostrea gigas and Saccostrea commercialis. Mar. Biol. 54, 157–169. Burton, R.S., Feldman, M.W., 1983. Physiological effects of an allozyme polymorphism: glutamate-pyruvate transaminase and response to hyperosmotic stress in the copepod Tigriopus californicus. Biochem. Genet. 21, 239–251. David, P., 1998. Heterozygosity-fitness correlations: new perspectives on old problems. Heredity 80, 531–537. Fujio, Y., 1982. A correlation of heterozygosity with growth rate in the Pacific oyster, Crassostrea gigas. Tohoku J. Agric. Res. 33, 66–75. Gaffney, P.M., Scott, T.M., 1984. Genetic heterozygosity and production traits in natural and hatchery populations of bivalves. Aquaculture 42, 289–302. Gaffney, P.M., Scott, T.M., Koehn, R.K., Diehl, W.J., 1990. Interrelationships of heterozygosity, growth rate and heterozygotes deficiencies in the coot clam, Mulinia lateralis. Genetics 124, 687–699. Gardner, J.P.A., Lobkov, I., 2005. A test for overdominance at the phosphoglucomutase-2 locus in Pacific oysters (Crassostrea gigas) from New Zealand. Aquaculture 244, 29–39. Gentili, M.R., Beaumont, A.R., 1988. Environmental stress, heterozygosity and growth rate in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 120, 145–153. Gosling, E.M., 1982. Genetic variability in hatchery-produced Pacific oysters (Crassostrea gigas Thunberg). Aquaculture 26, 273–287. Hedgecock, D., McGoldrick, D.J., Bayne, B.L., 1995. Hybrid vigor in Pacific oysters: an experimental approach using crosses among inbred lines. Aquaculture 137, 285–298. Hedgecock, D., McGoldrick, D.J., Manahan, D.T., Vavra, J., Appelmans, N., Bayne, B.L., 1996. Quantitative and molecular genetic analyses of heterosis in bivalve molluscs. J. Exp. Mar. Biol. Ecol. 203, 49–59.
80
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
Hedgecock, D., Langdon, C., Blouin, M., Allen, S.K., 1997. Genetic improvement of cultured Pacific oysters by selection. Special Report No. 968. Agricultural Experiment Station, Oregon State University, Corvallis, OR, USA. 40 pp. Hu, Y.-P., Foltz, D.W., 1996. Genetics of scnDNA polymorphisms in juvenile oysters (Crassostrea virginica): Part I. Characterizing the inheritance of polymorphisms in controlled crosses. Mol. Mar. Biol. Biotechnol. 5, 123–129. Koehn, R.K., Diehl, W.J., Scott, T.M., 1988. The differential contribution by individual enzymes of glycolysis and protein catabolism to the relationship between heterozygosity and growth rate in the coot clam, Mulinia lateralis. Genetics 118, 121–130. Launey, S., Hedgecock, D., 2001. High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159, 255–265. McGoldrick, D.J., Hedgecock, D., 1997. Fixation, segregation and linkage of allozyme loci in inbred families of the Pacific oyster Crassostrea gigas (Thunberg): implications for the causes of inbreeding depression. Genetics 146, 321–334. McGoldrick, D.J., Hedgecock, D., English, L.J., Baoprasertkul, P., Ward, R.D., 2000. The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea gigas): selection and null alleles. J. Shellfish Res. 19, 779–788. Mitton, J.B., Grant, M.C., 1984. Associations among protein heterozygosity, growth rate and developmental homeostasis. Ann. Rev. Ecolog. Syst. 15, 479–499.
Ozaki, H., Fujio, Y., 1985. Genetic differentiation in geographical populations of the Pacific oyster (Crassostrea gigas) around Japan. Tohoku J. Agric. Res. 36, 49–61. Pogson, G.H., 1989. Biochemical characterization of genotypes at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas. Biochem. Genet. 27, 571–589. Pogson, G.H., 1991. Expression of overdominance for specific activity at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas. Genetics 128, 133–141. Pogson, G.H., Zouros, E., 1994. Allozyme and RFLP heterozygosities as correlates of growth rate in the scallop Placopecten magellanicus—a test of the associative overdominance hypothesis. Genetics 137, 221–231. Rice, W.R., 1989. Analyzing tables of statistical tests. Evolution 43, 223–225. Smith, P.J., Ozaki, H., Fujio, Y., 1986. No evidence for reduced genetic variation in the accidentally introduced oyster Crassostrea gigas in New Zealand. N.Z. J. Mar. Freshw. Res. 20, 569–574. Thelen, G.C., Allendorf, F.W., 2001. Heterozygosity-fitness correlations in rainbow trout: effects of allozyme loci or associative overdominance? Evolution 55, 1180–1187. van Delden, W., 1982. The alcohol dehydrogenase polymorphism in Drosophila melanogaster: selection at an enzyme locus. Evol. Biol. 15, 187–222. Zouros, E., 1993. Associative overdominance: evaluating the effects of inbreeding and linkage disequilibrium. Genetica 89, 35–46.
