The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of te...
The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.
The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.
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제안 방법
After initial amplification, six polymorphic dinucleotide microsatellites were selected based on the observed heterozygosity and PCR product size to ensure clear separation, and grouped into two panels based on the size range of the PCR products for multiplex PCR. Panel 1 included the loci, ucdCg170, Cg108, Cg49, which produced PCR products of 89 base pairs (bp) to 201 bp.
PCR conditions were as follows: 11 min at 95℃, followed by 35 cycles of 1 min at 94℃, 1 min at the annealing temperature listed in Table 1, and 1 min at 72℃, with a final extension of 5 min at 72℃. Microsatellite polymorphisms were screened using an ABI PRISM 3130 XL automated DNA sequencer (Applied Biosystems, Boston), and alleles were designated according to PCR product size, relative to a molecular size marker (GENESCAN 400 HD [ROX]; PE Applied Biosystems, Boston).
PCR amplification was performed in a 10 μl reaction mixture containing 0.25 U Ex taq DNA polymerase (TaKaRa Biomedical Inc., Shiga, Japan), 1× PCR buffer, 0.2 mM dNTP mix, 10 pmol of each primer (forward primer of each pair was 5'-end-labeled with 6-FAM, NED, and HEX dyes; PE Applied Biosystems, Boston), and 100 ng template DNA, using a PTC 200 DNA Engine (MJ Research, Waltham, MA, USA).
gigas. The development of an accurate protocol for triploidy verification of C. gigas using appropriate polymorphic microsatellite markers and their multiplex PCR was analyzed in this study.
후속연구
However, tetraploid oyster production is not feasible in all oysters; tetraploid oyster production was unsuccessful in species such as the Sydney rock oyster (Nell, 2002). Therefore, application of the methods described in this study can be applied to the future development of triploid oysters and mollusks.
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