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Genetic diversity in oysters


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English, Louise,(Louise Jasmins) 2001 , 'Genetic diversity in oysters', PhD thesis, University of Tasmania.

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This project examined the effects on genetic diversity of oysters by hatchery
techniques and selective breeding. The edible oyster industry in Australia
comprises of two main species: Crassostrea gigas and Saccostrea glomerata,
which are produced by hatcheries or natural spatfall, respectively. This study
examined the levels of genetic diversity as a result of unintentional selection in C.
gigas, and as a result of intentional selection (a selective breeding program to
increase meat weight) in S. glomerata. This study has implications for aquaculture
species worldwide as it examines the levels of genetic diversity in introduced (eg
C. gigas) and a previously declining, native (S. glomerata) species, which
comprise two of the main groups of species utilised for aquaculture. The oyster aquaculture industry in Tasmania is based on Pacific oysters, C. gigas,
and derived from imports of Japanese oysters made some 40 years ago. Fears
were held that introduction and subsequent domestication had eroded the genetic
diversity. As the industry wished to embark on selective breeding programs, a
genetic audit of the hatchery stocks was required. Four Australian established (ie
non-hatchery produced) populations, and two of Japanese populations that
originally imported, were analysed to determine the amount of genetic diversity
present. Three different genetic techniques were employed - allozyme
electrophoresis, microsatellite DNA and mitochondrial DNA RFLP (restriction
fragment length polymorphism) analyses.
Using 17 allozyme loci, three hatchery and four naturalised populations of
Crassostrea gigas (Thunberg) in Australia were compared with one another and with two endemic Japanese populations. All populations showed a high degree of
genetic variability. The percent of polymorphic loci ranged from an average of
70.6% (hatcheries) through 73.5% (naturalised and Japan). Mean observed
heterozygosities ranged from 0.267 (naturalised) through 0.285 (hatcheries) to
0.291 (Japan). Mean numbers of alleles per locus ranged from 3.0 (hatcheries)
through 3.3 (naturalised) to 3.5 (Japan). Most loci and populations showed good
fits to Hardy-Weinberg expectations; the few significant exceptions were
heterozygote deficiencies. Tests of allele frequency differentiation among the nine
populations revealed that 11 of the 16 variable loci showed significant (a reduced
to 0.05/16 = 0.0031) inter-population heterogeneity after both x 2
. and GsT
analysis (Table 2.5). Four loci — GDA, 6PGDH, PEPS-I and PROT-2 — were
non-significant for both analyses, and EST-D was significant for x2 analysis (P <
0.001), but not after Bonferroni correction for GsT analysis (P = 0.004). Five
populations (BEA, BRI, DUN, SMI, SEN) conformed to Hardy-Weinberg
equilibrium for all loci. A few populations and loci did not conform (after
Bonferroni correction, using a = 0.0031): HIR (AK, DIA, PGI), NSW (PEPS-2),
PIT (DIA), and SWA (DIA) (data not shown). All the non-conforming samples
showed heterozygote deficiencies, which were significant in two cases: HR
(DIA: x2 = 35.67, P < 0.001; D — 0.314, P < 0.001); and SWA (DIA: X2
79.04, P = 0.001, D = - 0.397, P < 0.001). Allele-frequency differences among
populations were minor, although sometimes statistically significant: only about
1% of the allele frequency variation could be attributed to among-population
differences. The levels of homozygous excess observed in this study were lower
than that previously reported for this species, but may be due to the same reasons such as gel scoring errors, null alleles, selection, inbreeding, population admixture
or sampling error.
Four microsatellite loci (consisting of two dinucleotide, one tetranucleotide and
one tetranucleotide/dinucleotide motifs) were used to analyse the populations.
Primers were designed on clone sequence containing at least five repeats of a
microsatellite motif. Ten sets of primers were trialled but only four sets had
variability levels useful for population genetics analyses, as the others had
between 23-34 alleles or were monomorphic. High levels of genetic variability
were observed (mean polymorphism = 0.889, mean heterozygosity = 0.188).
Tests of allele frequency differentiation among the nine populations revealed
that three of the four variable loci showed significant (a reduced to 0.05/4 =
0.0125) inter-population heterogeneity after x2 analysis and Gsr analysis —
cmrCgl 7, BV59 and cmrCg61. The amount of differentiation among the
populations was, however, small. Across all loci, only 4% of the genetic
variation could be attributed to differences among populations. No population
conformed to Hardy-Weinberg equilibrium for all loci. Where populations did
not conform to Hardy-Weinberg equilibrium, a significant excess of
homozygotes was observed. Although null alleles have been previously
reported for the loci used in this study null alleles do not appear to explain the