No evidence for overdominance at the phosphoglucomutase-2 locus in Pacific oysters (Crassostrea gigas) from New Zealand Ann R. Wood ⁎, Jonathan P.A. Gardner Centre for Marine Environmental and Economic Research, School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand Received 28 November 2005; received in revised form 11 May 2006; accepted 15 May 2006
Abstract Overdominance for enzyme specific activity at the phosphoglucomutase-2 (PGM-2) locus has been demonstrated for wild, adult Pacific oysters (Crassostrea gigas) that are formed from heterozygous combinations of the allele at highest frequency and any other allele at this locus. Overdominant genotypes have been shown to have higher catalytic capacities than other genotypes, and consequently it was proposed that this effect might account for genotype-dependent weight differences among Pacific oysters. From an aquaculture perspective, it would be relatively easy for a selective breeding programme to exploit a genotype-dependent effect on production traits (growth rate, wet body weight) if this phenomenon could be demonstrated among hatchery-reared oysters. However, a previous test of this failed to detect either an overdominant effect or a genotype-dependent effect among wild juvenile oysters (9 months of age), among adults of this same batch 12 months later, or among juveniles (5 months of age) from 8 full-sib hatchery-reared families. In the present paper, we extend this work by examining the relationship between PGM-2 genotype and production traits in adult market-size Pacific oysters from five full-sib families specifically selected because they contain putatively overdominant and other genotypic combinations. Within-families, there were significant weight differences among genotypes, but there was no evidence of a consistent PGM-2 genotype-dependent effect, overdominant or otherwise, on wet tissue weight, dry shell weight or total weight (=wet tissue weight plus dry shell weight). Among families, tests of pooled data showed no significant difference between putative overdominant and other genotypes for wet tissue weight, dry shell weight or total weight. We conclude that there is no evidence on which to base a Pacific oyster-breeding programme using PGM-2 as a marker for increased production. © 2006 Elsevier B.V. All rights reserved. Keywords: Pacific oyster; Crassostrea gigas; Overdominance; Phosphoglucomutase; PGM-2; Growth
1. Introduction Positive correlations between allozyme heterozygosity and various fitness-related traits have been observed frequently in many species, and in particular among marine bivalves (e.g. Fujio, 1982; Gaffney and Scott, ⁎ Corresponding author. Tel.: +64 4 463 5578; fax: +64 4 463 5331. E-mail address: [email protected] (A.R. Wood). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.05.015
1984; Mitton and Grant, 1984; Gentili and Beaumont, 1988; Britten, 1996), and various suggestions have been advanced for genetic mechanisms by which this heterozygote advantage (=heterosis) can occur. Debate has centred around two main hypotheses: overdominance, which involves the direct action of selection on allozyme genotypes and superiority of heterozygotes at one or more loci influencing fitness, and associative overdominance, where allozyme loci are not under
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
selection directly but act as markers of selection against deleterious recessive alleles at linked loci which influence fitness (e.g., Koehn et al., 1988; Gaffney et al., 1990; Zouros, 1993; Pogson and Zouros, 1994; Thelen and Allendorf, 2001). Regardless of its mechanistic basis, the utility of the heterosis phenomenon to the aquaculture production of shellfish and other species has long been of interest (e.g., Gosling, 1982; Gaffney and Scott, 1984; Hedgecock et al., 1995, 1996, 1997). In Japanese populations of the Pacific oyster, Crassostrea gigas, Fujio (1982) described a positive correlation between body weight and heterozygosity at five enzyme loci, which translated into a pronounced growth advantage for heterozygotes compared to homozygotes. At the phosphoglucomutase-2 (PGM-2) locus, the mean body weight of heterozygotes across all 20 populations was 21% greater than that of homozygotes. Because multi-locus heterozygote deficiencies were also observed, Fujio (1982) suggested that inbreeding may have resulted in an excess of homozygotes and thus of individuals homozygous for deleterious genes. More recently, overdominance of enzyme specific activity in C. gigas was reported at the PGM-2 locus (Pogson, 1991). Heterozygotes for the most common allele and any other allele showed specific activities approximately 20% greater than those observed for all homozygotes and all other heterozygotes. Pogson (1991) suggested that the heterozygosity/growth correlation observed for C. gigas at the PGM-2 locus by Fujio (1982) might be explained by the greater specific activities of heterozygotes for the most common allele. If the specific-activity overdominance observed by Pogson (1991) translates to faster growth or larger market size, it would be relatively easy for a hatcherybased aquaculture programme to breed overdominant genotypes to take advantage of this. To investigate this possibility, Gardner and Lobkov (2005) looked, but found no support, for a relationship between PGM-2 genotype and body weight in juvenile hatcheryproduced New Zealand C. gigas. Gardner and Lobkov (2005) suggested that an overdominant effect might for some reason only be visible in adult oysters (as analysed by Fujio, 1982; Pogson, 1991) and not in juveniles. In the present study, we have investigated further this putative overdominant effect by analysing individual PGM-2 genotypes and body weights for adult marketsize Pacific oysters from five new hatchery-produced full-sib families, specifically selected for testing because they each contain putative overdominant and other
75
PGM-2 genotypes to facilitate within-family and among-family comparisons. 2. Materials and methods 2.1. Hatchery oysters During December 2003, 23 full-sib families were produced at the Cawthron Institute's Glenhaven hatchery, from wild parents collected from Kaipara Harbour in northern New Zealand. Tissue from the parents was stored at − 80 °C. Offspring were reared in the hatchery for the first month, transferred to nursery upwellers for a further 2 months, and then grown in intertidal grow-out bags at Orongo Bay (Bay of Islands, northern New Zealand) from March 2004 until they were harvested in December 2004. Particularly small larvae were lost from each family during routine sieving in the hatchery, but no further culling of oysters took place. Samples of digestive gland were taken from each parent and analysed for PGM-2 genotype as described below. As far as possible, the observed genotypes were matched to those found by Gardner and Lobkov (2005) and Pogson (1989, 1991). Allele frequencies for the parent oysters genotyped in this study were similar to those found in previous studies (Buroker et al., 1975, 1979; Ozaki and Fujio, 1985; Smith et al., 1986; Pogson, 1989, 1991; Gardner and Lobkov, 2005). It therefore seems unlikely that the most common allele observed in the present study (Pgm-23) is not the same as the most common allele observed by Pogson (1989, 1991). Based on parent genotypes, five families (F800, F805, F810, F816 and F818) that would allow within family comparison of putative overdominant and other genotypes were chosen for this study. In the present context, “overdominant” refers to the Pgm-23 allele in combination with any other allele, and “other” refers to all other PGM-2 genotypic combinations including heterozygous combinations not involving the Pgm-23 allele. 2.2. Sample processing Approximately 110 haphazardly selected 12-monthold oysters from each of the five families were couriered on ice to Victoria University of Wellington in December 2004 and stored at −20 °C for 1–2 months prior to analysis. A total of 538 oysters were analysed (mean ± SD of 107.6 ± 2.7 per family). Small batches of oysters were removed from the freezer and held on ice during processing. Shells were opened whilst the oysters were still frozen and a sample of digestive gland was taken for
76
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
allozyme electrophoresis. Dry shell weight and blotted wet soft tissue weight were determined to 0.001 g. The digestive gland sample weight was estimated after preparation of allozyme wicks (see below), by centrifuging the sample for 10 min at 13 000 rpm, removing the supernatant (comprising the added distilled water plus the original water content of the sample, minus the liquid absorbed by the wick), weighing the pelleted tissue left in the microtube and subtracting the weight of the empty microtube. An alternative method, weighing samples whilst they were still frozen, gave similar results (results not shown). 2.3. Allozyme electrophoresis Allozyme electrophoresis methods were essentially the same as those of Gardner and Lobkov (2005), using approximately 100 mg of digestive gland tissue for each individual. Alleles were numbered Pgm-21 to Pgm-25 with Pgm-21 being the most anodal, following Gardner and Lobkov (2005). 