heterozygote deficiencies observed in this study, based on analysis by the
NullTest program (W. Amos, pers. comm.) as the frequency of the proposed
null alleles seems too high. Unbiased (Nei, 1978) genetic distances over the
four loci were estimated between all pair-wise combinations of populations.
All pair-wise population distances are very small (Nei D<0.03). However, the standard errors of the distances are large (ranging to 0.0295±0.0867 between
the SWA/BEA/BRI/HIR/PIT/NSW/SMI/SEN and DUN clusters), indicating
that the groupings, based on only four loci, are not statistically meaningful.
There were no significant changes of genetic diversity between the
populations. Overall, the use of microsatellites confirmed the results of the
allozyme study of these populations. - the introduction of oysters from Japan to
Tasmania, and their subsequent domestication, have not substantially eroded
the genetic basis of the Tasmanian stock.
Very little variation was found using mitochondrial DNA (mtDNA) RFLP
analysis using 12 restriction enzymes in one hatchery and one endemic
population. The mtDNA fragment used was found to contain two conserved
genes explaining the lack of variability observed. The technical limitations and
lack of knowledge of the oyster mtDNA genome made this approach
inappropriate for population genetics analysis. However, the identification of
the proximity of the 16srRNA, and COIII genes observed in this study gives
further insight into the mtDNA gene order of C. gigas. Together with previous
findings of the mtDNA RE site map (Oohara and Mori, 1989), the location of
the cytochrome b gene . (Li and Hedgecock, 1998), this study demonstrates a
block of COIII, 16srRNA and cytochrome b mtDNA genes —a combination
unique to bivalves.
Two generations of a selected breeding line for increased whole weight (using
mass selection) and unselected group of the Sydney rock oyster Saccostrea
glomerata were examined using allozyme electrophoresis (a total of 14 loci analysed). Genetic variability levels were determined for each group - all were
high and similar to one another (mean percentage polymorphic, 66.7, mean
observed heterozygosity, 0.222; mean number of alleles, 2.5). Genotype
frequencies at all loci in all groups conformed to Hardy-Weinberg equilibrium,
except for the ESTD-2 locus in the second generation (P<0.001), which had a
large and significant excess of homozygotes (Selander index D = -0.451,
P<0.001). Thus significant allele frequency differences were observed at seven
loci between control and second generation, at eight loci between second and third
generation, and at only one locus for control and third generation. This suggests
that the second generation sample is responsible for most of the heterogeneity
observed. Despite the difference between actual and estimated broodstock
numbers, the expected numbers of alleles of the second and third generations of
the selected breeding line were very close to the observed numbers in all cases,
suggesting that random genetic drift (sampling variation) alone was the cause of
allelic variation between the groups. The results of this allozyme survey indicate
that the selective breeding for increased whole weight has not substantially eroded
levels of genetic variation. There are high levels of genetic variation present in the
control group and in the two generations of the selected breeding line. The
limited, but statistically significant, heterogeneity between the second generation
and other samples appears to be due to a sampling artifact; it is likely to be
biologically unimportant. The type of selective breeding program used (mass
selection) aided the retention of genetic diversity across the generations sampled.
By using oysters from 4 different areas and using large numbers of oysters for
spawning helped to achieve the levels of genetic diversity observed.
In summary, this study has not only substantially broadened the base of
knowledge in the two oysters species investigated, but also shown that intentional
(in the form of selective breeding) and unintentional (in the form of introduction
and subsequent domestication) selection of aquaculture species need not result in
major allele loss. Without major allele loss an aquaculture species then has the
potential for successful selective breeding for desired traits; as the genetic
diversity and hence likelihood of finding particular gene(s) of interest have not
been diminished significantly, and has every chance of success. That the two
commercially important edible oyster species (introduced and native) in this study
have not shown a large magnitude of genetic diversity loss despite either
introduction, naturalisation and domestication, or selective breeding serves as a
encouraging example to the oyster industries and researchers involved as well as
those in other countries and indeed other aquaculture species.

Item Type: Thesis - PhD
Authors/Creators:English, Louise,(Louise Jasmins)
Keywords: Oysters, Crassostrea, Sydney rock oyster
Copyright Holders: The Author
Copyright Information:

Copyright 2001 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (PhD)--University of Tasmania, 2001. Includes bibliographical references

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