2.4. Data analysis PGM-2 genotypes of parents were used to determine the expected genotypes of offspring in each family. The observed genotypic frequencies in the offspring were tested against expected frequencies calculated on assumptions of Mendelian inheritance (ratios of 1:1, 1:2:1 or 1:1:1:1), using a chi-square “goodness-of-fit” test. The software package SPSS (v 12.0, SPSS Inc., Chicago, Illinois, USA) was used for analysis of dry shell weight, blotted wet soft tissue weight (wet tissue weight) and total weight (dry shell weight+ wet tissue weight) estimates. For each family a one-way ANOVA was used to test for differences in the weight variables among genotypes. In all cases, Levene's test for homogeneity of variances among genotype classes gave non-significant results, and in all cases except one, the weight variables were normally distributed (except F810 — wet tissue weight (Kolmogorov-Smirnov test; P = 0.006) and total weight (P = 0.017)). For family F810, because of the nonnormally distributed weight variables, a non-parametric Kruskal-Wallis analysis of ranks was also used. In cases where ANOVA showed significant differences in weight variables among genotypes, Tukey's HSD post-hoc test was used to identify the location of significant differences. For families F805, F810 and F816 (which had more than 2 genotypes) and for pooled data from the five families, oysters were coded into two groups, “putative overdominant genotypes” and “all other genotypes”. t-tests were used to test for weight
differences between these two groups. Where appropriate, the sequential Bonferroni correction for multiple testing was employed (Rice, 1989). To allow for the possibility that our scoring did not match that from previous studies, rather than simply assuming that the allele at highest frequency in this study (our Pgm-23 allele) was the same as that observed by Pogson (1991), each allele in our study was individually tested for an overdominant effect. As appropriate, depending on homogeneity of variances and/or normal distribution of data, we used either a t-test (all alleles except Pgm-25) or the Mann Whitney U-test (only the Pgm-25 allele, for which Levene's test showed significant heterogeneity of variances) to test for classical overdominance. Heterozygotes for each allele in turn were tested against all other genotypes to examine the possible relationship between allelespecific overdominance and mean weight (dry shell, wet body, and total body weight). We tested the data from this study and that of Gardner and Lobkov (2005) for a difference in mean values for each weight variable between putative overdominant and other genotypes within each family using a randomization test procedure (Resampling Stats software, v5.0.2, Resampling Stats Inc., Arlington, Virginia, USA). This test assigns at random the individual weight values (dry shell, wet body or total body) to individual genotype scores. For each test, 100,000 randomizations of the data were performed to produce a distribution of the difference in mean values that should apply under the null hypothesis of no overdominant effect. Given this distribution, the probability of obtaining the observed difference between mean weights was calculated to permit a test of the effect (if any) of putative overdominant versus other genotypes on production characteristics. 3. Results PGM-2 genotypic frequencies observed in offspring were significantly different from those expected assuming Mendelian inheritance in three of the five families (Table 1). Family F800 exhibited a deficiency of the Pgm-23/4 and an excess of the Pgm-23/3 genotype. In F805, an excess of the Pgm-22/2 genotype was observed along with deficiencies of the Pgm-22/3 and Pgm-23/3 genotypes. In F816, genotypes Pgm-22/3 and Pgm-23/3 were approximately in accordance with expectations but there was an excess of the Pgm-22/4 and a deficit of the Pgm-23/4 genotypes. Genotype frequencies of families F810 and F818 were not significantly different from expectations.
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80 Table 1 Parent and offspring PGM-2 genotypes and results of χ2 test for observed vs. expected genotype frequencies in 5 hatchery-produced Crassostrea gigas families Family
PGM-2 genotype Dam
Sire
Offspring
800
3/4
3/3
805
2/3
2/3
810
3/4
4/5
816
3/4
2/3
818
3/3
1/3
3/3 3/4 2/2 2/3 3/3 3/4 3/5 4/4 4/5 2/3 2/4 3/3 3/4 1/3 3/3
Nexp
Nobs
χ2 test of genotype frequencies
54 54 27.5 55 27.5 24.5 24.5 24.5 24.5 25.8 25.8 25.8 25.8 54 54
78 30 63 32 15 26 19 28 25 26 41 29 7 52 56
χ2 = 21.333, P < 0.001 χ2 = 61.127, P < 0.001 χ2 = 1.837, P = 0.607
χ2 = 23.097, P < 0.001 χ2 = 0.148, P = 0.700
Nexp = number of oysters expected for each genotype assuming Mendelian inheritance. Nobs = number of oysters observed for each genotype. P values shown in bold typeface are statistically significant.
Three of the five families (F800, F805 and F810) exhibited mean values of dry shell weight, wet tissue weight and total body weight for putative overdom-
77
inant genotypes (those with allele Pgm-23) which were slightly greater than those for other genotypes, however, families F816 and F818 showed the opposite trend (Table 2). After within-family correction for multiple testing (Rice, 1989), only the test for wet tissue weight in F810 showed a statistically significant difference among genotypes. For F810 wet tissue weight, Tukey's HSD post-hoc test showed that the putative overdominant genotype Pgm-23/4 had significantly greater wet tissue weight than the other genotypes Pgm-24/4 and Pgm-24/5, but Pgm-23/5 was not significantly different from either Pgm-23/4, Pgm24/4 or Pgm-24/5. Within-family t-test comparisons of difference in mean weight between putative overdominant and other genotypes were significant in only two instances. For family F810 the mean wet tissue weight for putative overdominant genotypes was greater than the mean weight for other genotypes (Pgm-23/4 + Pgm-23/5 > Pgm24/4 + Pgm-24/5, P = 0.002), whereas for family F816 the mean total weight for other genotypes was greater than that for putatively overdominant genotypes (Pgm22/4 + Pgm-23/3 > Pgm-22/3 + Pgm-23/4 , P = 0.008). The same test on pooled data from all five families showed no significant difference between putative overdominant and other genotypes for mean dry shell weight (t = − 0.849, P = 0.397), mean wet tissue weight (t = − 0.704, P = 0.482) or mean total weight (t = − 1.068, P = 0.286).
Table 2 Mean estimates of dry shell weight, wet tissue weight and total weight for each genotype in 5 hatchery-produced Crassostrea gigas families Family
800 805
810
816
818
PGM-2 genotype
Class1
3/4 3/3 2/3 2/2 3/3 3/4 3/5 4/4 4/5 2/3 3/4 2/4 3/3 1/3 3/3
O N O N N O O N N O O N N O N
N2
30 78 32 (27) 63 (49) 15 (14) 26 (25) 19 28 (25) 25 (22) 26 7 (6) 41 (35) 29 (28) 52 56
Dry shell weight
Wet tissue weight
Total weight
Mean (g)
P value
Mean (g)
P value
Mean (g)
P value
26.648 24.725 22.528 22.229 21.886 23.308 21.546 21.786 21.129 20.730 20.059 25.206 22.326 23.173 24.617
PA = 0.126
12.592 11.425 8.577 8.459 8.529 9.173a 8.330a,b 7.698b 7.411b 7.381 7.686 9.137 8.614 8.480 9.232
PA = 0.092
39.240 36.151 31.085 31.116 30.606 32.410 29.876 29.459 28.423 28.110 27.264 34.692 30.913 31.653 33.849
PA = 0.091
PA = 0.923
PA = 0.477, PKW = 0.601
PA = 0.021
PA = 0.261
PA = 0.977 PA = 0.007, PKW = 0.007
PA = 0.109
PA = 0.193
PA = 0.972
PA = 0.199, PKW = 0.241
PA = 0.019
PA = 0.223
PA = P value from one-way ANOVA, PKW = P value from Kruskal-Wallis analysis of ranks. P values shown in bold typeface are statistically significant after within-family correction for multiple testing (Rice, 1989). a and b indicate the two homogeneous subsets identified by ANOVA post-hoc tests (Tukey HSD) for family 810 wet tissue weight. 1 Genotype class: O = putative overdominant genotypes, N = other genotypes. 2 Sample size. Numbers in parentheses indicate sample size for dry shell weight and total tissue weight where this differs from the sample size for wet tissue weight due to shells of several individuals being fused together.
78
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
To allow for the possibility that our scoring did not match that from previous studies, each allele in our study was individually tested for an overdominant effect. t-tests and Mann-Whitney U-tests on the complete data set showed no significant differences with the exception of the test for Pgm-25, where the putative overdominant genotypes weighed significantly less than the other genotypes (Mann-Whitney U-test, P = 0.024 for dry shell weight, P = 0.004 for wet tissue weight, P = 0.008 for total weight). Randomization tests of the difference in mean weight estimates (dry shell weight, wet body weight or total body weight) between putative overdominant and other genotypes within families were significant after correction for multiple testing for family F810 for wet tissue weight (Pgm-23/4 + Pgm-23/5 > Pgm-24/4 + Pgm-24/5, Pobserved = 0.0007, Pcritical = 0.0167) and for family F816 for wet tissue weight (Pgm-22/4 + Pgm23/3 > Pgm-22/3 + Pgm-23/4, Pobserved = 0.0036, Pcritical = 0.0167), dry shell weight (Pgm-22/4 + Pgm-23/3 > Pgm22/3 + Pgm-23/4, Pobserved = 0.0014, Pcritical = 0.0167) and total weight (Pgm-22/4 + Pgm-23/3 > Pgm-22/3 + Pgm-23/4, Pobserved = 0.0010, Pcritical = 0.0167). Randomization test results for families F800, F805 and F818 (present study) and for families F2, F15 and F20 (these three families contained putatively overdominant genotypes) from Gardner and Lobkov (2005) were not significant, indicating the absence of the putative overdominant effect. 4. Discussion Of the five C. gigas families analysed, three showed significant deviation from expected Mendelian segregation ratios. If overdominance for production traits is occurring at the PGM-2 locus, we would expect to see an excess of putative overdominant genotypes (heterozygotes with the most common allele, Pgm-23), associated with this fitness advantage. However, the observed deviations from Mendelian ratios were instead characterised by a deficit of these putative overdominant genotypes, and no consistent excess or deficit of any particular genotype was observed. Many previous studies of bivalves have reported deviations from Mendelian segregation ratios for both allozyme and DNA markers (e.g. Gaffney and Scott, 1984; Hu and Foltz, 1996; McGoldrick and Hedgecock, 1997; McGoldrick et al., 2000; Launey and Hedgecock, 2001). Segregation distortion increases with age (Gaffney and Scott, 1984; Launey and Hedgecock, 2001) is particularly pronounced in inbred families (McGoldrick and Hedgecock, 1997; Bierne et al., 1998), and can be
explained by selection against deleterious recessive mutations at loci closely linked to the markers (Launey and Hedgecock, 2001). In our study, the parent oysters were collected from the wild Kaipara Harbour population, the major site of spat collection for the New Zealand oyster industry (National Institute of Water and Atmospheric Research website: http://www.niwascience.co. nz/ncfa/aquaspecies/pacific). If there is a history of inbreeding it relates to the natural environment and has not been produced as a consequence of generations of hatchery inbreeding. We have no genetic data for the Kaipara population, but note that Smith et al. (1986) reported no evidence of reduced genetic variation among New Zealand oyster populations. A further possibility is that the loss of small larvae during routine hatchery sieving might, by chance, account for the observed deviations from expected Mendelian segregation ratios. The presence of a null allele at the PGM-2 locus could generate deficiencies of heterozygotes and lower the specific activity of the apparent homozygotes in which it was present relative to all fully expressed heterozygotes. Pogson (1991) observed no difference between homozygotes and those heterozygotes without the most common allele and concluded that it was unlikely that a null allele could explain his results. In our study the presence of a null allele (i.e., a “false” homozygote) was only possible in two families. Family 818 did not show distortion of Mendelian segregation ratios, but family 800 showed a significant deviation from expected ratios in the form of an excess of Pgm23/3 homozygotes and/or a deficiency of Pgm-23/4 heterozygotes. This pattern could result if the family 800 sire was heterozygote for a null allele (Pgm-23/null , observed as Pgm-23/3). However, the presence of a null allele would result in unexpected offspring genotypes (Pgm-24/null in this case, observed as Pgm-24/4), which we did not encounter. We therefore discount the likelihood that our observed genotypic frequencies are distorted by the presence of a null allele. We observed no consistent PGM-2 genotype-dependent effect on wet tissue weight, dry shell weight or total weight in the five families studied. We therefore conclude that there is no evidence for a PGM-2 genotype-dependent effect on growth in the New Zealand C. gigas studied and that this is not a useful marker for a hatchery-based breeding programme. In contrast to this situation, Pogson (1991) demonstrated overdominance for phosphoglucomutase specific activity in Pacific oysters introduced to Canada. Possible explanations for the absence of an overdominant effect on growth among the New Zealand oyster families
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
include differences in alleles or allele frequencies between Canadian and New Zealand C. gigas, variation in expression of overdominance in different environmental conditions, or a lack of translation of overdominance for enzyme specific activity into overdominance for growth. These three explanations are discussed below. 1. Given the similarity in allele frequencies across studies from Japan, USA, Canada and New Zealand (Buroker et al., 1975, 1979; Ozaki and Fujio, 1985; Smith et al., 1986; Pogson, 1989; Gardner and Lobkov, 2005; the present study) it seems unlikely that our Pgm23 allele does not match the overdominant Pgm-2100 allele described by Pogson (1989, 1991). However, we have taken a conservative approach by testing all five individual alleles for the overdominant effect. Overall, we found no evidence of an overdominant effect for any allele. 2. It is possible that environmental conditions in our study were not suited to expression of overdominance. However, the genotypic effect observed by Pogson (1991) was consistent in the sense that the specific activity of both groups (overdominant genotypes and all other genotypes) responded in the same way to changes in season and location. 3. Perhaps the simplest explanation is that the overdominance for enzyme specific activity observed by Pogson (1991) does not translate into overdominance for growth. Phosphoglucomutase functions in glycogen metabolism, catalysing the interconversion of glucose1-phosphate and glucose-6-phosphate (Pogson, 1989). Increased specific activity of PGM-2 is expected to confer on overdominant genotypes a higher catalytic capacity than other genotypes but, to the best of our knowledge, the physiological effect of this on glycogen metabolism has not been investigated. There are examples where variation in specific activity between genotypes at enzyme loci has been associated with differences in physiological performance (e.g. van Delden, 1982; Burton and Feldman, 1983) but this is not always the case (David, 1998). In conclusion, our findings confirm those of the previous investigation (Gardner and Lobkov, 2005) and indicate that the hatchery production of Pacific oysters is unlikely to benefit from selective breeding of particular PGM-2 genotypes because of the absence of overdominance for this locus among tested individuals. It would however be particularly interesting to determine why this phenomenon is absent among New Zealand Pacific oysters. Research looking at PGM-2 enzyme kinetics
79
and perhaps also involving a targeted proteomics-type approach might be particularly informative in this case. Acknowledgements We thank Achim Janke, Henry Kaspar and Rodney Roberts of the Cawthron Institute, Nelson, New Zealand, for providing the oysters. We are grateful to Lesley Milicich for technical assistance. This research was supported by a New Zealand Public Good Science Fund (PGSF) award, as part of The Cawthron Institute's PGSF funded shellfish aquaculture programme (contract CAWX0303). References Bierne, N., Launey, S., Naciri-Graven, Y., Bonhomme, F., 1998. Early effect of inbreeding as revealed by microsatellite analyses on Ostrea edulis larvae. Genetics 148, 1893–1906. Britten, H.B., 1996. Meta-analyses of the association between multilocus heterozygosity and fitness. Evolution 50, 2158–2164. Buroker, N.E., Hershberger, W.K., Chew, K.K., 1975. Genetic variation in the Pacific oyster, Crassostrea gigas. J. Fish. Res. Board Can. 32, 2471–2477. Buroker, N.E., Hershberger, W.K., Chew, K.K., 1979. Population genetics of the Family Ostreidae: I. Intraspecific studies of Crassostrea gigas and Saccostrea commercialis. Mar. Biol. 54, 157–169. Burton, R.S., Feldman, M.W., 1983. Physiological effects of an allozyme polymorphism: glutamate-pyruvate transaminase and response to hyperosmotic stress in the copepod Tigriopus californicus. Biochem. Genet. 21, 239–251. David, P., 1998. Heterozygosity-fitness correlations: new perspectives on old problems. Heredity 80, 531–537. Fujio, Y., 1982. A correlation of heterozygosity with growth rate in the Pacific oyster, Crassostrea gigas. Tohoku J. Agric. Res. 33, 66–75. Gaffney, P.M., Scott, T.M., 1984. Genetic heterozygosity and production traits in natural and hatchery populations of bivalves. Aquaculture 42, 289–302. Gaffney, P.M., Scott, T.M., Koehn, R.K., Diehl, W.J., 1990. Interrelationships of heterozygosity, growth rate and heterozygotes deficiencies in the coot clam, Mulinia lateralis. Genetics 124, 687–699. Gardner, J.P.A., Lobkov, I., 2005. A test for overdominance at the phosphoglucomutase-2 locus in Pacific oysters (Crassostrea gigas) from New Zealand. Aquaculture 244, 29–39. Gentili, M.R., Beaumont, A.R., 1988. Environmental stress, heterozygosity and growth rate in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 120, 145–153. Gosling, E.M., 1982. Genetic variability in hatchery-produced Pacific oysters (Crassostrea gigas Thunberg). Aquaculture 26, 273–287. Hedgecock, D., McGoldrick, D.J., Bayne, B.L., 1995. Hybrid vigor in Pacific oysters: an experimental approach using crosses among inbred lines. Aquaculture 137, 285–298. Hedgecock, D., McGoldrick, D.J., Manahan, D.T., Vavra, J., Appelmans, N., Bayne, B.L., 1996. Quantitative and molecular genetic analyses of heterosis in bivalve molluscs. J. Exp. Mar. Biol. Ecol. 203, 49–59.
80
A.R. Wood, J.P.A. Gardner / Aquaculture 259 (2006) 74–80
Hedgecock, D., Langdon, C., Blouin, M., Allen, S.K., 1997. Genetic improvement of cultured Pacific oysters by selection. Special Report No. 968. Agricultural Experiment Station, Oregon State University, Corvallis, OR, USA. 40 pp. Hu, Y.-P., Foltz, D.W., 1996. Genetics of scnDNA polymorphisms in juvenile oysters (Crassostrea virginica): Part I. Characterizing the inheritance of polymorphisms in controlled crosses. Mol. Mar. Biol. Biotechnol. 5, 123–129. Koehn, R.K., Diehl, W.J., Scott, T.M., 1988. The differential contribution by individual enzymes of glycolysis and protein catabolism to the relationship between heterozygosity and growth rate in the coot clam, Mulinia lateralis. Genetics 118, 121–130. Launey, S., Hedgecock, D., 2001. High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159, 255–265. McGoldrick, D.J., Hedgecock, D., 1997. Fixation, segregation and linkage of allozyme loci in inbred families of the Pacific oyster Crassostrea gigas (Thunberg): implications for the causes of inbreeding depression. Genetics 146, 321–334. McGoldrick, D.J., Hedgecock, D., English, L.J., Baoprasertkul, P., Ward, R.D., 2000. The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea gigas): selection and null alleles. J. Shellfish Res. 19, 779–788. Mitton, J.B., Grant, M.C., 1984. Associations among protein heterozygosity, growth rate and developmental homeostasis. Ann. Rev. Ecolog. Syst. 15, 479–499.
Ozaki, H., Fujio, Y., 1985. Genetic differentiation in geographical populations of the Pacific oyster (Crassostrea gigas) around Japan. Tohoku J. Agric. Res. 36, 49–61. Pogson, G.H., 1989. Biochemical characterization of genotypes at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas. Biochem. Genet. 27, 571–589. Pogson, G.H., 1991. Expression of overdominance for specific activity at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas. Genetics 128, 133–141. Pogson, G.H., Zouros, E., 1994. Allozyme and RFLP heterozygosities as correlates of growth rate in the scallop Placopecten magellanicus—a test of the associative overdominance hypothesis. Genetics 137, 221–231. Rice, W.R., 1989. Analyzing tables of statistical tests. Evolution 43, 223–225. Smith, P.J., Ozaki, H., Fujio, Y., 1986. No evidence for reduced genetic variation in the accidentally introduced oyster Crassostrea gigas in New Zealand. N.Z. J. Mar. Freshw. Res. 20, 569–574. Thelen, G.C., Allendorf, F.W., 2001. Heterozygosity-fitness correlations in rainbow trout: effects of allozyme loci or associative overdominance? Evolution 55, 1180–1187. van Delden, W., 1982. The alcohol dehydrogenase polymorphism in Drosophila melanogaster: selection at an enzyme locus. Evol. Biol. 15, 187–222. Zouros, E., 1993. Associative overdominance: evaluating the effects of inbreeding and linkage disequilibrium. Genetica 89, 35–46.